Synthesis and Evaluation of Anti-Influenza Activity of Amide Derivatives of 3-Spiro-1,2,4-trioxolanolithocholic Acid

Abstract

Objective: To develop novel steroidal 3-spiro-1,2,4-trioxolanes based on lithocholic acid amides with trifluoromethyl ketones and to evaluate their antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in vitro. Methods: The 3-spiro-1,2,4-trioxolanes were synthesized via co-ozonolysis of O-methyl ketoximes of lithocholic acid amides in the presence of trifluoromethyl ketones at 0°C in a cyclohexane–dichloromethane mixture. The structures of the products were confirmed by NMR spectroscopy, and individual diastereomers were isolated using column chromatography. Antiviral activity and cytotoxicity were assessed in MDCK cell cultures, determining IC₅₀, CC₅₀, and the selectivity index (SI). Results and discussion: A series of six new steroidal 3-spiro-1,2,4-trioxolanes was obtained with yields of 65–70%. Compounds with cyclohexyl or benzylamide fragments and a methyl/trifluoromethyl substituent in the trioxolane ring showed moderate activity with a selectivity index (SI) of 8 and 11, respectively. The lead compound, N-cyclohexyl-3,3′-spiro-[5′-trifluoromethyl-5′-phenyl-1,2,4-trioxolano-3-yl]-5β-cholan-24-amide, showed an IC₅₀ of 12.5 µM and an SI of 37. Structure–activity analysis indicated that the nature of substituents in the amide side chain and trioxolane ring significantly affects the balance between antiviral activity and cytotoxicity. Conclusion: The obtained 3-spiro-1,2,4-trioxolanes based on lithocholic acid amides demonstrate a promising profile for further preclinical studies and the development of antiviral agents against the influenza A H1N1 virus.