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Gamma-aminobutyric acid type A receptor (GABA A R) modulators are crucial in treating neurological and psychiatric disorders, including epilepsy, anxiety, insomnia, and depression. This review examines the synthetic approaches and clinical applications of representative small-molecule GABA A R modulators. Benzodiazepines, such as Diazepam, are well-known positive allosteric modulators (PAMs) that enhance GABA A R function, providing therapeutic effects but also associated with side effects like sedation and dependence. Non-benzodiazepine modulators, including Z-drugs like Zolpidem and Zaleplon, offer improved selectivity for the α1 subunit of GABA A R, reducing some of these side effects. Neurosteroids such as allopregnanolone and its synthetic analogs, including Brexanolone, have emerged as potent GABA A R modulators with applications in conditions like postpartum depression and refractory epilepsy. Advances in molecular biology and pharmacology have facilitated the development of isoform-specific modulators, potentially reducing off-target effects and enhancing therapeutic profiles. Additionally, combining GABA A R modulators with other therapeutic agents has shown promise in enhancing efficacy and minimizing side effects. This review highlights the design strategies, pharmacodynamics, clinical efficacy, and safety profiles of these compounds, emphasizing the opportunities for developing novel GABA A R modulators with improved therapeutic outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Zuranolone (SAGE-217) is a neuroactive steroid (γ-aminobutyric acid) A (GABA A ) receptor positive allosteric modulator (PAM) as the first oral drug approved by the FDA in 2023, which is used to treat patients with postpartum depression (PPD). SAGE-217 has a "black box" warning with impairing ability to drive or engage in other potentially hazardous activities. In addition, SAGE-217 can cause CNS depressant effects such as somnolence and confusion, suicidal thoughts and behavior and embryo-fetal toxicity. Based on the structure-activity relationship (SAR) of SAGE-217, a total of 28 neuroactive steroids with novel pharmacophore at C-21 modulated SAGE-217 derivatives were designed and synthesized. The biological activities were evaluated by both synaptic α1β2γ2 GABA A receptor and extrasynaptic α4β3δ GABA A receptor cell assays. The optimal compound S28 exhibited much more potent potency and similar efficacy at extrasynaptic GABA A receptor than SAGE-217. Different from above, compound S28 exhibited similar potency and lower efficacy at synaptic GABA A receptor than SAGE-217, which were consistent with the analysis of molecular docking and dynamics simulation results. The appropriate lower efficacy at synaptic GABA A receptor of compound S28 might contribute to reduce the side effects of excessive sedation. Furthermore, compound S28 was demonstrated to have excellent in vivo pharmacokinetic (PK) parameters, robust in vivo pharmacodynamic (PD) effects and good safety profiles. Therefore, compound S28 represents a potentially promising treatment of PPD candidate that warrants further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

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 GABA A receptors of the α1β2γ2L 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 GABA A 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 GABA A 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.

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 (GABA A ) chloride channel, the translocator protein (TSPO), the murine double minute 2 (MDM2) protein, the A 2B adenosine receptor (A 2B 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.

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 GABA A 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 GABA A receptor inhibitory postsynaptic currents with an IC 50 value of 7.9 μM. These results, by showing an ability to modulate the GABA A receptor function, cast fresh light on the biological activities of the secondary metabolites of W. somnifera roots.

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 ED 50 ), 285.02 and 293.42 mg/kg (scPTZ ED 50 ), and 389.11 and 412.16 mg/kg (TD 50 ), respectively. Docking studies were also performed for all synthesized compounds to get insight into the binding pattern toward the GABA A 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., (© 2017 Deutsche Pharmazeutische Gesellschaft.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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.

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.

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.

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.

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.

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.

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., (Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.

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.

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).

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.

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.

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.

The planar 5alpha-reduced steroid (3alpha,5alpha)-3-hydroxypregnan-20-one and the nonplanar 5beta-reduced steroid (3alpha,5beta)-3-hydroxypregnan-20-one act at GABA(A) receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABA(A) receptors have not been determined. To determine how structural modifications at the steroid C-6 and C-7 positions effect the actions of these anesthetic steroids, an axial or equatorial methyl group was introduced at these positions. The analogues were evaluated (1) in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5alpha- and 5beta-reduced series of compounds were strikingly similar. In both series, a 6beta-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6alpha-, 7beta- or 7alpha-Me substituent resulted in reduced potency for inhibition of radioligand binding, GABA(A) receptor modulation and tadpole anesthesia. Because of the similar effects of methyl group substitution in the two series of compounds and previous results from other studies showing that structural modifications in the steroid D ring/side chain region produce similar effects regardless of the stereochemistry of the A,B-ring fusion, we propose that either the 3alpha-hydroxyl groups of planar and nonplanar anesthetic steroids hydrogen bond to different amino acids on GABA(A) receptors or that this critical hydrogen bonding group interacts with membrane lipids instead of the receptor.

