A method for preparing terlipressin in solid phase
Through solid-phase synthesis method and liquid-phase oxidation technology, high-purity teripressin was prepared, which solved the problem of process level limiting product quality improvement in the existing technology, and achieved efficient and low-cost industrial production.
Patent Information
- Application Number
- CN202411984542.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-01
AI Technical Summary
The process level of existing terley vasopressin products limits its quality improvement, resulting in difficulty in improving yield and quality, limiting the prospect of commercial production.
The linear terivasopressin peptide resin was synthesized by solid phase synthesis, and purified by cleavage, cyclic oxidation and reverse phase high-performance liquid chromatography. Finally, high-purity terivasopressin was obtained by converting salt and lyophilization.
This method is simple to operate, has low production cost and high finished product yield. It is suitable for large-scale industrial production, with excellent product quality, high purity and low impurity content, and has good application prospects.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and particularly relates to a method for preparing terlipressin in a solid phase. Background Art
[0002] Terlipressin was first developed by Ferring and was first marketed in the Netherlands as a hemostatic drug in 1977 under the trade name Glypressin. It was later marketed in France, Germany, and the United Kingdom under the trade name Glypressine for the treatment of varicose bleeding in the digestive tract. The dosage form is lyophilized powder injection, with a specification of 1 mg / vial. my country imported this product from Ferring in 1999. There are two dosage forms, lyophilized powder injection and injection solution.
[0003] Terlipressin is used to treat complications of cirrhosis (esophageal and gastric varicose bleeding, hepatorenal syndrome, ascites). It is also suitable for bleeding in the urogenital tract and other abdominal organs, septic shock, burns, acute liver failure, cardiac arrest, etc.
[0004] Terlipressin is a new type of synthetic long-acting vasopressin preparation. It is a prodrug that is inactive itself. After the three glycine residues at the N-terminus are removed by amino acid enzymes in the body, it slowly "releases" active lysine vasopressin. Compared with vasopressin, it has a long-lasting effect and does not cause dangerous complications.
[0005] Terlipressin has the following structure:
[0006] In the existing reports, there are few synthetic methods that have good yield, quality and commercialization. Due to the limitation of technological level, it is difficult to significantly improve the quality of terlipressin products in my country. At present, there is an urgent need to improve the process in order to improve the overall quality and yield of the product and facilitate commercial production. Summary of the invention
[0007] The purpose of the present invention is to provide a method for preparing terlipressin with simple operation and high quality of finished product in view of the defects of the prior art.
[0008] The method for preparing terlipressin in the solid phase is: 1) Solid phase synthesis of linear terlipressin peptide resin; 2) Linear terlipressin peptide resin was cleaved to obtain crude linear terlipressin peptide; 3) the crude linear terlipressin peptide reacts with the cyclization oxidation solution to obtain the terlipressin cyclization reaction solution; 4) The terlipressin cyclization reaction solution is purified by reverse phase high performance liquid chromatography, converted to salt, and freeze-dried to obtain terlipressin.
[0009] In step 1), amino resin with a substitution degree of 0.4-0.9 mmol / g is used as a carrier, and amino acids with Fmoc protective groups are coupled one by one to synthesize a linear terlipressin peptide resin.
[0010] The amino resin is Rink Amide-AM resin, Rink Amide-MBHA resin or Rink Amide resin.
[0011] The amino acids to which the Fmoc protecting groups are coupled one by one are Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Gly-Gly-OH.
[0012] The cleavage agent is trifluoroacetic acid.
[0013] The cyclooxidation liquid is a 2-5% iodine methanol solution.
[0014] The preparation method of terlipressin of the present invention adopts solid phase synthesis method to synthesize linear terlipressin peptide resin, then cracks to obtain linear terlipressin crude peptide, then performs cyclization oxidation on the linear terlipressin crude peptide and cyclization oxidation liquid, then purifies by reverse phase high performance liquid chromatography, and finally converts salt to freeze-dry to obtain terlipressin. Compared with the prior art, the method has simple operation steps, low production cost, high finished product yield, well combines the advantages of solid phase synthesis and liquid phase oxidation, is suitable for large-scale industrial production, and the obtained terlipressin product has good quality, high purity, low impurity content, and has good application prospects. DETAILED DESCRIPTION
[0015] The following examples are given to specifically describe the present invention. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made to the present invention by technical personnel skilled in the art based on the content of the present invention still fall within the scope of protection of the present invention.
