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.

One of the long-standing issues surrounding cholesterol (Chol) relates to its two-faced character. In particular, the consequences of its having a rough β-face and a smooth α-face on its structural influence in cell membranes has remained elusive. In this study, direct comparisons have been made between cholesterol and a “smoothened” analog, DChol (i.e., 18,19-dinorcholesterol) using model membranes and a combination of nearest-neighbor recognition, differential scanning calorimetry, fluorescence, and monolayer measurements. Taken together, these results indicate that subtle differences exist between the interaction of these two sterols with the different states of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Chol has a greater condensing power than DChol, but only slightly so, i.e., on the order of a few tens of calories per mole.

Herein, we report the first synthesis of a demethylated form of cholesterol (18,19-di-nor-cholesterol), in which the C18 and C19 methyl groups of the β-face were eliminated. Recent molecular simulations modeling 18,19-di-nor-cholesterol have suggested that cholesterol’s opposing rough β-face and smooth α-face play necessary roles in cholesterol’s membrane condensing abilities and, additionally, that specific facial preferences are displayed as cholesterol interacts with different neighboring lipids and transmembrane proteins. Inspired by these poorly characterized biochemical interactions, an extensive 18-step synthesis was completed as part of a collaborative effort, wherein synthesizing a “smoothened” cholesterol analogue would provide a direct way to experimentally measure the significance of the β-face methyl groups. Starting from known perhydrochrysenone A, the synthesis of 18,19-di-nor-cholesterol was accomplished with an excellent overall yield of 3.5%. The use of the highly stereoselective Dieckmann condensation and the employment of Evans’ chiral auxiliary were both key to ensuring the success of this synthesis.

The enantiomer pair androsterone and ent-androsterone are positive allosteric modulators of γ-aminobutyric acid (GABA) type A receptors. Each enantiomer was shown to bind at the same receptor site. Binding orientations of the enantiomers at this site were deduced using enantiomer pairs containing OBn substituents at either C-7 or C-11. 11β-OBn-substituted steroids and 7α-OBn-substituted ent-steroids potently displace [35S]-tert-butylbicyclophosphorothionate, augment GABA currents, and anesthetize tadpoles. In contrast, 7β-OBn-substituted steroids and 11α-OBn-substituted ent-steroids have diminished actions. The results suggest that the binding orientations of the active analogues are inverted relative to each other with the 7α- and 11β-substituents similarly located on the edges of the molecules not in contact with the receptor surface. Analogue potentiation of the GABA current was abrogated by an α1 subunit Q241L mutation, indicating that the active analogues act at the same sites in α1β2γ2L receptors previously associated with positive neurosteroid modulation.

This study addresses the hypothesis that the lack of anesthetic activity for (3α,5α)-3-hydroxypregn-16-ene-11,20-dione (Δ16-alphaxalone) is explained by the steroid Δ16 double bond constraining the steroid 20-carbonyl group to a position that prevents it from favorably interacting with γ-aminobutyric acid type A (GABAA) receptors. A series of Δ16 and Δ17(20) analogues of Δ16-alphaxalone was prepared to evaluate this hypothesis in binding, electrophysiological, and tadpole anesthesia experiments. The results obtained failed to support the hypothesis. Instead, the results indicate that it is the presence of the C-21 methyl group in Δ16-alphaxalone, not the location of the constrained C-20 carbonyl group, that prevents Δ16-alphaxalone from interacting strongly with the GABAA receptor and having anesthetic activity. Consistent with this conclusion, a Δ17(20) analogue of Δ16-alphaxalone without a C-21 methyl group was found to be very similar to the anesthetic steroid (3α,5α)-3-hydroxypregnane-11,20-dione (alphaxalone) with regard to time of onset and rate of recovery from anesthesia when administered to mice by tail vein injection.

Expansion of the D-ring of 19-norsteroids with incorporation of the steroid C-18 methyl group into a newly formed six-membered ring provides easy access to the chrysene ring system. By taking advantage of the symmetry of the chrysene ring system and avoiding meso chrysene intermediates, four optically pure 2,8-difunctionalized (C-2 hydroxyl group and C-8 oxo group) hexadecahydrochrysene diastereomers, and their corresponding optically pure enantiomers were prepared from 19-nortestosterone. The eight chrysene stereoisomers are of interest as starting materials for preparing chrysene analogues of physiologically important neurosteroids.

Alphaxalone, a neuroactive steroid containing a 17β-acetyl group, has potent anesthetic activity in humans. This pharmacological activity is attributed to this steroid’s enhancement of γ-amino butyric acid-mediated chloride currents at γ-amino butyric acid type A receptors. The conversion of alphaxalone into Δ16-alphaxalone produces an analogue that lacks anesthetic activity in humans and that has greatly diminished receptor actions. By contrast, the corresponding 17β-carbonitrile analogue of alphaxalone and the Δ16-17-carbonitrile analogue both have potent anesthetic and receptor actions. The differential effect of the Δ16-double bond on the actions of alphaxalone and the 17β-carbonitrile analogue is accounted for by a differential effect on the orientation of the 17-acetyl and 17-carbonitrile substituents.

