Neurosteroid Analogues. 10. The Effect of Methyl Group Substitution at

steroid (3R,5β)-3-hydroxypregnan-20-one act at GABAA receptors to induce general anesthesia. The structural features of the binding sites for these a...
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J. Med. Chem. 2005, 48, 3051-3059

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Neurosteroid Analogues. 10. The Effect of Methyl Group Substitution at the C-6 and C-7 Positions on the GABA Modulatory and Anesthetic Actions of (3r,5r)and (3r,5β)-3-Hydroxypregnan-20-one Chun-min Zeng,† Brad D. Manion,‡ Ann Benz,§ Alex S. Evers,†,‡ Charles F. Zorumski,§,⊥ Steven Mennerick,§,⊥ and Douglas F. Covey*,† Departments of Molecular Biology and Pharmacology, Anesthesiology, Psychiatry, and Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, Missouri 63110 Received November 30, 2004

The planar 5R-reduced steroid (3R,5R)-3-hydroxypregnan-20-one and the nonplanar 5β-reduced steroid (3R,5β)-3-hydroxypregnan-20-one act at GABAA receptors to induce general anesthesia. The structural features of the binding sites for these anesthetic steroids on GABAA 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 [35S]-tert-butylbicyclophosphorothionate binding experiments, (2) in electrophysiological experiments using rat R1β2γ2L GABAA receptors expressed in Xenopus laevis oocytes, and (3) as tadpole anesthetics. The effects of methyl group substitution in the 5R- and 5β-reduced series of compounds were strikingly similar. In both series, a 6β-Me group gave compounds with actions similar to or greater than those of the parent steroids. A 6R-, 7β- or 7R-Me substituent resulted in reduced potency for inhibition of radioligand binding, GABAA 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 3R-hydroxyl groups of planar and nonplanar anesthetic steroids hydrogen bond to different amino acids on GABAA receptors or that this critical hydrogen bonding group interacts with membrane lipids instead of the receptor. Introduction

Chart 1

The steroids (3R,5R)- and (3R,5β)-3-hydroxypregnan20-one (1a and 2a, Chart 1) are potent anesthetics. It is now widely accepted that the molecular mechanism for steroid-induced anesthesia by these and other structurally related 3R-hydroxysteroids is enhancement of inhibitory GABAergic neurotransmission. Although pharmacological studies have shown that the binding sites for anesthetic steroids on GABAA receptors are distinct from the binding sites for benzodiazepines, barbiturates and picrotoxin, the location and number of binding sites for anesthetic steroids on these receptors have not been determined.1 Anesthetic steroid modulation of GABAA receptor function occurs for many subtypes of receptors composed of native R,β,γ,δ subunit isoforms.1,2 Although no receptor subtype specific anesthetic steroids have been yet identified, some differences in the potency and efficacy of analogues for different receptor subtypes have been reported.3,4 Initially, binding and GABAA receptor-mediated chloride flux studies suggested that multiple binding sites exist for anesthetic steroids on GABAA receptors.5,6 More recently, electrophysiological evidence for the existence * Corresponding author. Telephone: 314-362-1726. Fax: 314-3627058. Email: [email protected]. † Department of Molecular Biology and Pharmacology. ‡ Department of Anesthesiology. § Department of Psychiatry. ⊥ Department of Anatomy and Neurobiology.

of multiple binding sites has been presented.7 The presence of multiple binding sites for anesthetic steroids on GABAA receptors greatly complicates attempts to address one of the most striking structure-activity relationships (SAR) observed in the field of anesthetic steroid research. This SAR concerns the relative unimportance of the stereochemistry for the A,B-ring fusion at C-5. The anesthetic and GABAergic effects of these two steroids are very similar despite the fact that the 5R-reduced steroid 1a is planar and the 5β-reduced steroid 2a is not (Chart 1).8,9 Models for a common binding site for steroids 1a and 1b have been proposed.10,11 However, neither this model nor any other model proposed thus far completely explains all of the current SAR data. The results which are most difficult to explain by a common site model

10.1021/jm049027+ CCC: $30.25 © 2005 American Chemical Society Published on Web 03/25/2005

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are those results showing differences in the enantioselectivity for GABAA receptor modulation by steroids 1a and 2a12,13 and results reported for an antagonist that potently reverses the anesthetic and GABAergic actions of steroid 1a while having minimal effects on these actions of steroid 1b.14 To probe further for similarities and differences in the binding of 5R- and 5β-reduced steroids to binding sites on GABAA receptors, we performed SAR studies that utilized a series of C-6 and C-7 methyl substituted analogues (1b-e; 2b-e) of steroids 1a and 2a. This region of the steroid molecule was chosen for modification because it is relatively near to the A,B-ring fusion (the distinguishing feature of steroids 1a and 1b) and because several previous reports in the literature suggested that both active and inactive compounds might be obtained by the proposed modifications.15-19 Functional actions of the compounds at GABAA receptors were determined using electrophysiological methods and rat R1β2γ2L GABAA receptors expressed in Xenopus laevis oocytes. Binding interactions of the compounds with GABAA receptors were studied by measuring the noncompetitive displacement of [35S]TBPS from the picrotoxin site found on the heterogeneous GABAA receptors present in rat brain membranes. Anesthetic effects of the compounds were determined by measuring the loss of righting reflex (LRR) and loss of swimming reflex (LSR) in Xenopus laevis tadpoles. We found similar SAR for the C-6 or C-7 methylsubstituted analogues of steroids 1a and 2a. Analogues with the axial 6β-Me group retain high activity as modulators of GABAA receptor function and tadpole behavior. By contrast, analogues with either an equatorial 6R- or 7β-Me group, as well as those containing an axial 7R-Me group, have greatly reduced activities in these bioassays. The results suggest a striking similarity in the shape of GABAA receptor binding sites for steroids 1a and 2a in the C-6 and C-7 regions of these molecules. Chemistry. The 6-Me steroid analogues 1b, 1c and 2b, 2c were prepared as shown in Scheme 1. The starting material, (6β)-6-methylpregn-4-ene-3,20-dione (3), was prepared from commercially available pregnenolone acetate according to literature procedures.20 Catalytic hydrogenation of steroid 3 using 5% Pd/CaCO3 under basic conditions gave a 90% yield of a 3.4:1 mixture of the C-5 epimers 4a (5R-configuration) and 4b (5β-configuration). Under the same experimental conditions, hydrogenation of progesterone gives a product in which the epimer having the 5β-configuration is the major product.21 Presumably, the 6β-Me group present in steroid 3 makes the β-face of this steroid less accessible to the surface of the catalyst and is responsible for the hydrogenation results observed with steroid 3. The regio- and stereoselective reduction of the C-3 carbonyl group of steroid 4a using K-Selectride gave steroid 1b in 57% yield. Steroid 2b was prepared in 69% yield by the regio- and stereoselective reduction of the C-3 carbonyl group of steroid 4b using lithium tri-tertbutoxyaluminum hydride. Base-catalyzed isomerization of (6β)-6-methylpregn4-ene-3,20-dione gave (6R)-6-methylpregn-4-ene-3,20dione (5) in 77% yield as described previously.21 Cata-

Zeng et al.

Scheme 1a

a (a) KOH, MeOH; (b) H , 5% Pd/CaCO , KOH, EtOH; (c) 1) 2 3 K-Selectride, THF; 2) MeOH, 5 N NaOH, 30% H2O2-H2O; (d) LiAl(OCMe3)3H, THF, 0 °C.

lytic hydrogenation of steroid 5 using 5% Pd/CaCO3 gave a 57% yield of a 1:2.7 mixture of the C-5 epimers 6a (5R-configuration) and 6b (5β-configuration). Regio- and stereoselective reduction of the C-3 carbonyl group of steroid 6a using K-Selectride gave steroid 1c in 61% yield, and regio- and stereoselective reduction of the C-3 carbonyl group of steroid 6b using lithium tri-tertbutoxyaluminum hydride gave steroid 2c in 81% yield. The 7-Me steroid analogues 1d, 1e and 2d, 2e were prepared as shown in Schemes 2 and 3. The starting material, pregna-4,6-diene-3,20-dione (7), was prepared by DDQ oxidation of commercially available progesterone according to a literature procedure.22 Ketalization of both carbonyl groups of steroid 7 with ethylene glycol, followed by selective removal of the C-3 ketal group with MgSO4, gave steroid 8 in 75% yield. The 1,6-addition of (CH3)2CuLi to steroid 8, followed by base-catalyzed isomerization of the ∆5-double bond in the addition product, gave a 61% yield of a 2.9:1 mixture of the C-7 epimers 9 and 10. Reduction of the double bond of steroid 9 using Li in liquid NH3 gave a 66% yield of a 1:3.6 mixture of ketosteroid 11a and 3β-hydroxysteroid 11b. The 3βhydroxysteroid 11b was either converted quantitatively back into ketosteroid 11a by PDC oxidation (not shown) or converted into 3β-hydroxysteroid 13 (69% yield) by hydrolysis of the 20-ketal group (Scheme 3). K-Selectride reduction of steroid 11a gave a 3R-hydroxysteroid product, which after hydrolysis of the 20-ketal group,

