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Tetramethylenedisulfotetramine (TETS) is a so-called "caged" convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABA A ) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α 2 β 3 γ 2 GABA A receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α 2 β 3 γ 2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6' ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6' in either the α 2 or β 3 subunit reduces the IC 50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABA A receptor at a location that is overlapping but not identical to the picrotoxinin binding site. SIGNIFICANCE STATEMENT: Our study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α 2 β 3 γ 2 GABA receptor type A receptor at the so-called T6' ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel., (Copyright © 2020 by The Author(s).)

This paper focuses on determining the structural similarity of two molecules, i.e., the similarity of the interconnection of all the elementary cycles in the corresponding molecular graphs. In this paper, we propose and analyze an algorithmic approach based on the resolution of the Maximum Common Edge Subgraph (MCES) problem with graphs representing the interaction of cycles molecules. Using the ChEBI database, we compare the effectiveness of this approach in terms of structural similarity and computation time with two calculations of similarity of molecular graphs, one based on the MCES, the other on the use of different fingerprints (Daylight, ECFP4, ECFP6, FCFP4, FCFP6) to measure Tanimoto coefficient. We also analyze the obtained structural similarity results for a selected subset of molecules., Competing Interests: The authors have declared that no competing interests exist.

Technologies for mapping the spatial and temporal patterns of neural activity have advanced our understanding of brain function in both health and disease. An important application of these technologies is the discovery of next-generation neurotherapeutics for neurological and psychiatric disorders. Here, we describe an in vivo drug screening strategy that combines high-throughput technology to generate large-scale brain activity maps (BAMs) with machine learning for predictive analysis. This platform enables evaluation of compounds' mechanisms of action and potential therapeutic uses based on information-rich BAMs derived from drug-treated zebrafish larvae. From a screen of clinically used drugs, we found intrinsically coherent drug clusters that are associated with known therapeutic categories. Using BAM-based clusters as a functional classifier, we identify anti-seizure-like drug leads from non-clinical compounds and validate their therapeutic effects in the pentylenetetrazole zebrafish seizure model. Collectively, this study provides a framework to advance the field of systems neuropharmacology.

The 'neurotrophic sesquiterpenes' refer to a group of molecules derived from the Illicium genus of flowering plant. They display neurotrophic effects in cultured neuron preparations and have been suggested to be cognitive enhancers and potential therapeutics for neurodegenerative disorders and dementias. Recent synthetic advances generated sufficient quantities of jiadifenolide for in vivo investigation into its biological effects. Jiadifenolide did not induce convulsions in mice nor did it enhance or diminish convulsions induced by pentylenetetrazole. Other negative allosteric modulators of GABA A receptors, picrotoxin, tetramethylenedisulfotetramine (TETS), and bilobalide all induced convulsions. Either i.p. or i.c.v. dosing generated micromolar plasma and brain levels of jiadifenolide but only small effects on locomotion of mice. However, jiadifenolide decreased d-amphetamine-induced hyperlocomotion in mice, an antipsychotic-like drug effect. Jiadifenolide did not significantly alter body temperature or behavior in the forced-swim test in mice. Molecular simulation data suggested a potential site in the pore/M2 helix region that is at an overlapping, yet lower position than those observed for other 'cage convulsant' compounds such as TETS and picrotoxin. We hypothesize that a position nearer to the entrance of the pore channel may allow for easier displacement of jiadifenolide from its blocking location leading to lower potency and lower side-effect liability. Like jiadifenolide, memantine (Namenda), one of the few drugs used in the symptomatic treatment of dementias, occupies a unique site on the NMDA receptor complex that creates low binding affinity that is associated with its reduced side-effect profile. Given the potential therapeutic applications of jiadifenolide and its relatively inert effects on overt behavior, the possibility of clinical utility for jiadifenolide and related compounds becomes intriguing., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Drug-induced seizure liabilities produce significant compound attrition during drug discovery. Currently available in vitro cytotoxicity assays cannot predict all toxicity mechanisms due to the failure of these assays to predict sublethal target-specific electrophysiological liabilities. Identification of seizurogenic and other electrophysiological effects at early stages of the drug development process is important to ensure that safe candidate compounds can be developed while chemical design is taking place, long before these liabilities are discovered in costly preclinical in vivo studies. The development of a high throughput and reliable in vitro assay to screen compounds for seizure liabilities would de-risk compounds significantly earlier in the drug discovery process and with greater dependability. Here we describe a method for screening compounds that utilizes rat cortical neurons plated onto multiwell microelectrode array plates to identify compounds that cause neurophysiological disruptions. Changes in 12 electrophysiological parameters (spike train descriptors) were measured after application of known seizurogenic compounds and the response pattern was mapped relative to negative controls, vehicle control and neurotoxic controls. Twenty chemicals with a variety of therapeutic indications and targets, including GABAA antagonists, glycine receptor antagonists, ion channel blockers, muscarinic agonist, δ-opioid receptor agonist, dopaminergic D2/adrenergic receptor blocker and nonsteroidal anti-inflammatory drugs, were tested to assess this system. Sixteen of the seventeen seizurogenic/neurotoxic compounds tested positive for seizure liability or neurotoxicity, moreover, different endpoint response patterns for firing rate, burst characteristics and synchrony that distinguished the chemicals into groups relating to target and seizurogenic response emerged from the data. The negative and vehicle control compounds had no effect on neural activity. In conclusion, the multiwell microelectrode array platform using cryopreserved rat cortical neurons is a highly effective high throughput method for reliably screening seizure liabilities within an early de-risking drug development paradigm.

