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All-solid-phase preparation method for carbetocin

A solid-phase preparation technology for carbetocin, applied in the field of polypeptide drug preparation, can solve the problems of many impurities, many side reactions, and low yield

Inactive Publication Date: 2017-06-16
HYBIO PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The former class uses bromobutyric acid as raw material, because bromine is relatively active, there are more side reactions in the coupling, resulting in more impurities and low yield; the latter method increases the cost of raw materials due to the need to protect the carboxyl group first And the side reaction caused by decarboxylation protection, the impurity situation is not improved compared with the former method, but increases the cost and complexity of operation

Method used

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  • All-solid-phase preparation method for carbetocin
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  • All-solid-phase preparation method for carbetocin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: Preparation of carbetocin peptide resin

[0029] Weigh 52.8g (29.4mmol) of Rink Amide resin with a substitution degree of 0.557mmol / g, add it to a solid-phase reaction column, wash it twice with DMF, swell the resin with DMF for 30 minutes, deprotect the DBLK for 6min+8min, DMF Wash 6 times. Weigh 26.2g (88.2mmol) Fmoc-Gly-OH and 13.1g (97.0mmol) HOBT dissolved in DMF, add 15.2mL (106mmol) DIPCDI to activate for 3min, then add the mixture into the reaction column, react at room temperature for 2 hours, Use ninhydrin to detect the end of the reaction (if the resin is colorless and transparent, terminate the reaction; if the resin develops color, prolong the reaction for 1 hour).

[0030] After the reaction is over, wash the resin with DMF for 3 times, add DBLK for deprotection for 5min + 7min, wash the resin with DMF for 6 times, and detect the color of the resin with ninhydrin. Weigh 31.2g (88.2mmol) Fmoc-Leu-OH and 13.1g (97.0mmol) HOBT dissolved in DMF...

Embodiment 2

[0036] Embodiment 2: the preparation of carbetocin essence peptide

[0037] Add 78.5 grams of the carbetocin peptide resin obtained in Example 1 into a 2000ml three-necked bottle, and protect it with nitrogen. Add pre-made TFA:TIS:H 2 O=90:5:5 (V:V) 785ml, react at room temperature for 2 hours, filter the resin, and collect the filtrate. The resin was washed with a small amount of TFA, and the filtrates were combined. The filtrate was slowly added to 7850 ml of glacial ether for precipitation, centrifuged, washed twice with ether, and dried under reduced pressure to obtain 28.7 g of crude peptide with an HPLC purity of 87.23%. After preparation and purification by high-pressure liquid phase, 19.7 g of carbetocin refined peptide was obtained by lyophilization, with a purity of 99.42% and a maximum of 0.15%. The theoretical yield is 29.04g, and the total yield is 67.8%.

Embodiment 3

[0038] Embodiment 3: adopt bromobutyric acid as the comparative example of raw material

[0039] According to the method of Example 1, after changing 4-chlorobutyric acid to 4-bromobutyric acid for solid-phase coupling, BrCH 2 CH 2 CH 2 CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Mmt)-Pro-Leu-Gly-RinkAmide resin, followed by removal of the Mmt protecting group. In the solid-phase cyclization step, DIPEA was used for cyclization at room temperature for 3 hours, and the end of the reaction was detected by DTNB (if the resin is colorless and transparent, stop the reaction; if the resin develops color, prolong the reaction for 0.5 hours until the resin is colorless). After the reaction was completed, the reaction solution was drained, the resin was washed with DMF for 3 times, and the liquid was drained. Methanol shrinks the resin 3 times, and the peptide resin is vacuum-dried. The crude peptide was cleaved by the method of Example 3, and the purity of the crude peptide was 60.4%, wh...

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Abstract

The invention relates to an all-solid-phase preparation method for carbetocin. The method comprises the following steps: subjecting chlorobutyric acid to amide coupling with the amino group of tyrosine of a peptide chain; and then with DBU as alkali, carrying out cyclization under the condition of a solid phase so as to obtain crude peptide of carbetocin.

Description

technical field [0001] The invention belongs to the field of polypeptide medicine preparation, and in particular relates to a preparation method of carbetocin. Background technique [0002] Carbetocin is a synthetic long-acting nonapeptide analogue of oxytocin with agonist properties. A single dose intravenously may be administered immediately after cesarean section under epidural or spinal anesthesia to prevent uterine hypotonia and postpartum hemorrhage. [0003] The clinical and pharmacological properties of carbetocin are similar to those of naturally occurring oxytocin. Like oxytocin, carbetocin binds to the oxytocin receptors of uterine smooth muscle, causing rhythmic contractions of the uterus, increasing its frequency and increasing uterine tension on the basis of the original contractions. Oxytocin receptor levels in the uterus are low in the non-pregnant state, increase during pregnancy, and peak at parturition. Carbetocin therefore has no effect on the non-preg...

Claims

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Application Information

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IPC IPC(8): C07K7/16C07K1/06C07K1/04
CPCC07K1/04C07K1/06C07K7/16Y02P20/55
Inventor 姚志军马婧思伍柯瑾宓鹏程陶安进袁建成
Owner HYBIO PHARMA
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