Synthesis and Pharmacological Properties of Novel 8-Substituted Imidazobenzodiazepines: High-Affinity, Selective Probes for α5-Containing GABAAReceptors

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Abstract
The synthesis and pharmacological properties of imidazobenzodiazepines with both high affinity and selectivity for α5-containing GABAA receptors are described. Four of these compounds (5, 6, 8, and 9) inhibited [3H]flunitrazepam binding to recombinant α5β2γ2 GABAA receptors with IC50 values between ∼0.4 and 5 nM. These compounds were ≥24−75-fold more selective for recombinant receptors containing α5 subunits compared to other, “diazepam-sensitive” GABAA receptors containing either α1, α2, or α3 subunits. Imidazobenzodiazepine 9 (used as the prototypical α5 selective ligand) inhibited [3H]flunitrazepam binding to hippocampal membranes with high- and low-affinity components (IC50 0.6 ± 0.2 and 85.6 ± 13.1 nM, respectively), representing ∼16% and ∼84% of the receptor pool. Inhibition of [3H]flunitrazepam binding to cerebellar membranes with imidazobenzodiazepine 9 was best fitted to a single population of sites with an IC50 of 79.8 ± 18.3 nM. These imidazobenzodiazepines behaved as GABA negative ligands in recombinant GABAA receptors expressed in Xenopus oocytes and were convulsant in mice after parenteral administration. The relative potencies of flumazenil and zolpidem in blocking convulsions induced by 9 and DMCM, respectively, indicated that occupation of α5-containing GABAA receptors substantially contributed to the convulsant properties of acetylene analog 9. These 8-substituted imidazobenzodiazepines (5, 6, 8, and 9) should prove useful in examining the physiological roles of GABAA receptors bearing an α5 subunit and may also lead to the development of novel, subtype selective therapeutic agents.