Method for preparing crizotinib intermediate by using carbonyl reductase

A crizotinib and reductase technology, which is applied in the field of preparing crizotinib intermediates by using carbonyl reductase, can solve the problems of cheap preparation of unfavorable intermediates, unsuitable large-scale production, low product yield and the like, and achieves important industrial Application value, the effect of small amount of biocatalyst and high yield

Inactive Publication Date: 2017-05-10
ZHEJIANG OCEAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at present mainly based on chemical synthesis, but the method steps are cumbersome, the product yield is low, the pollution is big, and it is not suitable for large-scale production (US7858643B2)
The current bio-preparation method of (S)-2',6'-dichloro-3'-fluorophenylethanol requires the use of expensive coenzyme NADP and the use of glucose dehydrogenase for coenzyme regeneration in the catalytic process, which is not conducive to this intermediate cheap preparation of

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  • Method for preparing crizotinib intermediate by using carbonyl reductase
  • Method for preparing crizotinib intermediate by using carbonyl reductase
  • Method for preparing crizotinib intermediate by using carbonyl reductase

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Effect test

Embodiment 1

[0027] (1) Synthesis of the gene encoding carbonyl reductase: according to the original gene sequence of carbonyl reductase CM3 (genebankSequence ID: ABB91667.1), the codon optimization of the original gene sequence was carried out according to the codon analysis table of Escherichia coli and the synthesis of the target gene was carried out. Total synthesis, gene synthesis entrusted to Shanghai Jierui Biotechnology Co., Ltd., the obtained coding gene sequence is shown in SEQ ID NO.2, and the amino acid sequence of the translated carbonyl reductase is shown in SEQ ID NO.1. Both ends of the synthesized coding gene were cut with NdeI and XhoI restriction sites respectively and connected to pET26b(+) to obtain the recombinant expression vector pET26b-CM3 (see figure 1 ).

[0028] (2) Expression of carbonyl reductase:

[0029] Prepare Escherichia coli BL21(DE3) competent cells, transfer the recombinant expression vector pET26b-CM3 to the expression strain BL21(DE3) by the heat sho...

Embodiment 2

[0034] The difference between this embodiment and the embodiment lies in step (4):

[0035] 100 g of the substrate 2',6'-dichloro-3'-fluoroacetophenone was added to a 2.5 L reactor containing 850 mL of phosphate buffer (0.1 M, pH 7.0), followed by 150 mL of 100 mL of isopropyl Alcohol, 50mg NAD + , 10 g of carbonyl reductase in 0.1 M phosphate buffer (pH 7.0) was added to the reactor. The reaction was stirred magnetically at 30 °C and the pH of the reaction was adjusted with base to maintain it at around 7.0. Thin-layer chromatography was used to qualitatively detect the consumption of substrate and the formation of product. It was detected by HPLC that the substrate was completely consumed, and NaCl was added to saturation, extracted three times with ethyl acetate, and the combined organic phases were extracted. Anhydrous sodium sulfate was used to remove water, suction filtered, and concentrated under reduced pressure to obtain an oily liquid with a chiral ee value greate...

Embodiment 3

[0037] The difference between this embodiment and the embodiment lies in step (4):

[0038] 80 g of the substrate 2',6'-dichloro-3'-fluoroacetophenone was added to a 2.5 L reactor containing 850 mL of phosphate buffer (0.1 M, pH 7.0), followed by 150 mL of 70 mL of isopropyl Alcohol, 80mg NAD + , 3 g of carbonyl reductase in 0.1 M phosphate buffer (pH 7.0) was added to the reactor. The reaction was stirred magnetically at 30 °C and the pH of the reaction was adjusted with base to maintain it at around 7.0. Thin-layer chromatography was used to qualitatively detect the consumption of substrate and the formation of product. It was detected by HPLC that the substrate was completely consumed, and NaCl was added to saturation, extracted three times with ethyl acetate, and the combined organic phases were extracted. Anhydrous sodium sulfate was used to remove water, suction filtered, and concentrated under reduced pressure to obtain an oily liquid with a chiral ee value greater t...

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Abstract

The invention discloses a method for preparing a crizotinib intermediate by using carbonyl reductase. The method comprises the following steps: by taking 2',6'-dichloro-3'-fluoroacetophenone as a substrate and phosphate buffer as a reaction medium, adding the carbonyl reductase, a cofactor and a hydrogen donor to form a biological catalysis reaction system; performing a biological catalysis reaction to generate (S)-2',6'-dichloro-3'- fluorobenzene ethanol. In the method, the novel carbonyl reductase is used for catalyzing a reduction reaction, so that the method has the advantages of small using amount of a biological catalyst, mild reaction conditions, short reaction time, high yield, high optical purity and the like.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to a method for preparing an intermediate of crizotinib by using carbonyl reductase. Background technique [0002] Xalkori (crizotinib) is an ATP-competitive multi-target protein kinase inhibitor developed by Pfizer that inhibits Met / ALK / ROS. It has been confirmed that crizotinib has a significant clinical effect on humans in tumor patients with abnormal activity of ALK, ROS and MET kinases. Xalkori is one of the fastest drugs in the history of cancer drug development. It caused a sensation after it was launched in the United States in 2011. [0003] The chemical structural formula of crizotinib is as follows: [0004] [0005] (S)-2',6'-dichloro-3'-fluorophenylethanol is an important intermediate in the chemical synthesis of crizotinib. However, chemical synthesis is the main method at present, but the method has cumbersome steps, low product yield and high polluti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16
CPCC12P17/165
Inventor 唐云平丁国芳余方苗杨最素黄芳芳
Owner ZHEJIANG OCEAN UNIV
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