Benzodiazepine Binding Site Ligands Research Articles

Acute chloroquine poisoning: A comprehensive experimental toxicology assessment of the role of diazepam

Background and PurposeResurgence in the use of chloroquine as a potential treatment for COVID‐19 has seen recent cases of fatal toxicity due to unintentional overdoses. Protocols for the management of poisoning recommend diazepam, although there are uncertainties in its pharmacology and efficacy in this context. The aim was to assess the effects of diazepam in experimental models of chloroquine cardiotoxicity.Experimental ApproachIn vitro experiments involved cardiac tissues isolated from rats and incubated with chloroquine alone or in combination with diazepam. In vivo models of toxicity involved chloroquine administered intravenously to pentobarbitone‐anaesthetised rats and rabbits. Randomised, controlled treatment studies in rats assessed diazepam, clonazepam and Ro5‐4864 administered: (i) prior, (ii) during and (iii) after chloroquine and the effects of diazepam: (iv) at high dose, (v) in urethane‐anaesthetised rats and (vi) co‐administered with adrenaline.Key ResultsChloroquine decreased the developed tension of left atria, prolonged the effective refractory period of atria, ventricular tissue and right papillary muscles, and caused dose‐dependent impairment of haemodynamic and electrocardiographic parameters. Cardiac arrhythmias indicated impairment of atrioventricular conduction. Studies (i), (ii) and (v) showed no differences between treatments and control. Diazepam increased heart rate in study (iv) and as with clonazepam also prolonged the QTc interval in study (iii). Combined administration of diazepam and adrenaline in study (vi) improved cardiac contractility but caused hypokalaemia.Conclusion and ImplicationsNeither diazepam nor other ligands for benzodiazepine binding sites protect against or attenuate chloroquine cardiotoxicity. However, diazepam may augment the effects of positive inotropes in reducing chloroquine cardiotoxicity.Linked ArticlesThis article is part of a themed issue on The Pharmacology of COVID‐19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc

GABAA receptor subtypes in the mouse brain: Regional mapping and diazepam receptor occupancy by in vivo [18F]flumazenil PET

Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABAA receptors composed of two α, two β and one γ2 subunit. Their high affinity binding site is located at the interface between the γ2 and the adjacent α subunit. The α-subunit gene family consists of six members and receptors can be homomeric or mixed with respect to the α-subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABAA receptor subtypes, as defined by the benzodiazepine-binding α subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual α subunits across the CNS have been extensively characterized. However, as GABAA receptors may contain two different α subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [18F]flumazenil PET and in vitro [3H]flumazenil autoradiography in combination with GABAA receptor point-mutated mice to characterize the distribution of the two most prevalent GABAA receptor subtypes (α1 and α2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of α2-containing receptors were found in brain regions of the neuronal network of anxiety. The α1/α2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [18F]flumazenil PET to determine the degree of receptor occupancy at GABAA receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1–2mg/kg, in agreement with published data from ex vivo studies with wild type mice. In conclusion, we have resolved the quantitative distribution of α1- and α2-containing homomeric and mixed GABAA receptors in vivo at the millimeter scale and demonstrate that the regional drug receptor occupancy in vivo at these GABAA receptor subtypes can be determined by [18F]flumazenil PET. Such information should be valuable for drug development programs aiming for subtype-selective benzodiazepine site ligands for new therapeutic indications.

Endogenous ligands of benzodiazepine binding site have inverse agonistic properties

