Discovery of (<i>R</i>)‑<i>N</i>‑Benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide [<b>(<i>R</i>)-AS‑1</b>], a Novel Orally Bioavailable EAAT2 Modulator with Drug-like Properties and Potent Antiseizure Activity <i>In Vivo</i>

(R)-7 [(R)-AS-1] showed broad-spectrum antiseizure activity across in vivo mouse seizure models: maximal electroshock (MES), 6 Hz (32/44 mA), acute pentylenetetrazol (PTZ), and PTZ-kindling. A remarkable separation between antiseizure activity and CNS-related adverse effects was also observed. In vitro studies with primary glia cultures and COS-7 cells expressing the glutamate transporter EAAT2 showed enhancement of glutamate uptake, revealing a stereoselective positive allosteric modulator (PAM) effect, further supported by molecular docking simulations. (R)-7 [(R)-AS-1] was not active in EAAT1 and EAAT3 assays and did not show significant off-target activity, including interactions with targets reported for marketed antiseizure drugs, indicative of a novel and unprecedented mechanism of action. Both in vivo pharmacokinetic and in vitro absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) profiles confirmed the favorable drug-like potential of the compound. Thus, (R)-7 [(R)-AS-1] may be considered as the first-in-class small-molecule PAM of EAAT2 with potential for further preclinical and clinical development in epilepsy and possibly other CNS disorders.(R)-7 [(R)-AS-1] 在体内小鼠惊厥模型中展现出广谱抗惊厥活性，涵盖最大电惊厥（maximal electroshock, MES）、6 Hz（32/44 mA）电刺激、急性戊四氮（pentylenetetrazol, PTZ）以及PTZ点燃模型。该化合物同时在抗惊厥活性与中枢神经系统（central nervous system, CNS）相关不良反应之间呈现出显著的分离效应。针对原代胶质细胞培养物以及表达谷氨酸转运体EAAT2的COS-7细胞开展的体外研究显示，(R)-7 [(R)-AS-1]可增强谷氨酸摄取，揭示出其立体选择性正变构调节剂（positive allosteric modulator, PAM）的作用，该结果进一步得到分子对接模拟的佐证。(R)-7 [(R)-AS-1]在EAAT1与EAAT3检测中未显示活性，且未表现出显著的脱靶活性，包括与已上市抗惊厥药物所报道的靶点的结合，这表明其作用机制新颖且前所未闻。体内药代动力学以及体外吸收、分布、代谢、排泄与毒性（absorption, distribution, metabolism, excretion, toxicity, ADME-Tox）特性均证实了该化合物良好的类药成药性潜力。因此，(R)-7 [(R)-AS-1]可被视为首款靶向EAAT2的小分子正变构调节剂，有望进一步开展癫痫及其他可能的中枢神经系统疾病的临床前与临床开发。