Neurosteroid analogues. 17. Inverted binding orientations of androsterone enantiomers at the steroid potentiation site on γ-aminobutyric acid type A receptors

Article abstract of DOI:10.1021/jm2014925

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 α₁β₂γ_2L receptors previously associated with positive neurosteroid modulation.

Full text of DOI:10.1021/jm2014925

Article
pubs.acs.org/jmc
Neurosteroid Analogues. 17. Inverted Binding Orientations of
Androsterone Enantiomers at the Steroid Potentiation Site on γ-
Aminobutyric Acid Type A Receptors
†
‡
§
‡
‡
Kathiresan Krishnan, Brad D. Manion, Amanda Taylor, John Bracamontes, Joseph H. Steinbach,
∥
†,‡
§,⊥
§,⊥
David E. Reichert, Alex S. Evers, Charles F. Zorumski, Steven Mennerick,
,†
and Douglas F. Covey*
†
‡
§
∥
Department of Developmental Biology, Department of Anesthesiology, Department of Psychiatry, Department of Radiology, and
⊥
Department of Anatomy and Neurobiology, Washington University in St. Louis School of Medicine, St. Louis, Missouri 63110,
United States
*
S Supporting Information
ABSTRACT: The enantiomer pair androsterone and ent-
androsterone are positive allosteric modulators of γ-amino-
butyric 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
3
5
potently displace [ S]-tert-butylbicyclophosphorothionate, aug-
ment 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 α subunit Q241L mutation, indicating that the active analogues
1
act at the same sites in α β γ receptors previously associated with positive neurosteroid modulation.
1
2 2L
INTRODUCTION
their effects at GABA receptors, and much remains to be
A
learned about their SARs at this class of receptors.
In this study, we investigate SARs related to the
enantioselectivity observed for steroid modulation of GABAA
■
12−14
Endogenous steroids such as allopregnanolone (1) and
androsterone (2) as well as other endogenous 3α-hydroxyste-
roids and synthetic analogues potentiate γ-aminobutyric acid
receptors by naturally occurring steroid 2 and its unnatural
(
GABA)-mediated chloride currents at GABA type A (GABA )
A
1
−4
enantiomer ent-2. Both of these steroids have been shown
receptors (Chart 1).
One consequence of potentiation of
14
previously to potentiate GABA receptors. Unexpectedly, the
GABA receptor function is an increase in neuronal inhibition
A
A
activity of the unnatural enantiomer is greater than that of the
natural enantiomer. This same activity relationship (unnatural >
natural) is also true for the more potent spiroepoxide analogues
in the brain. Consequently, steroids having this action are of
interest as anxiolytic, anticonvulsant, sedative/hypnotic, and
5,6
anesthetic agents.
Site-directed mutagenesis experiments
14
3
and ent-3.
The first goal of this study was to establish that potentiators
have shown that the binding sites for this type of steroid
modulation are located in the transmembrane domains of the
α subunits of the pentameric α β γ subtype of the mouse and
such as steroids 2 and 3 and their enantiomers ent-2 and ent-3,
respectively, exerted their effects at the same potentiation site
1
1 2 2
7
,8
rat receptor. Similarly located sites exist on other forms of the
9
previously identified on the α subunits (one site on each of the
α subunits.
1
two α subunits) in the pentameric α β γ GABA receptor.
Because of the lack of GABA protein crystal structure data,
1
1
2 2L
A
A
The second goal was to gain a better understanding of how
active natural and enantiomeric steroids are oriented relative to
each other at these receptor sites. Our strategy for achieving
these two goals was to initially assume, and then later confirm,
that there was indeed a common binding site for potentiating
the molecular details of steroid binding to the receptor have not
been established. Nevertheless, numerous analogue studies
have established some general features of the structure−activity
1
,10,11
relationships (SARs) for steroid potentiation.
For
example, highly active steroids nearly always contain a 3α-
hydroxyl group on the steroid A-ring and, for the most active
compounds, a 17β-hydrogen bond acceptor group on the
steroid D-ring. Only a few ent-steroids have been evaluated for
Received: November 4, 2011
Published: December 20, 2011
©
2011 American Chemical Society
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Article
Chart 1
toxin binding site on GABAA receptors, potentiate GABA-
mediated chloride currents of rat α β γ GABA receptors
1
2 2L
A
expressed in Xenopus laevis oocytes, and cause loss of righting
reflex (LRR) and loss of swimming reflex (LSR) in tadpoles.
We then carried out site-directed mutagenesis studies to
determine if analogues that were active in all three bioassays
acted at the same sites on the GABA receptor.
A
We found that steroids 4 and 5, which have an 11β-
benzyloxy group, have high activity whereas their enantiomers
ent-4 and ent-5 do not. Conversely, we found that steroids 6
and 7, which have a 7β-benzyloxy group, have weak activity
whereas their enantiomers ent-6 and ent-7 have high activity.
We found the potentiation effects of the active steroids and ent-
steroids were either eliminated or greatly reduced when the
known potentiation sites on the α subunits were rendered
1
ineffective by the Q241L mutation that is known to disrupt
8
steroid actions at rat α β γ GABA receptors.
1
2 2L
A
CHEMISTRY
■
The synthesis of analogues 4 and 5 is summarized in Scheme 1.
The starting material was purchased triketone 8a. Regio- and
a
Scheme 1
steroids and ent-steroids on GABA receptors. This assumption
A
allowed us to design analogues that would inform us as to how
natural and enantiomeric steroids were aligned in a common
binding site.
It seemed likely to us that the active steroids and ent-steroids
would have the 3α-hydroxy group similarly positioned in the
receptor binding sites since this group is critical for high
potentiation activity. This is only possible if the active steroids
and active ent-steroids are bound upside down relative to each
other. Less immediately apparent is one consequence of this
relative orientation of a pair of enantiomers. When oriented in
this way, the C-6, C-7 edge of the natural steroids and the C-11,
C-12 edge of the ent-steroids can be located proximate to each
other (see the Discussion for the alignment model) and a
testable prediction can be made about the effect that
substituents at C-11 or C-7 will have on the activity of pairs
of enantiomeric analogues.
Accordingly, since it is known that a steroid analogue with
both an 11β-OBn and an 11α-Me substituent is a strong
a
Reagents: (a) (i) K(s-Bu) BH, THF, −78 °C; (ii) 3 N NaOH, 30%
3
potentiator at GABA receptors and steroid analogues with
A
H O (72%); (b) PTSA, HO(CH ) OH, benzene, reflux (70%); (c)
2
2
2 2
1
5,16
either a 7α- or a 7β-Me group are not,
it can be confidently
(MOM)Cl, (i-Pr) EtN, DMAP, CH Cl (91%); (d) LAH, Et O
2 2 2 2
predicted that analogues 4 and 5 will have high activity and
analogues 6 and 7 will not. Less confidently predicted, but of
more interest, are the activities of the enantiomers of these
analogues. If the ent-steroids are bound upside down relative to
the natural steroids, then the 11α-substituents in ent-4 and ent-5
will be located proximate to the 7β-substituents in steroids 6
and 7, and ent-4 and ent-5 are predicted to be inactive.
Conversely, the 7α-substituents in analogues ent-6 and ent-7
will be located proximate to the 11β-substituents in steroids 4
and 5, and ent-6 and ent-7 are predicted to be active.
(80%); (e) KH, BnBr, THF, reflux (75%); (f) MeOH, 6 N HCl
(89%); (g) Ac O, pyridine, DMAP (95%); (h) K CO , MeOH (96%);
2
2
3
(i) (CH
)
SI, DMF, KO-t-Bu (61%).
