Rational approaches for the design of various GABA modulators and their clinical progression.

GABA (γ-amino butyric acid) is an important inhibitory neurotransmitter in the central nervous system. Attenuation of GABAergic neurotransmission plays an important role in the etiology of several neurological disorders including epilepsy, Alzheimer's disease, Huntington's chorea, migraine, Parkinson's disease, neuropathic pain, and depression. Increase in the GABAergic activity may be achieved through direct agonism at the GABAA receptors, inhibition of enzymatic breakdown of GABA, or by inhibition of the GABA transport proteins (GATs). These functionalities make GABA receptor modulators and GATs attractive drug targets in brain disorders associated with decreased GABA activity. There have been several reports of development of GABA modulators (GABA receptors, GABA transporters, and GABAergic enzyme inhibitors) in the past decade. Therefore, the focus of the present review is to provide an overview on various design strategies and synthetic approaches toward developing GABA modulators. Furthermore, mechanistic insights, structure-activity relationships, and molecular modeling inputs for the biologically active derivatives have also been discussed. Summary of the advances made over the past few years in the clinical translation and development of GABA receptor modulators is also provided. This compilation will be of great interest to the researchers working in the field of neuroscience. From the light of detailed literature, it can be concluded that numerous molecules have displayed significant results and their promising potential, clearly placing them ahead as potential future drug candidates.

'Proximity frequencies' a new parameter to evaluate the profile of GABAAR modulators.

We reported the synthesis of new 8-methoxypyrazolo[1,5-a]quinazolines bearing an amide fragment at the 3-position. The final compounds, as aromatic (2a-i) and 4,5-dihydro derivatives (3a-i), have been evaluated in vitrofor their ability to modulate the chlorine current on recombinant GABAA receptors of the α1ß2γ2L type (expressed in frog oocytes of the Xenopus laevis species). From electrophysiological test two groups of compounds emerged: positive modulators agonist (2e, h, i and 3e, h) and null modulators antagonist (2a, b, d, f, g and 3a-d, f, g) of GABAA subtype receptor. Using a set of compounds (new derivatives, known products and GABAA subtype receptor ligands from our library) we identify the amino acids at the α+/γ- interface, which could be involved in the agonist or antagonist profile, using the 'Proximity Frequencies', namely the frequencies with which a ligand intercepts two or more binding-site amino acids during the molecular dynamic simulation. The linear discriminant analysis (LDA) evidences that the combination of amino acids αVAL203- γTHR142 and αTYR 160- γTYR 58 allowed to collocate 70.6% of agonists and 72.7% of antagonists in their respective class.

Exploiting the Indole Scaffold to Design Compounds Binding to Different Pharmacological Targets.

Several indole derivatives have been disclosed by our research groups that have been collaborating for nearly 25 years. The results of our investigations led to a variety of molecules binding selectively to different pharmacological targets, specifically the type A γ-aminobutyric acid (GABAA) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A2B adenosine receptor (A2B AR) and the Kelch-like ECH-associated protein 1 (Keap1). Herein, we describe how these works were conceived and carried out thanks to the versatility of indole nucleus to be exploited in the design and synthesis of drug-like molecules.

Ferulic Acid Esters and Withanolides: In Search of Withania somnifera GABAA Receptor Modulators.

Nine compounds, including two undescribed withanolides, withasomniferolides A and B (1 and 2), three known withanolides (3-5), a ferulic acid dimeric ester (6), and an inseparable mixture of three long alkyl chain ferulic acid esters (7-9), were isolated from a GABAA receptor positive activator methanol extract of the roots of Withania somnifera. The structures of the isolated compounds were elucidated based on NMR, MS, and ECD data analysis. In order to bioassay the single ferulic acid derivatives, compounds 6-9 were also synthesized. The most active compound, docosanyl ferulate (9), was able to enhance the GABAA receptor inhibitory postsynaptic currents with an IC50 value of 7.9 µM. These results, by showing an ability to modulate the GABAA receptor function, cast fresh light on the biological activities of the secondary metabolites of W. somnifera roots.

