WO2021185304A1 - Method for preparing long aliphatic chain diacid derivative and application thereof - Google Patents

Method for preparing long aliphatic chain diacid derivative and application thereof Download PDF

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Publication number
WO2021185304A1
WO2021185304A1 PCT/CN2021/081465 CN2021081465W WO2021185304A1 WO 2021185304 A1 WO2021185304 A1 WO 2021185304A1 CN 2021081465 W CN2021081465 W CN 2021081465W WO 2021185304 A1 WO2021185304 A1 WO 2021185304A1
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aliphatic chain
reaction
long aliphatic
chain diacid
hours
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PCT/CN2021/081465
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French (fr)
Chinese (zh)
Inventor
郑宝液
王海龙
郭林峰
杨福斌
饶万兵
叶艳影
刘玉婷
廖廷聪
王仲清
李文佳
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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 WO2021185304A1 publication Critical patent/WO2021185304A1/en

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    • 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
    • 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
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a method for preparing long aliphatic chain diacid derivatives and applications thereof.
  • 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. For this reason, the present invention provides a new method for preparing long aliphatic chain diacid derivatives.
  • the present invention proposes a method for preparing long aliphatic chain diacid derivatives.
  • the method includes: (1) cyclizing a long aliphatic chain diacid, the long aliphatic chain diacid having a structure represented by formula (A); (2) converting the long aliphatic chain The diacid cyclization product is reacted with benzyl alcohol to obtain long aliphatic chain diacid monobenzyl ester; (3) The long aliphatic chain diacid monobenzyl ester is esterified with N-hydroxysuccinimide to obtain Long fatty chain diacid succinimide benzyl ester; (4) The long fatty chain diacid succinimide benzyl ester and the compound represented by formula (B) are subjected to a nucleophilic addition amidation reaction to obtain the formula ( C) the compound represented by; (5) the compound represented by the formula (C) is again esterified with N-hydroxysucc
  • 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:
  • the cyclization reaction is carried out in the presence of an acid anhydride.
  • the acid anhydride is Boc 2 O anhydride.
  • the cyclization reaction is carried out in an aprotic polar reagent, for example, in one or at least two solvents of N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran ongoing.
  • an aprotic polar reagent for example, in one or at least two solvents of N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran ongoing.
  • the aprotic polar reagent is beneficial to the ionization of the catalyst and the occurrence of the nucleophilic addition cyclization reaction induced by Lewis acid.
  • the cyclization reaction is carried out under the catalysis of one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid. Furthermore, the conversion rate of the cyclization reaction of the long aliphatic chain diacid is further improved.
  • the acid anhydride and the above-mentioned catalyst are used to catalyze the formation of an acid anhydride from the long aliphatic chain diacid, which is then reacted with benzyl alcohol to obtain the target product, which can effectively improve the selectivity of the reaction.
  • the cyclization reaction is carried out at a temperature of 30°C to 80°C for 12 to 48 hours. This further improves the conversion rate of the reaction and reduces the generation of by-products.
  • the amount of the acid anhydride is excessive relative to the long aliphatic chain diacid. This further improves the conversion rate of the cyclization reaction.
  • the molar amount of the Boc 2 O acid anhydride is equivalent to 1.0 to 2.0 times the molar amount of the long aliphatic chain diacid. Furthermore, the full occurrence of the reaction can be ensured, the dosage of the long aliphatic chain diacid can be reduced, and the production cost and post-processing cost can be effectively reduced.
  • the molar amount of one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid is equivalent to the molar amount of the long aliphatic chain diacid 0.1 to 1.0 times of
  • step (2) the reaction is carried out in a non-polar hydrocarbon solvent at 25°C to 80°C for 12 to 48 hours.
  • the non-polar hydrocarbon solvent is one or at least two of n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, methyl tert-butyl ether, and toluene .
  • the molar amount of the benzyl alcohol is equivalent to 1.0 to 3.0 times the molar amount of the long aliphatic chain diacid.
  • step (2) of the present application benzyl protection is introduced, and subsequent direct hydrogen debenzylation is sufficient.
  • the amount of trifluoroacetic acid used is small, and the waste acid water is small.
  • step (3) the esterification reaction is carried out in one or at least two solvents among 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 comprises 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 (4) the reaction is performed in the solvent being 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 (5) it further comprises contacting the benzyl ester long aliphatic chain diacyl-L-Glu-OBn with dicyclohexylcarbodiimide.
  • step (5) 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 (6) the debenzylation reaction is performed in the condition 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.
  • 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.
  • 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 aforementioned method 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 fatty acid side chain modification.
  • the present invention overcomes the shortcomings and deficiencies of the prior art and provides a method for preparing long aliphatic chain diacid derivatives.
  • the method has cheap and easy to obtain raw materials and reagents, simple operation, low product-related impurities, simple intermediate purification, and High rate, less "solid waste” and low environmental protection pressure.
  • the prepared long aliphatic chain diacid derivatives can be used for the development of long-acting insulin drugs such as insulin degludec, GLP1 analogs and other drugs through the general protein modification technology.
  • the solvent A shown in step 1) is one or at least two of N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran;
  • the catalyst A shown in step 1) is one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid;
  • solvent B is one or at least two of non-polar hydrocarbon solvents such as n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, methyl tert-butyl ether, and toluene;
  • the solvent C is one or at least two of dichloromethane, ethyl acetate, and propyl acetate;
  • the solvent D is one or at least two of tetrahydrofuran, ethyl acetate and dichloromethane;
  • the solvent E is one or at least two of n-heptane, n-hexane, cyclohexane, petroleum ether or methyl tert-butyl ether;
  • the solvent F in step 4) is one or at least two of acetonitrile, N,N-dimethylformamide, tetrahydrofuran or N-methylpyrrolidone;
  • the organic base in step 4) is one or at least two of triethylamine, pyridine, DMAP, potassium carbonate or potassium hydroxide;
  • the solvent G in step 6) is one or at least two of acetone and tetrahydrofuran;
  • Pd/C in step 6 is a catalyst, wherein the palladium content is 5-15%;
  • the reaction temperature of the long aliphatic chain anhydride intermediate prepared by reacting the long aliphatic chain diacid, Boc 2 O anhydride and catalyst A in step 1) is 30°C to 80°C; the reaction time is 12 to 48 hours;
  • the reaction temperature between the long aliphatic anhydride intermediate prepared in step 2) and benzyl alcohol is 25°C to 80°C; the reaction time is 12 to 48 hours;
  • the temperature for cooling in step 3) is -10°C to 20°C;
  • step 3 The time described in step 3) for returning to 15-40°C and continuing the reaction is 3-24 hours;
  • the solvent used for recrystallization in step 3) is a mixed solvent, wherein the polar solvent is one or at least two of methanol, ethanol, and isopropanol, and the non-polar solvent is n-heptane, n-hexane, and cyclohexane.
  • the polar solvent is one or at least two of methanol, ethanol, and isopropanol
  • the non-polar solvent is n-heptane, n-hexane, and cyclohexane.
  • alkane and methyl tert-butyl ether is a mixed solvent, wherein the polar solvent is one or at least two of methanol, ethanol, and isopropanol, and the non-polar solvent is n-heptane, n-hexane, and cyclohexane.
  • alkane and methyl tert-butyl ether is one or at least two of alkane and methyl tert
  • the reaction temperature in step 4) is 25°C-40°C, and the reaction time is 6-24 hours;
  • the concentration of the hydrochloric acid solution described in step 4) is 0.5-2M;
  • the temperature for cooling in step 4) is -10°C to 10°C, and the time for crystallization and stirring is 1.0 to 3.0h;
  • the cooling temperature in step 5 is -10 to 10°C, and the time to resume the reaction when it is restored to 15°C to 40°C is 3-24 hours;
  • the recrystallization solvent in step 5) is one or at least two of ethanol, tert-butanol, and isopropanol;
  • step 6 the reaction temperature is 15-40°C, and the hydrodebenzylation time is 1 to 5 hours;
  • the time for beating the mixed solvent in step 6) is 1 to 3 hours.
  • the molar amount of Boc 2 O anhydride in step 1) is equivalent to 1.0 to 2.0 times the molar amount of the long aliphatic chain diacid;
  • the molar amount of the catalyst A in step 1) is equivalent to 0.1 to 1.0 times the molar amount of the long aliphatic chain diacid
  • the amount of solvent A described in step 1) is calculated according to the volume-to-mass ratio of the solvent A to the long aliphatic chain diacid 8mL-20mL:1g;
  • the molar amount of benzyl alcohol in step 2) is equivalent to 1.0 to 3.0 times the molar amount of the long aliphatic chain diacid
  • the amount of solvent B described in step 2) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid 8mL-20mL:1g;
  • the amount of solvent C described in step 2) is calculated based on the volume-to-mass ratio of the solvent C to the long aliphatic chain diacid 8mL-20mL:1g;
  • the amount of tap water mentioned in step 2) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid 20mL-40mL:1g;
  • the molar amount of the N-hydroxysuccinimide in step 3) is equivalent to 1.0 to 1.2 times the molar amount of the long aliphatic chain diacid benzyl ester;
  • the molar amount of dicyclohexylcarbodiimide in step 3) is equivalent to 1.0 to 1.4 times the molar amount of benzyl long aliphatic chain diacid;
  • the amount of solvent D described in step 3) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid benzyl ester 10mL-20mL:1g;
  • the amount of recrystallization solvent described in step 3) is calculated according to the volume-mass ratio of the long aliphatic chain diacid benzyl ester 10mL ⁇ 20mL:1g, wherein the volume ratio of the polar solvent to the non-polar solvent is 1:1 ⁇ 2.0;
  • the dosage of the agent E for leaching in step 3) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid benzyl ester 2mL ⁇ 5mL:1g;
  • the molar amount of L-glutamate-1-benzyl ester in step 4) is equivalent to 1.0 to 1.2 times the molar amount of long aliphatic chain diacid succinimide benzyl ester;
  • the molar amount of the organic base described in step 4) is equivalent to 0.8 to 1.5 times the molar amount of the long aliphatic chain diacid succinimide benzyl ester;
  • the amount of solvent F described in step 4) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid succinimide benzyl ester 6mL-20mL:1g;
  • the amount of the hydrochloric acid solution described in step 4) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid succinimide benzyl ester 9mL-15mL:1g;
  • the amount of tap water added during low-temperature crystallization in step 4) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid succinimide benzyl ester 6mL-15mL:1g;
  • the molar amount of N-hydroxysuccinimide in step 5) is equivalent to 1.0 to 1.2 times the molar amount of benzyl long aliphatic chain diacyl-L-Glu-OBn;
  • the molar amount of N-hydroxysuccinimide in step 5) is equivalent to 1.0 to 1.4 times the molar amount of benzyl long aliphatic chain diacyl-L-Glu-OBn;
  • the amount of solvent C in step 5) is calculated according to the volume-to-mass ratio of the benzyl long aliphatic chain diacyl-L-Glu-OBn 8mL-20mL:1g;
  • the amount of the recrystallization solvent in step 5) is calculated according to the volume-to-mass ratio of the benzyl long aliphatic chain diacyl-L-Glu-OBn 10mL-20mL:1g;
  • step 5 the amount of solvent E used for rinsing is calculated according to the volume-to-mass ratio of benzyl long aliphatic chain diacyl-L-Glu-OBn 2mL ⁇ 5mL:1g;
  • the amount of 10% Pd/C in step 6) is calculated as 5% to 15% of the mass of the benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn;
  • the amount of trifluoroacetic acid in step 6) is calculated to be equivalent to 0.05% to 1.0% of the mass of the benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn;
  • the amount of solvent G in step 6) is calculated based on the volume-to-mass ratio of the benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn 15mL ⁇ 25mL:1g;
  • the amount of the mixed solvent (solvent G and solvent E) used for beating described in step 6) is 15mL ⁇ 25mL:1g calculation, where the volume ratio of solvent G to solvent E is 1:2.0 ⁇ 4.0;
  • step (2) the yield of long aliphatic chain diacid benzyl ester is 35-45%, and the purity is 95-98%; in step (3) the yield of long aliphatic chain diacid succinimide benzyl ester is 80-95%, Purity 95-99%; step (4) benzyl long fatty chain diacyl-L-Glu-OBn yield 95-99%, purity 95-97%; step (5) benzyl long fatty chain diacyl-L- The yield of Glu(OSu)-OBn is 80-95% and the purity is 95-99%; step (6) the yield of the long aliphatic chain diacid derivative is 90-98% and the purity is 95-99%.
  • the yield of insulin degludec API used for the preparation is 55-68%, and the purity is 99-99.80%.
  • LysB29 N- ⁇ -tetradecanedioyl- ⁇ -glutamyl de(B30) human insulin, after purification, insulin analogues with purity higher than 99.60% can be obtained .
  • LysB29 N- ⁇ -octadecandioyl- ⁇ -glutamyl de(B30) human insulin, after purification, an insulin analog with a purity higher than 99.60% can be obtained
  • LysB29 N- ⁇ -eicosandioyl- ⁇ -glutamyl de(B30) human insulin, after purification, an insulin analog with a purity higher than 99.60% can be obtained
  • acidic cationic resins can make hexadecanedioic acid be used to selectively synthesize hexadecanedioic acid benzyl ester, but the Dowex50WX2-100 acidic cationic resin used in this method is relatively 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.
