Biochemical evidence for a new benzodiazepine receptor antagonist

1-(3-Chlorophenyl)-3-diethylcarbamoyl-1 H-1,2,4-triazole (CP-32,961) inhibited [³H]diazepam binding to rat cortical membrances and [³H]flunitrazepam binding to mouse brain in vivo. Its inhibition of the binding of [³H]Ro 15-1788 (benzodiazepine receptor antagonist) to these membranes was not facilitated by added GABA; CP-32,961 exhibited a GABA ratio of 0.84 compared to 2.44 for diazepam. Cerebellar cyclic GMP content in rats was raised by CP-32,961, which also further elevated the increased cyclic GMP levels induced by isoniazid. These neurochemical actions are similar to those shown by ethyl β-carboline-3-carboxylate (β-CCE) and suggest that CP-32,961 is a benzodiazepine receptor antagonist with inverse agonist activity.     

Tricyclic heteroaromatic systems. [1]benzopyranopyrrol-4-ones and [1]benzopyrano-1,2,3-triazol-4-ones as benzodiazepine receptor ligands. Synthesis and structure-activity relationships

The synthesis, ability to displace [3H]flunitrazepam binding from bovine brain membranes, and GABA ratio of some [1]benzopyranopyrroles 1a-i and [1]benzopyrano-1,2,3-triazoles 2a,b are reported. The GABA ratios of some previously synthesized pyrazoloquinolines A and [1]benzopyranopyrazoles C are also presented in order to draw some structure-activity relationships among our benzodiazepine receptor ligands. 1,3-Diarylpyrrole derivatives 1a-h show similar affinity and efficacy to that of diazepam, while the 1-aryltriazoles 2a,b have no receptor affinity. Comparison of the latter results with those on previously reported compounds suggests that there are several hydrophobic regions on the benzodiazepine recognition site whose occupation gives rise to different affinity and efficacy.     

Group-selective reagent modification of the benzodiazepine-gamma-aminobutyric acid receptor-ionophore complex reveals that low-affinity gamma-aminobutyric acid receptors stimulate benzodiazepine binding

The modification of membrane proteins with diethylpyrocarbonate (DEP) and diazotized sulfanilate was investigated on the binding of three benzodiazepine radioligands in three brain regions. Both of these reagents produced a dose-dependent inactivation of [3H] diazepam, [3H]flunitrazepam, and [3H]propyl beta-carboline-3-carboxylate binding to cortex, cerebellum, and hippocampus. Both DEP and diazotized sulfanilate decrease the Bmax of the benzodiazepine binding sites without altering the KD. The ability of muscimol and pentobarbital to enhance [3H]diazepam binding was not altered by DEP pretreatment in any of the three regions. Scatchard analysis indicated that, following the inactivation of 40-50% of [3H]diazepam binding by 1 mM DEP, pentobarbital and muscimol were still able to increase the affinity of [3H]diazepam binding in cortex, cerebellum, and hippocampus. In contrast, diazotized sulfanilate pretreatment abolishes the ability of muscimol and pentobarbital to enhance [3H]diazepam binding in these three regions. The effects of these reagents on [3H] gamma-aminobutyric acid (GABA) binding revealed that sulfanilate but not DEP eliminates the low-affinity GABA receptor sites in cortex and cerebellum. Thus, while both DEP and sulfanilate inactivate benzodiazepine binding sites, only sulfanilate abolishes the low-affinity GABA binding sites and the ability of the GABA agonists to enhance [3H]diazepam binding. These results suggest that the stimulation of benzodiazepine binding appears to be mediated by the low-affinity GABA receptors.     

Synthesis and benzodiazepine receptor binding of 5H-pyrido[2,1-C][1,4]benzothiazines

The pyrido[2,1-c][1,4]benzothiazines 1a-t and the pyrido[2,1-c][1,4]thiazines 7a,b were prepared and tested for their ability to displace specific [3H]diazepam binding from rat brain membranes. Some of the examined compounds of type 1 and 7b showed moderate binding affinity for the benzodiazepine receptor.     