Herein we describe the structure activity relationships uncovered in the pursuit of an mGluR5 positive allosteric modulator (PAM) for the treatment of schizophrenia. It was discovered that certain modifications of an oxazolidinone-based chemotype afforded predictable changes in the pharmacological profile to give analogs with a wide range of functional activities. The discovery of potent silent allosteric modulators (SAMs) allowed interrogation of the mechanism-based liabilities associated with mGluR5 activation and drove our medicinal chemistry effort toward the discovery of low efficacy (fold shift) PAMs devoid of agonist activity. This work resulted in the identification of dipyridyl 22 (BMS-952048), a compound with a favorable free fraction, efficacy in a rodent-based cognition model, and low potential for convulsions in mouse., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Covering: 1860-2016A mechanistic link may exist between convulsant plant substances typified by picrotoxinin, and 'neurotrophic' sesquiterpenes like jiadifenolide. Picrotoxinin elicits convulsion by anion blockade of the Cys-loop family of neurotransmitter-gated ion channels. These same receptors mediate neuronal development and neurite outgrowth prior to synapse formation. Due to its structural homology with picrotoxin and anisatin, it is possible that jiadifenolide enhances NGF-stimulated neurite outgrowth by modulation of the Cys-loop family of receptors.

Key Points: Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood. We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABAA receptors. In mice the S-enantiomer is a convulsant, but the R-enantiomer is an anticonvulsant. The convulsant S-enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R-enantiomer inhibits with equal affinity. In contrast, only the anticonvulsant R-enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective. The in vivo actions of the convulsant S-enantiomer are accounted for by its interactions with GABAA receptors., Abstract: Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABAA receptors (GABAA Rs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1-methyl-5-propyly-5-(m-trifluoromethyldiazirinyl) phenyl barbituric acid (mTFD-MPPB). In mice, S-mTFD-MPPB acted as a convulsant, whereas R-mTFD-MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α1 β3 γ2L GABAA Rs expressed in HEK cells, we found that S-mTFD-MPPB inhibited GABA-induced currents, whereas R-mTFD-MPPB enhanced them. S-mTFD-MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration-response curve to the right. R-mTFD-MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate-limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration-response curve to the left. Under conditions when most GABAA Rs were open, an inhibitory action of R-mTFD-MPPB was revealed that had a similar IC50 to that of S-mTFD-MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S-mTFD-MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABAA Rs underlie the enantiomers' different pharmacological activities in mice., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

