Structural Requirements for the Formation of Carbinolamines by Oxidative Metabolism of N-Methyl Containing Compounds

Abstract

A study was made of the structural requirements in an N-methyl containing molecule which predisposes it to form a characterizable N-hydroxymethyl compound during metabolic oxidative N-demethylation. The metabolism by mouse liver microsomes of hexamethyl melamine and derivatives, N,N-dimethylaminoantipyrine and its N-desmethyl derivative, and compounds of the general formula Ary1-X-N-(CH3) 2 where X is either -N=N- (3-aryl-1,1-dimethyltriazenes, -N=CH- (N'-ary1-N,N-dimethy1-formamidines), -NH-CO- (N'-aryl-N,N-dimethylureas) or is absent (aryldimethylamines) was studied using a colourimetric technique (Nash assay). Whereas the N-methyl moieties of the aryldimethyltriazenes, formamidines, amines and aminoantipyrine derivatives were metabolized to formaldehyde, aryldimethylureas and the N-methyl melamine derivatives except 2-azido-4, 6-bis-(dimethylamino)-1,3,5-triazine formed stable formaldehyde precursors during metabolism. The metabolism of the herbicide monuron [N'-(4-chloropheny1)-N,Ndimethylurea] was investigated using high pressure liquid chromatography. Two metabolites were observed on incubation of monuron with microsomes. One metabolite was characterized as the N-desmethyl derivative whilst the other was tentatively identified as N’-(4-chlorophenyl)-N-hydroxymethy1-N-methylurea. Certain N-hydroxymethyl compounds were so stable that they did not produce a positive response in the Nash assay. Two such N-methylols were N-hydroxymethylformamide, which was tentatively identified as a urinary metabolite of N-methylformamide in mice and man, and N-hydroxymethylbenzamide which was characterized as a metabolite of N-methylbenzamide in vitro and as a urinary metabolite. N-Hydroxymethyl-N-methylbenzamide was identified as a microsomal metabolite of N,N-dimethylbenzamide but, unlike N-hydroxymethylbenzamide, was a relatively unstable species and produced a positive response in the Nash assay. Both N-methylbenzamide and N-hydroxymethylbenzamide were metabolized to N-formylbenzamide in vivo and in hepatocytes. The conversion of N-hydroxymethylbenzamide to N-formylbenzamide in vitro was also catalyzed by 9000g and microsomal supernatants and by horse liver alcohol dehydrogenase, and was inhibited by pyrazole. N-Formylbenzamide was unstable and degraded to produce benzamide in Earl's buffer (pH=7.4, 37°C) with a half-life of 7.8 minutes. This suggests that N-demethylation need not be synchronous with formaldehyde production. Electron density calculations were consistent with metabolic evidence which indicated that the formation of stable N-hydroxymethyl compounds during metabolic N-demethylation was favoured if the nitrogen bearing the methyl group was situated in an electron withdrawing environment.