阅读说明：本技术 一种固相合成醋酸艾替班特的方法 (Solid-phase synthesis method of icatibant acetate ) 是由 闫雪峰 游军辉 郭彦亮 王俊亮 曹莹 于 2019-07-01 设计创作，主要内容包括：本发明涉及一种固相合成醋酸艾替班特的方法。本发明提供一种纯度高、杂质含量低、合成效率高且收率高的醋酸艾替班特的制备方法,所得产品质量稳定。本发明的方法反应条件温和,对设备损害低,工艺简单,绿色环保且成本低,能够确保连续生产出高质量的产品,具有较高的应用价值,适合产业化生产。(The invention relates to a method for solid-phase synthesis of icatibant acetate. The invention provides a preparation method of icatibant acetate, which has the advantages of high purity, low impurity content, high synthesis efficiency and high yield, and the obtained product has stable quality. The method disclosed by the invention is mild in reaction condition, low in damage to equipment, simple in process, green, environment-friendly and low in cost, can ensure that high-quality products can be continuously produced, has a high application value, and is suitable for industrial production.)

1. A preparation method of solid-phase synthesis icatibant acetate is characterized in that Fmoc-Arg (Pbf) -Wang Resin is sequentially coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thi-OH, Fmoc-Gly-OH, Fmoc-Hyp (tBu) -OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH and Fmoc-D-Arg (Pbf) -OH, and the cleavage product is subjected to purification, salt transfer and freeze-drying to obtain the icatibant acetate.

2. The process of claim 1, wherein the coupling reagent combination used in the coupling reaction is selected from one or more of DCC/HOBt, DIC/HOAt, DIC/HOBt, HBTU/DIEA, TBTU/DIEA and DEPBT/DIEA; preferably, the coupling reagent combination is as follows: DIC/HOBt, HBTU/DIEA or DEPBT/DIEA are selected as coupling reagents when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; selecting DEPBT/DIEA, HBTU/DIEA or DIC/HOBt as coupling reagents when coupling with Fmoc-Ser (tBu) -OH and Fmoc-Thi-OH; when the coupling reagent is coupled with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH and Fmoc-D-Arg (Pbf) -OH, HBTU/DIEA, DIC/HOBt or DEPBT/DIEA is selected as a coupling reagent;

more preferably, DIC/HOBt is selected as the coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; DEPBT/DIEA was chosen as coupling reagent for coupling with Fmoc-Ser (tBu) -OH and Fmoc-Thi-OH; HBTU/DIEA was selected as the coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH and Fmoc-D-Arg (Pbf) -OH.

3. The method according to claim 1, wherein the solvent used in the coupling reaction is one or two selected from dichloromethane and N, N-dimethylformamide, preferably N, N-dimethylformamide.

4. The process according to claim 1, wherein the temperature of the coupling reaction is 20 to 40 ℃, preferably 27 to 33 ℃.

5. The process according to claim 1, wherein the coupling reaction is carried out for a period of 2 to 12 hours, preferably 2 to 5 hours.

6. The method of claim 1, wherein the coupling reaction comprises a deprotection step, and the deprotection reagent is selected from one or two of a 20% diethylamine in N, N-dimethylformamide, a 20% piperidine in N, N-dimethylformamide, and preferably a 20% piperidine in N, N-dimethylformamide.

7. The process of claim 1, wherein the cracking reaction uses a settling solvent, and the settling solvent is one or two selected from diethyl ether, isopropyl ether and methyl tert-butyl ether, preferably isopropyl ether.

8. The method of claim 1, wherein a lysate of the cleavage reaction is trifluoroacetic acid, dithiothreitol, triisopropylsilane, thioanisole and water, preferably in a ratio of 30-40: 1:1:1:1, more preferably 36:1:1:1: 1.

9. The method of claim 1, wherein the purification is performed using a phosphate system.

10. The method of claim 1, the phosphate system comprising mobile phase a: 0.02M sodium dihydrogen phosphate buffer; mobile phase B: and (3) acetonitrile.

Technical Field

The invention relates to the field of medicine synthesis, and in particular relates to a preparation method of solid-phase synthesis icatibant acetate.

