Contribution of Artifacts to N-Methylated Piperazine Cyanide Adduct Formation In Vitro from N-Alkyl Piperazine Analogs

In the liver microsome cyanide (CN)-trapping assays, piperazine-containing compounds formed significant N-methyl piperazine CN adducts. Two pathways for the N-methyl piperazine CN adduct formation were proposed: 1) The alpha-carbon in the N-methyl piperazine is oxidized to form a reactive iminium ion that can react with cyanide ion; 2) N-dealkylation occurs followed by condensation with formaldehyde and dehydration to produce N-methylenepiperazine iminium ion, which then reacts with cyanide ion to form the N-methyl CN adduct. The CN adduct from the second pathway was believed to be an artifact or metabonate. In the present study, a group of 4'-N-alkyl piperazines and 4'-N-[C-13]methyl-labeled piperazines were used to determine which pathway was predominant. Following microsomal incubations in the presence of cyanide ions, a significant percentage of 4'-N-[C-13]methyl group in the CN adduct was replaced by an unlabeled natural methyl group, suggesting that the second pathway was predominant. For 4'-N-alkyl piperazine, the level of 4'-N-methyl piperazine CN adduct formation was limited by the extent of prior 4'-N-dealkylation. In a separate study, when 4'-NH-piperaziens were incubated with potassium cyanide and [C-13]labeled formaldehyde, 4'-N-[C-13]methyl piperazine CN-adduct was formed without NADPH or liver microsome suggesting a direct Mannich reaction is involved. However, when [C-13]-labeled methanol or potassium carbonate was used as the one-carbon donor, 4'-N-[C-13]methyl piperazine CN adduct was not detected without liver microsome or NADPH present. The biologic and toxicological implications of bioactivation via the second pathway necessitate further investigation because these one-carbon donors for the formation of reactive iminium ions could be endogenous and readily available in vivo.