A Baeyer-Villiger oxidation specifically catalyzed by human flavin-containing monooxygenase 5.
10-((4-Hydroxypiperidin-1-yl)methyl)chromeno[4,3,2-de]phthalazin-3(2H)-one (E7016), an inhibitor of poly(ADP-ribose) polymerase, is being developed for anticancer therapy. One of the major metabolites identified in preclinical animal studies was the product of an apparent oxidation and ring opening of the 4-hydroxypiperidine. In vitro, this oxidized metabolite could not be generated by incubating E7016 with animal or human liver microsomes. Further studies revealed the formation of this unique metabolite in hepatocytes. In a NAD(P)(+)-dependent manner, this metabolite was also generated by liver S9 fractions and recombinant human flavin-containing monooxygenase (FMO) 5 that was fortified with liver cytosol fractions. In animal and human liver S9, this metabolic pathway could be inhibited by 4-methylpyrazole, bis-p-nitrophenylphosphate (BNPP), or a brief heat treatment at 50°C. Based on these results, the overall metabolic pathway was believed to involve a two-step oxidation process: dehydrogenation of the secondary alcohol in liver cytosol followed by an FMO5-mediated Baeyer-Villiger oxidation in liver microsomes. The two oxidation steps were coupled via regeneration of NAD(P)(+) and NAD(P)H. To further confirm this mechanism, the proposed ketone intermediate was independently synthesized. In an NAD(P)H-dependent manner, the synthetic ketone intermediate was metabolized to the same ring-opened metabolite in animal and human liver microsomes. This metabolic reaction was also inhibited by BNPP or a brief heat treatment at 50°C. Methimazole, the substrate/inhibitor of FMO1 and FMO3, did not inhibit this reaction. The specificity of FMO5 toward catalyzing this Baeyer-Villiger oxidation was further demonstrated by incubating the synthetic ketone intermediate in recombinant enzymes.
Lai
W George
WG
Drug Metabolism and Pharmacokinetics-Andover, Biopharmaceutical Assessments, Eisai Inc., Andover, Massachusetts 01810-2441, USA. george_lai@eisai.com
Farah
Nadia
N
Moniz
George A
GA
Wong
Y Nancy
YN
D000818:Animals; D001578:Benzopyrans / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D055162:Biocatalysis; D004285:Dogs; D005260:Female; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000378:metabolism*; D010793:Phthalazines / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D051381:Rats
D001578:Benzopyrans; D010793:Phthalazines; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); C559134:10-((4-hydroxypiperidin-1-yl)methyl)chromeno(4,3,2-de)phthalazin-3(2H)-one