A kind of method for preparing Nesiritide

Abstract

The invention relates to a method for preparing nesiritide. The specific steps of the invention are: A) synthesizing the tripeptide fragment H-Arg(Boc)2-Arg(Boc)2-His(Trt)-OtBu by a liquid phase method; B) ) using solid-phase synthesis method, starting with 2-CTC-resin, and sequentially coupling amino acids with N-terminal Fmoc protection and side chain protection according to the peptide sequence of nesiritide main chain 1-29; C) in organic base Under certain conditions, iodine was used to fix the ring; D) cleavage to obtain the fully protected nesiritide main chain 1-29 peptide fragment; E) tripeptide fragment H-Arg(Boc)2-Arg(Boc)2-His(Trt) Coupling of ‑OtBu and fully protected nesiritide main chain 1‑29 peptide fragment; F) cleavage, purification, and lyophilization to obtain nesiritide. The invention provides a high-purity, low-cost nesiritide preparation process suitable for large-scale production. This process can not only effectively control the racemic peptide impurity D‑His32‑nesiritide and the missing peptide impurity Des‑Arg31‑nesiritide Peptide or Des‑Arg30‑nesiritide, the yield of nesiritide was also improved by optimizing the ring fixation conditions.

Description

technical field [0001] The invention belongs to the technical field of polypeptide drug preparation methods, and in particular relates to a method for preparing nesiritide. Background technique [0002] Nesiritide, English name: Nesiritide, the structural formula is as follows: [0003] H-Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gly-Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Ser-Ser-Gly-Leu- Gly-Cys-Lys-Val-Leu-Arg-Arg-His-OH (disulfide bond: 10-26) [0004] Nesiritide was isolated from pig brain tissue by Suhoh in 1988. It is a cyclic peptide composed of 32 amino acids. The 10-26 positions are connected by S-S bonds. It is exactly the same as the endogenous hormone and is mainly used for acute decompensation. The treatment of dyspnea in congestive heart failure has better effect and less side effects, and is a promising peptide drug. [0005] As for the preparation method of nesiritide, CN200410083873.X reports the preparation method of atrial peptide, which is not suitable for ...

AI Technical Summary

Problems solved by technology

Chinese patents CN200910104860.9 and CN200910104861.3 report a solid-phase preparation and purification method. On the one hand, HMPB-AM resin is used as the carrier resin. When HMPB-AM resin is esterified with the first protected amino acid, there is 50% Fmoc -His(Trt)-OH will undergo racemization, so there are racemic isomers in the product; on the other hand, due to the use of a low-concentration nesiritide linear peptide liquid ring, a large amount of waste liquid will be generated, causing large pollution, and Production efficiency is not high, not suitable for mass production

) reported nesiritide-CTC-resin with fully protected side chains of the iodine-fixed ring. Although the iodine-fixed ring avoids a large amount of waste liquid from the liquid ring, hydrogen iodide, a by-product of iodine oxidation, will cause the peptide to be released from the CTC-resin. cut off, affecting the total yield

In addition, the structure of this product contains an Arg-Arg fragment. During solid-phase coupling, since the side chain protection group of arginine is a macromolecular hydrophobic group, the arginine coupling is incomplete and generates Deletion peptide impurity Des-Arg 31 - Nesiritide (or Des-Arg 30 -nesiritide), due to the racemic isomer D-His 32 - Nesiritide and Deletion Peptide Des-Arg 31 - Nesiritide (or Des-Arg 30 -nesiritide) is similar in polarity to nesiritide, and it is difficult to remove during the purification process, so the total yield of this product in conventional solid-phase synthesis is very low, and it is difficult to achieve large-scale production

Images