WO2021197303A1 - 制备长链脂肪二酸单苄酯的方法及其应用 - Google Patents

制备长链脂肪二酸单苄酯的方法及其应用 Download PDF

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WO2021197303A1
WO2021197303A1 PCT/CN2021/083862 CN2021083862W WO2021197303A1 WO 2021197303 A1 WO2021197303 A1 WO 2021197303A1 CN 2021083862 W CN2021083862 W CN 2021083862W WO 2021197303 A1 WO2021197303 A1 WO 2021197303A1
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long
reaction
acid
diacid
ester
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PCT/CN2021/083862
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French (fr)
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杨福斌
李松
王海龙
王仲清
郑宝液
郭林峰
李平珠
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东莞市东阳光生物药研发有限公司
广东东阳光药业有限公司
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Publication of WO2021197303A1 publication Critical patent/WO2021197303A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/08—Preparation of carboxylic acid esters by reacting carboxylic acids or symmetrical anhydrides with the hydroxy or O-metal group of organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • 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
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/30—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/313—Preparation of carboxylic acid esters by modifying the acid moiety of the ester, such modification not being an introduction of an ester group by introduction of doubly bound oxygen containing functional groups, e.g. carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C07D207/48—Sulfur atoms

Definitions

  • the present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a method for preparing long-chain fatty diacid monobenzyl ester and its application.
  • Long-acting insulin is classified according to the time of action of insulin. It has a longer action time (24h or more) and is used for the treatment of type 1 and type 2 diabetes. Usually only 1 injection per day, there is no obvious peak of action in the body, mainly to provide basal insulin.
  • the mainstream long-acting insulins include insulin glargine, insulin detemir and insulin deglubber, the latter two are obtained by using long-chain fatty acid modification technology.
  • long aliphatic chain diacid derivatives can be used in the development of long-acting insulin drugs through general protein modification techniques
  • chemical reagents have high cost, poor selectivity, and side effects.
  • methods suitable for industrial scale-up have low yield, long reaction time, cumbersome post-processing, high preparation cost, large reagent loss, large amount of "solid waste", and high processing cost. problem.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention provides a method for preparing long fatty chain diacid monobenzyl ester, and long fatty chain diacid monobenzyl ester is a key intermediate for preparing long fatty chain diacid derivatives and long-acting insulin.
  • the method according to the embodiment of the present invention provides a new idea for the preparation of long aliphatic chain diacid derivatives and long-acting insulin.
  • the present invention proposes a method for preparing long-chain fatty diacid monobenzyl ester.
  • the method includes: 1) subjecting long aliphatic chain diacids to esterification reaction to obtain long aliphatic chain dibenzyl esters; 2) subjecting long aliphatic chain dibenzyl esters to hydrolysis reaction to obtain long aliphatic chain dibenzyl esters Chain aliphatic diacid monobenzyl ester; wherein, the long aliphatic chain diacid has a structure represented by formula (A), and X is an integer from 6 to 32.
  • the reaction reagents are cheap and easy to obtain, easy to operate, low in product-related impurities, simple intermediate purification, short production cycle, high yield, low cost, and "waste liquid” is generated. Less, no solid waste is generated, and environmental protection pressure is low.
  • the above method may further include at least one of the following additional technical features:
  • the esterification reaction is carried out by combining the long aliphatic chain diacid with benzyl alcohol, benzyl chloride, benzyl bromide, benzylamine, dibenzylhydrazine, benzyl formate, benzyl acetate, and benzaldehyde. At least one of the contacts is carried out.
  • the esterification reaction is carried out by contacting the long aliphatic chain diacid with benzyl alcohol.
  • the esterification reaction is carried out under the catalysis of a first catalyst
  • the first catalyst includes at least one selected from the group consisting of hydrochloric acid, sulfuric acid, p-toluenesulfonic acid and trifluoroacetic acid.
  • the first catalyst is p-toluenesulfonic acid and trifluoroacetic acid. Toluenesulfonic acid.
  • the esterification reaction is carried out in a first solvent
  • the first solvent includes at least one selected from the group consisting of methanol, ethanol, acetonitrile, toluene, tert-butanol, benzyl alcohol, DMF and DMSO 1.
  • the first solvent is toluene.
  • the hydrolysis reaction is acid hydrolysis or alkaline hydrolysis, preferably, the hydrolysis reaction is alkaline hydrolysis.
  • alkaline hydrolysis has higher selectivity for long aliphatic chain dibenzyl esters and can specifically hydrolyze long aliphatic chain dibenzyl esters, while the probability of hydrolysis of long aliphatic chain monobenzyl esters is small.
  • the inventor found that the long aliphatic chain dibenzyl ester can be hydrolyzed under alkaline conditions to obtain a high content of monobenzyl ester, and the content of monobenzyl ester can be as high as 95% or more.
  • the hydrolysis reaction is acid hydrolysis.
  • step 2) it further includes acid free treatment and extraction treatment to obtain an organic phase containing long-chain fatty diacid monobenzyl ester, which facilitates subsequent post-treatment and purification.
  • the long aliphatic chain dibenzyl ester is contacted with an acid to perform a hydrolysis reaction, and the acid includes at least one selected from the group consisting of hydrochloric acid, phosphoric acid, acetic acid, p-toluenesulfonic acid, and trifluoroacetic acid.
  • the acid hydrolysis is carried out in the presence of the above-mentioned acid, and the yield of the long aliphatic chain monobenzyl ester is higher.
  • the long aliphatic chain dibenzyl ester is contacted with a base to perform a hydrolysis reaction
  • the base includes selected from NaOH, KOH, K 2 CO 3 , t-BuOK, LiOH, KOAc, Cs 2 CO 3 , At least one of K 3 PO 4 , NaOAc, LiOH ⁇ H 2 O, and Ca(OAc) 2 ⁇ H 2 O.
  • the inventor found that the alkali hydrolysis is carried out in the presence of the above-mentioned alkali, and the yield of the long aliphatic chain monobenzyl ester is significantly improved.
  • the hydrolysis reaction is carried out in a second solvent
  • the second solvent includes selected from ethanol, methanol, isopropanol, tert-butanol, benzyl alcohol, acetone, butanone, 1, At least one of 4-dioxane, NMP, DMF, DMSO, toluene, and water.
  • the inventor found that the acid hydrolysis is carried out in the above-mentioned solvent, and the yield of long aliphatic chain monobenzyl ester is significantly improved.
  • step 1-1) and before step 1-2 it further includes subjecting the system after step 1-1) to crystallization and filtration treatment.
  • step 1-2 after step 1-2), it further includes subjecting the system after step 1-2) to crystallization and filtration treatment.
  • the crystallization treatment is performed by adding a reverse solvent to the reaction system in step 1-1) or the reaction system in step 1-2).
  • the reverse solvent is a non-polar hydrocarbon solvent.
  • the esterification reaction is carried out at a temperature of 50°C to 150°C for 2 to 48 hours.
  • the hydrolysis reaction is carried out at a temperature of 10 to 75° C. for 0.5 h to 48 h.
  • the molar ratio of the long aliphatic chain diacid to benzyl alcohol is 1: (2-5).
  • the molar ratio of the long aliphatic chain diacid to the first catalyst is 1: (0.1-1).
  • the molar ratio of the long aliphatic chain dibenzyl ester to acid or base is 1: (1-5).
  • the present invention proposes a method for preparing long aliphatic chain diacid derivatives.
