Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

Abstract

Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of 18 O from H 2 18O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]-benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979.

Metabolism of Strained Rings: Glutathione S-transferase–Catalyzed Formation of a Glutathione-Conjugated Spiro-azetidine without Prior Bioactivation

“…However, an important conclusion is also that the ring strain of the azetidine is not sufficient to promote spontaneous reactions with nucleophiles, e.g., hydrolysis or GSH adduction. The analogous situation has been observed for the oxetanyl moiety that undergoes ring-opening hydration only in the presence of microsomal epoxide hydrolase (Li et al, 2016). Thus, although the GSH conjugate was indeed formed, this finding does not suggest a general reactivity of this spiro moiety toward biomacromolecules.…”

“…The remaining three metabolites, the formation of which was not inhibited by 1-ABT, were subsequently characterized as an N-glucuronide (M11) and an N-oxide (M13) of AZD1979, with the biotransformation occurring on the spiro-ring system (data not shown). Full characterization of the third metabolite revealed its formation via microsomal epoxide hydrolase to the oxetane ring-opened diol (M1) (Li et al, 2016).…”

“…1254 Li et al solution can also be explained by the absence of either the protonated azetidiniumyl or the GS 2 species. Our previous studies (Li et al, 2016;Toselli et al, 2017) revealed that the oxetanyl rings in the spirooxetanylazetidinyl moiety in AZD1979 and its oxetane-containing analogs also undergo an unusual non-P450-catalyzed reaction with the formation of oxetanyl ring-opened diol mediated by microsomal epoxide hydrolase. All of these findings highlighted the potential reactivity of high-energy, ring-strained-containing xenobiotics that may be subject to unexpected metabolic reactions.…”

“…1) contains a spirooxetanylazetidinyl moiety that confers favorable physicochemical and pharmacokinetic properties in comparison with other analogs with simple 4-, 5-, or 6-membered ring systems (Johansson et al, 2016). Previous studies from this laboratory revealed a novel metabolic pathway on the spiro-ring system: the microsomal epoxide hydrolase catalyzed hydrolysis of the spiro-oxetanyl ring of AZD1979, forming the corresponding ring-opened diol product (Li et al, 2016). In the present study, we investigated an additional unusual non-cytochrome P450 (non-P450)-dependent metabolic pathway: glutathione (GSH) conjugation of the parent with the modification located again on the spiro-ring system.…”

Oxetane Substrates of Human Microsomal Epoxide Hydrolase

“…1) (Johansson et al, 2016). In subsequent studies using selective inhibitors in human liver fractions, hydrolytic ring opening of the spiro-oxetanyl ring system in this compound was observed and found to be a novel reaction catalyzed by the human microsomal epoxide hydrolase (mEH) (Li et al, 2016).…”