WO2021056754A1 - 一种酸法制备抗体药物偶联物中间体的方法及其应用 - Google Patents

一种酸法制备抗体药物偶联物中间体的方法及其应用 Download PDF

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WO2021056754A1
WO2021056754A1 PCT/CN2019/118446 CN2019118446W WO2021056754A1 WO 2021056754 A1 WO2021056754 A1 WO 2021056754A1 CN 2019118446 W CN2019118446 W CN 2019118446W WO 2021056754 A1 WO2021056754 A1 WO 2021056754A1
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add
mmaf
reaction
acid
pipette
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PCT/CN2019/118446
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French (fr)
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李乐乐
黄长江
孙友祥
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烟台迈百瑞国际生物医药股份有限公司
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Application filed by 烟台迈百瑞国际生物医药股份有限公司 filed Critical 烟台迈百瑞国际生物医药股份有限公司
Priority to CN201980004121.4A priority Critical patent/CN111328333B/zh
Priority to CA3076714A priority patent/CA3076714C/en
Priority to US16/650,273 priority patent/US11833219B2/en
Priority to KR1020217018833A priority patent/KR20210095167A/ko
Priority to JP2021525571A priority patent/JP7252333B2/ja
Priority to AU2019341067A priority patent/AU2019341067B2/en
Priority to EP19941775.9A priority patent/EP3828196A4/en
Publication of WO2021056754A1 publication Critical patent/WO2021056754A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • 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
    • A61K38/05—Dipeptides
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A61K47/545—Heterocyclic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin

Definitions

  • the invention relates to the field of antibody drug conjugates, in particular to a method for preparing an antibody drug conjugate intermediate and its application in preparing antibody drug conjugates.
  • ADC Antibody-Drug Conjugate
  • ADC Antibody-Drug Conjugate
  • the three components of ADC together form a targeted drug delivery system, where the antibody achieves targeting, and the linker ensures the stability of the ADC during blood transport, and after reaching the target point,
  • the toxin exerts a killing effect on cancer cells.
  • auristatin or maytansinoids are the two most widely used mitosis inhibitors in ADC development. They can bind to tubulin near the vinblastine binding site, causing G2/M cell cycle arrest and subsequent apoptosis. This cell killing mechanism is very effective in rapidly proliferating cells, but non-dividing and static cells may be less sensitive to the effects of drugs, leading to drug resistance. Because tumor cells divide faster than most normal cells, they are resistant to mitosis. The drug is particularly effective on cancer cells.
  • tubulin inhibitors such as maytansine and auristatin
  • tubulin inhibitors have been successfully used as clinically approved ADC drugs (brentuximabvedotin, trastuzumabemtansine, etc.).
  • Dolastatin is a class of linear polypeptide compounds with high cytotoxicity discovered from censored sea hares in the Indian Ocean. This type of cytotoxic drug can significantly inhibit the formation and polymerization of tubulin. However, donotoxin 10 has large toxic and side effects, poor pharmacokinetic properties, and narrow therapeutic window when used alone, which limits its development. Miyazaki et al.
  • MMAD monomethyl auristatin D
  • auristatin N-monomethyl substituted dorestatin derivative
  • MMAE Hu Xinyue, Li Yanping, Li Zhuorong. Research progress on warhead molecules of antibody-drug conjugates[J].China Medical Biotechnology,2017(6):549- 555.).
  • MMAF Monomethyl auristatin F
  • D10 dolastatin 10
  • MMAF monomethyl auristatin derivative among ADCs.
  • these small molecule drugs are expensive.
  • the market price of 10mg of MMAF is close to 5,000 yuan, and the intermediate Mc-MMAF used for coupling to antibodies is close to 6,000 yuan.
  • the high price of ADC intermediates is an important factor in the high production cost of related ADCs.
  • Mc-MMAF The structure of Mc-MMAF is as follows:
  • Mc-MMAE The structure of Mc-MMAE is as follows:
  • Mc-MMAD The structure of Mc-MMAD is as follows:
  • the Chinese patent with patent publication number CN1938046A discloses two methods for preparing Mc-MMAF (pages 206-207 of the specification and pages 207-208 of the specification), one of which is to synthesize Mc-MMAF with tert-butyl ester, and the other It uses dimethoxybenzyl ester to synthesize Mc-MMAF.
  • the process routes of these two methods are:
  • Mc-MMAF route using tert-butyl ester is to first synthesize Mc-MMAF-OtBu (ie Mc-MeVal-Val-Dil-Dap-Phe-OtBu), and then use trifluoro Acetic acid is deesterified to obtain Mc-MMAF (total yield is less than 60%).
  • the route of using dimethoxybenzyl ester to synthesize Mc-MMAF is to first synthesize Mc-MMAF-ODMB (ie Mc-MeVal-Val-Dil-Dap-Phe-ODMB), and then deesterify with trifluoroacetic acid to obtain Mc- MMAF.
  • the disadvantage is that in the two-step synthesis, the yield in the first step is only 57%, the yield in the second step is 73%, and the total yield is only 42%, and MMAF-ODMB is not easy to obtain.
  • the N-terminal valine of this route of MMAF has a methyl group on the N, which is sterically hindered.
  • the reaction speed It will be slower.
  • This route is used for the synthesis of MC-MMAF of less than 1 g, and finally high-pressure reverse phase preparation is used to remove isomeric impurities, and the yield is less than 50%.
  • the present invention provides a method for preparing an antibody drug conjugate intermediate by an acid method.
  • the technical scheme of the present invention is:
  • a method for preparing an antibody-drug conjugate intermediate by an acid method is as follows:
  • L is any linking group containing an acyl structure
  • L' is the residue of the linking group after covalently linking the monomethyl auristatin derivative
  • Solvent A and solvent B are either polar or non-polar solvents
  • the method includes the following steps:
  • the molar amount of the organic base added in the step 1) is greater than the molar amount of all free carboxyl groups in the reaction system of the step 3).
  • the acid is selected from one or more combinations of trifluoroacetic acid and sulfonic acid.
  • the monomethyl auristatin derivatives include MMAF, MMAE and MMAD.
  • reaction route of the method is as follows:
  • the condensing agent is DCC, EDCI, DIC, HATU, HBTU, HBPIPU, HBPyU, HCTU, HDMA, TATU, TBTU, TCTU, TCFH, TDBTU, TFFH, BTFFH, PyBOP, PyClOP, PyAOP, PyCIU, DEPBT
  • the condensing agent is HATU.
  • the organic base is N,N-diisopropylethylamine, triethylamine, pyridine, dimethylaminopyridine, triethylenediamine, N-methylmorpholine, 1,8-diazepine
  • the organic base is N, N-di Isopropylethylamine, triethylamine.
  • solvent A and solvent B are independently selected from DMF, DMA, NMP, dichloromethane, carbon tetrachloride, DMSO, chloroform, tetrahydrofuran, 1,4-dioxane, hexamethylphosphoryl
  • solvent A and solvent B can be the same or different; preferably, solvent A and solvent B are DMF, DMA , DMSO.
  • the sulfonic acid includes p-toluenesulfonic acid, benzenesulfinic acid, trifluoromethanesulfonic acid, (-)-10-camphorsulfonic acid, (+)-10-camphorsulfonic acid, methanesulfonic acid
  • the p-toluenesulfonic acid is preferably p-toluenesulfonic acid monohydrate.
  • said L is any linking group containing an acyl structure, and its structure is as shown in formula (I):
  • R 1 is selected from
  • R 2 is selected from And its salt.
  • said L is selected from the following structures: the following structures:
  • the L’-MMAF is selected from the following structures:
  • the molar ratio of the amount of the acid to the corresponding monomethyl auristatin derivative is preferably greater than or equal to 1 or greater than or equal to 2.
  • the method of adding the solution 1 to the solution 2 is uniform addition or dropwise addition.