  • the method includes: (1) processing long aliphatic chain diacids according to the aforementioned method to obtain the long aliphatic chain diacid monobenzyl ester, the long fatty chain diacid It has the structure represented by formula (A); (2) The long fatty chain diacid monobenzyl ester is esterified with N-hydroxysuccinimide to obtain the long fatty chain diacid succinimide benzyl ester (3) The long aliphatic chain diacid succinimide benzyl ester and the compound represented by formula (B) are subjected to nucleophilic addition amidation reaction to obtain the compound represented by formula (C); (4) The compound represented by the formula (C) again undergoes an esterification reaction with N-hydroxysuccinimide to obtain the compound represented by the formula (D); and (5) the compound represented by the formula (D) is
  • the raw materials are cheap and easy to obtain, the preparation cost is low; the impurities are less, the purification is easy, the reaction operation is simple, the post-treatment is convenient, the yield is high, and the industrialization is easy; The processing cost is low, and the environmental protection pressure is low.
  • the above method may further include at least one of the following additional technical features:
  • step (2) the esterification reaction is carried out in one or at least two solvents of tetrahydrofuran, ethyl acetate and dichloromethane at -10°C to 20°C. 2 hours, 15-40 °C continue to react under the condition of 3-24 hours.
  • it further includes contacting the long aliphatic chain diacid monobenzyl ester with dicyclohexylcarbodiimide (DCC). Furthermore, the conversion rate of the esterification reaction is further improved.
  • the initial stage of the DCC/HOSu coupling reaction of the carboxyl group should be reacted under appropriate low temperature conditions (-10°C ⁇ 20°C) to prevent the reaction from being too violent and causing the exotherm to increase and affecting the reaction.
  • the reaction at room temperature (15-40°C) generally takes within 3-24 hours, and more than 24 hours will cause the activated ester (HOSu group) to fall off and return to the raw material.
  • the molar amount of the N-hydroxysuccinimide is equivalent to 1.0 to 1.2 times the molar amount of the long aliphatic chain diacid benzyl ester.
  • the Y is 1, 2 or 3.
  • the compound represented by formula (B) is L-glutamic acid-1-benzyl ester, and the structure is
  • the compound represented by the formula (C) is a benzyl ester long aliphatic chain diacyl-L-Glu-Obn, and the structure is
  • the compound represented by formula (D) is a benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn with the structure
  • step (3) the reaction is carried out in one or at least two of acetonitrile, N,N-dimethylformamide, tetrahydrofuran or N-methylpyrrolidone, and temperature Perform 6-24 under the conditions of 25°C-40°C.
  • the molar amount of the L-glutamic acid-1-benzyl ester is equivalent to 1.0 to 1.2 times the molar amount of the long aliphatic chain diacid succinimide benzyl ester.
  • step (4) it further comprises contacting the benzyl ester long aliphatic chain diacyl-L-Glu-OBn with dicyclohexylcarbodiimide.
  • step (4) the esterification reaction is carried out in a solvent of one or at least two of dichloromethane, ethyl acetate, and propyl acetate, and a temperature of -10°C ⁇
  • the reaction was carried out at 10°C for 2 hours, and then the reaction was continued at a temperature of 15°C to 40°C for 3-24 hours.
  • step (5) the debenzylation reaction is performed in the conditions that the solvent is one or at least two of acetone and tetrahydrofuran, the catalyst is Pd/C, and the temperature is 15-40°C. Continue for 1 to 5 hours.
  • the inventor found that too high temperature or too long time of the debenzylation reaction will lead to: 1) the product removes the activated ester (HOSu group) into a long aliphatic chain triacid derivative; (2) it will also be caused by HOSu In the process of group removal, it reacts with the imine at the amide group to produce lactam impurities; (3) the chirality inversion of the glutamic acid fragment will cause the increase of enantiomeric impurities.
  • HOSu group activated ester
  • the present invention provides a method for preparing a hypoglycemic protein drug.
  • the method includes: modifying the protein with a long fatty chain diacid derivative to obtain the hypoglycemic protein drug, and the long fatty chain diacid derivative is based on the method described above get.
  • the hypoglycemic protein medicine obtained according to the method of the embodiment of the present invention has high yield and high purity.
  • the hypoglycemic protein drug includes at least one selected from insulin, GLP-1, insulin analogues and GLP-1 analogues.
  • the insulin analogue is insulin degludec.
  • the GLP-1 analog is liraglutide.
  • the modification is a fat side chain modification.
  • the catalysts and solvents used in the above reaction are as follows:
  • the catalyst a shown in step 1-1) is any one of hydrochloric acid, sulfuric acid, p-toluenesulfonic acid, trifluoroacetic acid, etc.;
  • the solvent b shown in step 1-1) is one or at least two of methanol, ethanol, acetonitrile, toluene, tert-butanol, benzyl alcohol, DMF or DMSO;
  • the solvent c is one or at least two of non-polar hydrocarbon solvents such as n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, etc.;
  • Step 1-2) Reagent d is hydrochloric acid, phosphoric acid, acetic acid, p-toluenesulfonic acid and trifluoroacetic acid or NaOH, KOH, LiOH, K 2 CO 3 , t-BuOK, KOAc, Cs 2 CO 3 , K 3 PO 4 , NaOAc, LiOH ⁇ H 2 O, Ca(OAc) 2 ⁇ H 2 O, etc.;
  • the solvent e is any one of ethanol, methanol, isopropanol, tert-butanol, benzyl alcohol, acetone, butanone, 1,4-dioxane, NMP, DMF, DMSO, toluene and water
  • the solvent e is any one of ethanol, methanol, isopropanol, tert-butanol, benzyl alcohol, acetone, butanone, 1,4-dioxane, NMP, DMF, DMSO, toluene and water
  • the reagent f is any one of hydrochloric acid, sulfuric acid, acetic acid and trifluoroacetic acid;
  • the solvent g is any one of dichloromethane, ethyl acetate, isopropyl acetate, toluene and methyl tert-butyl fan;
  • the solvent h is one or at least two of non-polar hydrocarbon solvents such as n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, and the like.
  • Step 1-1) The reaction temperature of the intermediate diacid A and benzyl alcohol to prepare the dibenzyl intermediate A" intermediate is 50°C to 150°C; the reaction time is 2 to 48 hours;
  • step 1-1 after the reaction, the temperature is lowered to room temperature, and the room temperature is 20-35°C, and the subsequent cooling and crystallization temperature is -10°C to 5°C; the holding time of the crystallization end point is 0.5 to 2 hours;
  • step 1-1 the baking temperature is 40°C ⁇ 70°C, and the baking time is 4h ⁇ 24h.
  • the reaction temperature of dibenzyl hydrolysis in step 1-2) is 10 ⁇ 75°C, and the reaction time is 0.5h ⁇ 48h;
  • the recrystallization temperature in step 1-2) is 15°C to 25°C; the holding time for the end point of crystallization is 0.5 to 2 hours;
  • Step 1-2 The temperature of the middle baking material is 40°C ⁇ 60°C, and the baking time is 4h ⁇ 24h.
  • the molar amount of benzyl alcohol in step 1-1) is equivalent to 2.0 to 5.0 times the molar amount of hexadecanedioic acid;
  • the molar amount of the catalyst c in step 1-1) is equivalent to 0.1 to 1.0 times the molar amount of the long aliphatic chain diacid
  • the amount of solvent b described in step 1-1) is calculated at a volume-to-mass ratio of 5mL-20ml/g to hexadecanedioic acid;
  • the amount of solvent c mentioned in step 1-1) is calculated according to the volume-to-mass ratio of hexadecanedioic acid 5mL-20ml/g;
  • the amount of solvent c for eluting the filter cake in step 1-1) is calculated according to the volume-mass ratio of hexadecanedioic acid to 1mL-3ml/g;
  • the molar amount of compound d in step 1-2) is equivalent to 1.0 to 5.0 times the molar amount of dibenzyl hexadecanedioic acid;
  • the amount of solvent e described in step 1-2) is calculated at a volume-to-mass ratio of 5mL-20ml/g to dibenzyl hexadecanedioate;
  • the amount of acid f described in step 1-2) is calculated at 2.0-10 times the molar amount ratio of dibenzyl hexadecanedioic acid;
  • the amount of solvent g described in step 1-2) is calculated according to the volume-to-mass ratio of 5mL-20ml/g to dibenzylhexadecanedioic acid;
  • the amount of the extraction solvent h described in step 1-2) is calculated at a volume-to-mass ratio of 5mL-20ml/g to dibenzyl hexadecanedioate;
  • the amount of the elution solvent h described in step 1-2) is calculated according to the volume-to-mass ratio of hexadecanoic acid dibenzyl ester of 1 mL to 3 mL/g.
