A newly developed anesthetic based on a unique chemical core.

Intravenous anesthetic agents are associated with cardiovascular instability and poorly tolerated in patients with cardiovascular disease, trauma, or acute systemic illness. We hypothesized that a new class of intravenous (IV) anesthetic molecules that is highly selective for the slow type of γ-aminobutyric acid type A receptor (GABAAR) could have potent anesthetic efficacy with limited cardiovascular effects. Through in silico screening using our GABAAR model, we identified a class of lead compounds that are N-arylpyrrole derivatives. Electrophysiological analyses using both an in vitro expression system and intact rodent hippocampal brain slice recordings demonstrate a GABAAR-mediated mechanism. In vivo experiments also demonstrate overt anesthetic activity in both tadpoles and rats with a potency slightly greater than that of propofol. Unlike the clinically approved GABAergic anesthetic etomidate, the chemical structure of our N-arylpyrrole derivative is devoid of the chemical moieties producing adrenal suppression. Our class of compounds also shows minimal to no suppression of blood pressure, in marked contrast to the hemodynamic effects of propofol. These compounds are derived from chemical structures not previously associated with anesthesia and demonstrate that selective targeting of GABAAR-slow subtypes may eliminate the hemodynamic side effects associated with conventional IV anesthetics.

Synthesis of 4,6-diphenylpyrimidin-2-ol derivatives as new benzodiazepine receptor ligands.

Benzodiazepines (BZDs) have been widely used in neurological disorders such as insomnia, anxiety, and epilepsy. The use of classical BZDs, e.g., diazepam, has been limited due to adverse effects such as interaction with alcohol, ataxia, amnesia, psychological and physical dependence, and tolerance. In the quest for new benzodiazepine agonists with more selectivity and low adverse effects, novel derivatives of 4,6-diphenylpyrimidin-2-ol were designed, synthesized, and evaluated. In this series, compound 2, 4-(2-(benzyloxy)phenyl)-6-(4-fluorophenyl)pyrimidin-2-ol, was the most potent analogue in radioligand binding assay with an IC50 value of 19 nM compared to zolpidem (IC50 = 48 nM), a nonbenzodiazepine central BZD receptor (CBR) agonist. Some compounds with a variety of affinities in radioligand receptor binding assay were selected for in vivo evaluations. Compound 3 (IC50 = 25 nM), which possessed chlorine instead of fluorine in position 4 of the phenyl ring, exhibited an excellent ED50 value in most in vivo tests. Proper sedative-hypnotic effects, potent anticonvulsant activity, appropriate antianxiety effect, and no memory impairment probably served compound 3, a desirable candidate as a benzodiazepine agonist. The pharmacological effects of compound 3 were antagonized by flumazenil, a selective BZD receptor antagonist, confirming the BZD receptors' involvement in the biological effects of the novel ligand.

GABAA Receptor-Mediated Sleep-Promoting Effect of Saaz-Saphir Hops Mixture Containing Xanthohumol and Humulone.

Hops contain flavonoids that have sedative and sleep-promoting activities such as α-acid, ß-acid, and xanthohumol. In this study, the sleep-enhancing activity of a Saaz-Saphir hops mixture was measured. In the caffeine-induced insomnia model, the administration of a Saaz-Saphir mixture increased the sleep time compared to Saaz or Saphir administration alone, which was attributed to the increase in NREM sleep time by the δ-wave increase. Oral administration of the Saaz-Saphir mixture for 3 weeks increased the γ-amino butyric acid (GABA) content in the brain and increased the expression of the GABAA receptor. As the GABA antagonists picrotoxin and bicuculline showed a decrease in sleep activity, it was confirmed that the GABAA receptor was involved in the Saaz-Saphir mixture activity. In addition, the GABAA receptor antagonist also reduced the sleep activity induced by xanthohumol and humulone contained in the Saaz-Saphir mixture. Therefore, xanthohumol and humulone contained in the Saaz-Saphir mixture showed sleep-promoting activity mediated by the GABAA receptors. The mixture of the Saaz and Saphir hop varieties may thus help mitigate sleep disturbances compared to other hop varieties.

A multiorganism pipeline for antiseizure drug discovery: Identification of chlorothymol as a novel γ-aminobutyric acidergic anticonvulsant.

