title: Metabolism-dependent mutagenicity of a compound containing a piperazinyl indazole motif: Role of a novel p450-mediated metabolic reaction involving a putative oxaziridine intermediate.
abstract: Compound 1a (6-chloro-5-{3-[4-(1H-indazol-3-yl)-piperazin-1-yl]-propyl}-3,3-dimethyl-1,3-dihydro-indol-2-one) was mutagenic to Salmonella typhimurium TA98 in the presence of rat liver S9 subcellular fraction. The metabolism of 1a in rat liver S9 or microsomes demonstrated that it underwent a P450-mediated N-deindazolation (loss of indazole ring) as a predominant metabolic pathway. To investigate a possible link between metabolism and mutagenicity, a structural analogue 1b (6-chloro-5-{3-[4-(1H-indazol-3-yl)-piperidin-1-yl]-propyl}-3,3-dimethyl-1,3-dihydro-indol-2-one), the cleaved product 2a (6-chloro-3,3-dimethyl-5-(3-piperazin-1-yl-propyl)-1,3-dihydro-indol-2-one), and the core motif 3a (3-piperazinyl indazole) were evaluated in the Ames assay. It was found that 1b was not mutagenic to Salmonella typhimurium TA98 in the absence or presence of a metabolic activating system. In contrast to 1a, 1b did not undergo the metabolic cleavage (loss of indazole ring). Marginal mutagenicity of 2a to TA98 was observed with rat liver S9, whereas 3a was shown to be a promutagen. It was further demonstrated that 1a inactivated P450 3A, the principle enzyme catalyzing the N-deindazolation reaction, in an NADPH-, time-, and concentration-dependent manner. The kinetics of inactivation was characterized by a K(I) of 8.1 microM and k(inact) of 0.114 min(-1). The differences in mutagenicity between 1a and 1b suggest that a chemical bond extending from the 3-position of the indazole to a heteroatom (as part of another cyclic ring) is a prerequisite for the toxicity. The metabolic process leading to the elimination of the indazole from the rest of the molecule apparently plays a key role in causing mutagenicity. It is postulated that the N-deindazolation of 1a proceeds via an oxaziridine intermediate, the formation of which is indirectly inferred from the presence of benzoic acid in microsomal incubations. Benzoic acid is thought to be derived from the hydrolysis of 3-indazolone, an unstable product generated from the oxaziridine. Evidence suggests that the electrophilic oxaziridine intermediate may be responsible for the mutagenicity and inactivation of P450 3A.
authors.affiliation: Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research & Development, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA. hao.chen@pfizer.com
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.forename: Joel
authors.initials: J
authors.lastname: Murray
authors.forename: Brian
authors.initials: B
authors.lastname: Kornberg
authors.forename: Lloyd
authors.initials: L
authors.lastname: Dethloff
authors.forename: David
authors.initials: D
authors.lastname: Rock
authors.forename: Sham
authors.initials: S
authors.lastname: Nikam
authors.forename: Abdul E
authors.initials: AE
authors.lastname: Mutlib
mesh_terms: D000818:Animals; D001388:Aziridines / Q000737:chemistry / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007191:Indazoles / Q000138:chemical synthesis / Q000737:chemistry* / Q000633:toxicity; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D015394:Molecular Structure; D016296:Mutagenesis / Q000235:genetics*; D010084:Oxidation-Reduction; D010879:Piperazines / Q000138:chemical synthesis / Q000737:chemistry* / Q000633:toxicity; D051381:Rats; D012486:Salmonella typhimurium / Q000187:drug effects / Q000235:genetics; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000737:chemistry / Q000378:metabolism
chemical_list: D001388:Aziridines; D007191:Indazoles; D010879:Piperazines; C516748:piperazinyl indazole; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System