  • n-heptane When the n-heptane is added dropwise, continue to heat and stir for 0.5h, and transfer the reaction system to a low temperature. Crystallization continued in the tank, and the temperature of the low-temperature tank was gradually reduced to -5°C. After the system was cooled to -5°C, the temperature was kept and stirred for 1 hour to allow the system to crystallize completely. After filtering and washing the filter cake with 200 ml of n-heptane, the filter cake was placed at 40° C. and dried in vacuum for 24 hours to obtain 154.8 g of white solid with a yield of 95% and a purity of 98.85% by HPLC.
  • n-heptane 40 ml was added dropwise to the organic phase, and a white solid began to precipitate out with the addition of n-heptane in the system.
  • Transfer the reaction system to a low temperature tank at 0°C, continue to cool down and crystallize. After the system is cooled to 0°C, more solids will precipitate in the reaction flask, and continue to heat and stir for 0.5h.
  • the wet product was vacuum dried at 50° C. for 17 hours to obtain 3.38 g of a white solid dry product with a yield of 89% and a purity of 96.3% by HPLC.
  • hexadecanedioic acid dibenzyl ester (1.0g, 2.14mmol), acetone (6ml), and 2ml water into a 50ml single-necked flask, and stir to dissolve the solid by r.t. stirring. Then use 2ml acetone and 0.6ml water to dissolve NaOH. (146.8.mg, 3.67mmol). Under stirring at room temperature, the acetone aqueous solution of NaOH was added dropwise to the acetone aqueous solution of hexadecanedioic acid. After the addition was completed, the reaction system was transferred to an oil bath at 35° C., and the mixture was stirred for 24 hours with heat preservation.
  • the reaction was stopped, and the pH of the reaction system was adjusted to 1-2 with 1M HCl.aq, 150ml of dichloromethane was added to the reaction system, filtered, and the aqueous phase was separated and discarded.
  • Under stirring at room temperature add 200ml n-heptane dropwise to the organic phase. With the dropwise addition of n-heptane, solids are precipitated in the system, and the solids gradually increase.
  • the filter cake was rinsed twice with 10ml to obtain a white solid wet product.
  • the wet product was vacuum dried at 50°C for 24 hours to obtain 7.26 g of a white solid dry product, with a yield of 90%, and a purity of 98.2% by HPLC.
  • Dibenzyl hexadecanedioate (10.0 g, 21.4 mmol) and THF (80 ml) were added to a 250 ml single-neck flask, and stirred at room temperature (r.t.) to dissolve the solid.
  • Dissolve potassium carbonate (5.92g, 42.8mmol) with 20ml of water. Under stirring at room temperature, the aqueous solution of potassium carbonate was added dropwise to the THF solution of hexadecanedioic acid. After the dropwise addition was completed, the reaction system was transferred to an oil bath at 70° C., and the mixture was kept warm and stirred for 2 hours.
  • the reaction was stopped, and the pH of the reaction system was adjusted to 1-2 with 1M HCl.aq, 150ml of dichloromethane was added to the reaction system, filtered, and the aqueous phase was separated and discarded.
  • Under stirring at room temperature add 200ml n-heptane dropwise to the organic phase. With the dropwise addition of n-heptane, solids are precipitated in the system, and the solids gradually increase.
  • the filter cake was rinsed twice with 10ml to obtain a white solid wet product.
  • the wet product was vacuum dried at 50° C. for 24 hours to obtain a white solid dry product of 2.42gg, with a yield of 30%, and an HPLC purity of 80%.
  • n-heptane 40 ml was added dropwise to the organic phase, and a white solid began to precipitate out with the addition of n-heptane in the system.
  • the reaction system was transferred to a low temperature tank at 0°C, and the temperature was continued to be lowered to crystallize. After the system was cooled to 0°C, more solids precipitated in the reaction flask, and the temperature was kept and stirred for 0.5h. It was filtered at low temperature and rinsed twice with n-heptane (10ml) to obtain a white solid wet product. The wet product was vacuum dried at 50° C. for 17 hours to obtain 1.10 g of a white solid dry product with a yield of 29% and a purity of 90.3% by HPLC.
  • the reaction was stopped, and the pH of the reaction system was adjusted to 1-2 with 1M HCl.aq, 15 ml of dichloromethane was added to the reaction system, filtered, and the aqueous phase was separated and discarded.
  • Under stirring at room temperature add 20ml n-heptane dropwise to the organic phase. With the dropwise addition of n-heptane, solids precipitate in the system, and the solids gradually increase.
  • the filter cake was rinsed twice with 2ml to obtain a white solid wet product.
  • the wet product was vacuum dried at 50° C. for 4 hours to obtain 0.39 g of a white solid dry product, with a yield of 49%, and a purity of 96.9% by HPLC.
  • acidic cationic resins can make hexadecanedioic acid to selectively synthesize benzyl hexadecanedioate, but the Dowex50WX2-100 acidic cationic resin used in this method is more expensive, and the amount of solvent used is large, and the reaction time is long , The yield is low, the post-processing operation is more troublesome, and the preparation cost is relatively high.
  • the existing production process is the optimized preparation of hexadecandioyl-L-Glu(OSu)-OH described in the preparation of hexadecandioyl–L-Glu(OSu)-OH in the optimized Novo Nordisk patent number WO2007104737A1 on page 26, paragraph 2 to prepare benzyl hexadecandioate.
  • Hexadecanedioic acid (160.00g, 559.03mmol) and Dowex 50wx2-100 (480.00g) were added to 2880ml of n-octane, heated to 110°C and refluxed, added benzyl formate (152.23g, 1118.06mmol), and reacted under reflux After 6h, benzyl formate (152.23g, 1118.06mmol) was added, and the reaction was continued with reflux and stirring for 64h. Cool to room temperature, filter with suction, and rinse the filter cake with 240.00ml n-octane to obtain a wet product.
  • One-time beating transfer the wet product to a 5000ml three-necked flask, add 2880ml of dichloromethane (18.00ml/g diacid) into the reaction flask, after beating at room temperature for 2.0h, filter with suction and rinse with 200.00ml of dichloromethane
  • the filter cake, the filtrate was concentrated in vacuo at 40°C to obtain a crude white solid
  • the second beating add 1440ml of dichloromethane (9.00ml/g diacid) to the crude product, and after beating for 2.0h at room temperature, filter with suction and use 200.00ml of two
  • the filter cake was eluted with methyl chloride, and the filtrate was concentrated in vacuo at 40°C to obtain a crude white solid
  • one recrystallization add 320ml isopropanol and 960ml n-heptane to the crude product, warm the system to 65°C and stir to clear, then keep stirring for 0.5 h, cooling down to
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features. In the description of the present invention, “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.

