Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.

Two naturally occurring metabolites of progesterone, 3 alpha-hydroxy-5 alpha- and 5 beta-pregnan-20-one (1 and 2), are potent allosteric modulators of the GABAA receptor. Their therapeutic potential as anxiolytics, anticonvulsants, and sedative/hypnotics is limited by rapid metabolism. To avoid these shortcomings, a series of 3 beta-substituted derivatives of 1 and 2 was prepared. Small lipophilic groups generally maintain potency in both the 5 alpha- and 5 beta-series as determined by inhibition of [35S]TBPS binding. In the 5 alpha-series, 3 beta-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3 beta-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. In the 5 beta-series, the unsubstituted parent 2 is a two-component inhibitor, whereas all of the 3 beta-substituted derivatives of 2 inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5 beta-sterols tested are full inhibitors of [35S]TBPS binding. Electrophysiological measurements using alpha 1 beta 2 gamma 2L receptors expressed in oocytes show that 3 beta-methyl- and 3 beta-(azidomethyl)-3 alpha-hydroxy-5 alpha-pregnan-20-one (6 and 22, respectively) are potent full efficacy modulators and that 3 alpha-hydroxy-3 beta-(trifluoromethyl)-5 alpha-pregnan -20-one (24) is a low-efficacy modulator, confirming the results obtained from [35S]TBPS binding. These results indicate that modification of the 3 beta-position in 1 and 2 maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, compound 6 (CCD 1042) is an orally active anticonvulsant, while the naturally occurring progesterone metabolites 1 and 2 are inactive when administered orally, suggesting that 3 beta-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

3 alpha-Hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970): a partial agonist at the neuroactive steroid site of the gamma-aminobutyric acidA receptor.

Neuroactive steroids bind to a unique site on the gamma-aminobutyric acidA (GABAA) receptor complex and allosterically modulate the binding of convulsant ([35S]t-butylbicyclophosphorothionate, [35S]TBPS), GABA ([3H]muscimol), and benzodiazepine ([3H]flunitrazepam) site ligands. In rat cortical membranes, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) is a full agonist at the steroid site, inhibiting 96% of specific [35S]TBPS binding and enhancing [3H]flunitrazepam and [3H]muscimol binding 95% and 69% above control levels, respectively. In contrast, the synthetic steroid 3 alpha-hydroxy-3 beta-trifluoromethyl-5 alpha-pregnan-20-one (Co 2-1970) has limited efficacy for modulating the binding of [35S]TBPS (44% inhibition), [3H]flunitrazepam (41% enhancement), and [3H]muscimol (< 10% enhancement). In competition experiments, Co 2-1970 (10 microM) reduced the apparent potency of 3 alpha, 5 alpha-P by 7-17-fold for modulating the binding of these radioligands in rat cortical membranes, suggesting that it has partial agonist properties. Because cortical membranes contain a heterogeneous population of receptors, Co 2-1970 was examined in recombinant GABAA receptors stably expressed in human embryonic kidney 293 cells. Co 2-1970 inhibited [35S]TBPS binding with limited efficacy (39-65% inhibition) in the five receptor combinations examined and, at 10 microM, reduced the apparent potency of 3 alpha, 5 alpha-P 57-fold for inhibiting [35S]TBPS binding to alpha 1 beta 1 gamma 2L receptors. To verify these findings functionally, the effects of 3 alpha, 5 alpha-P and Co 2-1970 were examined electrophysiologically in Xenopus oo-cytes expressing alpha 1 beta 1 gamma 2L receptors. Co 2-1970 showed limited efficacy potentiation of GABA-evoked chloride currents relative to 3 alpha, 5 alpha-P (28% and 86% of the GABA maximum current, respectively). Moreover, Co 2-1970 produced a concentration-dependent antagonism of the 3 alpha, 5 alpha-P-induced potentiation that was associated with a reduction in the apparent affinity of 3 alpha, 5 alpha-P (11-fold at 10 microM Co 2-1970). Taken together, these data indicate that Co 2-1970 is a partial agonist at the neuroactive steroid site associated with GABAA receptors.

Steroid inhibition of [3H]SR 95531 binding to the GABAA recognition site.

