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Biological preparation method for (1R, 2S)-2-(3,4-difluorophenyl) cyclopropylamine D-mandelate (I)

An amino acid and carbonyl technology, applied in the field of medicine, can solve the problems of harsh process operating conditions, severe exotherm, difficult product ee value, etc.

Active Publication Date: 2017-05-24
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the strict control of the reaction conditions in this reaction, it is found that the ee value of the product is difficult to reach 99% in the scale-up experiment, and it is stable at 96-97%. Hidden danger
[0012] The above-mentioned method is to obtain the chiral alcohol compound (V) through borane reduction of the carbonyl group under the catalysis of the chiral ligand. This method has the following disadvantages: the cost of the ligand is high, and it is sensitive to oxygen and water, so the process operating conditions are severe. Such as nitrogen protection, anhydrous and oxygen-free; solvent system complex toluene, tetrahydrofuran mixed system, etc.
Applied and ndEnvironmental Microbiology 2013, 79(4), p1378-1384 reported that the carbonyl reductase QNR derived from Mic robacterium luteolum JCM 9174 showed good activity for quinuclidone, but showed low activity for similar substrate alicyclic ketone Catalytic activity, so there is still no relevant literature report to use it for the reduction of aromatic ketone substrates, including for the substrate formula (Ⅵ) compound involved in the present invention

Method used

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  • Biological preparation method for (1R, 2S)-2-(3,4-difluorophenyl) cyclopropylamine D-mandelate (I)
  • Biological preparation method for (1R, 2S)-2-(3,4-difluorophenyl) cyclopropylamine D-mandelate (I)
  • Biological preparation method for (1R, 2S)-2-(3,4-difluorophenyl) cyclopropylamine D-mandelate (I)

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preparation example Construction

[0100] The invention provides a preparation method of a compound of formula V, comprising the steps of:

[0101] (a) In a liquid reaction system, the compound of formula VI is used as a substrate, in the presence of a coenzyme, under the catalysis of carbonyl reductase, to perform an asymmetric reduction reaction, thereby forming the compound of formula V;

[0102]

[0103] with

[0104] (b) optionally isolating the compound of formula V from the reaction system after the reaction in the previous step.

[0105] In the present invention, the above reaction can be coupled or not coupled with the coenzyme regeneration system.

[0106] Preferably, the above reactions are coupled with a coenzyme regeneration system in the same system, thereby further improving production efficiency, reducing production costs and increasing tolerance to substrates.

[0107] reaction system

[0108] The present invention also provides a reaction system used in the biological preparation method of...

Embodiment 1

[0166] Embodiment 1, the construction of carbonyl reductase engineering bacteria

[0167]The QNR target gene and alcohol dehydrogenase gene were entrusted to a commercial company to carry out the whole gene synthesis, cloned into the pET28a(+) vector, transformed into Escherichia coli DH5α competent cells, cultured on the plate, picked a single colony of positive transformants and extracted the plasmid After the sequencing is confirmed, the recombinant plasmid is extracted, introduced into the BL21 (DE3) strain, cultured in LB, and the genetically engineered bacteria capable of inducing the expression of the recombinant carbonyl reductase and alcohol dehydrogenase are obtained.

Embodiment 2

[0168] Embodiment 2, the preparation of recombinant carbonyl reductase, alcohol dehydrogenase

[0169] Inoculate the genetically engineered bacterium stored in glycerol in the previous step into the LB liquid medium containing kanamycin, cultivate for 13 hours at 37°C and 220rpm to obtain the seed medium, and inoculate the seed culture medium in a proportion of 1.5%. On the liquid medium containing 50ug / ml kanamycin resistance, then cultivate to OD at 37℃, 220rmp 600 If the value is >2.0, add lactose with a final concentration of 1.0%, cool down to 25°C, continue to cultivate for 3 hours, add lactose with a final concentration of 0.5%, and cultivate for 20 hours, put it in a tank, and centrifuge to obtain bacteria, which is ready for biotransformation.

[0170] The fermentation formula is as follows:

[0171] raw material Mass content (%) Yeast extract 2.4 soy peptone 1.2 Sodium chloride 0.3 glycerin 0.5 Dipotassium phosphate 0.2 ...

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PUM

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Abstract

The invention provides a biological preparation method for (1R, 2S)-2-(3,4-difluorophenyl) cyclopropylamine D-mandelate (I). Specifically, the method disclosed by the invention comprises the following steps: (a) taking a compound of a formula VI as a substrate in a liquid reaction system, and carrying out an asymmetric addition reaction in the presence of coenzymes under catalysis of carbonyl reductase so as to form a compound of a formula V; (b) enabling the compound of the formula V to react with triethyl phosphonoacetate so as to obtain a compound of a formula IV; and (c) performing ammonolysis and Hofmann degradation on the compound of the formula IV and salifying with D-mandelic acid, thereby obtaining the compound of the formula VI. The invention further provides a reaction system which comprises (i) an aqueous solvent; (ii) a substrate which is the compound of the formula IV; (iii) coenzymes; (iv) carbonyl reductase; (v) a cosubstrate; and (vi) enzymes used for coenzymes regeneration. The structural formula is as shown in the specification.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a preparation method of (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine D-mandelate. Background technique [0002] Ticagrelor, chemical name [1S-[1α,2α,3β(1S,2R),5β]]-3-[7-[2-(3,4-difluorophenyl)-cyclopropylamino ]-5-(propylthio)-3H-1,2,3-triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1 ,2-diol is an oral antiplatelet drug developed by AstraZeneca A B. The drug can reversibly act on the ADP P2Y12 receptor, has obvious inhibitory effect on platelet aggregation caused by ADP, and has a rapid onset of oral administration, and is clinically used to reduce the incidence of thrombotic cardiovascular events in patients with acute coronary syndrome. [0003] The structural formula of ticagrelor is as follows: [0004] [0005] (1R,2S)-2-(3,4-difluorophenyl)cyclopropylamine (Ⅱ) is the key intermediate in the synthesis of ticagrelor, because it is oily, wh...

Claims

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Application Information

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IPC IPC(8): C12P7/22C07C51/41C07C59/50C07C209/68C07C209/62C07C211/40C07C231/02C07C233/58C07C67/343C07C69/743
CPCC07C51/412C07C67/343C07C209/62C07C209/68C07C231/02C12P7/22C07C59/50C07C211/40C07C233/58C07C69/743
Inventor 张福利倪国伟陈少欣鞠佃文汤佳伟谭支敏邹杰郭翔王政文
Owner SHANGHAI INST OF PHARMA IND
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