5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic γ-aminobutyric acid receptors by binding to the 4′-ethynyl-4-n-propylbicycloorthobenzoate site

Abstract

Acyclic noncompetitive antagonists of ionotropic γ-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order CC(CH2)2COOH>CC(CH2)2COOCH3>CCH>Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less potent. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 μM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.