The hydrogen-bond-acceptor properties of the carbonyl moiety in the 17beta-acetyl group on the D-ring of the anesthetic steroids (3alpha,5alpha)- and (3alpha,5beta)-3-hydroxypregan-20-one form an important part of the anesthetic steroid pharmacophore. 13,24-Cyclo-18,21-dinorcholanes containing a ketone or conjugated ketone group at C-20, C-22, C-23, or C-24 were prepared as conformationally constrained analogues of these anesthetic steroids and were used to probe for alternate locations for the D-ring hydrogen-bond-accepting carbonyl group. The analogues were evaluated (1). in [(35)S]-tert-butylbicyclophosphorothionate binding experiments, (2). in electrophysiological experiments using rat alpha(1)beta(2)gamma(2L) GABA(A) receptors expressed in Xenopus laevis oocytes, and (3). as tadpole anesthetics. In the binding assay, the relative order of potencies for the analogues in the 5alpha- and 5beta-series is identical. For the ketones, the order is 24-one >or= 23-one > 20-one > 22-one. Likewise, for the enones, the order is delta(22)-24-one > delta(20(22))-23-one > delta(22)-20-one > delta(23)-22-one. Similar relative orders of potencies are also found in the other two bioassays. The activities of the 24-one and delta(22)-24-one compounds were expected to be very low, because the carbonyl group in these compounds is located over the steroid C-ring and oriented toward C-8. Instead, these compounds have the highest activities in their respective series, with the delta(22)-24-one compounds having activities comparable to those of the reference anesthetic steroids. The electrophysiology results obtained with the 24-oxo-cyclosteroids suggest that rat alpha(1)beta(2)gamma(2L) GABA(A) receptors contain more than one donor for the hydrogen-bond-acceptor group of anesthetic steroids. The family of cyclosteroids should be useful for future structure-activity relationship studies of steroid modulation of other GABA(A) receptor subtypes.

Various cyclic ether and other 3 alpha-hydroxyandrostane derivatives bearing a conformationally constrained hydrogen-bonding moiety were prepared. Their anesthetic potency and their binding affinity for GABA(A) receptors, measured by intravenous administration to mice and inhibition of [(35)S]TBPS binding to rat whole brain membranes, were compared with that of known anesthetic 3 alpha-hydroxypregnan-20-ones. Synthetic steroids with similar in vitro and in vivo activities to the endogenous 3 alpha-hydroxypregnan-20-ones all had an ether oxygen on the beta-face of the steroid D-ring. These results suggest that for optimal GABA(A) receptor modulation, the hydrogen bond-accepting substituent should be near perpendicular to the plane of the D-ring on the beta-face of the steroid.

A new method of [(11)C]-methylation is described, which attains the goals of simplicity, high radiochemical yields, speed, versatility, and automation. A standard high performance liquid chromatography (HPLC) injection loop on a standard HPLC injection valve is loaded with a solution (80 microL) of precursor (0.3-1.0 mg) in dimethyl formamide (DMF) or dimethyl sulfoxide (DMSO) (+ base if required). At ambient temperature [(11)C]-iodomethane is passed through the loop for 3-4 min with >90% trapping of activity. After a further 1-5 min, the contents of the loop are quantitatively injected onto the HPLC column for purification. Radiochemical yields are equal to or superior to conventional solution methods in all cases, even though no heat is applied. [(11)C]-labeled radiotracers that have been prepared by this method for human or animal studies include Raclopride, N-methylspiperone, Ro 15-1788, FLB 457, RTI-32, Rolipram, SCH 23390, and SKF 82957. Since no vials, transfer lines, cooling, heating, or sealing valves are required, no transfer losses occur, yields are high, and cleanup is minimal, this "loop method" is ideal for most radiopharmaceuticals prepared from [(11)C]-iodomethane.

The unnatural enantiomers of dehydroepiandrosterone sulfate (1), pregnenolone sulfate (2), and (3alpha,5beta)-3-hydroxypregnan-20-one sulfate (3), compounds 4-6, respectively, were prepared by total steroid synthesis. The enantioselectivity of the compounds as negative modulators of the GABAA receptors present in cultured rat hippocampal neurons was examined using electrophysiological methods. Enantioselectivity was found for the inhibitory actions of the dehydroepiandrosterone enantiomers. The IC50s for compounds 1 and 4 were 11 +/- 1 and 80 +/- 14 microM, respectively. Little, if any, enantioselectivity was found for the other two pairs of steroid sulfate inhibitors. The IC50s for compounds 2 and 5 were 82 +/- 12 and 76 +/- 27 microM, respectively. The IC50s for compounds 3 and 6 were 39 +/- 7 and 46 +/- 2 microM, respectively. The results suggest that the sites of action for the androstane and pregnane series of steroid sulfate blockers of GABA-mediated current are different. The observed enantioselectivity for the actions of dehydroepiandrosterone sulfate indicates that its inhibitory actions are mediated via a chiral recognition site and provides new evidence in support of the earlier hypothesis that there is a binding site for this compound on GABAA receptors. Conversely, the failure to observe enantioselectivity for the actions of pregnenolone sulfate and steroid sulfate 3 indicates that a chiral recognition site for these steroids does not exist on GABAA receptors and suggests that the effects of these compounds on this receptor's function may arise indirectly as a consequence of steroid-induced membrane perturbation.