In vitro metabolism of the Cox-2 inhibitor DFU, including a novel glutathione adduct rearomatization

The metabolic profile of DFU [5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsu lphonyl)phenyl-2(5H)-furanone], a potent and selective COX-2 inhibitor, was characterized using in vitro microsomal and hepatocyte incubations. A sing le product, corresponding to p-hydroxylation, p-OH-DFU [(5,5-dimethyl-3-(3- fluoro-4-hydroxyphenyl)-4-(4-methylsulphonyl)phenyl-2(5H)- furanone)], was produced in rat microsomal incubations of DFU. In contrast, three metabolit es were produced in incubations using suspensions of freshly isolated rat h epatocytes. Microsomal production of the p-O-glucuronide metabolite of DFU from synthetic p-OH-DFU was shown to have chromatographic and mass spectrom etric properties identical to the earliest eluting hepatocyte metabolite (M 1). The molecular weights of the other two hepatocyte metabolites were read ily obtained using capillary high-performance liquid chromatography continu ous-flow liquid secondary ion mass spectrometry (HPLC/CF-LSIMS); however, t he elemental composition of these metabolites was not. Unlike typical metab olic products, which produce readily identified increments in molecular wei ght, metabolites M2 and M3 produced molecular ions in positive- and negativ e-ion CF-LSIMS that were consistent with oxidation of DFU (+16 Da), followe d by addition of glutathione (+306 Da) and subsequent loss of 20 and 18 Da, respectively. Capillary HPLC/high-resolution CF-LSIMS was used to generate accurate mass data for M2 and M3 that provided evidence that the losses of 20 and 18 Da, respectively, corresponded to a rearomatization through loss of HF or H2O. Isolation and NMR characterization provided the definitive s tructural proof for these metabolites. Overall, the metabolism of DFU in ra t hepatocytes is proposed to proceed through an epoxide intermediate, which then either rearranges to the p-OH-DFU and is conjugated with glucuronic a cid, or is trapped with glutathione, followed by rearomatization with loss of HF (M2) or H2O (M3).