Azodicarboxamides:  A New Class of Cysteine Proteinase Inhibitor for Hepatitis A Virus and Human Rhinovirus 3C Enzymes

Hepatitis A virus (HAV) 3C and human rhinovirus (HRV) 3C cysteine proteinases have been shown to be essential for viral maturation and infectivity through the cleavage of a viral polyprotein precursor. A number of bis-hydrazides (e.g., 11−14), analogous to nanomolar inhibitors of cathepsin K, were synthesized and tested for effectiveness against HAV 3C and HRV 3C proteinases, but these compounds showed no significant inhibition of the viral enzymes. However, oxidation of the bis-hydrazides to the corresponding azodicarboxamides gave potent, irreversible inhibitors with IC50's in the low micromolar range. These compounds probably act by adding the active site thiol to the azo moiety in a Michael fashion to give a covalent complex, which was detected by electrospray mass spectrometry. Azodicarboxamide 16 was shown to have a rate constant (k inact/k I) of 35 644 M-1 min-1.