Design, Synthesis, and Docking Study of Pyrimidine-Triazine Hybrids for GABA Estimation in Animal Epilepsy Models.

A series of new pyrimidine-triazine hybrids (4a-t) was designed and synthesized, from which potent anticonvulsant agents were identified. Most of the compounds exhibited promising anticonvulsant activity against the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests, along with minimal motor impairment with higher safety compared to the standard drugs, phenytoin and carbamazepine. In the series, 5-(4-(4-fluorophenyl)-6-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4o) and 5-(6-(4-hydroxy-3-methoxyphenyl)-4-(4-hydroxyphenyl)-2-thioxo-5,6-dihydropyrimidin-1(2H)-yl)-1,2-dihydro-1,2,4-triazin-3(6H)-one (4s) emerged as most potent anticonvulsant agents with median doses of 22.54 and 29.40 mg/kg (MES ED50 ), 285.02 and 293.42 mg/kg (scPTZ ED50 ), and 389.11 and 412.16 mg/kg (TD50 ), respectively. Docking studies were also performed for all synthesized compounds to get insight into the binding pattern toward the GABAA receptor as a possible mechanism of their anticonvulsant action, and in silico ADME studies were carried out to predict the safety and stability of the molecules. The increased GABA level in the experimental animals in the neurochemical estimation assay confirmed their GABAergic modulating activity. The most potent compounds were also evaluated for their neurotoxic and hepatotoxic effects. Fortunately, they did not show any sign of neurotoxicity or hepatotoxicity, suggesting that they have a broad spectrum of anticonvulsant activity with a large safety margin. Together, this research suggested that 4o and 4s may serve as leads in the discovery and development of new anticonvulsant drugs.

Design, Synthesis and Evaluation of the Antidepressant and Anticonvulsant Activities of Triazole-Containing Benzo[d]oxazoles.

BACKGROUND: Epilepsy and depression are two of the common diseases seriously threatening life and health of human. A shared neurobiological substrate led to the bidirectional relationship and high comorbid occurrence of the two disorders. Recently, an increasing number of patients with epilepsy (PWE) require some form of antidepressant medication. However, most of the available antidepressants are inadequate for PWE for some reasons. So, the search for novel and increasingly effective drugs with anticonvulsant and antidepressant activities is necessary. METHODS: A series of 2-substituted-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazoles (5a-p) were designed and synthesized. Their anticonvulsant activities were evaluated using maximal electroshock shock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. Their antidepressant activities were screened with the forced swimming test (FST). RESULTS: All the compounds showed anti-MES activities in different degree, among which 5g and 5j were the most promising one with ED50 value of 31.7 and 12.7 mg/kg, respectively. What's more, 5g and 5j also exhibited nice anti-scPTZ activities and low neurotoxicity. Interestingly, these compounds also showed good antidepressant activities in FST. And the efficacy of 5g were also confirmed by a tail suspension test and a open field test. The pretreatment of thiosemicarbazide (an inhibitor of γ- aminobutyric acid synthesis enzyme) significantly increased the ED50 of 5g in MES and reversed the reductions in the immobility time of 5g in FST. CONCLUSION: Triazole-containing benzo[d]oxazole is a good skeleton to develop compounds with both anticonvulsant and antidepressant activities. We have got the compound 5g, which display remarkable antidepressant and anticonvulsant activities, and the GABAergic system was involved in the action mechanism of 5g.

Development of Novel, CNS Penetrant Positive Allosteric Modulators for the Metabotropic Glutamate Receptor Subtype 1 (mGlu1), Based on an N-(3-Chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide Scaffold, That Potentiate Wild Type and Mutant mGlu1 Receptors Found in Schizophrenics.