  • the present invention also provides the application of any one of the above methods in preparing antibody drug conjugates.
  • the acid method provided by the present invention prepares the antibody drug conjugate intermediate (specifically the linking group-MMAF covalent conjugate), which is based on the traditional preparation method (Patent Publication No. CN1938046A Chinese Patent Specification Page 2
  • the preparation method disclosed in row [0009] is based on the addition of acidic additives, so that MMAF becomes a salt and participates in the reaction. After a series of reactions, we were surprised to find that the addition of acidic additives can significantly increase the yield of the final product. , And the acid price is low (relative to MMAF can be ignored), which will greatly reduce the production cost of the final ADC product.
  • the preparation method provided by the present invention adopts a one-step preparation method, which not only has a higher yield of the final product, but also saves money in production. It reduces the cost of production consumables, labor, equipment, site, raw materials, etc., and also greatly reduces the production waste liquid, reduces production costs, improves production efficiency, and is suitable for industrial large-scale production.
  • Fig. 1 is a liquid chromatogram of the product with various sulfonic acid additives added in Example 2 of the present invention.
  • Figure 2 is the area ratio of the reaction product when adding different molar amounts of p-toluenesulfonic acid monohydrate in Example 3 of the present invention (the area ratio refers to the ratio of the peak area of the product in the chromatogram to the reference area, and the reference area uses acidic additives and MMAF
  • the peak area when the dosage molar ratio is equal to 1:1 is a broken line graph.
  • Fig. 3 is a liquid chromatogram of the reaction product of adding different molar amounts of N,N-diisopropylethylamine in Example 4 of the present invention.
  • HBPIPU (Benzotriazol-1-yloxy)dipiperidine carbohexafluorophosphate HBPyU O-(benzotriazol-1-yl)-N,N,N',N'-dipyrrolyl urea hexafluorophosphate
  • HBPyU O-(benzotriazol-1-yl)-N,N,N',N'-dipyrrolyl urea hexafluorophosphate
  • HDMA 1-[(Dimethylamino)(morpholine)methyl]-3-oxo-1H-[1,2,3]triazole[4,5-b]pyridine 3-hexafluorophosphate
  • TBTU O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluorobo
  • an antibody drug conjugate includes a combination of two or more antibody drug conjugates and the like.
  • linking group in the present invention refers to a molecule with bifunctional or multifunctional groups that can react with protein/antibody molecules and MMAF respectively, so it acts as a "bridge” to connect the protein/antibody with MMAF .
  • the linking group involved in the present invention specifically refers to those groups containing an acyl structure in the structure.
  • antibody drug conjugate intermediate in the present invention refers to a covalent conjugate of a linking group and MMAF.
  • the unit in the weight-volume percentage in the present invention is well-known to those skilled in the art, for example, refers to the weight of the solute in a 100 ml solution.
  • Mc-OH 84.6mg and HATU 144.6mg add them to a 50mL round bottom flask, use a 5mL syringe to take 5mL N, N-dimethylformamide into the round bottom flask, and then use a 200 ⁇ L pipette to take 165.4 ⁇ L N,N -Add diisopropylethylamine to the sample bottle, place it on a magnetic stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • the solvent is spin-dried with a rotary evaporator, and 5 mL of acetonitrile is added, and after suction with a 5 mL syringe, the organic phase needle filter is used to filter into a 10 mL sample bottle to prepare a liquid phase method for purification.
  • the preparation method is as follows: mobile phase A: H2O, 0.1% HCOOH, mobile phase B: MeCN, 0.1% HCOOH, flow rate 40 mL/min, gradient: 25% B-70% B, 30 min, and peak at 25.1 min.
  • Mc-MMAF preparation method 2 (adding trifluoroacetic acid)
  • Mc-OH 84.8mg and HATU 144.7mg add them to a 50mL round bottom flask, use a 5mL syringe to take 5mL N, N-dimethylformamide into the round bottom flask, and then use a 200 ⁇ L pipette to take 165.4 ⁇ L N,N -Add diisopropylethylamine to the sample bottle, place it on a magnetic stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • MMAF and 22.8mg of trifluoroacetic acid add them to a 25mL round-bottomed flask, use a 10mL syringe to take 10mL N,N-dimethylformamide into the sample bottle, add a stir bar, place it on the magnetic stirrer, add Stir in an ice bath. Under ice bath conditions, use a 5 mL plastic dropper to transfer the Mc-OH system into the MMAF solution, remove the ice bath on the magnetic stirrer, and continue stirring for 1 h.
  • the solvent is spin-dried with a rotary evaporator, and 5 mL of acetonitrile is added, and after suction with a 5 mL syringe, the organic phase needle filter is used to filter into a 10 mL sample bottle to prepare a liquid phase method for purification.
  • the preparation method is as follows: mobile phase A: H2O, 0.1% HCOOH, mobile phase B: MeCN, 0.1% HCOOH, flow rate 40 mL/min, gradient: 25% B-70% B, 30 min, and peak at 25.1 min.
  • Mc-MMAF preparation method 3 (addition of p-toluenesulfonic acid monohydrate)
  • Mc-OH 84.6mg and HATU 144.2mg add them to a 50mL round bottom flask, use a 5mL syringe to take 5mL N, N-dimethylformamide into the round bottom flask, and then use a 200 ⁇ L pipette to take 165.4 ⁇ L N,N -Add diisopropylethylamine to the sample bottle, place it on a magnetic stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • MMAF and 34.8mg of p-toluenesulfonic acid monohydrate add them to a 25mL round bottom flask, use a 10mL syringe to take 10mL N,N-dimethylformamide into the sample bottle, add a stir bar, and place it under magnetic stirring On the device, add an ice bath and stir. Under ice bath conditions, use a 5 mL plastic dropper to transfer the Mc-OH system into the MMAF solution, remove the ice bath on the magnetic stirrer, and continue stirring for 1 h.
  • the solvent is spin-dried with a rotary evaporator, and 5 mL of acetonitrile is added, and after suction with a 5 mL syringe, the organic phase needle filter is used to filter into a 10 mL sample bottle to prepare a liquid phase method for purification.
  • the preparation method is as follows: mobile phase A: H2O, 0.1% HCOOH, mobile phase B: MeCN, 0.1% HCOOH, flow rate 40 mL/min, gradient: 25% B-70% B, 30 min, and peak at 25.1 min.
  • Mc-MMAF preparation method 4 (addition of (-)-10-camphorsulfonic acid):
  • Mc-OH 84.2mg and HATU 144.4mg add them to a 50mL round bottom flask, use a 5mL syringe to take 5mL N, N-dimethylformamide into the round bottom flask, and then use a 200 ⁇ L pipette to take 165.4 ⁇ L N,N -Add diisopropylethylamine to the sample bottle, place it on a magnetic stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • the solvent is spin-dried with a rotary evaporator, and 5 mL of acetonitrile is added, and after suction with a 5 mL syringe, the organic phase needle filter is used to filter into a 10 mL sample bottle to prepare a liquid phase method for purification.
  • the preparation method is as follows: mobile phase A: H2O, 0.1% HCOOH, mobile phase B: MeCN, 0.1% HCOOH, flow rate 40 mL/min, gradient: 25% B-70% B, 30 min, and peak at 25.1 min.
  • Mc-MMAF preparation method 5 (adding trifluoromethanesulfonic acid)
  • Mc-OH 84.6mg and HATU 144.7mg add them to a 50mL round bottom flask, use a 5mL syringe to take 5mL N, N-dimethylformamide into the round bottom flask, and then use a 200 ⁇ L pipette to take 165.4 ⁇ L N,N -Add diisopropylethylamine to the sample bottle, place it on a magnetic stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • MMAF and 30.0mg of trifluoromethanesulfonic acid add them to a 25mL round bottom flask, use a 10mL syringe to take 10mL N,N-dimethylformamide into the sample bottle, add a stir bar, and place on the magnetic stirrer , Stir in an ice bath. Under ice bath conditions, use a 5 mL plastic dropper to transfer the Mc-OH system into the MMAF solution, remove the ice bath on the magnetic stirrer, and continue stirring for 1 h.
