Serine and Threonine β-Lactones:  A New Class of Hepatitis A Virus 3C Cysteine Proteinase Inhibitors

Hepatitis A virus (HAV) 3C enzyme is a cysteine proteinase essential for viral replication and infectivity and represents a target for the development of antiviral drugs. A number of serine and threonine β-lactones were synthesized and tested against HAV 3C proteinase. The d-N-Cbz-serine β-lactone 5a displays competitive reversible inhibition with a Ki value of 1.50 × 10-6 M. Its enantiomer, l-N-Cbz-serine β-lactone 5b is an irreversible inactivator with kinact = 0.70 min-1, KΙ = 1.84 × 10-4 M and kinact/KΙ = 3800 M-1 min-1. Mass spectrometry and HMQC NMR studies using 13C-labeled 5b show that inactivation of the enzyme occurs by nucleophilic attack of the cysteine thiol (Cys-172) at the β-position of the oxetanone ring. Although the N-Cbz-serine β-lactones 5a and 5b display potent inhibition, other related analogues with an N-Cbz side chain, such as the five-membered ring homoserine γ-lactones 14a and 14b, the four-membered ring β-lactam 33, 2-methylene oxetane 34, cyclobutanone 36, and 3-azetidinone 39, fail to give significant inhibition of HAV 3C proteinase, thus demonstrating the importance of the β-lactone ring for binding.