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Solid-phase synthesis method of liraglutide

A technology of liraglutide and solid-phase synthesis, which is applied to the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of high cost of reagents, many side reactions, production restrictions, etc. The effect of small quantity, beneficial to industrial production, and short synthesis cycle

Inactive Publication Date: 2013-05-08
刘卫 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Novo Nordisk’s method for preparing liraglutide is technically difficult and requires high cost and equipment. The intermediates of the synthesis methods reported in US6458924B2 and US6268343B1 need to be purified by liquid phase, the steps are cumbersome and have many side reactions, and the waste liquid produced is not good The cost of processing and reagent usage is also high
The domestically reported patent method for the solid-phase synthesis of liraglutide (CN102286092) uses Fmoc-Lys(Alloc)-OH for the 26-position lysine, and the selective removal of Alloc requires a heavy metal catalyst Pd(Ph3P)4, which generally requires nitrogen protection. reaction, industrial production will be limited

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Condensation of linear liraglutide

[0026] Weigh 370mg (0.1mmol) Fmoc-Gly-Wang resin (0.27mmol / g) into the reactor, and swell the DMF solution for 30min. Weigh the amino acids to be reacted in each step in advance, and the dosage is 5 times that of the resin (0.5mmol). The first amino acid Gly at the C-terminal is already on the resin, and the reaction starts from Arg. Reaction steps: 25% piperidine / DMF solution to deresin Fmoc protecting group twice, each time for 15min, wash the resin twice with DMF, methanol, DCM successively, drain, add the first step reactant Fmoc-Arg(Pbf) -OH and condensing agent: 1.1mL of Cl-HOBt and DIC solution (DIC solution: 8mL DIC plus DMF to 100mL; Cl-HOBt solution: 8.5g Cl-HOBt plus DMF to dissolve to 100mL;), shake the reaction at room temperature for 2h, DMF , methanol and DCM to wash the resin twice, and drain. According to the amino acid sequence of liraglutide straight chain His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Se...

Embodiment 2

[0027] Example 2 Removal of Mtt protecting group and condensation side chain g-glutamic acid and palmitic acid on Lys20

[0028] Add about 10 mL of newly prepared cleavage reagent A: (TFA 1 mL; Tis 5 mL; DCM 96 mL), shake at room temperature for 5 times, each time for 5 min, wash with DCM, methanol, DMF, DCM twice each, and drain. Add Fmoc-Glu-OtBu (0.213 g) and condensing agent (1.1 mL of Cl-HOBt and DIC solution) to react for 2 hours, de-Fmoc twice with 25% piperidine / DMF solution, drain, wash the resin with DMF, methanol and DCM for 2 hours Once, drained, add 10 times the amount of palmitic acid (0.256 g), 2.2 mL of Cl-HOBt and DIC solution to react for 3 h, wash and drain the polypeptide resin.

Embodiment 3

[0029] Example 3 Cutting the polypeptide from the resin

[0030]Liraglutide peptide resin, add cleavage reagent B: (TFA: 9mL; anisole: 0.25mL; phenol: 0.25g; Tis: 0.25mL; water: 0.25mL) at room temperature for 2 hours, take the filtrate to a 50mL centrifuge tube , wash the resin twice with a small amount of TFA (1 mL), and combine the filtrates. Add anhydrous diethyl ether (cooled in ice in advance) directly to the centrifuge tube to more than 45mL, and a slightly pinkish white precipitate can be seen immediately. After mixing thoroughly, place the centrifuge tube in ice to cool for 30min. After centrifugation, discard the supernatant, wash the crude peptide precipitate with ether for 2-3 times, and then dry it in the air. Finally, 332.7 mg of crude product was obtained, and the yield of crude peptide was 88.7%.

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Abstract

The invention discloses a method for synthesizing liraglutide. The method comprises the following steps of: 1, selecting Fmoc-Gly-Wang resin and N terminal Fmoc protected and side chain protected amino acid as raw materials, wherein lysine on a 26th site adopts Fmoc-Lys(Mtt)-OH or Fmoc-Lys(Mmt)-OH, histidine on an N terminal adopts Boc-His(Trt)-OH or Boc-His(Boc)-OH; 2, selectively removing a Mtt or Mmt protecting group on the lysine by adopting 1 percent TFA (Trifluoroacetic Acid); 3, sequentially coupling one g-glutamic acid and palmitic acid on a side chain; and 4, cutting resin by the TFA to obtain a crude product of the liraglutide, and carrying out preparative liquid chromatography purification and lyophilization to obtain a pure product of the liraglutide. The method for synthesizing the liraglutide is simple in steps, saves time and labor, is less in difficult sequences in a coupling process, simple and easy to operate during side chain de-protection, less in byproducts and high in yield, and is suitable for industrialized production.

Description

[0001] Technical Field The present invention relates to the field of pharmaceutical technology, especially to a method for synthesizing liraglutide. Background technique [0002] Type II diabetes mellitus is a common chronic disease. Currently, commonly used oral therapeutic drugs include: biguanides, sulfonylureas, α-glucosidase inhibitors, thiazolidinediones, DPP-4 inhibitors, etc. For patients who do not respond well to oral hypoglycemic agents, combined insulin therapy is often used. Since most of the current treatment plans focus on the control of blood sugar, while ignoring the protection of islet function, most patients will eventually experience severe decline in β-cell function, and the body's insulin sensitivity will gradually decrease. Drugs had no effect on the rate of beta cell damage. Liraglutide is a new type of hypoglycemic drug GLP-1 analogue. Many experiments have proved that this type of drug can stimulate the proliferation and differentiation of pancreatic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/605C07K1/06C07K1/04
CPCY02P20/55
Inventor 刘卫杨树耀
Owner 刘卫
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