The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.

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Twenty esters of 3 alpha- and 3beta-hydroxy(nor)tropanes and two amides of 3 alpha-aminotropane were prepared with substituted benzoic acids. These (nor)tropeines inhibited [(3)H]strychnine binding to glycine receptors in synaptosomal membranes of rat spinal cord. A ternary allosteric model was applied to determine the dissociation constants (K(A)) of the tropeines having strong negative cooperativities with [(3)H]strychnine binding (alpha > 10). K(A) values about 10 nM are well below those of known allosteric agents. Low concentrations (0.1K(A)) of the (nor)tropeines potentiated the displacing effects of glycine. Positive cooperativity with glycine (beta < 1) decreased with the increase in concentration and binding affinity of tropeines. Displacing potencies were also measured for [(3)H]granisetron binding to 5-HT(3) type serotonin receptors of rat cerebral cortex. Selectivities to glycine receptors versus 5-HT(3) receptors varied within 4 orders of magnitude. Nortropeines might serve as a lead to high-affinity selective allosteric modulators of glycine receptors.