Benzodiazepines have been widely used in clinical praxis for many decades. They act as GABAA receptor agonists and possess muscle-relaxant, hypnotic-sedative, anticonvulsant, and anxiolytic properties. Flumazenil acts as a benzodiazepine receptor antagonist (subunits α1, α2, α3, and α5) or partial agonist (subunits α4 and α6). It competitively inhibits the activity at the benzodiazepine recognition site on the GABA/benzodiazepine receptor complex, thereby reversing the effects of benzodiazepines. In our experiments, administration of flumazenil in rabbits was surprisingly associated with anxiolytic effects similar to those of midazolam. Additionally, flumazenil significantly and dose-dependently decreased the total number of vocalizations in rats, i.e. it was anxiolytic. These observations seem to be in contrast to the effect of flumazenil in humans, where it is believed to produce mainly anxiogenic effects. It seems that in individuals, who exhibit anxiogenic behavior or in individuals with anticipation anxiety, flumazenil acts as an anxiolytic agent, while in individuals without any signs of anxiety, flumazenil can also act as anxiogenic agent. Thus, we hypothesize that flumazenil is associated with decreased intensity of anticipatory anxiety due to occupancy of benzodiazepine binding sites by an endogenous ligand with inverse agonistic properties.

Assessment of luteolin (3′,4′,5,7-tetrahydroxyflavone) neuropharmacological activity

Since the discovery that certain flavonoids (namely flavones) specifically recognise the central BDZ receptors, several efforts have been made to identify naturally occurring GABA A receptor benzodiazepine binding site ligands. Flavonoid derivatives with a flavone-like structure such as apigenin, chrysin and wogonin have been reported for their anxiolytic-like activity in different animal models of anxiety. Luteolin (3′,4′,5,7-tetrahydroxyflavone) is a widespread flavonoid aglycon that was reported as devoid of specific affinity for benzodiazepine receptor (BDZ-R) binding site, but its psychopharmacological activity is presently unknown. Considering (1) the close structural similarity with other active flavones, (2) the activity of some of its glycosilated derivatives and (3) the complexity of flavonoid effects in the central nervous system, luteolin was submitted to a battery of tests designed to evaluate its possible activity upon the CNS and its ability to interact with the BDZ-receptor binding sites was also analysed. Luteolin apparently has CNS activity with anxiolytic-like effects despite the low affinity for the BDZ-R shown in vitro. Our findings suggest a possible interaction with other neurotransmitter systems but we cannot rule out the possibility that luteolin's metabolites might show a higher affinity for the BDZ-R in vivo, thus eliciting the evident anxiolytic-like effects through a GABAergic mechanism.

Up‐regulation of PK11195 binding in areas of axonal degeneration coincides with early microglial activation in mouse brain

Increased binding of the peripheral benzodiazepine binding site (PBBS) ligand [(3)H]PK11195 in the central nervous system of patients suffering from acute and chronic neuropathology has been associated with reactive microgliosis. However, it remains uncertain which stages of microglial activation occur in conjunction with the increased [(3)H]PK11195 binding. We used quantitative autoradiography for [(3)H]PK11195 and quantitative polymerase chain reaction for PBBS mRNA and markers of early and late microglial activation to investigate the time-course of cellular responses in the hippocampus of mice with degeneration of the entorhinal-hippocampal perforant path. The axonal lesion evoked an increase in the B(max) for [(3)H]PK11195 in hippocampus which peaked at 2 days post-lesion, remained elevated at day 5 and began to decline at 10 days post-lesion. These changes occurred in the absence of significant changes in affinity in vitro. Quantitative polymerase chain reaction analysis of isolated hippocampi using exon-specific primers indicated the presence of several splice variants of PBBS mRNA, which appeared to be affected differentially by the lesion. The changes in PBBS mRNA and CD11b mRNA levels correlated with the B(max) for [(3)H]PK11195 during 10 days post-lesion, suggesting that microglial activation couples with increases in mRNA levels for these markers. In addition, the onset of changes in PBBS mRNA levels coincided with the significantly elevated tumor necrosis factor mRNA levels present during early microglial activation at 2 days post-lesion. We conclude that up-regulation of [(3)H]PK11195 binding and PBBS mRNA levels coincided with early microglial activation, characterized by concomitantly increased microglial tumor necrosis factor mRNA levels, and persisted throughout the period with reactive microgliosis.

Synthesis of Pyrido[2,3‐d]pyridazines and Pyrazino[2,3‐d]‐pyridazines — Novel Classes of GABAA Receptor Benzodiazepine Binding Site Ligands.

