Dual competitive mechanisms for CYP3A4-catalyzed gefitinib oxidative defluorination: Steric-conformational coupling and enzymatic/non-enzymatic mechanistic divergence.

Gefitinib is a widely used fluorinated anticancer drug primarily metabolized by cytochrome P450 3A4 (CYP3A4). However, its oxidative defluorination can generate toxic quinone imine metabolites. The mechanistic origin of distinct defluorination pathways in enzymatic vs. non-enzymatic systems remains incompletely understood. Herein, we combine static DFT calculations with dynamic conformational analysis to elucidate the competitive oxidation and defluorination pathways of gefitinib. Strong hydrogen bonding stabilizes the initial hydrogen atom abstraction (HAT) pathway in both systems. However, the subsequent hydroxyl rebound-favorable in free solution-is prohibited within the enzyme active site due to stringent substrate conformational constraints. Consequently, enzymatic defluorination shifts to the energetically more favorable electrophilic attack pathway, followed by efficient 1,2‑fluorine migration. The consistency between static transition-state structures and dynamic conformational snapshots validates this mechanism. Notably, artificial catalysts lacking spatial confinement exhibit higher defluorination efficiency. This work reveals that biological spatial confinement reshapes reaction pathways and modulates activation barriers, thereby deepening the mechanistic understanding of fluorinated drug metabolism and supporting rational design for safer pharmaceuticals and environmentally benign detoxification strategies.