Mechanistic insights into the bioactivation of phenacetin to reactive metabolites: A DFT study

Abstract

The knowledge of biochemical mechanism of cancer induction and carcinogenicity by drugs is an essential requisite for drug metabolism and toxicity studies. Metabolic bioactivation of phenacetin leads to the generation of several reactive metabolites (RMs) and intermediates, with varied toxicological consequences. The carcinogenicity and mutagenecity of phenacetin have been known for several years; however, the molecular level details of the formation and reactivity of the RMs behind them are still elusive. Quantum chemical analysis was carried out to identify the critical RMs generated via three different metabolic pathways of phenacetin. DFT-based descriptors were utilized to obtain the electrophilicity parameters of all the RMs involved in the bioactivation pathway. The three metabolic pathways studied are: (i) O-dealkylation, (ii) N-hydroxylation, and (iii) N-deacetylation. It was observed that the O-dealkylation process leading to the formation of acetaminophen is energetically (ΔG = −77.34 kcal/mol) more favorable than N-hydroxylation (−20.13 kcal/mol) and deacetylation (−3.51 kcal/mol) reactions. The activation barrier, calculated using B3LYP/6-311+G(d) basis set and implicit solvent effect, for O-dealkylation (37.55 kcal/mol) was observed to be lower as compared to N-hydroxylation (42.38 kcal/mol) and N-deacetylation (55.63 kcal/mol). The O-ethyl-N-acetyl-p-benzoquinone imine (O–Et-NAPQI) was observed to be the most critical and electrophilic metabolite (global electrophilicity; ω = 19.43 eV), generated via initial N-hydroxylation (Phase I) and subsequent Phase II metabolism. The higher electrophilicty of O–Et-NAPQI (compared to NAPQI; 4.23 eV) accounts for its easy binding with nucleophiles such as macromolecules and DNA nucleotides, and higher reactivity (as compared to other RMs) leading to carcinogenicity. Therefore, the knowledge of the RMs, especially O–Et-NAPQI, involved in the carcinogenicity of phenacetin can be utilized in understanding the relevance of several crucial Phase I and II metabolic reactions.