SYNTHESIS AND BIOLOGICAL-ACTIVITY OF 7-ALKYLIDENECEPHEMS

Several 7-alkylidenecephalosporins were synthesized and biologically e valuated as beta-lactamase inhibitors. The three beta-lactamase enzyme s used in this study included two type C beta-lactamases, derived from Enterobacter cloacae P99 and E. cloacae SC12368, and one type A beta- lactamase, derived from Escherichia coli WC3310. Of the cephalosporins prepared, compound 7e, the sodium salt of 7-[(Z)-(2'-pyridyl)methylen e]cephalosporanic acid sulfone, was found to have excellent inhibitory properties against both type C enzymes. Also, compound 7f, the sodium salt of (Z)-(tert-butoxycarbonyl)methylene]cephalosporanic acid sulfo ne showed high activity as an inhibitor of the type A enzyme. The inhi bition kinetics of 7e were further explored. The IC50 value of 7e indi cated that this compound was approximately 20-fold more active than ta zobactam against the enzyme derived from E. cloacae P99 and 167-fold m ore active than tazobactam against the enzyme derived from E. cloacae SC12368. A plot of enzymatic activity vs incubation time with stoichio metric amounts of inhibitor reveals a rapid deactivation of the enzyme followed by an extremely slow reactivation. 7e exhibited a second-ord er rate constant of k(3)' = 5.3 x 10(6) L/mol.min, and a partition rat io of approximately 20:1 inhibitor:enzyme was determined for this inhi bitor. After separation of excess inhibitor with Sephadex filtration, a rate constant of enzyme reactivation was measured at k(reactiv) = 1. 0 x 10(-3) s(-1). Following 24 h of incubation of enzyme with a large excess of inhibitor and sephadex filtration to remove excess inhibitor , the enzyme was able to recover only 43% of its original activity, in dicating an irreversible component to the inhibition. Potential mechan isms of inhibition for both 7e and 7f are suggested.