3
3
stereoselective reduction of the 3-ketone using K(s-Bu) BH
3
yielded after workup predominately the 3α-hydroxysteroid 8b
(72%). The 17-ketone was then selectively protected as the
cyclic ketal in the standard manner (9a, 70%), and the 3α-OH
group was protected with a methoxymethyl (MOM) group (9b,
91%). The 11-ketone is then reduced with lithium aluminum
hydride (LAH) to yield the 11β-OH of steroid 10a (80%), and
the 11β-OH is alkylated with BnBr to yield steroid 10b (75%).
Removal of the MOM protecting group yielded target
compound 4 (89%), which contained an impurity that was
Hence, to accomplish our goals, we prepared steroids 4−7
and their enantiomers ent-4−ent-7. We evaluated these agents
3
5
for their ability to noncompetitively displace [ S]-tert-
3
5
butylbicyclophosphorothionate ([ S]TBPS) from the picro-
1
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Journal of Medicinal Chemistry
Article
not easily removed. Acetylation of the 3α-OH group (95%),
purification of the acetate derivative by flash column
chromatography, and removal of the acetyl group (96%)
allowed pure steroid 4 to be prepared. Epoxidation of the 17-
ketone using (CH ) SI, KO-t-Bu in dimethylformamide
the desired α configuration by a two-step procedure: PCC
oxidation (14b, 96%) followed by LAH reduction (14c, 95%).
Benzylation of the 11α-OH group gave ent-steroid 14d (69%),
and removal of the MOM protecting group gave the required
analogue ent-4 (92%). The minor impurity that complicated
purification of steroid 4 was not present in enantiomer ent-4,
making purification of the enantiomer possible without
conversion to its 3β-acetate derivative. Epoxidation of 17-
ketone ent-4 using (CH ) SI, KO-t-Bu in DMF gave the 17-
3
3
(
DMF) gave the 17-spiroepoxide 5 (61%). This epoxidation
is highly stereoselective with a minor amount (ca. 5%) of the
epimeric epoxide being removed by a combination of flash
column chromatography and recrystallization. The stereo-
chemistry at all new chiral centers formed in Scheme 1 and
synthetic Schemes 2−4 was assigned on the basis of well-
3
3
spiroepoxide ent-5 (61%).
The synthesis of analogues 6 and 7 is shown in Scheme 3.
Dehydroepiandrosterone was purchased, converted in the usual
1
known literature precedents and H NMR data.
The preparation of analogues ent-4 and ent-5 is presented in
Scheme 2. The starting material, ent-steroid 11, was prepared
a
Scheme 3
a
Scheme 2
a
Reagents: (a) (MOM)Cl, DMAP, (i-Pr) EtN, CH Cl (91%); (b)
2
2
2
7
1
0% aqueous t-BuOOH, NaClO , aqueous ACN, 50 °C (34%); (c)
2
0% Pd/C, EtOAc (80%); (d) LAH, Et O (86%); (e) KH, THF, BnBr
2
(
68%); (f) MeOH, 6 N HCl (91%); (g) PCC, CH Cl (86%); (h) (i)
a
2 2
Reagents: (a) Li, liquid NH , toluene, THF, −78 °C (81%); (b)
3
K(s-Bu) BH, THF, −78 °C; (ii) 3 N NaOH, 30% H O (67%); (i)
3
2
2
Jones reagent, acetone (92%); (c) (i) K(s-Bu) BH, THF, −78 °C; (ii)
3
3
(CH ) SI, DMF, KO-t-Bu (57%).
3
3
N NaOH, 30% H O (65%); (d) PPTS, HO(CH ) OH, benzene,
2 2 2 2
reflux (87%); (e) (MOM)Cl, DMAP, (i-Pr) EtN, CH Cl (97%); (f)
2
2
2
manner to its cyclic ketal derivative 15, and used as the starting
material. The 3β-OH group was protected with a MOM group
(
i) BH ·THF, THF, 0 °C; (ii) 3 N NaOH, 30% H O (67%); (g)
3 2 2
PCC, CH Cl (96%); (h) LAH, Et O (95%); (i) KH, THF, BnBr
2
2
2
5
(
69%); (j) MeOH, 6 N HCl (92%); (k) (CH ) SI, DMF, KO-t-Bu
to yield steroid 16 (91%). Allylic oxidation of the Δ double
3
3
5
(
61%).
bond using NaClO and aqueous t-BuOOH gave the Δ -6-
2
ketone 17 in adequate, but not high, yield (34%). Catalytic
hydrogenation of steroid 17 using Pd/C gave product 18a in
high yield (80%). LAH reduction of the C-7 ketone group gave
an inseparable mixture of epimeric 7α- and 7β-alcohols 18b
(86%). The decision was made to benzylate the 7-OH groups
(68%) to determine if the benzylated epimers were separable.
This turned out to be the case, and the desired 7β-OBn epimer
18c, which was formed by benzylation of the minor LAH
reduction product, was obtained after flash column chromatog-
raphy in 23% yield. The undesired 7α-OBn epimer 18d was
obtained in 45% yield. Removal of the MOM protecting group
from steroid 18c gave product 19a (91%), PCC oxidation of
1
7
by our previously described method. Li/liquid NH reduction
3
4
of the Δ -3-ketone group yielded the trans A,B ring fused
product 12a (81%). Jones oxidation of ent-steroid 12a gave the
diketone 12b (92%). The 3-ketone was then reduced with K(s-
Bu) BH to the 3β-OH group of ent-steriod 12c (97%). The 17-
ketone was next protected as the cyclic ketal to yield product
3
1
3a (87%), and the 3β-OH group was protected with a MOM
9
(11)
group (13b, 97%). The Δ
double bond was then removed
by hydroboration to give ent-steroid 14a (67%), which
contained the 9β-H and 11β-OH group. The stereochemistry
of the 11-OH group was changed from the β configuration to
1
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steroid 19a gave steroid 19b (86%), and K(s-Bu) BH reduction
of the 3-ketone group of steroid 19b gave target compound 6
a 7-ketone group would be the 7β-alcohol. Removal of the 3,17-
bis(cyclic ketal) groups from ent-steroid 22d gave product 23
3
(
67%). Epoxidation of the 17-ketone using (CH ) SI, KO-t-Bu
(89%), K(s-Bu) BH reduction of ent-steroid 23 gave target
3
3
3
in DMF gave the 17-spiroepoxide 7 (61%). As observed for the
epoxidation of the compounds described in Schemes 1 and 2,
this epoxidation was also highly stereoselective and yielded
almost exclusively the desired spiroepoxide product.
The preparation of analogues ent-6 and ent-7 is demonstrated
in Scheme 4. The starting material was ent-androstenedione
compound ent-6 (73%), and epoxidation of product ent-6 gave
target compound ent-7 (68%).
3
5
[ S]TBPS DISPLACEMENT RESULTS
■
The enantiomer pairs shown in Chart 1 were evaluated as
35
noncompetitive displacers of [ S]TBPS from the picrotoxin
binding site on the heterogeneous GABA receptors found in
A
a
Scheme 4
rat brain membranes. The IC displacement values are given in
50
Table 1. The values reported for the reference compounds 2,
3
5
Table 1. Inhibition of [ S]TBPS Binding by Steroids 1−7
a
and Steroid Enantiomers ent-2−ent-7
compd
IC50 (nM)
nHill
b
1
74 ± 7
0.89 ± 0.06
0.89 ± 0.20
1.00 ± 0.10
1.32 ± 0.16
4.28 ± 1.90
0.90 ± 0.09
c
2
410 ± 130
311 ± 36
105 ± 12
467 ± 93
285 ± 37
c
ent-2
c
3
c
ent-3
4
d
ent-4
5
>10 μM
15 ± 2
1.1 ± 0.1
ent-5
6
2050 ± 400
>10 μM
2.04 ± 0.7
e
ent-6
7
168 ± 8
6750 ± 1000
52 ± 5
1.10 ± 0.05
1.4 ± 0.2
1.12 ± 0.10
ent-7
a
The results presented are from duplicate experiments performed in
triplicate. Error limits are calculated as the standard error of the mean.
b
27
c
14 d
Literature values. Literature values. No inhibition was observed
e
at 10 μM. Partial inhibition (∼35%) was observed at 10 μM.
