SYNTHESIS AND MECHANISTIC EVALUATION OF 7-VINYLIDENECEPHEM SULFONES AS BETA-LACTAMASE INHIBITORS

Representative 7-vinylidenecephalosporins 1 were synthesized from 7-aminocephalosporanic acid and were biologically evaluated as beta-lactamase inhibitors. These chiral allenes were prepared stereospecifically from a cephalosporin-derived propargylic triflate using organocopper reagents. The sodium salts of a few such unsaturated cephalosporanates were evaluated as beta-lactamase inhibitors of the type C enzyme derived from Enterobacter cloacae strain P99. One compound, sodium 7-(2'alpha-tert-butylvinylidene)cephalasporanate sulfone (16),was found to be an excellent progressive inhibitor of this enzyme, exhibiting a second-order rate constant of inactivation of k(3)' = 1.7 x 10(6) 1/(mol.min) and a turnover number of 12. A potential mechanism of inhibition was investigated. The corresponding terminally deuterated allene sodium 7-(2'alpha-tert-butyl-2'-beta-deuteriovinylidene) sulfone (21) was prepared and biologically evaluated. The deuterated compound inhibited the enzyme with a rate constant of k(3)' = 2.7 x 10(5) 1/(mol.min), representing an isotope effect of 6.3. The deuterated compound had an IC50 value which was approximately twice that of the protio compound, and had a turnover number of 25. A mechanism of inhibition which is consistent with this data was proposed. The mechanism of inhibition involves an acyl enzyme which becomes stabilized toward hydrolysis through its conversion-to a vinylogous urethane (beta-aminoacrylate). This intermediate is formed by an elimination reaction which transforms the allene into an enyne. The inhibition disappears extremely slowly, presumedly due to hydrolysis of the stabilized acyl enzyme. This pattern of reactivity is further confirmed by a H-1 NMR study of the nonenzymatic hydrolysis of the inhibitor under basic conditions. A second type of enzymatic inhibition, which does not disappear with time, was also observed. This second (irreversible) type of inhibition required longer incubation times and higher ratios of inhibitor to enzyme and showed no isotope effect.