OBJECTIVE: Current medicines are ineffective in approximately one-third of people with epilepsy. Therefore, new antiseizure drugs are urgently needed to address this problem of pharmacoresistance. However, traditional rodent seizure and epilepsy models are poorly suited to high-throughput compound screening. Furthermore, testing in a single species increases the chance that therapeutic compounds act on molecular targets that may not be conserved in humans. To address these issues, we developed a pipeline approach using four different organisms. METHODS: We sequentially employed compound library screening in the zebrafish, Danio rerio, chemical genetics in the worm, Caenorhabditis elegans, electrophysiological analysis in mouse and human brain slices, and preclinical validation in mouse seizure models to identify novel antiseizure drugs and their molecular mechanism of action. RESULTS: Initially, a library of 1690 compounds was screened in an acute pentylenetetrazol seizure model using D rerio. From this screen, the compound chlorothymol was identified as an effective anticonvulsant not only in fish, but also in worms. A subsequent genetic screen in C elegans revealed the molecular target of chlorothymol to be LGC-37, a worm γ-aminobutyric acid type A (GABAA ) receptor subunit. This GABAergic effect was confirmed using in vitro brain slice preparations from both mice and humans, as chlorothymol was shown to enhance tonic and phasic inhibition and this action was reversed by the GABAA receptor antagonist, bicuculline. Finally, chlorothymol exhibited in vivo anticonvulsant efficacy in several mouse seizure assays, including the 6-Hz 44-mA model of pharmacoresistant seizures. SIGNIFICANCE: These findings establish a multiorganism approach that can identify compounds with evolutionarily conserved molecular targets and translational potential, and so may be useful in drug discovery for epilepsy and possibly other conditions.

Crystal structure of a human GABAA receptor.

Type-A γ-aminobutyric acid receptors (GABAARs) are the principal mediators of rapid inhibitory synaptic transmission in the human brain. A decline in GABAAR signalling triggers hyperactive neurological disorders such as insomnia, anxiety and epilepsy. Here we present the first three-dimensional structure of a GABAAR, the human ß3 homopentamer, at 3 Å resolution. This structure reveals architectural elements unique to eukaryotic Cys-loop receptors, explains the mechanistic consequences of multiple human disease mutations and shows an unexpected structural role for a conserved N-linked glycan. The receptor was crystallized bound to a previously unknown agonist, benzamidine, opening a new avenue for the rational design of GABAAR modulators. The channel region forms a closed gate at the base of the pore, representative of a desensitized state. These results offer new insights into the signalling mechanisms of pentameric ligand-gated ion channels and enhance current understanding of GABAergic neurotransmission.

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABAA Receptor.

Analgesic and anti-inflammatory properties mediated by the κ opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, µ opioid receptor (MOR), and δ opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC50 of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with 3H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABAAR) of the α1-3ß2-3γ1-2 subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABAARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors.

Design, synthesis, in silico ADMET profile and GABA-A docking of novel phthalazines as potent anticonvulsants.

A new series of 2-substituted-2,3-dihydrophthalazine-1,4-diones (2- 9) were designed and synthesized to evaluate their anticonvulsant activity. The neurotoxicity was assessed using the rotarod test. Molecular docking was performed for the synthesized compounds to assess their binding affinities as γ-aminobutyric acid A (GABA-A) receptor agonists as a possible mechanism of their anticonvulsant action, to rationalize their anticonvulsant activity in a qualitative way. The data obtained from the molecular modeling was strongly matched with that obtained from the biological screening, which revealed that compounds 5a , 9b , and 9h showed the highest binding affinities toward the GABA-A receptor and also showed the highest anticonvulsant activities with relative potencies of 1.66, 1.63, and 1.61, respectively, compared with diazepam. The most active compounds 5a , 9b , and 9h were further tested against maximal electroshock seizures. Compounds 5a and 9b showed 100% protection at a dose level of 125 µg/kg, while compound 9h exhibited 83.33% protection at the same dose level. A GABA enzymatic assay was performed for these highly active compounds to confirm the obtained results and to explain the possible mechanism for their anticonvulsant action. These agents exerted low neurotoxicity and a high safety margin compared with valproate as a reference drug. Most of our designed compounds exhibited a good ADMET profile.

Synthesis of New GABAA Receptor Modulator with Pyrazolo[1,5-a]quinazoline (PQ) Scaffold.

