Synthesis of pseudoxazolones and their inhibition of the 3C cysteine proteinases from hepatitis A virus and human rhinovirus-14

The hepatitis A virus (HAV) and human rhinovirus (HRV) are important pathogens that belong to the picornavirus family. All picornaviruses have a 3C proteinase that processes an initially biosynthesised precursor protein and is crucial for viral maturation and replication. Monophenyl and diphenyl pseudoxazolones were prepared by cyclisation-elimination of N-alpha-chloroacyl amino acids or by condensation of p-substituted benzamides with glyoxylic acid followed by dehydrative cyclisation. Such pseudoxazolones are good time-dependent inhibitors of the HAV and HRV 3C proteinases with IC50 values in the micromolar range. Mechanistic insights into the mode of inhibition of the pseudoxazolones were obtained from mass spectrometry and gHMQC NMR spectroscopy of the HAV 3C enzyme-inhibitor complex using a pseudoxazolone labelled at the alpha-carbon with C-13, 13b(alpha-C-13). The results indicate that HAV 3C proteinase was inactivated via the formation of a thioether bond by nucleophilic attack of the enzyme thiolate at the imine position of the pseudoxazolone 13b(alpha-C-13).