  • a kind of benzyl tridecanedioate ( The preparation method of compound 7) described in the literature, dissolve tridecanedioic acid (4.8g, 19.7mmol) in 150mL methanol, add 11mL-10% potassium hydroxide methanol solution dropwise with stirring at room temperature and gradually form anhydrous The salt precipitated solid. After stirring for 20 minutes, the system was concentrated to dryness to obtain potassium tridecane monocarboxylate, which was suspended in 50 mL of toluene. Tetra-tert-butylammonium bromide (546mg, 1.97mmol) and benzyl bromide (2.57 mL, 21.6mmol), reflux and react for 6.0 hours.
  • 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

A method for preparing a long aliphatic chain diacid derivative. The method comprises: (1) performing a cyclization reaction on long aliphatic chain diacid, the long aliphatic chain diacid having a structure as shown in formula (A); (2) reacting the cyclization product of the long aliphatic chain diacid with benzyl alcohol, so as to obtain long aliphatic chain diacid-benzyl ester; (3) performing an esterification reaction on the long aliphatic chain diacid-benzyl ester and N-hydroxysuccinimide, so as to obtain long aliphatic chain diacid-succinimide benzyl ester; (4) performing a nucleophilic addition amidation reaction on the long aliphatic chain diacid-succinimide benzyl ester and a compound represented by formula (B), so as to obtain a compound represented by formula (C); (5) performing an esterification reaction on the compound represented by formula (C) and N-hydroxysuccinimide again, so as to obtain a compound represented by formula (D); and (6) performing a debenzylation reaction on the compound represented by formula (D), so as to obtain a compound represented by formula (E).

Description

制备长脂肪链二酸衍生物的方法及其应用Method for preparing long aliphatic chain diacid derivative and its application 技术领域Technical field
本发明涉及生物制药领域，具体地，本发明涉及制备长脂肪链二酸衍生物的方法及其应用。The present invention relates to the field of biopharmaceuticals. Specifically, the present invention relates to a method for preparing long aliphatic chain diacid derivatives and applications thereof.
背景技术Background technique
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。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. At present, 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.
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。Although long aliphatic chain diacid derivatives can be used in the development of long-acting insulin drugs through general protein modification techniques, in the prior art, in the preparation process of long aliphatic chain diacid derivatives, chemical reagents have high cost, poor selectivity, and side effects. There are many products and it is difficult to purify, and it is difficult to achieve industrial scale-up. However, 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.
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。Therefore, the preparation method of long aliphatic chain diacid derivatives still needs further development and improvement.
发明内容Summary of the invention
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，本发明提供了一种全新的制备长脂肪链二酸衍生物的方法。The present invention aims to solve one of the technical problems in the related art at least to a certain extent. For this reason, the present invention provides a new method for preparing long aliphatic chain diacid derivatives.
在本发明的第一方面，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)使长脂肪链二酸发生环化反应，所述长脂肪链二酸具有式(A)所示的结构；(2)将长脂肪链二酸环化产物与苄醇反应，以便获得长脂肪链二酸一苄酯；(3)将所述长脂肪链二酸一苄酯与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；(4)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；(5)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(6)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；In the first aspect of the present invention, the present invention proposes a method for preparing long aliphatic chain diacid derivatives. According to an embodiment of the present invention, the method includes: (1) cyclizing a long aliphatic chain diacid, the long aliphatic chain diacid having a structure represented by formula (A); (2) converting the long aliphatic chain The diacid cyclization product is reacted with benzyl alcohol to obtain long aliphatic chain diacid monobenzyl ester; (3) The long aliphatic chain diacid monobenzyl ester is esterified with N-hydroxysuccinimide to obtain Long fatty chain diacid succinimide benzyl ester; (4) The long fatty chain diacid succinimide benzyl ester and the compound represented by formula (B) are subjected to a nucleophilic addition amidation reaction to obtain the formula ( C) the compound represented by; (5) the compound represented by the formula (C) is again esterified with N-hydroxysuccinimide to obtain the compound represented by the formula (D); and (6) the compound represented by the formula (D) The compound represented by formula (D) undergoes a debenzylation reaction to obtain the compound represented by formula (E);
Figure PCTCN2021081465-appb-000001
Figure PCTCN2021081465-appb-000001
其中，X为6～32的整数；Y为1～6的整数。Wherein, X is an integer of 6 to 32; Y is an integer of 1 to 6.
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。According to the above method of the embodiment of the present invention, 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.
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：According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的实施例，所述环化反应是在酸酐存在的条件进行的，优选地，所述酸酐为Boc 2O酸酐。发明人发现，所述环化反应在酸酐存在的条件下进行，尤其是在Boc 2O酸酐的条件下进行，长脂肪链二酸一苄酯的纯度显著提高。 According to an embodiment of the present invention, the cyclization reaction is carried out in the presence of an acid anhydride. Preferably, the acid anhydride is Boc 2 O anhydride. The inventor found that the cyclization reaction is carried out in the presence of acid anhydride, especially in the presence of Boc 2 O acid anhydride, and the purity of the long aliphatic chain diacid monobenzyl ester is significantly improved.
根据本发明的实施例，所述环化反应是在非质子极性试剂中进行的，例如，N,N-二甲基甲酰胺、乙腈、丙酮和四氢呋喃中的一种或至少两种溶剂中进行的。发明人发现，非质子极性试剂，有利于催化剂的电离和由路易斯酸诱导的亲核加成环化反应的发生。According to an embodiment of the present invention, the cyclization reaction is carried out in an aprotic polar reagent, for example, in one or at least two solvents of N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran ongoing. The inventor found that the aprotic polar reagent is beneficial to the ionization of the catalyst and the occurrence of the nucleophilic addition cyclization reaction induced by Lewis acid.
根据本发明的实施例，所述环化反应是在无水氯化镁、6水氯化镁、氯化铁、氯化锆、DMAP或浓硫酸中的一种或至少两种的催化下进行的。进而进一步提高长脂肪链二酸发生环化反应的转化率。According to an embodiment of the present invention, the cyclization reaction is carried out under the catalysis of one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid. Furthermore, the conversion rate of the cyclization reaction of the long aliphatic chain diacid is further improved.
发明人发现，长脂肪链二酸，如十六烷二酸的选择性较差，在反应的起始很容易形成双封端的杂质。本申请采用酸酐和上述催化剂催化先让长脂肪链二酸形成酸酐，后续再与苄醇反应得到目标产物，可有效提高反应的选择性。The inventor found that long aliphatic chain diacids, such as hexadecanedioic acid, have poor selectivity, and it is easy to form double-terminated impurities at the beginning of the reaction. In this application, the acid anhydride and the above-mentioned catalyst are used to catalyze the formation of an acid anhydride from the long aliphatic chain diacid, which is then reacted with benzyl alcohol to obtain the target product, which can effectively improve the selectivity of the reaction.
根据本发明的实施例，所述环化反应是在温度为30℃～80℃的条件下进行12～48小时。进而进一步提高了反应的转化率和降低了副产物的产生。According to an embodiment of the present invention, the cyclization reaction is carried out at a temperature of 30°C to 80°C for 12 to 48 hours. This further improves the conversion rate of the reaction and reduces the generation of by-products.
根据本发明的实施例，相对于所述长脂肪链二酸，所述酸酐的用量是过量的。进而进一步提高了环化反应的转化率。According to an embodiment of the present invention, the amount of the acid anhydride is excessive relative to the long aliphatic chain diacid. This further improves the conversion rate of the cyclization reaction.
根据本发明的实施例，所述的Boc 2O酸酐的摩尔用量相当于所述的长脂肪链二酸摩尔量的1.0～2.0倍。进而可保证反应的充分发生，降低长脂肪链二酸的用量，有效降低生产成本和后处理成本。 According to an embodiment of the present invention, the molar amount of the Boc 2 O acid anhydride is equivalent to 1.0 to 2.0 times the molar amount of the long aliphatic chain diacid. Furthermore, the full occurrence of the reaction can be ensured, the dosage of the long aliphatic chain diacid can be reduced, and the production cost and post-processing cost can be effectively reduced.
根据本发明的实施例，无水氯化镁、6水氯化镁、氯化铁、氯化锆、DMAP或浓硫酸中的一种或至少两种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。According to an embodiment of the present invention, the molar amount of one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid is equivalent to the molar amount of the long aliphatic chain diacid 0.1 to 1.0 times of
根据本发明的实施例，在步骤(2)中，所述反应是在非极性烃类溶剂中、在25℃～80℃的条件下进行12～48小时。According to an embodiment of the present invention, in step (2), the reaction is carried out in a non-polar hydrocarbon solvent at 25°C to 80°C for 12 to 48 hours.
根据本发明的实施例，所述非极性烃类溶剂为正己烷、石油醚、环己烷、正庚烷、正辛烷、甲基叔丁基醚、甲苯中的一种或至少两种。According to an embodiment of the present invention, the non-polar hydrocarbon solvent is one or at least two of n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, methyl tert-butyl ether, and toluene .
根据本发明的实施例，所述的苄醇的摩尔用量相当于所述的长脂肪链二酸摩尔量的 1.0～3.0倍。According to an embodiment of the present invention, the molar amount of the benzyl alcohol is equivalent to 1.0 to 3.0 times the molar amount of the long aliphatic chain diacid.
在本申请的第(2)步反应中，引入苄基保护，后续直接氢气脱苄基即可，三氟乙酸的使用量很少，产生废酸水少。发明人发现，后续进行脱苄基，相比于脱叔丁基，更容易脱完全，且活性酯(HOSU基团)在脱苄基过程中能保持较好的稳定性。In the reaction of step (2) of the present application, benzyl protection is introduced, and subsequent direct hydrogen debenzylation is sufficient. The amount of trifluoroacetic acid used is small, and the waste acid water is small. The inventor found that subsequent debenzylation is easier to complete debenzylation than tert-butyl, and the active ester (HOSU group) can maintain better stability during the debenzylation process.
根据本发明的实施例，在步骤(3)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。According to an embodiment of the present invention, in step (3), the esterification reaction is carried out in one or at least two solvents among tetrahydrofuran, ethyl acetate and dichloromethane, at -10°C to 20°C. 2 hours, 15-40 ℃ continue to react under the condition of 3-24 hours.
根据本发明的实施例，进一步包括将长脂肪链二酸一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。According to an embodiment of the present invention, it further comprises 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℃～20℃) 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.
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。According to an embodiment of the present invention, 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.
根据本发明的实施例，所述Y为1、2或3。According to an embodiment of the present invention, the Y is 1, 2 or 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 PCTCN2021081465-appb-000002
According to an embodiment of the present invention, the compound represented by formula (B) is L-glutamic acid-1-benzyl ester, and the structure is
Figure PCTCN2021081465-appb-000002
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 PCTCN2021081465-appb-000003
According to an embodiment of the present invention, the compound represented by the formula (C) is a benzyl ester long aliphatic chain diacyl-L-Glu-Obn, and the structure is
Figure PCTCN2021081465-appb-000003
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 PCTCN2021081465-appb-000004
According to an embodiment of the present invention, the compound represented by formula (D) is a benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn with the structure
Figure PCTCN2021081465-appb-000004
根据本发明的实施例，所述式(E)所示化合物的结构为
Figure PCTCN2021081465-appb-000005
According to an embodiment of the present invention, the structure of the compound represented by formula (E) is
Figure PCTCN2021081465-appb-000005
根据本发明的实施例，在步骤(4)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。According to an embodiment of the present invention, in step (4), the reaction is performed in the solvent being 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.
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。According to an embodiment of the present invention, 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.
根据本发明的实施例，在步骤(5)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。According to an embodiment of the present invention, in step (5), it further comprises contacting the benzyl ester long aliphatic chain diacyl-L-Glu-OBn with dicyclohexylcarbodiimide.
根据本发明的实施例，在步骤(5)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。According to an embodiment of the present invention, in step (5), 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.
根据本发明的实施例，在步骤(6)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。According to an embodiment of the present invention, in step (6), the debenzylation reaction is performed in the condition 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.
在本发明的第二方面，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。In the second aspect of the present invention, the present invention provides a method for preparing a hypoglycemic protein drug. According to an embodiment of the present invention, 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 aforementioned method get. The hypoglycemic protein medicine obtained according to the method of the embodiment of the present invention has high yield and high purity.
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。According to an embodiment of the present invention, the hypoglycemic protein drug includes at least one selected from insulin, GLP-1, insulin analogues and GLP-1 analogues.
根据本发明的实施例，所述胰岛素类似物为德谷胰岛素。According to an embodiment of the present invention, the insulin analogue is insulin degludec.
根据本发明的实施例，所述GLP-1类似物为利拉鲁肽。According to an embodiment of the present invention, the GLP-1 analog is liraglutide.
根据本发明的实施例，所述修饰为脂肪酸侧链修饰。According to an embodiment of the present invention, the modification is a fatty acid side chain modification.