Synthesis and interaction of 5-(substituted-phenyl)-3-methyl-6,7-dihydropyrazolo[4,3-e] [1,4]diazepin-8(7H)-ones with benzodiazepine receptors in rat cerebral cortex

On the basis of the anxiolytic property of ripazepam, 1-ethyl-4,6-dihydro-3- methyl-8-phenylpyrazolo[4,3-e][1,4]diazepin-5(1H)-one (1), a series of isomeric 5-(phenyl-substituted)pyrazolo[4,3-e][1,4] diazepin-8-ones 3a-f were prepared and tested for their ability to bind to the benzodiazepine receptor. All compounds 3a-f display affinities for the benzodiazepine receptor in the microM range of concentration; in particular 5-phenyl-3-methyl-6,7-dihydropyrazolo[4,3-e][1,4] diazepin-8(7H)-one (3a) is 2 orders of magnitude less potent in inhibiting [3H]flunitrazepam binding than diazepam and displays an affinity for the benzodiazepine receptor practically comparable to that of its structural isomer, ripazepam, and to that of chloriazepoxide.     

Synthesis and benzodiazepine receptor binding of 1H-pyrrolo[2,1-C][1,4]benzothiazines

The pyrrolo[2,1-c][1,4]benzothiazines 2a-n and the pyrrolo[2,1-c][1,4]thiazine 13 were prepared and tested for their ability to displace specific [3H]diazepam binding from rat brain membranes. Such compounds were found essentially devoid of binding affinity for the benzodiazepine receptor.     

Interaction of avermectins with [3H]beta-carboline-3-carboxylate ethyl ester and [3H]diazepam binding sites in rat brain cortical membranes

The binding of [3H] beta-carboline-3-carboxylate ethyl ester ([3H] beta-CCE), a ligand for the benzodiazepine receptor in the mammalian CNS, to rat cortical membranes, is enhanced by avermectin B1a and its therapeutic formulation, Ivermectin. In contrast to the effects of the avermectins on [3H]diazepam binding, which involve changes in both receptor affinity and number, increases in beta-CCE binding, which are much less than those observed for the benzodiazepine ligand, involve only alterations in receptor number. This Bmax increase is bicuculline insensitive whereas Ivermectin effects on benzodiazepine binding are partially antagonized by GABA antagonist. The data suggest a differential interaction by the avermectins on benzodiazepine and beta-CCE binding sites in rat cortical membranes and indicate that these macrolide anthelmintics may be a useful tool for characterizing benzodiazepine/anxiolytic receptor subtypes.     

Characterization of peripheral-type benzodiazepine binding sites in brain using [3H]Ro 5-4864

The binding of [3H]Ro 5-4864 to the peripheral-type benzodiazepine binding site in brain is characterized. The binding is saturable, high-affinity (KD = 1.6 nM), and reversible. The comparison of [3H]Ro 5-4864 and [3H]diazepam binding sites reveals major differences which include the following. There are about one-fourth as many peripheral-type binding sites than central sites in brain. Peripheral sites are present in many extranervous tissues and have a brain regional distribution distinct from that of the central-type receptor. The [3H]Ro 5-4864 binding site also is apparently highly localized in the nuclear membrane in contrast to the central-type receptor, which is synaptosomal. gamma-Aminobutyric acid has no effect on [3H]Ro 5-8464 binding, again in contrast to its marked effect on [3H]diazepam binding. Various putative benzodiazepine receptor ligands, such as purines, beta-carbolines, and kynurenamines, are also inactive as inhibitors of [3H]Ro 5-4864 binding. Blocking the benzodiazepine receptor by photoaffinity labeling decreases [3H]diazepam binding by more than 80% and has no effect on [3H]Ro 5-4864 binding. These results indicate that the peripheral-type benzodiazepine binding site in brain is a separate entity whose physiological function is probably distinct from that of the central-type benzodiazepine receptor.     

