Method for synthesizing chiral tofacitinib citrate intermediate by enzyme method

A technology for tofacitinib and enzymatic synthesis, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, enzymes, etc., and can solve the problems of complex operation, poor atom economy, and complicated procedures.

Active Publication Date: 2022-01-14
ZHEJIANG LEPU PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the existing chemical synthesis method involves many reagents, and the operation is complicated and the process is cumbersome. Especially, 50% of the product in the final resolution must be discarded, resulting in a very low yield and particularly poor atom economy, so the cost is high. And the target product with high optical purity cannot be obtained

Method used

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  • Method for synthesizing chiral tofacitinib citrate intermediate by enzyme method
  • Method for synthesizing chiral tofacitinib citrate intermediate by enzyme method
  • Method for synthesizing chiral tofacitinib citrate intermediate by enzyme method

Examples

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Effect test

Embodiment 1

[0022] The preparation of embodiment one transaminase thalline freeze-dried powder

[0023] The synthesized transaminase catalyst gene DNA fragment was double-digested with restriction endonuclease NdeI and AvrII at 35-37°C for 6h, purified by agarose gel electrophoresis, and the target fragment was recovered using an agarose gel DNA recovery kit (SEQ ID NO .1); Then, under the action of T4 DNA ligase, the target fragment was ligated with the plasmid pACYCDuet-B that was digested by NdeI and EcoRI overnight at 25-27°C to obtain a recombinant expression plasmid; transforming the recombinant expression plasmid To Escherichia coli competent cells, the transformation conditions are: 40-45°C, heat shock for 80-90 seconds, screen positive recombinants on a resistance plate containing chloramphenicol, pick a single clone, and culture Recombinant bacteria, after the plasmid is amplified, extract the plasmid, re-transform into competent cells, spread the transformation solution on an L...

Embodiment 2

[0024] Preparation of Example 2 TOF20-A

[0025]

[0026] Dissolve 100 g of isopropylamine in 100 ml of water, adjust the pH value to 7.0-8.0 with aqueous hydrochloric acid under cooling in an ice-water bath, and add 10 ml of dimethyl sulfoxide, then dilute to 700 ml with 0.1M Tris-HCl buffer, and pre- Heat to 30°C, then add 100ml of dimethyl sulfoxide solution containing 50g of 4-methyl-1-(phenylmethyl)-3-piperidone (TOF15), and finally add 1g of transaminase lyophilized powder and PLP (Pyridoxal Phosphate) 0.8g, use 20% isopropylamine aqueous solution to control the pH value of 7.0-8.0 for the reaction, convert the temperature at 15-25° C. for more than 12 hours, and monitor the completion of the reaction by TLC. The solid was removed by filtration, the mother liquor was extracted three times with dichloromethane, the combined organic phase was dried over anhydrous sodium sulfate, and concentrated to obtain 45 g of oily TOF20-A, with a yield of about 90%.

Embodiment 3

[0027] Preparation of Example Three (3R,4R)-1-benzyl-N,4-dimethylpiperidin-3-amine (TOF25-A)

[0028]

[0029] Dissolve 10g (about 50mmol) of the oil TOF20-A in Example 1 in 100ml of dichloromethane, add 7g (about 50mmol) of powdered potassium carbonate, cool in an ice-water bath to 0-5°C, and start Iodomethane (7.1 g, about 50 mmol) was added dropwise. After the dropwise addition was completed, the temperature was slowly raised to 15-25° C. and kept for 2 hours. The reaction was monitored by TLC. The solid was removed by filtration, the dichloromethane layer was washed three times with water, the dichloromethane phase was dried over anhydrous sodium sulfate, and concentrated to obtain 10 g of oily TOF25-A, with a yield of about 95%.

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Abstract

The invention discloses a method for synthesizing a chiral tofacitinib citrate intermediate by an enzymic method. The method comprises the following steps: by taking 4-methyl-1-(phenyl methyl)-3-piperidone (TOF15) as a raw material, performing biological catalysis to obtain a chiral intermediate TOF 20-A, and then performing methylation reaction to obtain an intermediate TOF 25-A, namely free (3R, 4R)-1-benzyl-N, 4-dimethyl piperidine-3-amine, wherein the intermediate TOF 25-A can be further made into hydrochloride, so that (3R, 4R)-1-benzyl-N, 4-dimethyl piperidine-3-amine dihydrochloride (TOF30) can be obtained. The method is a synthesis technology which is low in cost, high in yield and environment-friendly, is suitable for large-scale industrial production, and has huge market application value.

Description

technical field [0001] The invention relates to the technical field of enzyme catalysis, in particular to a transaminase catalyst, and to an enzymatic synthesis of a chiral tofacitinib citrate intermediate ((3R,4R)-1-benzyl-N,4 -Dimethylpiperidin-3-amine dihydrochloride), and a chiral tofacitinib citrate intermediate ((3R,4R)-1-benzyl-N,4-dimethyl The production method of piperidin-3-amine dihydrochloride). Background technique [0002] (3R,4R)-1-Benzyl-N,4-dimethylpiperidin-3-amine dihydrochloride is an important intermediate in medicine and organic synthesis, especially for the synthesis of tofacitinib citrate . [0003] Tofacitinib citrate, developed by Pfizer, is the first JAK pathway inhibitor with a mechanism of action and a new type of oral protein tyrosine kinase inhibitor. The product was first approved for marketing in the United States in 2012, and has been approved for marketing in more than 50 countries and regions around the world, including the United State...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P17/12C07D211/56
CPCC12N9/1096C12P17/12C07D211/56C12Y206/01
Inventor 成碟林义杨成钰陈普明
Owner ZHEJIANG LEPU PHARMA CO LTD
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