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Solid-phase fragment method for bivalirudin synthesis

A technology of bivalirudin and solid-phase synthesis, which is applied in the field of polypeptide drug preparation, can solve the problems of high risk, low total product yield, high difficulty in preparation and purification, and achieve the effect of reducing the content

Pending Publication Date: 2019-09-06
HAINAN ZHONGHE PHARM CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The technical problem to be solved in the present invention is: select a suitable synthetic route that can be produced industrially, and solve the following technical problems: (1) the liquid phase synthesis of bivalirudin process adopts highly toxic reagents, and the production process is dangerous, so it is not suitable for Large-scale production; (2) The purity of the crude peptide obtained by the existing solid-phase synthesis method is low, the preparation and purification are difficult, and the total yield of the product is not high

Method used

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  • Solid-phase fragment method for bivalirudin synthesis
  • Solid-phase fragment method for bivalirudin synthesis
  • Solid-phase fragment method for bivalirudin synthesis

Examples

Experimental program
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Effect test

Embodiment 1

[0095] Embodiment 1: One by one coupling method synthesis bivalirudin

[0096] Weigh 16.67g Wang resin (10mmol, substitution degree 0.6mmol / g) into the solid-phase reactor, add 70ml DMF to swell for 30min, and wash twice with DMF. Weigh 10.60g Fmoc-Leu-OH (3.0eq.), 0.37g DMAP (0.3eq) and 5.40g HOBt (4.0eq.) Dissolve in 50ml DMF, add 6.2ml DIC (4.0eq.) at 0-10℃ , pre-activated for 5-10min, added to the solid-phase reactor, and reacted with nitrogen gas for 5-7h. Drained and washed 3 times with DMF. Add 20% PIP / DMF to remove Fmoc protection twice (the time is 5min+10min respectively), and wash with DMF 6 times.

[0097] Weigh 13.78g Fmoc-Tyr(tBu)-OH (3.0eq) and 5.40g HOBt (4.0eq) and dissolve in 40ml DMF, add 6.20ml DIC (4.0eq) at 0-10°C, pre-activate for 5-10min, Add it into a solid-phase reactor, stir and react for 2 to 3 hours, and the ninhydrin test is negative. Drained and washed 3 times with DMF. Add 20% PIP / DMF to remove Fmoc protection twice (the time is 5min+10min ...

Embodiment 2

[0100] Embodiment 2: the synthesis of fragment-peptide resin

[0101] Weigh 125.0g Wang resin (100mmol, degree of substitution 0.8mmol / g) into the solid-phase reactor, add 700ml DMF to swell for 30min, and wash twice with DMF. Weigh 106.0g Fmoc-Leu-OH (3.0eq.), 3.67g DMAP (0.3eq) and 54.1g HOBt (4.0eq.) Dissolve in 500ml DMF, add 62.0ml DIC (4.0eq. ), pre-activated for 5-10 minutes, added to the solid-phase reactor, and stirred for 5-7 hours. Drained and washed 3 times with DMF. Add 20% PIP / DMF to remove Fmoc protection twice (the time is 5min+10min respectively), and wash with DMF 6 times.

[0102] Weigh 137.8g Fmoc-Tyr(tBu)-OH (3.0eq) and 54.1g HOBt (4.0eq) and dissolve in 400ml DMF, add 62.0ml DIC (4.0eq) at 0-10°C, pre-activate for 5-10min, Add it to a solid-phase reactor, blow nitrogen gas to react for 2-3 hours, and the ninhydrin test was negative. Drained and washed 3 times with DMF. Add 20% PIP / DMF to remove Fmoc protection twice (the time is 5min+10min respective...

Embodiment 3

[0104] Embodiment 3: the synthesis of fragment dipeptide resin

[0105] Weigh 200.0g of CTC-Cl resin (200mmol, substitution degree 1.0mmol / g) into the solid-phase synthesis reactor, add 1400ml of DCM to swell for 30min, and wash twice with DMF. Fmoc-Asn(Trt)-OH119.34g (1.0eq) and DIEA1020ml (3.0eq) were added, and stirred at room temperature for 3 hours. Filter and wash 3 times with DMF. Add 20% PIP / DMF to remove Fmoc protection twice (the time is 5min+10min respectively), and wash with DMF 6 times.

[0106] Weigh 281.2g Fmoc-Gly-Gly-Gly-Gly-OH (3.0eq) and 108.2g HOBt (4.0eq) and dissolve in 800ml DMSO, add 124.0ml DIC (4.0eq) at 0-10°C to preactivate 5- After 10 minutes, add it to the solid-phase reactor, stir and react for 2-3 hours, and the ninhydrin test is negative. Drained and washed 3 times with DMF. Add 20% PIP / DMF to remove Fmoc protection twice (the time is 5min+10min respectively), and wash with DMF 6 times.

[0107] Repeat the above steps, connect Fmoc-Pro-OH,...

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Abstract

The invention discloses a solid-phase fragment method for bivalirudin synthesis. The method includes: taking trityl chloride resin as starting resin, performing solid-phase synthesis of 1-9 full-protection peptide resin, cutting full-protection nonapeptide off resin, performing inoculation of 10-20 peptide resin in a solid phase, and cracking to obtain a bivalirudin crude product. The method has advantages that by replacement of a step-by-step method with the solid-phase fragment method for bivalirudin synthesis, deprotection times can be reduced, five-membered ring rearrangement side reactionof a 10Gly-9Asn peptide sequence structure under alkaline conditions is avoided, and the content of impurities including Asp9 and beta-Asp9 is effectively reduced. The method is simple in process operation and high in crude product purity, prepared product purity reaches 99.8%, the single impurity content is smaller than 0.1%, and a promising industrial production prospect is achieved.

Description

[0001] field of invention [0002] The invention belongs to the technical field of polypeptide drug preparation methods, in particular to a solid-phase fragment synthesis method of bivalirudin. Background technique [0003] Bivalirudin was approved for marketing by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) in 2000 and 2004, and is clinically used for perioperative percutaneous coronary intervention (PCI). period of anticoagulant therapy. Its effective anticoagulant component is a hirudin derivative fragment, which plays an anticoagulant effect by directly and specifically inhibiting the activity of thrombin. No matter whether thrombin is in the blood circulation or combined with thrombus, this product can bind to its catalytic site and anion binding site. Point (also known as substrate recognition site) specific binding, thereby directly inhibiting the activity of thrombin. Its action is different from that of heparin, and it does not...

Claims

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Application Information

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IPC IPC(8): C07K14/815C07K1/16C07K1/06C07K1/04
CPCC07K14/815Y02P20/55
Inventor 黄清炳陈建华蒋名更陈超凌振宏
Owner HAINAN ZHONGHE PHARM CO LTD
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