Novel NQO1 substrates bearing two nitrogen redox centers: Design, synthesis, molecular dynamics simulations, and...

By analyzing the crystal structure of NQO1, an additional binding region for the ligand was discovered. In this study, a series of derivatives with a novel skeleton bearing two nitrogen redox centers were designed by introducing amines or hydrazines to fit with the novel binding region of NQO1. Compound 24 with a (4-fluorophenyl)hydrazine substituent was identified as the most efficient substrate for NQO1 with the reduction rate and catalytic efficiency of 1972 ± 82 μmol NADPH/min/μmol NQO1 and 6.4 ± 0.4 × 10⁶ M^-1s^-1, respectively. Molecular dynamics (MD) simulation revealed that the distances between the nitrogen atom of the redox centers and the key Tyr128 and Tyr126 residues were 3.5 Å (N₁-Tyr128) and 3.4 Å (N₂-Tyr126), respectively. Compound 24 (IC₅₀/A549 = 0.69 ± 0.09 μM) showed potent antitumor activity against A549 cells both in vitro and in vivo through ROS generation via NQO1-mediated redox cycling, leading to a promising NQO1-targeting antitumor candidate.