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β-D-N4-Hydroxycytidine-5′′-iso-Pr ester is an investigational direct-acting antiviral agent that is under development for the treatment of COVID-19.Given the potential high demand for this compound, it was critical to develop a sustainable and efficient synthesis from commodity raw materials.A cost effective, com. viable two step synthesis of highly pure β-D-N⁴-Hydroxycytidine-5′′-iso-Pr ester from cytidine is described in this paper.The main contribution for enzymic synthesis is the cost of the enzyme.After understanding the reaction profile enzyme loading was reduced to one fourth i.e., 5% with respect to N-hydroxy cytidine from 20-200% i.e., 4-40 times higher enzyme loading as reported in and 50-200% i.e., 10-40 times higher enzyme loading as reported in for the formation of β-D-N⁴-Hydroxycytidine-5′′-iso-Pr ester.By this selective process Enzyme loading can be reduced to one fourth i.e., 5% with respect to N-hydroxy cytidine from 20-200% i.e., 4-40 times higher enzyme loading to afford good yield and enhanced good purity of β-D-N⁴-Hydroxycytidine-5′′-iso-Pr ester by controlling all other process related impurities less than 0.10% followed by purification techniques using ionic liquids, water and mixture of both.

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