  • Mc-MMAF preparation method 2 we made further explorations on the basis of the above-mentioned Mc-MMAF preparation method 1.
  • the MMAF was first converted into a salt by adding acid or alkali reagents and then the subsequent reaction was carried out.
  • Mc-MMAF preparation method 3 p-toluenesulfonic acid monohydrate
  • the product yield of Mc-MMAF has also been surprisingly improved.
  • the product yield of Mc-MMAF increased from 52% to 68%, and the absolute value of the yield increased by 16%.
  • the relative value of the yield after adding acid increased by 30.7% ((68 %-52%)/52%).
  • the Mc-MMAF product yields in the test group with (-)-10-camphorsulfonic acid and trifluoromethanesulfonic acid also reached 64% and 66%, respectively.
  • Test Example 1 Adding (-)-10-camphorsulfonic acid: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, use a 1 mL pipette to take 1 mL of N,N-dimethylformamide and add In the sample bottle, use a 100 ⁇ L pipette to take 22.3 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on a parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • MMAF 20.0mg and (-)-10-camphorsulfonic acid 6.3mg add to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add stirring Put it on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 2 Adding (+)-10-camphorsulfonic acid: Weigh 11.5mg of Mc-OH and 20.5mg of HATU, add them to a 5mL sample bottle, use a 1mL pipette to take 1mL of N,N-dimethylformamide and add In the sample bottle, use a 100 ⁇ L pipette to take 22.3 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on a parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • MMAF 20.0mg and (+)-10-camphorsulfonic acid 6.3mg add to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add stirring Put it on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 3 Adding 10-camphorsulfonic acid: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, use a 1 mL pipette to take 1 mL of N,N-dimethylformamide into the sample bottle, and then Use a 100 ⁇ L pipette to take 22.3 ⁇ L N,N-diisopropylethylamine into the sample bottle, place it on a parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • MMAF and 6.3 mg of 10-camphorsulfonic acid add them to another 5 mL sample bottle, use a 1 mL pipette to take two 1 mL N,N-dimethylformamide into the sample bottle, add a stir bar, and place On a magnetic stirrer, add an ice bath to stir. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 4 Adding methanesulfonic acid: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, use a 1 mL pipette to take 1 mL of N,N-dimethylformamide into the sample bottle, and then use Take 22.3 ⁇ L of N,N-diisopropylethylamine with a 100 ⁇ L pipette and add it to the sample bottle, place it on a parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • Test Example 5 Adding trifluoromethanesulfonic acid: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, use a 1 mL pipette to take 1 mL of N,N-dimethylformamide and add it to the sample bottle, and then Use a 100 ⁇ L pipette to take 22.3 ⁇ L N,N-diisopropylethylamine into the sample bottle, place it on a parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • MMAF and 4.1 mg of trifluoromethanesulfonic acid add them to another 5 mL sample bottle, use a 1 mL pipette to take two 1 mL N,N-dimethylformamide into the sample bottle, add a stir bar, and place On a magnetic stirrer, add an ice bath to stir. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 6 Adding p-toluenesulfonic acid monohydrate: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, use a 1 mL pipette to take 1 mL of N,N-dimethylformamide and add it to the sample bottle , Then use a 100 ⁇ L pipette to take 22.3 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • MMAF and 4.7mg of p-toluenesulfonic acid monohydrate add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add a stir bar, Place on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • test results are shown in Figure 1.
  • Various sulfonic acids are added, such as (-)-10-camphorsulfonic acid, (+)-10-camphorsulfonic acid, 10-camphorsulfonic acid, methylsulfonic acid and trifluoromethane
  • sulfonic acid additives to the reaction system can achieve a reaction promotion effect equivalent to that of adding p-toluenesulfonic acid monohydrate additives, and can effectively increase the product yield of Mc-MMAF.
  • Test Example 1 No acidic additives: Weigh 11.5 mg (0.055 mmol) of Mc-OH and 20.5 mg (0.054 mmol) of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformaldehyde The amide was added to the sample bottle, and then 35.7 ⁇ L (0.216 mmol) of N,N-diisopropylethylamine was added to the sample bottle with a 100 ⁇ L pipette, placed on a parallel reaction stirrer, added with a stir bar, and stirred at room temperature for 0.5 h.
  • Test Example 2 The molar ratio of acid additive to MMAF dosage is equal to 1:100: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • Test Example 3 The molar ratio of acidic additive to MMAF is 1:20: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • Test example 4 The molar ratio of acidic additives to MMAF is 1:10: Weigh 11.5mg of Mc-OH and 20.5mg of HATU, add them to a 5mL sample bottle, and use a 1mL pipette to take 1mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • Test Example 5 The molar ratio of acidic additives to MMAF is 1:5: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5h.
  • Test Example 6 The molar ratio of acidic additive to MMAF dosage is equal to 2:5: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • Test Example 7 The molar ratio of acidic additives to MMAF is 1:2: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • Test Example 8 The molar ratio of acid additive to MMAF dosage is equal to 1:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • MMAF 20.0mg and p-toluenesulfonic acid monohydrate 4.6mg (0.027mmol) add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, Add a stir bar, place it on a magnetic stirrer, and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 9 The molar ratio of acidic additives to MMAF is 2:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • Test Example 10 The molar ratio of acid additive to MMAF dosage is equal to 3:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • Test Example 11 The molar ratio of acidic additives to MMAF is 4:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL of N,N-dimethylformamide Add the sample bottle, and then use a 100 ⁇ L pipette to take 35.7 ⁇ L of N,N-diisopropylethylamine into the sample bottle, place it on the parallel reaction stirrer, add a stir bar, and stir at room temperature for 0.5 h.
  • DIPEA is taken as an example to further carry out related experiments.
  • Test Example 1 The molar ratio of alkaline additives (DIPEA) to acidic additives is 2:1: Weigh 11.5 mg (0.055 mmol) of Mc-OH and 20.5 mg (0.054 mmol) of HATU, add them to a 5 mL sample bottle, and transfer with 1 mL Take 1mL of N,N-dimethylformamide from the liquid gun and add it to the sample bottle, then use a 100 ⁇ L pipette to take 8.9 ⁇ L of N,N-diisopropylethylamine (0.055mmol) into the sample bottle, and place it in the parallel reaction stirrer Add a stirring bar and stir at room temperature for 0.5h.
  • DIPEA alkaline additives
  • Test Example 2 The molar ratio of alkaline additives (DIPEA) to acidic additives is 3:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL N,N- Add dimethylformamide to the sample bottle, then use a 100 ⁇ L pipette to take 13.4 ⁇ L of N,N-diisopropylethylamine (0.081mmol) into the sample bottle, place it on the parallel reaction stirrer, add the stir bar, and stir at room temperature 0.5h.
  • DIPEA alkaline additives
  • MMAF and 4.6mg of p-toluenesulfonic acid monohydrate add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add a stir bar, Place on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 3 The molar ratio of alkaline additive (DIPEA) to acidic additive is 4:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL N,N- Add dimethylformamide to the sample bottle, then use a 100 ⁇ L pipette to take 17.9 ⁇ L of N,N-diisopropylethylamine (0.108mmol) into the sample bottle, place it on the parallel reaction stirrer, add the stir bar, and stir at room temperature 0.5h.