Brain benzodiazepine receptors and their rapid changes after seizures in the Mongolian gerbil

To elucidate the physiological role of benzodiazepine receptors in convulsion, these receptors were studied in the brain of the Mongolian gerbil (Meriones unguiculatus), an animal model used for the study of epilepsy. Benzodiazepine binding sites in the gerbil brain were demonstrated using [3H]diazepam. The binding was saturable and stereospecific. Benzodiazepines inhibited [3H]diazepam binding to the membranes and their ability to inhibit the binding closely correlated with their potency as anticonvulsants. These results showed that the characteristics of the benzodiazepine receptors in the Mongolian gerbil were similar to those obtained from rat and human brain. Seizures increase the specific binding of [3H]diazepam to the membranes of all regions of the gerbil brain, the most remarkable increase seen in the striatum. Time course studies showed that the increase reached a maximum 10 min after the seizure and the binding returned to the control level within 20 min. The increase in specific [3H]diazepam binding was due almost entirely to shifts in the affinity of [3H]diazepam for its receptors. Thus, the seizures may not be due to changes in the benzodiazepine receptors, and seizures produce an increase in receptor binding, which may be related to a physiological modification of excessive excitation.     

Effects of tetrazole derivatives on [3H]diazepam binding in vitro: correlation with convulsant potency

A series of 8 tetrazole derivatives which differ more than one hundred-fold in their potencies as convulsants were tested for their abilities to inhibit [3H]diazepam binding to benzodiazepine receptors in vitro. The concentrations of drug necessary to inhibit 50% of specifically bound [3H]diazepam ranges from 18 micro M for undecamethylenetetrazole to 20 mM for trimethylenetetrazole. A comparison of the minimum convulsive doses for the 8 tetrazole derivatives tested with their relative potencies in displacing [3H]diazepam binding in vitro revealed a highly significant correlation (r = 0.98, P less than 0.001). In contrast, several representative tetrazole derivatives were found to have no inhibitory effects on beta-adrenergic, alpha-adrenergic or muscarinic cholinergic receptors in the same membrane preparation. These results suggest that pentamethylenetetrazole and related tetrazole derivatives may elicit their convulsant effects by interaction with the benzodiazepine receptor.     

Modification of the peripheral-type benzodiazepine receptor by arachidonate, diethylpyrocarbonate and thiol reagents

Peripheral type benzodiazepine receptors are differentially modified by arachidonate, diethylpyrocarbonate and thiol reagents, as evidenced by the finding that binding of a proposed agonist ([3H]R05-4864) and a proposed antagonist ([3H]PK 11195) to rat kidney mitochondrial membranes can be modified separately. (1) Arachidonate significantly lowered the affinity of the peripheral-type benzodiazepine receptor for R05-4864 and diazepam but did not alter the affinity for PK 11195 and only slightly altered for dipyridamole. In contrast, diethylpyrocarbonate inhibited [3H]PK 11195 binding by 43% while reducing [3H]R05-4864 binding only 16%. (2) Diethylpyrocarbonate treatment causes a reduction in affinity for both dipyridamole and PK 11195, did not change the affinities for R05-4864 and diazepam but increased the affinities for diuretics. PK 11195, R05-4864 and dipyridamole totally protected peripheral benzodiazepine receptors against inactivation by diethylpyrocarbonate. In contrast, diethylpyrocarbonate inactivation of [3H]PK 11195 binding was increased after preincubation of peripheral benzodiazepine receptors with metolazone. Arachidonate both lowers the affinity for [3H]R05-4864 and eliminated the ability of R05-4864 to protect peripheral benzodiazepine receptors against inactivation by diethylpyrocarbonate. (3) Among different thiol reagents, only dithiothreitol treatment resulted in 26 and 43% inhibition of [3H]PK 11195 and [3H]R05-4864 binding, respectively. These results indicate that the peripheral-type benzodiazepine receptor molecule possesses at least two different conformations or separate mutually exclusive sites for agonists and antagonists. Additionally, the interaction of the thiazide class of diuretics with peripheral-type benzodiazepine receptors seems to require binding sites on the receptor molecule in addition to those for specific agonist and antagonist, whereas dipyridamole appears to bind to the antagonist site or conformation.     

Specific inhibition of benzodiazepine receptor binding by some 1,2,3-triazole derivatives

Certain 1,2,3-triazole derivatives were prepared and tested for their ability to displace [3H]diazepam from bovine brain membranes. From these compounds, the quinolytriazole derivatives (14, 15, 16, 17) were clearly the most potent, while the naphthyl- and the naphthyridyl-triazoles were considerably less active. The p-nitrophenyl derivative (15) was the compound that bound with the highest affinity within the quinolyltriazole compounds class. The replacement of the p-nitrophenyl group with other substituents greatly decreased the binding activity. From a Lineweaver-Burk analysis of 11, it appears that the inhibition is competitive.     