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Abstract

本发明提出了一种制备长链脂肪二酸单苄酯的方法。该方法包括：1)将长脂肪链二酸进行酯化反应，以便获得长脂肪链二苄酯；2)将长脂肪链二苄酯进行水解反应，以便获得长链脂肪二酸单苄酯；其中，所述长脂肪链二酸具有式(A)所示的结构，X为6～32的整数。该方法，相对于现有技术，反应试剂廉价易得、操作简便、产品相关杂质含量低、中间体纯化简单，生产周期短，收率高，成本低，产生"废液"少，没有固废生成，环保压力低。

Description

制备长链脂肪二酸单苄酯的方法及其应用 技术领域
本发明涉及生物制药领域，具体地，本发明涉及制备长链脂肪二酸单苄酯的方法及其应用。
背景技术
长效胰岛素是按胰岛素作用时间分类，作用时间较长(24h或以上)的一类胰岛素，用于1型和2型糖尿病的治疗。通常每天只需1次注射，体内没有明显的作用高峰，主要是提供基础胰岛素。目前主流的长效胰岛素有甘精胰岛素，地特胰岛素和德谷胰岛素，后两者均通过采用长链脂肪酸修饰技术而获得。
虽然长脂肪链二酸衍生物可以通过蛋白修饰通用技术用于长效胰岛素药物的研发，而现有技术中，长脂肪链二酸衍生物的制备过程中，化学试剂成本高、选择性差、副产物多且难以提纯，难以实现工业放大；而适合工业化放大的方法存在产率低，反应时间长，后处理繁琐，制备成本耗费大，试剂损耗大，产生“固废”多，处理成本大的问题。
因此，对于长脂肪链二酸衍生物的制备方法仍需要进一步开发和改进。
发明内容
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明提供了一种制备长脂肪链二酸单苄酯的方法，而长脂肪链二酸单苄酯是制备长脂肪链二酸衍生物以及长效胰岛素的关键中间体。根据本发明实施例的方法，为长脂肪链二酸衍生物以及长效胰岛素的制备提供了新的思路。
在本发明的第一方面，本发明提出了一种制备长链脂肪二酸单苄酯的方法。根据本发明的实施例，所述方法包括：1)将长脂肪链二酸进行酯化反应，以便获得长脂肪链二苄酯；2)将长脂肪链二苄酯进行水解反应，以便获得长链脂肪二酸单苄酯；其中，所述长脂肪链二酸具有式(A)所示的结构，X为6～32的整数。
Figure PCTCN2021083862-appb-000001
根据本发明实施例的方法，相对于现有技术，反应试剂廉价易得、操作简便、产品相关杂质含量低、中间体纯化简单，生产周期短，收率高，成本低，产生“废液”少，没有固废生成，环保压力低。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述酯化反应是通过将所述长脂肪链二酸与包括苄醇、氯苄、溴苄、苄胺、二苄肼、甲酸苄酯、乙酸苄酯、苯甲醛的至少之一进行接触进行的。优选地，所述酯化反应是通过将所述长脂肪链二酸与苄醇进行接触进行的。
所述酯化反应是在第一催化剂催化下进行的，所述第一催化剂包括选自盐酸、硫酸、对甲苯磺酸和三氟乙酸的至少之一，优选地，所述第一催化剂为对甲苯磺酸。
根据本发明的实施例，所述酯化反应是在第一溶剂中进行的，所述第一溶剂包括选自甲醇、乙醇、乙腈、甲苯、叔丁醇、苄醇、DMF和DMSO的至少之一，优选地，所述第一溶剂为甲苯。
根据本发明的实施例，所述水解反应为酸水解或碱水解，优选地，所述水解反应为碱水解。发明人发现，碱水解对长脂肪链二苄酯的选择性更高，可特异性地使长脂肪链二苄酯水解，而长脂肪链单苄酯的水解几率小。发明人发现，长脂肪链二苄酯在碱性条件下进行水解，可得到高含量的单苄酯，单苄酯的含量可高达95％以上。
根据本发明的实施例，所述水解反应为酸水解，步骤2)之后进一步包括酸游离处理和萃取处理，以便获得含有长链脂肪二酸单苄酯的有机相，利于后续的后处理纯化。
根据本发明的实施例，长脂肪链二苄酯与酸进行接触，以进行水解反应，所述酸包括选自选自盐酸、磷酸、醋酸、对甲苯磺酸和三氟乙酸的至少之一。发明人发现，所述酸水解在上述酸存在的条件下进行，长脂肪链单苄酯的收率较高。
根据本发明的实施例，长脂肪链二苄酯与碱进行接触，以进行水解反应，所述碱包括选自NaOH、KOH、K 2CO 3、t-BuOK、LiOH、KOAc、Cs 2CO 3、K 3PO 4、NaOAc、LiOH·H 2O和Ca(OAc) 2·H 2O的至少之一。发明人发现，所述碱水解在上述碱存在的条件下进行，长脂肪链单苄酯的收率显著提高。
根据本发明的实施例，所述水解反应是在第二溶剂中进行的，所述第二溶剂包括选自乙醇、甲醇、异丙醇、叔丁醇、苄醇、丙酮、丁酮、1,4-二氧六环、NMP、DMF、DMSO、甲苯和水的至少之一。发明人发现，所述酸水解在上述溶剂中进行，长脂肪链单苄酯的收率显著提高。
根据本发明的实施例，步骤1-1)之后，步骤1-2)之前，进一步包括使步骤1-1)反应后的体系进行析晶和过滤处理。
根据本发明的实施例，步骤1-2)之后，进一步包括使步骤1-2)反应后的体系进行析晶和过滤处理。
根据本发明的实施例，所述析晶处理是通过向步骤1-1)反应后的体系中或步骤1-2)反应后的体系中加入反向溶剂进行的。
根据本发明的实施例，所述反向溶剂为非极性烃类溶剂。
根据本发明的实施例，所述酯化反应是在温度为50℃～150℃的条件下进行2～48小时。
根据本发明的实施例，所述水解反应是在温度为10～75℃的条件下进行0.5h～48h。
根据本发明的实施例，所述长脂肪链二酸与苄醇的摩尔比为1：(2～5)。
根据本发明的实施例，所述长脂肪链二酸与第一催化剂的摩尔比为1：(0.1～1)。
根据本发明的实施例，所述长脂肪链二苄酯与酸或碱的摩尔比为1：(1～5)。
在本发明的第二方面，本发明提出了一种制备长脂肪链二酸衍生物的方法。根据本发明的实施例，所述方法包括：(1)将长脂肪链二酸依据前面所述的方法进行处理，以便获得所述长脂肪链二酸单苄酯，所述长脂肪链二酸具有式(A)所示的结构；(2)将所述长脂肪链二酸单苄酯与N-羟基琥珀酰亚胺发生酯化反应，以便获得长脂肪链二酸琥珀酰亚胺苄酯；(3)将所述长脂肪链二酸琥珀酰亚胺苄酯与式(B)所示化合物进行亲核加成酰胺化反应，以便获得式(C)所示化合物；(4)将所述式(C)所示化合物再次与N-羟基琥珀酰亚胺发生酯化反应，以便获得式(D)所示化合物；以及(5)将所述式(D)所示化合物进行脱苄基反应，以便获得式(E)所示化合物。
Figure PCTCN2021083862-appb-000002
Figure PCTCN2021083862-appb-000003
其中，X为6～32的整数；Y为1～6的整数。
根据本发明实施例的上述方法，原料廉价易得，制备成本少；产生杂质少，容易纯化，同时反应操作简单和后处理方便，收率高，容易实现工业化；产生“固废”少，后处理成本小，环保压力低。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，在步骤(2)中，所述酯化反应是在四氢呋喃、乙酸乙酯和二氯甲烷中的一种或至少两种溶剂中，在-10℃～20℃下反应2小时，15-40℃继续反应3-24小时的条件下进行的。
根据本发明的实施例，进一步包括将长脂肪链二酸单苄酯与二环己基碳二亚胺(DCC)进行接触。进而进一步提高酯化反应转化率。羧基的DCC/HOSu耦合反应起始阶段应该在适当的低温条件(-10℃～20℃)下反应，防止反应过于剧烈导致放热增大而影响反应，如杂质增多，后期稳定后可以回复到室温(15-40℃)反应，时间一般在3-24小时以内，超过24小时则导致活化酯(HOSu基团)脱落而变回原料。
根据本发明的实施例，所述的N-羟基琥珀酰亚胺的摩尔用量相当于所述的长脂肪链二 酸苄酯摩尔量的1.0～1.2倍。