The therapeutic potential of selective mGlu1 activation is vastly unexplored relative to the other group I mGlu receptor, mGlu5; therefore, our lab has focused considerable effort toward developing mGlu1 positive allosteric modulators (PAMs) suitable as in vivo proof of concept tool compounds. Optimization of a series of mGlu1 PAMs based on an N-(3-chloro-4-(1,3-dioxoisoindolin-2-yl)phenyl)-3-methylfuran-2-carboxamide scaffold provided 17e, a potent (mGlu1 EC50 = 31.8 nM) and highly CNS penetrant (brain to plasma ratio (Kp) of 1.02) mGlu1 PAM tool compound, that potentiated not only wild-type human mGlu1 but also mutant mGlu1 receptors derived from deleterious GRM1 mutations found in schizophrenic patients. Moreover, both electrophysiological and in vivo studies indicate the mGlu1 ago-PAMs/PAMs do not possess the same epileptiform adverse effect liability as mGlu5 ago-PAMs/PAMs and maintain temporal activity suggesting a broader therapeutic window.

Activation of the γ-Aminobutyric Acid Type B (GABA(B)) Receptor by Agonists and Positive Allosteric Modulators.

Since the discovery of the GABA(B) agonist and muscle relaxant baclofen, there have been substantial advancements in the development of compounds that activate the GABA(B) receptor as agonists or positive allosteric modulators. For the agonists, most of the existing structure-activity data apply to understanding the role of substituents on the backbone of GABA as well as replacing the carboxylic acid and amine groups. In the cases of the positive allosteric modulators, the allosteric binding site(s) and structure-activity relationships are less well-defined; however, multiple classes of molecules have been discovered. The recent report of the X-ray structure of the GABA(B) receptor with bound agonists and antagonists provides new insights for the development of compounds that bind the orthosteric site of this receptor. From a therapeutic perspective, these data have enabled efforts in drug discovery in areas of addiction-related behavior, the treatment of anxiety, and the control of muscle contractility.

Synthesis and biological applications of phosphinates and derivatives.

This review first outlines general considerations on phosphinic acids and derivatives as bioisosteric groups. The next sections present key aspects of phosphinic acid-based molecules and include a brief description of the biological pathways involved for their activities. The synthetic aspects and the biological activities of such compounds reported in the literature between 2008 and 2013 are also described.

Neurosteroid analogues. 18. Structure-activity studies of ent-steroid potentiators of γ-aminobutyric acid type A receptors and comparison of their activities with those of alphaxalone and allopregnanolone.

A model of the alignment of neurosteroids and ent-neurosteroids at the same binding site on γ-aminobutyric acid type A (GABAA) receptors was evaluated for its ability to identify the structural features in ent-neurosteroids that enhance their activity as positive allosteric modulators of this receptor. Structural features that were identified included: (1) a ketone group at position C-16, (2) an axial 4α-OMe group, and (3) a C-18 methyl group. Two ent-steroids were identified that were more potent than the anesthetic steroid alphaxalone in their threshold for and duration of loss of the righting reflex in mice. In tadpoles, loss of righting reflex for these two ent-steroids occurs with EC50 values similar to those found for allopregnanolone. The results indicate that ent-steroids have considerable potential to be developed as anesthetic agents and as drugs to treat brain disorders that are ameliorated by positive allosteric modulators of GABAA receptor function.

Surface active benzodiazepine-bromo-alkyl conjugate for potential GABAA-receptor purification.

A conjugable analogue of the benzodiazepine 5-(2-hydroxyphenyl)-7-nitro-benzo[e][1,4]diazepin-2(3H)-one containing a bromide C(12)-aliphatic chain (BDC) at nitrogen N1 was synthesized. One-pot preparation of this benzodiazepine derivative was achieved using microwave irradiation giving 49% yield of the desired product. BDC inhibited FNZ binding to GABA(A)-R with an inhibition binding constant K(i) = 0.89 µM and expanded a model membrane packed up to 35 mN m(-1) when penetrating in it from the aqueous phase. BDC exhibited surface activity, with a collapse pressure π = 9.8 mN m(-1) and minimal molecular area A(min) = 52 Å(2)/molecule at the closest molecular packing, resulted fully and non-ideally mixed with a phospholipid in a monolayer up to a molar fraction x≅ 0.1. A geometrical-thermodynamic analysis along the π-A phase diagram predicted that at low x(BDC) (<0.1) and at all π, including the equilibrium surface pressures of bilayers, dpPC-BDC mixtures dispersed in water were compatible with the formation of planar-like structures. These findings suggest that, in a potential surface grafted BDC, this compound could be stabilize though London-type interactions within a phospholipidic coating layer and/or through halogen bonding with an electron-donor surface via its terminal bromine atom while GABA(A)-R might be recognized through the CNZ moiety.