14
ent-2, 3, and ent-3 are our previously reported values. Relative
to steroid 2, the reported IC values indicate that the axial 11β-
a
50
Reagents: (a) PTSA, HO(CH ) OH, benzene, reflux (83%); (b) 70%
2
2
OBn substituent present in steroid 4 slightly increases the
displacement potency whereas the equatorial 7β-OBn sub-
stituent present in steroid 6 drastically decreases the displace-
ment potency. Relative to compound ent-2, the 11α-OBn group
present in enantiomer ent-4 drastically reduces the binding
potency and the 7α-OBn substituent present in enantiomer ent-
6 increases the binding potency.
aqueous t-BuOOH, NaClO , aqueous ACN, 50 °C (31%); (c) 10%
2
Pd/C, H (50 psi), EtOAc (76%); (d) Raney Ni, H (60 psi), i-PrOH
2
2
(
(
(
96%); (e) KH, THF, BnBr (72%); (f) MeOH, 6 N HCl (89%); (g)
i) K(s-Bu) BH, THF, −78 °C; (ii) 3 N NaOH, 30% H O (73%);
3
2
2
h) (CH ) SI, DMF, KO-t-Bu (68%).
3
3
1
8
(
20), which was prepared as we described earlier. This ent-
5
steroid was first converted into its Δ -3,17-bis(cyclic ketal) 21a
in the usual manner (83%) and subjected to allylic oxidation, in
the manner described earlier for the preparation of steroid 17,
to obtain ent-steroid 21b (31%). Catalytic hydrogenation of
enone 21b gave the 7-ketone product 22a (76%). Because the
LAH reduction of the 7-ketone group of steroid 18a described
in Scheme 3 gave, after benzylation, the undesired 7α-OBn
product 18b as the major product, a different method was used
for the reduction of the 7-ketone in ent-steroid 22a in an
attempt to increase the amount of the desired epimer. Raney Ni
Binding results obtained with the 17-spiroepoxide enan-
tiomer pairs 5, ent-5 and 7, ent-7 followed the same pattern
observed for the binding results obtained with enantiomer pairs
4, ent-4 and 6, ent-6. Relative to steroid 3, the 11β-OBn
substituent present in steroid 5 increases the displacement
potency whereas the 7β-OBn substituent present in steroid 7
decreases the displacement potency. Relative to analogue ent-3,
the 11α-OBn substituent present in analogue ent-5 reduces the
displacement potency whereas the 7α-OBn substituent present
in analogue ent-7 increases the displacement potency. Both
compounds 5 and ent-7 are more potent displacers of
1
9
reduction of the 7-ketone in ent-steroid 22a gave product 22b
96%) as an inseparable mixture of 7α- and 7β-alcohols, and
benzylation (72%) gave the readily separated 7β-OBn product
2c (22%) and 7α-OBn product 22d (50%). Presumably,
35
[
S]TBPS than the endogenous neurosteroid 1.
(
ELECTROPHYSIOLOGY RESULTS
2
■
steroid approach to the surface of the Raney Ni from the ent-
steroid β face (i.e., opposite the C-19 Me group on the α face)
explains why the desired 7α-OH product was the major epimer
formed in the Raney Ni reduction. For natural steroids, this
means that the major alcohol product of Raney Ni reduction of
Enantiomer pairs were evaluated for their ability to potentiate
chloride currents mediated by 2 μM GABA at rat α β γ
1
2 2L
GABA receptors expressed in X. laevis oocytes (Table 2). This
A
GABA concentration gates, on average, ∼4% of the maximum
response recorded from an oocyte. The maximum achievable
1
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Table 2. Modulation of Rat α β γ GABA Receptor
Function by Steroids 1−7 and Steroid Enantiomers ent-2−
ent-7
We next addressed the question of whether the steroid and
ent-steroid potentiators act at the potentiation site previously
identified for neurosteroid 1 by site-directed mutagenesis
1
2 2L
A
8
studies. We did this to rule out the possibility that the steroid
a
oocyte electrophysiology
and ent-steroid potentiators act at different sites on the rat
α β γ GABA receptor. Accordingly, residue 241 in the α
1
compd
0.1 μM
1 μM
10 μM
10 μM (gating)
1
2 2L
A
b
1
1.26 ± 0.14
0.97 ± 0.02
1.27 ± 0.29
3.11 ± 0.17
2.62 ± 0.29
1.08 ± 0.03
0.89 ± 0.03
2.99 ± 0.45
1.01 ± 0.05
0.97 ± 0.02
1.25 ± 0.03
0.83 ± 0.05
3.47 ± 0.13
3.89 ± 1.34
1.41 ± 0.01
3.66 ± 0.89
21.92 ± 1.30
15.89 ± 3.89
2.96 ± 0.14
0.85 ± 0.02
21.19 ± 2.59
1.30 ± 0.09
0.90 ± 0.04
5.74 ± 0.94
0.88 ± 0.05
20.29 ± 2.75
9.65 ± 3.87
5.44 ± 0.19
18.87 ± 2.38
33.73 ± 2.04
26.28 ± 7.90
24.37 ± 1.47
0.60 ± 0.01
31.15 ± 2.87
3.21 ± 0.65
0.76 ± 0.02
25.23 ± 7.16
0.77 ± 0.20
32.39 ± 5.42
0.37 ± 0.07
0.02 ± 0.01
0.03 ± 0.21
0.22 ± 0.02
0.10 ± 0.02
0.20 ± 0.03
0.00 ± 0.01
0.42 ± 0.06
0.05 ± 0.03
0.00 ± 0.02
0.11 ± 0.02
0.09 ± 0.16
0.22 ± 0.03
subunit was changed from glutamine to leucine. As reported
c
20
2
previously and shown in Figure 1, this amino acid change
c
ent-2
c
3
c
ent-3
4
ent-4
5
ent-5
6
ent-6
7
ent-7
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 to 2 μM GABA current. Error limits are
b
calculated as the standard error of the mean (N ≥ 4). Literature
27
c
14
values. Literature values.
potentiation of currents by an analogue varies for different
batches of oocytes. Hence, since the different compounds in
Table 2 were evaluated on different batches of oocytes, it is not
possible to confidently compare the absolute potentiation
caused by each analogue shown in Table 2. The low aqueous
solubility of these compounds also prevented us from
determining the maximal potentiation. Inspection of the
concentration effect data in Table 2 suggests that there may
be differences in both potency and efficacy for the different
compounds. However, the conclusions we draw from the
results presented in Table 2 do not require a comparison of
absolute potentiation values.
