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(R)-2-(2,5-difluorophenyl)pyrrolidine, preparation method and applications thereof

A difluorophenyl, pyrrolidine technology, applied in the field of biocatalysis, can solve the problems of high cost, serious pollution, poor stereoselectivity, etc., and achieve the effect of low cost and environmental friendliness

Pending Publication Date: 2020-04-24
ABIOCHEM BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The technical problem to be solved by the present invention is aimed at the defects of high cost, serious pollution, and poor stereoselectivity in the method for preparing (R)-2-(2,5-difluorophenyl)pyrrolidine in the prior art, Provide a kind of (R)-2-(2,5-difluorophenyl)pyrrolidine and its preparation method and application

Method used

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  • (R)-2-(2,5-difluorophenyl)pyrrolidine, preparation method and applications thereof
  • (R)-2-(2,5-difluorophenyl)pyrrolidine, preparation method and applications thereof
  • (R)-2-(2,5-difluorophenyl)pyrrolidine, preparation method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0058] The preparation of embodiment 1 imine reductase

[0059] 1.1 Acquisition of imine reductase gene

[0060] According to the gene sequence SEQ ID NO.2, 4, 6, 8, 10 of the reported imine reductase SEQ ID NO.1, 3, 5, 7, 9, 11, 13, 15, 17, 19 encoded on NCBI , 12, 14, 16, 18, 20 whole gene synthesis imine reductase gene.

[0061] The information of each gene is shown in Table 1 below.

[0062] Table 1

[0063]

[0064]

[0065] 1.2 Preparation of imine reductase

[0066] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 121°C for 20min, ready for use.

[0067] The synthetic imine reductase gene was connected to the pET28a vector, and the enzyme-linked vector was transformed into the host E.coli BL21 (DE3) competent cells, and the engineered strain containing the IRED gene was obtained.

[0068] After the engineering bacteria containing the imine reductase gene were ac...

Embodiment 2

[0074] Example 2 Acquisition and expression of glucose dehydrogenase gene

[0075] The glucose dehydrogenase gene was fully synthesized according to the glucose dehydrogenase gene sequence derived from Bacillus subtilis 168 (NCBI accession number: NP_388275.1).

[0076] Glucose dehydrogenase gene is connected to pET28a, restriction site NdeI&HindIII, and the enzyme-linked vector is transformed into host E.coli BL21(DE3) competent cells to obtain an engineering strain containing glucose dehydrogenase gene. After the engineered bacteria containing the glucose dehydrogenase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 ml LB liquid medium containing 50 μg / ml kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer to 50ml fresh LB liquid medium also containing 50μg / ml kanamycin according to 2v / v% inoculum amount, and shake to OD at 37°C 600 When it reaches about 0.8, add IPTG to its final concentration of 0.5mM, and indu...

Embodiment 3

[0077] The preparation of embodiment 3 glucose dehydrogenase enzyme crude enzyme liquid and enzyme activity assay

[0078] Wash 3 g of the bacteria collected in Example 2 twice with 0.1M pH 7.0 phosphate buffer, then resuspend the bacteria in 15 mL of pH 7.0 phosphate buffer, homogeneously break at low temperature and high pressure, and centrifuge the broken solution The precipitate is removed, and the obtained supernatant is a crude enzyme solution containing recombinant glucose dehydrogenase.

[0079] Enzyme activity detection method: the total reaction volume is 1mL. Take a cuvette with an optical path of 1.0 cm, add 880 μL of 100 mM phosphate buffer (pH 7.0) containing 400 mM glucose, and 100 μL of 25 mM NADP + Solution, placed in the colorimetric tank, turn on the heat preservation device on the ultraviolet spectrophotometer, 25, keep warm for 10min, dilute the prepared enzyme solution 200 times with deionized water, take 20μL and immediately add it to the colorimetric c...

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Abstract

The invention discloses (R)-2-(2,5-difluorophenyl)pyrrolidine, a preparation method and applications thereof. The preparation method comprises: carrying out a hydrogenation reduction reaction on 5-(2,5-difluorophenyl)-3,4-dihydro-2H-pyrrole in the presence of imine reductase and reduced coenzyme NADPH to obtain the product. According to the invention, the ee value of the prepared (R)-2-(2,5-difluorophenyl)pyrrolidine can at least reach 94%, and the conversion rate can at least reach 84% through HPLC detection; the industrial production requirements of (R)-2-(2,5-difluorophenyl)pyrrolidine andlarotrectinib are met; and compared with the chemical method, the preparation method of the invention is low in cost and environment-friendly.

Description

technical field [0001] The invention belongs to the field of biocatalysis, and specifically relates to (R)-2-(2,5-difluorophenyl)pyrrolidine, a preparation method and application thereof. Background technique [0002] Larotrectinib (Chinese name larotrectinib) is a potent, oral, selective inhibitor of tropomyosin receptor kinase (TRK), a genetic abnormality that occurs when the TRK gene in cancer cells fuses with one of the other genes product of. Larotrectinib was developed by Array BioPharma and conducted clinical research by Loxo Oncology. On June 4, 2017, the annual meeting of the American Society of Clinical Oncology (ASCO) announced the clinical trial results of larotrectinib. In the trial, 76% of patients achieved remission after treatment with larotrectinib, and the remission of larotrectinib was more durable, with 79% of patients remission lasting 12 months after starting treatment. At present, larotrectinib is expected to become the first targeted drug approved ...

Claims

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Application Information

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IPC IPC(8): C12P17/10C12N9/06C07D487/04
CPCC12P17/10C12N9/0028C07D487/04
Inventor 王舒丁少南程占冰田振华肖宇焦琦汪媚妮
Owner ABIOCHEM BIOTECH CO LTD
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