Method for preparing carbetocin through solid phase and liquid combining method

A carbetocin-binding technique, applied in the fields of peptide preparation, chemical instruments and methods, organic chemistry, etc., can solve the problems of difficult sample purification, cumbersome steps, inconvenient operation, etc., and avoid glycine-deficient peptide impurities. , The process is easy to operate, and the effect of reducing production costs

Inactive Publication Date: 2016-02-24
JINAN KANGHE MEDICAL TECH
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Problems solved by technology

[0005] At present, the preparation method of carbetocin has been widely reported at home and abroad: Spanish patent ES2115543 discloses a preparation method of carbetocin: using Fmoc-Cys(Trt)-OH and chlorobutyric acid as raw materials to synthesize carbetocin Betocin linear peptide resin, after cleavage to get linear peptide, then dissolve it in the solution, through NaOH/LiOH/DIEA/DMAP/NaHCO 3 Carbetocin is obtained by cyclization with alkaline reagents; this method uses pure solid-phase synthesis of carbetocin linear peptides, the cost is high, and the purity of crude peptide samples is low
[0006] In addition, Czech Patent CS8605461 discloses a method for synthesizing Z-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH by solid-phase method 2 Linear peptide, hydrogenated to give Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH 2 Then react with bromobutyric acid, and then cyclize to obtain carbetocin. Due to the existence of hydrogenation operation in this method, the safety risk is large, and it is not suitable for industrial production
[0007] In China, patents CN104262464A, CN102796178A, CN104086631A, CN101555272B and CN102260326A, CN103467573A respectively disclose a kind of solid-phase synthesis of carbetocin linear peptide resin, and then carry out cyclization reaction on the solid phase. Glycine-deficient peptide impurities are easily produced during the synthesis process, and amino resins are generally used, which has high production costs and is not suitable for large-scale production
[0008] In Chinese patent CN103833831A, a method for solid-phase synthesis of carbetocin by fragment method, the method adopt...

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  • Method for preparing carbetocin through solid phase and liquid combining method

Examples

Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Preparation of Fmoc-Cys(Trt)-Wangresin

[0056] The carrier Wanggresin550.0g (sub=1.0mmol / g) was placed in a synthesis column, washed twice with 4LDMF, and swelled with 4LDCM for 30min; after the DCM was filtered off, Fmoc-Cys(Trt)-OH / DIC / HOBt was added Mixed solution [Weigh 644g (1100mmol) Fmoc-Cys(Trt)-OH and 164g (1210mmol) HOBt into a dissolving bottle, add 2L of DMF and DCM mixed solution with a volume ratio of 1:1 and stir to dissolve, add under ice-water bath 190ml (1210mmol) DIC, activated for 5min], add 6.7g (55mmol) DMAP after reacting for 10min; react for 5h, remove the reaction solution, wash 3 times with 4LDMF, add end-capping reagent 4L (1L acetic anhydride and 0.85L pyridine dissolved in 2.15 in DMF) reacted for 1 h, filtered off the reaction solution, washed with DMF, DCM, and methanol three times, and dried in vacuum to obtain 856.0 g of Fmoc-Cys(Trt)-Wangresin; the degree of substitution was determined to be 0.614 mmol / g by sampling. The...

Embodiment 2

[0057] Embodiment 2: Preparation of Fmoc-Cys(Trt)-CTCresin

[0058] Weigh CTCresin63.0g (sub=1.0mmol / g) and place it in a synthesis column, wash it twice with 500mL DMF, add 500mL DCM to swell for 30min; after filtering off the DCM, add 74g (126mmol) of Fmoc-Cys(Trt)- OH DCM / DMF (volume ratio 3:1) solution 300mL, after stirring, add DIEA40ml (250mmol), react for 5h, remove the reaction solution, add DCM / CH 3 400ml of OH / DIEA (volume ratio 17:2:1) mixed solution was capped for 1h; then washed three times with DMF, DCM and methanol, and dried in vacuum to obtain 100.0g of Fmoc-Cys(Trt)-CTCresin. The measured degree of substitution was 0.632mmol / g. The total synthesis scale was calculated to be 61 mmol.

Embodiment 3

[0059] Example 3: Fragment I Peptide Resin:

[0060] Br-(CH 2 ) 3 Preparation of -CO-Tyr(Me)-Ile-Gln(Trt)-Asn(Trt)-Cys(Trt)-Wangresin

[0061] Accurately weigh Fmoc-Cys(Trt)-Wangresin 823g (synthetic scale 500mmol) with a degree of substitution of 0.614mmol / g in Example 1 and place it in a synthesis column, add 4LDCM to swell for 30min; after filtering off the DCM, wash with 800mlDMF twice, Add 4L of 20% piperidine / DMF solution for deprotection twice, react for 10min and 10min respectively; then wash with DMF 6 times, 4L each time; 2.5 LDMF was dissolved, pre-cooled in an ice-water bath for 15-20 minutes, then added DIC170ml (1100mmol) to activate for 5 minutes, added the solution to the resin, and reacted for 2 hours. solution, washed 6 times with DMF, 4L each time; then deprotected. Repeated cyclic operation in this way, the protected amino acids Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Tyr(Me)-OH, Br(CH 2 ) 3 -COOH, to obtain the fragment I peptide resin with fully protect...

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Abstract

The invention belongs to the field of polypeptide synthesis, and particularly relates to a method for preparing carbetocin through a solid phase and liquid combining method. A solid-phase synthesis method is firstly adopted for preparing an X-(CH2)3-CO-Tyr(Me)-Ile-Gln-Asn-Cys-OH fragment which is subjected to a coupled reaction with an H-Pro-Leu-Gly-NH2 fragment prepared through a liquid-phase method, carbetocin linear peptide is obtained, and then a cyclization reaction of linear peptide is carried out in the liquid phase to prepare carbetocin. According to the method, the operation process is easy and convenient, production cost is low, the situation that glycine is in lack of peptide impurities can be avoided, the subsequent purifying process is facilitated, and the method is suitable for large-scale production.

Description

technical field [0001] The invention relates to the field of polypeptide synthesis, in particular to a method for preparing carbetocin by a solid-liquid phase combination method. Background technique [0002] Carbetocin, whose English name is Carbetocin, is a long-acting oxytocin nonapeptide analog with agonist properties developed and synthesized by Ferring Inc. of Canada. It was launched in China in 2009. Its structure is as follows: [0003] [0004] Carbetocin is used for the prevention of uterine atony and postpartum hemorrhage after elective epidural or spinal anesthesia after caesarean section. Like oxytocin, carbetocin binds to the oxytocin receptor of uterine smooth muscle, causing rhythmic contraction of the uterus, increasing its frequency and increasing uterine tension on the basis of the original contraction; in the non-pregnant state, the uterine Oxytocin receptor levels are low, increase during pregnancy, and peak at parturition; carbetocin therefore has ...

Claims

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Application Information

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IPC IPC(8): C07K1/10C07K1/06C07K1/04C07K1/02
Inventor 张颖王德龙王仁友李同金石鑫磊
Owner JINAN KANGHE MEDICAL TECH
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