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Scheme 2a

Table 1. Inhibition of [35S]TBPS by Steroids 1a-e, 2a-e,13 and 14 compd

IC50 (µM)a

nHill

1a 1b ,6β-Me 1c, 6R-Me 1d, 7R-Me 1e, 7β-Me 13, 7R-Me

5R-Steroids 0.074 ( 0.0074 0.0057 ( 0.0014 2.94 ( 0.21 5.99 ( 0.06 1.24 ( 0.15 12 ( 0.19

0.89 ( 0.06 1 ( 0.12 1.17 ( 0.09 1.26 ( 0.13 0.74 ( 0.05 0.99 ( 0.16

2a 2b, 6β-Me 2c, 6R-Me 2d, 7R-Me 2e, 7β-Me 14 ,7R-Me

5β-Steroids 0.071 ( 0.018 0.045 ( 0.004 3.08 ( 0.36 1.04 ( 0.08 1.24 ( 0.13 10.8 ( 1.2

0.57 ( 0.06 0.78 ( 0.04 2.33 ( 0.71 0.63 ( 0.03 0.75 ( 0.05 0.9 ( 0.09

a Results presented are from duplicate experiments performed in triplicate. Error limits are calculated as standard error of the mean.

a (a) 1) HOCH CH OH, TsOH, benzene; 2) MgSO ; (b) 1) CuI, 2 2 4 MeLi; 2) KOH, 80% CH3OH-H2O; (c) Li/NH3, THF, -78 °C; (d) H2, 5% Pd/CaCO3, KOH, EtOH.

Scheme 3a

a (a) 1) K-Selectride, THF; 2) MeOH, 5 N NaOH, 30% H O 2 2 H2O; 3) TsOH, acetone; (b) TsOH, acetone; (c) 1) LiAl(OCMe3)3H, THF, 0 °C; 2) TsOH, acetone.

gave steroid 1d in 84% yield. Catalytic hydrogenation of steroid 9 using 5% Pd/CaCO3 under basic conditions gave an 80% yield of 5β-reduced steroid 11c (Scheme 2). Lithium tri-tert-butoxyaluminum hydride reduction of steroid 11c, followed by removal of the 20-ketal group under acidic conditions, gave the C-3 epimers 2d and 14 in yields of 70% and 12%, respectively (Scheme 3).

Because steroid 10 proved to be difficult to separate from steroid 9, a mixture of these two compounds was used for the preparation of steroids 1e and 2e. Lithium/ liquid NH3 reduction of a mixture of compounds 9 and 10 gave a mixture of intermediates 11a and 12a. These intermediates were not separated, and intermediate 12a was not characterized. The mixture of intermediates 11a and 12a was reduced using K-Selectride to give a mixture of steroids 1d and 1e from which product 1e was obtained by HPLC. Similarly, catalytic hydrogenation of a mixture of steroids 9 and 10 gave a mixture of unseparated intermediates 11c and 12b (uncharacterized) which was then reduced using lithium tri-tertbutoxyaluminum hydride to give a mixture of products 2d and 2e. Pure steroid 2e was obtained by column chromatography. [35S-TBPS] Displacement. The results of the binding experiments are summarized in Table 1. In comparison to steroid 1a, an added 6β-Me group (1b) causes a 13-fold enhancement in binding. By contrast an additional 6R-, 7β- or 7R-Me group (1c-e) causes a decrease in binding. The largest decrease in binding was found for 7R-Me steroid 1d (81-fold). Additionally, inhibition of [35S-TBPS] binding by steroid 1d was only 2-fold more potent than that observed for the corresponding 3β-hydroxysteroid 13. The Hill slopes are not markedly different for any of the 3R-hydroxysteroids in the series, a result which is consistent with the conclusion that all analogues are binding to a similar number of binding sites. In comparison to steroid 2a, the additional 6β-Me group found in steroid 2b had little effects on its potency as an inhibitor of [35S-TBPS] binding. However, as in the 1c-e series, an additional 6R-, 7β- or 7R-Me group (2c-e) caused large decreases in binding potency with the 6R-Me group having the most deleterious effect. The 3R- and 3β-hydroxysteroids with the 7R-Me group differed in inhibition potency by a factor of 10 (2d > 14). Except for steroid 2c, which has a Hill slope of 2.33, the remaining steroids in the series have Hill slopes that are similar in value and generally are less steep than those found in the 5R-reduced series 1a-e. Electrophysiology. The electrophysiological evaluations were carried out on Xenopus laevis oocytes expressing rat R1β2γ2L type GABAA receptors and are summarized in Table 2. Compounds were evaluated at

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Table 2. Modulation of Rat R1β2γ2L GABAA Receptor Function by Steroids 1a-e, 2a-e, 13 and 14 oocyte electrophysiologya compd

0.1 µM

1 µM

10 µM

1ab 1b, 6β-Me 1c, 6R-Me 1d, 7R-Me 1e, 7β-Me 13, 7R-Me

1.26 ( 0.14 2.50 ( 0.33 1.01 ( 0.04 0.87 ( 0.10 0.95 ( 0.12 0.84 ( 0.01

5R-Steroids 3.89 ( 1.34 9.65 ( 3.87 7.70 ( 1.56 10.04 ( 2.16 1.17 ( 0.04 3.24 ( 0.14 1.19 ( 0.32 1.30 ( 0.20 1.32 ( 0.06 1.51 ( 0.07 0.81 ( 0.04 0.94 ( 0.03

2a 5β-DHP 2b, 6β-Me 2c, 6R-Me 2d, 7R-Me 2e, 7β-Me 14, 7R-Me

1.20 ( 0.10 1.48 ( 0.09 0.96 ( 0.08 1.06 ( 0.13 1.09 ( 0.11 0.95 ( 0.02

5β-Steroids 2.82 ( 0.51 9.77 ( 2.15 4.36 ( 0.63 22.5 ( 1.44 1.02 ( 0.03 2.51 ( 0.10 1.08 ( 0.16 2.09 ( 0.26 1.00 ( 0.12 3.51 ( 0.49 0.98 ( 0.06 0.93 ( 0.04

(gating) 10 µM 0.37 ( 0.07 0.59 ( 0.18 0.06 ( 0.02 0.01 ( 0.01 0.00 ( 0.02 0.01 ( 0.02 0.06 ( 0.03 0.62 ( 0.30 0.15 ( 0.03 -0.04 ( 0.03 0.02 ( 0.03 0.00 ( 0.01

a The GABA concentration used for the control response was 2 µM. Each compound was evaluated on at least four different oocytes at the concentrations indicated, and the results reported are the ratio of currents measured in the presence/absence of added compound. Gating represents direct current gated by 10 µM compound in the absence of GABA, and this current is reported as the ratio of compound only current/2 µM GABA current. Error limits are calculated as standard error of the mean (N g 4). b Values reported are from ref 25.

three different concentrations for their ability to augment currents mediated by 2 µM GABA. Direct gating effects of each compound (10 µM) in the absence of GABA also were determined. This screening method is adequate for making qualitative distinctions between compounds with enhanced or reduced activities. However, since all compounds were not tested on the same oocyte, and because GABAA receptor steroid sensitivity varies within and between preparations of oocytes, quantitative comparisons are not possible. Nevertheless, it is clear that only substitution with the 6β-Me group leads to compounds with potentiation or gating actions similar to or greater than those of the parent unsubstituted steroids. Additionally, it appears that 2b is more efficacious than 2a at the highest concentration tested (10 µM), although receptor desensitization may limit our ability to accurately estimate maximum potentiation. The 3β-hydroxysteroids 13 and 14 were unable to potentiate GABA-mediated currents. Quantitative comparisons were made for the 6β-Me steroids 1b and 2b in subsequent experiments wherein each compound was applied along with the correspond-

