Method for preparing carbetocin

A carbetocin and resin technology, which is applied in the field of solid-phase synthesis of peptides, can solve problems such as unfavorable large-scale production, high equipment requirements, high price, etc., and achieves the advantages of being beneficial to industrialized large-scale production, ensuring production safety, and responding mild effects

Active Publication Date: 2015-01-07
ZHEJIANG PEPTITES BIOTECH CO LTD
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  • Abstract
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Problems solved by technology

[0005] Patent No. ZL200910106889.0 discloses a method for preparing carbetocin in solid phase, using amino resin as the starting material, connecting amino acids with Fmoc protecting groups in sequence, removing half of the carbetocin with tetrakis(triphenylphosphine) palladium Cystine side chain allyl protecting group, cyclization, cleavage, purification and lyophilization to obtain refined carbetocin, the reagent for removing the side chain allyl protecting group in this method is expensive, and the cleavage reagent used is The mixed solution of trifluoroacetic acid, water, phenol, ethanedithiol and sulfide anisole is a high-concentration strong corrosive acid, which is easy to burn the human body, has high requirements on the equipment used, causes great environmental pollution, increases c

Method used

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  • Method for preparing carbetocin

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Embodiment 1

[0039] Embodiment 1: Preparation of Fmoc-Gly-PAM resin

[0040] Add 22.2g of PAM resin (degree of substitution: 0.9mmol / g) into the solid-phase reaction column, swell with DMF for 30 minutes, put 11.88g of Fmoc-Gly-OH, 5.4g of HOBT, 0.122g of DMAP into the container, and keep the mixture at room temperature Shake for 2.5-3 hours, filter the reaction solution, wash 3 times with 200ml of DMF, then wash with 200ml of pyridine / Ac 2 O / DMF= 6:5:50 (v / v / v) solution was reacted for 2 hours, after the reaction was completed, the DMF was filtered off, washed 3 times with DMF (250 ml), washed 3 times with DCM (250 ml), washed with Methanol (250 ml) was washed 3 times and filtered to obtain 27.81 g of Fmoc-Gly-PAM resin, the measured substitution degree was 0.69 mmol / g, and the yield was 95.94%.

[0041]

Embodiment 2

[0042] Example 2: BrCH 2 CH 2 CH 2 Preparation of CONH-Tyr(OMe)-Ile-Gln-Asn-Cys(MMt)-Pro-Leu-Gly-PAM Resin (Intermediate Ⅰ)

[0043] Weigh 1.45 g of the prepared Fmoc-Gly-PAM resin and add it to the reactor, swell it with DMF for 30 minutes, remove the Fmoc protection with 20% DBLK for 30 minutes, and connect Fmoc-Leu-OH after washing 5 times with DMF (0.71g Fmoc -Leu-OH, 0.27g HOBT was put into the container in an ice-water bath and dissolved with DMF, added 0.32ml DIC to activate for 10 minutes and then added to the reactor), reacted for about 1h. The end point of the reaction was determined by the ninhydrin method. Repeat the above steps, and use the amino acids of the Fmoc protecting group to complete the remaining connections in turn to obtain BrCH 2 CH 2 CH 2 CONH-Tyr(OMe)-Ile-Gln-Asn-Cys(MMt)-Pro-Leu-Gly-PAM Resin (Intermediate I).

[0044]

Embodiment 3

[0045] Example 3: Preparation of BrCH2CH2CH2CONH-Tyr(OMe)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-PAM Resin (Intermediate II)

[0046] Use 50 ml of DCM:TIS:TFA=95:4:1 (v / v / v) solution to remove the MMt protecting group on Cys from the intermediate I prepared in Example 2 for 10 minutes, and repeat 3 times to obtain BrCH 2 CH 2 CH 2 CONH-Tyr(OMe)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-PAM Resin (Intermediate II).

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Abstract

The invention relates to a solid-phase synthesis method of carbetocin, which comprises the following steps: reacting a PAM (polyacrylamide) resin and Fmoc-Gly-OH to obtain an Fmoc-Gly-PAM resin; sequentially connecting amino acid with Fmoc protective group by solid-phase synthesis to obtain a BrCH2CH2CH2CONH-Tyr(OMe)-Ile-Gln-Asn-Cys(Mmt)-Pro-Leu-Gly-PAM resin; removing the Mmt protective group in the Cys; carrying out cyclization reaction by using DMAP (dimethylaminopyridine) as a cyclization reagent to obtain Cyclo[CH2CH2CH2CO-Tyr(OMe)-Ile-Gln-Asn-Cys]-Pro-Leu-Gly-PAM; carrying out ammonolysis on the cyclization product with an ammonia methanol solution to obtain a carbetocin crude product; and purifying and freeze-drying to obtain the carbetocin. The total yield is up to higher than 45%. Compared with the conventional preparation method, the method provided by the invention avoids using abundant high-concentration strongly-corrosive acid and flammable and explosive aether, has the advantages of high yield, low cost, mild reaction conditions and small environmental pollution, and is beneficial to industrialized production.

Description

technical field [0001] The invention relates to a method for solid-phase synthesis of polypeptides, in particular to a method for synthesizing carbetocin. Background technique [0002] Carbetocin (Carbetocin) was developed by Swedish Ferring Pharmaceutical Co., Ltd. and was used clinically in 1990. It is a synthetic long-acting oxytocin nonapeptide analogue with agonist properties, used for selective hard After cesarean section under extramembranous or spinal anesthesia, it is used to prevent uterine atony and postpartum hemorrhage, with quick onset, long duration of action, and safe and effective clinical application. [0003] The molecular formula of carbetocin is C45H69N11O12S, the molecular weight is 998.17, the chemical name is: desammonium-2-oxo-methyltyrosine-1-к oxytocin, and the structure is: [0004] [0005] Patent No. ZL200910106889.0 discloses a method for preparing carbetocin in solid phase, using amino resin as the starting material, connecting amino acids...

Claims

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Application Information

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IPC IPC(8): C07K7/16C07K1/06C07K1/04
CPCY02P20/55
Inventor 纪东亮秦德志施跃英龚裕录
Owner ZHEJIANG PEPTITES BIOTECH CO LTD
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