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

Representative 7-vinylidenecephalosporins 1 were synthesized from 7-am inocephalosporanic acid and were biologically evaluated as beta-lactam ase inhibitors. These chiral allenes were prepared stereospecifically from a cephalosporin-derived propargylic triflate using organocopper r eagents. The sodium salts of a few such unsaturated cephalosporanates were evaluated as beta-lactamase inhibitors of the type C enzyme deriv ed from Enterobacter cloacae strain P99. One compound, sodium 7-(2'alp ha-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 prep ared and biologically evaluated. The deuterated compound inhibited the enzyme with a rate constant of k(3)' = 2.7 x 10(5) 1/(mol.min), repre senting an isotope effect of 6.3. The deuterated compound had an IC50 value which was approximately twice that of the protio compound, and h ad a turnover number of 25. A mechanism of inhibition which is consist ent 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 interme diate 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 reac tivity is further confirmed by a H-1 NMR study of the nonenzymatic hyd rolysis of the inhibitor under basic conditions. A second type of enzy matic inhibition, which does not disappear with time, was also observe d. This second (irreversible) type of inhibition required longer incub ation times and higher ratios of inhibitor to enzyme and showed no iso tope effect.