Together with NOE and J coupling, one-bond residual dipolar coupling (RDC), which reports on the three-dimensional orientation of an internuclear vector in the molecular frame, plays an important role in the conformation and configuration analysis of small molecules in solution by NMR spectroscopy. When the molecule has few C-H bonds, or too many bonds are in parallel, the available RDCs may not be sufficient to obtain the alignment tensor used for structure elucidation. Long-range RDCs that connect nuclei over multiple bonds are normally not parallel to the single bonds and therefore complement one-bond RDCs. Herein we present a method for extracting the long-range RDC of a chosen proton or group of protons to all remotely connected carbon atoms, including non-protonated carbon atoms. Alignment tensors fitted directly to the total long-range couplings (T=J+D) enabled straightforward analysis of both the long-range and one-bond RDCs for strychnine., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The density functional theoretical (DFT) computations were performed at the B3LYP/6-311G++(d, p) level to calculate the equilibrium geometry, vibrational wave numbers, intensities, and various other molecular properties of brucine and strychnine, which were found in satisfactory agreement with the experimental data. The out-of-phase stretching modes of aromatic rings and carbonyl stretching modes in combination with CH stretching modes at stereogenic centers generate VCD signals, which are remarkably efficient configuration markers for these chiral molecular systems. NBOs analysis reveals that the large values of second order perturbation energy (47.24kcal/mol for brucine and 46.93kcal/mol for strychnine) confirms strong hyperconjugative interaction between the orbital containing the lone pair of electron of nitrogen and the neighboring CO antibonding orbital. The molecular electrostatic potential map of strychnine molecule, with no polar groups other than the lone keto group, shows less polarization, which accounts for its lower susceptibility towards electrophilic attack as compared to brucine., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A series of ring-constrained phenylpropyloxyethylamines, partial opioid structure analogs and derivatives of a previously studied sigma (σ) receptor ligand, was synthesized and evaluated at σ and opioid receptors for receptor selectivity. The results of this study identified several compounds with nanomolar affinity at both σ receptor subtypes. Compounds 6 and 9 had the highest selectivity for both σ receptor subtypes, compared to μ opioid receptors. In addition, compounds 6 and 9 significantly reduced the convulsive effects of cocaine in mice, which would be consistent with antagonism of σ receptors., (Copyright © 2013. Published by Elsevier Ltd.)

A user-friendly 2D NMR approach denoted as CLIP-HSQMBC is proposed for the very easy, direct and accurate measurement of long-range proton-carbon coupling constants in organic molecules and natural products. The J value can be extracted directly from the analysis of resolved in-phase (1)H multiplets that show an additional splitting arising from the proton-carbon coupling. In cases of unresolved peaks, a simple fitting analysis using the internal satellite lines as a reference is performed. Addition of a spin-lock period results in a CLIP-HSQMBC-TOCSY experiment that is suitable for the measurement of very small coupling values or to observe correlations from overlapped resonances.

Oenanthotoxin (OETX) and dihydro-OETX are polyacetylenic diols occurring in Oenanthe crocata and are known to exert proconvulsant effects. We have recently demonstrated that these compounds downregulated GABAergic currents (Appendino et al., 2009) and that OETX induced open channel block and allosterically modulated GABA(A) receptors (Wyrembek et al., 2010). O. crocata also contains several minor OETX analogues and in the present study we tested whether their effect on GABA(A) receptors depends on the compounds' polarity. We investigated a series of five polyacetylenes characterized by a higher lipophylicity than OETX, (1-acetyl-2,3-dihydrooenanthotoxin - X1, 14-acetyloenanthotoxin-X2, 1-deoxyoenanthotoxin - X3, 14-deoxyoenanthotoxin - X4, 14-dehydro-1-deoxyOETX - X5, polarity sequence: X1>X2>X3>X4>X5). Their effects were tested first on miniature inhibitory postsynaptic currents (mIPSCs). All but X3, significantly decreased the mIPSC amplitudes while X1, X2, X4 decreased, and X3 and X5 increased the mIPSC frequency. The lack of a clear correlation between the compounds' polarity and their effect on mIPSCs might result from their presynaptic effects. We thus considered their impact on current responses to exogenous GABA applications. Amplitude reduction of current responses was most prominent for X1 and virtually absent for X5 indicating a dependence on the compound's polarity. Only X1 and X2 showed open channel block, while the kinetics of currents were affected only by X1 which further supports a dependence of the drug's effects on their polarity. In conclusion, GABA(A) receptors are inhibited and allosterically modulated by naturally occurring OETX analogues (except X5) and these effects are positively correlated with the compounds' polarity., (Copyright © 2012. Published by Elsevier B.V.)

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3' and 5' RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of robust and predictive QSAR models is highly dependent on the use of molecular descriptors that contain information relevant to the property being modelled. Selection of these relevant features from a large pool of possibilities is difficult to achieve effectively. Modern Bayesian methods provide substantial advantages over conventional feature selection methods for feature selection and QSAR modelling. We illustrate the importance of descriptor choice and the beneficial properties of Bayesian methods to select context-dependent relevant descriptors and build robust QSAR models, using data on anaesthetics. Our results show the effectiveness of Bayesian feature selection methods in choosing the best descriptors when these are mixed with less informative descriptors. They also demonstrate the efficacy of the Abraham descriptors and identify deficiencies in ParaSurf descriptors for modelling anaesthetic action.