Figure 1. Effects of a steroid-insensitive α (Q241L) subunit on
potentiation of GABA currents by analogues. (A) Oocytes expressing
1
wild-type (WT) α β γ GABAA receptor subunits were exposed to
1
2 2L
GABA alone or GABA coapplied with the indicated analogues at a
membrane potential of −70 mV. The concentrations of the
compounds were as follows: GABA, 2 μM; compound 1, 0.5 μM;
compound 4, 1 μM, compound 5, 0.5 μM; compound ent-6, 1 μM;
compound ent-7, 0.5 μM. The concentrations were adjusted to
partially account for different potencies observed in initial screening
and to thereby yield reasonably similar potentiation values. (B) Same
sequence of compound presentation in oocytes injected with RNA
As reported previously, steroids 2 and 3 and their
enantiomers ent-2 and ent-3 all cause a concentration-
1
4
dependent increase in GABA-gated chloride currents.
Steroids 4 and 5, which contain 11β-OBn substituents, also
cause a concentration-dependent increase in chloride currents.
Steroids 6 and 7, which contain 7β-OBn substituents, do not.
For the analogues ent-4 and ent-5, the 11α-OBn substituents
either do not potentiate chloride currents (ent-4) or only
weakly potentiate at the highest concentration, 10 μM (ent-5).
Analogues ent-6 and ent-7, which contain 7α-OBn substituents,
both cause a concentration-dependent increase in GABA-gated
chloride currents. The active benzyloxy-substituted compounds
encoding a point mutation (Q241L) in the α subunit that renders
1
8
receptors insensitive to compound 1. WT β RNA and γ RNA were
2
2L
coinjected. (C) Summary of normalized responses of four WT oocytes
and four α (Q241L)β γ oocytes in experiments like those depicted
1
2 2L
in (A) and (B). The normalizing response to GABA alone is denoted
by a dotted line at y = 1. The asterisk indicates p < 0.05 by an
independent sample t test.
eliminates the potentiation caused by neurosteroid 1. It also
eliminates or nearly eliminates the potentiation caused by the
active analogues prepared in this study. The results support the
conclusion that the potentiation caused by all the active
compounds is mediated through a common site of action on
(4, 5, ent-6, ent-7) at a concentration of 10 μM also directly
gate a chloride current in the absence of GABA, a well-known
effect of active neurosteroid analogues.
The results reported in Table 2 correlate with the results
presented in Table 1. Compounds that potently displace
35
the GABA receptor. Figure 1 also provides information on the
[
S]TBPS also strongly potentiate GABA-mediated chloride
A
relative activity of the potentiating compounds. The 17-
spiroepoxides 5 (0.5 μM) and ent-7 (0.5 μM) potentiate
more than the 17-ketones 4 (1 μM) and ent-6 (1 μM) when
compared on the same oocyte. This result is consistent with the
currents. Compounds that either weakly displace or do not
35
displace [ S]TBPS at a concentration of 10 μM either weakly
potentiate or do not potentiate GABA-mediated chloride
currents.
1
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Journal of Medicinal Chemistry
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3
5
greater potency of the 17-spiroepoxides for [ S]TBPS
displacement (Table 1).
substituent also causes both LRR and LSR at concentrations
below 10 μM. However, this analogue is also weakly active in
the other two bioassays (Tables 1 and 2), so its ability to cause
LRR and LSR in tadpoles is not surprising.
TADPOLE LRR AND LSR RESULTS
■
The enantiomeric analogues ent-6 and ent-7 (7α-OBn
substituents) both cause, as expected, LRR and LSR in
tadpoles. The enantiomeric 17-ketoanalogue ent-6 is less potent
than the enantiomeric 17-spiroepoxide analogue ent-7. This
order of potency for their anesthetic effects correlates with their
The anesthetic effects in tadpoles of the compounds are
reported in Table 3. The EC values for LRR and LSR
5
0
Table 3. Effects of Steroids 1−7 and Steroid Enantiomers
ent-2−ent-7 on Tadpole Righting and Swimming Reflexes
a
35
order of potency for [ S]TBPS displacement (Table 1) and
tadpole LRR
EC50 (μM)
tadpole LRR
tadpole LSR
EC50 (μM)
tadpole LSR
electrophysiological actions at GABA receptors (Figure 1).
b
A
compd
nHill
nHill
c
1
0.42 ± 0.04
3.38 ± 0.9
−1.83 ± 0.32
−2.83 ± 2.66
−2.17 ± 0.48
−3.69 ± 0.08
−18.1 ± 1.3
−2.61 ± 0.07
−3.27 ± 0.07
−1.58 ± 0.21
−22.8 ± 1.0
5.5 ± 0.5
none
−7.5 ± 1.1
DISCUSSION
d
e
■
2
The prior observation that steroids 2 and 3 and their
enantiomers ent-2 and ent-3 all potentiated GABA-mediated
chloride currents at GABA receptors raised two interesting
questions: Are the pairs of enantiomers (steroid and
corresponding ent-steroid) acting at the same sites on the
d
ent-2
1.42 ± 0.18
1.35 ± 0.01
1.08 ± 0.01
0.20 ± 0.00
1.96 ± 0.02
0.07 ± 0.01
2.64 ± 0.01
5.48 ± 0.12
2.76 ± 0.01
2.71 ± 0.01
−33.3 ± 0.1
−21.1 ± 0.7
−21.8 ± 1
−20.4 ± 0.4
−21.1 ± 0
−21.4 ± 0.5
−23.2 ± 0
d
3
d
A
ent-3
f
4
0.87 ± 0
g
ent-4
5
3.45 ± 0
h
GABA receptor? How are the pairs of enantiomers aligned
A
0.27 ± 0
i
relative to each other if they act at the same sites on the
receptors? The goal of this study was to provide answers to
these two questions.
ent-5
6
2.74 ± 0
j
e
>10
none
ent-6
7
1.21 ± 0.4
3.22 ± 0.03
0.31 ± 0.04
−4.27 ± 6.44
−15.6 ± 1.8
−2.19 ± 0.61
2.93 ± 0
7.94 ± 0
−22.0 ± 0.5
−27.3 ± 0
−33.3 ± 0.1
k
An analogue approach, used in conjunction with site-directed
mutagenesis methodology, was chosen to address both
questions. The analogues chosen were designed on the basis
of an initial assumption that all enantiomer pairs acted at a
common site on the receptor and on previous steroid SAR
results. It was shown previously that modifying neurosteroid 1
by introducing an 11α-Me substituent together with an 11β-
OBn substituent gave an analogue with increased potentiation
ent-7
0.55 ± 0.01
a
Error limits are calculated as the standard error of the mean (N = 10
or more tadpoles at each of five or more different concentrations
ranging from 0.01 to 30 μM). Unless stated otherwise, all tadpoles
(
b
regained LRR and LSR after overnight recovery. LSR for compounds
with weak activity typically has a very steep concentration−response
curve. When n values are in the range of −20 to −33, the n values
Hill
Hill
15
reflect the fact that at one concentration nearly all tadpoles had a
swimming reflex and at the next highest concentration tested nearly all
14
activity. Hence, we expected that new steroid analogues
having 11β-OBn substituents (4, 5) would have activity. Since
it was also shown previously that adding a group as small as
either a 7α- or 7β-Me group to neurosteroid 1 produced an
c
27
d
or all tadpoles did not. Literature values.
