Bioactivation of a dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide) scaffold to a putative p-chlorophenyl isocyanate in rat liver microsomes and in vivo in rats.
Compound I (4,5-dihydropyrazole-1,5-dicarboxylic acid-1-[(4-chlorophenyl)-amide] 5-[(2-oxo-2 H-[1,3']bipyridinyl-6'-yl)-amide] was found to undergo metabolic activation in rat liver microsomes in the presence of NADPH. A reactive intermediate, postulated to be p-chlorophenyl isocyanate (CPIC), was trapped by GSH in vitro and characterized by liquid chromatography tandem mass spectrometry (LC/MS/MS). Subsequently, the structure of the GSH conjugate was confirmed by a comparison with a synthetic standard. The GSH conjugate was also found in the bile of rats that received an oral dose (10 mg/kg) of compound I. Further analyses of rat bile and urine using online electrochemical derivatization coupled to LC/MS demonstrated the presence of p-chlorophenyl aniline (CPA), a hydrolytic product of the intermediate isocyanate. This provided further evidence for the potential existence of CPIC. Approximately 7% of the dose was accounted by the products of CPIC, which included the GSH conjugate and CPA excreted in bile and urine. Multiple rat cytochrome P450 enzymes, including P450 1A, P450 2C, and P450 3A, appeared to be responsible for the activation of compound I to CPIC. The activation kinetics of compound I to CPIC in male rat liver microsomes exhibited a biphasic profile, indicative of at least two contributing P450 enzymes. One enzyme showed a small value of K m at 42 microM and a low V max of 66 pmol min (-1) mg (-1), while the other exhibited a large value of K m at 148 microM and a high V max of 1200 pmol min (-1) mg (-1). The formation of a putative CPIC intermediate, a carbamoylating species known to be capable of covalent binding to macromolecules, suggests a potential liability associated with the compound, particularly the dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide) scaffold, which appears to be responsible for the generation of CPIC. The mechanism of bioactivation to the putative CPIC is postulated to involve an initial P450-mediated hydroxylation of the pyrazoline at the 3 position followed by subsequent decomposition to CPIC. This mechanistic insight into the bioactivation allowed for the development of a rational structural modification strategy to mitigate or minimize the reactive metabolite formation. One of the approaches included the introduction of a metabolically stable substituent with electron-donating character at the 3 position of pyrazoline to block CPIC formation.
Chen
Hao
H
Departments of Pharmacokinetics, Dynamics, and Metabolism, and Chemistry, Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA. chenh10@wyeth.com
Zhang
Yanhua
Y
Edmunds
Jeremy
J
Bigge
Christopher
C
Mutlib
Abdul
A
D000818:Animals; D002722:Chlorobenzenes / Q000737:chemistry / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004789:Enzyme Activation; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D017953:Isocyanates / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000187:drug effects* / Q000378:metabolism; D015394:Molecular Structure; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats
D002722:Chlorobenzenes; D017953:Isocyanates; D011720:Pyrazoles; C530175:dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide); D003577:Cytochrome P-450 Enzyme System; D005978:Glutathione; C452229:chlorophenyl isocyanate