Differential Oxidation of Two Thiophene-Containing Regioisomers to Reactive Metabolites by Cytochrome P450 2C9

Abstract

The uricosuric diuretic agent tienilic acid (TA) is a thiophene-containing compound that is metabolized by P450 2C9 to 5-OH-TA. A reactive metabolite of TA also forms a covalent adduct to P450 2C9 that inactivates the enzyme and initiates immune-mediated hepatic injury in humans, purportedly through a thiophene-S-oxide intermediate. The 3-thenoyl regioisomer of TA, tienilic acid isomer (TAI), is chemically very similar and is reported to be oxidized by P450 2C9 to a thiophene-S-oxide, yet it is not a mechanism-based inactivator (MBI) of P450 2C9 and is reported to be an intrinsic hepatotoxin in rats. The goal of the work presented in this manuscript was to identify the reactive metabolites of TA and TAI by the characterization of products derived from P450 2C9-mediated oxidation. In addition, in silico approaches were used to better understand both the mechanisms of oxidation of TA and TAI and/or the structural rearrangements of oxidized thiophene compounds. Incubation of TA with P450 2C9 and NADPH yielded the well-characterized 5-OH-TA metabolite as the major product. However, contrary to previous reports, it was found that TAI was oxidized to two different types of reactive intermediates that ultimately lead to two types of products, a pair of hydroxythiophene/thiolactone tautomers and an S-oxide dimer. Both TA and TAI incorporated 18O from 18O2 into their respective hydroxythiophene/thiolactone metabolites indicating that these products are derived from an arene oxide pathway. Intrinsic reaction coordinate calculations of the rearrangement reactions of the model compound 2-acetylthiophene-S-oxide showed that a 1,5-oxygen migration mechanism is energetically unfavorable and does not yield the 5-OH product, but instead yields a six-membered oxathiine ring. Therefore, arene oxide formation and subsequent NIH-shift rearrangement remains the favored mechanism for formation of 5-OH-TA. This also implicates the arene oxide as the initiating factor in TA induced liver injury via covalent modification of P450 2C9. Finally, in silico modeling of P450 2C9 active site ligand interactions with TA using the catalytically active iron-oxo species revealed significant differences in the orientations of TA and TAI in the active site which correlated well with experimental results showing that TA was oxidized only to a ring carbon hydroxylated product, whereas TAI formed both ring carbon hydroxylated products and an S-oxide.

Mechanistic Studies of the Cationic Binding Pocket of CYP2C9 In Vitro and In Silico: Metabolism of Nonionizable Analogs of Tienilic Acid

“…The ions at m/z 247 and 189 arose from the 4-keto-2,3 dichlorophenoxy acetic acid and phenol moieties, respectively, and indicated (in all subsequent cases) that attachment of GSH or oxygen occurred on the R1 side of the molecule. An oxidative metabolite (+16 Da) also formed in this incubation and was consistent with oxidation to 5-OH TA with fragment ions at m/z 247 and 189, consistent with previous reports (Mansuy et al, 1984;Dansette et al, 1991;Rademacher et al, 2012).…”

“…Using 5-hydroxylase activity is limiting because thiophene-S-oxides are not capable of converting to 5-hydroxy thiophenes (Rademacher et al, 2012), and affinity assays are limiting because they are not indicative of metabolic activity. A LC-UV-MSbased metabolic profiling approach with rCYP2C9, NADPH, and GSH allowed for a broad assessment of metabolite formation.…”

“…The 2-[2,3-dichloro-4-(thiophene-2-carbonyl)phenoxy]acetic acid (TA), 2-[2,3-dichloro-4-(thiophene-3-carbonyl)phenoxy]acetic acid (TAI), 2-[2,3-dichloro-4-(5-methylthiophene-2-carbonyl)phenoxy]acetic acid (5-MeTA), 2-[2,3-dichloro-4-(5-chlorothiophene-2-carbonyl)phenoxy]acetic acid (5-ClTA), ethyl 2-[2,3-dichloro-4-(thiophene-2-carbonyl)phenoxy]acetate (TA-EE), 2-[2,3-dichloro-4-(thiophene-2-carbonyl)phenoxy]-N,N-diethylacetamide (TA-DiEA), and 2-[2,3-dichloro-4-(thiophene-2-carbonyl)phenoxy]-N-ethylacetamide (TA-EA) were all synthesized according to previously published procedures and were 95-99% pure by liquid chromatography (LC)/UV, LC/ mass spectrometry (MS), and 1 H NMR (Thuillier et al, 1974;Rademacher et al, 2012). The compounds (2,3-dichloro-4-(3-hydroxypropoxy)phenyl)(thiophen-2-yl)methanone (TA-OnPA) and (2,3-dichloro-4-methoxyphenyl)(thiophen-2-yl)methanone (TA-OMe) were purchased from Cayman Chemical (Ann Arbor, MI; Lot 165277-166307) and Toronto Research Chemical (Ontario, Canada; Lot 7-SWZ-43-1), respectively.…”

“…The difference in the inhibitory properties of TA versus TAI was originally thought to be a result of differences in the reactivity of the proposed CYP2C9-generated thiophene S-oxides (Mansuy, 1997). More recent studies, however, indicate that TA only forms a thiophene epoxide, whereas TAI forms two reactive metabolites, as follows: a thiophene epoxide and a thiophene-S-oxide (Bonierbale et al, 1999;Rademacher et al, 2012). Reasons for why the reactive arene oxide or S-oxide metabolites of TAI do not lead to the inactivation of CYP2C9 are unknown.…”

Biotransformations Leading to Toxic Metabolites

“…In the case of tienilic acid, the electrophilic reactive species is unknown but is believed to be either thiophene sulfoxide, as has been demonstrated for its 3-isomer [78], or a thiophene epoxide (Figure 25.26) [30,79]. In both cases, the electrophilic character of the intermediate is enhanced by the presence of an activating 2-keto group.…”