具体实施方式Detailed ways
本发明在于克服现有技术的缺点和不足，提供一种制备长脂肪链二酸衍生物的方法，该方法原料试剂价廉易得、操作简便、产品相关杂质含量低、中间体纯化简单，收率高，产生“固废”少，环保压力低。所制备的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如德谷胰岛素、GLP1类似物等药物的研发。The present invention overcomes the shortcomings and deficiencies of the prior art and provides a method for preparing long aliphatic chain diacid derivatives. The method has cheap and easy to obtain raw materials and reagents, simple operation, low product-related impurities, simple intermediate purification, and High rate, less "solid waste" and low environmental protection pressure. The prepared long aliphatic chain diacid derivatives can be used for the development of long-acting insulin drugs such as insulin degludec, GLP1 analogs and other drugs through the general protein modification technology.
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。The embodiments of the present invention are described in detail below. The embodiments described below are exemplary, and are intended to explain the present invention, but should not be construed as limiting the present invention.
1)将长脂肪链二酸、Boc 2O酸酐、催化剂A和溶剂A制备得到长脂肪链酸酐中间体， 其中Boc 2O酸酐稍过量，反应结束真空浓缩至干除掉未参与反应的Boc 2O酸酐得到固体粗品，粗品不经过提纯直接进行下一步反应； 1) The long aliphatic chain diacid, Boc 2 O acid anhydride, catalyst A and solvent A are prepared to obtain the long aliphatic chain anhydride intermediate, in which Boc 2 O anhydride is slightly excessive, and the reaction is completed and concentrated in vacuo to dryness to remove Boc 2 that has not participated in the reaction. O acid anhydride obtains a solid crude product, and the crude product is directly subjected to the next reaction without purification;
Figure PCTCN2021081465-appb-000006
Figure PCTCN2021081465-appb-000006
2)往体系中加入溶剂B和苄醇置于30℃-80℃中反应8-24h，反应结束加入适量自来水并于室温搅拌，过滤收集沉淀，将沉淀置于单口烧瓶中并加入适量溶剂C于室温打浆，过滤收集固体沉淀，再将固体沉淀溶于适量二氯甲烷中打浆，过滤取滤液，减压蒸馏至干即可得到长脂肪链二酸苄酯；2) Add solvent B and benzyl alcohol to the system and react for 8-24 hours at 30°C-80°C. At the end of the reaction, add an appropriate amount of tap water and stir at room temperature. The precipitate is collected by filtration. The precipitate is placed in a single-neck flask and an appropriate amount of solvent C is added. Beat at room temperature, filter and collect the solid precipitate, then dissolve the solid precipitate in an appropriate amount of dichloromethane to make a slurry, filter the filtrate, and distill under reduced pressure to dryness to obtain the long aliphatic chain diacid benzyl ester;
Figure PCTCN2021081465-appb-000007
Figure PCTCN2021081465-appb-000007
3)将长脂肪链二酸苄酯溶解于溶剂D中，降温至-10℃～20℃，加入N-羟基琥珀酰亚胺和二环己基碳二亚胺，于-10℃～20℃下继续反应2小时，恢复至15-40℃继续反应3-24小时，过滤除掉沉淀，减压蒸馏浓缩至干得固体，重结晶，用溶剂E淋洗滤饼，得到长脂肪链二酸琥珀酰亚胺苄酯；3) Dissolve the long aliphatic chain diacid benzyl ester in solvent D, lower the temperature to -10℃～20℃, add N-hydroxysuccinimide and dicyclohexylcarbodiimide, and keep at -10℃～20℃ Continue the reaction for 2 hours, return to 15-40°C and continue the reaction for 3-24 hours, filter to remove the precipitate, concentrate under reduced pressure to dry to obtain a solid, recrystallize, rinse the filter cake with solvent E to obtain long fatty chain diacid succinate Benzyl imide
Figure PCTCN2021081465-appb-000008
Figure PCTCN2021081465-appb-000008
4)将长脂肪链二酸琥珀酰亚胺苄酯与L-谷氨酸-1-苄酯溶解于溶剂F中，在有机碱存在的条件下置于25℃～40℃过夜搅拌，依次用0.5-2.0mol/L盐酸溶液和水，所述盐酸溶液和水的体积比为1：5～1：10，降温至-10℃～10℃中搅拌析晶，过滤并用适量水洗滤饼至中性，即可得到苄基长脂肪链二酰基-L-Glu-OBn；4) Dissolve long aliphatic chain diacid succinimidyl benzyl ester and L-glutamate-1-benzyl ester in solvent F, and stir overnight at 25°C to 40°C in the presence of an organic base. 0.5-2.0mol/L hydrochloric acid solution and water, the volume ratio of the hydrochloric acid solution and water is 1:5～1:10, cool to -10℃～10℃, stir and crystallize, filter and wash the filter cake with appropriate amount of water to the middle , You can get the benzyl long aliphatic chain diacyl-L-Glu-OBn;
Figure PCTCN2021081465-appb-000009
Figure PCTCN2021081465-appb-000009
5)将苄基长脂肪链二酰基-L-Glu-OBn溶解于溶剂C中，降温至-10℃～10℃，加入N-羟基琥珀酰亚胺和二环己基碳二亚胺，于-10℃～10℃下继续反应2小时，恢复至15℃～40℃继续反应3-24小时，过滤除掉沉淀，减压蒸馏浓缩至干得固体，重结晶，过滤，用溶剂E淋洗滤饼，得到苄基长脂肪链二酰基-L-Glu(OSu)-OBn；5) Dissolve the benzyl long aliphatic chain diacyl-L-Glu-OBn in solvent C, lower the temperature to -10℃～10℃, add N-hydroxysuccinimide and dicyclohexylcarbodiimide, Continue the reaction at 10℃～10℃ for 2 hours, return to 15℃～40℃ and continue the reaction for 3-24 hours, filter to remove the precipitate, concentrate by distillation under reduced pressure to dryness to obtain a solid, recrystallize, filter, and rinse with solvent E Cake to obtain benzyl long fatty chain diacyl-L-Glu(OSu)-OBn;
Figure PCTCN2021081465-appb-000010
Figure PCTCN2021081465-appb-000010
6)将苄基长脂肪链二酰基-L-Glu(OSu)-OBn溶于溶剂G中，加入适量三氟乙酸和适量10％Pd/C，于15～40℃进行氢化脱苄反应，反应结束过滤除掉钯碳，减压蒸馏浓缩至干得到固体粗品，加入适量溶剂G和溶剂E进行打浆，抽滤，用溶剂E淋洗滤饼，得到长脂肪链二酸衍生物。6) Dissolve benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn in solvent G, add appropriate amount of trifluoroacetic acid and appropriate amount of 10% Pd/C, and perform hydrogenation debenzylation reaction at 15-40°C. After filtration, the palladium carbon is removed, the crude solid product is obtained by distillation under reduced pressure and concentrated to dryness. Appropriate amount of solvent G and solvent E are added for pulping, filtered with suction, and the filter cake is rinsed with solvent E to obtain a long fatty chain diacid derivative.
Figure PCTCN2021081465-appb-000011
Figure PCTCN2021081465-appb-000011
其中，in,
步骤1)中所示的溶剂A为N,N-二甲基甲酰胺、乙腈、丙酮和四氢呋喃中的一种或至少两种；The solvent A shown in step 1) is one or at least two of N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran;
步骤1)中所示的催化剂A为无水氯化镁、6水氯化镁、氯化铁、氯化锆、DMAP或浓硫酸中的一种或至少两种；The catalyst A shown in step 1) is one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid;
步骤2)中溶剂B为正己烷、石油醚、环己烷、正庚烷、正辛烷、甲基叔丁基醚、甲苯等非极性烃类溶剂中的一种或至少两种；In step 2), solvent B is one or at least two of non-polar hydrocarbon solvents such as n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, methyl tert-butyl ether, and toluene;
步骤2)中溶剂C为二氯甲烷、乙酸乙酯、乙酸丙酯中的一种或至少两种；In step 2), the solvent C is one or at least two of dichloromethane, ethyl acetate, and propyl acetate;
步骤3)中溶剂D为四氢呋喃、乙酸乙酯和二氯甲烷中的一种或至少两种；In step 3), the solvent D is one or at least two of tetrahydrofuran, ethyl acetate and dichloromethane;
步骤3)中溶剂E为正庚烷、正己烷、环己烷、石油醚或甲基叔丁基醚中的一种或至少两种；In step 3), the solvent E is one or at least two of n-heptane, n-hexane, cyclohexane, petroleum ether or methyl tert-butyl ether;
步骤4)中的溶剂F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；The solvent F in step 4) is one or at least two of acetonitrile, N,N-dimethylformamide, tetrahydrofuran or N-methylpyrrolidone;
步骤4)中的有机碱为三乙胺、吡啶、DMAP、碳酸钾或氢氧化钾中的一种或至少两种；The organic base in step 4) is one or at least two of triethylamine, pyridine, DMAP, potassium carbonate or potassium hydroxide;
步骤6)中的溶剂G为丙酮、四氢呋喃中的一种或至少两种；The solvent G in step 6) is one or at least two of acetone and tetrahydrofuran;
步骤6)中的Pd/C是催化剂，其中钯含量为5～15％；Pd/C in step 6) is a catalyst, wherein the palladium content is 5-15%;
其中，in,
步骤1)中所述的将长脂肪链二酸、Boc 2O酸酐和催化剂A反应制备的长脂肪链酸酐中间体的反应温度为30℃～80℃；反应时间为12～48小时； The reaction temperature of the long aliphatic chain anhydride intermediate prepared by reacting the long aliphatic chain diacid, Boc 2 O anhydride and catalyst A in step 1) is 30°C to 80°C; the reaction time is 12 to 48 hours;
步骤2)中所述的制备的长脂肪链酸酐中间体与苄醇的反应温度为25℃～80℃；反应时间为12～48小时；The reaction temperature between the long aliphatic anhydride intermediate prepared in step 2) and benzyl alcohol is 25°C to 80°C; the reaction time is 12 to 48 hours;
步骤3)中所述的降温的温度为-10℃～20℃；The temperature for cooling in step 3) is -10°C to 20°C;
步骤3)中所述的恢复至15～40℃继续反应的时间为3-24小时；The time described in step 3) for returning to 15-40°C and continuing the reaction is 3-24 hours;
步骤3)中所述的重结晶所用的溶剂为混合溶剂，其中极性溶剂为甲醇、乙醇、异丙醇中一种或至少两种，非极性溶剂为正庚烷、正己烷、环己烷、甲基叔丁基醚中的一种或至少两种；The solvent used for recrystallization in step 3) is a mixed solvent, wherein the polar solvent is one or at least two of methanol, ethanol, and isopropanol, and the non-polar solvent is n-heptane, n-hexane, and cyclohexane. One or at least two of alkane and methyl tert-butyl ether;
步骤4)中所述的反应温度为25℃～40℃，反应时间为6～24小时；The reaction temperature in step 4) is 25°C-40°C, and the reaction time is 6-24 hours;
步骤4)中所述的盐酸溶液的浓度为0.5-2M；The concentration of the hydrochloric acid solution described in step 4) is 0.5-2M;
步骤4)中所述的降温的温度为-10℃～10℃，其中析晶搅拌的时间为1.0～3.0h；The temperature for cooling in step 4) is -10°C to 10°C, and the time for crystallization and stirring is 1.0 to 3.0h;
步骤5)中所述的降温的温度为-10～10℃，恢复至15℃～40℃继续反应的时间为3-24h；The cooling temperature in step 5) is -10 to 10°C, and the time to resume the reaction when it is restored to 15°C to 40°C is 3-24 hours;
步骤5)中所述的重结晶溶剂为乙醇、叔丁醇、异丙醇中的一种或至少两种；The recrystallization solvent in step 5) is one or at least two of ethanol, tert-butanol, and isopropanol;
步骤6)中所述的反应温度为15～40℃，氢化脱苄时间为1～5小时；In step 6), the reaction temperature is 15-40°C, and the hydrodebenzylation time is 1 to 5 hours;
步骤6)中所述的混合溶剂打浆的时间为1～3小时。The time for beating the mixed solvent in step 6) is 1 to 3 hours.
