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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. / Pharmaceutical Sciences, Faculty of / Graduate

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. Because NAE levels in seeds decline during imbibition similar to ABA, a physiological role was predicted for these metabolites in Arabidopsis thaliana seed germination and seedling development. There is also a corresponding increase of AtFAAH (fatty acid amide hydrolase), transcript levels and activity, which metabolizes NAE to ethanolamine and free fatty acids. Based on whole genome microarray studies it was determined that a number of up-regulated genes that were responsive to NAE were also ABA responsive. NAE induced gene expression in these ABA responsive genes without elevating endogenous levels of ABA. It was also determined that many of these NAE/ABA responsive genes were associated with an ABA induced secondary growth arrest, including ABI3. ABI3 is a transcription factor that regulates the transition from embryo to seedling growth, the analysis of transcript levels in NAE treated seedlings revealed a dose dependent, inverse relationship between ABI3 transcript levels and growth, high ABI3 transcript levels were associated with growth inhibition. Similar to ABA, NAE negatively regulated seedling growth within a narrow window of early seedling establishment. When seedlings are exposed to NAE or ABA within the window of sensitivity, the induction of genes normally associated with the ungerminated desiccation tolerant state resumed. The NAE tolerant FAAH overexpressor and the NAE sensitive FAAH knockout both had a NAE/ABA sensitive window similar to the wild type A. thaliana. The abi3-1 ABA insensitive mutant does not undergo growth arrest upon exposure to ABA, but NAE did induce growth arrest when treated within the sensitivity window. This evidence showed that although NAE functions within an ABA dependent pathway, it also functions in an ABA independent signaling pathway. The FAAH overexpressor is tolerant to NAE through its ability to quickly metabolize NAE from the growth media, yet it is hypersensitive to ABA. The FAAH overexpressor also displayed hypersensitivity to GA, which improved its delayed germination in non-stratified seed, while the FAAH knock out showed GA insensitivity. Overall, these results showed that NAE functions as a negative regulator of germinating seed and seedling growth in ABA dependent and independent signaling pathways, and that altered NAE metabolism may interfere with ABA/GA perception in germinating seed.