The convulsant activity of 48 compounds studied by Eger et al. has been analyzed using an Abraham solvation equation. Four compounds identified by Eger et al. as more potent than expected were similarly identified, and for the remaining 44 compounds, the equation had R(2)=0.978 and a standard deviation of 0.17 log units. The structural features of compounds that lead to convulsant activity or to anesthetic activity are uncovered, and used to explain which compounds will act as convulsants and which compounds will act as anesthetics. The present equation for convulsant activity and our previous equation for inhalation anesthetic activity can be used to predict convulsant pressure and anesthetic pressure. For all 48 compounds, the predicted convulsant pressure is less than the predicted anesthetic pressure, in agreement with experiment. Although convulsants tend to have a small hydrogen bond acidity, this is not the only factor that influences the effect. The difference between the compound pressure needed to produce convulsions and that needed to produce anesthesia is quite small for many of the 48 compounds, and so minor structural features can be significant. These structural features are incorporated into our equations that successfully predict convulsant/anesthetic effects for the 48 compounds.

Recently we identified (R,S)-2-acetyl-1-(4'-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (6) as a potent non-competitive AMPA receptor antagonist able to prevent epileptic seizures. We report here the optimized synthesis of compound 6, its resolution by chiral preparative HPLC, and the absolute configuration of (R)-enantiomer established by X-ray diffractometry. The biological tests of the single enantiomers revealed that higher anticonvulsant and antagonistic effects reside in (R)-enantiomer as also suggested by molecular modeling studies.

It has been reported that 2-(4-substituted thiazol-2-ylthio)-1beta-methyl-carbapenems exhibit potent activity against methicillin-resistant staphylococci (MRS) and vancomycin-resistant enterococci (VRE). In order to develop a novel broad-spectrum carbapenem, the structure-activity relationships of a series of 2-(4-tetrahydropyridinylthiazol-2-ylthio)-1beta-methylcarbapenems and 4-dihydropyrrolyl thiazole analogs were investigated with regard to their activity against Gram-positive and especially Gram-negative bacteria and also their convulsant activity, which is a major side effect concern of carbapenems. The introduction of substituent(s) on the dihydropyrrole moiety did not cause remarkable changes in anti-MRS and VRE activities, but tended to lower the anti-Gram-negative bacterial activity except in some cases of methyl group introduction. These substitutions did however cause a reduction of the convulsant activity, which was affected by the size and also the configuration of the substituent. In the case of SM-216601 (6), introduction of a methyl group brought about significant reduction in neurotoxicity while maintaining favorable anti-Gram-negative bacterial activity.

Two glutamic acid analogs, (+)-(S)- and (-)-(R)-4-(2,2-diphenyl-1,3,2-oxazaborolidin-5-oxo)propionic acid ((+)-(S)- and (-)-(R)-Trujillon, respectively), were prepared. The stereospecific activity of their pharmacological properties was studied. The median convulsant dose (CD(50)) and median lethal dose (LD(50)) were analyzed in female Swiss Webster mice and their effects in vivo on unitary electrical activity in globus pallidus neurons were elucidated in male Wistar rats. Compounds were characterized by (1)H, (13)C, and (11)B nuclear magnetic resonance. The LD(50) of (+)-(S)-Trujillon was 449.08 mg/kg and it increased spontaneous motor activity, while with (-)-(R)-Trujillon there was no mortality up to 1,000 mg/kg and it decreased spontaneous motor activity. The CD(50) in experiments with (+)-(S)-Trujillon was 199.34 mg/kg. Unitary recording in globus pallidus neurons showed i.v. administration (+)-(S)-Trujillon (50 mg/kg) increased frequency 79.0 +/- 23.0% in relation to basal response. (-)-(R)-Trujillon and (+)-(S)-glutamate (50 mg/kg each) did not provoke changes in spontaneous basal firing. Local infusion of (+)-(S)-Trujillon (1 nMol) increased spontaneous firing in most neurons tested by 269.0 +/- 83.0% in relation to basal values. Intrapallidal infusion of (-)-(R)-Trujillon (1 nMol) and saline solution did not cause statistically significant changes in globus pallidus spiking. Results showed that (+)-(S)-Trujillon crosses the blood-brain barrier and has stereospecific activity., ((c) 2004 Wiley-Liss, Inc.)

