title: Characterization of cytochrome P450-mediated bioactivation of a compound containing the chemical scaffold, 4,5-dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide), to a chemically reactive p-chlorophenyl isocyanate intermediate in human liver microsomes.
abstract: Compound A (Cmpd A) was previously reported to form p-chlorophenyl isocyanate (CPIC), which was trapped by GSH to yield S- (N- [p-chlorophenyl] carbamoyl) glutathione adduct (SCPG) in the presence of human liver microsomes. In this study, P450 3A4 and 2C9 were demonstrated to be the enzymes mediating the activation of Cmpd A to CPIC in human liver microsomes based on inhibitory and correlation studies. Enzyme kinetics studies indicated that P450 3A4 was the primary enzyme involved in the activation of Cmpd A. In silico P450 3A4 active site docking of Cmpd A exhibited a low energy pose that orientated the pyrazole ring proximate to the heme iron atom, in which the distance between the C-3 and potential activated oxygen species was shown to be 3.4 A. Quantum molecular calculations showed that the electron density on C-3 was relatively higher than those on C-4 and C-5. These measurements suggested that the C-3 of Cmpd A was the preferred site of oxidation and hence predisposed Cmpd A in forming CPIC as previously proposed. The in silico prediction was corroborated by studies with the C-3 substituted analogue (methyl at C-3), which showed minimal conversion to CPIC in human liver microsomes. These results demonstrated a pivotal role for P450 3A4 in bioactivating Cmpd A by oxidizing at C-3 of the pyrazoline, hence facilitating the CPIC formation. Evidence of the bioactivation to CPIC in vivo was obtained by liquid chromatography-mass spectrometry (LC/MS) analysis of urine samples from human subjects administered a structural analogue of Cmpd A. The presence of S-(N-[p-chlorophenyl] carbamoyl) N-acetyl l-cysteine (SCPAC) as well as p-chlorophenyl aniline (CPA) was unequivocally demonstrated in the urine samples. The chemical scaffold, 4,5-dihydropyrazole-1-carboxylic acid-[(4-chlorophenyl)-amide], was demonstrated to possess potential metabolic liability in forming a reactive intermediate, CPIC, in humans. Bioactivation to CPIC may cause undesirable side effects through its reactivity and subsequent conversion to CPA, an established rodent carcinogen.
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA. chenh10@wyeth.com
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Michael
authors.initials: M
authors.lastname: Zientek
authors.forename: Mehran
authors.initials: M
authors.lastname: Jalaie
authors.forename: Yanhua
authors.initials: Y
authors.lastname: Zhang
authors.forename: Christopher
authors.initials: C
authors.lastname: Bigge
authors.forename: Abdul
authors.initials: A
authors.lastname: Mutlib
mesh_terms: D020134:Catalytic Domain; D002722:Chlorobenzenes / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D003198:Computer Simulation; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D017953:Isocyanates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism*; D011720:Pyrazoles / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D011753:Pyrones / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D011789:Quantum Theory; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: C545880:4,5-dihydropyrazole-1,5-dicarboxylic acid 1-((4-chlorophenyl)-amide) 5-(2-oxo-2H-(1,3')bipyridinyl-6'-yl)-amide; D002722:Chlorobenzenes; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D017953:Isocyanates; D011720:Pyrazoles; D011753:Pyrones; C530175:dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide); D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C452229:chlorophenyl isocyanate