Figure 1. Comparison of endogenous compounds 1a and 2a with corresponding 6β-Me steroids (1b and 2b). A. Current response of a Xenopus oocyte expressing recombinant rat R1β2γ2L subunit combination. Traces represent superimposed responses to 2 µM GABA, 2 µM GABA plus 0.5 µM compound 1a, and 2 µM GABA plus 0.5 µM compound 1b. Note that compound 1b is a better potentiator of GABA responses compared with compound 1a at this concentration. B. Summary of effects of compound 1a and 1b in 7 oocytes as in panel A. Potentiation was calculated as Rs/RG-1, where Rs is the response in the combined presence of GABA and modulator and RG is the response to GABA alone. C and D. Same as A and B, but for the 5β-reduced pair of endogenous (2a) and 6βMe (2b) steroids. For C, responses are from the same cell as those in A. For D, N ) 11 oocytes. Note the comparable potentiation by the two compounds at equimolar concentration.

ing reference steroids 1a and 1b to the same oocyte. At 0.5 µM, compound 1b enhances GABA-mediated currents about twice as well as steroid 1a (Figure 1A, 1B). This result is consistent with the screening results obtained with different oocytes (Table 2) for these two compounds at concentrations of 0.1µM and 1 µM. For steroids 2a and 2b, the relative effects of the two compounds when evaluated at 0.5 µM are equal (Figure 1C, 1D). These results are also consistent with the smaller qualitative differences in potentiation reported for low concentrations (0.1 µM and 1 µM) of these compounds in Table 2. We recently reported that (3R,5R)-17-phenylandrost16-en-3-ol (15) effectively antagonizes the GABAergic and anesthetic actions of steroid 1a, but not those of steroid 2a.14 Hence, if the binding sites that recognize steroids 1a and 2a are the same sites at which the 6βMe steroids 1b and 2b are recognized, we expect that

Table 3. Effects of Steroids 1a-e, 2a-e,13 and 14 on Tadpole Righting and Swimming Responses compd

tadpole LRRa ED50 (µM)

tadpole LRR(nHill)

1a 1b, 6β-Me 1c, 6R-Me 1d, 7R-Me 1e, 7β-Me 13, 7R-Me

0.42 ( 0.04 0.51 ( 0.14 1.40 ( 1.45 2.68 ( 0.78 1.34 ( 0.09 1.63 ( 0.03

5R-Steroids -1.83 ( 0.32 -2.62 ( 0.11 -0.91 ( 0.47 -1.85 ( 0.75 -2.27 ( 0.27 -2.85 ( 0.08

2a 2b, 6β-Me 2c, 6R-Me 2d, 7R-Me 2e, 7β-Me 14, 7R-Me

0.06 ( 0.01 0.40 ( 0.11 2.66 ( 2.29 1.67 ( 0.01 1.36 ( 0.81 1.25 ( 1.42

5β-Steroids -1.54 ( 0.12 -4.97 ( 4.94 -1.35 ( 0.93 -3.73 ( 0.02 -1.18 ( 0.56 -1.00 ( 0.72

tadpole LSRb ED50 (µM)

tadpole LSR(nHill)

5.5 ( 0.5 1.05 ( 0.01 8.9 ( 0.0 5.5 ( 0.1 5.5 ( 0.1 5.5 ( 0.1

-7.5 ( 1.1 -17.3 ( 1.0 -19.5 ( 0.0 -33.5 ( 0.1 -33.5 ( 0.1 -33.5 ( 0.1

0.30 ( 0.01 0.55 ( 0.01 5.5 ( 0.01 5.5 ( 0.1 5.5 ( 0.1 10 ( 0.1

-6.9 ( 0.5 -33.5 ( 0.1 -18.1 ( 0.01 -33.5 ( 0.1 -33.5 ( 0.1 -17.0 ( 0.1

a LRR ) loss of righting response. Error limits are calculated as standard error of the mean (N ) 10 animals at each of five or more different concentrations). b LSR ) loss of swimming response. Error limits are calculated as standard error of the mean (N ) 10 animals at each of five or more different concentrations).

Neurosteroid Analogues

Figure 2. Effect of the steroid antagonist 15 on potentiation by 6β-Me steroids. A. Raw traces show an oocyte response to 2 µM GABA, 2 µM GABA plus 0.5 µM compound 1b, and 2 µM GABA plus 0.5 µM compound 1b plus 10 µM antagonist 15. B. Summary of response of 4 oocytes. C and D. Similar to A and B but for the 5β-reduced steroid 2b. Note the weaker effect of antagonist 15 versus the 5β-reduced steroid. For C, responses are from the same cell as A. For D, N ) 4 oocytes (same oocytes as panel B).

this selective antagonist will more effectively antagonize the GABAergic actions of steroid 1b than those of steroid 2b. As shown in Figure 2, this proved to be the case. The result suggests that 6β-Me group substitution has no major effect on the selectivity of 5R- or 5βreduced steroids for their respective binding sites. Tadpole Behavior. In comparison to steroid 1a, the 13-fold potency increase for binding to GABAA receptors found for steroid 1b was not accompanied by a corresponding increase in potency for tadpole LRR. However, compound 1b did have 5-fold increased potency for tadpole LSR. Steroids 1c-e were three to 6-fold less potent than steroid 1a at causing tadpole LRR. These compounds were still able to cause tadpole LSR at concentrations similar to those required for steroid 1a to produce this behavior. The 3β-hydroxysteroid 13 also was similar in potency to steroids 1c-e for both behavioral endpoints. That steroid 13 causes LRR and LSR, even though it is inactive in the electrophysiological bioassay, presumably results from modulation of non-GABAergic mechanisms that affect tadpole behavior. As is generally the case, the slopes of the concentration-response curves for LSR are very steep. Although there was a general correspondence in binding and swimming behavior for the 2a-e series, steroid 2b was not as potent at causing LRR as would have been expected from the binding or electrophysiology results. Steroid 2b was found to be about 7-fold less potent than steroid 2a for tadpole LRR. However, it had about the same potency as steroid 2a for causing LSR. Just as steroids 2c-e were all less potent than steroid 2a in displacing [35S-TBPS], they were also less potent in causing either tadpole LRR or LSR. As in the 5Rseries of compounds, the 3β-hydroxysteroid 14 was also able to induce LRR and LSR. Discussion As described in the Introduction there are now several lines of experimental evidence suggesting the presence of multiple binding sites for anesthetic steroids on GABAA receptors. The extent to which 5R-reduced anesthetic steroids bind to one set of binding sites and

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5β-reduced anesthetic steroids bind to a separate set of binding sites remains undetermined. The discovery of an antagonist that is selective for 5R-reduced steroids strongly suggests that there are differences in the way, and possibly the sites, that steroids 1a and 2a modulate GABAA receptor function.14 In this context, we prepared the C-6 and C-7 methyl-substituted analogues of steroids 1a and 2a to investigate the possibility that the pharmacological actions of these 5R- or 5β-reduced compounds might be more divergent than those of the parent steroids 1a and 2a. We found that the effects of C-6 or C-7 methyl group substitution were strikingly similar for the two series of 5R- or 5β-reduced anesthetic steroids. Incorporating an axial 6β-Me group into either steroid 1a or steroid 2a yielded analogues with comparable or increased pharmacological activities in the binding, electrophysiological and behavorial assays. Since it was previously shown that the presence or absence of a C-19 methyl group in 5R- or 5β-reduced anesthetic steroids had little effect on pharmacological activity,3,8,9,11,23 it is perhaps not surprising that the 6β-Me groups of steroids 1b and 2b are well tolerated. Recently, it was shown that the 6β-oxa substituent found in (3R,6β)-3-hydroxy-6,19-oxidopregn-4-en-20-one, a steroid which is conformationally similar to steroid 2a, yields an analogue which is more potent than steroid 2a at enhancing GABA-induced chloride flux in a synaptoneurosome preparation.24 We found that the electropositive 6β-Me group found in steroid 2b has a similar effect on GABA receptor function. That either a carbon or ether type oxygen is well tolerated as a 6βsubstituent is somewhat surprising since, as described below, replacing the C-6 carbon of steroid 2a with an oxygen to produce the corresponding 6-oxa analogue leads to a compound with greatly diminished GABAergic activity.15 We also investigated the possibility that the enhanced activities of steroids 1b or 2b might result from a crossover in the binding of steroid 1b to sites for steroid 2a or the binding of steroid 2b to sites for steroid 1a. This did not seem to be the case since compound 15, which is a selective antagonist for 5R-reduced steroid 1a, reduced the electrophysiological actions of 5Rreduced steroid 1b more effectively than those of steroid 2b. Previously, it was shown that the addition of a 5RMe group to steroid 1a had a negative effect on activity.11 Since there is no equivalent 5R-Me group substitution possible for a 5β-reduced steroid, it was unclear from that previous study if an axial methyl group on the R face of a 5β-reduced anesthetic steroid would lead to compounds with reduced activity. To investigate the effect of hydrophobic bulk on the R face of both 5R- and 5β-reduced anesthetic steroids in the region adjacent to C-5, analogues 1d and 2d were prepared. In both cases, the axial 7R-Me subsituent reduced activity indicating that hydrophobic bulk on the steroid R face is incompatible with high anesthetic activity when placed in this region of 5R- or 5β-reduced steroids. Compounds 1c and 2c, which contain an equatorial 6R-Me group, and compounds 1e and 2e, which contain an equatorial 7β-Me group, were also found to have diminished pharmacological activity. Thus, regardless

