A series of imidazo[1,5-a][1,4]benzodiazepine esters have been synthesized with varying ester side chains and 8-position substituents. The affinities of these compounds were evaluated at both "diazepam-insensitive" (DI) and diazepam-sensitive (DS) subtypes of the benzodiazepine receptor (BZR). A profound steric effect of the 3-position ester side chain moiety was observed on ligand affinity at DI. In contrast, ester size had a less robust effect on ligand affinity at DS. The tert-butyl ester compound 8 displayed the highest affinity (Ki = 1.7 nM) for DI within a series of 8-chloro esters. Furthermore, halogens at the 8-position resulted in an enhancement of both ligand affinity and selectivity at DI among the series of tert-butyl esters examined. The 8-nitro derivative 23 and 8-isothiocyanato congener 25 had high affinities for both DI and DS but exhibited little subtype selectivity (10.8 and 2.7 nM at DI versus 14 and 3.7 nM at DS, respectively). The 8-azido tert-butyl ester 29 exhibited a significantly higher affinity (Ki = 0.43 nM) and selectivity (DI/DS ratio of 0.2) than the corresponding ethyl ester, the prototypic DI ligand 1 (Ro 15-4513). Among the compounds synthesized, 29 is the highest affinity ligand for DI described to date while its 8-bromo analog 18 is the most selective ligand (DI/DS ratio of 0.17) for this novel BZR subtype.
The induction of generalized tonic-clinic seizures in mice by the methylxanthine stimulant caffeine is described. These seizures are indistinguishable in quality from those induced by pentylenetetrazol (PTZ), and pretreatment with low doses of caffeine potentiates PTZ-induced seizures. Benzodiazepines inhibit caffeine-induced seizures with a rank order potency that parallels their affinities for the central nervous system (CNS) benzodiazepine receptor in vitro. Inosine, a purine that has recently been shown to be a competitive inhibitor of [3H] diazepam binding in vitro, antagonizes caffeine-induced seizures, while 7-methyl-inosine, a purine that lacks receptor binding inhibitory activity, has no effect on seizures. Since the benzodiazepines, inosine, caffeine, and pentylenetetrazol all competitively inhibit [3H] diazepam binding and have marked effects on inducing or antagonizing seizures, further study of this receptor-ligand system may provide additional insights that concern possible biochemical mechanisms of seizures.
p-, m-, and o-isothiocyanate derivatives (1-3, respectively) of tert-butylbicycloorthobenzoate (TBOB) were synthesized from 3-tert-butyloxetane-3-methanol (4) as the starting material. While 2 was readily obtained in four steps via catalytic hydrogenation of the m-nitro-tert-butylbicycloorthobenzoate (9) intermediate, 1 and 3 could not be obtained this way. 1 and 3 were instead synthesized by an alternative four-step approach while made use of the stability of the isothiocyanate moiety to strong Lewis acids such as boron trifluoride etherate, conditions that would isomerize isothiocyanato oxetane ester intermediates to their corresponding orthoesters. The p-isothiocyanate derivative of TBOB, compound 1, inhibited [35S]-tert-butylbicyclophosphorothionate (TBPS) binding to rat cortical membranes with a potency (IC50 62 nM) comparable to the parent compound while 2 and 3 were approximately 10-fold less potent (IC50 values 570 and 609 nM, respectively). Preincubating tissue with radioligand further reduced the potencies of 2 and 3 by approximately 1 order of magnitude (IC50 values 5400 and 7500 nM, respectively) while the potency of 1 (IC50 90 nM) was only marginally affected by this procedure. Pretreatment of membranes with 1 and 2 followed by extensive washing resulted in a concentration-dependent inhibition of [35S]TBPS binding. In contrast, preincubating tissues with up to 2.4 microM of 3 did not elicit an apparent acylation of [35S]TBPS binding sites. Molecular modeling of the effective diameters of 1-3 in their thermodynamically most stable conformations indicates a relationship between these diameters and their relative efficacies as site-directed acylators; the smaller the diameter, the more potent the acylator. This hypothesis explains both the relative potencies of these compounds and their differential abilities to acylate the TBPS binding site.
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