Literature values.
e
“
None” indicates that all tadpoles had a swimming response at 10
μM. Recovery from LRR and/or LSR was partial at concentrations ≥1
μM. At 10 μM, 10 of 10 tadpoles died without recovering overnight
from LSR. At 10 μM, 10 of 10 tadpoles died without recovering
overnight from LSR. At 10 μM, 5 of 10 tadpoles died without
recovering overnight from LSR. At 10 μM, 6 of 10 tadpoles died
without recovering overnight from LSR. Some tadpoles had LRR at
f
16
analogue with greatly diminished activity, we also expected
that new steroid analogues having 7β-OBn substituents (6, 7)
would have little, if any, activity.
g
h
i
Expected activity outcomes for the enantiomeric compounds
ent-4−ent-7 depend on the choice made to align each steroid
with its corresponding enantiomer. We made this alignment
choice on the basis of the assumption that the critical 3-OH
groups will be similarly positioned in three-dimensional space if
enantiomer pairs are bound to the same GABAA receptor
binding sites. To satisfy this assumption, active ent-steroids
must be oriented upside down relative to active steroids. When
so oriented, an 11β-OBn substituent in a natural steroid and a
j
k
1
0 μM, but the EC50 was above this value. At 10 μM, 4 of 10 tadpoles
died without recovering overnight from LSR.
reported in Table 3 for steroids 2 and 3 and their enantiomers
ent-2 and ent-3 are the values we reported previously. Steroids
and 5, which contain axial 11β-OBn substituents, are both
more potent in causing LRR and LSR in tadpoles than the
reference steroids 2 and 3, respectively. The 17-ketosteroid 6
14
4
7
α-OBn substituent in an ent-steroid can occupy similar regions
of three-dimensional space (Figure 2A). Hence, we expected
that new ent-steroids having 7α-OBn substituents (ent-6, ent-7)
would, like the 11β-OBn steroids 4 and 5, have potentiation
activity. Similarly, since a 7β-OBn substituent in a natural
steroid and an 11α-OBn substituent in an upside down
oriented ent-steroid can occupy similar regions of three-
dimensional space (Figure 2B), we expected that new ent-
steroid analogues having 11α-OBn substituents (ent-4, ent-5)
would, like the 7β-OBn steroids 6 and 7, have greatly
diminished potentiation activity.
(
7β-OBn substituent) does not have an EC value of <10 μM
50
for either LRR or LSR. The results correlate with the actions of
this analogue in the other two bioassays (Tables 1 and 2). The
corresponding 17-spiroepoxide 7 with the 7β-OBn substituent
does have weak anesthetic activity as it produces both LRR
(
EC50 = 3.22 μM) and LSR (EC50 = 7.94 μM). This result for
analogue 7 is consistent with its weak ability to displace
3
5
[
S]TBPS (Table 1), but not with its failure to potentiate
GABA-mediated chloride currents (Table 2).
The enantiomeric 17-ketosteroid ent-4 (11α-OBn substitu-
Other factors also influenced our choice of analogues chosen
for this study. First, since the enantiomer of neurosteroid 1 is
3
5
ent), which neither displaces [ S]TBPS (Table 1) nor
potentiates GABA-mediated chloride currents (Table 2) at
concentrations up to 10 μM, unexpectedly causes both LRR
and LSR at concentrations below 10 μM. The corresponding
enantiomeric 17-spiroepoxysteroid ent-5 with the 11α-OBn
12
essentially inactive, these studies could not have been done
with analogues of neurosteroid 1 containing benzyloxy groups
at C-7 or C-11 since no active enantiomers of neurosteroid 1
bearing benzyloxy groups at either position would be possible.
1
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Journal of Medicinal Chemistry
Article
block of GABAA receptors that is removed by a V256S
23
mutation of the α subunit. Future studies will be required to
1
determine whether the effects of ent-4, 6, and 7 can be
explained by this mechanism, which is independent of
potentiation.
This enantioselectivity study examined only 5α-reduced
analogues. An earlier study examined the effect of a Q241
mutation in the α subunit on the actions of a 5β-reduced
1
24
steroid potentiator and its active enantiomer. It was reported
that mutation of the Q241 residue eliminated the actions of the
5
β-reduced steroid and reduced the actions of its enantiomer,
consistent with the present results. Because weak potentiation
remained in the previous study and was statistically significant
for analogue ent-6 in the present study, the possibility of
additional binding sites cannot be excluded.
Figure 2. (A) Molecular superimpositions of active compounds 4
(
green) and ent-6 (yellow) and (B) inactive compounds 6 (green) and
ent-4 (yellow). In both panels a low-energy conformation of the
analogue with the absolute configuration of natural steroids was used
as the template and is shown in green, and the best result from fitting a
library of conformers of the analogues with the absolute configuration
opposite that of natural steroids (ent-steroids) is shown in yellow. The
alignments maximize the overlap of the molecular volumes as well as
structural features such as hydrogen bond donors and acceptors. Edge
views are shown on the left, and views from above the molecules are
shown on the right. Hydrogens other than that of the hydroxyl group
are removed for clarity. Alignments were made using the software
program ROCS (OpenEye Scientific Software).
The modeling results in Figure 2 were obtained with the
program ROCS (OpenEye Scientific Software), which max-
imizes the volume overlap between a query molecule (low-
energy conformers of compounds 4 and ent-6) and a library of
other molecules or conformers while also maximizing the
overlap of selected chemical features such as ring centroids and
hydrogen bond donors and acceptors. The alignments should
not be interpreted to imply the precise arrangements shown, as
the modeling is only intended to demonstrate that when
steroids and ent-steroids are oriented as shown in Figure 2, the
substituents that adversely affect activity are located along one
edge of the aligned molecules and those that do not adversely
affect activity are located on the opposite edge of the aligned
molecules. Data are currently insufficient to interpret the
alignments in a more precise manner.
25
Second, 11β-substituents in the steroids have the axial
configuration, whereas the 7α-subsituents in the ent-steroids
have the equatorial configuration. Thus, a relatively large and
flexible group such as a benzyloxy group was considered
advantageous for maximizing possible overlap of substituents
having different configurations at the C-7 and C-11 positions.
Third, we considered it important to have the C-11- and C-7-
substituents be identical to avoid significant differences in log P
for the analogues used in the study. log P can have a major
CONCLUSION
On the basis of studies of newly prepared enantiomeric pairs of
steroid analogues, we conclude that androsterone and ent-
■
androsterone bind at the same sites on GABA receptors. We
3
5
A
effect on the observed [ S]TBPS displacement and potentia-
tion activity because the amount of steroid that accumulates in
the cell membrane will determine its effective concentration at
further conclude that ent-androsterone is bound in an upside
down orientation relative to androsterone at these sites. We
also provide the first information describing the effects of C-7-
or C-11-substituents on the GABAergic effects of ent-steroids.
The results obtained will be useful for the design of new
the transmembrane steroid binding sites located on the GABA
A
2
1,22
receptor.
3
5
As shown by the [ S]TBPS displacement results (Table 1)
and electrophysiology results (Table 2), our expected outcomes
were observed in these two bioassays. Two unexpected
outcomes were found in the tadpole behavioral bioassay
potentiators of GABA receptors with a structure based on that
A
of an ent-steroid.
EXPERIMENTAL SECTION
■
(
Table 3). The analogues ent-4 and 7 both caused LRR and
General Methods. Solvents were either used as purchased or dried
and purified by standard methodology. Extraction solvents were dried
with anhydrous Na SO and, after filtration, removed on a rotary
LSR at concentrations below 10 μM even though the
3
5
compounds neither effectively displace [ S]TBPS nor
potentiate GABA-mediated chloride currents. We suggest that
the anomalous behavioral effects of these two compounds
2
4
evaporator. Flash column chromatography was performed using silica
gel (32−63 μm) purchased from Scientific Adsorbents (Atlanta, GA).
Melting points were determined on a Kofler micro hot stage and are
uncorrected. FT-IR spectra were recorded as films on a NaCl plate.
result from actions on receptors other than GABA receptors.