  • DIPEA alkaline additive
  • MMAF and 4.6mg of p-toluenesulfonic acid monohydrate add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add a stir bar, Place on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 4 The molar ratio of alkaline additives (DIPEA) to acid additives is 5:1: Weigh 11.5 mg of Mc-OH and 20.5 mg of HATU, add them to a 5 mL sample bottle, and use a 1 mL pipette to take 1 mL N,N- Add dimethylformamide to the sample bottle, then use a 100 ⁇ L pipette to take 22.3 ⁇ L of N,N-diisopropylethylamine (0.135mmol) into the sample bottle, place it on the parallel reaction stirrer, add the stir bar, and stir at room temperature 0.5h.
  • DIPEA alkaline additives
  • MMAF and 4.6mg of p-toluenesulfonic acid monohydrate add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add a stir bar, Place on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 5 The molar ratio of alkaline additive (DIPEA) to acidic additive is 6:1: Weigh 11.5mg of Mc-OH and 20.5mg of HATU, add them to a 5mL sample bottle, and use a 1mL pipette to take 1mL N,N- Add dimethylformamide to the sample bottle, then use a 100 ⁇ L pipette to take 26.8 ⁇ L of N,N-diisopropylethylamine (0.162 mmol) into the sample bottle, place it on the parallel reaction stirrer, add the stir bar, and stir at room temperature 0.5h.
  • DIPEA alkaline additive
  • MMAF and 4.6mg of p-toluenesulfonic acid monohydrate add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add a stir bar, Place on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.
  • Test Example 6 The molar ratio of alkaline additives (DIPEA) to acidic additives is 7:1: Weigh 11.5mg of Mc-OH and 20.5mg of HATU, add them to a 5mL sample bottle, and use a 1mL pipette to take 1mL N,N- Add dimethylformamide to the sample bottle, then use a 100 ⁇ L pipette to take 31.3 ⁇ L of N,N-diisopropylethylamine (0.189 mmol) into the sample bottle, place it on the parallel reaction stirrer, add the stir bar, and stir at room temperature 0.5h.
  • DIPEA alkaline additives
  • MMAF and 4.6mg of p-toluenesulfonic acid monohydrate add them to another 5mL sample bottle, use a 1mL pipette to take two 1mL N,N-dimethylformamide into the sample bottle, add a stir bar, Place on a magnetic stirrer and stir in an ice bath. Under ice bath conditions, use a 5mL plastic dropper to transfer the Mc-OH system into the MMAF solution, place the reaction flask on the parallel reactor, and continue stirring for 1 hour.

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Abstract

一种酸法制备抗体药物偶联物中间体的方法，该方法采用酸性添加剂，使得单甲基auristatin衍生物变成盐之后参与反应，酸性添加剂的加入能够显著的提高最终产物的产率，且酸的价格低廉，大大降低最终ADC产物的生产成本。

Description

一种酸法制备抗体药物偶联物中间体的方法及其应用 技术领域
本发明涉及抗体药物偶联物领域，具体涉及一种抗体药物偶联物中间体的制备方法及其在制备抗体药物偶联物中的应用。
背景技术
抗体偶联药物(Antibody-Drug Conjugate，ADC)作为一种新型的生物导弹，实现了单抗靶向作用和小分子药物细胞毒性的强强联合，现已成为肿瘤靶向治疗发展最快的领域之一。ADC的三个构件(抗体、细胞毒素和连接子)共同组成靶向药物递送系统，其中抗体实现靶向性，连接子保证在ADC在血液转运过程中的稳定性，而到达作用靶点后，毒素发挥对癌细胞的杀伤作用。
目前auristatin或maytansinoids是ADC开发中最广泛使用的两种有丝分裂抑制剂，它们能在长春碱结合位点附近与微管蛋白结合，引起G2/M细胞周期阻滞和随后的细胞凋亡。这种细胞杀伤机制在快速增殖的细胞中非常有效，但非分裂和静态细胞可能对药物作用不太敏感，导致产生耐药性，因肿瘤细胞分裂速度比大多数正常细胞更快，所以抗有丝分裂药物对癌细胞特别有效。由于这种固有的选择性，高效的微管蛋白抑制剂，如美登素和auristatin，已成功用作临床批准的ADC药物(brentuximabvedotin和trastuzumabemtansine等)。
海兔毒素(dolastatin)是从印度洋生物截尾海兔中发现的一类具有高细胞毒性的线性多肽类化合物，这类细胞毒药物能够显著抑制微管蛋白的形成和聚合。然而海兔毒素10在单独使用时存在毒副作用大、药代动力学性质差、治疗窗口窄等问题限制了其开发。Miyazaki等发现N端为仲胺的单甲基奥瑞他汀D(monomethyl auristatin D，MMAD)毒性与海兔毒素10相当，将这类N-单甲基取代的海兔毒素衍生物命名为奥瑞他汀类(auristatin)，它们可实现与连接子的有效连接，新一代的高效ADC也由此产生。目前，ADC研发常用的弹头分子为奥瑞他汀类化合物MMAE和MMAF(胡馨月,李艳萍,李卓荣.抗体药物偶联物的弹头分子研究进展[J].中国医药生物技术,2017(6):549-555.)。
MMAF(Monomethyl auristatin F)是海兔毒素10(dolastatin10，D10)的衍生物，可以抑制细胞有丝分裂，具有很强的抗肿瘤活性。目前已有多 个处于临床阶段的ADC产品偶联的细胞毒分子即为MMAF，也是目前ADC中选择最多的一种单甲基auristatin衍生物。但是，这些小分子药物价格昂贵。
MMAF市场售价10mg的价格接近5000元，而用于偶联于抗体的中间体Mc-MMAF 1mg即接近6000元，从MMAF制备到Mc-MMAF中间体后的价格提升十多倍(MMAF、Mc-MMAF的价格可参见如下网址：https://www.medchemexpress.cn/mmaf-hydrochloride.html？src＝360-prod uct、https://www.medchemexpress.cn/McMMAF.html)。ADC中间体价格高昂是相关ADC生产成本高的一个重要因素。
相关ADC中间体(连接子-药物共价偶联物)价格相对于相应细胞毒分子价格急剧提升的一个重要原因是抗体药物偶联物中间体(即制备过程的收率较低。因此，提高相关ADC中间体的收率将能有效的控制ADC药物大规模生产时的成本。
Mc-MMAF结构如下所示：
Figure PCTCN2019118446-appb-000001
Mc-MMAE结构如下所示：
Figure PCTCN2019118446-appb-000002
Mc-MMAD结构如下所示：
Figure PCTCN2019118446-appb-000003
专利公开号为CN1938046A的中国专利公开了两种制备Mc-MMAF的方法(说明书第206-207页和说明书第207-208页)，其中一种是利用叔丁酯合成Mc-MMAF，另一种是利用二甲氧基苄基酯合成Mc-MMAF，这两种方法的工艺路线分别为：
(1)利用叔丁酯合成Mc-MMAF
Figure PCTCN2019118446-appb-000004