Nonselective inverse agonists at the gamma-aminobutyric acid(A) (GABA-A) benzodiazepine binding site have cognition-enhancing effects in animals but are anxiogenic and can precipitate convulsions. Herein, we describe novel GABA-A alpha5 subtype inverse agonists leading to the identification of 16 as an orally active, functionally selective compound that enhances cognition in animals without anxiogenic or convulsant effects. Compounds of this type may be useful in the symptomatic treatment of memory impairment associated with Alzheimer's disease and related dementias.

Acute onset convulsive disorders in the canine may result from exposure to a variety of toxicants including strychnine, insecticides, metaldehyde, zinc phosphide, methylxanthines, drugs of abuse, bromethalin, and the tremorgenic mycotoxins (roquefortine and penitrem A). Although several of the above can be identified in a single gas chromatography-mass spectrometry (GC-MS) screen most have to be determined by separate tests. This report describes a modification of the strychnine extraction procedure, which allows thin layer chromatographic (TLC) identification of strychnine, bromethalin, roquefortine, and penitrem A in suspect baits, stomach contents or vomitus, and extends the identification to a wide variety of drugs, pesticides, and environmental contaminants by GC-MS. Samples were mixed with base, extracted into CH2Cl2 and the organic fraction back-extracted with acid. The organic fraction (neutrals) was purified by gel permeation chromatography (GPC) and analyzed by TLC to determine penitrem A and bromethalin. The acidic aqueous fraction was adjusted to pH > 9 and extracted into CH2Cl2. The resulting CH2Cl2 layer (bases) was then analyzed by TLC to determine strychnine and roquefortine. The organic basic and neutral fractions were recombined with a late eluting GPC fraction and analyzed by GC-MS. Of 312 samples analyzed by TLC from 1995 to 2001, 35 were positive for strychnine alone, 58 were positive for both roquefortine and penitrem A, 4 were positive for roquefortine alone, and 1 was positive for bromethalin. None of the samples were positive for penitrem A alone. Samples negative by TLC were analyzed by the GC-MS extended procedure since mid-1999, and 14 have shown positive for a wide variety of compounds with convulsant activity.

New convulsive compounds, brasiliamides A (1) and B (2), were isolated by activity-guided fractionation from okara fermented with a soil isolate of Penicillium brasilianum Batista JV-379. Their structures were elucidated on the basis of spectral and chemical evidence and by X-ray crystallography of the hydrogenated product of 2. In the 1H- and 13C-NMR spectra of 2, the signals were complicated, all being doubled or broadened in several deuterated solvents at room temperature. The conformational change of 2 was clarified as the rotational isomerization of amide bonds in solution by NMR measurements at various temperatures. Four rotamers of 2 at two amide bonds were presented at -60 degrees C in CDCl3, whereas only two isomers were apparent at room temperature, owing to rapid rotation of one of the amide bonds. Brasiliamides A and B respectively showed convulsive activity against silkworms with ED50 values of 300 and 50 microg/g of diet.

A number of analogues of the low-efficacy partial GABA(A) agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA(A) receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA(B) receptor sites or GABA uptake, they did show affinity for the GABA(A) receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K(i) = 9.1 microM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K(i) = 0.074 microM and K(i) = 0.049 microM). In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA(A) agonist isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC(50) = 0.37 microM and IC(50) = 0.02 microM) comparable with or markedly higher than that of the standard GABA(A) antagonist 4 (IC(50) = 0.24 microM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED(50) = 0.024 micromol/kg) and 7s (ED(50) = 0.21 micromol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA(A) receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o,m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.

A novel series of 3-heteroaryl-5,6-bis(aryl)-1-methyl-2-pyridones were developed with high affinity for the benzodiazepine (BZ) binding site of human gamma-aminobutyric acid (GABA(A)) receptor ion channels, low binding selectivity for alpha 2- and/or alpha 3- over alpha 1-containing GABA(A) receptor subtypes and high binding selectivity over alpha 5 subtypes. High affinity appeared to be associated with a coplanar conformation of the pyridone and sulfur-containing 3-heteroaryl rings resulting from an attractive S.O intramolecular interaction. Functional selectivity (i.e., selective efficacy) for alpha 2 and/or alpha 3 GABA(A) receptor subtypes over alpha1 was observed in several of these compounds in electrophysiological assays. Furthermore, an alpha 3 subtype selective inverse agonist was proconvulsant and anxiogenic in rodents while an alpha 2/alpha 3 subtype selective partial agonist was anticonvulsant and anxiolytic, supporting the hypothesis that subtype selective BZ site agonists may provide new anxiolytic therapies.