A
The site-directed mutagenesis study (Figure 1) provides
evidence that the two active steroids (4, 5) and the two active
ent-steroids (ent-6, ent-7) are acting at the same GABAA
receptor binding sites. The Q241L mutation abolishes,
completely or nearly completely, the activity of all four active
compounds at a concentration of either 0.5 μM (the 17-
NMR spectra were recorded in CDCl at ambient temperature at 300
3
1
13
MHz ( H) or 74 MHz ( C). A purity of >95% was determined for all
evaluated compounds by combustion analysis for C, H performed by
M-H-W Laboratories (Phoenix, AZ). Steroids 8a and dehydroepian-
drosterone were purchased from Steraloids (Newport, RI). K(s-
Bu)
Milwaukee, WI).
3α,5α,11β)-11-(Benzyloxy)-3-hydroxyandrostan-17-one (4).
BH (K-Selectride) was purchased from Aldrich Chemical Co.
3
(
spiroepoxides) or 1 μM (the 17-ketones). The GABA receptor
A
(
sites of action were not determined for the analogues ent-4, ent-
A mixture of steroid 10b (242 mg, 0.5 mmol), MeOH (15 mL), and 6
N HCl (3 mL) was stirred at room temperature for 36 h. The MeOH
was removed under reduced pressure, and the residual solution was
extracted with EtOAc, dried, and concentrated to give a viscous liquid
which was purified by flash column chromatography (silica gel eluted
with 30−40% EtOAc in hexanes) to give crude product 4 (176 mg,
5
[
, 6, and 7. These analogues neither strongly displace
S]TBPS nor strongly potentiate GABA-mediated currents.
3
5
It is interesting to note that compounds ent-4, 6, and 7 appear
to weakly inhibit GABA-mediated currents. We have observed
previously that 3β-hydroxysteroids can show a use-dependent
1
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Journal of Medicinal Chemistry
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8
9%). This crude product was acetylated (95% yield) in the standard
give an off-white solid which was purified by flash column
manner (Ac O, pyridine, 4-(dimethylamino)pyridine (DMAP), room
temperature) and purified by column chromatography to give the pure
chromatography (silica gel eluted with 20−40% EtOAc in hexanes)
2
2
0
to give product 6 (40 mg, 67%): mp 130−132 °C; [α] +131.5 (c
−1 1
D
acetate derivative: mp 117−119 °C; IR ν 2927, 2856, 1736, 1452,
0.04, CHCl ); IR ν 3400, 2928, 2858, 1736, 1453, 1361 cm ; H
3 max
max
−1 1
1
4
2
359, 1260, 1244 cm ; H NMR δ 7.40−7.20 (m, 5H), 5.00 (m, 1H),
.65 (d, 1H, J = 11.3 Hz), 4.26 (d, 1H, J = 11.3 Hz), 4.01 (m, 1H),
.55−2.35 (m, 2H), 2.04 (s, 3H), 1.08 (s, 3H), 1.01 (s, 3H); C NMR
δ 219.9, 170.5, 138.4, 128.1 (2C), 127.7(2C), 127.2, 74.6, 70.2, 69.8,
NMR δ 7.40−7.22 (m, 5H), 4.62 (d, 1H, J = 11 Hz), 4.38 (d, 1H, J =
13
11 Hz), 4.08 (br s, 1H), 3.26 (m, 1H), 0.88 (s, 3H), 0.83 (s, 3H);
C
13
NMR δ 221.7, 138.6, 128.4 (2C), 127.8 (2C), 127.5, 82.0, 70.2, 66.2,
53.2, 51.4, 48.4, 41.0, 36.1, 36.0, 35.7 (2C), 33.5, 32.2, 31.6, 29.1, 25.0,
20.3, 14.2, 11.3. Anal. (C H O ) C, H.
5
2
8.6, 53.2, 47.1, 40.8, 36.1, 35.3, 32.8, 32.7, 32.3, 31.3 (2C), 27.5, 25.7,
1.5, 21.4, 14.9, 14.3.
27
36
3
(3β,5β,7α,8α,9β,10α,13α,14β)-7-(Benzyloxy)-3-hydroxyan-
drostan-17-one (ent-6). Compound 23 (105 mg, 0.27 mmol) was
converted into compound ent-6 using the procedure reported for the
preparation of steroid 6. Flash column chromatography (silica gel
eluted with 20−40% EtOAc in hexanes) gave product ent-6 (78 mg,
The acetate derivative was then hydrolyzed using K CO /MeOH to
2
3
2
0
give product 4 (152 mg, 96% yield): mp 62−65 °C; [α] +82.9 (c 0.1,
D
−
1
1
CHCl ); IR ν 3436, 2922, 1738, 1452, 1354 cm ; H NMR δ
3
max
7
.40−7.20 (m, 5H), 4.65 (d, 1H, J = 11.3 Hz), 4.26 (d, 1H, J = 11.3
2
0
Hz), 4.03 (br s, 1H), 4.02 (br s, 1H), 2.51−2.37 (m, 2H), 1.07 (s, 3H),
73%): mp 136−138 °C; [α] −135.5 (c 0.07); IR ν 3401, 2927,
−1 1
D
max
0
7
3
.99 (s, 3H); 13C NMR δ 220.1, 138.5, 128.1 (2C), 127.6 (2C), 127.1,
4.7, 70.2, 66.2, 58.7, 53.2, 47.2, 39.9, 36.3, 35.3, 35.3, 32.9, 32.0, 31.5,
1.3, 28.6, 27.6, 21.5, 15.0, 14.2. Anal. (C H O ) C, H.
2857, 1736, 1453, 1361 cm ; H NMR δ 7.40−7.20 (m, 5H), 4.62 (d,
H, J = 11 Hz), 4.38 (d, 1H, J = 11 Hz), 4.08 (br s, 1H), 3.26 (1H, m),
0.88 (s, 3H), 0.83 (s, 3H); C NMR δ 221.8, 138.6, 128.4 (2C), 127.8
1
13
26
36
3
(
3β,5β,8α,9β,10α,11α,13α,14β)-11-(Benzyloxy)-3-hydroxyan-
(
(
(
2C), 127.5, 82.0, 70.2, 66.2, 53.2, 51.4, 48.4, 41.0, 36.14, 36.06, 35.7
2C), 35.6, 33.5, 32.1, 31.6, 29.1, 25.0, 20.3, 14.2, 11.3. Anal.
C H O ) C, H.
drostan-17-one (ent-4). A mixture of compound 14d (121 mg, 0.25
mmol), MeOH (15 mL), and 6 N HCl (3 mL) was stirred at room
temperature for 36 h. The MeOH was removed under reduced
pressure, and the residual solution was extracted with EtOAc, dried,
and concentrated to give an oily liquid which was purified by flash
column chromatography (silica gel eluted with 30−40% EtOAc in
27
36
3
(
3α,5α,7β,17β)-Spiro[7-(benzyloxy)androstane-17,2′-oxir-
an]-3-ol (7). Steroid 6 (60 mg, 0.15 mmol) was converted into steroid
using the procedure reported for the preparation of steroid 5. Flash
7
column chromatography (silica gel eluted with 20−35% EtOAc in
2
0
hexanes) to give product ent-4 (92 mg, 92%): mp 62−64 °C; [α]
20
D
D
hexanes) gave product 7 (35 mg, 57%): mp 146−148 °C; [α] +55.5
−1 1
−
84.6 (c 0.08, CHCl ); IR ν 3401, 2921, 1738, 1452, 1354 cm ; H
NMR δ 7.40−7.20 (m, 5H), 4.66 (d, 1H, J = 11.2 Hz), 4.27 (d, 1H, J =
3 max
(c 0.06, CHCl ); IR ν 3436, 2927, 2855, 1497, 1454, 1358, 1265
3
max
−1 1
cm ; H NMR δ 7.40−7.20 (m, 5H), 4.58 (d, 1H, J = 11 Hz), 4.37 (d,
H, J = 11 Hz), 4.07 (br s, 1H), 3.18 (m, 1H), 2.90 (d, 1H, J = 5.2
Hz), 2.59 (d, 1H, J = 5.2 Hz), 0.89 (s, 3H), 0.82 (s, 3H); C NMR δ
1
3
1
3
1.2 Hz), 4.04 (br s, 1H), 4.03 (br s, 1H), 2.54−2.37 (m, 2H), 1.08 (s,
H), 1.01 (s, 3H); 13C NMR δ 220.1, 138.5, 128.1 (2C), 127.6 (2C),
27.1, 74.7, 70.2, 66.1, 58.7, 53.2, 47.1, 39.9, 36.3, 35.3, 35.2, 32.8,
2.0, 31.4, 31.3, 28.6, 27.6, 21.5, 14.9, 14.2. Anal. (C H O ) C, H.