GABAergic dysfunction in essential tremor: an 11C-flumazenil PET study.

UNLABELLED: Essential tremor is the most common movement disorder, but the underlying pathophysiology is not well understood. A primary overactivity of cerebellothalamic output pathways is the most conspicuous finding, as indicated by animal and human studies. It has been argued that this overactivity may be due to impaired central inhibition, and converging evidence points toward a potential role of gamma-aminobutyric acid (GABA) dysfunction in tremor generation. METHODS: Using (11)C-flumazenil and PET, we calculated the distribution volume, an index of availability of benzodiazepine receptor sites of the GABA(A) complex, in a group of 8 patients with bilateral essential tremor, as compared with 11 healthy controls. RESULTS: Significant increases in binding of (11)C-flumazenil at the benzodiazepine receptor site of the GABA(A) receptor in the cerebellum, the ventrolateral thalamus, and the lateral premotor cortex were identified in the essential tremor group. CONCLUSION: Essential tremor is associated with reduced GABAergic function and increased availability of benzodiazepine receptor sites in brain regions implicated specifically in tremor genesis. This finding is thought to reflect overactivity of cerebellothalamic circuits and, hence, lends support to the "GABA hypothesis" of essential tremor.

New 1,5-benzodiazepine compounds: activity at native GABA(A) receptors.

Various new 1,5-benzodiazepine compounds were synthesized and tested for their biological activity in terms of effects on GABA(A) receptors of rat cerebellar granules in culture. Their effects were compared to those of a 1,4-benzodiazepine agonist, flunitrazepam and the already known 1,5-benzodiazepine antiepileptic clobazam. The effects were evaluated for the two different GABA(A) receptor populations present in these neurons, one mediating phasic inhibition and the other one mediating tonic inhibition. Many such compounds display a profile of inverse agonist to both GABA(A) receptor populations. One of them presents a profile of full agonist at the component mediating phasic inhibition. Interestingly, substitution of just one oxygen atom in that compound with sulphur in a specific position of a morpholine ring resulted in a remarkable change of activity from full agonist to a probable inverse agonist. This indicates such a position as a proton accepting one for the ligand within the benzodiazepine binding pocket of the relevant GABA(A) receptors. In addition, that position appears to be critical for the pharmacological activity.

Allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one) derivatives with a polar chain in position 16alpha: synthesis and activity.

The lipophilic nature of allopregnanolone prevents its user-friendly application in human medicine. On inspiration by previously prepared allopregnanolone with a 16alpha-bound tetraethylammonium salt, an attempt was made to produce allopregnanolone analogues with polar groups introduced into position 16alpha with the goal of increasing water solubility, brain accessibility, and potency of neuroactive steroids. The Michael addition to derivatives of pregn-16-en-20-one was the key reaction step. The link between the steroid skeleton and the new side chain was either a methylene group (when diethyl malonate was added) or an oxygen atom (when a hydroxy derivative was added). [(35)S]TBPS displacement was used to evaluate the products. Several carbamates (but not their parent alcohols) displaced TBPS from the picrotoxin binding site on GABA(A) receptors. Although none of them was more potent than the above ammonium salt, which stimulated this study, their nonionic nature should not prevent their passage into the brain.

Total synthesis of valerenic acid, a potent GABAA receptor modulator.

The first total synthesis of the sesquiterpenoid valerenic acid, a constituent of Valeriana officinalis, is described. The compound is a potent modulator of the GABA(A) receptor and may thus be useful in the treatment of various dysfunctions of the central nervous system. The synthesis is enantio-, diastereo-, and regiocontrolled and utilizes an enyne-RCM, a metal-coordinated Diels-Alder reaction, a hydroxy-directed Crabtree hydrogenation, and a Negishi methylation as key steps.