Background

Icatibant acetate (frazyr) is a bradykinin B2 receptor antagonist having the structural formula shown below:

icatibant acetate was originally developed by Sanofi, a company Jerini 11 months 2001, to which icatibant acetate was developed. 9 months in 2008 are first marketed in germany and the uk, 3 months in 2009 in european union and 10 months in 2011 in the us. FDA approved for acute onset treatment of Hereditary Angioedema (HAE) in adults 18 years and older, which is caused by low protein levels or dysfunction of a known C1 inhibitor, unpredictable paroxysmal edema and swelling of the hands, feet, face, larynx and abdomen, leading to disfigurement, disability or death. Icatibant treats the embolized local swelling of acute HAE by inhibiting the effects of bradykinin associated with embolized local swelling, inflammation, pain symptoms of HAE.

However, the existing preparation method of the icatibant acetate has the advantages of low product purity, high impurity content, low synthesis efficiency and low yield, cannot ensure the continuous production of products with stable quality, is not suitable for industrial production, and has low application value.

Patent CN102532267A reports a method for direct solid-phase synthesis of icatibant, which uses 2-chloro-triphenyl-chloro resin as an initial carrier, and because of the sensitivity of this kind of resin to alkali, part of peptide chains will fall off from the branches during the synthesis process, resulting in lower final yield. In addition, the coupling reaction of the amino acid derivatives in the route needs to be carried out in an ice bath, the reaction condition requirement is high, the operation is complex, the method is not suitable for industrial production, and the application value is low; meanwhile, HOAt (N-hydroxy-7-azabenzotriazole), HATU (2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate) and other expensive coupling agents are required to be used in the route, so that the production cost is high.

139-152 utilizes Wang resin as solid phase carrier, Fmoc-AA-OH as starting amino acid raw material, DCC (N, N-dicyclohexylcarbodiimide)/HOBt (1-hydroxybenzotriazole) as coupling agent to synthesize icatiban acetate. Because DCU (1, 3-dicyclohexylurea) which is a byproduct generated in the reaction process of DCC is difficult to dissolve in an organic solvent and remove, the DCU is inconvenient for scale-up production.

In addition, in the prior art, a method for synthesizing the icatibant acetate by taking Boc-Arg (tos) -Merrifield-Resin as a solid phase carrier exists, and a large amount of strong corrosive solvents such as anhydrous hydrogen fluoride and the like are required in the synthesis process, so that the method has high requirements and damages on equipment, and is not beneficial to environmental protection and labor protection.

Disclosure of Invention

Aiming at the problems, the invention aims to provide a preparation method of the icatibant acetate, which has the advantages of high purity of solid phase synthesis, low impurity content, high synthesis efficiency, mild reaction conditions, simple process, environmental friendliness, low cost and high yield.

The above object of the present invention is achieved by the following technical solutions:

Fmoc-Arg (Pbf) -Wang Resin is sequentially coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH, Fmoc-Ser (tBu) -OH, Fmoc-Thi-OH, Fmoc-Gly-OH, Fmoc-Hyp (tBu) -OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH and Fmoc-D-Arg (Pbf) -OH, and then cracked to obtain a cracked product, and the cracked product is purified, transferred to salt and freeze-dried to obtain the icatibant acetate.

Preferably, a coupling solvent is used in the preparation process, and the coupling solvent is N, N-dimethylformamide.

Preferably, the deprotection solvent is 20% piperidine N, N-dimethylformamide (V/V) used in the preparation process.

Preferably, the coupling temperature is from 27 to 33 ℃.

Preferably, the coupling time is 3 to 5 hours.

Preferably, the lysis solution used for the lysis reaction is trifluoroacetic acid: triisopropylsilane: dithiothreitol: thioanisole: the ratio of water to water is 30-40: 1:1:1:1, and more preferably 36:1:1:1: 1.

Preferably, when the coupling reagent is selected from the group consisting of DCC/HOBt, DIC/HOAt, HBTU/DIEA, TBTU/DIEA and DEPBT/DIEA, and particularly when the coupling reagent comprises two or more of the above-mentioned combinations, the purity and yield of the product can be effectively improved.