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Imidazo[1,2- a]pyrimidines as functionally selective GABA A ligands

Imidazo[1,2- a]pyrimidines are GABA A receptor benzodiazepine binding site ligands which can exhibit functional selectivity for the α 3 subtype over the α 1 subtype. SAR studies to optimize this functional selectivity are described.

Pyrazolopyridinones as functionally selective GABA A ligands

2,5-Dihydro-3 H-pyrazolo[4,3- c]pyridin-3-ones are GABA A receptor benzodiazepine binding site ligands, which can exhibit functional selectivity for the α 3 subtype over the α 1 subtype. SAR studies to optimize this functional selectivity are described.

Bidirectional effects of benzodiazepine binding site ligands on active avoidance acquisition and retention: differential antagonism by flumazenil and β-CCt

The pharmacological approach, using subtype selective ligands, complements genetic studies on the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. The aim of this study was to examine the relative significance of alpha1-containing GABA(A) receptors in the effects of modulators at the benzodiazepine site on anxiety and memory processes. We tested the effects of the nonselective antagonist flumazenil, the preferential alpha1-subunit selective antagonist beta-carboline-3-carboxylate-t-butyl ester (beta-CCt), the nonselective agonist midazolam, the preferential alpha1-subunit selective agonist zolpidem, and the nonselective inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in a two-way active avoidance task in rats. The influence of flumazenil (10.0 mg/kg) and beta-CCt (30.0 mg/kg) on the effects of the two agonists were also examined. In the schedule 2 x 30 trials, drugs were administered i.p. 20 min before the training session. Avoidance responses in the training session are an anxiety-mediated behavior, whereas performance in the retention session relates to the effects on memory. Flumazenil and beta-CCt did not affect behavior. Midazolam (2.0 mg/kg) facilitated acquisition performance, while DMCM (1.0 and 2.0 mg/kg) induced the opposite effect. Flumazenil antagonized both effects. Beta-CCt potentiated the effect of midazolam, and partly antagonized the effect of DMCM. Midazolam (0.5 and 1.0 mg/kg) and zolpidem (1.0-3.0 mg/kg) impaired, while DMCM (0.1 mg/kg) facilitated the subjects' performance in the retention test. The amnesic effects were attenuated but not fully reversed, while the effect of DMCM was counteracted by both antagonists. The results indicate the alpha1-subunit interferes with the anxiolytic effect of a benzodiazepine site agonist and may contribute to the DMCM-induced anxiogenic effect. It is also substantially involved in the bidirectional memory processing in the active avoidance paradigm.

Bidirectional effects of benzodiazepine binding site ligands in the passive avoidance task: differential antagonism by flumazenil and β-CCt

Recent research on genetically modified mice has attributed the amnesic effect of benzodiazepines mainly to the α 1-containing GABA A receptor subtypes. The pharmacological approach, using subtype selective ligands, is needed to complement genetic studies. We tested the effects of the non-selective antagonist flumazenil (0–20.0 mg/kg), the preferential α 1-subunit selective antagonist β-carboline-3-carboxylate- t-butyl ester (β-CCt) (0–30.0 mg/kg), the non-selective agonist midazolam (0–2.0 mg/kg), the preferential α 1-subunit selective agonist zolpidem (0–3.0 mg/kg), and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM) (0–2.0 mg/kg) in the one-trial step-through passive avoidance task in rats. The compounds were administered intraperitoneally, before the acquisition test. Flumazenil and β-CCt did not affect retention performance. Midazolam and zolpidem induced amnesia in a dose-dependent manner. The complete reversal of amnesia was unattainable. The effects of zolpidem were significantly attenuated by the both, flumazenil (10.0 mg/kg) and β-CCt (30.0 mg/kg); by contrast, only flumazenil was considerably effective when combined with midazolam. DMCM exerted promnesic effects at 0.2 mg/kg, in an inverted U-shape manner. Both antagonists tended to abolish this action. The results indicate that some other α-subunit(s), in addition to the α 1-subunit, contribute to the amnesic actions of non-selective benzodiazepine site agonists in the passive avoidance task. On the other hand, a significant part of the DMCM-induced promnesic effect could involve the α 1-subunit and/or other putative β-CCt-sensitive binding site(s).

