Application of LC-NMR for the study of the volatile metabolite of MK-0869, a substance P receptor antagonist

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 NK1 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[C-14]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 NaBH4 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 M I and M2 in NADPH-dependant reactions catalyzed by microsomes containing recombinant human CYP2C19, CYP1A2 or CYP3A4. (C) 2002 Elsevier Science B.V. All rights reserved.