其中，in,
步骤1)中所述的Boc 2O酸酐的摩尔用量相当于所述的长脂肪链二酸摩尔量的1.0～2.0倍； The molar amount of Boc 2 O anhydride in step 1) is equivalent to 1.0 to 2.0 times the molar amount of the long aliphatic chain diacid;
步骤1)中所述的催化剂A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；The molar amount of the catalyst A in step 1) is equivalent to 0.1 to 1.0 times the molar amount of the long aliphatic chain diacid;
步骤1)中所述的溶剂A的用量按其与所述的长脂肪链二酸体积质量比8mL～20mL:1g计算；The amount of solvent A described in step 1) is calculated according to the volume-to-mass ratio of the solvent A to the long aliphatic chain diacid 8mL-20mL:1g;
步骤2)中所述的苄醇的摩尔用量相当于所述的长脂肪链二酸摩尔量的1.0～3.0倍；The molar amount of benzyl alcohol in step 2) is equivalent to 1.0 to 3.0 times the molar amount of the long aliphatic chain diacid;
步骤2)中所述的溶剂B的用量按其与所述的长脂肪链二酸体积质量比8mL～20mL:1g计算；The amount of solvent B described in step 2) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid 8mL-20mL:1g;
步骤2)中所述的溶剂C的用量按其与所述的长脂肪链二酸体积质量比8mL～20mL:1g计算；The amount of solvent C described in step 2) is calculated based on the volume-to-mass ratio of the solvent C to the long aliphatic chain diacid 8mL-20mL:1g;
步骤2)中所述的自来水的用量按其与所述的长脂肪链二酸体积质量比20mL～40mL:1g计算；The amount of tap water mentioned in step 2) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid 20mL-40mL:1g;
步骤3)中所述的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；The molar amount of the N-hydroxysuccinimide in step 3) is equivalent to 1.0 to 1.2 times the molar amount of the long aliphatic chain diacid benzyl ester;
步骤3)中所述的二环己基碳二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1.0～1.4倍；The molar amount of dicyclohexylcarbodiimide in step 3) is equivalent to 1.0 to 1.4 times the molar amount of benzyl long aliphatic chain diacid;
步骤3)中所述的溶剂D的用量按其与所述的长脂肪链二酸苄酯体积质量比10mL～20mL:1g计算；The amount of solvent D described in step 3) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid benzyl ester 10mL-20mL:1g;
步骤3)中所述的重结晶溶剂用量按其与所述的长脂肪链二酸苄酯体积质量比10mL～20mL:1g计算，其中极性溶剂与非极性溶剂的体积比为1:1～2.0；The amount of recrystallization solvent described in step 3) is calculated according to the volume-mass ratio of the long aliphatic chain diacid benzyl ester 10mL～20mL:1g, wherein the volume ratio of the polar solvent to the non-polar solvent is 1:1 ~2.0;
步骤3)中所述的淋洗用的剂E的用量按其与所述的长脂肪链二酸苄酯体积质量比2mL～5mL:1g计算；The dosage of the agent E for leaching in step 3) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid benzyl ester 2mL～5mL:1g;
步骤4)中所述的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；The molar amount of L-glutamate-1-benzyl ester in step 4) is equivalent to 1.0 to 1.2 times the molar amount of long aliphatic chain diacid succinimide benzyl ester;
步骤4)中所述的有机碱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0.8～1.5倍；The molar amount of the organic base described in step 4) is equivalent to 0.8 to 1.5 times the molar amount of the long aliphatic chain diacid succinimide benzyl ester;
步骤4)中所述的溶剂F的用量按其与所述的长脂肪链二酸琥珀酰亚胺苄酯体积质量比6mL～20mL:1g计算；The amount of solvent F described in step 4) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid succinimide benzyl ester 6mL-20mL:1g;
步骤4)中所述的盐酸溶液的用量按其与所述的长脂肪链二酸琥珀酰亚胺苄酯体积质量比9mL～15mL:1g计算；The amount of the hydrochloric acid solution described in step 4) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid succinimide benzyl ester 9mL-15mL:1g;
步骤4)中所述的低温析晶时加入自来水的用量按其与所述的长脂肪链二酸琥珀酰亚胺苄酯体积质量比6mL～15mL:1g计算；The amount of tap water added during low-temperature crystallization in step 4) is calculated according to the volume-to-mass ratio of the long aliphatic chain diacid succinimide benzyl ester 6mL-15mL:1g;
步骤5)中所述的N-羟基琥珀酰亚胺摩尔用量相当于所述的苄基长脂肪链二酰基-L-Glu-OBn摩尔量的1.0～1.2倍；The molar amount of N-hydroxysuccinimide in step 5) is equivalent to 1.0 to 1.2 times the molar amount of benzyl long aliphatic chain diacyl-L-Glu-OBn;
步骤5)中所述的N-羟基琥珀酰亚胺摩尔用量相当于所述的苄基长脂肪链二酰基-L-Glu-OBn摩尔量的1.0～1.4倍；The molar amount of N-hydroxysuccinimide in step 5) is equivalent to 1.0 to 1.4 times the molar amount of benzyl long aliphatic chain diacyl-L-Glu-OBn;
步骤5)中所述溶剂C的用量按其与所述的苄基长脂肪链二酰基-L-Glu-OBn体积质量比8mL～20mL:1g计算；The amount of solvent C in step 5) is calculated according to the volume-to-mass ratio of the benzyl long aliphatic chain diacyl-L-Glu-OBn 8mL-20mL:1g;
步骤5)中所述重结晶溶剂的用量按其与所述的苄基长脂肪链二酰基-L-Glu-OBn体积质量比10mL～20mL:1g计算；The amount of the recrystallization solvent in step 5) is calculated according to the volume-to-mass ratio of the benzyl long aliphatic chain diacyl-L-Glu-OBn 10mL-20mL:1g;
步骤5)中所述淋洗用的溶剂E的用量按其与所述的苄基长脂肪链二酰基-L-Glu-OBn体积质量比2mL～5mL:1g计算；In step 5), the amount of solvent E used for rinsing is calculated according to the volume-to-mass ratio of benzyl long aliphatic chain diacyl-L-Glu-OBn 2mL～5mL:1g;
步骤6)中所述的10％Pd/C的用量相当于所述的苄基长脂肪链二酰基-L-Glu(OSu)-OBn质量的5％～15％计算；The amount of 10% Pd/C in step 6) is calculated as 5% to 15% of the mass of the benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn;
步骤6)中所述的三氟乙酸的用量相当于所述的苄基长脂肪链二酰基-L-Glu(OSu)-OBn质量的0.05％～1.0％计算；The amount of trifluoroacetic acid in step 6) is calculated to be equivalent to 0.05% to 1.0% of the mass of the benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn;
步骤6)中所述溶剂G的用量按其与所述的苄基长脂肪链二酰基-L-Glu(OSu)-OBn体积质量比15mL～25mL:1g计算；The amount of solvent G in step 6) is calculated based on the volume-to-mass ratio of the benzyl long aliphatic chain diacyl-L-Glu(OSu)-OBn 15mL～25mL:1g;
步骤6)中所述的用于打浆用的混合溶剂(溶剂G和溶剂E)的用量按其与所述的苄 基长脂肪链二酰基-L-Glu(OSu)-OBn体积质量比15mL～25mL:1g计算，其中溶剂G与溶剂E的体积比为1:2.0～4.0；The amount of the mixed solvent (solvent G and solvent E) used for beating described in step 6) is 15mL~ 25mL:1g calculation, where the volume ratio of solvent G to solvent E is 1:2.0～4.0;
经统计，步骤(2)中长脂肪链二酸苄酯收率35-45％，纯度95-98％；步骤(3)长脂肪链二酸琥珀酰亚胺苄酯收率80-95％，纯度95-99％；步骤(4)苄基长脂肪链二酰基-L-Glu-OBn收率95-99％，纯度95-97％；步骤(5)苄基长脂肪链二酰基-L-Glu(OSu)-OBn收率80-95％，纯度95-99％；步骤(6)长脂肪链二酸衍生物收率90-98％，纯度95-99％。According to statistics, in step (2), the yield of long aliphatic chain diacid benzyl ester is 35-45%, and the purity is 95-98%; in step (3) the yield of long aliphatic chain diacid succinimide benzyl ester is 80-95%, Purity 95-99%; step (4) benzyl long fatty chain diacyl-L-Glu-OBn yield 95-99%, purity 95-97%; step (5) benzyl long fatty chain diacyl-L- The yield of Glu(OSu)-OBn is 80-95% and the purity is 95-99%; step (6) the yield of the long aliphatic chain diacid derivative is 90-98% and the purity is 95-99%.
用于制备的德谷胰岛素API收率55-68％，纯度99-99.80％。The yield of insulin degludec API used for the preparation is 55-68%, and the purity is 99-99.80%.
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。The present invention will be further explained below in conjunction with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1Example 1
十六烷二酸一苄酯的合成(a)Synthesis of hexadecanedioic acid monobenzyl ester (a)
Figure PCTCN2021081465-appb-000012
Figure PCTCN2021081465-appb-000012
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、Boc 2O酸酐(7.62g，34.91mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到十六烷酸酐固体粗品，加入苄醇(9.44g，87.36mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体5.26g，收率40％，HPLC检测纯度96.81％。 Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), Boc 2 O anhydride (7.62g, 34.91mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and the reaction solvent that did not participate in the reaction, to obtain the crude hexadecanoic anhydride solid product, add benzyl alcohol (9.44g, 87.36mmol) and 70mL tetrahydrofuran, place at 40℃ and stir for 24h. , Add 350mL tap water to the system, stir at room temperature for 0.5 hours to precipitate a solid, collect the precipitate by filtration, add 180mL n-octane, put the slurry at room temperature for 1.0h, filter, 20mL n-octane rinse the wet product to obtain a wet product, Add 180mL of dichloromethane to the wet product, beat at room temperature for 1.0h, filter and collect the filtrate, place it at 35°C and concentrate to dryness to obtain a solid, place it at 40°C and vacuum dry to constant weight to obtain white hexadecanedioic acid monobenzyl ester The solid was 5.26 g, the yield was 40%, and the purity by HPLC was 96.81%.
十六烷酸酐固体粗品MS测试：ESI-MS m/z：269.2[M+H] +，与理论值相符。 MS test of crude solid hexadecanoic anhydride: ESI-MS m/z: 269.2 [M+H] + , which is consistent with the theoretical value.
十六烷二酸一苄酯MS测试：ESI-MS m/z：377.2[M+H] +，与理论值符合 MS test of hexadecanedioic acid monobenzyl ester: ESI-MS m/z: 377.2[M+H] + , which accords with the theoretical value
十六烷二酸一苄酯的合成(b)Synthesis of hexadecanedioic acid monobenzyl ester (b)
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、Boc 2O酸酐(7.62g， 34.91mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(9.44g，87.36mmol)和70mL四氢呋喃，置于80℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体5.00g，收率38％，HPLC检测纯度97.26％。 Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), Boc 2 O anhydride (7.62g, 34.91mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove Boc 2 O anhydride and reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (9.44g, 87.36mmol) and 70mL tetrahydrofuran, and place it at 80°C and stir for 24h to react to the system. Add 350mL tap water, stir at room temperature for 0.5 hours to precipitate a solid, filter to collect the precipitate, add 180mL n-octane, pulp 1.0h at room temperature, filter, 20mL n-octane rinse wet product, get wet product, add to the wet product 180mL of dichloromethane, slurried at room temperature for 1.0h, filtered and collected the filtrate, placed at 35°C and concentrated to dryness to obtain a solid, placed at 40°C under vacuum and dried to constant weight to obtain 5.00g of white solid of hexadecanedioic acid monobenzyl ester. The yield was 38%, and the purity as determined by HPLC was 97.26%.
ESI-MS m/z：377.2[M+H] +，与理论值符合 ESI-MS m/z: 377.2[M+H] + , consistent with the theoretical value
十六烷二酸一苄酯的合成(c)Synthesis of hexadecanedioic acid monobenzyl ester (c)
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、Boc 2O酸酐(6.10g，27.93mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(9.44g，87.36mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体3.25g，收率24.71，HPLC检测纯度96.50％。 Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), Boc 2 O anhydride (6.10g, 27.93mmol) and 100mL of tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (9.44g, 87.36mmol) and 70mL tetrahydrofuran. Place it at 40°C and stir for 24h, then add it to the system. Add 350mL tap water, stir at room temperature for 0.5 hours to precipitate solids, filter to collect the precipitate, add 180mL n-octane, slurry 1.0h at room temperature, filter, 20mL n-octane rinse wet product, get wet product, add to the wet product 180mL of dichloromethane, slurried at room temperature for 1.0h, filtered and collected the filtrate, placed at 35°C and concentrated to dryness to obtain a solid, placed at 40°C under vacuum and dried to constant weight to obtain 3.25g of white solid of monobenzylhexadecanedioic acid. The yield was 24.71, and the purity by HPLC was 96.50%.
ESI-MS m/z：377.2[M+H] +，与理论值符合 ESI-MS m/z: 377.2[M+H] + , consistent with the theoretical value
十六烷二酸一苄酯的合成(d)Synthesis of hexadecanedioic acid monobenzyl ester (d)
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、Boc 2O酸酐(11.42g，52.37mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(9.44g，87.36mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体5.70g，收率43.35％，HPLC检测纯度96.30％。 Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), Boc 2 O anhydride (11.42g, 52.37mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (9.44g, 87.36mmol) and 70mL tetrahydrofuran. Place it at 40°C and stir for 24h, then add it to the system. Add 350mL tap water, stir at room temperature for 0.5 hours to precipitate solids, filter to collect the precipitate, add 180mL n-octane, slurry 1.0h at room temperature, filter, 20mL n-octane rinse wet product, get wet product, add to the wet product Make 180mL of dichloromethane, beat at room temperature for 1.0h, filter and collect the filtrate, place it at 35°C and concentrate to dryness to obtain a solid, and place it at 40°C to vacuum dry to constant weight to obtain 5.70g of white solid of hexadecanedioic acid monobenzyl ester. The yield was 43.35%, and the purity detected by HPLC was 96.30%.
