RESUMEN
Foslevodopa (FLD, levodopa 4'-monophosphate, 3) and foscarbidopa (FCD, carbidopa 4'-monophosphate, 4) were identified as water-soluble prodrugs of levodopa (LD, 1) and carbidopa (CD, 2), respectively, which are useful for the treatment of Parkinson's disease. Herein, we describe asymmetric syntheses of FLD (3) and FCD (4) drug substances and their manufacture at pilot scale. The synthesis of FLD (3) employs a Horner-Wadsworth-Emmons olefination reaction followed by enantioselective hydrogenation of the double bond as key steps to introduce the α-amino acid moiety with the desired stereochemistry. The synthesis of FCD (4) features a Mizoroki-Heck reaction followed by enantioselective hydrazination to install the quaternary chiral center bearing a hydrazine moiety.
Asunto(s)
Enfermedad de Parkinson , Preparaciones Farmacéuticas , Carbidopa , Humanos , Hidrogenación , Levodopa/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológicoRESUMEN
INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality. RESULTS: Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23-79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99%. CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.