Studies on the synthesis of condensed pyridazine derivatives. III. Synthesis and benzodiazepine receptor binding studies of condensed triazolopyridazine derivatives

A series of 6H-(1)benzothiopyrano[3,4-e][1,2,4]triazolo[4,3- b]pyridazines and 6,7-dihydro-(1)benzothiepino[4,5- e][1,2,4]triazolo [4,3-b]pyridazines were prepared and tested for their ability to displace [3H] diazepam from rat brain membranes. An approximately planar shape of these molecules was essential for high affinity to the benzodiazepine receptor. Among them, 11-aryl compounds in the latter series were found to have high affinity to the benzodiazepine receptor. 11-Phenyl- and 11-thienyl- 6,7-dihydro-(1)benzothiepino[4,5-e][1,2,4]triazolo[4,3- b]pyridazine (3b-5 and 3b-11 respectively) showed the potent affinity comparable to that of diazepam. The structure-activity relationships are also discussed.     

Increased benzodiazepine receptor number elicited in vitro by a novel purine, EMD 28422

EMD 28422 (N6-[2-(4-chlorophenyl)-bicyclo-2.2.2.octyl-(3)]-adenosine) was demonstrated to increase the number of binding sites for [3H]diazepam (Bmax) in vitro without an accompanying increase in receptor affinity (KD). The increase in receptor number was observed in both crude synaptosomal preparations (P2) and thrice-washed membrane preparations with and without the addition of 50 microM GABA. Furthermore, this effect appeared to be independent of the concentration of chloride ion, since the increases in Bmax were observed in both Tris-HCl and Tris-maleate buffer. The effects of EMD 28422 were stereospecifically antagonized by the GABA antagonist bicuculline, despite the lack of effect of EMD 28422 on [3H]muscimol binding at concentrations which markedly increased benzodiazepine receptor number. Neither EMD 39011 nor adenosine, the two parent moieties of EMD 28422, increased [3H]diazepam binding at concentrations of up to 1 mM. The increases in benzodiazepine receptor number observed with EMD 28422 in vitro suggests that this compound induces a conformational change in the benzodiazepine receptor which may cause the dissociation of an endogenous noncompetitive inhibitor of [3H]diazepam binding from the membrane, thus 'unmasking' binding sites. The stereospecific antagonism of this effect by bicuculline and the apparent inability of GABA to alter the action of EMD 28422 suggests the presence of a novel type or different functional state of GABA receptor which may play a permissive role in the rapid modulation of benzodiazepine receptor number in vitro.     

Benzodiazepine receptor binding: the interactions of some non-benzodiazepine drugs with specific [3H] diazepam binding to rat brain synaptosomal membranes

Summary The interaction of several non-benzodia-zepine drugs with [³H] diazepam binding to benzodiazepine receptors in rat brain synaptosomal membranes was investigated. Baclofen, benzoctamine, hydroxyzine, chlorpromazin, haloperidol, imipramine, and amitriptyline displace specific [³H] diazepam binding, but the concentrations needed are too high to explain pharmacological effects of these drugs by an interaction with benzodiazepine receptors. The most potent non-benzodiazepine drug for inhibiting specific [³H] diazepam binding was methaqualone (IC₅₀ value of 150 M). It is suggested that interactions with benzodiazepine receptors may account for the anxiolytic and anticonvulsive side effects of this drug. The analeptic drug pentylenetetrazole interacts with benzodiazepine receptor binding with an IC₅₀ value of about 1 mM, which is possibly too high to explain its convulsive properties by an antagonism at the benzodiazepine receptor.     

Enhancement of GABA binding by the benzodiazepine partial agonist CGS9896

Compounds have been reported that act on the benzodiazepine receptor as full agonists (diazepam and CL218872), full antagonists (CGS8216 and RO15-1788), on partial agonists (CGS9896). We examined the effect of these compounds on [3H]GABA binding to membrane fragments from rat brain. Incubations were performed at 37 degrees C in a buffer containing EGTA to reduce free calcium ion levels. Centrifugation was then used to separate bound from free [3H]GABA. Diazepam caused a 20-45% enhancement of [3H]GABA binding and this effect was inhibited by 5 mM CaCl2. The magnitude of the enhancement of [3H]GABA by CL218872 was similar to that of diazepam. In contrast, the benzodiazepine antagonists, RO15-1788 and CGS8216 caused little enhancement of [3H]GABA binding. Finally, the partial agonist CGS9896 was distinguishable from both the benzodiazepine antagonists and full agonists by an intermediate level of enhancement of [3H]GABA binding. The extent of enhancement of [3H]GABA binding appears to be predictive of the pharmacological efficacy of compounds acting at the benzodiazepine receptor.     