ESI-MS m/z：377.2[M+H] +，与理论值符合 ESI-MS m/z: 377.2[M+H] + , consistent with the theoretical value
十六烷二酸一苄酯的合成(e)Synthesis of hexadecanedioic acid monobenzyl ester (e)
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、Boc 2O酸酐(11.42g，52.37mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(4.15g，38.40mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体4.62g，收率35.16％，HPLC检测纯度95.68％。 Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), Boc 2 O anhydride (11.42g, 52.37mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and the reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (4.15g, 38.40mmol) and 70mL tetrahydrofuran, and place it at 40°C and stir for 24h to react to the system. Add 350mL tap water, stir at room temperature for 0.5 hours to precipitate a solid, filter to collect the precipitate, add 180mL n-octane, pulp 1.0h at room temperature, filter, 20mL n-octane rinse wet product, get wet product, add to the wet product 180mL of dichloromethane, be slurried at room temperature for 1.0h, filtered and collected the filtrate, placed at 35°C and concentrated to dryness to obtain a solid, placed at 40°C under vacuum and dried to constant weight to obtain 4.62g of white solid of hexadecanedioic acid monobenzyl ester. The yield was 35.16%, and the purity by HPLC was 95.68%.
ESI-MS m/z：377.2[M+H] +，与理论值符合 ESI-MS m/z: 377.2[M+H] + , consistent with the theoretical value
十六烷二酸一苄酯的合成(f)Synthesis of hexadecanedioic acid monobenzyl ester (f)
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、Boc 2O酸酐(11.42g，52.37mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(9.44g，87.36mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，真空浓缩至干得到固体，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体2.61g，收率19.85％，HPLC检测纯度90.15％。 Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), Boc 2 O anhydride (11.42g, 52.37mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24 hours, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (9.44 g, 87.36 mmol) and 70 mL tetrahydrofuran. Place the reaction at 40° C. and stir for 24 hours, then concentrate in vacuo to Dry to obtain a solid, add 180mL of n-octane, put it at room temperature for 1.0h, filter, 20mL of n-octane rinse the wet product to obtain a wet product, add 180mL of dichloromethane to the wet product, beat at room temperature for 1.0h, filter and collect the filtrate. Placed at 35°C and concentrated to dryness in vacuum to obtain a solid, placed at 40°C and vacuum-dried to constant weight to obtain 2.61 g of white solid of hexadecanedioic acid monobenzyl ester, with a yield of 19.85% and a purity of 90.5% by HPLC.
ESI-MS m/z：377.2[M+H] +，与理论值符合 ESI-MS m/z: 377.2[M+H] + , consistent with the theoretical value
实施例2 十六烷二酸琥珀酰亚胺苄酯的合成Example 2 Synthesis of hexadecanedioic acid succinimidyl benzyl ester
Figure PCTCN2021081465-appb-000013
Figure PCTCN2021081465-appb-000013
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％。Add hexadecanedioic acid monobenzyl ester (10.0g, 25.56mmol) and 130mL of dichloromethane into a 250mL single-necked flask, cool to -5°C, add N-hydroxysuccinimide (3.24g, 28.12mmol) and two Cyclohexylcarbodiimide (6.32g, 30.67mmol), continue the reaction at -5°C for 2 hours, return to 30°C and continue the reaction for 16 hours, filter to remove the precipitate, distill and concentrate to dryness to obtain a crude solid product, add 60mL isopropyl Recrystallize the alcohol and 60 mL n-heptane, filter and rinse the wet product with 20 mL n-heptane, and dry to constant weight under reduced pressure to obtain 11.62 g of hexadecanedioic acid succinimidyl benzyl ester as a white solid, with a yield of 92.35%, which is detected by HPLC The purity is 98.80%.
ESI-MS m/z：474.1[M+H] +，与理论值符合 ESI-MS m/z: 474.1[M+H] + , consistent with the theoretical value
实施例3 苄基十六烷二酰基-L-Glu-OBn的合成Example 3 Synthesis of benzylhexadecane diacyl-L-Glu-OBn
Figure PCTCN2021081465-appb-000014
Figure PCTCN2021081465-appb-000014
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％。Dissolve hexadecanedioic acid succinimidyl benzyl ester (10.0g, 21.11mmol) and L-glutamate-1-benzyl ester (5.26g, 22.17mmol) in 100mL acetonitrile, add triethylamine (3.2g , 31.67mmol) was placed at 30°C and stirred for 12 hours. After the reaction, 120mL 1M hydrochloric acid solution and 90mL tap water were added. After vacuum drying at ℃ to constant weight, 12.09 g of benzylhexadecandioyl-L-Glu-OBn pale yellow solid was obtained, the yield was 96.12%, and the purity by HPLC was 97.53%.
ESI-MS m/z：596.4[M+H] +，与理论值符合 ESI-MS m/z: 596.4[M+H] + , consistent with the theoretical value
实施例4 苄基十六烷二酰基-L-Glu(OSu)-OBn的合成Example 4 Synthesis of benzylhexadecandioyl-L-Glu(OSu)-OBn
Figure PCTCN2021081465-appb-000015
Figure PCTCN2021081465-appb-000015
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％。Dissolve benzylhexadecandioyl-L-Glu-OBn (10.0g, 16.79mmol) in 130mL of dichloromethane, cool to -5°C, and add N-hydroxysuccinimide (2.13g, 18.47mmol) And dicyclohexylcarbodiimide (4.15g, 20.14mmol), continue to react at -5°C for 2 hours, return to 30°C and continue to react for 8 hours, filter to remove the precipitate, concentrate by distillation under reduced pressure to obtain a solid, add Recrystallize with 120 mL of absolute ethanol, filter, rinse the filter cake with 20 mL of n-heptane, and place the filter cake at 45°C under vacuum drying to constant weight to obtain benzylhexadecandioyl-L-Glu(OSu)- OBn white solid 10.38g, the yield is 89.26%, and the purity detected by HPLC is 98.35%.
ESI-MS m/z：693.4[M+H] +，与理论值符合 ESI-MS m/z: 693.4[M+H] + , consistent with the theoretical value
实施例5 十六烷二酰基-L-Glu(OSu)的合成Example 5 Synthesis of hexadecane diacyl-L-Glu (OSu)
Figure PCTCN2021081465-appb-000016
Figure PCTCN2021081465-appb-000016
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％。Dissolve benzylhexadecane diacyl-L-Glu(OSu)-OBn (10g, 14.43mmol) in 200mL acetone, add trifluoroacetic acid (0.1g, 0.8mmol) and 0.8g 10% Pd/C, set Hydrodebenzylation was carried out at 30°C for 3.0 hours. At the end of the reaction, the palladium carbon was removed by filtration, and the crude solid product was obtained by distillation under reduced pressure. 55mL acetone and 138mL n-heptane were added to make slurry at room temperature for 2.0 hours, filtered with suction, and 30mL n-heptane The filter cake was eluted with alkane, and the filter cake was vacuum-dried to constant weight at 40°C to obtain 7.05 g of hexadecandioyl-L-Glu(OSu) white solid, with a yield of 95.34% and a purity of 98.78% by HPLC .
ESI-MS m/z：513.3[M+H] +，与理论值符合 ESI-MS m/z: 513.3[M+H] + , consistent with the theoretical value
实施例6 十六烷二酰基-L-Glu(OSu)在德谷胰岛素中的制备应用Example 6 Preparation and application of hexadecandioyl-L-Glu (OSu) in insulin deglu
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。Dissolve 500 mg of Des (B30) human insulin in 10 mL of water, and add triethylamine to adjust the pH of the system to 11.00. According to the molar ratio of hexadecandioyl L-Glu (OSu): Des (B30) human insulin of 1.2:1, weigh out 53 mg of hexadecandioyl-L-Glu (OSu) and dissolve in 2mL NMP(N-methylpyrrolidone) ), and then added to the human insulin solution. After stirring for 30 minutes, the reaction was terminated by adding 6.5 mL of diluted HCl to adjust the pH of the system to 0.2M ethanolamine at 9.0. HPLC analysis showed that 65% of insulin degludec was formed: LysB29 (N-ε-hexadecandioyl-γ-glutamyl) de(B30) human insulin, after purification, insulin degludec with a purity higher than 99.60% can be obtained .
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 ESI-MS m/z: 1526.8[M+4H] 4+ ,1221.7[M+5H] 5+ , 1218.1[M+6H] 6+ , which accords with the theoretical value
实施例7 十四烷二酸一苄酯的合成Example 7 Synthesis of monobenzyl tetradecanedioate
Figure PCTCN2021081465-appb-000017
Figure PCTCN2021081465-appb-000017
将十四烷二酸(10.0g，38.70mmol)、无水氯化镁(0.37g，3.87mmol)、Boc 2O酸酐(12.67g，58.05mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(10.46g，96.84mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得 到十四烷二酸一苄酯白色固体5.98g，收率44.36％，HPLC检测纯度97.63％。 Add tetradecanedioic acid (10.0g, 38.70mmol), anhydrous magnesium chloride (0.37g, 3.87mmol), Boc 2 O anhydride (12.67g, 58.05mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and the reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (10.46g, 96.84mmol) and 70mL tetrahydrofuran, place it at 40°C and stir for 24h, then add to the system. Add 350mL tap water, stir at room temperature for 0.5 hours to precipitate solids, filter to collect the precipitate, add 180mL n-octane, slurry 1.0h at room temperature, filter, 20mL n-octane rinse wet product, get wet product, add to the wet product 180mL of dichloromethane, be slurried at room temperature for 1.0h, filtered and collected the filtrate, placed at 35°C and concentrated to dryness to obtain a solid, placed at 40°C under vacuum and dried to constant weight to obtain 5.98g of white solid of monobenzyl myristate. The yield was 44.36%, and the purity as determined by HPLC was 97.63%.
ESI-MS m/z：349.3[M+H] +，与理论值符合 ESI-MS m/z: 349.3[M+H] + , consistent with the theoretical value
实施例8 十四烷二酸琥珀酰亚胺苄酯的合成Example 8 Synthesis of succinimide benzyl myristate
Figure PCTCN2021081465-appb-000018
Figure PCTCN2021081465-appb-000018
将十四烷二酸一苄酯(10.0g，28.70mmol)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(3.63g，31.57mmol)和二环己基碳二亚胺(7.10g，34.44mmol)，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，得到十四烷二酸琥珀酰亚胺苄酯固体11.53g，收率90.12％，HPLC检测纯度为96.75％。Add tetradecanedioic acid monobenzyl ester (10.0g, 28.70mmol) and 130mL of dichloromethane into a 250mL single-necked flask, cool to -5°C, add N-hydroxysuccinimide (3.63g, 31.57mmol) and two Cyclohexylcarbodiimide (7.10g, 34.44mmol), continue the reaction at -5°C for 2 hours, return to 30°C and continue the reaction for 16 hours, filter to remove the precipitate, distill and concentrate to dryness to obtain a crude solid product, add 60mL isopropyl Recrystallize the alcohol and 60 mL n-heptane, filter and rinse the wet product with 20 mL n-heptane, and dry under reduced pressure to a constant weight to obtain 11.53 g of succinimide benzyl myristate as a solid. The yield is 90.12%. The purity is determined by HPLC. It is 96.75%.
ESI-MS m/z：446.4[M+H] +，与理论值符合 ESI-MS m/z: 446.4[M+H] + , consistent with the theoretical value
实施例9 苄基十四烷二酰基-L-Glu-OBn的合成Example 9 Synthesis of benzyltetradecane diacyl-L-Glu-OBn
Figure PCTCN2021081465-appb-000019
Figure PCTCN2021081465-appb-000019
将十四二酸琥珀酰亚胺苄酯(10.0g，22.44mmol)与L-谷氨酸-1-苄酯(5.59g，23.56mmol)溶解于100mL乙腈中，加入三乙胺(3.41g，33.66mmol)置于30℃搅拌12小时，反应结束一次加入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，即可得到苄基十四烷二酰基-L-Glu-OBn淡黄色固体12.00g，收率94.23％，HPLC检测纯度为96.60％。Dissolve succinimidyl myristate (10.0g, 22.44mmol) and L-glutamate-1-benzyl ester (5.59g, 23.56mmol) in 100mL acetonitrile, add triethylamine (3.41g, 33.66mmol) was placed at 30°C and stirred for 12 hours. After the reaction, 120mL 1M hydrochloric acid solution and 90mL tap water were added at once, cooled to -5°C and stirred for crystallization for 2.0 hours. Filtered and washed the filter cake with an appropriate amount of water until it became neutral. The filter cake was placed at 45°C. After vacuum drying at ℃ to constant weight, 12.00 g of benzyltetradecanedioyl-L-Glu-OBn pale yellow solid can be obtained, the yield is 94.23%, and the purity detected by HPLC is 96.60%.
