CHARACTERIZATION OF NOVEL LIGANDS FOR WILD-TYPE AND NATURAL MUTANT DIAZEPAM-INSENSITIVE BENZODIAZEPINE RECEPTORS

A series of benzodiazepine receptor ligands with different chemical st ructures were evaluated for their affinities at diazepam-sensitive and diazepam-insensitive binding sites for [H-3]Ro 15-4513 4H-imidazo-[1, 5a][1,4]benzodiazepine-3-carboxylate in cerebellar GABA(A) receptors. Rats of Wistar strain and of alcohol-sensitive (ANT) and alcohol-insen sitive (AT) lines were used. The ANT rats possess a single point mutat ion in their GABA(A) receptor alpha 6 subunit, which makes their diaze pam-insensitive sites sensitive to benzodiazepine agonists, unlike tho se of AT and Wistar rats. All compounds evaluated displayed high-affin ity binding to diazepam-sensitive sites (K-i < 50 nM). In contrast, a wider range of affinities were observed at diazepam-insensitive sites which depended upon the basic structure and substitutions. The 7- and 8-halogen substituted imidazobenzodiazepines and 12-halogen substitute d diimidazoquinazolines displayed the highest affinities (K-i < 15 nM) , while intermediate to low affinities (100 < K-i < 4000 nM) were disp layed by imidazoquinazolines, thienopyrimidines, one oxoimidazoquinoxa line, and some cyclopyrrolones. The imidazoquinoxalines evaluated disp layed the lowest affinity (K-i > 10000 nM). The oxoimidazoquinoxaline, ihydro-5-isopropyl-4-oxo-imidazo[1,5-a]quinoxaline (NNC 14-0578) and suriclone represent the first benzodiazepine receptor full agonists to bind with relatively high affinity (K-i similar to 100 nM) to diazepa m-insensitive sites. The 5 position substituted methoxybenzyl, dimethy lallyl, and 4-fluorobenzyl oxoimidazoquinoxaline analogs demonstrated a 58-336-fold higher affinity for ANT than AT diazepam-insensitive sit es. Classical benzodiazepines having a 5-phenyl substituent have demon strated a similar preference for ANT sites, suggesting that all these structures bind to diazepam-insensitive sites in the same orientation. The other compounds evaluated demonstrated only a more modest selecti vity (1-12-fold), indicating different structural requirements for bin ding to mutant ANT and wild-type AT and Wistar receptors. These result s expand the range of ligands which display high affinity for diazepam -insensitive sites. Such compounds should be helpful in determining in trinsic actions of high-affinity ligands at these sites and in assessi ng the contribution of these sites in enhanced sedative sensitivity of cerebellar function in the ANT rats.