Bidirectional effects of benzodiazepine binding site ligands in the elevated plus-maze: differential antagonism by flumazenil and β-CCt

Recent research using genetically modified mice has pointed to the specific contribution of individual receptor subtypes to the various effects of benzodiazepines. The aim of this study was to examine the relative significance of α 1-containing GABA A receptors in the effects of modulators at the benzodiazepine site in the elevated plus-maze (EPM) under dim red light in rats. We tested the effects of the non-selective antagonist flumazenil (0–20.0 mg/kg), the preferential α 1-subunit selective antagonist β-carboline-3-carboxylate- t-butyl ester (β-CCt, 0–30.0 mg/kg), the non-selective agonist midazolam (0–2.0 mg/kg), the preferential α 1-subunit selective agonist zolpidem (0–2.0 mg/kg) and the non-selective inverse agonist methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, 0–2.0 mg/kg). The influence of flumazenil (10.0 mg/kg) and β-CCt (30.0 mg/kg) on the effects of both kinds of agonists were also examined. The standard spatio-temporal parameters reflecting anxiety (percentage of open arm entries and time) and locomotion (closed and total arm entries) were analyzed. β-CCt did not affect behavior, while flumazenil at the highest dose (20.0 mg/kg) decreased indices of open arm activity and total arm entries. Midazolam at the dose of 1.0 mg/kg significantly increased the percentage of open arm time, whereas at 2.0 mg/kg both anxiety-related parameters were increased. In contrast to the open arm entries, the open arm time was independent of the decreased closed arm entries, observed at 2.0 mg/kg. Flumazenil abolished these effects, whereas β-CCt partially potentiated the anxiolytic actions of midazolam. Zolpidem significantly increased both open-arm indices at 1.0 mg/kg, but the effect was dependent on the decreased closed arm entries. The selectivity of the anxiolytic-like effects of zolpidem was further checked under brighter white illumination. In these settings, the influence on anxiety-related, but not activity-related parameters, was absent. All of the activity-related effects of midazolam and zolpidem were mainly counteracted by both antagonists. DMCM produced significant anxiogenic effects at 1.0 mg/kg (open arm time) and 2.0 mg/kg (both parameters). β-CCt (30.0 mg/kg) and flumazenil at higher dose (20.0 mg/kg) antagonized the effects of DMCM. The results indicate the anxiolytic effects of a non-selective benzodiazepine site agonist involve a predominant role of subunits other than α 1, whereas the behavioral indices of the anxiolytic-like properties of an α 1-selective ligand, if observed, depend on the experimental settings and the changes in locomotor activity, and hence were behaviorally non-specific. The present results generally correspond well to the behavioral findings with the genetically modified mice. On the other hand, the relative significance of the α 1-subunit in the anxiogenic effects of DMCM could not be clearly deduced.

Affinity of various benzodiazepine site ligands in mice with a point mutation in the GABA A receptor γ2 subunit

The benzodiazepine binding site of GABA A receptors is located at the interface of the α and γ subunits. Certain point mutations in these subunits have been demonstrated to dramatically reduce the affinity of benzodiazepine binding site ligands for these receptors. Recently, mice were generated with a phenylalanine (F) to isoleucine (I) substitution at position 77 in the γ2 subunit of GABA A receptors. Here we tested the potency of 24 benzodiazepine binding site ligands from 16 different structural classes for inhibition of [ 3H]flunitrazepam binding to brain membranes of these γ2F77I mice. Results indicate that the potency of the classical 1,4-benzodiazepines, of the 1,4-thienodiazepine clotiazepam, the 1,5-benzodiazepine clobazam, or the pyrazoloquinoline CGS 9896 is only 2–7-fold reduced by this γ2F77I point mutation. The potency of the imidazopyrimidines Ru 32698, Ru 33203, and Ru 33356, of the imidazoquinoline Ru 31719, or the pyrazolopyridine CGS 20625 is reduced 10–20-fold, whereas the potency of some imidazobenzodiazepines, β-carbolines, cyclopyrrolones, imidazopyridines, triazolopyridazines, or quinolines is 100–1000-fold reduced. Interestingly, the extent of potency reduction induced by the γ2F77I point mutation varied within the structural classes of compounds. Results support and significantly extend previous observations indicating that the residue γ2F77 is important for high affinity binding of some, but not all benzodiazepine site ligands.

