Discovery of fluoxakalner, a novel and preferential Kv7.2/7.3 channel opener for antinociception in mice.

Activating neuronal M-current (Kv7.2/7.3) channels constitutes a promising therapeutic strategy for antinociception and epilepsy through hyperpolarizing membrane potential and reducing neuronal hyperexcitability. However, retigabine (RTG), the first-in-class Kv7 opener, was withdrawn from clinical use due to its oxidative metabolites that cause tissue discoloration and toxicity. To mitigate this clinical risk, we synthesized the N-{4-[(4-fluorobenzyl)oxy]-2,6-dimethylphenyl}-2-phenylacetamide, designated fluoxakalner, by modifying the primary amine moiety of RTG through introducing two methyl groups onto the benzene ring and replacing the -NH- linkage with O bridge. Whole-cell patch clamp recordings demonstrated that fluoxakalner is a potent Kv7.2/7.3 opener with an EC50 of 0.36 ± 0.06 μM and 38.45 ± 5.80 mV leftward shift (ΔV1/2) in channel activation. Among other channel subtypes, fluoxakalner exclusively accelerates the activation kinetics of the Kv7.2/7.3 channel, reducing the activation time from 99.38 ± 24.68 ms to 54.19 ± 14.28 ms. Fluoxakalner exhibits a preference for Kv7.2/7.3 channels over other Kv7 subtypes. Fluoxakalner at 5 mg/kg exerted antinociceptive effects without sedative impact on locomotion and without significant inhibition of cardiac Kv7.1, hERG, or Nav1.5 channels. Notably, RTG undergoes N-acetylation to form NAMR, followed by oxidation to quinone diimine intermediates and RTG/NAMR-derived dimers associated with ocular pigmentation and skin discoloration. In contrast, metabolism in rat liver assays revealed fluoxakalner avoids the formation of both ortho- and para-quinone diimine species linked to RTG toxicity. Taken together, these findings identify fluoxakalner as a novel Kv7.2/7.3 opener and a promising lead compound for further development of effective and safe antinociceptive agents.