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.

Cyclopropylamine inactivation of cytochromes P450: Role of metabolic intermediate complexes

“…This mechanism is supported by direct observations of ring-opened products in chemical model systems [2][3][4][5], and in the oxidation of cyclopropylanilines by peroxidases [6][7][8]. While the formation of cinnamaldehyde as a metabolite of an N-(2-phenylcyclopropyl)amine has been reported, direct evidence for the formation of ring-opened metabolites of a simple N-cyclopropylamine has not been demonstrated for a P450 system.…”

“…Liver microsomes were prepared from untreated and phenobarbital-treated male Sprague-Dawley rats (UT-and PB-microsomes, respectively) following literature protocols [7,24]. Isolation of CYP2B1/2 from PB-microsomes was performed as previously reported [25].…”

“…CYP2C11 and CYP2B1/2 (0.5-1.0 nmol) were reconstituted with reductase (1.0-2.0 nmol) and DLPC (30 µg) for 40 min at 0 ºC [7]. CYP2E1 (1.0 nmol) was reconstituted with reductase (3.0 nmol), cytochrome b 5 (5.0 nmol) and DLPC (30 µg) for 40 min at 0 ºC [29].…”

“…While the formation of cinnamaldehyde as a metabolite of an N-(2-phenylcyclopropyl)amine has been reported, direct evidence for the formation of ring-opened metabolites of a simple N-cyclopropylamine has not been demonstrated for a P450 system. On the contrary, P450 systems have shown only formation of cyclopropanone hydrate from the N-dealklylation of cyclopropylanilines [7]. NCyclopropylanilines, however, do not inactivate P450, which leaves open the possibility that they are oxidized differently from aliphatic cyclopropylamines such as 1.…”

A DFT Study of the Heme Role in the N-Demethylation of Theophylline Mediated by Compound I of Cytochrome P450

“…8,9) The HAT mechanism is supported by the identification of carbinolamines in the N-dealkylation of particular substrates and by the fact that their oxygen atom originates from molecular oxygen. [10][11][12] On the other hand the SET mechanism, supported by the majority of authors 8,13) may also involve the formation of a carbinolamine resulting from the nucleophilic addition of the hydroxo ligand of Fe III OH onto the iminium intermediate within the active site of the enzyme.…”