1
13
1
4
1
38.8, 128.3 (2C), 127.8 (2C), 127.3, 82.4, 70.2, 66.2, 53.7, 53.1, 52.8,
1.5, 40.9, 36.2 (2C), 35.7 (2C), 34.0, 33.6, 32.2, 29.4, 29.1, 26.6, 20.4,
4.7, 11.3. Anal. (C H O ) C, H.
26
36
3
(
3α,5α,11β,17β)-Spiro[11-(benzyloxy)androstane-17,2′-oxir-
27
38
3
an]-3-ol (5). Trimethylsulfonium iodide (40.5 mg, 0.2 mmol)
followed by KO-t-Bu (16.5 mg, 0.15 mmol) was added to steroid 4
dissolved in DMF (4 mL), and the mixture was stirred at room
temperature for 2.5 h. Brine was added, and the product was extracted
into EtOAc (20 mL × 3). The combined extracts were dried and
concentrated to give a viscous liquid which was purified by flash
column chromatography (silica gel eluted with 30−40% EtOAc in
(
3β,5β,7α,8α,9β,10α,13α,14β,17α)-Spiro[7-(benzyoxy)-
androstane-17,2′-oxiran]-3-ol (ent-7). Compound ent-6 (39 mg,
.1 mmol) was converted into compound ent-7 using the procedure
0
reported for the preparation of steroid 5. Flash column chromatog-
raphy (silica gel eluted with % EtOAc in hexanes) gave product ent-7
2
0
(
28 mg, 68%): mp 145−148 °C; [α] −59.5 (c 0.05, CHCl ); IR ν
D
3
max
−1 1
2
0
3435, 2926., 2855, 1454, 1358, 1265 cm ; H NMR δ 7.40−7.20 (m,
H), 4.59 (d, 1H, J = 11 Hz), 4.37 (d, 1H, J = 11 Hz), 4.08 (br s, 1H),
3.18 (m, 1H), 2.91 (d, 1H, J = 5 Hz), 2.60 (d, 1H, J = 5 Hz), 0.89 (s,
H), 0.82 (s, 3H); 13C NMR δ 138.8, 128.3 (2C), 127.8 (2C), 127.4,
82.4, 70.2, 66.3, 53.7, 53.1, 52.8, 41.5, 40.9, 36.2 (2C), 35.7 (2C), 34.0,
3.6, 32.2, 29.5, 29.1, 26.6, 20.4, 14.7, 11.3. Anal. (C H O ) C, H.
hexanes) to give product 5 (25 mg, 61%): mp 144−146 °C; [α]
D
5
+
49.6 (c 0.05, CHCl ); IR ν 3400, 2921, 2854, 1587, 1455, 1355
3 max
−
1 1
cm ; H NMR δ 7.40−7.20 (m, 5H), 4.60 (d, 1H, J = 11 Hz), 4.17 (d,
3
1
H, J = 11 Hz), 4.02 (br s, 1H), 3.96 (m, 1H), 2.90 (d, 1H, J = 4.9
13
Hz), 2.60 (d, 1H, J = 4.9 Hz), 1.11 (s, 3H), 0.98 (s, 3H); C NMR δ
3
27
38
3
1
5
2
38.8, 128.1 (2C), 127.7 (2C), 127.1, 74.6, 71.1, 70.2, 66.4, 58.5, 54.7,
3.7, 40.1, 39.5, 36.3, 35.4, 35.1, 32.19, 32.09, 32.03, 28.8, 28.7, 27.8,
3.4, 15.7, 14.2. Anal. (C H O ) C, H.
(
3α,5α)-3-Hydroxyandrostane-11,17-dione (8b). Steroid 8a
(
3.18 g, 10.4 mmol) was converted into compound 8b using the
27
38
3
procedure reported for the preparation of steroid 6. After flash column
chromatography (silica gel eluted with 20−40% EtOAc in hexanes)
product 8b (2.28, 72%) gave the following data: mp 146−149 °C; IR
(
3β,5β,8α,9β,10α,11α,13α,14β,17α)-Spiro[11-(benzyloxy)-
androstane-17,2′-oxiran]-3-ol (ent-5). Compound ent-4 (40 mg,
.10 mmol) was converted into steroid ent-5 using the procedure
0
−
1 1
ν
1
5
3461, 2923, 1741, 1705, 1454, 1233 cm ; H NMR δ 4.05 (br s,
reported for the preparation of steroid 5. Flash column chromatog-
raphy (silica gel eluted with 20−35% EtOAc in hexanes) gave product
max
13
H), 1.02 (s, 3H), 0.82 (s, 3H); C NMR δ 217.5, 209.0, 66.1, 64.9,
0.6 (2C), 50.4, 38.9, 36.1, 36.0, 35.9, 35.2, 31.3, 30.8, 28.8, 27.6, 21.5,
2
0
ent-5 (25 mg, 61%): mp 143−146 °C; [α] −44.5 (c 0.05, CHCl );
D
3
−1 1
IR ν 3369, 2921, 2853, 1595, 1454, 1357 cm ; H NMR δ 7.40−
.20 (m, 5H), 4.61 (d, 1H, J = 11.3 Hz), 4.18 (d, 1H, J = 11.3 Hz),
.03 (s, 1H) 3.97 (m, 1H), 2.91 (d, 1H, J = 5.0 Hz), 2.61 (d, 1H, J =
.0 Hz), 1.11 (s, 3H), 0.98 (s, 3H); C NMR δ 138.8, 128.1 (2C),
27.7 (2C), 127.1, 74.6, 71.1, 70.2, 66.5, 58.5, 54.7, 53.7, 40.1, 39.5,
6.3, 35.4, 35.1, 32.1, 32.0, 32.0, 28.8, 28.7, 27.8, 23.4, 15.7, 14.2. Anal.
14.6, 11.0. Anal. (C19H O ) C,H.