根据本发明的实施例，所述Y为1、2或3。
根据本发明的实施例，所述式(B)所示化合物为L-谷氨酸-1-苄酯，结构为
Figure PCTCN2021083862-appb-000004
根据本发明的实施例，所述式(C)所示化合物为苄酯长脂肪链二酰基-L-Glu-Obn，结构为
Figure PCTCN2021083862-appb-000005
根据本发明的实施例，所述式(D)所示化合物为苄基长脂肪链二酰基-L-Glu(OSu)-OBn,结构为
Figure PCTCN2021083862-appb-000006
根据本发明的实施例，所述式(E)所示化合物的结构为
Figure PCTCN2021083862-appb-000007
根据本发明的实施例，在步骤(3)中，所述反应是在溶剂为乙腈、N,N-二甲基甲酰胺、四氢呋喃或N-甲基吡咯烷酮中的一种或至少两种、温度为25℃～40℃的条件下进行6～24。
根据本发明的实施例，所述的L-谷氨酸-1-苄酯的摩尔用量相当于所述的长脂肪链二酸琥珀酰亚胺苄酯摩尔量的1.0～1.2倍。
根据本发明的实施例，在步骤(4)中，进一步包括将苄酯长脂肪链二酰基-L-Glu-OBn与二环己基碳二亚胺进行接触。
根据本发明的实施例，在步骤(4)中，所述酯化反应是在溶剂为二氯甲烷、乙酸乙酯、乙酸丙酯中的一种或至少两种的、温度为-10℃～10℃的条件进行2小时，后在温度为15℃～40℃继续反应3-24小时。
根据本发明的实施例，在步骤(5)中，所述脱苄基反应是在溶剂为丙酮、四氢呋喃中的一种或至少两种，催化剂为Pd/C，温度为15～40℃的条件下进行1～5小时。发明人发现，脱苄基反应的温度过高或时间过长将会导致：1)产物脱掉活化酯(HOSu基团)变为长脂肪链三酸衍生物；(2)同时也会由于HOSu基团脱掉的过程中与酰胺基团处的亚胺反应产生内酰胺杂质；(3)谷氨酸片段的手性反转会导致对映异构体杂质的增多。
在本发明的第三方面，本发明提出了一种制备降血糖蛋白药物的方法。根据本发明的实施例，所述方法包括:采用长脂肪链二酸衍生物对蛋白进行修饰，以便获得所述降血糖蛋白药物，所述长脂肪链二酸衍生物是依据前面所述的方法获得。根据本发明实施例的方法所获得的降血糖蛋白药物，收率高、纯度高。
根据本发明的实施例，所述降血糖蛋白药物包括选自胰岛素、GLP-1、胰岛素类似物和GLP-1类似物的至少之一。
根据本发明的实施例，所述胰岛素类似物为德谷胰岛素。
根据本发明的实施例，所述GLP-1类似物为利拉鲁肽。
根据本发明的实施例，所述修饰为脂肪侧链修饰。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
根据本发明实施例的制备长链脂肪二酸单苄酯的方法可如下所述：
Figure PCTCN2021083862-appb-000008
1-1)化合物A、苄醇、催化剂a在溶剂b中制备得到中间体A’，反应结束，降温至室温，然后向反应体系中加入反向溶剂c，继续降温至0℃左右析晶，过滤，用反向溶剂c淋洗滤饼，得纯度95％以上的化合物B，烘干即可进行下一步使用。
1-2)化合物A’、(酸)碱d在溶剂e中搅拌反应，反应结束，(若反应用酸水解可跳过酸游离和溶剂萃取)酸f游离后，溶剂g萃取，室温下向有机相中加入反向溶剂h析晶，过滤，反向溶剂h淋洗滤饼，滤饼烘干后得95％以上纯度的产品A”。
其中，上述反应的所用催化剂和溶剂如下：
步骤1-1)中所示的催化剂a为盐酸、硫酸、对甲苯磺酸和三氟乙酸等中的任意一种；
步骤1-1)中所示的溶剂b为甲醇、乙醇、乙腈、甲苯、叔丁醇、苄醇、DMF或DMSO中的一种或至少两种；
步骤1-1)中溶剂c为正己烷、石油醚、环己烷、正庚烷、正辛烷、等非极性烃类溶剂中的一种或至少两种；
步骤1-2)中试剂d为盐酸、磷酸、醋酸、对甲苯磺酸和三氟乙酸或NaOH、KOH、LiOH、K 2CO 3、t-BuOK、KOAc、Cs 2CO 3、K 3PO 4、NaOAc、LiOH·H 2O、Ca(OAc) 2·H 2O等中的任意一种；
步骤1-2)中溶剂e为乙醇、甲醇、异丙醇、叔丁醇、苄醇、丙酮、丁酮、1,4-二氧六环、NMP、DMF、DMSO、甲苯和水中的任意一种或两种；
步骤1-2)中试剂f为盐酸、硫酸、醋酸和三氟乙酸中的任意一种；
步骤1-2)中溶剂g为二氯甲烷、乙酸乙酯、醋酸异丙酯、甲苯和甲基叔丁基迷中的任意一种；
步骤1-2)中溶剂h为正己烷、石油醚、环己烷、正庚烷、正辛烷、等非极性烃类溶剂中的一种或至少两种。
其中，上述反应的温度和时间条件如下所述：
步骤1-1)中二酸A与苄醇制备二苄中间体A”中间体的反应温度为50℃～150℃；反应时间为2～48小时；
步骤1-1)中反应结束后降温至室温，室温温度为20～35℃，后续的继续降温析晶温度为-10℃～5℃；析晶终点保温时间为0.5～2小时；
步骤1-1)中烘料温度为40℃～70℃，烘料时间4h～24h。
步骤1-2)中二苄水解的反应温度为10～75℃，反应时间为0.5h～48h；
步骤1-2)中的重结晶温度为15℃～25℃；析晶终点保温时间为0.5～2小时；
步骤1-2)中烘料温度为40℃～60℃，烘料时间4h～24h。
其中，上述反应的物质用量如下所述：
步骤1-1)中所述的苄醇的摩尔用量相当于所述的十六碳二酸摩尔量的2.0～5.0倍；
步骤1-1)中所述的催化剂c的摩尔用量相当于所述的长脂肪链二酸摩尔量的0.1～1.0倍；
步骤1-1)中所述的溶剂b的用量按其与十六碳二酸体积质量比5mL～20ml/g计算；
步骤1-1)中所述的溶剂c的用量按其与所述的十六碳二酸体积质量比5mL～20ml/g计算；
步骤1-1)中所述的淋洗滤饼的溶剂c的用量按其与十六碳二酸体积质量比1mL～3ml/g计算；
步骤1-2)中所述的化合物d的摩尔用量相当于所述的十六碳二酸双苄酯摩尔量的1.0～5.0倍；
步骤1-2)中所述的溶剂e的用量按其与十六碳二酸双苄酯体积质量比5mL～20ml/g计算；
步骤1-2)中所述的酸f的用量按其与十六碳二酸双苄酯摩尔用量比2.0～10倍计算；
步骤1-2)中所述的溶剂g的用量按其与十六碳二酸双苄酯体积质量比5mL～20ml/g计算；
步骤1-2)中所述的萃取溶剂h的用量按其与十六碳二酸双苄酯体积质量比5mL～20ml/g计算；
步骤1-2)中所述的淋洗溶剂h的用量按其与十六碳二酸双苄酯体积质量比1mL～3ml/g计算。
当X为16，即式(A)所示化合物为十六烷二酸，所得十六碳二酸单苄酯的收率80-90％，纯度95-99％。
下面将结合具体实施例对本发明进行进一步解释说明。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1十六烷二酸双苄酯的合成
Figure PCTCN2021083862-appb-000009
将十六烷二酸(100.0g，349.4mmol)、苄醇(167.4g，1547.8mmol)、TsOH(6.0g，34.9mmol)和1000mL甲苯加入3000mL三口烧瓶中置于120℃搅拌反应24h，反应结束，降温至室温，用0.5M Na 2CO 3.aq(35ml，17mmol)洗涤反应体系，分液弃水相。搅拌下向有机相中加入1000ml的正庚烷，体系中随着正庚烷的加入，体系中逐渐析出固体，当正庚烷滴加完后，继续保温搅拌0.5h，将反应体系转移至低温槽中继续析晶，低温槽梯度降温至-5℃，当体系降温至-5℃后，保温搅拌1h让体系析晶彻底。过滤，并用200ml正庚烷洗涤滤饼，将滤饼置于40℃下真空干燥24h，得白色固体154.8g，收率95％，HPLC检测纯度98.85％。
