Method for preparing nesiritide

Abstract

The invention relates to a method for preparing nesiritide. The method comprises the following specific steps: (A) synthesizing a tripeptide segment H-Arg(Boc)2-Arg(Boc)2-His(Trt)-OtBu by virtue of a liquid phase method; (B) sequentially coupling amino acids protected by N-terminal Fmoc and side chains according to peptide sequence of a nesiritide main chain 1-29 by taking 2-CTC- resin as initial resin by virtue of a solid phase synthesis method; (C) fixing rings by virtue of iodine in the presence of organic alkali; (D) carrying out schizolysis, so as to obtain a full-protected nesiritide main chain 1-29 peptide segment; (E) coupling the tripeptide segment H-Arg(Boc)2-Arg(Boc)2-His(Trt)-OtBu with the full-protected nesiritide main chain 1-29 peptide segment; and (F) carrying out schizolysis, purification and freeze-drying, so as to obtain nesiritide. A nesiritide preparation process provided by the invention is high in purity, low in cost and suitable for scale production; a racemized peptide impurity D-His32-nesiritide and a deleted peptide impurity Des-Arg31-nesiritide or Des-Arg30-nesiritide can be effectively controlled, furthermore, ring fixation conditions are optimized, and the yield of nesiritide is increased.

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 ...

Examples

Embodiment 1

[0089] Embodiment 1: Fmoc-Arg(Boc) 2 Synthesis of -OSu Activated Ester

[0090] Weigh 596.67g Fmoc-Arg(Boc) 2 -OH (1.0mol), 138.10g HOSu (1.2mol) was added to 3000ml THF, 247.56g DCC (1.2mol) was added in an ice-water bath, reacted for 1 hour, warmed up to room temperature and reacted for 3 hours, the reaction solution was filtered, and the mother liquor was spin-dried, Add DCM to dissolve, filter, wash with saturated sodium bicarbonate 3 times, pure water 2 times, back-extract 2 times, combine organic phases, dry with anhydrous sodium carbonate, spin dry, recrystallize with ice ethanol 3 times, filter, and pull dry with solid oil pump Obtained 617.45g Fmoc-Arg(Boc) 2 -OSu activated ester, yield 89%.

Embodiment 2

[0091] Embodiment 2: Fmoc-Arg(Boc) 2 -Arg(Boc) 2 Synthesis of -OH

[0092] Weigh 187.33g H-Arg(Boc) 2 -OH (0.5mol) and 79.50g Na 2 CO 3 (0.75mol) was added to the mixed solution of 500ml water and 500ml THF to dissolve, and weighed 346.88g Fmoc-Arg(Boc) 2 -OSu (0.5mol) was added to 500ml THF, dissolved and added dropwise to the above mixed solution, reacted overnight at room temperature, adjusted the pH to 7 with 10% dilute hydrochloric acid, removed THF by rotary evaporation, and then adjusted the pH to 3. A large white precipitate was obtained which was filtered. The resulting white precipitate was recrystallized from ice ethanol. The solid oil pump was dried to obtain 414.70g Fmoc-Arg(Boc) 2 -Arg(Boc) 2 -OH, yield 87%.

Embodiment 3

[0093] Embodiment three: Fmoc-Arg (Boc) 2 -Arg(Boc) 2 Synthesis of -OSu Activated Ester

[0094] Weigh 381.34g Fmoc-Arg(Boc) 2 -Arg(Boc) 2-OH (0.4mol), 55.24g HOSu (0.48mol) was added to 800ml THF, 99.02g DCC (0.48mol) was added in an ice-water bath, reacted for 1 hour, warmed up to room temperature and reacted for 3 hours, the reaction liquid was filtered, and the mother liquor was spin-dried, Add DCM to dissolve, filter, wash with saturated sodium bicarbonate 3 times, pure water 2 times, back-extract 2 times, combine organic phases, dry with anhydrous sodium carbonate, spin dry, recrystallize with ice ethanol 3 times, filter, and pull dry with solid oil pump Obtained 373.95g Fmoc-Arg(Boc) 2 -Arg(Boc) 2 -OSu activated ester, yield 89%.

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 ...

Examples

Embodiment 1

[0089] Embodiment 1: Fmoc-Arg(Boc) 2 Synthesis of -OSu Activated Ester

[0090] Weigh 596.67g Fmoc-Arg(Boc) 2 -OH (1.0mol), 138.10g HOSu (1.2mol) was added to 3000ml THF, 247.56g DCC (1.2mol) was added in an ice-water bath, reacted for 1 hour, warmed up to room temperature and reacted for 3 hours, the reaction solution was filtered, and the mother liquor was spin-dried, Add DCM to dissolve, filter, wash with saturated sodium bicarbonate 3 times, pure water 2 times, back-extract 2 times, combine organic phases, dry with anhydrous sodium carbonate, spin dry, recrystallize with ice ethanol 3 times, filter, and pull dry with solid oil pump Obtained 617.45g Fmoc-Arg(Boc) 2 -OSu activated ester, yield 89%.

Embodiment 2

[0091] Embodiment 2: Fmoc-Arg(Boc) 2 -Arg(Boc) 2 Synthesis of -OH

[0092] Weigh 187.33g H-Arg(Boc) 2 -OH (0.5mol) and 79.50g Na 2 CO 3 (0.75mol) was added to the mixed solution of 500ml water and 500ml THF to dissolve, and weighed 346.88g Fmoc-Arg(Boc) 2 -OSu (0.5mol) was added to 500ml THF, dissolved and added dropwise to the above mixed solution, reacted overnight at room temperature, adjusted the pH to 7 with 10% dilute hydrochloric acid, removed THF by rotary evaporation, and then adjusted the pH to 3. A large white precipitate was obtained which was filtered. The resulting white precipitate was recrystallized from ice ethanol. The solid oil pump was dried to obtain 414.70g Fmoc-Arg(Boc) 2 -Arg(Boc) 2 -OH, yield 87%.

Embodiment 3

[0093] Embodiment three: Fmoc-Arg (Boc) 2 -Arg(Boc) 2 Synthesis of -OSu Activated Ester

[0094] Weigh 381.34g Fmoc-Arg(Boc) 2 -Arg(Boc) 2-OH (0.4mol), 55.24g HOSu (0.48mol) was added to 800ml THF, 99.02g DCC (0.48mol) was added in an ice-water bath, reacted for 1 hour, warmed up to room temperature and reacted for 3 hours, the reaction liquid was filtered, and the mother liquor was spin-dried, Add DCM to dissolve, filter, wash with saturated sodium bicarbonate 3 times, pure water 2 times, back-extract 2 times, combine organic phases, dry with anhydrous sodium carbonate, spin dry, recrystallize with ice ethanol 3 times, filter, and pull dry with solid oil pump Obtained 373.95g Fmoc-Arg(Boc) 2 -Arg(Boc) 2 -OSu activated ester, yield 89%.

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