title: Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.
abstract: N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more electrophilic sulfinic acid species that is trapped intramolecularly by the pendant alcohol motif in compound 1.
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Heather
authors.initials: H
authors.lastname: Eng
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Raman
authors.initials: R
authors.lastname: Sharma
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Angela
authors.initials: A
authors.lastname: Wolford
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Li
authors.initials: L
authors.lastname: Di
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Roger B
authors.initials: RB
authors.lastname: Ruggeri
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Leonard
authors.initials: L
authors.lastname: Buckbinder
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Edward L
authors.initials: EL
authors.lastname: Conn
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts.
authors.forename: Deepak K
authors.initials: DK
authors.lastname: Dalvie
authors.affiliation: Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., Groton, Connecticut (H.E., R.S., A.W., L.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department, Pfizer Inc., La Jolla, California (D.K.D.); Pharmacokinetics, Pharmacodynamics, and Metabolism Department (A.S.K.), Worldwide Medicinal Chemistry (E.L.C., R.B.R.), and Cardiovascular and Metabolic Research Unit (L.B.), Pfizer Inc., Cambridge, Massachusetts amit.kalgutkar@pfizer.com.
authors.forename: Amit S
authors.initials: AS
authors.lastname: Kalgutkar
mesh_terms: D061605:Administration, Intravenous; D000818:Animals; D001711:Biotransformation; D004285:Dogs; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000097:blood / Q000493:pharmacokinetics*; D006207:Half-Life; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008297:Male; D008657:Metabolic Clearance Rate; D008862:Microsomes, Liver / Q000201:enzymology; D008954:Models, Biological; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009195:Peroxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017208:Rats, Wistar; D013045:Species Specificity; D013889:Thiouracil / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000097:blood / Q000493:pharmacokinetics*
chemical_list: D004791:Enzyme Inhibitors; D013889:Thiouracil; D009195:Peroxidase; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)