十六烷二酸双苄酯MS测试：ESI-MS m/z：467.3[M+H] +，与理论值相符。
实施例2
十六烷二酸单苄酯的合成(a)
Figure PCTCN2021083862-appb-000010
将十六烷二酸双苄酯(4.7g，10mmol)、苄醇(25ml)加入100mL单口烧瓶中，室温搅拌溶解后，室温搅拌下，向反应体系中滴加KOH(0.55g，11mmol)的苄醇(10ml)溶液，滴加完后，室温搅拌过夜。用1M的HCl.aq(25ml)调节反应体系pH：1～2，过滤，分液弃水相。室温下，向有机相中滴加正庚烷(40ml)，体系中随着正庚烷的加入开始析出白色固体。将反应体系转移至0℃低温槽中，继续降温析晶，体系降温至0℃后反应瓶中析出更 多固体，继续保温搅拌0.5h。趁低温过滤，并用正庚烷(10ml)淋洗两次，得白色固体湿品。将湿品于50℃下真空干燥17h，得白色固体干品3.38g，收率89％，送检HPLC纯度96.3％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
十六烷二酸单苄酯的合成(b)
将十六烷二酸双苄酯(1.0g，2.14mmol)、丙酮(6ml)、水2ml加入50ml单口瓶中，r.t.搅拌使固体溶解。再用2ml丙酮和0.6ml水溶解NaOH.(146.8.mg，3.67mmol)。室温搅拌下，将NaOH的丙酮水溶液滴加到十六烷二酸的丙酮水溶液中，滴加完后，将反应体系转移至35℃的油浴锅中，保温搅拌24h。停止反应，用1M的HCl.aq调反应体系pH：1～2，向反应体系中加入15ml二氯甲烷，过滤，分液弃水相。室温搅拌下，向有机相中滴加20ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷2ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥4h，得白色固体干品0.65g，收率81％，送检HPLC纯度97.2％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
十六烷二酸单苄酯的合成(c)
将十六烷二酸双苄酯(1.0g，2.14mmol)、甲苯(6ml)、水2ml加入50ml单口瓶中，rt搅拌使固体溶解。再用2ml甲苯和0.6ml水溶解LiOH·H 2O(102.7mg，2.44mmol)。室温搅拌下，将LiOH的甲苯水溶液滴加到十六烷二酸的甲苯水溶液中，滴加完后，将反应体系转移至70℃的油浴锅中，保温搅拌2h。停止反应，用1M的HCl.aq调反应体系pH：1～2，向反应体系中加入15ml二氯甲烷，过滤，分液弃水相。室温搅拌下，向有机相中滴加20ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷2ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥4h，得白色固体干品0.71g，收率89％，送检HPLC纯度96.8％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
十六烷二酸单苄酯的合成(d)
将十六烷二酸双苄酯(10.0g，21.4mmol)、甲醇(80ml)250ml单口瓶中，r.t.搅拌使固体溶解。再用20ml水溶解碳酸钾(5.92g，42.8mmol)。室温搅拌下，将碳酸钾的水溶液滴加到十六烷二酸的甲醇溶液中，滴加完后，将反应体系转移至70℃的油浴锅中，保温搅拌2h。停止反应，用1M的HCl.aq调反应体系pH：1～2，向反应体系中加入150ml二氯甲烷，过滤，分液弃水相。室温搅拌下，向有机相中滴加200ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷10ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥24h，得白色固体干品7.26g，收率 90％，送检HPLC纯度98.2％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
十六烷二酸单苄酯的合成(e)
将十六烷二酸双苄酯(1.0g，2.14mmol)、DMF(10ml)、浓盐酸(1ml，12mmol)加入25ml单口瓶中，室温搅拌2h。停止反应，室温搅拌下，向有机相中滴加20ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷2ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥2h，得白色固体干品0.69g，收率86％，送检HPLC纯度97.8％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
对比例2-1：十六烷二酸单苄酯的合成(f)
将十六烷二酸双苄酯(10.0g，21.4mmol)、THF(80ml)加入到250ml单口瓶中，室温(r.t.)搅拌使固体溶解。再用20ml水溶解碳酸钾(5.92g，42.8mmol)。室温搅拌下，将碳酸钾的水溶液滴加到十六烷二酸的THF溶液中，滴加完后，将反应体系转移至70℃的油浴锅中，保温搅拌2h。停止反应，用1M的HCl.aq调反应体系pH：1～2，向反应体系中加入150ml二氯甲烷，过滤，分液弃水相。室温搅拌下，向有机相中滴加200ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷10ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥24h，得白色固体干品2.42gg，收率30％，送检HPLC纯度80％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
改变了案例(d)中溶剂，将甲醇换为THF，反应效果变差，产物单苄酯纯度和收率均下降较多。
对比例2-2：十六烷二酸单苄酯的合成(g)
将十六烷二酸双苄酯(4.7g，10mmol)、乙腈(25ml)加入100mL单口烧瓶中，室温搅拌溶解后，室温搅拌下，向反应体系中滴加KOH(0.55g，11mmol)的乙腈(10ml)溶液，滴加完后，室温搅拌过夜。用1M的HCl.aq(25ml)调节反应体系pH：1～2，过滤，分液弃水相。室温下，向有机相中滴加正庚烷(40ml)，体系中随着正庚烷的加入开始析出白色固体。将反应体系转移至0℃低温槽中，继续降温析晶，体系降温至0℃后反应瓶中析出更多固体，继续保温搅拌0.5h。趁低温过滤，并用正庚烷(10ml)淋洗两次，得白色固体湿品。将湿品于50℃下真空干燥17h，得白色固体干品1.10g，收率29％，送检HPLC纯度90.3％。
改变了案例a中溶剂，将苄醇换为乙腈，反应效果变差，产物单苄酯纯度和收率均下降较多。
对比例2-3：十六烷二酸单苄酯的合成(h)
将十六烷二酸双苄酯(10.0g，21.4mmol)、甲醇(80ml)250ml单口瓶中，r.t.搅拌使固体溶解。再用20ml水溶解碳酸氢钠(3.60g，42.8mmol)。室温搅拌下，将碳酸氢钠的水溶液滴加到十六烷二酸的甲醇溶液中，滴加完后，将反应体系转移至70℃的油浴锅中，保温搅拌2h。停止反应，用1M的HCl.aq调反应体系pH：1～2，向反应体系中加入150ml二氯甲烷，过滤，分液弃水相。室温搅拌下，向有机相中滴加200ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷10ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥24h，得白色固体干品4.03g，收率50％，送检HPLC纯度97.9％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
