Computational insights into the diverse mechanisms in cytochrome P450 2D6-mediated primaquine metabolism

Primaquine (PQ) is a representative 8-aminoquinoline antimalarial drug whose clinical use is limited by hemotoxic side effects, particularly those associated with the toxic metabolite 5-hydroxyprimaquine. In this study, human CYP2D6 was employed as a metabolic enzyme to investigate the metabolic mechanism of PQ by multiple computational methods. The hydrophobic and π-π interactions between PQ and residues Phe120 and Phe483 are the key factors for its strong substrate-binding affinity and stability. Electrostatic potential analysis further suggests that the C5 position is electronically predisposed to electrophilic attack, which may contribute to the observed regioselectivity in CYP2D6-mediated oxidation. Four feasible hydroxylation pathways are identified through QM and kinetic calculations, among which the NIH shift and epoxidation pathways are the dominant and competing routes. In both cases, the shared rate-limiting electrophilic addition at C5 adopts the face-on orientation with an energy barrier of 24.8 kcal mol^-1, highlighting the key role of the local electronic environment. Introducing exocyclic electron-withdrawing substituents to modulate the C5 electronic environment may represent a potential strategy for reducing the formation of the hemotoxic metabolite 5-hydroxyprimaquine. Collectively, these insights will advance the understanding of CYP2D6-mediated PQ metabolism and offer a theoretical foundation for the rational design of safer PQ derivatives with reduced hemotoxic potential.