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Figure 3. A modeling figure that emphasizes the similarities found in the SAR studies of B and D ring/side chain modified analogues of steroids 1a (blue) and 2a (orange). If either the hydroxyl groups on the steroid A rings interact differently with the steroid binding sites on GABAA receptors, or the hydroxyl groups interact with membrane lipids instead of the receptor, then the SAR for the remaining parts of the steroids could be very similar. The steroid B, C and D rings are superimposed in the figure. The figure is not intended to infer that steroids 1a and 2a are aligned this way in a common binding site. Such an interpretation is incorrect since 5R-Me substituted analogues of steroid 1a, which would be expected to be highly active if this interpretation is correct, enhance GABA-mediated current poorly or not at all.11

of the stereochemistry of the A,B-ring fusion, hydrophobic substituents in the plane of the steroid are not well tolerated along the C-6, C-7 edge of the steroid. Since it has previously been shown that the 6-oxa analogues of steroids 1a and 2a have greatly diminished GABAergic actions,15 it appears that factors other than hydrophobic bulk also have important effects on activity when made at the C-6 position of the parent steroids. Finally, it should be mentioned that despite considerable synthetic efforts, structural features other than the difference in the A,B-ring fusion (or the presence of a 5R-Me group in steroid 1a) that could contribute in a decisive way to the selective binding of 5R- and 5βreduced steroids to separate binding sites on GABAA receptors have not been identified. Previously, analogues identically modified in the D-ring/side chain regions of steroids 1a and 2a were also found to have similar SAR.25-27 What accounts for this strikingly similar SAR for 5R- and 5β-reduced anesthetic steroids in parts of the molecules distant from the A,B-ring fusion? The answer to this question remains elusive. To date, however, the reported SAR results are compatible with the hypothesis that the structural features of 5R- and 5β-reduced steroid binding sites on GABAA receptors are quite similar for those portions of the receptor in contact with the steroid B, C and D rings. The binding sites would then presumably discriminate between 5R- and 5β-reduced steroid by the altered position of the steroid A ring relative to the steroid B, C and D rings (Figure 3). The critical features of this hypothesis are implicit in the way that previous SAR results (molecular modeling results that superimpose the B, C and D rings of steroids 1a, 2a or their analogues) have been discussed (no one has explicitly concluded that their SAR results prove this model).4,26,27 Such a model would, however, predict that if the 3Rhydroxyl groups of 5R- and 5β-reduced steroids are directly involved in hydrogen bond interactions with the amino acids of GABAA receptors, such interactions are likely to be different for the two steroids. In this regard, it should be noted that although the importance of the

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3R-hydroxyl groups as hydrogen bond donors is clear, it has been assumed that the hydrogen bond interactions occur with the receptor. There is no direct evidence for this steroid interaction with the receptor. It still remains possible that the 3R-hydroxyl groups remain exposed to membrane lipid while other parts of the anesthetic steroids are bound to the receptor. This hypothesis would suggest that the critical role of the 3R-hydroxyl groups is to hydrogen bond to the polar headgroups of lipids thereby ensuring that the anesthetic steroids have the correct mobility and orientation in the membrane to permit their access to the steroid binding sites on the receptors. There are NMR data regarding the mobility of these anesthetic steroids in model membranes that distinguish them from their inactive 3β-hydroxysteroid epimers in support of this hypothesis.28 Additionally, this hypothesis also explains why the SAR for a series of 3β-substituted analogues29-31 of steroids 1a and 2a are so similar, since the 3R-hydroxyl groups (and any appended 3β-substituents) could remain exposed to the membrane lipid and need not be constrained to the same location within the steroid binding sites. The same hypothesis could also apply to the SAR reported for 2β-substituted anesthetic steroid analogues.8,9,32 Consequently, this hypothesis suggests that the SAR for these 2β- and 3β-substituted compounds is an SAR that is more concerned with lipid than receptor interactions for these compounds. Future synthetic and biophysical experiments are needed to address this issue. Conclusion In this study, we examined the SAR for methyl group substitution at the C-6 and C-7 positions of 5R- and 5βreduced anesthetic steroids at GABAA receptors. The SAR for the analogues in each series of C-5 epimers were strikingly similar. Analogues containing a 6β-Me group were highly active and those containing either a 6R-, 7β- or 7R-Me group had greatly diminished activity. Although it may be possible to change the 6β-substituent and obtain additional compounds with high activity, we suspect that such additional analogues will not further clarify the reasons for the similar SAR of 5Rand 5β-reduced anesthetic steroids. Experimental Section General Methods. Melting points were determined on a Kofler micro hot stage and are uncorrected. NMR spectra were recorded at ambient temperature at 300 MHz (1H) or 75 MHz (13C). IR spectra were recorded as films for liquids or KBr disks for solids. Elemental analyses were carried out by M-H-W laboratories, Phoenix, AZ. Solvents were used either as purchased or dried and purified by standard methodology. Organic extracts were dried over anhydrous Na2SO4. Column chromatography was performed using silica gel (32-63 microns) purchased from Scientific Adsorbents, Atlanta, GA. (5r,6β)-6-Methylpregnane-3,20-dione (4a) and (5β,6β)6-Methylpregnane-3,20-dione (4b). The steroid (6β)-6-methypregn-4-ene-3,20-dione (3, 563 mg, 1.72 mmol) prepared according to the literature methods20 was dissolved in EtOH (120 mL) containing 5% Pd/CaCO3 (150 mg) and KOH (240 mg, 4.29 mmol) dissolved in water (1 mL). The mixture was hydrogenated at room temperature under H2 (50 psi) for 6 h. The reaction mixture was filtered and the filtrate diluted with water and extracted with Et2O. The extract was washed with

