The Synthesis and Evaluation of Fluoro-, Trifluoromethyl-, and Iodomuscimols as GABA Agonists.

Halogenated analogues of the neurotoxic alkaloid muscimol were prepared with fluorine, iodine or trifluoromethyl at the 4 position of the isoxazole ring system. These compounds were investigated as agonists for GABAA receptors. Only the C-4 fluorine-containing analogue proved to be an active compound in these assays. The fluoro analogue was less active than muscimol, however it showed differential activity between synaptic (α1 ß2 γ2 ) and extrasynaptic (α4 ß2 γ) GABAA receptors, having a similar potency to the neurotransmitter GABA for the extrasynaptic (α4 ß2 γ) receptor.

Synthesis and biological evaluation of 4-(aminomethyl)-1-hydroxypyrazole analogues of muscimol as γ-aminobutyric acid(a) receptor agonists.

A series of bioisosteric 4-(aminomethyl)-1-hydroxypyrazole (4-AHP) analogues of muscimol, a GABA(A) receptor agonist, has been synthesized and pharmacologically characterized at native and selected recombinant GABA(A) receptors. The unsubstituted 4-AHP analogue (2a) (EC(50) 19 µM, R(max) 69%) was a moderately potent agonist at human α(1)ß(2)γ(2) GABA(A) receptors, and in SAR studies substitutions in the 3- and/or 5-position were found to be detrimental to binding affinities. Ligand-receptor docking in an α(1)ß(2)γ(2) GABA(A) receptor homology model along with the obtained SAR indicate that 2a and muscimol share a common binding mode, which deviates from the binding mode of the structurally related antagonist series based on 4-(piperidin-4-yl)-1-hydroxypyrazole (4-PHP, 1). Selectivity for α(1)ß(2)γ(2) over ρ(1) GABA(A) receptors was observed for the 5-chloro, 5-bromo, and 5-methyl substituted analogues of 2a illustrating that even small differences in structure can give rise to subtype selectivity.

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.

A novel route to 5-substituted 3-isoxazolols. Cyclization of N, O-DiBoc beta-keto hydroxamic acids synthesized via acyl Meldrum's acids.

3-Isoxazolols are most often synthesized from a beta-keto ester and hydroxylamine. This cyclization typically gives rise to a major byproduct, the corresponding 5-isoxazolone. We have found that N, O-diBoc-protected beta-keto hydroxamic acids can be synthesized and cyclized to 5-substituted 3-isoxazolols without formation of any byproduct. We present a novel and versatile three-step procedure in which carboxylic acid derivatives are converted into acyl Meldrum's acids which, upon aminolysis with N, O-bis(tert-butoxycarbonyl)hydroxylamine, lead to the N, O-diBoc-protected beta-keto hydroxamic acids. These hydroxamic acid analogues were then, upon treatment with hydrochloric acid, cyclized to the corresponding 5-substituted 3-isoxazolols.

Condensation of muscimol or thiomuscimol with aminopyridazines yields GABA-A antagonists.

Ten analogs of muscimol and thiomuscimol in which the amino function was delocalized in an amidinic system were prepared by N2 alkylation of 6-aryl-3-aminopyridazines with (chloromethyl)isoxazole or (chloromethyl)isothiazole derivatives. These muscimol and thiomuscimol derivatives show potent binding properties for GABA-A receptors (they displace [3H]GABA and [3H]gabazine) and provoke convulsions after iv injections. They fit well with the model pharmacophore proposed by our group for the GABA-A antagonists and show similar structure-activity profiles to that of the pyridazinyl-GABAs.