U-93631 causes rapid decay of gamma-aminobutyric acid-induced chloride currents in recombinant rat gamma-aminobutyric acid type A receptors.

We discovered the ability of U-93631 (4-dimethyl-3-t-butylcarboxyl-4,5- dihydro[1,5-a]imidazoquinoxaline) to accelerate decay of gamma-aminobutyric acid (GABA)-induced currents, and we explored its mechanism in human embryonic kidney cells (HEK-293) stably expressing the alpha 1 beta 2 gamma 2 subtype of GABAA receptors. Inward currents (Cl- efflux) induced by 5 microM GABA at the holding potential of -60 mV (under a symmetrical Cl- gradient) decayed with an exponential time course with a mean time constant (tau) of 222 +/- 25 sec, as examined with the whole-cell configuration of the patch-clamp technique. The monoexponential decay was greatly accelerated in the presence of U-93631 at 5 microM, with the mean tau value being 5.2 +/- 0.5 sec. The tau values were dependent on the concentration of U-93631, with an estimated Kd of approximately 2 microM. Outward currents at the holding potential of +60 mV decayed with a similar tau value in the presence of the drug, suggesting the voltage independence of the drug action. The initial amplitude of the GABA (5 microM)-induced Cl- current was not affected by preincubation with U-93631 (5 microM) or GABA (200 nM) alone but was reduced by preincubation with the combination of the two. In the presence of U-93631 at 5 microM, the peak amplitude decreased as a function of GABA concentration, with the half-maximal inhibitory concentration being approximately 100 nm, which is close to the Kd for the high affinity GABA site (85 nM). It appears that the drug interacts with GABA-bound receptors (at least monoliganded) and accelerates receptor desensitization, rather than acting as an open channel blocker. The binding site for U-93631 on GABAA receptors seems not to overlap with GABA, barbiturate, or benzodiazepine sites, because the drug effect persisted in the presence of excess ligands for those sites. With cloned GABAA receptors composed of only alpha 1 beta 2, beta 2 gamma 2, or alpha 1 gamma 2 subunits, U-93631 also accelerated the decay rate. This lack of subtype selectivity raises the possibility that the compound interacts with a region common among the three subunits, probably a novel modulatory site, which can possibly be exploited as a novel therapeutic target.

Substituted pyrazinones, a new class of allosteric modulators for gamma-aminobutyric acidA receptors.

We discovered substituted pyrazinones as a new class of allosteric modulators of gamma-aminobutyric acid (GABA)A receptors. Prototype pyrazinones, U-92813 [1-(furfuryl)-3,5-dichloro-6-phenylpyrazinone] and U-94863 [1-benzyl-3,5-dichloro-6-(2-chlorophenyl)pyrazinone], potentiated GABA-mediated Cl- currents in cloned GABAA receptors with certain subtype selectivity. The drugs markedly enhanced the GABA response in the alpha 1 beta 2 gamma 2 and alpha 1 beta 2 subtypes but not in the alpha 1 gamma 2 and beta 2 gamma 2 subtypes expressed in human kidney cells. The dose-response profile of U-94863 in the alpha 1 beta 2 subtype was largely indistinguishable from that in the alpha 1 beta 2 gamma 2 subtype, suggesting no critical role for the gamma 2 subunit in potentiation of the GABA response by the pyrazinones. The drugs also potentiated the GABA response in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes, indicating their nonselectivity toward the alpha isotypes. With respect to subtype selectivity, the pyrazinones differ not only from ligands for benzodiazepine receptors, which interact only with the subtypes containing alpha beta gamma subunits, but also from barbiturates and neurosteroids, which interact with all the subtypes tested in this study. The unique binding site for U-92813 on GABAA receptors was confirmed by the insensitivity of its action to Ro 15-1788, a classical benzodiazepine antagonist, and by the additive nature of its agonistic activity with that of barbiturates and neurosteroids. With respect to the mechanism of potentiation, the pyrazinones are similar to the other allosteric modulators, in that they potentiate the GABA response more effectively at low GABA concentrations than at high GABA concentrations. We propose that substituted pyrazinones represent a novel class of allosteric modulators of GABAA receptors, with their binding site probably located between the alpha and beta subunits.

Synthesis and biological evaluation of antiplatelet 2-aminochromones.

The synthesis and biological evaluation of a series of antiplatelet 2-morpholinylchromones has been described. Modification of the C-7 phenylmethoxy group of 8-methyl-7-(phenylmethoxy)-2-(4-morpholinyl)-4H-1-benzopyran-4-one (2) has led to the discovery of a series of 7-[(amino-ethyl)oxy]-8-methyl derivatives which are potent inhibitors of ADP-induced platelet aggregation. Several members of this class proved active in preventing platelet-dependent thrombus formation in the dog, including 8-methyl-7-[2-(4-methyl-1-piperazinyl)ethoxy]-2-(4- morpholinyl)-4H-1-benzopyran-4-one (39) which was devoid of hemodynamic effects at the effective antithrombotic dose.