Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.

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Studies with our screening lead 5 and the literature compound 6 led to the identification of 6-benzyloxy-3-(4-methoxy)phenyl-1,2,4-triazolo[3,4-a]phthalazine 8 as a ligand with binding selectivity for the gamma-aminobutyric acid-A (GABA-A) alpha 3- and alpha 5-containing receptor subtypes over the GABA-A alpha 1 subtype (K(i): alpha 2 = 850 nM, alpha 3 = 170 nM, alpha 5 = 72 nM, alpha 1 = 1400 nM). Early optimization studies identified the close analogue 10 (K(i): alpha 2 = 16 nM, alpha 3 = 41 nM, alpha 5 = 38 nM, alpha 1 = 280 nM) as a suitable lead for further study. High-affinity ligands were identified by replacing the 6-benzyloxy group of compound 10 with 2-pyridylmethoxy (compound 29), but binding selectivity was not enhanced (K(i): alpha 2 = 1.7 nM, alpha 3 = 0.71 nM, alpha 5 = 0.33 nM, alpha 1 = 2.7 nM). Furthermore, on evaluation in xenopus oocytes,(22) 29 was discovered to be a weak to moderate inverse agonist at all four receptor subtypes (alpha 1, -7%; alpha 2, -5%; alpha 3, -16%; alpha 5, -5%). Replacement of the 3-phenyl group of 29 with alternatives led to reduced affinity, and smaller 3-substituents led to reduced efficacy. Methyl substitution of the benzo-fused ring of 29 at the 7-, 8-, and 10-positions resulted in increased efficacy although selectivity was abolished. Increased efficacy and retention of selectivity for alpha 3 over alpha 1 was achieved with the 7,8,9,10-tetrahydro-(7,10-ethano)-phthalazine 62. Compound 62 is currently one of the most binding selective GABA-A alpha 3-benzodiazepine-site partial agonists known, and although its selectivity is limited, its good pharmacokinetic profile in the rat (33% oral bioavailability after a 3 mg/kg dose, reaching a peak plasma concentration of 179 ng/mL; half-life of 1 h) made it a useful pharmacological tool to explore the effect of a GABA-A alpha 2/alpha 3 agonist in vivo.

A series of high-affinity GABA(A) agonists with good oral bioavailability in rat and dog and functional selectivity for the GABA(A)alpha2 and -alpha3 subtypes is reported. The 7-trifluoromethylimidazopyrimidine 14g and the 7-propan-2-olimidazopyrimidine 14k are anxiolytic in both conditioned and unconditioned animal models of anxiety with minimal sedation observed at full BZ binding site occupancy.

There is increasing evidence that compounds with selectivity for gamma-aminobutyric acid(A) (GABA(A)) alpha2- and/or alpha3-subtypes may retain the desirable anxiolytic activity of nonselective benzodiazepines but possess an improved side effect profile. Herein we describe a novel series of GABA(A) alpha2/alpha3 subtype-selective agonists leading to the identification of the development candidate 17, a nonsedating anxiolytic in preclinical animal assays.