Synthesis of GABAA receptor agonists and evaluation of their alpha-subunit selectivity and orientation in the GABA binding site

Drugs used to treat various disorders target GABA A receptors. To develop Alpha 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 [(3)H]muscimol binding and in patch-clamp experiments with heterologously expressed GABA A Alpha ibeta 3gamma 2 receptors (i = 1-6). The effects of 5-aminomethyl-3 H-[1,3,4]oxadiazol-2-one 5d were comparable to GABA for all Alpha subunit isoforms. 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-one 5a and 5-piperidin-4-yl-3 H-[1,3,4]oxadiazol-2-thione 6a were weak agonists at Alpha 2-, Alpha 3-, and Alpha 5-containing receptors. When coapplied with GABA, they were antagonistic in Alpha 2-, Alpha 4-, and Alpha 6-containing receptors and potentiated Alpha 3-containing receptors. 6a protected GABA binding site cysteine-substitution mutants Alpha 1F64C and Alpha 1S68C from reacting with methanethiosulfonate-ethylsulfonate. 6a specifically covalently modified the Alpha 1R66C thiol, in the GABA binding site, through its oxadiazolethione sulfur. These results demonstrate the feasibility of synthesizing Alpha subtype selective GABA mimetic drugs.