The crystal structure of the second monoclinic P2(1)/c form of the beta-carboline-3-carboxylate, C13H10N2O2, has been determined. Very small changes in the packing scheme lead to a different unit cell; the role of weak C-H...O hydrogen bonds seems to be crucial.

The molecular structure of 2alpha-hydroxyneoanisatin, a positional isomer of the potent neurotoxin anisatin, was determined by X-ray crystallographic analysis. This compound and four further seco-prezizaane type sesquiterpene lactones previously isolated from Illicium floridanum, which represent different structural types with respect to the mode of cyclisation, did not induce anisatin/picrotoxinin-like convulsions in mice. Based on these results and literature data for other seco-prezizaanes, structural requirements for convulsant activity are discussed. Comparison of the three dimensional molecular shape and electrostatic properties of active and inactive seco-prezizaane type lactones with compounds of the picrotoxane type resulted in the identification of a common pharmacophore structure for these different skeletal classes of convulsant natural products.

Regioisomeric 3-carboxyisoxazolinyl prolines [CIP-A (+/-)-6 and CIP-B (+/-)-7] and 3-hydroxyisoxazolinyl prolines [(+/-)-8 and (+/-)-9] were synthesized and assayed for glutamate receptor activity. The tests were carried out in vitro by means of receptor binding techniques, second messenger assays, and the rat cortical wedge preparation. CIP-A showed a good affinity for both 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and kainic acid (KAIN) receptors. These results were confirmed in the cortical slice model where CIP-A displayed an EC(50) value very close to that of AMPA. The convulsant properties of all the compounds were evaluated in vivo on DBA/2 mice after icv injection. CIP-A showed a convulsant activity, measured as tonus and clonus seizures, 18-65 times higher than that produced by AMPA. It was also quite active after ip administration, since it induced seizures in mice at doses as low as 3.2 nmol/mouse. On the basis of the above-reported results we prepared and tested the enantiomers of CIP-A and CIP-B, obtained by reacting (S)-3,4-didehydroproline and (R)-3,4-didehydroproline, respectively, with ethoxycarbonylformonitrile oxide. In all the tests the S-form, CIP-AS [(-)-6], emerged as the eutomer evidencing common stereochemical requirements with the reference compounds AMPA and KAIN. Through modeling studies, carried out on CIP-A, AMPA, and KAIN, active conformations for CIP-AS and AMPA at AMPA receptors as well as for CIP-AS and KAIN at KAIN receptors are suggested.

Unlabelled: Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are sensitive to many anesthetics, including barbiturates, which suggests that these receptors are potential sites for anesthetic action. Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity. Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic action, S(+)MPPB exhibits pure excitatory effects, including convulsion. 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid is another example of a convulsant barbiturate. We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current to determine whether inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. Whole cell nicotine-induced currents were recorded in PC12 derived from rat pheochromocytoma, using the conventional whole cell patch clamp technique in the presence and absence of barbiturates. Both depressant and convulsant barbiturates inhibited the nicotine-induced inward current reversibly and in a dose-dependent manner when co-applied with nicotine. All barbiturates accelerated the current decay. There was no significant difference between the concentrations for 50% inhibition for MPPB isomers. There was no correlation between inhibition of ganglionic nAchRs and anesthetic effects of the barbiturates. These results strongly oppose the idea that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates., Implications: We found that both convulsant and depressant barbiturates inhibit the current mediated through ganglionic nicotinic acetylcholine receptors in PC12 cells. This finding suggests that the inhibition of neuronal nicotinic acetylcholine receptors does not contribute to the anesthetic action of barbiturates.

This study reports the results of the detection and quantitation of strychnine in formalin-fixed tissues and in the formalin solutions in which the tissues were fixed. The toxicological analyses were performed on formalin-fixed liver and kidney samples and formalin solutions (10% buffered pH 7) in which the same samples from a case of acute strychnine poisoning were preserved. The analyses carried out at the time of autopsy on body fluid and tissues (bile, 2.40 mg/L; stomach contents, 14.2 mg; liver, 6.68 mg/kg; kidney, 2.68 mg/kg) allowed the identification of this substance as cause of death. The tissue samples were preserved in formalin solutions for 8 weeks. The analyses performed on formalin-fixed tissues (liver and kidney) and on formalin solutions, in which the same tissues were preserved, permitted the detection and quantitation of strychnine (liver, 1.59 mg/kg; formalin from the liver, 1.80 mg/L; kidney, 0.98 mg/kg; formalin from the kidney, 1.11 mg/L). The results indicate that this particular toxic substance also shows good stability in biological specimens subjected to chemical fixation.