Although the structural features of binding sites for neuroactive steroids on γ-aminobutryic acid type A (GABAA) receptors are still largely unknown, structure–activity studies have established a pharmacophore for potent enhancement of GABAA 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 GABAA 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 GABAA 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 (α1Q241L mutation and α1N407A/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).

Allopregnanolone (1) and pregnanolone (2), steroids containing a 17β-acetyl group, are potent enhancers of GABA (γ-aminobutyric acid) action at GABAA receptors. Their effects are enantioselective with the non-naturally occurring enantiomers (ent-1 and ent-2) being less potent. Androsterone (3) and etiocholanolone (4), steroids with a C-17 carbonyl group, are weak enhancers of GABA action at GABAA receptors. Unexpectedly, their enantiomers (ent-3 and ent-4) have been found to have enhanced, not diminished, activity at GABAA receptors. Furthermore, the C-17 spiro-epoxide analogues (ent-5 and ent-6) of ent-3 and ent-4, respectively, have activities comparable to those of steroids 1 and 2. The results indicate that some ent-steroids are potent modulators of GABAA receptors and might have clinical potential as GABAergic drugs of the future.

Cellular cholesterol homeostasis is regulated by oxygenated cholesterol metabolites called oxysterols. While the importance of oxysterols in the acute regulation of cholesterol homeostasis is known, the precise molecular mechanisms through which oxysterols exert their effects remain to be elucidated. LY295427 (1) is a known antagonist of the cholesterol-homeostatic effects of 25-hydroxycholesterol (25-HC), a biologically active oxysterol. In order to examine the mechanism of action of this antagonism, and to further explore recent evidence suggesting that the membrane effects of 25-HC contribute to acute cholesterol regulation, we synthesized the enantiomer of LY295427 (ent-LY295427). ent-LY295427 (2) will serve as a unique probe to provide insight into the role of transcription-independent mechanisms in regulation of cholesterol homeostasis.Highlights► Synthesis of the enantiomer of LY295427. ► Tool for mechanistic studies of LY295427-dependent inhibition of 25-hydroxycholesterol. ► Total steroid synthesis.

•Synthesis of oxysterol enantiomers.•Cross-metathesis reactions of Δ22 steroids.•Enantioselective synthesis.A synthetic route that utilizes a cross-metathesis reaction with Δ22 steroids has been developed to prepare sterols with varying C-27 side-chains. Natural sterols containing hydroxyl groups at the 25 and (25R)-26 positions were prepared. Enantiomers of cholesterol and (3β,25R)-26-hydroxycholesterol (27-hydroxycholesterol) trideuterated at C-19 were prepared for future biological studies.

Membrane receptors are often modulated by steroids and it is necessary to distinguish the effects of steroids at these receptors from effects occurring at nuclear receptors. Additionally, it may also be mechanistically important to distinguish between direct effects caused by binding of steroids to membrane receptors and indirect effects on membrane receptor function caused by steroid perturbation of the membrane containing the receptor. In this regard, ent-steroids, the mirror images of naturally occurring steroids, are novel tools for distinguishing between these various actions of steroids. The review provides a background for understanding the different actions that can be expected of steroids and ent-steroids in biological systems, references for the preparation of ent-steroids, a short discussion about relevant forms of stereoisomerism and the requirements that need to be fulfilled for the interaction between two molecules to be enantioselective. The review then summarizes results of biophysical, biochemical and pharmacological studies published since 1992 in which ent-steroids have been used to investigate the actions of steroids in membranes and/or receptor-mediated signaling pathways.

Estrone and 17β-estradiol are phenolic steroids that are known to be neuroprotective in multiple models of neuronal injury. Previous studies have identified the importance of their phenolic steroid A-ring for neuroprotection and have identified ortho substituents at the C-2 and C-4 positions on the phenol ring that enhance this activity. To investigate the importance of the steroid ring system for neuroprotective activity, phenolic compounds having the cyclopent[b]anthracene, cyclopenta[b]phenanthrene, benz[f]indene, benz[e]indene, indenes linked to a phenol, and a phenolic spiro ring system were prepared. New synthetic methods were developed to make some of the cyclopent[b]anthracene analogues as well as the spiro ring system. Compounds were evaluated for their ability to protect HT-22 hippocampal neurons from glutamate neurotoxicity and their activity relative to a potent neuroprotective analogue of 17β-estradiol was determined. An adamantyl substituent placed ortho to the phenolic hydroxyl group gave neuroprotective analogues in all ring systems studied.

Expansion of the D-ring of 19-norsteroids with incorporation of the steroid C-18 methyl group into a newly formed six-membered ring provides easy access to the chrysene ring system. By taking advantage of the symmetry of the chrysene ring system and avoiding meso chrysene intermediates, four optically pure 2,8-difunctionalized (C-2 hydroxyl group and C-8 oxo group) hexadecahydrochrysene diastereomers, and their corresponding optically pure enantiomers were prepared from 19-nortestosterone. The eight chrysene stereoisomers are of interest as starting materials for preparing chrysene analogues of physiologically important neurosteroids.