Synthesis of GABA<sub>A</sub> Receptor Agonists and Evaluation of their α-Subunit Selectivity and Orientation in the GABA Binding Site

Drugs used to treat various disorders target GABA<sub>A</sub> receptors. To develop α subunit selective compounds, we synthesized 5-(4-piperidyl)-3-isoxazolol (4-PIOL) derivatives. The 3-isoxazolol moiety was substituted by 1,3,5-oxadiazol-2-one, 1,3,5-oxadiazol-2-thione, and substituted 1,2,4-triazol-3-ol heterocycles with modifications to the basic piperidine substituent as well as substituents without basic nitrogen. Compounds were screened by [<sup>3</sup>H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA<sub>A</sub> α<sub>i</sub>β<sub>3</sub>γ<sub>2</sub> receptors (<i>i</i> = 1−6). The effects of 5-aminomethyl-3<i>H</i>-[1,3,4]oxadiazol-2-one <b>5d</b> were comparable to GABA for all α subunit isoforms. 5-piperidin-4-yl-3<i>H</i>-[1,3,4]oxadiazol-2-one <b>5a</b> and 5-piperidin-4-yl-3<i>H</i>-[1,3,4]oxadiazol-2-thione <b>6a</b> were weak agonists at α<sub>2</sub>-, α<sub>3</sub>-, and α<sub>5</sub>-containing receptors. When coapplied with GABA, they were antagonistic in α<sub>2</sub>-, α<sub>4</sub>-, and α<sub>6</sub>-containing receptors and potentiated α<sub>3</sub>-containing receptors. <b>6a</b> protected GABA binding site cysteine-substitution mutants α<sub>1</sub>F64C and α<sub>1</sub>S68C from reacting with methanethiosulfonate-ethylsulfonate. <b>6a</b> specifically covalently modified the α<sub>1</sub>R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing α subtype selective GABA mimetic drugs.