Abstract
In the liver microsome cyanide trapping assays piperazine containing compounds were found to form significant N-methyl piperazine cyanide (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 which 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 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 KCN 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 biological 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.
- adverse drug reactions
- bioactivation
- drug-induced hepatotoxicity
- hepatotoxicity
- HPLC
- mechanism-based inhibition
- microsomes
- reactive intermediate
- reactive metabolites
- Received December 3, 2012.
- Accepted February 19, 2013.
- The American Society for Pharmacology and Experimental Therapeutics