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Process for preparing an intermediate of sitagliptin via enzymatic conversion

A sequence and carrier technology, applied in the field of intermediates for the preparation of sitagliptin through enzymatic conversion, can solve problems such as consumption, ineffective and low-cost preparation, and adverse environmental effects

Inactive Publication Date: 2013-07-31
CADILA HEALTHCARE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Therefore, these chemistries are not efficient for the low-cost preparation of compounds of formula (I) because they consume expensive solvents and additionally other chemicals that are difficult to handle on a large scale and are not environmentally friendly
[0017] In addition, a major disadvantage of these chemical methods is that in the resolution step only 50% of the total starting material can theoretically be isolated as the pure enantiomer from the racemic mixture
Thus, the loss of 50% of undesired raw materials makes the process expensive and has an adverse impact on the environment
Furthermore, recycling of the wrong isomer requires additional unit operations and costs

Method used

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  • Process for preparing an intermediate of sitagliptin via enzymatic conversion
  • Process for preparing an intermediate of sitagliptin via enzymatic conversion
  • Process for preparing an intermediate of sitagliptin via enzymatic conversion

Examples

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preparation example Construction

[0252] According to the present invention, the method for the preparation of formula (I) or any optically active (S) or (R) form thereof or an enantiomeric excess mixture of any of said forms can be carried out by various methods, including: using recombinant host cells, obtaining Desired enzymes from cell-free extracts / crude lysates of recombinant host cells, isolated from cell-free extracts / crude lysates or isolated from suitable organisms.

[0253] When the product is formed at the end of the reaction, it is then isolated from the reaction mixture using techniques well known in the art.

[0254] The (S) or (R)-3-hydroxyl-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a obtained above ]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one or its enantiomeric excess mixture is suitable as an intermediate for the preparation of sitagliptin body.

[0255] By reaction with methanesulfonyl chloride, (S)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a...

Embodiment 1

[0263] Example 1 Cloning and gene expression analysis of chemically synthesized oxidoreductase and cofactor regenerating enzymes

[0264] The DNA sequences deduced from the polypeptide sequences shown in Sequences Id No. 1, 2, 3, 4, 5 and 7 were codon optimized for expression in E. coli and cloned into the pET11a plasmid vector. In each case, the ligated DNA was further transformed into competent E. coli cells and the transformation mixture was plated on Luria agar plates containing ampicillin. Positive clones were identified based on their growth on petri plates as described above with ampicillin resistance and further restriction digests of plasmid DNA from them. Clones with the expected fragment lengths of the digested plasmid DNA samples were selected as putative positive clones. For each DNA sequence, one of such putative positive clones was subjected to nucleotide sequence analysis and found to have 100% homology to the sequence used for chemical synthesis. These pET1...

Embodiment 2

[0266] Example 2. Cloning and expression analysis of oxidoreductases derived from genomic DNA

[0267] The DNA sequences deduced according to the polypeptide sequences shown in the sequence Id No.6, 8, 9, 10 and 13 of Table no.1 were amplified by PCR using the primers from Saccharomyces cerevisiae according to Table no.1B, and the DNA sequences from the large intestine The respective primers for Bacillus were PCR amplified with those of sequences Id No. 11 and 12 for expression in E. coli. These amplified PCR products were purified and subjected to restriction digestion with internal digestive enzymes to check the PCR products. PCR products of the correct band sizes corresponding to sequences Id No. 9, 11, 12 and 13 were subjected to restriction digestion with cloning enzyme NdeI-BamHI to be ligated to NdeI-BamHI digest vector pET27b, corresponding to sequences Id No. 6, PCR products of the correct band size for 8 and 10 were to be ligated into a blunt-ended vector digested...

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Abstract

The invention provides a process for preparing 3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one (Formula I), into its racemic (R / S) form or any of its optically active (S) or (R) forms or enantiomeric excess mixture of any of the forms comprising: a) reacting 4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one of formula (III) with a suitable oxidoreductase enzymes or its suitable variants in the presence of suitable conditions and co-factor; and b) isolating 3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one, into its racemic (R / S) form or any of its optically active (S) or (R) forms or enantiomeric excess mixture of any of the forms.

Description

technical field [0001] The present invention relates to the preparation of 3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo[4,3-a]pyrazine-7 Process for the enzymatic reduction of (8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one. In particular, the invention relates to the preparation of (S) or (R)-3-hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]triazolo Method for the stereoselective enzymatic reduction of [4,3-a]pyrazin-7(8H)-yl)-4-(2,4,5-trifluorophenyl)butan-1-one. The invention provides amino acid sequences of polypeptides having relevant oxidoreductase activity. In addition, the present invention provides polynucleotide sequences encoding polypeptides having oxidoreductase activity. The present invention also discloses a cofactor regeneration system through a substrate-based or enzyme-based system to regenerate the cofactor during the target enzymatic reduction process. Background technique [0002] 3-Hydroxy-1-(3-(trifluoromethyl)-5,6-dihydro-[1,2,4]t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D487/04C12N9/02C12P17/18
CPCC12P17/182C07D487/04C12N9/0004C12P41/002C12R1/19C12N1/205C12R2001/19
Inventor 桑吉夫·库马尔·门迪拉塔比平·潘迪茹帕尔·乔希乌曼·特里维迪马扬克·G·达弗希曼舒·M·科塔里巴温·舒克拉
Owner CADILA HEALTHCARE LTD
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