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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.

Various cyclic ether and other 3 alpha-hydroxyandrostane derivatives bearing a conformationally constrained hydrogen-bonding moiety were prepared. Their anesthetic potency and their binding affinity for GABA(A) receptors, measured by intravenous administration to mice and inhibition of [(35)S]TBPS binding to rat whole brain membranes, were compared with that of known anesthetic 3 alpha-hydroxypregnan-20-ones. Synthetic steroids with similar in vitro and in vivo activities to the endogenous 3 alpha-hydroxypregnan-20-ones all had an ether oxygen on the beta-face of the steroid D-ring. These results suggest that for optimal GABA(A) receptor modulation, the hydrogen bond-accepting substituent should be near perpendicular to the plane of the D-ring on the beta-face of the steroid.