Kinetics of Action of a Two-Stage Pro-Inhibitor of Serine β‑Lactamases

β-Lactamase inhibitors are important in medicine in the protection of β-lactam antibiotics from β-lactamase-catalyzed destruction. The most effective inhibitors of serine β-lactamases covalently modify the enzyme active site. We have recently studied O-acyl and O-phosphyl hydroxamates as a new class of such inhibitors. In this paper, we describe our studies of the N-acyl derivatives of a cyclic O-acyl hydroxamic acid, 3H-benzo­[d]­[1,2]­oxazine-1,4-dione, and, in particular, the N-tert-butoxycarbonyl derivative. This compound is not a β-lactamase inhibitor itself but undergoes spontaneous hydrolysis in aqueous solution, yielding an O-phthaloyl hydroxamic acid, which is a β-lactamase inhibitor. This compound spontaneously, but reversibly, cyclizes in solution to form phthalic anhydride, which is also a β-lactamase inhibitor. Both inhibitors react to form the same transiently stable phthaloyl–enzyme complex. Thus, we have a two-step cascade, beginning with a pro-inhibitor, in which each step leads to a different inhibitor, presumably with different enzyme specificities. The kinetics of these transformations have been elucidated in detail. The phthaloyl derivatives, where the free carboxylate is important for facile reaction with the enzyme, represent a new lead for serine β-lactamase inhibitors. Analogues can be conveniently constructed in situ by reaction of nucleophiles with phthalic anhydrides and then screened for activity. Active hits may then become new leads.