Quinazolines and 1,4-benzodiazepines. 92. Conformational recognition of the receptor by 1,4-benzodiazepines

We propose that the conformation of 1,4-benzodiazepines that is recognized by the binding site on the benzodiazepine receptor complex is one in which the planes formed by the fused benzene ring and the methylene group (and the two adjoining atoms) of the diazepine ring are in the R configuration. The derivation of this conformation was based on comparisons of computer-generated 3-dimensional structures obtained from single-crystal X-ray data for diazepam, (R)- and (S)-1,3-dimethyl-5-(2-fluorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2- one, and the structurally rigid ethyl (S)-7-chloro-11,12,13,13 alpha-tetrahydro-9-oxo-9H-imidazo[1,5-alpha] pyrrolo[2,1-d][1,4]benzodiazepine-1-carboxylate. The affinity of ligands for the benzodiazepine binding site was determined using the [3H]-diazepam binding assay.     

Studies on the synthesis of condensed pyridazine derivatives. I. Synthesis and benzodiazepine receptor binding studies of 2-substituted-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3(2H)-ones and related compounds

A series of 2-substituted-4,4a,5,6-tetrahydrobenzo[h]cinnolin-3(2H)-ones and related compounds were synthesized and tested for their ability to displace [3H]diazepam from rat brain membranes. Among them, compounds bearing 4-methoxyphenyl, 4-chlorophenyl, or 4-methylphenyl group at the position-2 were found to have high affinity to the benzodiazepine receptor. 2-(4-Methoxyphenyl)-9-methyl- and 2-(4-methoxyphenyl)-9-methoxy-4,4a,5,6-tetrahydrobenzo[h]cinnolin- 3(2H)-ones (8b-14 and 8b-15, respectively) showed a potent affinity comparable to that of diazepam. These results suggest that a topographical planarity or pseudoplanarity of these molecules is essential for high affinity to the benzodiazepine receptor. The structure-activity relationships are discussed.     

3H]diazepam specific binding to rat cortex in vitro is enhanced by oleic, arachidonic and docosahexenoic acid isolated from pig brain

Substances were found in purified fractions from pig brain that enhanced the specific binding of [3H]diazepam to membranes from rat brain in vitro. These substances were identified as oleic acid, arachidonic acid and docosahexenoic acid. Oleic acid (10(-5) - 10(-4)M) increased the affinity for agonists binding to the benzodiazepine receptor, whereas the binding of antagonists to this receptor was only slightly enhanced. The number of [3H]muscimol binding sites was increased, whereas binding of [3H]SR 95331, a GABA receptor antagonist, was unchanged. The effect of oleic acid was additional to the GABA-induced enhancement of [3H]diazepam binding.     

Specific and potent interactions of carbamazepine with brain adenosine receptors

Carbamazepine, a drug effective in pain, seizure, and affective disorders, was screened for its ability to interact with a variety of neurotransmitter and neuromodulator binding sites on brain membranes. The most potent effect was observed on adenosine antagonist ( [3H]DPX) binding to the adenosine receptor (KI = 3.5 +/- 0.4 microM) followed by adenosine agonist ( [3H]CHA) binding (KI = 24.5 +/- 3.6 microM). Lower potency effects were observed on benzodiazepine receptors, and no inhibition was seen in a variety of other systems. The inhibition of adenosine receptor binding by carbamazepine was competitive. No correlation was observed between the potency of a series of carbamazepine analogs as inhibitors of either ( [3H]DPX, [3H]CHA or [3H]diazepam binding and their ability to inhibit electroshock-induced convulsions, suggesting that the anticonvulsant properties of these agents are not mediated by the adenosine receptor, but raising the possibility that other clinical effects of carbamazepine may relate to its ability to act at the adenosine receptor.