Design, synthesis, and biological evaluation of novel Nav1.2 inhibitors as antiepileptic agents.
Abstract
Voltage-gated sodium channel Nav1.2 is a validated target for anticonvulsant therapy, yet Nav1.2 inhibitors remain scarce. To explore the unexplored N-4 position of the [1,2,4]triazolo[1,5-a]pyrimidine scaffold, we designed and synthesized two series of novel derivatives bearing diverse substituents at this position. Their Nav1.2 inhibitory activities were evaluated using FLIPR membrane potential and whole-cell patch-clamp assays, while their anticonvulsant efficacy and neurotoxicity were assessed in MES/ sc-PTZ seizure models and rotarod test, respectively. Structure-activity relationship (SAR) analysis revealed that hydrophobic volume complementarity, rather than aromaticity, is the primary driver of Nav1.2 potency. Compound 3b, bearing a simple n-butyl group at N-4, emerged as the most promising lead, exhibiting moderate Nav1.2 inhibition (88.9% at 30 μM; FLIPR IC50 = 11.55 μM; patch-clamp IC50 = 7.62 μM for the inactivated state) and exceptional in vivo efficacy (ED50 = 9.17 mg/kg, TD50 > 813.40 mg/kg, PI >88.70) in the sc-PTZ model. Compound 3y also demonstrated moderate Nav1.2 inhibition (69.8%, IC50 = 12.19 μM) and potent anticonvulsant activity (ED50 = 13.7 mg/kg, PI >23.1). Patch-clamp electrophysiology confirmed that 3b and 3y inhibit the inactivated state of Nav1.2 (IC50 = 7.62 μM and 8.31 μM, respectively) with negligible effect on the resting state. Molecular docking and MD simulations revealed that 3b binds within the Nav1.2 pore via hydrogen bonds, π-π stacking, and hydrophobic contacts. Tissue distribution studies demonstrated efficient blood-brain barrier penetration (brain-to-plasma ratio of 1.65 at 1 h post-dose). Although 3b showed limited stability in human and rat liver microsomes (T1/2 = 12.20 and 10.65 min, respectively), its potent in vivo efficacy and exceptionally high protective index support its continued optimization and preclinical development as a promising candidate for epilepsy therapy.
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