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Segment method based solid-phase synthesis method of ganirelix acetate

A technique of Ganirelix and solid-phase synthesis, applied in peptide preparation methods, chemical instruments and methods, organic chemistry, etc., can solve problems such as unfavorable production scale-up, long synthesis cycle, industrial scale-up, etc., and achieve industrial scale-up The effect of large-scale production, reducing the difficulty of synthesis, and reducing the cost of purification

Inactive Publication Date: 2017-08-18
JINAN KANGHE MEDICAL TECH
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AI Technical Summary

Problems solved by technology

[0008] At present, the synthetic methods of ganirelix acetate mainly include the traditional Boc solid-phase synthesis method and the Fmoc solid-phase synthesis method. The method disclosed in US4801577, US5212288, and US5767082 is the Boc solid-phase synthesis method, which is backward in technology, high in cost and low in yield. , and need to use HF for cracking, which pollutes the environment very much and is not conducive to production amplification; the method disclosed in CN102584945A is the Fmoc solid-phase synthesis method, which uses Fmoc-HArg(Et) 2 -OH and Fmoc-D-HArg(Et) 2 -OH is used as a raw material, deprotected after amino acid coupling is completed, and acetylation reaction is carried out to obtain ganirelix acetate resin, which is cracked and purified to obtain ganirelix acetate; the methods disclosed in CN104017058A and CN104231055A are Fmoc solid-phase synthesis methods , Fmoc-Lys(Dde)-OH, Fmoc-Lys(Alloc)-OH and Fmoc-D-Lys(Dde)-OH, Fmoc-D-Lys(Alloc)-OH protected by different side chain protecting groups Instead of Fmoc-HArg(Et) 2 -OH and Fmoc-D-HArg(Et) 2 -OH, the Fmoc protecting group is removed after the amino acid coupling, and then the N-terminal is acetylated, and the side chains of lysine and D-lysine are modified to obtain ganirelix acetate resin to form Et Modified guanidine group, after cleavage and purification treatment, ganirelix acetate can be obtained; CN104844694A proposes to replace Fmoc-HArg(Et) with Fmoc-Lys(Boc)-OH and Fmoc-D-Lys(Boc)-OH respectively 2 -OH and Fmoc-D-HArg(Et) 2 -OH, cleavage after the coupling to obtain the precursor of ganirelix acetate, and modify the precursor of ganirelix acetate with water instead of organic solvent as the reaction solvent to obtain the crude peptide of ganirelix acetate, and obtain acetic acid after purification Garni Rick
[0009] The Boc solid-phase synthesis strategy in the above patents uses highly toxic HF, which limits mass production and restricts industrial scale-up; the Fmoc solid-phase synthesis strategy adopts the method of coupling amino acids one by one in the process of synthesizing peptide chains. Long synthesis cycle and low purity of linear crude peptide

Method used

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  • Segment method based solid-phase synthesis method of ganirelix acetate

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Preparation of Fmoc-D-Ala-Rink Amide Resin

[0056] Accurately weigh 20.0g of Rink Amide Resin (sub=0.65mmol / g) into the synthesis column, wash twice with 160mL DMF, add 160mL DCM to swell for 30min, filter off the DCM, wash twice with 160mL DMF; add 20% Deprotect 160ml of piperidine / DMF solution twice, react for 10min and 15min respectively; then wash twice with 160ml DMF, DCM, DMF respectively; filter off DMF, add Fmoc-D-Ala-OH / DIC / HOBT mixed DMF Solution [weigh 8.09g (26.00mmol) Fmoc-D-Ala-OH and 3.81g (28.20mmol) HOBT into a conical flask, add 80mL DMF solution and stir to dissolve, add 4.37ml (28.20 mmol) DIC, activated for 3min; reacted for 2h, took out the reaction solution, washed twice with 160mL DMF, added 250mL of end-capping reagent (50ml acetic anhydride, 42.5ml pyridine, 157.5mL DCM) for reaction for 2h, filtered off the reaction solution, respectively Wash 3 times with DMF, DCM, and methanol, and dry in vacuo to obtain 20.84 g of Fmoc-D-Ala...