ESI-MS m/z：568.2[M+H] +，与理论值符合 ESI-MS m/z: 568.2[M+H] + , consistent with the theoretical value
实施例10 苄基十四烷二酰基-L-Glu(OSu)-OBn的合成Example 10 Synthesis of benzyltetradecanedioyl-L-Glu(OSu)-OBn
Figure PCTCN2021081465-appb-000020
Figure PCTCN2021081465-appb-000020
将苄基十四烷二酰基-L-Glu-OBn(10.0g，17.61mmol)溶解于130mL二氯甲烷中，降 温至-5℃，加入N-羟基琥珀酰亚胺(2.23g，19.37mmol)和二环己基碳二亚胺(4.36g，21.13mmol)，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，即可得到苄基十四烷二酰基-L-Glu(OSu)-OBn白色固体10.21g，收率87.23％，HPLC检测纯度为98.16％。Dissolve benzyltetradecanedioyl-L-Glu-OBn (10.0g, 17.61mmol) in 130mL of dichloromethane, cool to -5°C, and add N-hydroxysuccinimide (2.23g, 19.37mmol) And dicyclohexylcarbodiimide (4.36g, 21.13mmol), continue to react at -5°C for 2 hours, return to 30°C and continue to react for 8 hours, filter to remove the precipitate, distill under reduced pressure and concentrate to dryness to obtain a solid, add Recrystallize with 120 mL of absolute ethanol, filter, rinse the filter cake with 20 mL of n-heptane, and place the filter cake at 45°C under vacuum drying to a constant weight to obtain benzyltetradecane diacyl-L-Glu(OSu)- OBn white solid 10.21g, the yield is 87.23%, and the purity detected by HPLC is 98.16%.
ESI-MS m/z：665.4[M+H] +，与理论值符合 ESI-MS m/z: 665.4[M+H] + , consistent with the theoretical value
实施例11 十四烷二酰基-L-Glu(OSu)的合成Example 11 Synthesis of Tetradecandiacyl-L-Glu (OSu)
Figure PCTCN2021081465-appb-000021
Figure PCTCN2021081465-appb-000021
将苄基十四烷二酰基-L-Glu(OSu)-OBn(10g，15.04mmol)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，即可得到十四烷二酰基-L-Glu(OSu)白色固体6.72g，收率92.18％，HPLC检测纯度为98.12％。Dissolve benzyltetradecane diacyl-L-Glu(OSu)-OBn (10g, 15.04mmol) in 200mL acetone, add trifluoroacetic acid (0.1g, 0.8mmol) and 0.8g 10% Pd/C, set Hydrodebenzylation was carried out at 30°C for 3.0 hours. At the end of the reaction, the palladium carbon was removed by filtration, and the crude solid product was obtained by distillation under reduced pressure. 55mL acetone and 138mL n-heptane were added to make slurry at room temperature for 2.0 hours, filtered with suction, and 30mL n-heptane The filter cake was eluted with alkane, and the filter cake was vacuum-dried at 40°C to a constant weight to obtain 6.72 g of white solid tetradecane diacyl-L-Glu (OSu) with a yield of 92.18% and a purity of 98.12% by HPLC .
ESI-MS m/z：485.4[M+H] +，与理论值符合 ESI-MS m/z: 485.4[M+H] + , consistent with the theoretical value
实施例12 十四烷二酰基-L-Glu(OSu)在胰岛素类似物中的制备应用Example 12 Preparation and application of tetradecanedioyl-L-Glu (OSu) in insulin analogues
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。按照十四烷二酰基L-Glu(OSu)：Des(B30)人胰岛素摩尔比1.2：1称取十四烷二酰基-L-Glu(OSu)50.96mg于2mL NMP(N-甲基吡咯烷酮)中，随后加入人胰岛素溶液中，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68％的德谷胰岛素：LysB29(N-ε-十四烷二酰基-γ-谷氨酰基)脱(B30)人胰岛素，纯化后可得纯度高于99.60％的胰岛素类似物。Dissolve 500 mg of Des (B30) human insulin in 10 mL of water, and add triethylamine to adjust the pH of the system to 11.00. According to the molar ratio of myristyl diacyl L-Glu (OSu): Des (B30) human insulin of 1.2:1, weigh 50.96 mg of myristyl diacyl-L-Glu (OSu) into 2 mL NMP (N-methylpyrrolidone) Then, it was added to the human insulin solution. After 30 minutes, the reaction was terminated by adding 6.5 mL of diluted HCl to adjust the pH of the system to 0.2M ethanolamine at 9.0. HPLC analysis showed that 68% of insulin degludec was formed: LysB29 (N-ε-tetradecanedioyl-γ-glutamyl) de(B30) human insulin, after purification, insulin analogues with purity higher than 99.60% can be obtained .
ESI-MS m/z：1519.8[M+4H] 4+,1216.1[M+5H] 5+，1013.2[M+6H] 6+，与理论值符合 ESI-MS m/z: 1519.8[M+4H] 4+ ,1216.1[M+5H] 5+ , 1013.2[M+6H] 6+ , consistent with the theoretical value
实施例13 十八烷二酸一苄酯的合成Example 13 Synthesis of monobenzyl octadecanedioate
Figure PCTCN2021081465-appb-000022
Figure PCTCN2021081465-appb-000022
将十八烷二酸(10.0g，31.80mmol)、无水氯化镁(0.30g，3.18mmol)、Boc 2O酸酐(10.41g，47.70mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(8.59g，79.50mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到十八烷二酸一苄酯白色固体4.91g，收率38.13％，HPLC检测纯度97.43％。 Add octadecanedioic acid (10.0g, 31.80mmol), anhydrous magnesium chloride (0.30g, 3.18mmol), Boc 2 O anhydride (10.41g, 47.70mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and the reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (8.59g, 79.50mmol) and 70mL tetrahydrofuran, and place it at 40°C and stir for 24h, then add to the system. Add 350mL tap water, stir at room temperature for 0.5 hours to precipitate solids, filter to collect the precipitate, add 180mL n-octane, slurry 1.0h at room temperature, filter, 20mL n-octane rinse wet product, get wet product, add to the wet product 180mL of dichloromethane, slurried at room temperature for 1.0h, filtered and collected the filtrate, placed at 35°C and concentrated to dryness to obtain a solid, placed at 40°C under vacuum and dried to constant weight to obtain 4.91g of white solid of monobenzyl octadecanedioate. The yield was 38.13%, and the purity as determined by HPLC was 97.43%.
ESI-MS m/z：405.3[M+H] +，与理论值符合 ESI-MS m/z: 405.3[M+H] + , consistent with the theoretical value
实施例14 十八烷二酸琥珀酰亚胺苄酯的合成Example 14 Synthesis of octadecanedioic acid succinimidyl benzyl ester
Figure PCTCN2021081465-appb-000023
Figure PCTCN2021081465-appb-000023
将十八烷二酸一苄酯(10.0g，24.72mmol)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(3.13g，27.19mmol)和二环己基碳二亚胺(6.12g，29.66mmol)，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，得到十八烷二酸琥珀酰亚胺苄酯固体11.28g，收率91.00％，HPLC检测纯度为98.10％。Add octadecanedioic acid monobenzyl ester (10.0g, 24.72mmol) and 130mL of dichloromethane into a 250mL single-necked flask, cool to -5°C, add N-hydroxysuccinimide (3.13g, 27.19mmol) and two Cyclohexylcarbodiimide (6.12g, 29.66mmol), continue the reaction at -5°C for 2 hours, return to 30°C and continue the reaction for 16 hours, filter to remove the precipitate, distill and concentrate to dryness to obtain a crude solid product, add 60mL isopropyl Recrystallize the alcohol and 60 mL n-heptane, filter and rinse the wet product with 20 mL n-heptane, and dry under reduced pressure to constant weight to obtain 11.28 g of solid succinimidyl octadecanedioate. The yield is 91.00%. The purity is determined by HPLC. Is 98.10%.
ESI-MS m/z：502.3[M+H] +，与理论值符合 ESI-MS m/z: 502.3[M+H] + , in accordance with the theoretical value
实施例15 苄基十八烷二酰基-L-Glu-OBn的合成Example 15 Synthesis of benzyl octadecane diacyl-L-Glu-OBn
Figure PCTCN2021081465-appb-000024
Figure PCTCN2021081465-appb-000024
将十八二酸琥珀酰亚胺苄酯(10.0g，19.93mmol)与L-谷氨酸-1-苄酯(4.97g，20.93mmol)溶解于100mL乙腈中，加入三乙胺(3.03g，29.90mmol)置于30℃搅拌12小时，反应结束一次加入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，即可得到苄基十八烷二酰基-L-Glu-OBn淡黄色固体12.00g，收率96.52％，HPLC检测纯度为95.62％。Dissolve octadecanoic acid succinimidyl benzyl ester (10.0g, 19.93mmol) and L-glutamate-1-benzyl ester (4.97g, 20.93mmol) in 100mL acetonitrile, and add triethylamine (3.03g, 29.90mmol) was placed at 30°C and stirred for 12 hours. After the reaction, 120mL 1M hydrochloric acid solution and 90mL tap water were added at once. The temperature was cooled to -5°C and stirred for crystallization for 2.0 hours. Filtered and washed the filter cake with an appropriate amount of water until it became neutral. Place the filter cake at 45°C. After vacuum drying at ℃ to constant weight, 12.00 g of benzyl octadecane diacyl-L-Glu-OBn light yellow solid can be obtained, with a yield of 96.52% and a purity of 95.62% by HPLC.
ESI-MS m/z：624.6[M+H] +，与理论值符合 ESI-MS m/z: 624.6[M+H] + , consistent with the theoretical value
实施例16 苄基十八烷二酰基-L-Glu(OSu)-OBn的合成Example 16 Synthesis of benzyl octadecane diacyl-L-Glu(OSu)-OBn
Figure PCTCN2021081465-appb-000025
Figure PCTCN2021081465-appb-000025
将苄基十八烷二酰基-L-Glu-OBn(10.0g，16.03mmol)溶解于130mL二氯甲烷中，降温至-5℃，加入N-羟基琥珀酰亚胺(2.03g，17.63mmol)和二环己基碳二亚胺(3.97g，19.24mmol)，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，即可得到苄基十八烷二酰基-L-Glu(OSu)-OBn白色固体9.59g，收率83.00％，HPLC检测纯度为97.85％。Dissolve benzyl octadecane diacyl-L-Glu-OBn (10.0 g, 16.03 mmol) in 130 mL of dichloromethane, lower the temperature to -5°C, and add N-hydroxysuccinimide (2.03 g, 17.63 mmol) And dicyclohexylcarbodiimide (3.97g, 19.24mmol), continue to react at -5°C for 2 hours, return to 30°C and continue to react for 8 hours, filter to remove the precipitate, concentrate under reduced pressure and distill to dryness to obtain a solid, add Recrystallize with 120 mL of absolute ethanol, filter, rinse the filter cake with 20 mL of n-heptane, and place the filter cake at 45°C under vacuum and dry to constant weight to obtain benzyl octadecane diacyl-L-Glu(OSu)- OBn white solid 9.59g, yield 83.00%, HPLC purity 97.85%.
ESI-MS m/z：721.2[M+H]+，与理论值符合ESI-MS m/z: 721.2[M+H]+, consistent with the theoretical value
实施例17 十八烷二酰基-L-Glu(OSu)的合成Example 17 Synthesis of octadecane diacyl-L-Glu (OSu)
Figure PCTCN2021081465-appb-000026
Figure PCTCN2021081465-appb-000026
将苄基十八烷二酰基-L-Glu(OSu)-OBn(10g，13.87mmol)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，即可得到十八烷二酰基-L-Glu(OSu)白色固体7.16g，收率95.48％，HPLC检测纯度为97.83％。Dissolve benzyl octadecane diacyl-L-Glu(OSu)-OBn (10g, 13.87mmol) in 200mL acetone, add trifluoroacetic acid (0.1g, 0.8mmol) and 0.8g 10% Pd/C, set Hydrodebenzylation was carried out at 30°C for 3.0 hours. At the end of the reaction, the palladium carbon was removed by filtration, and the crude solid product was obtained by distillation under reduced pressure. 55mL acetone and 138mL n-heptane were added to make slurry at room temperature for 2.0 hours, filtered with suction, and 30mL n-heptane The filter cake was rinsed with alkane, and the filter cake was vacuum-dried at 40°C to a constant weight to obtain 7.16 g of octadecane diacyl-L-Glu (OSu) white solid, with a yield of 95.48% and a purity of 97.83% by HPLC .