We previously published a series of 8-methoxypirazolo[1,5-a]quinazolines (PQs) and their 4,5-dihydro derivatives (4,5(H)PQ) bearing the (hetero)arylalkylester group at position 3 as ligands at the γ-aminobutyric type A (GABAA) subtype receptor. Continuing the study in this field, we report here the design and synthesis of 3-(hetero)arylpyrazolo[1,5-a]quinazoline and 3-(hetero)aroylpyrazolo[1,5-a]quinazoline 8-methoxy substituted as interesting analogs of the above (hetero)arylalkylester, in which the shortening or the removal of the linker between the 3-(hetero)aryl ring and the PQ was performed. Only compounds that are able to inhibit radioligand binding by more than 80% at 10 µM have been selected for electrophysiological studies on recombinant α1ß2γ2L GABAA receptors. Some compounds show a promising profile. For example, compounds 6a and 6b are able to modulate the GABAAR in an opposite manner, since 6b enhances and 6a reduces the variation of the chlorine current, suggesting that they act as a partial agonist and an inverse partial agonist, respectively. The most potent derivative was 3-(4-methoxyphenylcarbonyl)-8-methoxy-4,5-dihydropyrazolo[1,5-a] quinazoline 11d, which reaches a maximal activity at 1 µM (+54%), and it enhances the chlorine current at ≥0.01 µM. Finally, compound 6g, acting as a null modulator at α1ß2γ2L, shows the ability to antagonize the full agonist diazepam and the potentiation of CGS 9895 on the new α+/ß- 'non-traditional' benzodiazepine site.

Structure-Dependent Activity of Natural GABA(A) Receptor Modulators.

GABA(A) receptors are ligand-gated ion channels consisting of five subunits from eight subfamilies, each assembled in four hydrophobic transmembrane domains. This pentameric structure not only allows different receptor binding sites, but also various types of ligands, such as orthosteric agonists and antagonists, positive and negative allosteric modulators, as well as second-order modulators and non-competitive channel blockers. A fact, that is also displayed by the variety of chemical structures found for both, synthetic as well as nature-derived GABA(A)-receptor modulators. This review covers the literature for natural GABA(A)-receptor modulators until the end of 2017 and discusses their structure-activity relationship.

Efficient synthesis of the GABAA receptor agonist trans-4-aminocrotonic acid (TACA).

Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.

Discovery of the first low-shift positive allosteric modulators for the muscarinic M1 receptor.

Positive modulation of the muscarinic M1-receptor has for a long time attracted scientists and drug developers for the potential treatment of Alzheimer's disease or Schizophrenia. The precognitive potential of M1 activation has however not been clinically demonstrated as a result of side effects associated both with agonists and positive allosteric modulators (PAM's) of the M1-receptor. To avoid excessive activation of the M1-receptor we have designed a new screening format and developed the first low-shift positive allosteric modulators for the M1 receptor. Low-shift PAM's offer the potential of "use-dependent" attenuation of transmitter-signaling while avoiding pseudo-agonistic behavior in vivo as a common limitation of the so far described high-shift PAM's. With these novel M1-PAM's, the M1 receptor is potentially the first GPCR for which both, high- and low shift PAM's have become available.

Arginine derivatives of dicarboxylic acids from the parotid gland secretions of common toad Bufo bufo-New agonists of ionotropic γ-aminobutyric acid receptors.

Compounds activating γ-aminobutyric acid type A receptor were isolated from the toad Bufo bufo venom as a result of chromatographic separation. Analysis of the structure of these compounds by mass spectrometry and nuclear magnetic resonance showed that they are arginine derivatives of dicarboxylic acids and represent suberylarginine, pimeloylarginine, and adipoylarginine.

Synthesis, biological evaluation and molecular modeling study of some new thiazolodiazepine analogs as CNS active agents.

New derivatives of ethyl 8-oxo-5,6,7,8-tetrahydro-thiazolo[3,2-a][1,3]diazepin-3-carboxylate (HIE-124, 3), were synthesized as continuation to our previous patented efforts. Compounds 15 and 20 showed marginal hypnotic potency compared to 3. Compounds 15 (0.78mmol/kg) and 20 (0.39mmol/kg) showed remarkable 100% protection against PTZ induced convulsions with two and four fold increase in activity than sodium valproate (1.38mmol/kg), respectively. Molecular modeling studies showed hydrogen bonding interaction between 15 and Thr56 residues at the binding site of GABAA. Superposition, flexible alignment and surface mapping of 15, 20 and diazepam supports their biological resemblance where ADMET study suggested that those compounds could be used as oral anticonvulsants.

Moderate concentrations of 4-O-methylhonokiol potentiate GABAA receptor currents stronger than honokiol.

