Synthesis and Characterization of N-Demethylated Metabolites of Malachite Green and Leucomalachite Green

• Published in: Chemical research in toxicology 2003-03, Vol.16 (3), p.285-294
• Main Authors: Cho, Bongsup P, Yang, Tianle, Blankenship, Lonnie R, Moody, Joanna D, Churchwell, Mona, Beland, Frederick A, Culp, Sandra J
• Format: Article
• Language: English
• Subjects: 4-(Dimethylamino)benzophenone; 4-Nitrobenzophenone; Aniline Compounds - chemical synthesis; Aniline Compounds - chemistry; Aniline Compounds - metabolism; Benzophenones - chemistry; Fungicides, Industrial - chemistry; Fungicides, Industrial - metabolism; Leucomalachite green; Magnetic Resonance Spectroscopy; Malachite green; Mass Spectrometry - methods; Methylation; Rosaniline Dyes - chemical synthesis; Rosaniline Dyes - chemistry; Rosaniline Dyes - metabolism; Spectrophotometry, Ultraviolet; triphenylmethane dye
• ISSN: 0893-228X; 1520-5010
• DOI: 10.1021/tx0256679

Malachite green (MG), a triphenylmethane dye used to treat fungal and protozoan infections in fish, undergoes sequential oxidation to produce various N-demethylated derivatives (monodes-, dides(sym)-, dides(unsym)-, trides-, and tetrades-) both before and after reduction to leucomalachite green (LMG). The close structure resemblance of the metabolites with aromatic amine carcinogens implicates a potential genotoxicity from exposure to MG. The availability of the synthetic standards is important for metabolic and DNA adduct studies of MG. This paper describes a simple and versatile method for the synthesis of MG, LMG, and their N-demethylated metabolites. The synthesis involves a coupling of 4-(dimethylamino)benzophenone or 4-nitrobenzophenone with the aryllithium reagents derived from appropriately substituted 4-bromoaniline derivatives, followed by treatment with HCl in methanol. The resulting cationic MG and their leuco analogues showed systematic UV/vis spectral and tandem mass fragmentation patterns consistent with sequential N-demethylation. The extensive 1H and 13C spectral assignments of the metabolites were aided by the availability of 13C7-labeled MG and LMG. The results indicate the existence of a resonance structure with the cationic charge located in the central methane carbon (C7). The synthetic procedure is general in scope so that it can be extended to the preparation of N-demethylated metabolites of other structurally related N-methylated triphenylmethane dyes.