The interaction of three types of steroids with the GABAA recognition site labeled by the antagonist ligand [3H]SR 95531 was evaluated in rat brain cortical membranes. The first type is the GABA site antagonist RU 5135, which potently (IC50 7 nM) but also incompletely (Imax 82%) displaced [3H]SR 95531. RU 5135 probably binds only to high affinity [3H]SR 9553] sites recognized by GABA and unlabelled SR 95531. The second type are the neuroactive steroids which act as positive allosteric modulators, including 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P) and 5 beta-tetrahydrodeoxycorticosterone (5 beta-THDOC), which inhibited [3H]SR 95531 binding with limited efficacy (IC50 460 nM and 1.4 microM, Imax 41 and 31%, respectively). In contrast, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) was inactive. The third type are the neurosteroids acting as negative allosteric modulators, such as pregnenolone sulfate, which inhibited [3H]SR 95531 binding with limited efficacy (IC50 10 microM, Imax 23%). In the presence of a saturating concentration of pregnenolone sulfate, 3 alpha, 5 beta-P further inhibited [3H]SR 95531 binding suggesting that these two steroids act through different sites or, possibly, at different populations of GABAA receptors. The allosteric modulation was selective for steroids since benzodiazepines and barbiturates were inactive up to 100 microM. Taken together, these data suggest that 3 alpha, 5 beta-P and 5 beta-THDOC modulate [3H]SR 95531 binding by interacting with a unique site on the GABAA receptor complex distinct from the sites for 3 alpha, 5 alpha-P, pregnenolone sulfate, GABA, benzodiazepines, and barbiturates.

Correlation of neuroactive steroid modulation of [35S]t-butylbicyclophosphorothionate and [3H]flunitrazepam binding and gamma-aminobutyric acidA receptor function.

Neuroactive steroids, including endogenously occurring metabolites of progesterone and deoxycorticosterone as well as their synthetic derivatives, are positive allosteric modulators of the gamma-aminobutyric acid (GABA)A receptor complex. They inhibit the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS), enhance the binding of [3H]flunitrazepam, and potentiate GABA-evoked chloride currents and agonist-stimulated 36Cl- uptake. The structure-activity relationship for 31 neuroactive steroids and related compounds was explored by examining their relative ability to inhibit [35S]TBPS binding in rat brain cortical membranes. A free 3 alpha-hydroxy group is necessary for high potency inhibition. Whereas hydroxylation in the 21-position and subsequent esterification maintain activity, 11 alpha- or 12 alpha-hydroxylation greatly reduces activity. The rank order of potency for 17-position substitutions in the 5 alpha-reduced series is 17 beta-acetyl > 17 beta-cyano > 17 beta-methoxycarbonyl > 17 alpha-acetyl > 17-one > or = 17-oxime > or = 17 alpha-cyano. Introduction of a double bond between the 9- and 11-positions reduces potency, whereas a double bond in the 4-position reduces the maximal extent of inhibition. Comparing the activities of these neuroactive steroids and related compounds in the [35S]TBPS and [3H]flunitrazepam assays, there is a strong correlation between potency (r = 0.90, n = 17) and magnitude of modulation (r = 0.95, n = 31), indicating that the neuroactive steroid binding site is similarly coupled to the TBPS and benzodiazepine sites in rat cortex. However, there is a weaker correlation (r = 0.74-0.78, n = 31) between the degree of modulation in either binding assay and potentiation of muscimol-stimulated 36Cl- uptake in rat cortical synaptoneurosomes. Using an electrophysiological approach, stronger correlations (r = 0.89-0.94, n = 15) were observed between the magnitude of modulation in the binding assays and potentiation of GABA-evoked chloride currents in Xenopus oocytes expressing human alpha 1 beta 1 gamma 2L receptor complexes. Thus, neuroactive steroid modulation of [35S]TBPS and [3H]flunitrazepam binding is predictive of functional activity at the GABAA receptor complex.

The neuroactive steroid 3 alpha-hydroxy-5 beta-pregnan-20-one is a two-component modulator of ligand binding to the GABAA receptor.

Neuroactive steroids allosterically inhibit [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and enhance [3H]flunitrazepam binding to the GABAA receptor complex. In the presence of 5 microM GABA, 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P) inhibits [35S]TBPS binding with high- (IC50 21-32 nM) and low- (IC50 24-63 microM) affinity components in bovine cortical, cerebellar, and hippocampal membranes. The percentage of high-affinity sites ranges from 53% in cortex to 65% in cerebellum and hippocampus. However, 3 alpha, 5 beta-P is a single-site inhibitor in thalamus (IC50 43 nM). In the absence of GABA, similar affinities for the high- and low-affinity components were detected, although the percentages of high-affinity sites were reduced. Similarly, 3 alpha, 5 beta-P enhances [3H]flunitrazepam binding with high- (EC50 44-58 nM) and low- (EC50 2-13 microM) affinity components which account for 71-77% and 23-29% of the sites, respectively, in cortex, cerebellum and hippocampus. 3 alpha, 5 beta-P is a single-site enhancer in thalamus (EC50 80 nM). In contrast to 3 alpha,5 beta-P, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) is a single site modulator of [35S]TBPS and [3H]flunitrazepam binding in all regions examined. These data provide pharmacological evidence consistent with receptor heterogeneity for neuroactive steroids.