BACKGROUND: Magnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABAA receptors. 4-O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABAA receptors and its GABAA receptor subtype-specificity was established. METHODS: Different recombinant GABAA receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4-O-methylhonokiol. RESULTS: 3µM 4-O-methylhonokiol is shown here to potentiate responses of the α1ß2γ2 GABAA receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4-O-methylhonokiol is also detailed for 12 GABAA receptor subtypes to assess GABAA receptor subunits that are responsible for the potentiating effect. CONCLUSION: The much higher potentiation of GABAA receptors at identical concentrations of 4-O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4-O-methylhonokiol over honokiol. GENERAL SIGNIFICANCE: The results point to the use of 4-O-methylhonokiol as a lead for GABAA receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine.

Nitrogenated honokiol derivatives allosterically modulate GABAA receptors and act as strong partial agonists.

In traditional Asian medicinal systems, preparations of the root and stem bark of Magnolia species are widely used to treat anxiety and other nervous disturbances. The biphenyl-type neolignan honokiol together with its isomer magnolol are the main constituents of Magnolia bark extracts. We have previously identified a nitrogen-containing honokiol derivative (3-acetylamino-4'-O-methylhonokiol, AMH) as a high efficient modulator of GABAA receptors. Here we further elucidate the structure-activity relation of a series of nitrogenated biphenyl-neolignan derivatives by analysing allosteric modulation and agonistic effects on α1ß2γ2S GABAA receptors. The strongest IGABA enhancement was induced by compound 5 (3-acetamido-4'-ethoxy-3',5-dipropylbiphenyl-2-ol, Emax: 123.4±9.4% of IGABA-max) and 6 (5'-amino-2-ethoxy-3',5-dipropylbiphenyl-4'-ol, Emax: 117.7±13.5% of IGABA-max). Compound 5 displayed, however, a significantly higher potency (EC50=1.8±1.1 µM) than compound 6 (EC50=20.4±4.3 µM). Honokiol, AMH and four of the derivatives induced significant inward currents in the absence of GABA. Strong partial agonists were honokiol (inducing 78±6% of IGABA-max), AMH (63±6%), 5'-amino-2-O-methylhonokiol (1) (59±1%) and 2-methoxy-5'-nitro-3',5-dipropylbiphenyl-4'-ol (3) (52±1%). 3-N-Acetylamino-4'-ethoxy-3',5-dipropyl-biphenyl-4'-ol (5) and 3-amino-4'-ethoxy-3',5-dipropyl-biphenyl-4'-ol (7) were less efficacious but even more potent (5: EC50=6.9±1.0 µM; 7: EC50=33.2±5.1 µM) than the full agonist GABA.

Novel agonists of benzodiazepine receptors: design, synthesis, binding assay and pharmacological evaluation of 1,2,4-triazolo[1,5-a]pyrimidinone and 3-amino-1,2,4-triazole derivatives.

Agonists of benzodiazepine (BZD) binding site in GABA receptors are widely used in clinical practice. In spite of their benefits they have several side effects, so synthesis of new agonists of these receptors to get more specific effect and better profile of adverse drug reactions is still continued. Novel BZD agonists were designed based on the pharmacophore/receptor model of BZD binding site of GABAA receptor. Energy minima conformers of the designed compounds and estazolam, a known BZD receptor agonist, were well superimposed in conformational analysis. Docking studies revealed that the carbonyl group of the compound 4c, 3-(2-chlorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one, was near the nitrogen moiety of triazole ring of estazolam providing the hydrogen bond acceptor in proper direction in the BDZ-binding site of GABAA receptor model (α1ß2ϒ2). The designed compounds were synthesized and their in vitro affinity for the central BZD receptor was determined. Most of the novel compounds had better affinity for the BZD site of action on GABAA receptor complex than diazepam. Finally, the novel compound 4c with the best affinity in radioligand receptor binding assay (Ki=0.42 nM and IC50=0.68 nM) was selected as candidate for in vivo evaluation. This compound showed significant hypnotic activity and weak anticonvulsant effect with no impairment on learning and memory performance in mouse. The pharmacological effects of the compound 4c were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects of the novel ligand.

Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands.

Benzodiazepines exert their anxiolytic, anticonvulsant, muscle-relaxant and sedative-hypnotic properties by allosterically enhancing the action of GABA at GABA(A) receptors via their benzodiazepine-binding site. Although these drugs have been used clinically since 1960, the molecular basis of this interaction is still not known. By using multiple homology models and an unbiased docking protocol, we identified a binding hypothesis for the diazepam-bound structure of the benzodiazepine site, which was confirmed by experimental evidence. Moreover, two independent virtual screening approaches based on this structure identified known benzodiazepine-site ligands from different structural classes and predicted potential new ligands for this site. Receptor-binding assays and electrophysiological studies on recombinant receptors confirmed these predictions and thus identified new chemotypes for the benzodiazepine-binding site. Our results support the validity of the diazepam-bound structure of the benzodiazepine-binding pocket, demonstrate its suitability for drug discovery and pave the way for structure-based drug design.

