title: Formation of cyclopropanone during cytochrome P450-catalyzed N-dealkylation of a cyclopropylamine.
abstract: The role of single electron transfer (SET) in P450-catalyzed N-dealkylation reactions has been studied using the probe substrates N-cyclopropyl-N-methylaniline (2a) and N-(1'-methylcyclopropyl)-N-methylaniline (2b). In earlier work, we showed that SET oxidation of 2a by horseadish peroxidase leads exclusively to products arising via fragmentation of the cyclopropane ring [Shaffer, C. L.; Morton, M. D.; Hanzlik, R. P. J. Am. Chem. Soc. 2001, 123, 8502-8508]. In the present study, we found that liver microsomes from phenobarbital pretreated rats (which contain CYP2B1 as the predominant isozyme) oxidize [1'-(13)C, 1'-(14)C]-2a efficiently (80% consumption in 90 min). Disappearance of 2a follows first-order kinetics throughout, indicating a lack of P450 inactivation by 2a. HPLC examination of incubation mixtures revealed three UV-absorbing metabolites: N-methylaniline (4), N-cyclopropylaniline (6a), and a metabolite (M1) tentatively identified as p-hydroxy-2a, in a 2:5:2 mole ratio, respectively. 2,4-Dinitrophenylhydrazine trapping indicated formation of formaldehyde equimolar with 6a; 3-hydroxypropionaldehyde and acrolein were not detected. Examination of incubations of 2a by (13)C NMR revealed four (13)C-enriched signals, three of which were identified by comparison to authentic standards as N-cyclopropylaniline (6a, 33.6 ppm), cyclopropanone hydrate (11, 79.2 ppm), and propionic acid (12, 179.9 ppm); the fourth signal (42.2 ppm) was tentatively determined to be p-hydroxy-2a. Incubation of 2a with purified reconstituted CYP2B1 also afforded 4, 6a, and M1 in a 2:5:2 mole ratio (by HPLC), indicating that all metabolites are formed at a single active site. Incubation of 2b with PB microsomes resulted in p-hydroxylation and N-demethylation only; no loss or ring-opening of the cyclopropyl group occurred. These results effectively rule out the participation of a SET mechanism in the P450-catalyzed N-dealkylation of cyclopropylamines 2a and 2b, and argue strongly for the N-dealkylation of 2a via a carbinolamine intermediate formed by a conventional C-hydroxylation mechanism.
authors.affiliation: Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-7582, USA.
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Shaffer
authors.forename: Shawn
authors.initials: S
authors.lastname: Harriman
authors.forename: Yakov M
authors.initials: YM
authors.lastname: Koen
authors.forename: Robert P
authors.initials: RP
authors.lastname: Hanzlik
mesh_terms: D000478:Alkylation; D000814:Aniline Compounds / Q000378:metabolism; D003521:Cyclopropanes / Q000378:metabolism*; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D003521:Cyclopropanes; C067351:cyclopropylamine; D003577:Cytochrome P-450 Enzyme System; D019362:Cytochrome P-450 CYP2B1