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water, brine and dried. After solvent removal, the residue was purified by silica gel column chromatography to afford steroid 4a (394 mg, 70%) and steroid 4b (117 mg, 20%). Steroid 4a was obtained as a white solid: mp 180-182 °C (lit.33 mp 179-181 °C); IR (KBr) 2935, 1711, 1691, 1354, 1222 cm-1; 1H NMR (CDCl3) δ 2.13 (3H, s), 1.10 (3H, s), 0.93 (3H, d, J ) 7.5 Hz), 0.67 (3H, s); 13C NMR (CDCl3) δ 212.69, 209.58, 63.69, 56.09, 54.07, 48.30, 44.05, 43.22, 41.05, 38.77, 38.74, 38.09, 36.04, 32.91, 31.35, 30.43, 24.42, 22.63, 21.20, 15.55, 14.75, 13.31. Anal. (C22H34O2) C, H. Steroid 4b was obtained as a white solid: mp 112-114 °C; IR (KBr) 2922, 2860, 1715, 1698, 1392, 1154 cm-1; 1H NMR (CDCl3) δ 2.51-2.61 (2H, m), 2.11 (3H, s), 1.12 (3H, d, J ) 7.5 Hz), 1.07 (3H, s), 0.64 (3H, s); 13C NMR (CDCl3) δ 213.10, 209.38, 63.55, 56.38, 49.65, 44.43, 44.01, 41.75, 38.77, 37.32, 36.16, 35.05, 33.48, 32.60, 31.28, 30.76, 24.96, 24.25, 22.58, 22.14, 20.85, 13.19. Anal. (C22H34O2) C, H. (3r,5r,6β)-3-Hydroxy-6-methylpregnan-20-one (1b). KSelectride (0.4 mmol, 0.4 mL of a 1 M solution in THF) was added to a stirred solution of steroid 4a (132 mg, 0.40 mmol) in dry THF (15 mL) at -78 °C under nitrogen. Upon completion of addition, the cold bath was removed and the resultant solution was stirred at room temperature until the starting material disappeared completely. The reaction mixture was then cooled to 0 °C before it was sequentially treated with MeOH (0.5 mL), 5 N NaOH (0.5 mL) and 30% H2O2 (0.2 mL). The cold bath was removed and the mixture was stirred overnight. The mixture was extracted with Et2O, which was washed with water, brine and dried. After solvent removal, the residue was purified by silica gel column chromatography to give product 1b (75 mg, 57%) as a colorless solid: mp 139141 °C; IR (KBr) 3317, 2928, 1708, 1385, 1229, 1004 cm-1; 1H NMR (CDCl3) δ 4.15 (1H, m), 2.57 (1H, t, J ) 8.7 Hz), 2.14 (3H, s), 0.89 (3H, s), 0.88 (3H, d, J ) 6.9 Hz), 0.65 (3H, s); 13C NMR (CDCl3) δ 209.96, 66.67, 63.81, 56.42, 54.52, 44.16, 40.35, 39.08, 38.94, 36.59, 34.95, 34.31, 33.23, 31.40, 30.53, 28.97, 24.37, 22.56, 20.53, 15.48, 15.33, 13.34. Anal. (C22H36O2) C, H. (3r,5β,6β)-3-Hydroxy-6-methylpregnan-20-one (2b). Lithium tri-tert-butoxyaluminum hydride (70 mg, 0.28 mmol) in THF (0.7 mL) was added to a stirred solution of steroid 4b (76 mg, 0.23 mmol) in dry THF (15 mL) at 0 °C under nitrogen. The resulting mixture was stirred at 0 °C for 4 h and quenched by adding acetone (1 mL). The mixture was extracted with Et2O, which was washed with water, brine and dried. After solvent removal, the residue was purified by silica gel column chromatography to give product 2b (52.9 mg, 69%) as a white solid: mp 125-127 °C; IR (KBr) 3413, 2935, 1704, 1446, 1361 cm-1; 1H NMR (CDCl3) δ 3.60 (1H, m), 2.55 (1H, t, J ) 9.0 Hz), 2.12 (3H, s), 1.14 (3H, d, J ) 7.8 Hz), 0.99 (3H, s), 0.62 (3H, s); 13C NMR (CDCl3) δ 209.91, 71.16, 63.77, 56.53, 48.06, 44.16, 40.73, 39.24, 38.98, 36.19, 34.86, 34.74, 32.96, 31.37, 31.05, 29.82, 26.15, 24.39, 22.94, 22.64, 20.47, 13.26. Anal. (C22H36O2) C, H. (5r,6r)-6-Methylpregnane-3,20-dione (6a) and (5β,6R)6-Methylpregnane-3,20-dione (6b). Steroid 5 (359 mg, 1.09 mmol), prepared by a literature procedure20 from compound 3, was dissolved in EtOH (100 mL) containing 5% Pd/CaCO3 (159 mg) and KOH (130 mg, 2.32 mmol) dissolved in water (1 mL). The mixture was hydrogenated (H2, 55 psi) in a Parr hydrogenator for 8 h. The mixture was then filtered and the filtrate was diluted with water and extracted with Et2O. The extract was washed with water, brine and dried. After solvent evaporation, the residue was purified by silica gel column chromatography to give products 6a (56 mg, 15%) and 6b (150 mg, 42%). Steroid 6a was obtained as a white solid: mp 147-149 °C (lit.33 mp 152-153 °C; lit.34 mp 153-154 °C); IR (KBr) 2942, 1711, 1382, 1351 cm-1; 1H NMR (CDCl3) δ 2.54 (1H, t, J ) 9.0 Hz), 2.12 (3H, s), 1.02 (3H, s), 0.81 (3H, d, J ) 6.6 Hz), 0.63 (3H, s); 13C NMR (CDCl3) δ 212.48, 209.61, 63.55, 56.24, 53.46, 52.54, 43.93, 41.11, 40.99, 38.76, 38.18, 37.63, 35.68, 34.75, 31.72, 31.34, 24.16, 22.63, 21.23, 19.67, 13.23, 12.23. Anal. (C22H34O2) C, H.

Steroid 6b was obtained as a white solid: mp 102-104 °C (lit.34 mp 99-102 °C); 1H NMR (CDCl3) δ 2.57 (1H, t, J ) 9.0 Hz), 2.13 (3H, s), 1.02 (3H, s), 0.79 (3H, d, J ) 6.9 Hz), 0.63 (3H, s); 13C NMR (CDCl3) δ 213.36, 209.52, 63.55, 56.35, 49.46, 44.11, 40.12, 38.86, 36.83, 36.59, 35.54, 35.36, 33.61, 31.32, 29.26, 24.13, 22.67, 22.55, 20.96, 19.06, 13.22. (3r,5r,6r)-3-Hydroxy-6-methylpregnan-20-one (1c). Using a procedure similar to that reported for the reduction of steroid 4a, compound 6a (42 mg, 0.13 mmol) was reduced with K-Selectride (0.13 mL, 0.13 mmol, 1 M in THF) to give product 1c (26 mg, 61%) as a white solid: mp 121-123 °C; IR (KBr) 3406, 2935, 1704, 1354, 1008 cm-1; 1H NMR (CDCl3) δ 4.08 (1H, m), 2.54 (1H, t, J ) 9.0 Hz), 2.12 (3H, s), 0.80 (3H, d, J ) 6.3 Hz), 0.79 (3H, s), 0.60 (3H, s); 13C NMR (CDCl3) δ 210.03, 66.24, 63.74, 56.62, 54.08, 45.22, 44.10, 41.59, 39.00, 36.09, 34.87, 32.19, 31.47, 31.44, 30.64, 28.45, 24.19, 22.63, 20.65, 20.06, 13.32, 12.00. Anal. (C22H36O2) C, H. (3r,5β,6r)-3-Hydroxy-6-methylpregnan-20-one (2c). Using a procedure similar to that reported for the reduction of steroid 4b, compound 6b (150 mg, 0.46 mmol) was reduced with lithium tri-tert-butoxyaluminum hydride (147 mg, 0.58 mmol). Product 2c (123 mg, 81%) was obtained as a white solid: mp 197-199 °C; IR (KBr) 3427, 2935, 1694, 1446, 1368, 1069 cm-1; 1H NMR (CDCl3) δ 3.60 (1H, m), 2.54 (1H, t, J ) 9.0 Hz), 2.12 (3H, s), 0.91 (3H, s), 0.82 (3H, d, J ) 6.6 Hz), 0.59 (3H, s); 13C NMR (CDCl3) δ 209.93, 71.67, 63.69, 56.47, 47.45, 44.21, 39.86, 39.02, 35.66, 35.36, 35.27, 34.22, 31.34, 30.03, 29.45, 28.98, 24.17, 23.23, 22.64, 20.58, 19.61, 13.20. Anal. (C22H36O2) C, H. Pregna-4,6-diene-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (8). A mixture of pregna-4,6-diene-3,20-dione (7, 2.70 g, 8.64 mmol), ethylene glycol (5.0 mL), p-TsOH (100 mg, 0.52 mmol) and benzene (150 mL) was stirred and heated under reflux for 6 h. Water formed during the reaction was removed by a Dean-Stark trap. The cooled reaction mixture was diluted with Et2O (600 mL) and sequentially washed with saturated aqueous NaHCO3, water and brine. The Et2O extract was stirred and dried over anhydrous MgSO4 (35 g, 0.29 mol) for several h until TLC showed that the ketal group at C-3 was hydrolyzed completely. The mixture was filtered and the solvent was evaporated. The residue was purified by silica gel column chromatography to give product 8 (2.32 g, 75%) as a white solid: mp 164-166 °C; IR 2949, 1667, 1616, 1049 cm-1; 1 H NMR (CDCl3) δ 6.15 (1H, d, J ) 11.4 Hz), 6.10 (1H, d, J ) 11.4 Hz), 5.67 (1H, s), 3.86-4.03 (4H, m), 1.31 (3H, s), 1.12 (3H, s), 0.86 (3H, s); 13C NMR (CDCl3) δ 199.80, 164.07, 141.44, 127.96, 123.62, 111.74, 65.13, 63.16, 57.92, 53.28, 50.62, 42.72, 39.12, 37.13, 35.98, 33.86, 33.81, 24.45, 23.16, 22.82, 20.35, 16.16, 12.68. Anal. (C23H32O3) C, H. (7r)-7-Methylpregn-4-ene-3,20-dione, cyclic 20-(1,2ethanediyl acetal) (9) and (7β)-7-Methylpregn-4-ene-3,20-dione, cyclic 20-(1,2-ethanediyl acetal) (10). Methyllithium (32.3 mmol, 23 mL of a 1.4 M solution in Et2O) was added to a stirred suspension of CuI (3.25 g, 17.1 mmol) in anhydrous Et2O (150 mL) at 0 °C under N2. The resulting solution was stirred at 0 °C for 1 h. Then, a solution of steroid 8 (977 mg, 2.74 mmol) in anhydrous THF (30 mL) was added over a 10 min period. Stirring was continued for an additional 1 h at 0 °C and then at room-temperature overnight. The reaction mixture was quenched by adding saturated aqueous NH4Cl (100 mL) at 0 °C with vigorous stirring. Et2O (500 mL) was added and the organic layer washed with saturated aqueous NH4Cl, water, brine and dried. After solvent removal, the residue was stirred with KOH (298 mg, 5.33 mmol) dissolved in 80% aqueous MeOH (300 mL) at room temperature for 3 h. The product was extracted with Et2O, and the extract was washed with water, brine and dried. After solvent evaporation, the residue was purified by silica gel column chromatography to give pure product 9 (304 mg, 30%) and a mixture (1:1) of products 9 and 10 (314 mg, 30%). Steroid 9 was obtained as a colorless solid: mp 123-125 °C; IR 2942, 2874, 1670, 1613, 1212 cm-1; 1H NMR (CDCl3) δ 5.72 (1H, s), 3.85-4.01 (4H, m), 1.29 (3H, s), 1.20 (3H, s), 0.82 (3H, s), 0.77 (3H, d, J ) 7.2 Hz); 13C NMR (CDCl3) δ 198.94,