改变了案例(d)中的碱，将K 2CO 3换为NaHCO 3，反应效果变差，产物纯度影响不大，但是收率下降较大。
对比例2-4：十六烷二酸单苄酯的合成(i)
将十六烷二酸双苄酯(1.0g，2.14mmol)、甲苯(6ml)、水2ml加入50ml单口瓶中，r.t.搅拌使固体溶解。室温搅拌下，向十六烷二酸的甲苯水溶液中加入TEA(246.4mg，2.44mmol)，滴加完后，将反应体系转移至70℃的油浴锅中，保温搅拌2h。停止反应，用1M的HCl.aq调反应体系pH：1～2，向反应体系中加入15ml二氯甲烷，过滤，分液弃水相。室温搅拌下，向有机相中滴加20ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷2ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥4h，得白色固体干品0.39g，收率49％，送检HPLC纯度96.9％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
改变了案例(c)中的碱，将LiOH·H 2O换为TEA，反应效果变差，产物纯度影响不大，但是收率下降较大。
对比例2-5：十六烷二酸单苄酯的合成(J)
将十六烷二酸双苄酯(1.0g，2.14mmol)、DMF(10ml)、浓H 2SO 4(1ml，12mmol)加入25ml单口瓶中，室温搅拌2h。停止反应，室温搅拌下，向有机相中滴加20ml正庚烷，随着正庚烷的滴加，体系中析出固体，且固体逐渐增多，滴加完后，室温搅拌2.0h，过滤，并用正庚烷2ml淋洗滤饼2次，得白色固体湿品。将湿品于50℃下真空干燥2h，得白色固体干品0.17g，收率21％，送检HPLC纯度87.8％。
ESI-MS m/z：377.2[M+H] +，与理论值符合
改变了案例(e)中的酸，将浓盐酸换为浓硫酸，反应效果变差，产物纯度影响不大， 但是收率下降较大。
实施例3十六烷二酸琥珀酰亚胺苄酯的合成
Figure PCTCN2021083862-appb-000011
将十六烷二酸一苄酯(10.0g，25.56mmol)和130mL二氯甲烷加入250mL单口烧瓶中，降温至-5℃，加入N-羟基琥珀酰亚胺(3.24g，28.12mmol)和二环己基碳二亚胺(6.32g，30.67mmol)，于-5℃下继续反应2小时，恢复至30℃继续反应16小时，过滤除掉沉淀，蒸馏浓缩至干得到固体粗品，加入60mL异丙醇和60mL正庚烷重结晶，过滤并用20mL正庚烷淋洗湿品，减压干燥至恒重，得到十六烷二酸琥珀酰亚胺苄酯白色固体11.62g，收率92.35％，HPLC检测纯度为98.80％。
ESI-MS m/z：474.1[M+H] +，与理论值符合
实施例4苄基十六烷二酰基-L-Glu-OBn的合成
Figure PCTCN2021083862-appb-000012
将十六烷二酸琥珀酰亚胺苄酯(10.0g，21.11mmol)与L-谷氨酸-1-苄酯(5.26g，22.17mmol)溶解于100mL乙腈中，加入三乙胺(3.2g，31.67mmol)置于30℃搅拌12小时，反应结束加入120mL 1M盐酸溶液和90mL自来水，降温至-5℃中搅拌析晶2.0小时，过滤并用适量水洗滤饼至中性，滤饼置于45℃中真空干燥至恒重，即可得到苄基十六烷二酰基-L-Glu-OBn淡黄色固体12.09g，收率96.12％，HPLC检测纯度为97.53％。
ESI-MS m/z：596.4[M+H] +，与理论值符合
实施例5苄基十六烷二酰基-L-Glu(OSu)-OBn的合成
Figure PCTCN2021083862-appb-000013
将苄基十六烷二酰基-L-Glu-OBn(10.0g，16.79mmol)溶解于130mL二氯甲烷中，降温至-5℃，加入N-羟基琥珀酰亚胺(2.13g，18.47mmol)和二环己基碳二亚胺(4.15g，20.14mmol)，于-5℃下继续反应2小时，恢复至30℃继续反应8小时，过滤除掉沉淀，减压蒸馏浓缩至干得固体，加入120mL无水乙醇重结晶，过滤，用20mL正庚烷淋洗滤饼， 滤饼置于45℃中真空干燥至恒重，即可得到苄基十六烷二酰基-L-Glu(OSu)-OBn白色固体10.38g，收率89.26％，HPLC检测纯度为98.35％。
ESI-MS m/z：693.4[M+H] +，与理论值符合
实施例6十六烷二酰基-L-Glu(OSu)的合成
Figure PCTCN2021083862-appb-000014
将苄基十六烷二酰基-L-Glu(OSu)-OBn(10g，14.43mmol)溶于200mL丙酮中，加入三氟乙酸(0.1g，0.8mmol)和0.8g 10％Pd/C，置于30℃进行氢化脱苄反应3.0小时，反应结束过滤除掉钯碳，减压蒸馏浓缩至干得到固体粗品，加入55mL丙酮和138mL正庚烷于室温打浆2.0小时，抽滤，用30mL正庚烷淋洗滤饼，滤饼置于40℃中真空干燥至恒重，即可得到十六烷二酰基-L-Glu(OSu)白色固体7.05g，收率95.34％，HPLC检测纯度为98.78％。
ESI-MS m/z：513.3[M+H] +，与理论值符合
实施例7十六烷二酰基-L-Glu(OSu)在德谷胰岛素中的制备应用
将Des(B30)人胰岛素500mg溶解于10mL水中，加入三乙胺调体系pH为11.00。按照十六烷二酰基L-Glu(OSu)：Des(B30)人胰岛素摩尔比1.2：1称取十六烷二酰基-L-Glu(OSu)53mg，溶解于2mL NMP(N-甲基吡咯烷酮)中，随后加入人胰岛素溶液中，搅拌30分钟后，通过添加6.5mL稀HCl调节体系pH为9.0的0.2M乙醇胺终止反应。HPLC分析表明形成了65％的德谷胰岛素：LysB29(N-ε-十六烷二酰基-γ-谷氨酰基)脱(B30)人胰岛素，纯化后可得纯度高于99.60％的德谷胰岛素。
ESI-MS m/z：1526.8[M+4H] 4+,1221.7[M+5H] 5+，1218.1[M+6H] 6+，与理论值符合
对比例1
Figure PCTCN2021083862-appb-000015
按照诺和诺德公司专利号WO2007104737A1中第26页第2段中preparation of hexadecandioyl–L-Glu(OSu)-OH中所描述制备十六烷二酸苄酯的方法进行制备：将十六烷二酸(200.0g，0.7mol)、Dowex50WX2-100酸性阳离子树脂(700g)、正辛烷(3.6L)和甲酸苄酯(95g，0.7mol)，升温至91℃并将甲酸苄酯(340g，2.5mol)于9小时内逐步滴加入体系中，滴毕后91℃保持搅拌回流反应48小时，反应结束冷却至室温，过滤除掉溶 剂得到固体，固体溶于3L丙酮中并升温至40℃搅拌0.5小时，过滤，用1.5L丙酮淋洗滤饼，收集滤液真空浓缩至约1.5L后过滤并用-18℃丙酮淋洗得到固体湿品，溶于2L二氯甲烷并于室温搅拌0.5小时，过滤得滤液并减压蒸馏得到固体粗品，溶于900mL异丙醇中进行重结晶得到固体，真空烘干至恒重后得到固体约73g，收率27.8％。采用酸性阳离子树脂能够使得十六烷二酸选择性地合成十六烷二酸苄酯，但是该方法中所使用的Dowex50WX2-100酸性阳离子树脂比较昂贵，而且使用溶剂量较大，反应时间较长，收率低，后处理操作比较麻烦，导致制备成本较大。
