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-methylsulphonyl)phenyl-2(5H)-furanone], a potent and selective COX-2 inhibitor, was characterized using in vitro microsomal and hepatocyte incubations. A single 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 metabolites were produced in incubations using suspensions of freshly isolated rat hepatocytes. Microsomal production of the p-O-glucuronide metabolite of DFU from synthetic p-OH-DFU was shown to have chromatographic and mass spectrometric properties identical to the earliest eluting hepatocyte metabolite (M1). The molecular weights of the other two hepatocyte metabolites were readily obtained using capillary high-performance liquid chromatography continuous-flow liquid secondary ion mass spectrometry (HPLC/CF-LSIMS); however, the elemental composition of these metabolites was not. Unlike typical metabolic products, which produce readily identified increments in molecular weight, metabolites M2 and M3 produced molecular ions in positive- and negative-ion CF-LSIMS that were consistent with oxidation of DFU (+16 Da), followed 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 H(2)O. Isolation and NMR characterization provided the definitive structural proof for these metabolites. Overall, the metabolism of DFU in rat hepatocytes is proposed to proceed through an epoxide intermediate, which then either rearranges to the p-OH-DFU and is conjugated with glucuronic acid, or is trapped with glutathione, followed by rearomatization with loss of HF (M2) or H(2)O (M3).
Yergey
J A
JA
Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada. james_yergey@merck.com
Trimble
L A
LA
Silva
J
J
Chauret
N
N
Li
C
C
Therien
M
M
Grimm
E
E
Nicoll-Griffith
D A
DA
D000818:Animals; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism*; D005663:Furans / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008861:Microsomes / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
C106876:5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5H)-furanone; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D005663:Furans; D007527:Isoenzymes; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D005978:Glutathione