Unlabelled: Some inhaled compounds cause convulsions. To better appreciate the physical basis for this property, we correlated the partial pressures that produced convulsions in rats with the lipophilicity (nonpolarity) and hydrophilicity (polarity) of 45 compounds: 3 n-alkanes, 18 n-haloalkanes, 3 halogenated aromatic compounds, 3 cycloalkanes and 3 halocycloalkanes, 13 halogenated ethers, and 2 noble gases (He and Ne). In most cases, convulsions were quantified by averaging the alveolar partial pressures just below the pressures that caused and slightly higher pressures that did cause clonic convulsions (ED50). The ED50 did not correlate with hydrophilicity (the saline/gas partition coefficient), nor was there an obvious correlation with molecular structure. For 80% of compounds (36 of 45), the ED50 correlated closely (r2 = 0.99) with lipophilicity (the olive oil/gas partition coefficient). Perhaps because they block the effect of GABA on GABA(A) receptors, five compounds were more potent than would be predicted from their lipophilicity. Conversely, four compounds may have been less potent than would be predicted because they (like conventional inhaled anesthetics) enhance the effect of GABA on GABA(A) receptors., Implications: Nonimmobilizers and transitional compounds may produce convulsions by two mechanisms. One correlates with lipophilicity (nonpolarity), and the other correlates with an action on GABA(A) receptors.

A series of N'-phenylindol-3-ylglyoxylohydrazides, isosters of the N-benzylindol-3-ylglyoxylamide derivatives previously described by us, were synthesized and tested for their ability to displace [3H]Ro 15-1788 from bovine brain membranes. These compounds were designed with the aim of obtaining products which could exert an in vivo activity, thanks to a higher hydrosolubility and consequently a better bioavailability. Affinity was restricted to the derivatives unsubstituted in the 5 position of the indole nucleus (1, 6, 9, 12, 15, 18, 23, and 26), with Ki values ranging from 510 to 11 nM. The most active compounds (6, 9, 23, and 29) proved to be effective in antagonizing pentylenetetrazole-induced seizures. Molecular modeling studies were performed to rationalize the lack of affinity of hydrazides with a chloro or a nitro group in the 5 position of the indole nucleus. It was hypothesized that the conformational preference of the hydrazide side chain, characterized by a gauche disposition of lone pairs and substituents about the N-N bond, prevents all hydrazides from binding to the receptor similarly to other classes of indole analogues previously investigated. The potency of 5-H hydrazides was attributed to a binding mode which is not feasible for 5-Cl and 5-NO2 counterparts. This theoretical model of ligand-receptor interaction permitted a more stringent interpretation of structure-affinity relationships of hydrazides and of recently described benzylamide derivatives (Da Settimo et al. J. Med. Chem. 1996, 39, 5083-5091).

We examined the effects of 4 corymine-related compounds on glycine-induced chloride current in Xenopus oocytes. Dihydrocorymine, N-demethyl-3-epi-dihydrocorymine and deformylcorymine dose-dependently decreased the glycine current with IC50 values of 34, 37 and 55 microM, respectively. The effect of these compounds on the glycine current was more potent than that of pleiocarpamine (IC50 > 1 mM). N-demethyl-3-epi-dihydrocorymine and dihydrocorymine, at 100 microM, also decreased the gamma-aminobutyric acid-induced current by 65% and 22%, respectively, whereas deformylcorymine and pleiocarpamine failed. The inhibitory action of deformylcorymine on the glycine current was noncompetitive. These results suggest that deformylcorymine is a novel specific noncompetitive glycine receptor antagonist. The structure-activity relationship of these compounds was discussed.

1H NMR studies were performed on two beta-carboline derivatives interacting with human serum albumin. The spin-lattice relaxation rates of the two derivatives, having side chains of different length and polarity, were used to demonstrate a diverse motional behavior in solution together with slightly different relaxation pathways. Single- and double-selective excitation made it possible to evaluate dynamics in the free and protein-bound states. Occurrence of a relatively long hydrophilic chain interacting with the proton-acceptor nitrogen of the beta-carboline moiety was shown to yield lower association constants, slower dissociation rates, and diverse interacting modes with the indole hydrophobic site of the protein., (Copyright 1998 Academic Press.)