对比例2
现有生产工艺，为优化后的诺和诺德公司专利号WO2007104737A1中第26页第2段中preparation of hexadecandioyl–L-Glu(OSu)-OH中所描述制备十六烷二酸苄酯的方法进行制备：
十六碳二酸(160.00g，559.03mmol)和Dowex 50wx2-100(480.00g)加入到2880ml的正辛烷中，升温至110℃回流，加入甲酸苄酯(152.23g，1118.06mmol)，回流反应6h后，再加入甲酸苄酯(152.23g，1118.06mmol)，继续回流搅拌反应64h。降温至室温，抽滤，240.00ml正辛烷淋洗滤饼，得湿品。一次打浆：将湿品转移至5000ml的三口瓶中，向反应瓶中加入2880ml的二氯甲烷(18.00ml/g二酸)，室温打浆2.0h后，抽滤，并用200.00ml二氯甲烷淋洗滤饼，滤液于40℃下真空浓缩得到白色固体粗品；二次打浆：向粗品中加入1440ml的二氯甲烷(9.00ml/g二酸)，室温打浆2.0h后，抽滤，并用200.00ml二氯甲烷淋洗滤饼，滤液于40℃下真空浓缩得到白色固体粗品；一次重结晶：向粗品中加入320ml异丙醇和960ml正庚烷，将体系升温至65℃搅拌溶清后，保温搅拌0.5h，降温至室温析出固体，再降温至0℃搅拌析晶1.0h，抽滤，215ml正庚烷淋洗，得到固体湿品；二次重结晶：向粗品中加入320ml异丙醇和960ml正庚烷，将体系升温至65℃搅拌溶清后，保温搅拌0.5h，降温至室温析出固体，再降温至0℃搅拌析晶1.0h，抽滤，215ml正庚烷淋洗，得到固体湿品；干燥：湿品于45～55℃下真空干燥16h，得固体94.5g，收率45％，HPLC检测纯度98.45％。
对比例3
Figure PCTCN2021083862-appb-000016
文献(Novel Acyl r-Pyronoids,Dictyopyrone A,B,and C,fromDictyostelium Cellular Slime Molds.J.Org.Chem.2000,65,985-989)中描述的一种十三烷二酸苄酯(文献所描述的化合物7)的制备方法，将十三烷二酸(4.8g，19.7mmol)溶解于150mL甲醇中，于室温搅 拌下滴加11mL 10％的氢氧化钾甲醇溶液并逐步形成无水的盐沉淀固体，搅拌20分钟后，将体系浓缩至干得到十三烷单羧酸钾盐，悬浮于50mL甲苯中，加入四叔丁基溴化铵(546mg，1.97mmol)和苄溴(2.57mL,21.6mmol)，回流反应6.0小时，反应结束冷却至室温，加入100mL 0.5M盐酸溶液并用乙酸乙酯萃取三次，有机相依次用水、饱和食盐水洗涤，无水硫酸钠干燥，过滤收集滤液并减压真空浓缩，最后采用层析硅胶柱进行过柱提纯(正己烷：乙酸乙酯＝19:1,4:1和2:1)，收集淋洗液减压真空浓缩至干得到无色针状固体十三烷二酸苄酯3.42g，收率52％。该工艺方法产生杂质比较多，后处理提纯困难，难以实现工业化。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种制备长链脂肪二酸单苄酯的方法，其特征在于，包括：
    1-1)将长脂肪链二酸进行酯化反应，以便获得长脂肪链二苄酯；
    1-2)将长脂肪链二苄酯进行水解反应，以便获得长链脂肪二酸单苄酯；
    其中，所述长脂肪链二酸具有式(A)所示的结构，
    Figure PCTCN2021083862-appb-100001
    X为6～32的整数。
  2. 根据权利要求1所述的方法，其特征在于，所述水解反应为酸水解或碱水解，优选地，所述水解反应为碱水解；
    任选地，所述水解反应为酸水解，步骤1-2)之后进一步包括酸游离处理和萃取处理，以便获得含有长链脂肪二酸单苄酯的有机相。
  3. 根据权利要求2所述的方法，其特征在于，长脂肪链二苄酯与酸进行接触，以进行水解反应，所述酸包括选自选自盐酸、磷酸、醋酸、对甲苯磺酸和三氟乙酸的至少之一；
    优选地，长脂肪链二苄酯与碱进行接触，以进行水解反应，所述碱包括选自NaOH、KOH、LiOH、K 2CO 3、t-BuOK、KOAc、Cs 2CO 3、K 3PO 4、NaOAc、LiOH·H 2O和Ca(OAc) 2·H 2O的至少之一。
  4. 根据权利要求1所述的方法，其特征在于，所述水解反应是在第二溶剂中进行的，所述第二溶剂包括选自乙醇、甲醇、异丙醇、叔丁醇、苄醇、丙酮、丁酮、1,4-二氧六环、NMP、DMF、DMSO、甲苯和水的至少之一。
  5. 根据权利要求1所述的方法，其特征在于，所述酯化反应是通过将所述长脂肪链二酸与包括苄醇、氯苄、溴苄、苄胺、二苄肼、甲酸苄酯、乙酸苄酯、苯甲醛的至少之一进行接触进行的；
    任选地，所述酯化反应是在第一催化剂催化下进行的，所述第一催化剂包括选自盐酸、硫酸、对甲苯磺酸和三氟乙酸的至少之一，优选地，所述第一催化剂为对甲苯磺酸；
    任选地，所述酯化反应是在第一溶剂中进行的，所述第一溶剂包括选自甲醇、乙醇、乙腈、甲苯、叔丁醇、苄醇、DMF和DMSO的至少之一，优选地，所述第一溶剂为甲苯。
  6. 根据权利要求1所述的方法，其特征在于，步骤1-1)之后，步骤1-2)之前，进一 步包括使步骤1-1)反应后的体系进行析晶和过滤处理；
    任选地，步骤1-2)之后，进一步包括使步骤1-2)反应后的体系进行析晶和过滤处理；
    任选地，所述析晶处理是通过向步骤1-1)反应后的体系中或步骤1-2)反应后的体系中加入反向溶剂进行的；
    任选地，所述反向溶剂为非极性烃类溶剂。
  7. 根据权利要求1所述的方法，其特征在于，所述酯化反应是在温度为50℃～150℃的条件下进行2～48小时；
    任选地，所述水解反应是在温度为10～75℃的条件下进行0.5h～48h。
  8. 根据权利要求1～4任一项所述的方法，其特征在于，所述长脂肪链二酸与苄醇的摩尔比为1：(2～5)；
    优选地，所述长脂肪链二酸与第一催化剂的摩尔比为1：(0.1～1)；
    任选地，所述长脂肪链二苄酯与酸或碱的摩尔比为1：(1～5)。
  9. 一种制备长脂肪链二酸衍生物的方法，其特征在于，包括：
    (1)将长脂肪链二酸依据权利要求1～8任一项所述的方法进行处理，以便获得所述长脂肪链二酸单苄酯，所述长脂肪链二酸具有式(A)所示的结构；
    (2)将所述长脂肪链二酸单苄酯与N-羟基琥珀酰亚胺发生酯化反应，以便获得长脂肪链二酸琥珀酰亚胺苄酯；
    (3)将所述长脂肪链二酸琥珀酰亚胺苄酯与式(B)所示化合物进行亲核加成酰胺化反应，以便获得式(C)所示化合物；
    (4)将所述式(C)所示化合物再次与N-羟基琥珀酰亚胺发生酯化反应，以便获得式(D)所示化合物；以及
    (5)将所述式(D)所示化合物进行脱苄基反应，以便获得式(E)所示化合物；
    Figure PCTCN2021083862-appb-100002
    Figure PCTCN2021083862-appb-100003
    其中，X为6～32的整数；Y为1～6的整数。
  10. 一种制备降血糖蛋白药物的方法，其特征在于，包括:采用长脂肪链二酸衍生物对蛋白进行修饰，以便获得所述降血糖蛋白药物，所述长脂肪链二酸衍生物是依据权利要求9所述的方法获得；
    任选地，所述降血糖蛋白药物包括选自胰岛素、GLP-1、胰岛素类似物和GLP-1类似物的至少之一。
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