[0016] The meanings of the abbreviations used in the specification and claims are listed in the following table: Example 1
[0017] (1) Preparation of Fmoc-Gly-Rink Amide resin: Add 45g Rink Amide resin (substitution degree is 0.70mmol / g) to a 2L reactor, pretreat the resin first, and then remove the Fmoc protecting group from the resin with piperidine / DMF solution. Wash with DMF no more than 10 times, dissolve Fmoc-Gly-OH (95mmol) and TBTU (95mmol) with an appropriate amount of DMF, and then add DIEA (95mmol) at -10℃~10℃. After activation, add it to the reactor containing the resin and stir at room temperature for 3~6h. After the reaction is completed, remove the reaction solution, wash with DMF no more than 10 times, and detect the degree of reaction with Kaiser reagent.
[0018] (2) Preparation of linear terlipressin peptide resin: Remove the Fmoc protecting group from the Fmoc-Gly-Rink Amide resin prepared in step (1) with piperidine / DMF (V:V) solution. Wash with DMF 3 to 6 times. Dissolve Fmoc-Lys(Boc)-OH (95 mmol), TBTU (95 mmol), and DIEA (95 mmol) in an appropriate amount of DMF and add them to the above reactor. Stir with nitrogen at room temperature for 3 to 6 hours. After the reaction is completed, detect with Kaiser reagent, remove the reaction solution and wash with DMF no more than 10 times. According to the method of coupling Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Gly-Gly-OH are coupled in sequence. Finally, a fully protected terlipressin linear peptide resin is obtained. The obtained fully protected terlipressin linear peptide resin is air-dried to obtain a peptide resin.
[0019] (3) Cleavage of the linear terlipressin peptide resin to obtain linear terlipressin: Add the linear peptide resin obtained in step (2) into a 2 L reaction bottle, add 450 ml of the prepared cleavage solution (trifluoroacetic acid), react at room temperature for 3 to 6 h, filter, wash the resin with TFA 2 to 3 times, 100 mL each time, combine the filtrates, add the filtrates to 4.5 L of methyl tert-butyl ether, stir for 2 h, then centrifuge, wash and dry to obtain linear terlipressin.
[0020] (4) The crude linear terlipressin peptide is reacted with a cyclization oxidation solution to obtain a terlipressin cyclization reaction solution: the crude terlipressin peptide obtained in step (3) is added to purified water, poured into a 5 L beaker, and stirred for 30 min. The solution is transferred into a 10 L reactor, the stirring is turned on, and the prepared 5% iodine / methanol solution is added, and stirred for 0.5 to 1 h to obtain a terlipressin cyclization reaction solution.
[0021] (5) The terlipressin cyclization reaction liquid is purified by reverse phase high performance liquid chromatography, desalted and lyophilized to obtain terlipressin: The terlipressin cyclization reaction liquid obtained in step (4) is purified and desalted using a reverse phase chromatography column with octadecylsilane bonded silica gel (C18, 10 μm) as the stationary phase, and then the column is desalted using ammonium acetate. The column is then lyophilized at -25°C and finally heated to room temperature to obtain the product.
[0022] The above embodiments are only used to help understand the method and core idea of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing terlipressin in a solid phase, characterized in that: The specific steps of the method are: 1) Solid phase synthesis of linear terlipressin peptide resin; 2) Linear terlipressin peptide resin was cleaved to obtain crude linear terlipressin peptide; 3) the crude linear terlipressin peptide reacts with the cyclization oxidation solution to obtain the terlipressin cyclization reaction solution; 4) The terlipressin cyclization reaction solution is purified by reverse phase high performance liquid chromatography, converted to salt, and freeze-dried to obtain terlipressin.
2. The method according to claim 1, characterized in that: In step 1), amino resin with a substitution degree of 0.4-0.9 mmol / g is used as a carrier, and amino acids with Fmoc protective groups are coupled one by one to synthesize a linear terlipressin peptide resin.
3. The method according to claim 2, characterized in that The amino resin is Rink Amide-AM resin, Rink Amide-MBHA resin or Rink Amide resin.
4. The method according to claim 2, characterized in that: The amino acids to which the Fmoc protecting groups are coupled one by one are Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, and Fmoc-Gly-Gly-OH.
5. The method according to claim 1, characterized in that The cleavage agent is trifluoroacetic acid.
6. The method according to claim 1, characterized in that The cyclooxidation liquid is a 2-5% iodine methanol solution.