Enhancement of GABAergic activity: neuropharmacological effects of benzodiazepines and therapeutic use in anesthesiology.

GABA is the major inhibitory neurotransmitter in the central nervous system (CNS). The type A GABA receptor (GABA(A)R) system is the primary pharmacological target for many drugs used in clinical anesthesia. The α1, ß2, and γ2 subunit-containing GABA(A)Rs located in the various parts of CNS are thought to be involved in versatile effects caused by inhaled anesthetics and classic benzodiazepines (BZD), both of which are widely used in clinical anesthesiology. During the past decade, the emergence of tonic inhibitory conductance in extrasynaptic GABA(A)Rs has coincided with evidence showing that these receptors are highly sensitive to the sedatives and hypnotics used in anesthesia. Anesthetic enhancement of tonic GABAergic inhibition seems to be preferentially increased in regions shown to be important in controlling memory, awareness, and sleep. This review focuses on the physiology of the GABA(A)Rs and the pharmacological properties of clinically used BZDs. Although classic BZDs are widely used in anesthesiological practice, there is a constant need for new drugs with more favorable pharmacokinetic and pharmacodynamic effects and fewer side effects. New hypnotics are currently developed, and promising results for one of these, the GABA(A)R agonist remimazolam, have recently been published.

Identification of the metabolites of lesogaberan using linear trap quadrupole orbitrap mass spectrometry and hydrophilic interaction liquid chromatography.

1. In this study, hydrophilic interaction liquid chromatography (HILIC), radiochemical activity monitoring and linear trap quadrupole orbitrap mass spectrometry (MS) and tandem mass spectrometry (MS/MS) were used to identify the metabolites of a highly polar novel γ-aminobutyric acid type-B receptor agonist, lesogaberan, in rats. 2. Urine was collected from three male Wistar rats for 24 h after dosing with (14)C-labelled lesogaberan (170 mg/kg, 10 MBq/kg); plasma samples were taken 2 and 24 h after dosing. Pooled samples were separated by HILIC and eluents were analysed by radiochemical activity monitoring, MS and MS/MS. 3. Only the parent compound was detected in plasma, but six metabolites (M1-M6) were detected in urine. Analysis of MS and MS/MS data and comparison with synthetic reference standards enabled the identification of the structure of each metabolite. M1 was identified as the N-acetylated species [(2R)-3-acetamido-2-fluoropropyl]-phosphinic acid, and M6 as [(2R)-3-amino-2-fluoropropyl]-phosphonic acid. Metabolites M2 and M5 were the alcohol and carboxylic acid species 3-hydroxypropyl-phosphinic acid and 3-hydroxyphosphonoyl-propanoic acid, respectively, both of which had lost the fluorine atom present in the parent compound. M3 was the corresponding carboxylic acid species retaining the fluorine atom, (2R)-2-fluoro-3-hydroxyphosphonoyl-propanoic acid. Finally M4 was identified as [(2R)-2-fluoro-3-guanidino-propyl]-phosphinic acid.

Early pharmaceutical evaluation of a crystalline and hygroscopic GABAB receptor agonist.

Lesogaberan is a potent gamma amino butyric acid agonist and has been evaluated for its utility in treatment of gastroesophageal reflux disease. Lesogaberan is a crystalline substance that absorbs considerable amounts of water above 65% relative humidity (RH) where it also liquifies. As a result of the hygroscopicity of the zwitterionic form an investigation of different salt forms was performed. Since the test compound is polar and lacks ultraviolet (UV) chromophore, conventional separation and detection techniques could not be used to characterise the test compound and the impurities. The analytical techniques are described, focusing on the capillary electrophoresis method with indirect UV detection for purity, the liquid chromatographic method for enantiomeric separation with derivatisation with UV chromophore and two complementary nuclear magnetic resonance (NMR) approaches (¹9F-NMR and ¹H-NMR) for impurities. The stability study in solution showed that solutions between pH 5 and 7 were the most stable ones, but after some time degradation occurred at room temperature. When bulk lesogaberan was stored at 25°C/60% RH no chemical degradation was observed after 1 year. At 40°C/75% RH, where the compound liquefies, a significant degradation was observed after 1 month. However, in a closed container (= 40°C) or as a napsylate salt, no degradation of lesogaberan was observed at 40°C/75% RH.