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 beta-lactones were synthesized and tested against HAV 3C proteinase. The D-N-Cbz-serine beta-lactone 5a displays competitive reversible inhibition with a K-i value of 1.50 x 10(-6) M. Its enantiomer, L-N-Cbz-serine beta-lactone 5b is an irreversible inactivator with 0.70 min(-1), K, = 1.84 x 10(-4) M and k(inact)/K-I = 3800 M-1 min(-1). Mass spectrometry and HMQC NMR studies using C-13-labeled 5b show that inactivation of the enzyme occurs by nucleophilic attack of the cysteine thiol (Cys-172) at the beta-position of the oxetanone ring. Although the N-Cbz-serine beta-lactones 5a and 5b display potent inhibition, other related analogues with an N-Cbz side chain, such as the five-membered ring homoserine gamma-lactones 14a and 14b, the four-membered ring beta-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 beta-lactone ring for binding.