(2)利用二甲氧基苄基酯合成Mc-MMAF
Figure PCTCN2019118446-appb-000005
这两个路线都采用两步合成的方法，利用叔丁酯合成Mc-MMAF路线是先合成Mc-MMAF-OtBu(即Mc-MeVal-Val-Dil-Dap-Phe-OtBu)，再用三氟乙酸脱酯基，得到Mc-MMAF(总产率低于60％)。利用二甲氧基苄基酯合成Mc-MMAF路线是先合成Mc-MMAF-ODMB(即Mc-MeVal-Val-Dil-Dap-Phe-ODMB)，再用三氟乙酸脱酯基，得到Mc-MMAF。缺点是两步合成，第一步产率只有57％，第二步产率73％， 总产率仅为42％，而且MMAF-ODMB不易获得。
专利公开号为CN109824759A的中国专利也公开了一种制备Mc-MMAF的方法(说明书第2页第[0009]行)，它是利用Mc与MMAF直接反应，其工艺路线为：
Figure PCTCN2019118446-appb-000006
该路线MMAF的N终端缬氨酸的N上带有一个甲基，位阻较大，在这种情况下，将马来酰亚氨基正己酸(Mc-hex-Acid)接到MMAF上反应速度就会较慢。该路线用于小于1g的MC-MMAF合成，最后要应用高压反相制备除去异构杂质，产率低于50％。
发明内容
本发明提供了一种酸法制备抗体药物偶联物中间体的方法，本发明的技术方案为：
一种酸法制备抗体药物偶联物中间体的方法，所述方法的反应路线如下：
Figure PCTCN2019118446-appb-000007
其中：
L为任意包含酰基结构的连接基团；
L’为共价连接单甲基auristatin衍生物后的连接基团残基；
溶剂A和溶剂B为任一极性或非极性溶剂；
所述方法包括以下步骤：
1)将连接基团L和缩合剂、有机碱溶解在溶剂A中，形成溶液1；
2)将单甲基auristatin衍生物和酸溶解在溶剂B中，形成溶液2；
3)将溶液1加到溶液2中，L与单甲基auristatin衍生物通过缩合反应得到L’-单甲基auristatin衍生物；
所述步骤1)中加入的有机碱的摩尔量大于步骤3)的反应体系中所有游离羧基的摩尔量。
进一步的，所述的酸选自三氟乙酸、磺酸中的一种或多种组合。
进一步的，所述单甲基auristatin衍生物包括MMAF、MMAE和MMAD。
进一步的，所述方法的反应路线如下：
Figure PCTCN2019118446-appb-000008
Figure PCTCN2019118446-appb-000009
进一步的，所述的缩合剂为DCC、EDCI、DIC、HATU、HBTU、HBPIPU、HBPyU、HCTU、HDMA、TATU、TBTU、TCTU、TCFH、TDBTU、TFFH、BTFFH、PyBOP、PyClOP、PyAOP、PyCIU、DEPBT、EEDQ中的一种或多种，优选的，所述的缩合剂为HATU。
进一步的，所述的有机碱为N，N-二异丙基乙胺、三乙胺、吡啶、二甲氨基吡啶、三乙烯二胺、N-甲基吗啉、1,8-二氮杂双环[5.4.0]-7-十一碳烯、N-甲基咪唑、奎宁环、三甲基吡啶中的一种或几种，优选的，所述的有机碱为N，N-二异丙基乙胺、三乙胺。
进一步的，所述的溶剂A和溶剂B独立地选自DMF、DMA、NMP、二氯甲烷、四氯化碳、DMSO、氯仿、四氢呋喃、1,4-二氧六环、六甲基磷酰三胺、N，N-二甲基丙烯基脲、乙二醇二甲醚的一种或几种，溶剂A和溶剂B可以相同也可以不同；优选的，溶剂A和溶剂B为DMF、DMA、DMSO。
更进一步的，所述的磺酸包括对甲苯磺酸、苯亚磺酸、三氟甲磺酸、(-)-10-樟脑磺酸、(+)-10-樟脑磺酸、甲基磺酸中的一种或几种，所述的对甲苯磺酸优选为对甲苯磺酸一水化合物。
优选的，所述的L为任意包含酰基结构的连接基团，其结构如式(I)所示：
Figure PCTCN2019118446-appb-000010
其中：
R 1选自
Figure PCTCN2019118446-appb-000011
Figure PCTCN2019118446-appb-000012
R 2选自
Figure PCTCN2019118446-appb-000013
Figure PCTCN2019118446-appb-000014
及其盐。
更优选的，所述的L选自如下结构：如下结构：
Figure PCTCN2019118446-appb-000015
Figure PCTCN2019118446-appb-000016
更优选的，所述的L’-MMAF选自如下结构：
Figure PCTCN2019118446-appb-000017
更进一步的，所述酸与相应单甲基auristatin衍生物用量摩尔比优选大于或等于1或大于或等于2。
更进一步的，所述溶液1加入溶液2的方式为均匀加入或滴加。
本发明还提供了上述任一项所述的方法在制备抗体药物偶联物中的 应用。
本发明提供的酸法制备抗体药物偶联物中间体(具体为连接基团-MMAF共价偶联物)的方法，是在传统的制备方法(专利公开号为CN1938046A的中国专利说明书第2页第[0009]行公开的制备方法)基础上添加了酸性添加剂，使得MMAF变成盐之后参与反应，经过一系列的反应，我们惊喜的发现，酸添加剂的加入能够显著的提高最终产物的产率，且酸的价格低廉(相对于MMAF可以忽略不计)，这将大大降低最终ADC产物的生产成本。除此之外，相较于专利公开号为CN1938046A公开的两种方法来说，本发明提供的制备方法采用的是一步制备方法，除具有更高的终产物产率外，在生产中既节省了生产耗材、人工、设备、场地、原料等成本，也大大降低了产生的生产废液，降低了生产成本，提高了生产效率，适用于工业大规模生产。
附图说明
图1是本发明实施例2中加入各种磺酸类添加剂的产物的液相色谱图。
图2是本发明实施例3中加入不同摩尔量对甲苯磺酸一水合物时反应产物的面积比(面积比指色谱图中产物的峰面积与基准面积的比值，基准面积采用酸性添加剂与MMAF用量摩尔比等于1：1时的峰面积)折线图。
图3是本发明实施例4中加入不同摩尔量N,N-二异丙基乙胺反应产物的液相色谱图。
具体实施方式
[缩写]
除非另有说明，本发明使用的所有缩写具有本领域普通技术人员所理解的相同含义。如本发明所用，常用的缩写及其定义如下所示：
缩写 定义
DCC N,N'-二环己基碳二亚胺
EDCI 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐
DIC N,N'-二异丙基碳二亚胺
HATU 2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯
HBTU 苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐
HBPIPU (苯并三氮唑-1-基氧基)二哌啶碳六氟磷酸盐
HBPyU O-(苯并三唑-1-基)-N,N,N',N'-二吡咯基脲六氟磷酸盐
HCTU 6-氯苯并三氮唑-1,1,3,3-四甲基脲六氟磷酸酯
HDMA 1-[(二甲基氨基)(吗啡啉)甲基]-3-氧代-1H-[1,2,3]三氮唑[4,5-b]并吡啶3-六氟磷酸盐
TATU 2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲四氟硼酸盐
TBTU O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸盐
TCTU O-(6-氯-1H-苯并三唑-1-基)-N,N,N',N'-四甲基脲四氟硼酸盐
TCFH N,N,N',N'-四甲基氯甲脒六氟磷酸盐
TDBTU N,N,N',N'-四甲基-O-(4-羰基-3,4-二氢-1,2,3-苯并三嗪-3-基)脲四氟硼酸盐
TFFH 氟代-N,N,N',N'-四甲基脲六氟磷酸盐
BTFFH N,N,N',N'-双(四亚甲基)氟代甲脒六氟磷酸盐
TSTU 2-琥珀酰亚胺基-1,1,3,3-四甲基脲四氟硼酸酯
PyBOP 1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐
PyCIOP 氯代三吡咯烷基六氟磷酸盐
PyAOP (3H-1,2,3-三唑并[4,5-b]吡啶-3-氧基)三-1-吡咯烷基六氟磷酸盐
PyCIU 1-(氯-1-吡咯烷基亚甲基)吡咯烷六氟磷酸盐
DEPBT 3-(二乙氧基邻酰氧基)-1,2,3-苯并三嗪-4-酮
EEDQ 2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉
Mc 马来酰亚胺基己酰基
MMAF MonomethylauristatinF
DMF 二甲基甲酰胺
DMA 二甲基乙酰胺
NMP NMP
DMSO 二甲基亚砜
[定义]
与说明书的各方面相关的各种术语在说明书和权利要求书中通篇使 用。除非另外指明，否则此类术语被赋予本领域的普通含义。其它具体定义的术语应按照与本文所提供的定义相符的方式理解。
如本文所用，术语“一个”和“一种”和“所述”是按照标准惯例使用的并且意指一个或多个，除非上下文另有指示。因此例如，对“一种抗体药物偶联物”的提及包括两个或更多个抗体药物偶联物的组合等等。
应当理解，无论何处在本文中用语言“包含”描述方面，除此之外还提供了以“由......组成”和/或“基本上由......组成”描述的类似方面。
尽管本发明的广义范围所示的数字范围和参数近似值，但是具体实施例中所示的数值尽可能准确的进行记载。然而，任何数值本来就必然含有一定的误差，其是由它们各自的测量中存在的标准偏差所致。另外，本文公开的所有范围应理解为涵盖其中包含的任何和所有子范围。例如记载的“1至10”的范围应认为包含最小值1和最大值10之间(包含端点)的任何和所有子范围；也就是说，所有以最小值1或更大起始的子范围，例如1至6.1，以及以最大值10或更小终止的子范围，例如5.5至10。另外，任何称为“并入本文”的参考文献应理解为以其整体并入。
本发明中所用的
Figure PCTCN2019118446-appb-000018
是指含
Figure PCTCN2019118446-appb-000019
的基团在此处与其他基团通过化学键连接。
本发明中的术语“连接基团”是指一种具有双官能团或多官能团的分子，可分别与蛋白/抗体分子和MMAF反应，因此做为一种“桥梁”将蛋白/抗体与MMAF连接起来。本发明涉及的连接基团特指那些结构中含有酰基结构的基团。