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169.89, 125.47, 111.48, 64.81, 62.86, 57.77, 51.63, 45.72, 41.50, 40.53, 38.73, 38.36, 37.18, 35.52, 33.66, 30.99, 24.16, 22.70, 22.50, 20.45, 17.37, 12.46, 12.31. Anal. (C24H36O3) C, H. (5r,7r)-7-methylpregnane-3,20-dione, cyclic 20-(1,2ethanediyl acetal) (11a) and (3β,5r,7r)-3-Hydroxy-7-methylpregnan-20-one, cyclic-(1,2-ethanediyl acetal) (11b). Lithium (80 mg, 11.43 mmol) and a glass-coated stir bar were placed in a flame-dried, three-necked, round-bottomed flask fitted with a dry ice condenser under nitrogen. Liquid ammonia (40 mL) was collected in the flask at -78 °C to form a deep blue solution and anhydrous THF (15 mL) was added. A solution of steroid 9 (183 mg, 0.49 mmol) and t-BuOH (60 µL, 0.63 mmol) in anhydrous THF (15 mL) was added dropwise to the deep blue solution. Upon completion of the addition, the resultant blue solution was stirred for 1 h before it was quenched with saturated aqueous NH4Cl (10 mL). Then, the cold bath was removed and the solution was allowed to warm to room temperature. After extraction with Et2O, the combined organic layers were washed with saturated aqueous NH4Cl, water, saturated aqueous NaCl, dried, filtered, and concentrated. After column chromatography on silica gel, products 11a (27 mg, 15%) and 11b (95 mg, 51%), which can be easily converted into steroid 11a by PDC oxidation in CH2Cl2, were obtained. Steroid 11a was obtained as a white solid: mp 133-135 °C; IR (KBr) 2942, 1711, 1045 cm-1; 1H NMR (CDCl3) δ 3.783.95 (4H, m), 1.22 (3H, s), 0.95 (3H, s), 0.81 (3H, d, J ) 6.9 Hz), 0.71 (3H, s); 13C NMR (CDCl3) δ 212.43, 111.97, 65.09, 63.14, 58.11, 51.70, 45.27, 44.63, 41.87, 39.97, 39.12, 38.61, 38.15, 36.80, 36.44, 36.21, 29.76, 24.43, 23.20, 22.78, 20.91, 13.22, 12.70, 10.70. Anal. (C24H38O3) C, H. Steroid 11b was obtained as a white solid: 1H NMR (CDCl3) δ 3.77-3.94 (4H, m), 3.53 (1H, m), 1.22 (3H, s), 0.81 (3H, d, J ) 7.2 Hz), 0.74 (3H, s), 0.68 (3H, s); 13C NMR (CDCl3) δ 112.00, 71.20, 65.06, 63.10, 58.12, 51.79, 45.74, 41.85, 39.20, 38.06, 37.68, 37.03, 36.83, 36.25, 36.04, 31.35, 29.79, 24.42, 23.16, 22.72, 20.67, 13.41, 12.67, 11.53. (5β,7r)-7-Methylpregnane-3,20-dione, cyclic 20-(1,2ethanediyl acetal) (11c). Steroid 9 (467 mg, 1.25 mmol) was dissolved in EtOH (90 mL) containing 5% Pd/CaCO3 (79 mg) and KOH (306 mg, 5.46 mmol) dissolved in water (0.6 mL). The mixture was hydrogenated in a Parr hydrogenator (H2, 40 psi) at room temperature for 8.5 h. The reaction mixture was filtered and the filtrate concentrated, diluted with water and extracted with Et2O. The extract was washed with water, brine and dried. After solvent evaporation, the residue was purified by silica gel column chromatography to afford product 11c (374 mg, 80%) as a white solid: mp 172-174 °C; IR (KBr) 2942, 2874, 1711, 1052 cm-1; 1H NMR (CDCl3) δ 3.85-4.01 (4H, m), 2.73 (1H, t, J ) 14.4 Hz), 1.30 (3H, s), 1.06 (3H, s), 0.99 (3H, d, J ) 7.2 Hz), 0.79 (3H, s); 13C NMR (CDCl3) δ 213.37, 111.75, 64.94, 63.01, 57.88, 51.40, 45.37, 42.32, 41.67, 39.06, 36.57, 36.24 (2 × C), 35.27, 35.11, 34.05, 29.11, 24.32, 23.40, 22.67, 21.67, 20.52, 16.86, 12.49. (C24H38O3) C, H. (3r,5r,7r)-3-Hydroxy-7-methylpregnan-20-one (1d). KSelectride (0.4 mL, 0.4 mmol, 1 M in THF) was added to a stirred solution of steroid 11a (70.2 mg, 0.19 mmol) in anhydrous THF (10 mL) at -78 °C under nitrogen. Upon completion of addition, the cold bath was removed and the resultant solution was stirred at room temperature for 3 h. The reaction mixture was then cooled to 0 °C before it was sequentially treated with MeOH (0.5 mL), 5 N NaOH (0.5 mL) and 30% H2O2 (0.2 mL). The cold bath was removed and the mixture was stirred overnight, then extracted with Et2O. The extract was washed with water, brine and dried. After solvent removal, the residue was treated with p-TsOH (10.0 mg, 0.05 mmol) in acetone (15 mL). The mixture was stirred at room temperature for 24 h and extracted with Et2O. The extract was washed with saturated aqueous NaHCO3, water, brine and dried. The residue was purified by silica gel column chromatography to give product 1d (52 mg, 84%) as a white solid: mp 135-137 °C; IR (KBr) 3515, 2935, 1691, 1382, 1358, 1008 cm-1; 1H NMR (CDCl3) δ 4.05 (1H, m), 2.53 (1H, t, J ) 8.7 Hz), 2.11 (3H, s), 0.92 (3H, d, J ) 7.2 Hz), 0.79 (3H, s),

