Abstract

3-Acyloxy-, 3-methoxy-, and 3-alkyl-substituted derivatives of the benzodiazepine (BZ) agonist desmethyl-diazepam (DMD) were resolved, and the stereochemical properties of binding to central BZ receptors were investigated in synaptosomal membrane preparations of rat brain. Decreasing potency and stereoselectivity of 3-methyl, 3-ethyl, and 3-isopropyl derivatives in displacement of [3H]diazepam binding can be attributed to differential susceptibilities for steric hindrance of 3-axial versus 3-equatorial substituents of the binding conformation M. Chirality in the alpha-methyl-beta-phenyl-propionic acyl moiety of oxazepam, the 3-OH-derivative of DMD, was noncritical in binding, whereas the beta-phenyl substituent selectively increased the binding of the 3S-stereoisomer. Changing the pH from 7.4 to 5.6 significantly increased the IC50 of (3R)-oxazepam acetate but not those of (3R)-methyl-DMD and diazepam. Binding data led to a steric model of the BZ binding site with the postulation of an additional hydrogen-bond-donating moiety, probably histidine in the "ceiling" of the receptor cavity, that binds the 3-carbonyloxy groups and hinders the 3-alkyl ones. In vitro efficacies of 3-substituted BZs were estimated by allosteric binding interactions within the gamma-aminobutyric acidA (GABAA) receptor-ionophore complex. Non-equilibrium enhancement of t-butyl-bicyclophosphoro[35S]thionate binding by the BZ agonist oxazepam was stereoselectively antagonized by (3S)-oxazepam-(S)-alpha-methyl-beta-phenyl-propionate, suggesting a mixed agonist-antagonist character. GABA enhanced the [3H]diazepam-displacing potencies of the 3S-enantiomers of the acetate, hemisuccinate, and (S)-alpha-methyl-beta-phenyl-propionate esters of oxazepam by a factor of about 1.5-1.6, whereas the GABA shifts for 3R-esters were about 1.2. UV affinity labeling with flunitrazepam resulted in a significantly smaller decrease in the displacing potency of (3R)-oxazepam acetate than in that of the 3S-enantiomer. GABA shifts of successively 3-methylated DMD derivatives were also compared. The GABA shifts of DMD and its (3S)-methyl and 3,3-dimethyl derivatives were all characteristic of full agonists (2.4-2.7), whereas that of (3R)-methyl-DMD was 1.5. The 3-methoxy enantiomers of DMD displayed stereoselectivity and GABA shift values intermediate between those of 3-methyl and 3-acetoxy derivatives. These allosteric interactions suggest that 3-carbonyloxy derivatives in general, as well as (3R)-BZ enantiomers bound with axial 3-alkyl and 3-alkyloxy groups, decrease the agonist efficacies of 1,4-BZs to modulate the GABAA receptor complex.