Synthesis Of A Key Intermediate For Tofacitinib Via Enzymatic Dynamic Kinetic Resolution-Asymmetric Reductive Amination

Tofacitinib is an oral protein tyrosine kinase inhibitor approved for the treatment of rheumatoid arthritis,active psoriatic arthritis and ulcerative colitis,while its efficient production remains a challenge due to the two consecutive stereogenic centers associated to the piperidine ring.This project aims to develop an enzymatic dynamic kinetic resolution-asymmetric reductive amination to prepare enantiomerically complementary cis-1-benzyl-N,4-dimethylpiperidin-3-amine and its derivatives.First,we established analytical-scale conversion reactions for laboratory imine reductases using 1-benzyl-4-methyl-3-one hydrochloride(2)and methylamine hydrochloride(a)as model substrates.The library and the newly mined imine reductases were screened,and finally 43 enzymes were obtained that were active on the model substrates,of which IR89 and IR174 could catalyze the synthesis of enantiomerically complementary(3R,4R)and(3S,4S)-2a,and has excellent enantioselectivity and diastereoselectivity.In the stability analysis of enzyme-catalyzed reaction,high substrate concentration,overacid or overbase reaction environment will adversely affect the activity and stereoselectivity of the enzyme.Interestingly,IR89 exhibits differences in enantioselectivity under different p H conditions,a phenomenon that seems to be rare in this enzyme family.In the preparative reaction,the enantiomerically complementary1-benzyl-N,4-dimethylpiperidin-3-amine(2a)was prepared from rac-2 in high conversion(93%)with excellent stereoselectivity stability(97% and >99% ee)and diastereoselectivity(>99% dr).Purified IR110 was used for cyclopropylamine(b)and IR174 was used for allylamine(c)and propargylamine(d)to provide the corresponding(3S,4S)-N-substituted-1-Benzyl-4-methylpiperidin-3-amine in isolated yields of 44-90% with high stereoselectivity(>99% ee)and diastereoselectivity(>99% dr).we selected IR89 that can obtain the target configuration for enzyme engineering optimization,and finally obtained the mutant L177 A,whose specific enzyme activity was2.2 times higher than that of the wild type.However,enzyme stability was affected.In order to investigate the mechanism of enzymatic catalysis,the cis-configuration products were docked into the active pockets of IR174.We found that the selected amino acid residues interacted with the phenyl ring and the 3-position amine group.It was speculated that the anchoring effect of the amine group and the phenyl ring of the product may be the key to control the configuration change.In conclusion,we developed a new method for the synthesis of key intermediates of tofacitinib by enzymatic asymmetric reduction,which lays a solid foundation for the efficient green production of tofacitinib.