本发明中的术语“抗体药物偶联物中间体”是指连接基团和MMAF共价偶联物。
[具体实施例]
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商建议的条件进行，未注明具体来源的试剂，为市场购买的常规试剂。除非另外说明，否则所有的百分数、比率、比例、或份数按重量计。
本发明中的重量体积百分比中的单位是本领域技术人员所熟知的，例如是指在100毫升的溶液中溶质的重量。
除非另行定义，文中所使用的所有专业与科学用于与本领域熟悉人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明办法中。文中所述的较佳实施方式与材料仅作示范之用。
实施例1 Mc-MMAF制备方法中酸性试剂加入效果对照
Mc-MMAF制备方法1(不加酸)
Figure PCTCN2019118446-appb-000020
称取Mc-OH 84.6mg和HATU 144.6mg，加入50mL圆底烧瓶中，用5mL注射器取5mL N,N-二甲基甲酰胺加入圆底烧瓶，再用200μL移液枪取165.4μL N,N-二异丙基乙胺加入样品瓶，放置于磁力搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 146.4mg加入一个25mL圆底烧瓶中，用10mL注射器取10mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将磁力搅拌器上的冰浴撤去，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
反应完成后，用旋蒸仪将溶剂旋干，加入5mL乙腈，用5mL注射器吸取后，用有机相针式滤器过滤至10mL进样瓶中，制备液相法纯化。制备方法为流动相A：H2O，0.1％HCOOH，流动相B：MeCN，0.1％HCOOH，流速40mL/min，梯度：25％B-70％B，30min，在25.1min出峰。第一针进样5mL，得到45mL制备液，向样品瓶中加入5mL乙腈，第二针进样5mL，得到制备液45mL。将得到的制备液合并到250mL圆底烧瓶中，放-80℃冰箱冷冻3h，放冻干机冷冻干燥后，得到产品Mc-MMAF纯品102.7mg。收率52％。LC-MS：(M+H)+：924.4；(M-H)-：922.9。
Mc-MMAF制备方法2(添加三氟乙酸)
Figure PCTCN2019118446-appb-000021
称取Mc-OH 84.8mg和HATU 144.7mg，加入50mL圆底烧瓶中，用5mL注射器取5mL N,N-二甲基甲酰胺加入圆底烧瓶，再用200μL移液枪取165.4μL N,N-二异丙基乙胺加入样品瓶，放置于磁力搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 146.4mg和三氟乙酸22.8mg，加入一个25mL圆底烧瓶中，用10mL注射器取10mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将磁力搅拌器上的冰浴撤去，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
反应完成后，用旋蒸仪将溶剂旋干，加入5mL乙腈，用5mL注射器吸取后，用有机相针式滤器过滤至10mL进样瓶中，制备液相法纯化。制备方法为流动相A：H2O，0.1％HCOOH，流动相B：MeCN，0.1％HCOOH，流速40mL/min，梯度：25％B-70％B，30min，在25.1min出峰。第一针进样5mL，得到45mL制备液，向样品瓶中加入5mL乙腈，第二针进样5mL，得到制备液45mL。将得到的制备液合并到250mL圆底烧瓶中，放-80℃冰箱冷冻3h，放冻干机冷冻干燥后，得到产品Mc-MMAF纯品114.1mg。收率62％。LC-MS：(M+H)+：924.4；(M-H)-：922.9。
Mc-MMAF制备方法3(添加对甲苯磺酸一水合物)
Figure PCTCN2019118446-appb-000022
称取Mc-OH 84.6mg和HATU 144.2mg，加入50mL圆底烧瓶中，用5mL注射器取5mL N,N-二甲基甲酰胺加入圆底烧瓶，再用200μL移液枪取165.4μL N,N-二异丙基乙胺加入样品瓶，放置于磁力搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 146.4mg和对甲苯磺酸一水合物34.8mg，加入一个25mL圆底烧瓶中，用10mL注射器取10mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将磁力搅拌器上的冰浴撤去，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
反应完成后，用旋蒸仪将溶剂旋干，加入5mL乙腈，用5mL注射器吸取后，用有机相针式滤器过滤至10mL进样瓶中，制备液相法纯化。制备方法为流动相A：H2O，0.1％HCOOH，流动相B：MeCN，0.1％HCOOH，流速40mL/min，梯度：25％B-70％B，30min，在25.1min出峰。第一针进样5mL，得到45mL制备液，向样品瓶中加入5mL乙腈，第二针进样5mL，得到制备液45mL。将得到的制备液合并到250mL圆底烧瓶中，放-80℃冰箱冷冻3h，放冻干机冷冻干燥后，得到产品Mc-MMAF纯品125.9mg。收率68％。LC-MS：(M+H)+：924.4；(M-H)-：922.9。
Mc-MMAF制备方法4(添加(-)-10-樟脑磺酸)：
Figure PCTCN2019118446-appb-000023
称取Mc-OH 84.2mg和HATU 144.4mg，加入50mL圆底烧瓶中，用5mL注射器取5mL N,N-二甲基甲酰胺加入圆底烧瓶，再用200μL移液枪取165.4μL N,N-二异丙基乙胺加入样品瓶，放置于磁力搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 147.0mg和(-)-10-樟脑磺酸46.8mg，加入一个25mL圆底烧瓶中，用10mL注射器取10mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴 条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将磁力搅拌器上的冰浴撤去，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
反应完成后，用旋蒸仪将溶剂旋干，加入5mL乙腈，用5mL注射器吸取后，用有机相针式滤器过滤至10mL进样瓶中，制备液相法纯化。制备方法为流动相A：H2O，0.1％HCOOH，流动相B：MeCN，0.1％HCOOH，流速40mL/min，梯度：25％B-70％B，30min，在25.1min出峰。第一针进样5mL，得到45mL制备液，向样品瓶中加入5mL乙腈，第二针进样5mL，得到制备液45mL。将得到的制备液合并到250mL圆底烧瓶中，放-80℃冰箱冷冻3h，放冻干机冷冻干燥后，得到产品Mc-MMAF纯品117.4mg。收率64％。LC-MS：(M+H)+：924.4；(M-H)-：922.9。
Mc-MMAF制备方法5(添加三氟甲磺酸)
Figure PCTCN2019118446-appb-000024
称取Mc-OH 84.6mg和HATU 144.7mg，加入50mL圆底烧瓶中，用5mL注射器取5mL N,N-二甲基甲酰胺加入圆底烧瓶，再用200μL移液枪取165.4μL N,N-二异丙基乙胺加入样品瓶，放置于磁力搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 146.5mg和三氟甲磺酸30.0mg，加入一个25mL圆底烧瓶中，用10mL注射器取10mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将磁力搅拌器上的冰浴撤去，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
反应完成后，用旋蒸仪将溶剂旋干，加入5mL乙腈，用5mL注射器 吸取后，用有机相针式滤器过滤至10mL进样瓶中，制备液相法纯化。制备方法为流动相A：H2O，0.1％HCOOH，流动相B：MeCN，0.1％HCOOH，流速40mL/min，梯度：25％B-70％B，30min，在25.1min出峰。第一针进样5mL，得到45mL制备液，向样品瓶中加入5mL乙腈，第二针进样5mL，得到制备液45mL。将得到的制备液合并到250mL圆底烧瓶中，放-80℃冰箱冷冻3h，放冻干机冷冻干燥后，得到产品Mc-MMAF纯品121.2mg。收率66％。LC-MS：(M+H)+：924.4；(M-H)-：922.9。
在前期试验中，我们在上述Mc-MMAF制备方法1的基础上作了进一步的探索，在配制MMAF反应液时，通过加入酸或碱试剂使MMAF先转换成盐后再进行后续反应。我们试验添加了三氟乙酸、叔丁醇钾等试剂进行初步试验，发现在配制MMAF反应试剂时，加入三氟乙酸后(Mc-MMAF制备方法2)，Mc-MMAF的产品收率有了显著的提升。
后续进一步探索了加入其他酸的效果。其中，加入多种酸均对产品收率起到的惊奇的提升效果。如加入对甲苯磺酸一水合物(Mc-MMAF制备方法3)进行比较对照试验，Mc-MMAF的产品收率也有了惊奇的提升。Mc-MMAF的产品收率从52％提升到了68％，收率绝对值提升了16个百分点，相对于未加酸的制备方法1，加入酸后的收率相对值提高了30.7％((68％-52％)/52％)。此外，加入(-)-10-樟脑磺酸及三氟甲磺酸的试验组中的Mc-MMAF产品收率也分别达到了64％和66％。
试验结果表明，通过在反应体系中加入酸性试剂，可以有效提升Mc-MMAF的产品收率。
实施例2 加入各种磺酸类添加剂的试验结果
Figure PCTCN2019118446-appb-000025