ESI-MS m/z：541.5[M+H] +，与理论值符合 ESI-MS m/z: 541.5[M+H] + , consistent with the theoretical value
实施例18 十八烷二酰基-L-Glu(OSu)在胰岛素类似物中的制备应用Example 18 Preparation and application of octadecane diacyl-L-Glu (OSu) in insulin analogues
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。按照十八烷二酰基L-Glu(OSu)：Des(B30)人胰岛素摩尔比1.2：1称取十八烷二酰基-L-Glu(OSu)56.87mg于2mL NMP(N-甲基吡咯烷酮)中，随后加入人胰岛素溶液中，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66％的德谷胰岛素：LysB29(N-ε-十八烷二酰基-γ-谷氨酰基)脱(B30)人胰 岛素，纯化后可得纯度高于99.60％的胰岛素类似物Dissolve 500 mg of Des (B30) human insulin in 10 mL of water, and add triethylamine to adjust the pH of the system to 11.00. According to the molar ratio of octadecane diacyl L-Glu (OSu): Des (B30) human insulin 1.2:1, weigh 56.87 mg of octadecane diacyl-L-Glu (OSu) into 2 mL NMP (N-methylpyrrolidone) Then, it was added to the human insulin solution. After 30 minutes, the reaction was terminated by adding 6.5 mL of diluted HCl to adjust the pH of the system to 0.2M ethanolamine at 9.0. HPLC analysis showed that 66% of insulin degludec was formed: LysB29 (N-ε-octadecandioyl-γ-glutamyl) de(B30) human insulin, after purification, an insulin analog with a purity higher than 99.60% can be obtained
ESI-MS m/z：1533.6[M+4H] 4+,1227.1[M+5H] 5+，1022.7[M+6H] 6+，与理论值符合 ESI-MS m/z: 1533.6[M+4H] 4+ ,1227.1[M+5H] 5+ , 1022.7[M+6H] 6+ , which accords with the theoretical value
实施例19 二十烷二酸一苄酯的合成Example 19 Synthesis of eicosandioic acid monobenzyl ester
Figure PCTCN2021081465-appb-000027
Figure PCTCN2021081465-appb-000027
将二十烷二酸(10.0g，29.20mmol)、无水氯化镁(0.27g，2.92mmol)、Boc 2O酸酐(9.56g，43.80mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，反应结束减压蒸馏除去未参与反应的Boc 2O酸酐和反应溶剂，得到固体粗品，加入苄醇(7.89g，73mmol)和70mL四氢呋喃，置于40℃中搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到二十烷二酸一苄酯白色固体4.36g，收率34.53％，HPLC检测纯度97.23％。 Add eicosanedioic acid (10.0g, 29.20mmol), anhydrous magnesium chloride (0.27g, 2.92mmol), Boc 2 O anhydride (9.56g, 43.80mmol) and 100mL of tetrahydrofuran into a 250mL single-necked flask and place it at 40°C with stirring. After 24h, the reaction was completed under reduced pressure to remove the Boc 2 O acid anhydride and the reaction solvent that did not participate in the reaction to obtain a crude solid product. Add benzyl alcohol (7.89g, 73mmol) and 70mL tetrahydrofuran. Place it at 40°C and stir for 24h, then add it to the system. 350mL of tap water, stirred at room temperature for 0.5 hours to precipitate solids, filtered and collected the precipitate, added 180mL of n-octane, beaten at room temperature for 1.0h, filtered, 20mL of n-octane rinse the wet product, get wet product, add 180mL to the wet product Dichloromethane was slurried at room temperature for 1.0h. The filtrate was collected by filtration, and concentrated to dryness at 35°C to obtain a solid. Placed at 40°C for vacuum drying to constant weight, 4.36g of white solid eicosanedioic acid monobenzyl ester was obtained. The rate is 34.53%, and the purity detected by HPLC is 97.23%.
ESI-MS m/z：433.2[M+H] +，与理论值符合 ESI-MS m/z: 433.2[M+H] + , consistent with the theoretical value
实施例20 二十烷二酸琥珀酰亚胺苄酯的合成Example 20 Synthesis of Eicosandioic Acid Succinimidyl Benzyl Ester
Figure PCTCN2021081465-appb-000028
Figure PCTCN2021081465-appb-000028
将二十烷二酸一苄酯(10.0g，23.11mmol)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(2.93g，25.42mmol)和二环己基碳二亚胺(5.72g，27.73mmol)，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，得到二十烷二酸酸琥珀酰亚胺苄酯固体11.03g，收率90.18％，HPLC检测纯度为98.78％。Add eicosanedioic acid monobenzyl ester (10.0g, 23.11mmol) and 130mL of dichloromethane into a 250mL single-necked flask, cool to -5°C, add N-hydroxysuccinimide (2.93g, 25.42mmol) and two Cyclohexylcarbodiimide (5.72g, 27.73mmol), continue the reaction at -5°C for 2 hours, return to 30°C and continue the reaction for 16 hours, filter to remove the precipitate, distill and concentrate to dryness to obtain a crude solid product, add 60mL isopropyl Recrystallize the alcohol and 60 mL n-heptane, filter and rinse the wet product with 20 mL n-heptane, and dry to constant weight under reduced pressure to obtain 11.03 g of solid eicosanedioic acid succinimidyl benzyl ester, with a yield of 90.18%, detected by HPLC The purity is 98.78%.
ESI-MS m/z：530.6[M+H] +，与理论值符合 ESI-MS m/z: 530.6[M+H] + , in accordance with the theoretical value
实施例21 苄基二十烷二酰基-L-Glu-OBn的合成Example 21 Synthesis of benzyl eicosandioyl-L-Glu-OBn
Figure PCTCN2021081465-appb-000029
Figure PCTCN2021081465-appb-000029
将二十烷二酸琥珀酰亚胺苄酯(10.0g，18.88mmol)与L-谷氨酸-1-苄酯(4.70g，19.82mmol)溶解于100mL乙腈中，加入三乙胺(2.87g，28.32mmol)置于30℃搅拌12小时，反应结束一次加入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，即可得到苄基二十烷二酰基-L-Glu-OBn淡黄色固体12.31g，收率97.00％，HPLC检测纯度为96.03％。Dissolve eicosanedioic acid succinimidyl benzyl ester (10.0g, 18.88mmol) and L-glutamate-1-benzyl ester (4.70g, 19.82mmol) in 100mL acetonitrile, add triethylamine (2.87g , 28.32mmol) was placed at 30°C and stirred for 12 hours. After the reaction, 120mL 1M hydrochloric acid solution and 90mL tap water were added at one time. After vacuum drying at 45°C to constant weight, 12.31 g of benzyl eicosandioyl-L-Glu-OBn pale yellow solid can be obtained, with a yield of 97.00% and a purity of 96.03% by HPLC.
ESI-MS m/z：651.7[M+H] +，与理论值符合 ESI-MS m/z: 651.7[M+H] + , consistent with the theoretical value
实施例22 苄基二十烷二酰基-L-Glu(OSu)-OBn的合成Example 22 Synthesis of Benzyl Eicosane Diacyl-L-Glu(OSu)-OBn
Figure PCTCN2021081465-appb-000030
Figure PCTCN2021081465-appb-000030
将苄基二十烷二酰基-L-Glu-OBn(10.0g，15.34mmol)溶解于130mL二氯甲烷中，降温至-5℃，加入N-羟基琥珀酰亚胺(1.94g，16.84mmol)和二环己基碳二亚胺(3.80g，18.41mmol)，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，即可得到苄基二十烷二酰基-L-Glu(OSu)-OBn白色固体9.77g，收率85.00％，HPLC检测纯度为98.20％。Dissolve benzyleicosane diacyl-L-Glu-OBn (10.0g, 15.34mmol) in 130mL of dichloromethane, cool to -5°C, and add N-hydroxysuccinimide (1.94g, 16.84mmol) And dicyclohexylcarbodiimide (3.80g, 18.41mmol), continue to react at -5°C for 2 hours, return to 30°C and continue to react for 8 hours, filter to remove the precipitate, concentrate by distillation under reduced pressure to dryness to obtain a solid, add Recrystallize with 120 mL of absolute ethanol, filter, rinse the filter cake with 20 mL of n-heptane, and place the filter cake at 45°C under vacuum drying to a constant weight to obtain benzyl eicosandioyl-L-Glu(OSu)- The white solid of OBn is 9.77 g, the yield is 85.00%, and the purity detected by HPLC is 98.20%.
ESI-MS m/z：721.2[M+H] +，与理论值符合 ESI-MS m/z: 721.2[M+H] + , consistent with the theoretical value
实施例23 二十烷二酰基-L-Glu(OSu)的合成Example 23 Synthesis of Eicosane Diacyl-L-Glu (OSu)
Figure PCTCN2021081465-appb-000031
Figure PCTCN2021081465-appb-000031
将苄基二十烷二酰基-L-Glu(OSu)-OBn(10g，13.35mmol)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， 即可得到十八烷二酰基-L-Glu(OSu)白色固体7.40g，收率97.50％，HPLC检测纯度为98.80％。Dissolve benzyl eicosandioyl-L-Glu(OSu)-OBn (10g, 13.35mmol) in 200mL acetone, add trifluoroacetic acid (0.1g, 0.8mmol) and 0.8g 10% Pd/C, set Hydrodebenzylation was carried out at 30°C for 3.0 hours. At the end of the reaction, the palladium carbon was removed by filtration, and the crude solid product was obtained by distillation under reduced pressure. 55mL acetone and 138mL n-heptane were added to make slurry at room temperature for 2.0 hours, filtered with suction, and 30mL n-heptane The filter cake was eluted with alkane, and the filter cake was vacuum-dried to constant weight at 40°C to obtain 7.40 g of octadecane diacyl-L-Glu(OSu) white solid, with a yield of 97.50% and a purity of 98.80% by HPLC .
ESI-MS m/z：568.4[M+H] +，与理论值符合 ESI-MS m/z: 568.4[M+H] + , consistent with the theoretical value
实施例24 二十烷二酰基-L-Glu(OSu)在胰岛素类似物中的制备应用Example 24 Preparation and application of eicosane diacyl-L-Glu (OSu) in insulin analogues
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。按照十八烷二酰基L-Glu(OSu)：Des(B30)人胰岛素摩尔比1.2：1称取十八烷二酰基-L-Glu(OSu)59.81mg于2mL NMP(N-甲基吡咯烷酮)中，随后加入人胰岛素溶液中，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-二十烷二酰基-γ-谷氨酰基)脱(B30)人胰岛素，纯化后可得纯度高于99.60％的胰岛素类似物Dissolve 500 mg of Des (B30) human insulin in 10 mL of water, and add triethylamine to adjust the pH of the system to 11.00. According to the molar ratio of octadecane diacyl L-Glu (OSu): Des (B30) human insulin 1.2:1, weigh 59.81 mg of octadecane diacyl-L-Glu (OSu) into 2 mL NMP (N-methylpyrrolidone) Then, it was added to the human insulin solution. After 30 minutes, the reaction was terminated by adding 6.5 mL of diluted HCl to adjust the pH of the system to 0.2M ethanolamine at 9.0. HPLC analysis showed that 65% of insulin degludec was formed: LysB29 (N-ε-eicosandioyl-γ-glutamyl) de(B30) human insulin, after purification, an insulin analog with a purity higher than 99.60% can be obtained
ESI-MS m/z：1540.9[M+4H] 4+,1232.6[M+5H] 5+，1027.3[M+6H] 6+，与理论值符合 ESI-MS m/z: 1540.9[M+4H] 4+ , 1232.6[M+5H] 5+ , 1027.3[M+6H] 6+ , consistent with the theoretical value
对比例1 十六烷二酸一苄酯的合成Comparative Example 1 Synthesis of hexadecanedioic acid monobenzyl ester
将十六烷二酸(10.0g，34.91mmol)、无水氯化镁(0.34g，3.57mmol)、苄醇(9.44g，87.36mmol)和100mL四氢呋喃加入250mL单口烧瓶中置于40℃搅拌反应24h，往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到极少量固体，置于40℃真空干燥至恒重，得到十六烷二酸一苄酯白色固体0.01g，收率0.08％，HPLC检测纯度89.12％。Add hexadecanedioic acid (10.0g, 34.91mmol), anhydrous magnesium chloride (0.34g, 3.57mmol), benzyl alcohol (9.44g, 87.36mmol) and 100mL tetrahydrofuran into a 250mL single-necked flask and place it at 40°C and stir for 24h. Add 350mL tap water to the system, stir at room temperature for 0.5 hours to precipitate a solid, collect the precipitate by filtration, add 180mL n-octane, put the slurry at room temperature for 1.0h, filter, 20mL n-octane rinse the wet product to obtain a wet product, wet Add 180mL of dichloromethane to the product, beat at room temperature for 1.0h, filter and collect the filtrate, place it at 35°C and concentrate to dryness to obtain a very small amount of solid, place it at 40°C and vacuum dry to constant weight to obtain hexadecanedioic acid monobenzyl ester The white solid is 0.01 g, the yield is 0.08%, and the purity detected by HPLC is 89.12%.
