Theoretical Study of N-Demethylation of Substituted N,N-Dimethylanilines by Cytochrome P450:  The Mechanistic Significance of Kinetic Isotope Effect Profiles

Abstract

The mechanism of N-demethylation of N,N-dimethylanilines (DMAs) by cytochrome P450, a highly debated topic in mechanistic bioinorganic chemistry (Karki, S. B.; Dinnocenczo, J. P.; Jones, J. P.; Korzekwa, K. R. J. Am. Chem. Soc. 1995, 117, 3657), is studied here using DFT calculations of the reactions of the active species of the enzyme, Compound I (Cpd I), with four para-(H, Cl, CN, NO2) substituted DMAs. The calculations resolve mechanistic controversies, offer a consistent mechanistic view, and reveal the following features: (a) the reaction pathways involve C-H hydroxylation by Cpd I followed by a nonenzymatic carbinolamine decomposition. (b) C-H hydroxylation is initiated by a hydrogen atom transfer (HAT) step that possesses a "polar" character. As such, the HAT energy barriers correlate with the energy level of the HOMO of the DMAs. (c) The series exhibits a switch from spin-selective reactivity for DMA and p-Cl-DMA to two-state reactivity, with low- and high-spin states, for p-CN-DMA and p-NO2-DMA. (d) The computed kinetic isotope effect profiles (KIEPs) for these scenarios match the experimentally determined KIEPs. Theory further shows that the KIEs and TS structures vary in a manner predicted by the Melander-Westheimer postulate: as the substituent becomes more electron withdrawing, the TS is shifted to a later position along the H-transfer coordinate and the corresponding KIEs increases. (e) The generated carbinolaniline can readily dissociate from the heme and decomposes in a nonenzymatic environment, which involves water assisted proton shift.

Recent density functional theory model calculations of drug metabolism by cytochrome P450

“…It is seen from Table 1 that the calculated [102] KIE LS correlate nicely with the experimental values, increasing with the electronwithdrawing ability of the p-X substituents, while the KIE HS are almost constant, irrespective of the substituent variation. Fig.…”

“…A theoretical study by our group [102] on a series of p-X-DMAs which were used in experimental studies also supported the HAT mechanism and explained the experimental observations that had been considered to be typically associated with the SET mechanism on the basis of HAT process. This DFT study resolves the HAT-SET controversy and provides a consistent mechanistic view on the mechanism of the N-dealkylation reaction of amines.…”

“…The activation energy for C H hydroxylation of p-X-DMAs increases as the C H bond undergoes more stretching in the TS H structures. Table 1 shows the calculated [102] KIEs for the C H hydroxylation of the p-(H, Cl, CN, NO 2 )-DMAs on the HS and LS state, alongside the experimental [92,97] determined KIE data. The low KIE values reflect the asymmetry of the transition states along the H-transfer coordinate, C· · ·H· · ·O (Fig.…”

Quantum-Mechanical Studies of Reactions Performed by Cytochrome P450 Enzymes

“…If the reactive carbon atom is not in an alcohol group, the reorganization results in an O-or N-dealkylation ( Figure 1). 71,74,76 Consequently, from a theoretical perspective, this reaction is identical to the aliphatic hydroxylation, described in the previous section, i.e. with hydrogen-abstraction and rebound steps, of which the first is rate limiting.…”

“…with hydrogen-abstraction and rebound steps, of which the first is rate limiting. Because of the importance of understanding these reaction types in drug degradation, a number of theoretical studies have been performed, in several cases including the non-enzymatic reaction, 62,71,72,[74][75][76][77] as are summarized in Figure 5.…”