ABSTRACT
LC-NMR was applied to identify the polar volatile metabolite of MK-0869. MK-0869, a morpholine-based compound containing a triazolone ring, is a very potent NK(1) receptor antagonist. Currently, it is in development as an anti-emesis agent in chemotherapy treatments. The primary metabolites of MK-0869, M1 and M2, are non-polar and lack the triazolone ring. Incubation of [14C]M1 with liver microsomes from male rats produced a very polar and volatile metabolite, M3. Analysis was not possible by LC-MS or by conventional NMR because of poor ionization, small molecular weight and volatility, leaving chemical derivatization and LC-NMR as alternative methods. Reduction of M3 with NaBH(4) resulted in a derivative that had the same retention time as p-fluorophenylethylene glycol on HPLC. A small aliquot of the solution containing M3 was passed through the LC of the LC-NMR system, which was connected on-line with a radioactivity detector. The simultaneous UV and radioactivity chromatograms thus identified the chromatographic UV peak that was associated with the metabolite. Analysis was carried out by stop-flow on another portion of this fraction. From the chemical derivatization and the analysis by LC-NMR, M3 is shown to be p-fluoro-alpha-hydroxyacetophenone. Further studies using LC-NMR showed that M3 could be generated from both M1 and M2 in NADPH-dependant reactions catalyzed by microsomes containing recombinant human CYP2C19, CYP1A2 or CYP3A4.
Subject(s)
Antiemetics/analysis , Antiemetics/metabolism , Magnetic Resonance Spectroscopy/methods , Morpholines/antagonists & inhibitors , Neurokinin-1 Receptor Antagonists , Triazoles/antagonists & inhibitors , Animals , Antiemetics/chemistry , Aprepitant , Chromatography, Liquid/methods , Cytochrome P-450 Enzyme System/metabolism , Humans , Isoenzymes/metabolism , Microsomes, Liver/enzymology , Rats , Rats, Sprague-DawleyABSTRACT
[3R,5R,6S]-3-(2-cyclopropyloxy-5-trifluoromethoxyphenyl)-6-phenyl-1-oxa-7-azaspiro[4.5]decane is a substance P (Neurokinin 1 receptor) antagonist. Substance P antagonists are proven in concept to have excellent potential for the treatment of major depression, and they allow superior and sustained protection from acute and delayed chemotherapy-induced emesis. The metabolism of this compound was investigated in rat hepatocytes, and circulating rat plasma metabolites were identified following oral and intravenous dosing. The turnover in rat hepatocytes within 4 h was about 30%, and the major metabolites were identified as two nitrones and a lactam associated with the piperidine ring. Although these metabolites were also observed in rat plasma, the major circulating metabolite was a keto acid following oxidative de-amination of the piperidine ring. Liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance were used to confirm the structure of the latter metabolite. A mechanism leading to the formation of the keto acid metabolite has been suggested, and most intermediates were observed in rat plasma.
