We report on the optimization of 4H-1,2,4-triazole derivatives to increase their activity and selectivity as glycine transporter 1 (GlyT1) inhibitors. Structure-activity relationship exploration resulted in the identification of a 3-[3-ethyl-5-(6-phenylpyridin-3-yl)-4H-1,2,4-triazol-4-yl]-2-methylbenzonitrile (14u) compound with markedly higher selectivity for GlyT1. Physiochemical studies revealed that 14u exists as a stable pair of atropisomers under physiological conditions. We successfully separated the atropisomers to obtain active enantiomer (R)-14u, which displayed favorable pharmacokinetic properties, as well as positive results in the mice Y-maze test.