TY - JOUR T1 - Contribution of Artifacts to <em>N</em>-Methylated Piperazine Cyanide Adduct Formation In Vitro from <em>N</em>-Alkyl Piperazine Analogs JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 1023 LP - 1034 DO - 10.1124/dmd.112.050450 VL - 41 IS - 5 AU - Minli Zhang AU - Christina M. Resuello AU - Jian Guo AU - Mark E. Powell AU - Charles S. Elmore AU - Jun Hu AU - Karthick Vishwanathan Y1 - 2013/05/01 UR - http://dmd.aspetjournals.org/content/41/5/1023.abstract N2 - 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 α-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-[13C]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-[13C]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 [13C]-labeled formaldehyde, 4′-N-[13C]methyl piperazine CN-adduct was formed without NADPH or liver microsome suggesting a direct Mannich reaction is involved. However, when [13C]-labeled methanol or potassium carbonate was used as the one-carbon donor, 4′-N-[13C]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. ER -