0.60 (3H, s); 13C NMR (CDCl3) δ 209.99, 66.38, 63.63, 52.28, 45.63, 44.10, 38.61, 37.29, 36.63, 35.93, 35.81, 32.16, 31.90, 31.41, 29.94, 28.80, 23.76, 22.52, 20.39, 13.55, 13.06, 10.33. Anal. (C22H36O2) C, H. (3β,5r,7r)-3-Hydroxy-7-methylpregnan-20-one (13). Treatment of steroid 11b (32 mg, 0.08 mmol) with p-TsOH (7.0 mg, 0.04 mmol) in acetone (10 mL) gave product 13 (19 mg, 69%) as a white solid: mp 169-171 °C; IR (KBr) 3488, 2928, 1687, 1382, 1062 cm-1; 1H NMR (CDCl3) δ 3.58-3.65 (1H, m), 2.52 (1H, t, J ) 8.7 Hz), 2.11 (3H, s), 0.89 (3H, d, J ) 6.9 Hz), 0.81 (3H, s), 0.60 (3H, s); 13C NMR (CDCl3) δ 209.90, 71.22, 63.71, 52.26, 45.74, 44.13, 38.65, 38.04, 37.68, 37.38, 37.06, 36.15, 36.09, 31.43, 31.37, 29.91, 23.85, 22.61, 20.91, 13.44, 13.09, 11.55. Anal. (C22H36O2) C, H. (3r,5β,7r)-3-Hydroxy-7-methylpregnan-20-one (2d) and (3β,5β,7r)-3-Hydroxy-7-methylpregnan-20-one (14). Lithium tri-tert-butoxyaluminum hydride (329 mg, 1.29 mmol) in anhydrous THF (3 mL) was added to a solution of steroid 11c (370 mg, 0.99 mmol) in anhydrous Et2O (35 mL) and anhydrous THF (9 mL) at 0 °C under nitrogen. The resulting mixture was stirred at 0 °C for 3 h and quenched by adding acetone (1 mL). The mixture was extracted with Et2O, which was washed with water, brine and dried. After solvent removal, the residue was mixed with acetone (40 mL) and p-TsOH (62 mg, 0.32 mmol). The mixture was stirred at room temperature for 15 h and diluted with Et2O. The Et2O extract was washed with saturated aqueous NaHCO3, water, brine and dried. After solvent evaporation, the residue was purified by silica gel column chromatography to give products 2d (229 mg, 70%) and 14 (39 mg, 12%). Steroid 2d was obtained as a white solid: mp 188-190 °C; IR (KBr) 3529, 2908, 1698, 1079 cm-1; 1H NMR (CDCl3) δ 3.49 (1H, m), 2.54 (1H, t, J ) 8.7 Hz), 2.12 (3H, s), 1.02 (3H, d, J ) 7.2 Hz), 0.94 (3H, s), 0.60 (3H, s); 13C NMR (CDCl3) δ 209.94, 71.75, 63.49, 51.98, 44.01, 42.28, 40.02, 38.59, 37.27, 35.40, 35.31, 34.54, 32.57, 31.41, 30.61, 29.42, 24.22, 22.96, 22.59, 20.39, 17.87, 12.91. Anal. (C22H36O2) C, H. Steroid 14 was obtained as a white solid: mp 157-159 °C; IR (KBr) 3530, 2908, 1704 cm-1; 1H NMR (CDCl3) δ 4.06 (1H, b), 2.53 (1H, t, J ) 8.7 Hz), 2.12 (3H, s), 0.99 (3H, s), 0.98 (3H, d, J ) 7.5 Hz), 0.61 (3H, s); 13C NMR (CDCl3) δ 209.93, 66.77, 63.58, 52.04, 44.08, 38.67, 37.09, 36.88, 36.75, 35.96, 34.22, 32.35, 31.44, 29.99, 29.47, 27.85, 24.34, 23.26, 22.64, 20.77, 17.92, 12.96. Anal. (C22H36O2) C, H. (3r,5r,7β)-3-Hydroxy-7-methylpregnan-20-one (1e). Steroid 1e was prepared from the mixture of steroids 9 and 10 by the same reaction sequence described for preparation of steroid 1d. For the mixture, the concurrent reaction sequences are 9 f 11a f 1d and 10 f 12a f 1e. Intermediate 12a was not characterized. The final mixture was purified by HPLC on a silica gel column eluted with 15% ethyl acetate in hexanes. Steroid 1e was obtained as a colorless solid: mp 151153 °C; IR (KBr) 3474, 2922, 1687, 1358 cm-1; 1H NMR (CDCl3) δ 4.04 (1H, m), 2.48 (1H, t, J ) 9.3 Hz), 2.12 (3H, s), 0.95 (3H, d, J ) 6.3 Hz), 0.74 (3H, s), 0.62 (3H, s); 13C NMR (CDCl3) δ 210.32, 66.33, 63.04, 57.33, 54.83, 45.34, 42.92, 39.44, 39.41, 37.86, 36.48, 35.59 35.52, 32.26, 31.64, 28.94, 28.03, 23.28, 23.20, 21.09, 13.69, 11.20. Anal. (C22H36O2) C, H. (3r,5β,7β)-3-Hydroxy-7-methylpregnan-20-one (2e). Steroid 2e was prepared from a mixture of steroids 9 and 10 using the reaction sequence described for the preparation of steroid 2d. For the mixture, the concurrent reaction sequences are 9 f 11c f 2d and 10 f 12b f 2e. Intermediate 12b was not characterized. The product mixture was purified by silica gel column chromatography. Product 2e was obtained as a white solid: mp 110-112 °C; IR (KBr) 3481, 2928, 2853, 1684, 1361, 1042 cm-1; 1H NMR (CDCl3) δ 3.64 (1H, m), 2.48 (1H, t, J ) 9.3 Hz), 2.12 (3H, s), 0.92 (3H, d, J ) 6.0 Hz), 0.89 (3H, s), 0.62 (3H, s); 13C NMR (CDCl3) δ 210.25, 71.60, 63.02, 57.17, 45.48, 43.23, 42.19, 40.67, 39.61, 38.15, 36.78, 35.48, 34.13, 31.65, 31.17, 30.27, 28.06, 23.38, 23.26, 21.24, 13.61. Anal. (C22H36O2) C, H. [35S]TBPS Binding Methods. The methods used were as described previously.27

Neurosteroid Analogues

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 8 3059

Xenopus Oocyte Electrophysiological Methods. The methods used were as described previously.27 Tadpole Behavioral Methods. The methods used were as described previously.27

(17) Phillips, G. H.; Ewan, G. Steroid Derivatives. Ger. Offen. Patent 2715078, 1977, 117 pp. Chem. Abstr. 88, 38078. (18) Phillips, G. H.; May, P. J.; Ayres, B. E. Pregnane and Androstane Derivatives. Ger. Offen. Patent 2700267, 1977, 110 pp. Chem. Abstr. 87, 184774. (19) Phillipps, G. H.; Ewan, G. B. 11R-Amino-3R-hydroxy Steroids. Br. Pat. 1581235, 1980, 22 pp. Chem. Abstr. 95, 62504. (20) Burn, D.; Ellis, B.; Petrow, V.; Stuart-Webb, I. A.; Williamson, D. M. Modified Steroid Hormones. Part IV. 6-Methylpregnane Derivatives. J. Chem. Soc. 1957, 4092-4098. (21) Adeoti, S. B.; Charpentier, B.; Montagnac, A.; Chiaroni, A.; Riche, C.; Pais, M. Introduction of a 14β-amino function into a steroid nucleus. Application to the cardioactive 14β-amino-5β-pregnane3β,20β-diol starting from progesterone and deoxycholic acid. Tetrahedron 1989, 45, 3717-3730. (22) Turner, A. B.; Ringold, H. Application of high-potential quinones. Part I. The mechanism of dehydrogenation of steroidal ketones by 2,3-dichloro-5,6-dicyano-benzoquinone. J. Chem. Soc. Sect. C. 1967, 1720-1730. (23) Gyermek, L.; Iriarte, J,; Crabbe, P. Steroids. CCCX. StructureActivity Relationship of Some Steroidal Hypnotic Agents. J. Med. Chem. 1968, 11, 117-125. (24) Veleiro, A. S.; Rosenstein, R. E.; Jaliffa, C. O.; Grilli, M. L.; Speroni, F.; Burton, G. Synthesis and GABAA Activity of a 6, 19-Oxido Analogue of Pregnanolone. Bioorg. Med. Chem. Lett. 2003, 13, 343-346. (25) Covey, D. F.; Han, M.; Kumar, A. S.; de la Cruz, M. A. M.; Meadows, E. S.; Hu, Y.; Tonnies, A.; Nathan, D.; Coleman, M.; Benz, A.; Evers, A. S.; Zorumski, C. F.; Mennerick, S. Neurosteroid Analogues. 8 Structure-Activity Studies of N-Acylated 17a-Aza-D-homosteroid Analogues of the Anesthetic Steroids (3R,5R)- and (3R,5β)-3-Hydroxypregnan-20-one. J. Med. Chem. 2000, 43, 3201-3204. (26) Anderson, A.; Boyd, A. C.; Clark, J. K.; Fielding, L.; Gemmell, D. K.; Hamilton, N. M.; Maidment, M. S.; May, V.; McGuire, R.; McPhail, P.; Sansbury, F. H.; Sundaram, H.; Taylor, R. Conformationally Constrained Anesthetic Steroids That Modulate GABAA Receptors. J. Med. Chem. 2000, 43, 4118-4125. (27) Jiang, X.; Manion, B. D.; Benz, A.; Rath, N. P.; Evers, A. S.; Zorumski, C. F.; Mennerick, S.; Covey, D. F. Neurosteroid analogues. 9. Conformationally constrained pregnanes: structure-Activity Studies of 13,24-Cyclo-18,21-dinorcholane Analogues of the GABA Modulatory and Anesthetic Steroids (3R,5R)and (3R,5β)-3-Hydroxypregnan-20-one. J. Med. Chem. 2003, 46, 5334-5348. (28) Makriyannis, A.; DiMeglio, C. M.; Fesik, S. W. Anesthetic Steroid Mobility in Model Membrane Preparations As Examined by High-Resolution 1H and 2H NMR Spectroscopy. J. Med. Chem. 1991, 34, 1700-1703. (29) Hogenkamp, D. J.; Tahir, S. H.; Hawkinson, J. E.; Upasani, R. B.; Alauddin, M.; Kimbrough, C. L.; Acosta-Burruel, M.; Whittemore, E. R.; Woodward, R. M.; Lan, N. C.; Gee, K. W.; Bolger, M. B. Synthesis and In Vitro Activity of 3β-Substituted-3RHydroxypregnan-20-ones: Allosteric Modulators of the GABAA Receptor. J. Med. Chem. 1997, 40, 61-72. (30) Upasani, R. B.; Yang, K. C.; Acosta-Burruel, M., Konkoy, C. S.; McLellan, J. A.; Woodward, R. M.; Lan, N. C.; Carter, R. B.; Hawkinson, J. E. 3R-Hydroxy-3β-(phenylethynyl)-5β-pregnan20-ones: Synthesis and Pharmacological Activity of Neuroactive Steroids with High Affinity for GABAA Receptors. J. Med. Chem. 1997, 40, 73-84. (31) Hawkinson, J. E.; Acosta-Burruel, M.; Yang, K. C.; Hogenkamp, D. J.; Chen, J.-S.; Lan, N. C.; Drewe, J. A.; Whittemore, E. R.; Woodward, R. M.; Carter, R. B.; Upasani, R. B. Substituted 3βPhenylethynyl Derivatives of 3R-Hydroxy-5R-pregnan-20-one: Remarkably Potent Neuroactive Steroid Modulators of γAminobutyric acidA Receptors. J. Pharmacol. Exp. Ther. 1998, 287, 198-207. (32) Anderson, A.; Boyd, A. C.; Byford, A.; Campbell, A. C.; Gemmell, D. K.; Hamilton, N. M.; Hill, D. R.; Hill-Venning, C.; Lambert, J. J.; Maidment, M. S.; May, V.; Marshall, R. J.; Peters, J. A.; Rees, D. C.; Stevenson, D.; Sundaram, H. Anesthetic Activity of Novel Water-Soluble 2β-Morpholinyl Steroids and Their Modulatory Effects at GABAA Receptors. J. Med. Chem. 1997, 40, 1668-1681. (33) Beal, P. F.; Rebenstorf, M. A.; Pike, J. E. Preparation of 6-Methyl Steroids by the “Oxo Reaction”. J. Am. Chem. Soc. 1959, 81, 1231-1234. (34) Cocker, J. D.; Elks, J.; May, P. J.; Nice, F. A.; Phillipps, G. H.; Wall, W. F. Action of Some Steroids on the Central Nervous System of the Mouse. I. Synthetic Methods. J. Med. Chem. 1965, 8, 417-425.