2,5-Dihydropyrazolo[4,3- c]pyridin-3-ones: functionally selective benzodiazepine binding site ligands on the GABA A receptor

2,5-Dihydropyrazolo[4,3- c]pyridin-3-ones are GABA A receptor benzodiazepine binding site ligands with functional selectivity for the α3 subtype over the α1 subtype. SAR studies to optimise this functional selectivity are described.

2,5-Dihydropyrazolo64,3-c9pyridin-3-ones: functionally selective benzodiazepine binding site ligands on the GABAA receptor

Graphic 2,5-Dihydropyrazolo[4,3- c ]pyridin-3-ones are GABA A receptor benzodiazepine binding site ligands with functional selectivity for the α3 subtype over the α1 subtype. SAR studies to optimise this functional selectivity are described.

On the Benzodiazepine Binding Pocket in GABAA Receptors

Benzodiazepines are used for their sedative/hypnotic, anxiolytic, muscle relaxant, and anticonvulsive effects. They exert their actions through a specific high affinity binding site on the major inhibitory neurotransmitter receptor, the gamma-aminobutyric acid, type A (GABA(A)) receptor channel, where they act as positive allosteric modulators. To start to elucidate the relative positioning of benzodiazepine binding site ligands in their binding pocket, GABA(A) receptor residues thought to reside in the site were individually mutated to cysteine and combined with benzodiazepine analogs carrying substituents reactive to cysteine. Direct apposition of such reactive partners is expected to lead to an irreversible site-directed reaction. We describe here the covalent interaction of alpha(1)H101C with a reactive group attached to the C-7 position of diazepam. This interaction was studied at the level of radioactive ligand binding and at the functional level using electrophysiological methods. Covalent reaction occurs concomitantly with occupancy of the binding pocket. It stabilizes the receptor in its allosterically stimulated conformation. Covalent modification is not observed in wild type receptors or when using mutated alpha(1)H101C-containing receptors in combination with the reactive ligand pre-reacted with a sulfhydryl group, and the modification rate is reduced by the binding site ligand Ro15-1788. We present in addition evidence that gamma(2)Ala-79 is probably located in the access pathway of the ligand to its binding pocket.

6-Methylapigenin and hesperidin: new valeriana flavonoids with activity on the CNS

Valerian is an ancient tranquillizing drug obtained from the underground organs of several Valeriana species. Its active principles were assumed to be terpenoids in the form of valepotriates and/or as components of the essential oil. However, unknown active compounds were not discarded and synergic effects were suspected. We have recently isolated 6-methylapigenin (MA) from Valeriana wallichii and proved that it is a benzodiazepine binding site (BDZ-bs) ligand [Planta Med. 68 (2002) 934]. The present paper is the first report of the presence of 2 S(−)-hesperidin in valeriana and describes that it has sedative and sleep-enhancing properties. MA, in turn, was found to have anxiolytic properties and was able to potentiate the sleep-enhancing properties of hesperidin (HN). MA and HN are new members of the growing family of natural flavonoids with activity on the CNS, and their properties suggest that they are promising drug leads in the field.

