Suvorexant key intermediate enzymatic preparation method

A technique of asana and reductase, which is applied in the field of medicine, can solve the problems of unfavorable separation and purification, by-products, and the theoretical yield is only 50%.

Active Publication Date: 2019-09-24
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Application Information

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Problems solved by technology

[0004] 5-Chloro-2-((R)-5-methyl-[1,4]diazepan-1-yl)benzoxazole is a key intermediate in the synthesis of Suvorexan, and the patent US20130331379A1 uses tartaric acid 5-Chloro-2-((R)-5-methyl-[1,4]diazepan-1-yl)benzoxazole was prepared by dibenzoate resolution method, the disadvantages of this method The highest theoretical yield is only 50%, and multiple resolution crystallizations are required to obtain optically pure products
[0005] Literature (N.A.Strotman et al., J.Am.Chem.Soc.2011,133,8362) reported the synthesis of 5-chloro-2-((R)-5-methyl-[1 ,4]diazepan-1-yl)benzoxazole, but the ee value is only 94.5% and there may be heavy metal residues
[0006] Literature (Ian K.Mangion, et al., Org.Lett.2012,14,3458) utilizes transaminase method to prepare 5-chloro-2-((R)-5-methyl-[1,4]diazepine Cyclohept-1-yl) benzoxazole realizes the green synthesis of the intermediate, but the method yield is low (62%), and by-products are produced, which is unfavorable for separation and purification

Method used

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  • Suvorexant key intermediate enzymatic preparation method
  • Suvorexant key intermediate enzymatic preparation method
  • Suvorexant key intermediate enzymatic preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the acquisition of highly expressed genetically engineered bacteria

[0024] The whole gene synthesis was completed by Shanghai Xuguan Company.

[0025] According to the gene StIR (WP_023587323.1) of Streptomyces thermolilacinus, the gene KcIR (WP_020388085.1) of Kribbellacatacumbae, the gene SiIR (WP_044567941.1) of Streptomyces iranensis, the gene LmIR (local1039zeet98) of Leishmania major strain Friedlin, the gene Mdiumset98 MsIR (WP_026609689.1) and Microbulbifer gene MbIR (WP_067084177.1) were respectively codon-optimized in order to enable the gene to be expressed in the E. coli expression host. And add corresponding enzyme cutting sites at both ends of the gene, and construct them into corresponding vectors to obtain genetically engineered bacteria IR1, IR2, IR3, IR4, IR5, and IR6.

[0026] Transform the prepared recombinant vector into Escherichia coli BL21, Rosetta or Origami by conventional methods to construct a genetically engineered bacterium...

Embodiment 2

[0027] The cultivation of embodiment 2 genetically engineered bacteria and the preparation of resting cells

[0028] Pick a single colony on the plate and inoculate it into 5ml of fermentation medium containing corresponding antibiotics, cultivate it for about 15 hours as a seed solution, inoculate it into 600ml of fermentation medium according to the inoculation amount of 1%, and cultivate it on a shaker at 37°C and 200rpm to OD 600 = about 0.6-0.8, add IPTG with a final concentration of 0.1 mM to induce for more than 10 h, and collect the bacteria by centrifuging the culture solution at 8000 rpm.

Embodiment 3

[0029] Example 3 Asymmetric reductive amination of mesylate of formula (1) catalyzed by resting cells of IR1

[0030] Take 2.5g of IR1 resting cells and resuspend in 100mL sodium phosphate buffer (100mM, pH 7.2), add glucose (1 g), NADP + (5mg), GDH enzyme powder (50mg), 4-[(2-aminoethyl)(5-chloro-2-benzoxazolyl)amino]-2-butanone-bis(methanesulfonate)( 0.5g), use 10% sodium carbonate solution to control pH 7.0, react at 30°C for 24 hours, thin-layer chromatography detection shows that the reaction is complete, adjust pH to 9.0 with 10% sodium carbonate solution, extract with ethyl acetate (100mL*3) , dried over anhydrous sodium sulfate and spin-dried, separated and purified by column chromatography to obtain 5-chloro-2-((R)-5-methyl-[1,4]diazepan-1-yl)benzo Oxazole 0.252g, yield 90%, ee value>96%. 1H NMR (600MHz, CDCl 3 ):δ7.22(s,1H),7.06(d,J=8.4Hz,1H),6.88(d,J=8.4Hz,1H),3.79-3.91(m,2H),3.59-3.72(m ,2H),3.25(d,J=13.9Hz,1H), 2.96(t,J=11.4Hz,1H),2.84-2.92(m,1H),1.94-2.02(m,1...

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Abstract

The present invention discloses an imine reductase StIR (WP_023587323.1) derived from streptomyces thermolilacinus or an imine reductase KcIR (WP_020388085.1) from kribbella catacumbae or an imine reductase SiIR (WP_044567941.1) from streptomyces iranensis or an imine reductase LmIR (CAJ03998.1) from leishmania major strain friedlin or an imine reductase MsIR (WP_026609689.1) from methylocaldum szegediense or an imine reductase MbIR (WP_067084177.1) from microbulbifer, and the imine reductase is also used as a biological catalyst to prepare anti-insomnia drug suvorexant intermediate 5-chloro-2-((R)-5-methyl-[1,4] diazacyclic heptyl-1-yl)benzoxazole. The corresponding imine reductase can catalyze a substrate of 5-100 g/L and has a conversion rate more than 99%. The method has characteristics of mild reaction conditions, no pollution, simple process routes, etc., and has relatively great industrial application prospects.

Description

technical field [0001] The invention belongs to the field of medical technology, and relates to a preparation method of an important pharmaceutical intermediate, in particular to 5-chloro-2-((R)-5-methyl-[1,4]diazepan-1-yl ) Enzymatic preparation method of benzoxazole. Background technique [0002] Suvorexant (trade name Belsomra) is an anti-insomnia drug developed by Merck, which was approved by the FDA in August 2014. This is the first orexin receptor antagonist for the treatment of difficulty falling asleep or staying asleep , its structural formula is as follows: [0003] [0004] 5-Chloro-2-((R)-5-methyl-[1,4]diazepan-1-yl)benzoxazole is a key intermediate in the synthesis of Suvorexan, and the patent US20130331379A1 uses tartaric acid 5-Chloro-2-((R)-5-methyl-[1,4]diazepan-1-yl)benzoxazole was prepared by dibenzoate resolution method, the disadvantages of this method The highest theoretical yield is only 50%, and multiple resolution crystallizations are required ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/16
CPCC12P17/16
Inventor 姚培圆徐泽菲于珊珊吴洽庆朱敦明
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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