试验例1)添加(-)-10-樟脑磺酸：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺加入样品瓶， 放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和(-)-10-樟脑磺酸6.3mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例2)添加(+)-10-樟脑磺酸：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和(+)-10-樟脑磺酸6.3mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例3)添加10-樟脑磺酸：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和10-樟脑磺酸6.3mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例4)添加甲基磺酸：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺加入样品瓶，放置于平 行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和甲基磺酸2.6mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例5)添加三氟甲磺酸：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和三氟甲磺酸4.1mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例6)添加对甲苯磺酸一水合物：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物4.7mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
检测的结果如图1所示，加入各类磺酸，如(-)-10-樟脑磺酸、(+)-10-樟脑磺酸、10-樟脑磺酸、甲基磺酸及三氟甲磺酸添加剂到反应体系中，能起到与加入对甲苯磺酸一水合物添加剂相当的反应促进效果，均能够有 效提升Mc-MMAF的产品收率。
实施例3 酸性添加剂添加用量的影响试验
试验例1)不加酸性添加剂：称取Mc-OH 11.5mg(0.055mmol)和HATU 20.5mg(0.054mmol)，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL(0.216mmol)N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg(0.027mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例2)酸性添加剂与MMAF用量摩尔比等于1：100：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物46μg(2.7*10 -4mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例3)酸性添加剂与MMAF用量摩尔比等于1：20：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物0.23mg(13.5*10 -4mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。 在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例4)酸性添加剂与MMAF用量摩尔比等于1：10：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物0.46mg(2.7*10 -3mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例5)酸性添加剂与MMAF用量摩尔比等于1：5：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物0.92mg(5.4*10 -3mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例6)酸性添加剂与MMAF用量摩尔比等于2：5：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。 称取MMAF 20.0mg和对甲苯磺酸一水合物1.84mg(0.011mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例7)酸性添加剂与MMAF用量摩尔比等于1：2：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物2.3mg(0.014mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例8)酸性添加剂与MMAF用量摩尔比等于1：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物4.6mg(0.027mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例9)酸性添加剂与MMAF用量摩尔比等于2：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。 称取MMAF 20.0mg和对甲苯磺酸一水合物9.3mg(0.054mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例10)酸性添加剂与MMAF用量摩尔比等于3：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物13.9mg(0.081mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例11)酸性添加剂与MMAF用量摩尔比等于4：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取35.7μL N,N-二异丙基乙胺加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物18.6mg(0.108mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
检测结果如图2所示，酸性添加剂与MMAF用量摩尔比大于1时，对于反应有显著的促进效果。
实施例4 溶液1中有机碱加入量对于反应体系的影响
为验证溶液1(含连接基团的溶液)中使用的有机碱的量是否影响反应的结果，本实施例中以DIPEA为例进一步开展了相关试验。
试验例1)碱性添加剂(DIPEA)与酸性添加剂用量摩尔比等于2：1：称取Mc-OH 11.5mg(0.055mmol)和HATU 20.5mg(0.054mmol)，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取8.9μL N,N-二异丙基乙胺(0.055mmol)加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg(0.027mmol)和对甲苯磺酸一水合物4.6mg(0.027mmol)，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例2)碱性添加剂(DIPEA)与酸性添加剂用量摩尔比等于3：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取13.4μL N,N-二异丙基乙胺(0.081mmol)加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物4.6mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例3)碱性添加剂(DIPEA)与酸性添加剂用量摩尔比等于4：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取17.9μL N,N-二异丙基乙胺(0.108mmol)加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一 水合物4.6mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例4)碱性添加剂(DIPEA)与酸性添加剂用量摩尔比等于5：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取22.3μL N,N-二异丙基乙胺(0.135mmol)加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物4.6mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例5)碱性添加剂(DIPEA)与酸性添加剂用量摩尔比等于6：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取26.8μL N,N-二异丙基乙胺(0.162mmol)加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物4.6mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
