Preparation method of cyano reductase and gabapentin

A gabapentin and reductase technology, applied in biochemical equipment and methods, enzymes, enzymes, etc., can solve the problems of high risk factor, expensive, low efficiency, etc., achieve mild reaction conditions, increase overall yield, and reduce reaction steps Effect

Active Publication Date: 2021-08-10
SHENZHEN READLINE BIOTECH CO LTD
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0008] As can be seen from the traditional preparation route of gabapentin above, routes I and II require five-step reactions to complete, involving the use of heavy metal titanium tetrachloride, high-temperature hydrolysis of sulfuric acid, Hofmann rearrangement with the participation of liquid bromine, Raney nickel Hydrogenation and other expensive, low-efficiency, and high-risk chemicals are used, so the environmental and safety costs of large-scale production are high
Although Route III has added the use of cyanohydrolase, which shortens the preparation route to four steps, due to the use of heavy metal titanium tetrachloride, hazardous chemicals sodium cyanide and Raney nickel high-pressure hydrogenation, this route is compared with route I and II have no obvious advantages

Method used

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  • Preparation method of cyano reductase and gabapentin
  • Preparation method of cyano reductase and gabapentin
  • Preparation method of cyano reductase and gabapentin

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preparation example Construction

[0054] Preparation of related enzymes:

[0055] The genes required for the above enzymes were directly synthesized by the company (Anhui General Biology), and then subcloned into the pET28a plasmid with NdeI / XhoI restriction sites. The constructed plasmid was transferred into E.coli (BL21) strain (General Biology), and after plate culture, a single clone was picked and transferred to 5 mL of LB medium (37°C) containing 50 μM kanamycin for culture. When the cells grew to the right After several phases (OD ~ 0.6), 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce protein expression for 4 hours, and finally the supernatant was obtained by cell collection, disruption and high-speed centrifugation, and then Use sodium dodecylsulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm that the protein expression is correct, and then the bacteria can be gradually inserted into a 5L culture fermenter for growth at 37°C and induced expression with 0.5mM IPTG ...

Embodiment 1

[0058] Embodiment 1: Synthesis of 2-cyano-2-cyclohexane ethyl acetate

[0059]

[0060] First, cyclohexylcyanide (5.45g, 50mmol) and anhydrous tetrahydrofuran (200mL) were mixed into a three-necked round-bottomed flask, cooled to -10°C in an ice-salt bath, and then solid sodium hydride (1.44g, 62mmol) was added in batches to maintain After the mixture was stirred at low temperature (-10°C) for 10 minutes, ethyl 2-bromoacetate (9.7 mL, 75 mmol) was slowly added dropwise. Stir at low temperature for 15 minutes, then rise to room temperature and stir for 60 minutes, add ice water (200 mL) to terminate the reaction, extract the product with ethyl acetate, dry over sodium sulfate, concentrate and remove the solvent to obtain a solid crude product, and the final product is in ethyl acetate / ethanol=1: 3 (volume ratio) crystallized to obtain 7.2 g of white solid, yield 74%.

Embodiment 2

[0061] Embodiment 2: the synthesis of 2-cyano group-2-cyclohexyl acetic acid

[0062]

[0063] Add sodium hydroxide (2.0g, 50mmol) to the mixed solvent of ionized water (200mL) and ethanol (50mL) to dissolve completely, then add ethyl 2-cyano-2-cyclohexyl acetate (5.86g, 30mmol), The mixed solution was heated and stirred at 50°C for 2 hours, then cooled to room temperature, and finally extracted with ethyl acetate, dried, and concentrated to obtain 5.4 g of crude product, which was directly used in the next step without purification.

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Abstract

The invention relates to the technical field of medicine synthesis, in particular to a preparation method of cyano reductase and gabapentin. The method comprises the following steps: by taking sodium hydride as a catalyst, carrying out condensation reaction on cyclohexyl methyl cyanide and 2-ethyl bromoacetate to generate 2-cyano-2-cyclohexyl ethyl acetate; under an alkaline condition, carrying out hydrolysis reaction on 2-cyano-2-cyclohexyl ethyl acetate to obtain 2-cyano-2-cyclohexyl acetic acid; and converting the 2-cyano-2-cyclohexyl acetic acid into gabapentin under the action of the cyano reductase. Aiming at the defects (the problem of traditional chemical preparation of gabapentin) of a literature scheme, cyano reductase is introduced, and a route is systematically optimized, so that reaction steps are reduced, and meanwhile, reaction conditions are milder and more environment-friendly. According to the route, the overall yield of gabapentin can be remarkably increased, and meanwhile, the gabapentin better meets the requirements of current green production.

Description

technical field [0001] The invention relates to the technical field of drug synthesis, in particular to a preparation method of gabapentin. Background technique [0002] Gabapentin is a drug for the treatment of partial epilepsy, and it is also the drug of choice for the treatment of neuropathic pain such as diabetic neuritis and post-herpetic severe pain. [0003] Traditional gabapentin preparation method is as follows: [0004] [0005] Route I: This preparation method has been reported in many articles and patents, Dipak Varal et al {Chem.Sci.Trans, 5,442-446 (2016)}, Ashok Kumar mubai et al (Patent US20080103334A1), Delogu Pietro et al (Patent EP 2368872A1), Wolfram Geibel et al. (Patent US005091567A). Cyclohexanone is first condensed with ethyl cyanoacetate, then hydrolyzed under strong acid and high temperature, acetic anhydride forms ester, ammonolysis, and finally Hofmann rearranges five steps to obtain gabapentin; [0006] Route II: People such as Gareth Griff...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12P13/00
CPCC12N9/0044C12P13/005Y02P20/55
Inventor 赵弘于铁妹潘俊锋刘建
Owner SHENZHEN READLINE BIOTECH CO LTD
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