Discovery of Brain-Penetrative Negative AllostericModulators of NMDA Receptors Using FEP-Guided Structure Optimizationand Membrane Permeability Prediction

<i>N</i>-Methyl-d-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors in the central nervous system (CNS), have garnered attention for their role in brain disorders. Specifically, GluN2A-containing NMDA receptors have emerged as a potential therapeutic target for the treatment of depressive disorders and epilepsy. However, the development of GluN2A-containing NMDA receptor-selective antagonists, represented by <i>N</i>-(4-(2-benzoylhydrazine-1-carbonyl)benzyl)-3-chloro-4-fluorobenzenesulfonamide (TCN-201) and its derivatives, faces a significant challenge due to their limited ability to penetrate the blood–brain barrier (BBB), hampering their <i>in vivo</i> characterization and further advancement. In this study, we reported a series of 2-((5-(phemylamino)-1,3,4-thiadiazol-2-yl)thio)-<i>N</i>-(cyclohexylmethyl)acetamide derivatives, achieved through a structure-guided optimization strategy using free energy perturbation (FEP) and BBB permeability estimation. Through systematic exploration of various phenyl substitutions, compound <b>1f</b> emerged as a standout compound, demonstrating substantially enhanced inhibitory activity compared with the lead compound TCN-213. Compound <b>1f</b> not only displayed satisfactory BBB permeability but also showed antidepressant-like potency in the hydrocortisone-induced zebrafish depression-like model. All results position it as a promising candidate for developing innovative therapeutics for NMDA receptor-related disorders.