We examined the anesthetic and convulsant properties of 16 unfluorinated to completely fluorinated aromatic compounds, having six to nine carbon atoms (e.g., benzene to 1,3,5-tris(trifluoromethyl)benzene), and four cycloalkanes (cyclopentane to cyclooctane). Benzene, fluorobenzene, toluene, p-xylene, ethylbenzene, and cyclopentane caused excitation (twitching, jerking, and hyperactivity), and three aromatic compounds (perfluorotoluene, p-difluorotoluene and 1,3,5-tris(trifluoromethyl)benzene) and three cycloalkanes (cyclohexane, cycloheptane, and cyclooctane) produced convulsions. Cyclooctane and 1,3,5-tris(trifluoromethyl)benzene were nonanesthetics. Except for nonanesthetics and perfluorotoluene (too toxic to test for anesthetic potency), all compounds produced anesthesia or decreased the minimum alveolar anesthetic concentration of desflurane. Aromatic compounds were more potent and lipid-soluble than n-alkanes (data from previous report) and cycloalkanes. All three series increasingly disobeyed the Meyer-Overton hypothesis as molecular size increased. For a particular number of carbons (e.g., cyclohexane, n-hexane, and benzene), the deviation was cycloalkanes > or = normal alkanes > aromatic compounds. These results suggest that molecular shape (including "bulkiness") and size provide limited clues to the structure of the anesthetic site of action.

The effects of low and clinically relevant concentrations of inhalation anaesthetics and related compounds on the firing behaviour of nerve axons from a number of species are described. The observation of substantial, if sometimes transient, changes in excitability is contrasted with the view that axonal ion channels are insensitive to many general anaesthetics. Taking the squid giant axon as a well-studied example, we show that a full understanding of these effects can only be achieved through a detailed investigation of the actions of a range of compounds on a number of ion channels. Small alterations in the properties of individual channel types can in combination result in major changes in the behaviour of a multi-channel system such as an axon. Finally, as an example of the spectrum of activity of structurally related compounds, we compare the effects of inhalation anaesthetics with those of volatile convulsants.

To further study the putative gamma-butyrolactone site of the GABAA/chloride channel complex, constrained derivatives of convulsant and anticonvulsant alpha,alpha-disubstituted gamma-butyrolactones (alpha-spirocyclopropyl- and alpha-spirocyclopentyl-gamma-butyrolactones) were synthesized and evaluated biologically. Most of the spirocyclopropyl agents were anticonvulsants when tested against pentylenetetrazole-induced seizures in mice. These agents effectively displaced 35[S]-tert-butylbicyclophosphorothionate (35[S]-TBPS), a ligand for the picrotoxin binding site of the GABAA/chloride channel, from rat neuronal membranes and affected the GABA-mediated current in hippocampal neurons. The monomethyl-substituted spirocyclopropyl agent with a methyl group cis to the carbonyl (15) potentiates GABA-induced current whereas the trans derivative (16) blocks the current. The only anticonvulsant in the spirocyclopentyl series was the unsubstituted spirocyclopentyl compound 2. All the other substituted spirocyclopentyl targets were inactive in vivo at the highest dose tested except for convulsant 9, which has a trans 2,5-dimethyl-substituted cyclopentyl ring. All the spirocyclopentyl derivatives displaced 35[S]-TBPS from rat neuronal membranes very effectively, and they also all potentiated GABA-induced chloride current except for convulsant 9 which blocked the current. From the data obtained in this investigation, it appears that when the volume occupied above and below the lactone ring is as large as that occupied by spirocyclopentyl agent 9, convulsant activity is observed. Groups with less volume in these areas either are inactive in the behavioral test or have anticonvulsant activity. When bound to the GABAA/chloride channel, the larger molecules may stabilize the closed state of the channel whereas the smaller molecules may stabilize the open state.

Spontaneously occurring spike-wave discharges (SWD) in rats are used as a model for absence epilepsy in humans. In vitro, the binding parameters of 3H-Ro 5-4864, a ligand labelling the peripheral benzodiazepine receptor, were determined for brain membranes of WAG/Rij rats, an inbred strain showing SWD, and for ACI rats, an inbred strain showing no SWD. No difference in the Kd but a small difference in the Bmax values between the strains were found. Recently, other investigators reported a correlation between a decrease in affinity for 3H-Ro 5-4864 and the occurrence of SWD. Our results suggest however that it is doubtful that a change in Kd of the peripheral benzodiazepine receptor is causal in the etio-pathology of the spontaneous absence like phenomena in rats.