Synthesis and biological activity of 7-alkylidenecephems

Article abstract of DOI:10.1021/jm00006a022

Several 7-alkylidenecephalosporins were synthesized and biologically evaluated as β-lactamase inhibitors. The three β-lactamase enzymes used in this study included two type C β-lactamases, derived from Enterobacter cloacae P99 and E. cloacae SC12368, and one type A β-lactamase, derived from Escherichia coli WC3310. Of the cephalosporins prepared, compound 7e, the sodium salt of 7-[(Z)-(2'-pyridyl)methylene]cephalosporanic acid sulfone, was found to have excellent inhibitory properties against both type C enzymes. Also, compound 7f, the sodium salt of 7-[(Z)-(tert- butoxycarbonyl)methylene]cephalosporanic acid sulfone showed high activity as an inhibitor of the type A enzyme. The inhibition kinetics of 7e were further explored. The IC₅₀ value of 7e indicated that this compound was approximately 20-fold more active than tazobactam against the enzyme derived from E. cloacae P99 and 167-fold more active than tazobactam against the enzyme derived from E. cloacae SC12368. A plot of enzymatic activity vs incubation time with stoichiometric amounts of inhibitor reveals a rapid deactivation of the enzyme followed by an extremely slow reactivation. 7e exhibited a second-order rate constant of k₃' = 5.3 x 10⁶ L/mol · min, and a partition ratio of approximately 20:1 inhibitor:enzyme was determined for this inhibitor. 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, indicating an irreversible component to the inhibition. Potential mechanisms of inhibition for both 7e and 7f are suggested.