Analogues of chloramphenicol as mechanism-based inactivators of rat liver cytochrome P-450: modifications of the propanediol side chain, the p-nitro group, and the dichloromethyl moiety.

The importance of the p-nitro group, the propanediol side chain, and the dichloromethyl moiety of chloramphenicol in regulating its effectiveness and selectivity as a mechanism-based inactivator of rat liver cytochromes P-450 has been examined. 1-p-Nitrophenyl-2-dichloroacetamidoethane, 1-p-nitrophenyl-2-dibromoacetamidoethane, and 1-phenyl-2-dichloroacetamidoethane were as effective as chloramphenicol at inactivating the major phenobarbital-inducible isozyme of rat liver cytochrome P-450, whereas 1-p-nitrophenyl-2-difluoroacetamidoethane caused no enzyme inactivation. Unlike chloramphenicol, 1-p-nitrophenyl-2-dichloroacetamidoethane and 1-phenyl-2-dichloroacetamidoethane also inactivated the major beta-naphthoflavone-inducible isozyme of rat liver cytochrome P-450. Alkaline hydrolysis of the adducts formed upon in vitro incubation of liver microsomes from phenobarbital- and beta-naphthoflavone-induced rats with [14C]-1-p-nitrophenyl-2-dichloroacetamidoethane resulted in the release of 4-nitro-1-phenethyl-1,2-dicarboxylic acid amide and oxalic acid. Enzymatic digests of the radio-labeled protein produced by incubation of a reconstituted system containing the major isozymes induced by beta-naphthoflavone or phenobarbital with [14C]-1-p-nitrophenyl-2-dichloroacetamidoethane led to the release of 4-nitro-1-phenethyl-1,2-dicarboxylic acid amide and 4-nitro-1-phenethyl oxamyl lysine. These results suggest that a single oxamyl chloride intermediate is responsible for the covalent modification and, hence, inactivation of both isozymes by 1-p-nitrophenyl-2-dichloroacetamidoethane.