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

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-α-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.