CP-533,536, (3-{[(4-tert-butyl-benzyl)-(
pyridine-3-sulfonyl)-amino]-methyl}-phenoxy)-
acetic acid (1), an EP2 receptor-selective
prostaglandin E2 agonist, is being developed to aid in the healing of
bone fractures. To support the development of this program, in vitro metabolism of 1 was investigated in human liver microsomes and major recombinant human
cytochrome P450 (P450)
isoforms. 1 was metabolized in vitro by at least three recombinant human P450s:
CYP3A4,
CYP3A5, and
CYP2C8. The turnover of 1 was
NADPH-dependent and was completely inhibited by
ketoconazole and
quercetin in the
CYP3A4/5 and
CYP2C8 incubations, respectively. The major metabolic pathways were caused by oxidation of the tert-butyl moiety to form the omega-hydroxy metabolite (M4), oxidation of the
pyridine moiety, and/or N-dealkylation of the methylphenoxy
acetic acid moiety. The alcohol metabolite M4 was further oxidized to the corresponding
carboxylic acid M3. In addition to these pathways, three unusual metabolites (M22, M23, and M26) resulting from C-demethylation of the tert-butyl group were identified using high-resolution liquid chromatography/tandem mass spectrometry and liquid chromatography/mass spectrometry/NMR. The C-demethylated metabolites were not detected on incubation of
carboxylic acid metabolite M3 with either human liver microsomes or
CYP3A/2C8
isoforms, suggesting that these metabolites were not derived from decarboxylation of M3. A possible mechanism for C-demethylation may involve the oxidation of M4 to form an
aldehyde metabolite (M24), followed by P450-mediated deformylation, to give an unstable
carbon-centered radical and
formic acid. The
carbon-centered radical intermediate then undergoes either
oxygen rebound to form an alcohol metabolite M23 or
hydrogen abstraction leading to an
olefin metabolite M26.