ESI-MS m/z：377.2[M+H] +，与理论值符合 ESI-MS m/z: 377.2[M+H] + , consistent with the theoretical value
对比例2 十六烷二酸一苄酯的合成Comparative Example 2 Synthesis of hexadecanedioic acid monobenzyl ester
将十六烷二酸(10.0g，34.91mmol)和无水醋酸酐(70.30g，650.56mmol)加入250mL单口烧瓶中置于100℃搅拌反应24h，反应结束后减压蒸馏浓缩至干得到淡黄色油状物，置于室温则慢慢变为淡黄色蜡状固体，加入苄醇(9.44g，87.35mmol)和100mL四氢呋喃并置于40℃中搅拌反应24h，反应结束往体系中加入350mL自来水，于室温下搅拌反应0.5小时析出固体，过滤收集沉淀，加入180mL正辛烷，置室温下打浆1.0h，过滤，20mL正辛烷淋洗湿品，得到湿品，湿品中加入180mL二氯甲烷，室温打浆1.0h，过滤收集滤液，置于35℃中真空浓缩至干得到固体，置于40℃真空干燥至恒重，得到淡黄色蜡状固体7.06g，收率53.69％，HPLC检测中可见十六烷二酸一苄酯纯度仅为7.76％。Add hexadecanedioic acid (10.0g, 34.91mmol) and anhydrous acetic anhydride (70.30g, 650.56mmol) into a 250mL single-necked flask and place at 100°C for 24 hours with stirring. After the reaction is completed, vacuum distillation is concentrated to dryness and a pale yellow is obtained The oily substance slowly turns into a pale yellow waxy solid at room temperature. Add benzyl alcohol (9.44g, 87.35mmol) and 100mL tetrahydrofuran and place at 40°C and stir for 24h. When the reaction is over, add 350mL tap water to the system. Stir the reaction at room temperature for 0.5 hours to precipitate a solid, filter and collect the precipitate, add 180 mL of n-octane, put it at room temperature for 1.0 h, filter, 20 mL of n-octane rinse the wet product to obtain a wet product, add 180 mL of dichloromethane to the wet product, Beat at room temperature for 1.0h, filter and collect the filtrate, place it at 35°C and concentrate to dryness to obtain a solid, and place it at 40°C to vacuum dry to constant weight to obtain 7.06g of pale yellow waxy solid with a yield of 53.69%. The purity of hexadecanedioic acid monobenzyl ester is only 7.76%.
对比例3Comparative example 3
Figure PCTCN2021081465-appb-000032
Figure PCTCN2021081465-appb-000032
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。The preparation of benzyl hexadecandioyl-L-Glu(OSu)-OH described in the preparation of hexadecandioyl–L-Glu(OSu)-OH in Novo Nordisk’s patent number WO2007104737A1 on page 26, paragraph 2: Acid (200.0g, 0.7mol), Dowex50WX2-100 acidic cation resin (700g), n-octane (3.6L) and benzyl formate (95g, 0.7mol), heated to 91℃ and benzyl formate (340g, 2.5 mol) was gradually added dropwise to the system within 9 hours. After dripping, keep stirring and reflux for 48 hours at 91°C. After the reaction, cool to room temperature and filter to remove the solvent to obtain a solid. The solid is dissolved in 3L of acetone and heated to 40°C and stirred for 0.5 After hours, filter, rinse the filter cake with 1.5L acetone, collect the filtrate under vacuum and concentrate to about 1.5L, then filter and rinse with -18°C acetone to obtain a solid wet product, which is dissolved in 2L dichloromethane and stirred at room temperature for 0.5 hours, filtered to obtain The filtrate was distilled under reduced pressure to obtain a crude solid product, which was dissolved in 900 mL of isopropanol for recrystallization to obtain a solid, and dried in a vacuum to a constant weight to obtain a solid about 73 g, with a yield of 27.8%. The use of acidic cationic resins can make hexadecanedioic acid be used to selectively synthesize hexadecanedioic acid benzyl ester, but the Dowex50WX2-100 acidic cationic resin used in this method is relatively 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.
对比例4Comparative example 4
Figure PCTCN2021081465-appb-000033
Figure PCTCN2021081465-appb-000033
文献(Takaya et al.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。A kind of benzyl tridecanedioate ( The preparation method of compound 7) described in the literature, dissolve tridecanedioic acid (4.8g, 19.7mmol) in 150mL methanol, add 11mL-10% potassium hydroxide methanol solution dropwise with stirring at room temperature and gradually form anhydrous The salt precipitated solid. After stirring for 20 minutes, the system was concentrated to dryness to obtain potassium tridecane monocarboxylate, which was suspended in 50 mL of toluene. Tetra-tert-butylammonium bromide (546mg, 1.97mmol) and benzyl bromide (2.57 mL, 21.6mmol), reflux and react for 6.0 hours. After the reaction, cool to room temperature, add 100mL 0.5M hydrochloric acid solution and extract three times with ethyl acetate. Wash the organic phase with water and saturated brine successively, dry with anhydrous sodium sulfate, filter and collect the filtrate. Concentrated under reduced pressure and vacuum, and finally purified by column chromatography on silica gel column (n-hexane: ethyl acetate = 19:1, 4:1 and 2:1). Collect the eluent and concentrate under reduced pressure to dryness to obtain colorless needles. 3.42 g of solid benzyl tridecanedioate with a yield of 52%. This process method produces a lot of impurities, it is difficult to purify after treatment, and it is difficult to realize industrialization.
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。In addition, the terms "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.
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。In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" etc. mean specific features described in conjunction with the embodiment or example , Structures, materials or features are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the described specific features, structures, materials or characteristics can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without contradicting each other.
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。Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can comment on the above-mentioned embodiments within the scope of the present invention. The embodiment undergoes changes, modifications, substitutions, and modifications.

Claims (10)

  1. 一种制备长脂肪链二酸衍生物的方法，其特征在于，包括：A method for preparing a long aliphatic chain diacid derivative, which is characterized in that it comprises:
    (1)使长脂肪链二酸发生环化反应，所述长脂肪链二酸具有式(A)所示的结构；(1) Making a long aliphatic chain diacid undergo a cyclization reaction, and the long aliphatic chain diacid has a structure represented by formula (A);
    (2)将长脂肪链二酸环化产物与苄醇反应，以便获得长脂肪链二酸一苄酯；(2) Reacting the long aliphatic chain diacid cyclization product with benzyl alcohol to obtain long aliphatic chain diacid monobenzyl ester;
    (3)将所述长脂肪链二酸一苄酯与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) Carrying out the esterification reaction between the long fatty chain diacid monobenzyl ester and N-hydroxysuccinimide, so as to obtain the long fatty chain diacid succinimide benzyl ester;
    (4)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) Performing the nucleophilic addition amidation reaction between the long aliphatic chain diacid succinimide benzyl ester and the compound represented by formula (B) to obtain the compound represented by formula (C);
    (5)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) The compound represented by formula (C) is esterified again with N-hydroxysuccinimide to obtain the compound represented by formula (D); and
    (6)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；(6) subjecting the compound represented by formula (D) to a debenzylation reaction to obtain the compound represented by formula (E);
    Figure PCTCN2021081465-appb-100001
    Figure PCTCN2021081465-appb-100001
    Figure PCTCN2021081465-appb-100002
    Figure PCTCN2021081465-appb-100002
    其中，X为6～32的整数；Y为1～6的整数。Wherein, X is an integer of 6 to 32; Y is an integer of 1 to 6.
  2. 根据权利要求1所述的方法，其特征在于，所述环化反应是在酸酐存在的条件下进行的，优选地，所述酸酐为Boc 2O酸酐。 The method according to claim 1, wherein the cyclization reaction is carried out in the presence of an acid anhydride, and preferably, the acid anhydride is Boc 2 O anhydride.
  3. 根据权利要求1所述的方法，其特征在于，所述环化反应是在非质子极性试剂中进行的；The method of claim 1, wherein the cyclization reaction is carried out in an aprotic polar reagent;
    任选地，所述非质子极性试剂包括选自N,N-二甲基甲酰胺、乙腈、丙酮和四氢呋喃中的一种或至少两种；Optionally, the aprotic polar reagent includes one or at least two selected from the group consisting of N,N-dimethylformamide, acetonitrile, acetone and tetrahydrofuran;
    优选地，所述环化反应是在无水氯化镁、6水氯化镁、氯化铁、氯化锆、DMAP或浓硫酸中的一种或至少两种的催化下进行的。Preferably, the cyclization reaction is carried out under the catalysis of one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid.
  4. 根据权利要求1所述的方法，其特征在于，所述环化反应是在温度为30℃～80℃的条件下进行12～48小时。The method of claim 1, wherein the cyclization reaction is carried out at a temperature of 30°C to 80°C for 12 to 48 hours.
  5. 根据权利要求2或3所述的方法，其特征在于，相对于所述长脂肪链二酸，所述酸酐的用量是过量的；The method according to claim 2 or 3, wherein the amount of the acid anhydride is excessive relative to the long aliphatic chain diacid;
    优选地，所述的Boc2O酸酐的摩尔用量相当于所述的长脂肪链二酸摩尔量的1.0～2.0倍；Preferably, the molar amount of the Boc2O acid anhydride is equivalent to 1.0 to 2.0 times the molar amount of the long aliphatic chain diacid;
    优选地，无水氯化镁、6水氯化镁、氯化铁、氯化锆、DMAP或浓硫酸中的一种或至少两种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。Preferably, the molar amount of one or at least two of anhydrous magnesium chloride, hexahydrate magnesium chloride, ferric chloride, zirconium chloride, DMAP or concentrated sulfuric acid is equivalent to 0.1-1.0 of the molar amount of the long aliphatic chain diacid. Times.
  6. 根据权利要求1所述方法，其特征在于，在步骤(2)中，所述反应是在非极性烃类溶剂中、在25℃～80℃的条件下进行12～48小时；The method according to claim 1, wherein in step (2), the reaction is carried out in a non-polar hydrocarbon solvent at 25°C to 80°C for 12 to 48 hours;
    任选地，所述非极性烃类溶剂为正己烷、石油醚、环己烷、正庚烷、正辛烷、甲基叔丁基醚、甲苯中的一种或至少两种；Optionally, the non-polar hydrocarbon solvent is one or at least two of n-hexane, petroleum ether, cyclohexane, n-heptane, n-octane, methyl tert-butyl ether, and toluene;
    优选地，所述的苄醇的摩尔用量相当于所述的长脂肪链二酸摩尔量的1.0～3.0倍。Preferably, the molar amount of the benzyl alcohol is equivalent to 1.0 to 3.0 times the molar amount of the long aliphatic chain diacid.
  7. 根据权利要求1所述的方法，其特征在于，在步骤(3)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；The method according to claim 1, characterized in that, in step (3), the esterification reaction is carried out in one or at least two solvents among tetrahydrofuran, ethyl acetate and dichloromethane. The reaction is carried out at ℃～20℃ for 2 hours, and the reaction is continued at 15-40℃ for 3-24 hours;
    任选地，进一步包括将长脂肪链二酸一苄酯与二环己基碳二亚胺进行接触；Optionally, it further comprises contacting a long aliphatic chain diacid monobenzyl ester with dicyclohexylcarbodiimide;
    任选地，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。Optionally, 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.
  8. 根据权利要求1所述的方法，其特征在于，所述Y为1、2或3；The method according to claim 1, wherein the Y is 1, 2 or 3;
    任选地，所述式(B)所示化合物为L-谷氨酸-1-苄酯，结构为
    Figure PCTCN2021081465-appb-100003
    Optionally, the compound represented by formula (B) is L-glutamate-1-benzyl ester, and the structure is
    Figure PCTCN2021081465-appb-100003
    任选地，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 PCTCN2021081465-appb-100004
    Optionally, the compound represented by formula (C) is benzyl ester long aliphatic chain diacyl-L-Glu-OBn, the structure is
    Figure PCTCN2021081465-appb-100004
    任选地，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 PCTCN2021081465-appb-100005
    Optionally, the compound represented by formula (D) is a benzyl long aliphatic chain diacyl group-L-Glu(OSu)-OBn, and the structure is
    Figure PCTCN2021081465-appb-100005
    任选地，所述式(E)所示化合物的结构为
    Figure PCTCN2021081465-appb-100006
    Optionally, the structure of the compound represented by formula (E) is
    Figure PCTCN2021081465-appb-100006
  9. 根据权利要求8所述的方法，其特征在于，在步骤(4)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；The method according to claim 8, characterized in that, in step (4), the reaction is performed in one of acetonitrile, N,N-dimethylformamide, tetrahydrofuran or N-methylpyrrolidone in the solvent Or at least two, and the temperature is 25℃～40℃ for 6～24;
    任选地，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；Optionally, the molar amount of the L-glutamate-1-benzyl ester is equivalent to 1.0 to 1.2 times the molar amount of the long aliphatic chain diacid succinimide benzyl ester;
    任选地，在步骤(5)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；Optionally, in step (5), further comprising contacting the benzyl ester long aliphatic chain diacyl-L-Glu-OBn with dicyclohexylcarbodiimide;
    任选地，在步骤(5)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；Optionally, in step (5), 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 to 10°C. The conditions are carried out for 2 hours, and then the reaction is continued at a temperature of 15°C to 40°C for 3-24 hours;
    任选地，在步骤(6)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。Optionally, in step (6), the debenzylation reaction is carried out under 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. ~5 hours.
  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1～9任一项所述的方法获得；A method for preparing a hypoglycemic protein drug, which is characterized in that it comprises: 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 right Obtained by the method described in any one of claims 1-9;
    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。Optionally, the hypoglycemic protein drug includes at least one selected from insulin, GLP-1, insulin analogues and GLP-1 analogues.
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