Embodiment 2

[0057] Embodiment 2: Preparation of Fmoc-D-Ala-Rink Amide AM Resin

[0058] Accurately weigh 20.0g of Rink Amide AM Resin (sub=0.71mmol / g) into the synthesis column, wash twice with 200mL DMF, add 200mL DCM to swell for 30min, filter out the DCM, wash twice with 200mL DMF; 200ml of 20% piperidine / DMF solution was deprotected twice, reacted for 10min and 15min respectively; then washed twice with 200ml DMF, DCM, DMF respectively; DMF was filtered off, and Fmoc-D-Ala-OH / DIC / HOBT Mix DMF solution [weigh 8.84g (28.40mmol) Fmoc-D-Ala-OH and 4.22g (31.24mmol) HOBT into a conical flask, add 100mL DMF solution and stir to dissolve, add 4.84ml at low temperature (0°C) (31.24mmol) DIC, activated for 3min; reacted for 2h, took out the reaction solution, washed twice with 200mL DMF, added 300mL of capping reagent (60ml acetic anhydride, 51ml pyridine, 189mL DCM) to react for 2h, suction filtered off the reaction solution, respectively Wash 3 times with DMF, DCM, and methanol, and dry in ...

Embodiment 3

[0059] Embodiment 3: Preparation of Fmoc-Tyr(tBu)-CTC Resin

[0060] Weigh 80.0g (sub=1.00mmol / g) of CTC resin and place it in a synthesis column, wash twice with 500mL DMF, add 600mL DCM to swell for 30min; Tyr(tBu)-OH DCM solution 350ml, add 48.61ml (640.00mmol) DIPEA at low temperature (0°C), react for 60min, remove the reaction solution, add DCM / methanol / DIPEA (volume ratio 17:2:1) and mix 480ml of the solution was capped for 30min; then washed three times with DMF, DCM and methanol, and dried in vacuum to obtain 109.49g of Fmoc-Tyr(tBu)-CTC Resin; the degree of substitution was 0.63mmol / g.

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Abstract

The invention belongs to the field of polypeptide synthesis and relates to a segment method based solid-phase synthesis method of ganirelix acetate. The method comprises steps as follows: firstly, a peptide resin segment A [6-10] and an intermediate segment B [1-5] of ganirelix acetate are synthesized with a solid phase method; secondly, the intermediate segment B [1-5] is connected to the peptide resin A [6-10], pyrolysis is performed, and a ganirelix acetate crude peptide is obtained; finally, a ganirelix acetate refined peptide is obtained through purification. According to the technical scheme, synthesis period can be greatly shortened, crude peptide purity is improved to 93% or higher, purification difficulty and production cost are reduced, total yield of products is increased to 77% or higher, production cost is further reduced, and industrial large-scale production is facilitated.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for solid-phase synthesis of ganirelix acetate by a fragment method. [0002] technical background [0003] Ganirelix acetate injection (trade name: ), developed and produced by Merck (known as Merck in the United States and Canada), was approved by the State Food and Drug Administration of China in 2013 for marketing in China, and is used to prevent premature luteinizing hormone (LH) peaks. The incidence of infertility in the world reaches 9%, and the use of gonadotropin-releasing hormone (GnRH) agonist regulation in the controlled superovulation protocol (COH) during in vitro fertilization and embryo transfer has been routinely adopted. Ganirelix acetate injection is a third-generation GnRH antagonist, which can rapidly and reversibly inhibit the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the body through competitive binding t...

Claims

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Application Information

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IPC IPC(8): C07K7/23C07K1/06C07K1/04
CPCC07K7/23Y02P20/55
Inventor 张颖王品王仁友李同金石鑫磊
Owner JINAN KANGHE MEDICAL TECH
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