Characterization of novel glutathione adducts of a non-nucleoside reverse transcriptase inhibitor, (S)-6-chloro-4-(cyclopropylethynyl)-4-(trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), in rats.: Possible formation of an oxirene metabolic intermediate from a disubstituted alkyne

The postulated formation of oxirene-derived metabolites from rats treated with a disubstituted alkyne, (S)-6-chloro-4-(cyclopropylethynyl)-4-( trifluoromethyl)-3,4-dihydro-2(1H)-quinazolinone (DPC 961), is described. The reactivity of this postulated oxirene intermediate led to the formation of novel glutathione adducts whose structures were confirmed by LC/MS and by two-dimensional NMR experiments. These metabolites were either excreted in rat bile or degraded to mercapturic acid conjugates and eliminated in urine. To demonstrate the oxidation of the triple bond, an analogue of DPC 961 was synthesized, whereby the two carbons of the alkyne moiety were replaced with C-13 Stable isotope labels. Rats were orally administered [C-13]DPC 961 and glutathione adducts isolated from bile. The presence of an oxygen atom on one of the C-13 labels of the alkyne was demonstrated unequivocally by NMR experiments. Administration of C-14-labeled DPC 961 showed that biliary elimination was the major route of excretion with the 8-OH glucuronide conjugate (M1) accounting for greater than 90% of the eliminated radioactivity. On the basis of radiochemical profiling, the glutathione-derived metabolites were minor in comparison to the glucuronide conjugate. Studies with cDNA-expressed rat enzymes, polyclonal antibodies, and chemical inhibitors pointed to the involvement of P450 3A1 and P450 1A2 in the formation of the postulated oxirene intermediate. The proposed mechanism shown in Scheme 1 begins with P450-catalyzed formation of an oxirene, rearrangement to a reactive cyclobutenyl ketone, and a 1,4-Michael addition with endogenous glutathione to produce two isomeric adducts, GS-1 and GS-2. The glutathione adducts were subsequently catabolized via the mercapturic acid pathway to cysteinylglycine, cysteine, and N-acetylcysteine adducts. The transient existence of the alpha,beta-unsaturated cyclobutenyl ketone was demonstrated by incubating the glutathione adduct in the presence of N-acetylcysteine and monitoring the formation of N-acetylcysteine adducts by LC/MS. Epimerization of GS-1 to GS-2 was also observed when N-acetylcysteine was omitted from the incubation.