Acknowledgment. This work was supported by NIH Grant GM 47969 (D. F. C., A. S. E., C. F. Z.), NIH Grant AA 12952 (S. M.), NIH Grant MH 45493 (C. F. Z.), the Klingenstein Foundation (S. M.), and the Bantly Foundation (C. F. Z.). Supporting Information Available: Elemental analysis results for target compounds 1b-1e, 2b-2e, 13, and 14. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Olsen, R. W.; Cagetti, E.; Wallner, M. Subunit Specificity of Steroid Modulation of GABAA Receptors. In Neurosteroid Effects in the Central Nervous System: the Role of the GABAA Receptor; Smith, S., Ed.; CRC Press: New York, 2004; pp 47-61. (2) Belelli, D.; Casula, A.; Ling, A.; Lambert, J. J.; The Influence of Subunit Composition on the Interaction of Neurosteroids with GABAA Receptors. Neuropharmacology 2002, 43, 651-661. (3) Vanover, K. E.; Rosenzweig-Lipson, S.; Hawkinson, J. E.; Lan, N. C.; Belluzzi, J. D.; Stein, L.; Barrett, J. E.; Wood, P. L.; Carter, R. B. Characterization of the Anxiolytic Properties of a Novel Neuroactive Steroid, Co 2-6749 (GMA-839; WAY-141839; 3R, 21-Dihydroxy-3β-trifluoromethyl-19-nor-5β-pregnan-20-one), a Selective Modulator of γ-Aminobutyric AcidA Receptors. J. Pharmacol. Exp. Ther. 2000, 295, 337-345. (4) Pillai, G. V.; Smith, A. J.; Hunt, P. A.; Simpson, P. B. Multiple Structural Features of Steroids Mediate Subtype-selective Effects on Human R4β3δ GABAA Receptors. Biochem. Pharmacol. 2004, 68, 819-831. (5) Morrow, A. L.; Pace, J. R.; Purdy, R. H.; Paul, S. M. Characterization of Steroid Interactions with γ-Aminobutyric Acid Receptor-Gated Chloride Ion Channels: Evidence for Multiple Steroid Recognition Sites. Mol. Pharmacol. 1990, 37, 263-270. (6) Hawkinson, J. E.; Kimbrough, C. L.; McCauley, L. D.; Bolger, M. B.; Lan, N. C.; Gee, K. W. The Neuroactive Steroid 3R-Hydroxy-5β-pregnan-20-one is a Two-Component Modulator of Ligand Binding to the GABAA Receptor. Eur. J. Pharmacol. 1994, 269, 157-163. (7) Akk, G.; Bracamontes, J. R.; Covey, D. F.; Evers, A.; Dao, T.; Steinbach, J. H. Neuroactive Steroids Have Multiple Actions to Potentiate GABAA Receptors. J. Physiol. 2004, 558(Pt 1), 5974. (8) Phillipps, G. H. Structure-Activity Relationships in Steroidal Anesthetics. In Molecular Mechanisms in General Anaesthesia; Halsey, M. J., Millar, R. A.. Sutton, J. A., Eds.; Churchill Livingston: New York, 1974; pp 32-47. (9) Phillips, G. H. Structure-Activity Relationships in Steroidal Anesthetics. J. Steroid Biochem. 1975, 6, 607-613. (10) Purdy, R. H.; Morrow, A. L.; Blinn, J. R.; Paul, S. M. Synthesis, Metabolism, and Pharmacological Activity of 3R-Hydroxy Steroids Which Potentiate GABA-Receptor-Mediated Chloride Ion Uptake in Rat Cerebral Cortical Synaptoneurosomes. J. Med. Chem. 1990, 33, 1572-1581. (11) Han, M.; Zorumski, C. F.; Covey, D. F. Neurosteroid Analogues. 4. The Effect of Methyl Substitution at the C-5 and C-10 Positions of Neurosteroids on Electrophysiological Activity at GABAA Receptors. J. Med. Chem. 1996, 39, 4218-4232. (12) Wittmer, L. L.; Hu, Y.; Kalkbrenner, M.; Evers, A. S.; Zorumski, C. F.; Covey, D. F. Enantioselectivity of steroid-induced γaminobutyric acidA receptor modulation and anesthesia. Mol. Pharmacol. 1996, 50, 1581-1586. (13) Covey, D. F.; Nathan, D.; Kalkbrenner, M.; Nilsson, K. R.; Hu, Y.; Zorumski, C. F.; Evers, A. S. Enantioselectivity of Pregnanolone-Induced γ-Aminobutyric AcidA Receptor Modulation and Anesthesia. J. Pharmacol. Exp. Ther. 2000, 293, 1009-1016. (14) Mennerick, S.; He, Y.; Jiang, X.; Manion, B. D.; Wang, M.; Shute, A.; Benz, A.; Evers, A. S.; Covey, D. F.; Zorumski, C. F. Selective Antagonism of 5R-Reduced Neurosteroid Effects at GABAA Receptors. Mol. Pharmacol. 2004, 65, 1191-1197. (15) Nicoletti, D.; Ghini, A. A.; Furtmu¨ller, R.; Sieghart, W.; Dodd, R. H.; Burton, G. Synthesis and GABAA Receptor Activity of 6-Oxa Analogs of Neurosteroids. Steroids 2000, 65, 349-356. (16) Darbandi-Tonkabon, R.; Hastings, W. R.; Zeng, C.-M.; Akk, G.; Manion, B. D.; Bracamontes, J. R.; Steinbach, J. H.; Mennerick, S. J.; Covey, D. F.; Evers, A. S. Photoaffinity Labeling with a Neuroactive Steroid Analogue. 6-Azi-pregnanolone Labels Voltage-dependent Anion Channel-1 in Rat Brain. J. Biol. Chem. 2003, 278, 13196-13206.

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