Neurosteroid analogues. 14. Alternative ring system scaffolds: GABA modulatory and anesthetic actions of cyclopenta[b]phenanthrenes and cyclopenta[b]anthracenes.

Although the structural features of binding sites for neuroactive steroids on gamma-aminobutryic acid type A (GABA A) receptors are still largely unknown, structure-activity studies have established a pharmacophore for potent enhancement of GABA A receptor function by neuroactive steroids. This pharmacophore emphasizes the importance of the position and stereochemistry of hydrogen-bonding groups on the steroid. However, the importance of the steroid ring system in mediating hydrophobic interactions with the GABA A receptor is unclear. We have taken the cyclopenta[ b]phenanthrene (tetracyclic compounds with a nonlinear ring system different from that of steroids) and cyclopenta[ b]anthracene (tetracyclic molecules with a linear 6-6-6-5 carbocyclic ring system) ring systems and properly substituted them to satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids. We have found these cyclopenta[ b]phenanthrene and cyclopenta[ b]anthracene analogues to have potent activity at the GABA A receptor, rivaling that of the most potent steroid modulators. Single-channel analysis of electrophysiological data indicates that similarly substituted analogues in the different ring systems affect the kinetic components of macroscopic currents in different ways. Mutations to the hydrogen bonding amino acids at the putative steroid binding site (alpha1Q241L mutation and alpha1N407A/Y410F double mutation) produce similar effects on macroscopic current amplitude by the different ring system analogues suggesting that the different kinetic effects are explained by the precise interactions of each analogue with the same binding site(s).

Neurosteroid analogues. 11. Alternative ring system scaffolds: gamma-aminobutyric acid receptor modulation and anesthetic actions of benz[f]indenes.

Benz[f]indenes are tricyclic compounds with a linear 6-6-5 fused carbocyclic ring system. When properly substituted, benz[f]indenes can satisfy the pharmacophore requirements of the critical hydrogen-bond donor and acceptor groups found in neuroactive steroids that modulate gamma-aminobutyric acidA (GABAA) receptor function. Thus, the benz[f]indene ring system provides an opportunity to extend the previously well-studied GABAA receptor structure-activity relationships (SAR) of neuroactive steroids to a different ring system. Depending on whether the stereochemistry of the 6-6-5 ring fusions are trans-trans or cis-trans, either planar or nonplanar benz[f]indenes are obtained. We found that the planar trans-trans benz[f]indenes are active, but less active than the steroids they were designed to mimic, whereas the nonplanar cis-trans compounds have little, if any, activity. The results provide new insight into the importance of the steroid framework for the actions of neuroactive steroids at GABAA receptors.

Activity of B-nor analogues of neurosteroids on the GABA(A) receptor in primary neuronal cultures.

A GABA(A) receptor study of several B-nor analogues of allopregnanolone and pregnanolone has been carried out. B-norallopregnanolone (i.e., 3alpha-hydroxy-7-nor-5alpha-pregnan-20-one) was found comparable to allopregnanolone when measured with labeled TBPS. Analogous results were obtained from their effect on neurons in culture: this time, both 3alpha-hydroxy-7-nor-5xi-pregnan-20-ones (5 and 6) were found to stimulate [3H]flunitrazepam binding and GABA-induced 36Cl- influx. These effects were inhibited by GABA(A) receptor antagonists. Other analogues carrying electronegative substituents (epoxides 9 and 10 and ketone 12) in the B ring were inactive. Similarly, B-normal ketones 17, and 18 and 6-azasteroids 20 and 21 were also inactive. B-Nor analogues 5 and 6 did not induce neurotoxicity at relevant concentrations. A computational analysis of active and inactive neurosteroid analogues allowed the proposal of a 3D pharmacophoric hypothesis of their interaction with the GABA(A) receptor.

Novel 17beta-substituted conformationally constrained neurosteroids that modulate GABA A receptors.

The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.