5,7-Dihydroxy-6-methoxyflavone, a benzodiazepine site ligand isolated from Scutellaria baicalensis Georgi, with selective antagonistic properties

As part of an effort to identify naturally occurring GABA A receptor benzodiazepine binding site (BDS) ligands from traditional medicinal herbs, we previously reported that flavonoid derivatives isolated from Scutellaria baicalensis ( S. baicalensis) Georgi exhibited significant affinities for the BDS. The present study describes the characterization of 5,7-dihydroxy-6-methoxyflavone (oroxylin A), one of the major components of the herbal extract. Oroxylin A inhibited [ 3 H ]flunitrazepam binding to rat cerebral cortical membrane with a ic 50 value of 1.09±0.07 μM. A GABA ratio of 1.09±0.04 suggests that oroxylin A interacts as an antagonist at the recognition site. In neuropharmacological studies, oral administration of oroxylin A (3.75–60 mg kg −1) did not result in significant changes in animal models routinely employed for benzodiazepine (BD) evaluation. However, oroxylin A selectively abolished the anxiolytic, myorelaxant and motor incoordination, but not the sedative and anticonvulsant effects elicited by diazepam, a BDS agonist. These results add oroxylin A to the list of CNS active flavonoids, and as the first naturally occurring member endowed with selective antagonistic actions via the BDS.

SSR180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide), a peripheral benzodiazepine receptor ligand, promotes neuronal survival and repair.

In the present study, we have investigated the potential neuroprotective effects of a novel peripheral benzodiazepine binding site (PBR) ligand, 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]indole-1-acetamide (SSR180575), in models of central and peripheral neurodegeneration in vivo and its effect on steroid concentrations in plasma and nervous tissue. SSR180575 shows high affinity (IC(50), 2.5-3.5 nM) and selectivity for the rat and human PBR and potently inhibits the in vivo binding of [(3)H]alpidem to PBR in the rat brain and spleen after oral or i.p. administration (ID(50), 0.1-0.3 mg/kg). In an experimental model of motoneuron degeneration induced by facial nerve axotomy in the immature rat, SSR180575 given i.p. or orally for 8 days rescued facial motoneurons, increasing their survival by 40 to 72% at 6 and 10 mg/kg p.o. b.i.d. Moreover, in this model, SSR180575 (10 mg/kg p.o. b.i.d.) increased by 87% the number of motoneurons immunoreactive to peripherin, a type III intermediate filament, whose expression is up-regulated during nerve regeneration. SSR180575 also improved functional recovery in acrylamide-induced neuropathy in the rat when given therapeutically at 2.5 to 10 mg/kg/day p.o. Furthermore, SSR180575 (3 mg/kg i.p. b.i.d.) accelerated functional recovery of the blink reflex after local injury of the facial nerve in the rat. SSR180575 increased pregnenolone accumulation in the brain and sciatic nerve (+100% at 3 mg/kg i.p.), suggesting that its neuroprotective effects are steroid-mediated. These results indicate that PBR ligands (e.g., SSR180575) promote neuronal survival and repair in axotomy and neuropathy models and have potential for the treatment of neurodegenerative diseases (e.g., peripheral neuropathies or amyotrophic lateral sclerosis).

Influence of benzodiazepine binding site ligands on fear-conditioned contextual memory

Eight compounds that bind to the benzodiazepine binding site on the γ-amino butyric acid A (GABA A) receptor were assessed for their influence on contextual memory, an aspect of memory affected in various cognitive disorders including Alzheimer's disease. Using a Pavlovian fear-conditioning paradigm, each ligand was evaluated in C57Bl/6 mice in regards to its direct affect on contextual memory and whether the ligand could attenuate scopolamine-induced contextual memory impairment. Of the eight ligands tested, one impaired contextual memory (agonist), six attenuated scopolamine-induced contextual memory impairment (inverse agonists), and one antagonized the ability of an inverse agonist to attenuate scopolamine-induced contextual memory impairment. Hence, further demonstrating the bi-directional influence benzodiazepine binding site ligands are able to exert on memory modulation. This study serves as an initial starting point in the development of pharmacological tools to be used in deciphering how GABA A receptors influence contextual memory.

6,3′-Dibromoflavone and 6-Nitro-3′-bromoflavone: New Additions to the 6,3′-Disubstituted Flavone Family of High-Affinity Ligands of the Brain Benzodiazepine Binding Site with Agonistic Properties

6,3′-Dibromoflavone and 6-nitro-3′-bromoflavone inhibited [3H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with Ki values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [3H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3′-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.