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Metabolism of tertiary arylaliphatic amines and formamides in rats Slatter, John Gregory


The metabolites of the basic tertiary arylaliphatic amine N,N,α-trimethyl-7-phenylbenzenepropanamine (RecipavrinR) from male Wistar rats were characterized by gas chromatography-mass spectrometry (GCMS). The work was undertaken in an attempt to determine the source of a novel metabolite, N-(1-methyl-3,3-diphenylpropyl) formamide. The formamide metabolite was isolated from the bile of recipavrin dosed rats only after hydrolysis with the enzyme β-glucuronidase, suggesting that it arose from a glucuronide conjugated precursor. Recipavrin was chosen for the study based on structural similarity to the narcotic analgesic methadone which was shown to give rise to a similar metabolite, 6-formamido-4,4-diphenyl-3-heptanone. The secondary formamide was not a plausible candidate for a β-glucuronidase liberated metabolite of recipavrin, suggesting that a labile aglycone was responsible for the GCMS observation of the formamide metabolite. Labile isomeric compounds, α-methyl-(N-methylene)-7-phenylbenzenepropanamine N-oxide, N-(α-methyl-7-phenylbenzenpropylidene) methylamine N-oxide, and 2-(4',4'-diphenyl-but-2'-yl) oxaziridine were synthesized as possible precursors of the formamide. N-hydroxy-a-methyl-7-phenylbenzenepropanamine, and N-hydroxy-N,α-dimethyl-7-phenylbenzenepropanamine were synthesized as candidates for labile β-glucuronidase liberated aglycone precursors of the nitrones. The biliary nonconjugated and conjugated metabolites of recipavrin were characterized in detail. In addition to the formamide, 15 different metabolites representing the N- dealkylation, oxidative deamination, N-oxidation and phenyl ring oxidation pathways were identified by GCMS. To determine if thermal decomposition of the methylene nitrone in the GC inlet was responsible for the GCMS observation of the formamide metabolite, liquid chromatography-mass spectrometry (LCMS) was used to show that the formamide and not the isomeric methylene nitrone was present in bile prior to GCMS analysis. Although the synthetic methylene nitrone was shown to degrade in the GC inlet to the formamide, the LCMS experiment ruled out the thermal generation of the biliary formamide from a nitrone precursor. The nonconjugated and conjugated metabolites of the recipavrin metabolite, norrecipavrin were characterized in detail by GCMS. Since the secondary formamide metabolite was observed in the β-glucuronidase hydrolyzed bile extract, norrecipavrin was implicated as an intermediate in the biotransformation of recipavrin to the formamide. The possibility of solvent mediated formylation or free radical oxidation of desalkyl metabolites to afford the formamides was ruled out. The methylene nitrone was shown to afford the formamide metabolite under simulated workup conditions. An alkali catalyzed Beckmann rearrangement of nitrone to amide was used to account for this transformation. The secondary hydroxylamine was shown to give rise to the methylene nitrone under simulated workup conditions. It was concluded that the oxidation of a β-glucuronidase liberated secondary hydroxylamine metabolite to the methylene nitrone followed by Beckmann rearrangement of the nitrone to the formamide was the probable source of the formamide observed by GCMS in extracts of bile from recipavrin dosed rats. The metabolism of N-methyl-N-(1-methyl-3,3- diphenylpropyl) formamide was investigated in detail to determine whether the carbinolamide, N-hydroxymethyl-N-(1-methyl-3,3-diphenylpropyl) formamide was involved in the genesis of the formamide metabolite of recipavrin. The above carbinolamide and N-(1-hydroxy-1-methyl-3,3-diphenylpropyl) formamide were identified by GCMS along with 16 other metabolites representing the metabolic pathways N-deformylation, N-dealkylation, N-oxidation and phenyl ring oxidation. The carbinolamides were not found in bile from recipavrin dosed rats, ruling out the possibility of a carbinolamide glucuronide precursor of the recipavrin formamide metabolite. This was the first report of the isolation of stable dealkylation intermediates of a high molecular weight formamide. The hepatotoxicity of the anticancer agent N-methyl formamide and the solvent dimethylformamide, suggests that the recipavrin formamides could also be metabolized to toxic carbinolamide or glutathione related metabolites.

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