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Method For The Manufacture Of Degarelix

A technology of degarelix and a manufacturing method, applied in the field of synthetic peptide manufacturing, can solve the problems of polluting the environment, high disposal cost, dangerous manufacturing personnel, etc.

Active Publication Date: 2012-04-25
POLYPEPTIDE LAB AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of TFA is that it is highly toxic to humans, which puts manufacturing personnel at risk
Another disadvantage of TFA is that it is toxic to the environment, which leads to high disposal costs and pollutes the environment if it is not disposed of properly

Method used

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  • Method For The Manufacture Of Degarelix
  • Method For The Manufacture Of Degarelix
  • Method For The Manufacture Of Degarelix

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Hydantoin formation in the synthesis of degarelix. During the production of degarelix, a rearrangement of the hydrogenated orotic acid group to the hydantoin acetyl group was observed in two stages and under two alkaline conditions.

[0063] The first rearrangement occurs in the fragment Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-ILys(Boc)-Pro-D-Ala-NH 2 during the alkaline extraction process. The pH was adjusted to 9.1 with concentrated NaOH solution in an organic / aqueous biphasic system, whereby 4.5% by weight of the hydantoin analog was formed. The mechanism appears to consist of two steps: (a) hydrolysis of the six-membered hydroorotic acid moiety under basic conditions followed by ring closure under acidic conditions to the five-membered hydantoin analog.

[0064] The second rearrangement occurs during the evaporation of the fragment Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-OH DCHA. After the above extraction, Z-Ser(tBu)-4Aph(Hor)-D-4Aph(tBu-Cbm)-Leu-OH was dissol...

Embodiment 2

[0067] Stability of degarelix in DBL VDMF and piperidine / DMF. The stability of degarelix was determined under conditions corresponding to the conditions used to hide the Fmoc group during SPPS. It is known that the 5th amino acid residue in the degarelix sequence, ie, the hydroorotic acid group on the side chain of 4Aph (Hor), is sensitive to alkali and rearranges into a hydantoin acetyl group. All SPPS processes known to the inventors are based on Boc-chemistry.

[0068] Degarelix samples were dissolved in 20% piperidine / DMF, 2% DBU in DMF, and 2% DBU+5% water in DMF, respectively. Samples were analyzed by HPLC after 20 hours to determine the amount of hydantoin analogs.

[0069] 2% DBU / DMF resulted in 1.8% hydantoin formation. This amount was increased to 7% if 5% water was also present (simulated aqueous DMF). Surprisingly, the use of 20% piperidine in DMF did not result in the formation of any hydantoin analogs, suggesting that this mixture may be useful for Fmoc-based...

Embodiment 3

[0071] Synthesis and Purification of Degarelix Using Fmo- / Rink Amide AM Resin

[0072] Step 1. Fmoc-Rink amide AM resin (64 g; substitution 0.67 mmol / g) was placed in the reactor and washed with 1.9 L of DMF. A DMF solution of 20% piperidine was added to 250 ml of the swollen resin, and stirred for 20 minutes. The reactor was evacuated so that the reactor was vented through the bottom filter and a second treatment was performed with 250 ml of 20% piperidine in DMF for 20 minutes. The reactor was vented again by evacuating it, and the peptide resin was washed with 2 L of DMF. The reactor was then vented by evacuating it. The peptide resin is ready for step 2.

[0073] Step 2. A solution of 27.0 g of Fmoc-D-AIa-OH (2 equivalents), 14.3 g of HOBt and 13.2 ml of DIC was dissolved in 250 ml of DMF, activated for 15 minutes, and then poured into the resin containing peptide of the reactor. After the reaction time reached 1 hour, 2.2ml of NMM was added to the solution, and the r...

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Abstract

In a step-wise synthesis of degarelix comprising 0.3 % by weight or less of 4-([2-(5-hydantoyl)]acetylamino)-phenylalanine analog on (solid support)-NH2 a step comprises providing a solution of an amino acid or peptide of which the a-amino group is protected by Fmoc; contacting the support with the solution in the presence of reagent for forming a peptide bond between a carboxyl group of the amino acid or peptide and (solid support)-NH2; removing Fmoc by contacting the support with an organic base, in particular piperidine, in an organic solvent. Also disclosed is degarelix of high purity prepared by the method of the invention and the use of Fmoc in the synthesis of degarelix.

Description

field of invention [0001] The present invention relates to a method for producing a synthetic peptide, in particular to a method for producing a decapeptide degarelix. Background of the invention [0002] There are many known methods for peptide synthesis. A typical method is liquid-phase peptide synthesis (LPPS), which is the preferred method for large-scale production of peptides. Another method of peptide synthesis that is commonly used today is solid-phase peptide synthesis (SPPS), in which growing peptide chains are covalently bound to a resin on a solid support until they reach the desired length and sequence before being cleaved. In these methods, the reactive side chains of the incorporated amino acids need to be protected in order to prevent reactions other than the formation of new peptide bonds required by the growing peptide. In addition, in order to prevent side reactions between the added amino acids and the incorporation of various amino acids in each step, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/23
CPCC07K7/23A61P5/06A61K38/09C07K7/06
Inventor H·张J·弗姆斯加德G·斯泰卡尔
Owner POLYPEPTIDE LAB AS
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