试验例6)碱性添加剂(DIPEA)与酸性添加剂用量摩尔比等于7：1：称取Mc-OH 11.5mg和HATU 20.5mg，加入5mL样品瓶中，用1mL移 液枪取1mL N,N-二甲基甲酰胺加入样品瓶，再用100μL移液枪取31.3μL N,N-二异丙基乙胺(0.189mmol)加入样品瓶，放置于平行反应搅拌器上，加入搅拌子，室温搅拌0.5h。称取MMAF 20.0mg和对甲苯磺酸一水合物4.6mg，加入另一个5mL样品瓶中，用1mL移液枪取两次1mL N,N-二甲基甲酰胺加入样品瓶，加入搅拌子，置于磁力搅拌器上，加冰浴搅拌。在冰浴条件下，用5mL塑料滴管将Mc-OH体系转移入MMAF的溶液中，将反应瓶放置平行反应器上，继续搅拌1h。停止反应，用10μL移液枪取样10μL加入1.5mL离心管，再用1mL移液枪取1mL乙腈加入离心管，再用1mL注射器吸取后用有机相针式滤器过滤后，送样，通过LC-MS检测反应情况。
在整个反应体系中，MMAF的投入量为0.027mmol时，投入的Mc-OH为0.055mmol摩尔，酸试剂为0.027mmol摩尔，反应体系中总的存在的游离羧基为0.109mmol。而根据图3的检测结果，显示当DIPEA投入的摩尔量小于或等于0.108mmol时，没有反应或反应率低；当DIPEA摩尔量大于0.108mmol后，即体系中有机碱的摩尔量大于反应体系中游离羧基的总摩尔数后，反应促进效果显著。
通过以上实施例不难看出，相较于不添加酸性添加剂(实施例1中制备方法1)而言，通过利用本发明提供的制备方法(即酸法制备抗体药物偶联物中间体)制备的Mc-MMAF收率明显提高，具有意料不到的技术效果。
由此可见，根据上述以Mc-MMAF合成为试验例所验证的试验原理，可以推知通过加入酸试剂使单甲基auristatin衍生物先转换成盐后再进行后续反应均可以显著促进反应的进行，极大的提高产率。并且在生产中既节省了生产耗材、人工、设备、场地、原料等成本，也大大降低了产生的生产废液，降低了生产成本，提高了生产效率，适用于工业大规模生产。
本发明已通过各个具体实施例作了举例说明。但是，本领域普通技术人员能够理解，本发明并不限于各个具体实施方式，普通技术人员在本发明的范围内可以作出各种改动或变型，并且在本说明书中各处提及的各个技术特征可以相互组合，而仍不背离本发明的精神和范围。这样的改动和变型均在本发明的范围之内。

Claims (14)

  1. 一种酸法制备抗体药物偶联物中间体的方法，所述方法的反应路线如下：
    Figure PCTCN2019118446-appb-100001
    其中：
    L为任意包含酰基结构的连接基团；
    L’为共价连接单甲基auristatin衍生物后的连接基团残基；
    溶剂A和溶剂B为任一极性或非极性溶剂；
    所述方法包括以下步骤：
    1)将连接基团L和缩合剂、有机碱溶解在溶剂A中，形成溶液1；
    2)将单甲基auristatin衍生物和酸溶解在溶剂B中，形成溶液2；
    3)将溶液1加到溶液2中，L与单甲基auristatin衍生物通过缩合反应得到L’-单甲基auristatin衍生物；
    步骤1)中加入的有机碱的摩尔量大于步骤3)的反应体系中所有游离羧基的摩尔量。
  2. 根据权利要求1所述的方法，所述的酸选自三氟乙酸、磺酸中的一种或多种组合。
  3. 根据权利要求1或2所述的方法，其中所述单甲基auristatin衍生物包括MMAF、MMAE和MMAD。
  4. 根据权利要求1－3中任一项所述的方法，所述方法的反应路线如下：
    Figure PCTCN2019118446-appb-100002
  5. 根据权利要求1－4中任一项所述的方法，其特征在于，所述的缩合剂为DCC、EDCI、DIC、HATU、HBTU、HBPIPU、HBPyU、HCTU、HDMA、 TATU、TBTU、TCTU、TCFH、TDBTU、TFFH、BTFFH、PyBOP、PyClOP、PyAOP、PyCIU、DEPBT、EEDQ中的一种或多种，优选的，所述的缩合剂为HATU。
  6. 根据权利要求1－5中任一项所述的方法，其特征在于，所述的有机碱为N，N-二异丙基乙胺、三乙胺、吡啶、二甲氨基吡啶、三乙烯二胺、N-甲基吗啉、1,8-二氮杂双环[5.4.0]-7-十一碳烯、N-甲基咪唑、奎宁环、三甲基吡啶中的一种或几种，优选的，所述的有机碱为N，N-二异丙基乙胺、三乙胺。
  7. 根据权利要求1－6中任一项所述的方法，其特征在于，所述的溶剂A和溶剂B独立地选自DMF、DMA、NMP、二氯甲烷、四氯化碳、DMSO、氯仿、四氢呋喃、1,4-二氧六环、六甲基磷酰三胺、N，N-二甲基丙烯基脲、乙二醇二甲醚的一种或几种，溶剂A和溶剂B可以相同也可以不同；优选的，溶剂A和溶剂B为DMF、DMA、DMSO。
  8. 根据权利要求1－7中任一项所述的方法，其特征在于，所述的磺酸包括对甲苯磺酸、苯亚磺酸、三氟甲磺酸、(-)-10-樟脑磺酸、(+)-10-樟脑磺酸和甲基磺酸的一种或几种；所述的对甲苯磺酸优选为对甲苯磺酸一水化合物。
  9. 根据权利要求1－8任一项所述的方法，其特征在于，所述的L为任意包含酰基结构的连接基团，其结构如式(I)所示：
    Figure PCTCN2019118446-appb-100003
    其中：
    R 1选自
    Figure PCTCN2019118446-appb-100004
    Figure PCTCN2019118446-appb-100005
    R 2选自
    Figure PCTCN2019118446-appb-100006
    Figure PCTCN2019118446-appb-100007
    及其盐。
  10. 根据权利要求9所述的方法，其特征在于，所述的L选自如下结构：
    Figure PCTCN2019118446-appb-100008
  11. 根据权利要求10任一项所述的方法，其特征在于，所述的L’-MMAF选自如下结构：
    Figure PCTCN2019118446-appb-100009
  12. 根据权利要求1－11任一项所述的方法，其特征在于，所述酸与相应单甲基auristatin衍生物用量摩尔比大于或等于1。
  13. 根据权利要求1－12任一项所述的方法，其特征在于，所述溶液1加入溶液2的方式为均匀加入，进一步优选为滴加。
  14. 权利要求1－13任一项所述的方法在制备抗体药物偶联物中的应用。
PCT/CN2019/118446 2019-09-29 2019-11-14 一种酸法制备抗体药物偶联物中间体的方法及其应用 WO2021056754A1 (zh)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1938046A (zh) 2003-11-06 2007-03-28 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物
CN105744935A (zh) * 2013-11-27 2016-07-06 雷德伍德生物科技股份有限公司 肼基-吡咯并化合物及用于生成缀合物的方法
CN106573074A (zh) * 2014-06-02 2017-04-19 里珍纳龙药品有限公司 生物活性分子偶联物、试剂和制备方法及其治疗用途
CN108727466A (zh) * 2012-06-07 2018-11-02 Ambrx公司 前列腺特异性膜抗原抗体药物结合物
CN109824759A (zh) 2019-03-08 2019-05-31 联宁（苏州）生物制药有限公司 一种用于抗体药物偶联物的药物-连接子mc-mmaf的制备方法及其中间体

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9044518B2 (en) * 2011-03-30 2015-06-02 Arizona Board Of Regents, A Body Corporate Of The State Of Arizona Acting For And On Behalf Of Arizona State University Auristatin tyramine phosphate salts and auristatin aminoquinoline derivatives and prodrugs thereof
HUE038285T2 (hu) * 2012-10-23 2018-10-29 Synaffix Bv Módosított antitest, antitest-konjugátum és eljárás ezek elõállítására
CN106573956A (zh) * 2014-06-13 2017-04-19 诺华股份有限公司 澳瑞他汀衍生物及其缀合物
US20190209704A1 (en) * 2014-10-20 2019-07-11 Igenica Biotherapeutics, Inc. Novel antibody-drug conjugates and related compounds, compositions and methods of use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1938046A (zh) 2003-11-06 2007-03-28 西雅图基因公司 能够与配体偶联的单甲基缬氨酸化合物
CN108727466A (zh) * 2012-06-07 2018-11-02 Ambrx公司 前列腺特异性膜抗原抗体药物结合物
CN105744935A (zh) * 2013-11-27 2016-07-06 雷德伍德生物科技股份有限公司 肼基-吡咯并化合物及用于生成缀合物的方法
CN106573074A (zh) * 2014-06-02 2017-04-19 里珍纳龙药品有限公司 生物活性分子偶联物、试剂和制备方法及其治疗用途
CN109824759A (zh) 2019-03-08 2019-05-31 联宁（苏州）生物制药有限公司 一种用于抗体药物偶联物的药物-连接子mc-mmaf的制备方法及其中间体

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3828196A4

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