The basic process of solid phase polypeptide synthesis is to link the carboxyl group of C-terminal amino acid of target peptide with solid phase carrier (resin) in the form of covalent bond, and then to take the amino group of the amino acid as synthesis starting point to make it generate carboxyl group with adjacent amino acid (amino protection)Acylation reaction to form peptide bond. Deprotecting the amino group of the resin peptide containing the two amino acids, and reacting with the carboxyl group of the next amino acid, and repeating the process until the target peptide is formed. Racemization of amino acids is one of the most common problems during polypeptide synthesis; the 6 th L-Thi side chain in the icatibant amino acid sequence is a thiophene ring with aromaticity, and racemization is likely to occur in the synthetic process to be changed into D-Thi, thereby forming D-Thi ⁶-icatibant; in addition, Ser is susceptible to racemization during solid phase peptide synthesis, resulting in D-Ser ⁷-icatibant production; to reduce racemic impurities (D-Thi) in the crude product ⁶Icatmint and D-Ser ⁷Icatmint) using the DEPBT/DIEA strategy for coupling these two amino acids (positions 6 and 7).

To reduce the missing impurities (Des-Gly) ⁵Icatmint), a strong condensing agent HBTU/DIEA strategy is adopted for coupling the amino acid at the position, and the coupling reaction time of different coupling strategies is compared, so that the reaction time of the amino acids (D-Arg, Arg and Pro) at the 1 st, 2 nd and 3 rd positions can be shortened by adopting the HBTU/DIEA strategy, the coupling period is shortened, and the HBTU/DIEA strategy is also adopted for the coupling strategy of the amino acid at the three positions.

The rest secondary amino acids (Hyp, D-Tic, Oic and the like) are secondary amines, are not easy to racemize in the condensation process, so racemization impurities are difficult to generate, the coupling reaction time and the like are basically consistent in different coupling strategies, and the DIC/HOBt can meet the coupling requirements, so the DIC/HOBt coupling strategy is still adopted.

Preferably, DIC/HOBt is selected as a coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; DEPBT/DIEA was chosen as the coupling reagent for coupling with Fmoc-Ser (tBu) -OH, Fmoc-Thi-OH; HBTU/DIEA was selected as a coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-D-Arg (Pbf) -OH.

Preferably, HBTU/DIEA is selected as a coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; DEPBT/DIEA was selected as the coupling reagent for coupling with Fmoc-Ser (tBu) -OH and Fmoc-Thi-OH, and DIC/HOBt was selected as the coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH and Fmoc-D-Arg (Pbf) -OH.

Preferably, DEPBT/DIEA is selected as a coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; HBTU/DIEA was selected as a coupling reagent for coupling with Fmoc-Ser (tBu) -OH or Fmoc-Thi-OH, and DIC/HOBt was selected as a coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH or Fmoc-D-Arg (Pbf) -OH.

Preferably, DIC/HOBt is selected as a coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH, Fmoc-Hyp (tBu) -OH; HBTU/DIEA was selected as a coupling reagent for coupling with Fmoc-Ser (tBu) -OH or Fmoc-Thi-OH, and DEPBT/DIEA was selected as a coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH or Fmoc-D-Arg (Pbf) -OH.

Preferably, DEPBT/DIEA is selected as a coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; DIC/HOBt was selected as the coupling reagent for coupling with Fmoc-Ser (tBu) -OH and Fmoc-Thi-OH, and HBTU/DIEA was selected as the coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH and Fmoc-D-Arg (Pbf) -OH.

Preferably, HBTU/DIEA is selected as a coupling reagent when Fmoc-Arg (Pbf) -Wang Resin is coupled with Fmoc-Oic-OH, Fmoc-D-Tic-OH and Fmoc-Hyp (tBu) -OH; DIC/HOBt was chosen as coupling reagent for coupling with Fmoc-Ser (tBu) -OH, Fmoc-Thi-OH, DEPBT/DIEA was chosen as coupling reagent for coupling with Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Arg (Pbf) -OH, Fmoc-D-Arg (Pbf) -OH.

Preferably, the molar ratio of each amino acid to the coupling agent combination is 1: 2-10, preferably 1: 2-5.

Preferably, the purification step is a single purification step using a phosphate system. Preferably, purification is carried out using a phosphate system (mobile phase A: 0.02M sodium dihydrogen phosphate buffer, mobile phase B: acetonitrile 0.1-0.25% gradient slope). The salt conversion process is referred to patent CN 107417770A.

According to the technical scheme, the preparation of the acetate icatibant with high purity, low impurity content, high synthesis efficiency and high yield in solid phase synthesis is realized, the obtained product has stable quality, mild reaction conditions, low damage to equipment, simple process, environmental protection and low cost, the high-quality product can be continuously produced, and the method has high application value and is suitable for industrial production.

Detailed Description

The following detailed description of the invention is merely illustrative or explanatory thereof and is not to be construed as limiting the invention in any way.