A free radical mechanism of prostaglandin synthase-dependent aminopyrine demethylation.

The mechanism of prostaglandin synthase-dependent N-dealkylation has been investigated using an enzyme preparation derived from ram seminal vesicles. Incubation of an N-alkyl substrate, aminopyrine, with enzyme and arachidonic acid, 15-hydroperoxyarachidonic acid, or tert-butyl hydroperoxide resulted in the formation of the transient aminopyrine free radical species. Formation of this radical species, which was detected by electron paramagnetic resonance spectroscopy and/or absorbance at 580 nm, was maximal approximately 30 s following initiation of the reaction and declined thereafter. Free radical formation corresponded closely with formaldehyde formation in this system, in terms of dependence upon substrate and cofactor concentration, as well as in terms of time course. Both aminopyrine free radical and formaldehyde formation were inhibited by indomethacin and flufenamic acid, inhibitors of prostaglandin synthase. The results suggest that the aminopyrine free radical is an intermediate in the prostaglandin synthase-dependent aminopyrine N-demethylase pathway. The aminopyrine free radical electron paramagnetic resonance spectrum revealed that this species is a one-electron oxidized cation radical of the parent compound. A reaction mechanism has been proposed in which aminopyrine undergoes two sequential one-electron oxidations to an iminium cation, which is then hydrolyzed to the demethylated amine and formaldehyde. Accordingly, the oxygen atom of the aldehyde product is derived from neither molecular nor hydroperoxide oxygen, but from water.