28 3
max
7
4
5
1
3
(3α,5α)-3-Hydroxyandrostane-11,17-dione, Cyclic 17-(1,2-
Ethanediyl acetal) (9a). A mixture of 8b (609 mg, 2 mmol),
ethylene glycol (1 mL), and p-toluenesulfonic acid (PTSA; 100 mg) in
benzene (100 mL) was heated at reflux using a Dean−Stark apparatus
for 16 h. The reaction mixture was cooled and basified with aqueous
13
(
C H O ) C, H.
saturated NaHCO and extracted with EtOAc (50 mL × 4). The
3
2
7
38
3
(
3α,5α,7β)-7-(Benzyloxy)-3-hydroxyandrostan-17-one (6).
combined extracts were dried and concentrated to give a white solid
which was purified by flash column chromatography (silica gel eluted
with 20−35% EtOAc in hexanes) to give product 9a (488 mg, 70%):
mp 92−95 °C; IR νmax 3420, 2923, 1704, 1456, 1385, 1313, 1275,
Steroid 19b (59 mg, 0.15 mmol) was dissolved in THF (5 mL) and
cooled to −78 °C, and a 1 M solution of K-Selectride in THF (0.15
mL) was added. The reaction was stirred at −78 °C for 1.5 h and
quenched by adding a few drops of water. Then 3 N aqueous NaOH
5 mL) followed by 30% H O (5 mL) was added, and stirring was
continued at room temperature for 1.5 h. The product was extracted
−
1 1
1203 cm ; H NMR δ 4.03 (br s, 1H), 4.00−3.78 (m, 4H), 2.62 (d,
1H, J = 11.5 Hz), 1.00 (s, 3H), 0.79 (s, 3H); 13C NMR δ 211.3, 117.8,
66.2, 65.3, 64.5, 64.3, 50.2, 49.7, 49.5, 39.0, 37.1, 35.7, 35.3, 34.3, 31.9,
30.9, 28.8, 27.8, 22.1, 14.8, 10.9; HRMS (EI) m/z calcd for C H O
4
(
2 2
with Et O (2 × 30 mL) followed by EtOAc (2 × 40 mL). The
combined extracts were washed with brine, dried, and concentrated to
2
21 32
348.2301, found 348.2296.
1
341
dx.doi.org/10.1021/jm2014925 | J. Med. Chem. 2012, 55, 1334−1345

Journal of Medicinal Chemistry
Article
(
3α,5α)-3-[[(Methyloxy)methyl]oxy]androstane-11,17-dione,
(5β,8α,10α,13α,14β)-Androst-9(11)-ene-3,17-dione (12b).
Compound 12a (1.9 g, 6.6 mmol) was dissolved in acetone (50
mL) and stirred. Jones reagent was added dropwise until the orange
color persisted, and stirring was continued for 1 h. The excess Jones
reagent was quenched by adding a few drops of isopropyl alcohol, and
the acetone was removed under reduced pressure to give a green
liquid. Water was added, and the resulting solution was extracted with
EtOAc. The EtOAc was dried and removed to give a white solid. Flash
column chromatography (silica gel eluted with 20−35% EtOAc in
Cyclic 17-(1,2-Ethanediyl acetal) (9b). Steroid 9a (452 mg, 1.3
mmol), chloromethyl methyl ether (0.53 mL, 7 mmol), N,N-
diisopropylethylamine (1.74 mL, 10 mmol), and a catalytic amount
of DMAP (40 mg) dissolved in CH Cl (15 mL) were stirred at room
2
2
temperature for 16 h. The reaction was poured into an aqueous
saturated NaHCO solution and extracted with CH Cl (100 mL × 3).
3
2
2
The combined extracts were dried and concentrated to give a viscous
liquid which was purified by flash column chromatography (silica gel
eluted with 20−25% EtOAc in hexanes) to give product 9b (464 mg,
hexanes) yielded product 12b (1.73 g, 92%): mp 147−148 °C; IR ν
max
−
1 1
9
1
4
3
4
1
4
1%) as an oil: IR ν 2924, 1705, 1455, 1385, 1365, 1314, 1275,
2925, 2852, 1739, 1713, 1595, 1450, 1404, 1370, 1244, 1210 cm ; H
max
−1
1
13
204 cm ; H NMR δ 4.64 (dd, 2H, J = 8.5, 6.6 Hz), 3.98−3.75 (m,
H), 3.35 (s, 3H), 2.61 (d, 1H, J = 11.8 Hz), 1.01 (s, 3H), 0.79 (s,
H); 13C NMR δ 211.0, 117.6, 94.4, 71.3, 65.2, 64.4, 64.2, 54.9, 50.0,
9.5, 49.4, 39.5, 36.9, 35.4, 34.2, 33.1, 31.7, 31.4, 27.7, 25.9, 22.0, 14.7,
1.0; HRMS (FAB) m/z calcd for C H O Na 415.2460, found
NMR δ 5.43 (m, 1H), 1.18 (s, 3H), 0.85 (s, 3H); C NMR δ 221.4,
211.1, 146.4, 116.2, 48.8, 45.9, 44.8, 44.6, 38.1, 36.9, 36.2 (3C), 33.3,
31.7, 28.5, 22.7, 17.3, 13.8. Anal. (C19H O ) C, H.
26 2
(3β,5β,8α,10α,13α,14β)-3-Hydroxyandrost-9(11)-en-17-one
12c). Compound 12b (1.69 g, 5.94 mmol) was converted into
(
23
36
5
compound 12c using the procedure reported for the preparation of
steroid 6. After flash column chromatography (silica gel eluted with
20−40% EtOAc in hexanes) product 12c (1.1 g, 65%) gave the
following data: mp 186−188 °C; IR νmax 3401, 2921, 2854, 1738,
15.2466.
3α,5α,11β)-3-[[(Methyloxy)methyl]oxy]-11-hydroxyandro-
stan-17-one, Cyclic 17-(1,2-Ethanediyl acetal) (10a). Steroid 9b
392 mg, 1 mmol) dissolved in dry Et O was added to an ice cold,
(
(
2
−1 1
stirred suspension of LAH (114 mg, 3 mmol) in Et O. The reaction
1596, 1451, 1370, 1279, 1211 cm ; H NMR δ 5.38 (m, 1H), 4.06 (br
2
s, 1H), 0.94 (s, 3H), 0.82 (s, 3H); 13C NMR δ 222.1, 148.0, 114.7,
was warmed to room temperature, and stirring was continued for 2 h.
Water (0.5 mL) was added, then a drop of aqueous 5 N NaOH (0.5
mL) was added, and the mixture was stirred for 0.5 h. Water (1 mL)
was again added, and stirring was continued for another 0.5 h. The
Et O was decanted, and the remaining solid was washed with Et O.
6
2
6.3, 49.1, 46.0, 38.6, 37.7, 36.3, 36.2, 36.1, 33.3, 32.2, 30.6, 29.1, 28.1,
2.7, 17.0, 13.8. Anal. (C H O ) C, H.
19 28
2
(
3β,5β,8α,10α,13α,14β)-3-Hydroxyandrost-9(11)-en-17-one,
Cyclic 17-(1,2-Ethanediyl acetal) (13a). Compound 12c (1 g, 3.49
mmol) was converted into compound 13a using the procedure
reported for the preparation of steroid 9a (except that pyridinium p-
toluenesulfonate (PPTS) was used as the catalyst instead of PTSA).
Flash column chromatography (silica gel eluted with 20−40% EtOAc
2
2
The combined Et O solutions were dried and concentrated to give
product 10a as a liquid (315 mg, 80%): IR ν 3503, 2927, 1454,
279, 1212 cm ; H NMR δ 4.58 (dd, 2H, J = 6.3, 5.5 Hz), 4.31 (br s,
H), 3.90−3.70 (m, 4H), 3.30 (s, 3H), 1.01 (s, 3H), 0.98 (s, 3H);
2
max
−1 1
1
1
13
C
in hexanes) gave product 13a (1 g, 87%): mp 138−140 °C; IR ν
max
NMR δ 119.3, 94.4, 71.3, 67.8, 64.9, 64.3, 57.8, 55.0, 51.8, 45.0, 40.4,
9.5, 35.9, 33.9, 32.9, 32.4, 31.5, 31.3, 27.8, 25.9, 22.5, 16.8, 14.3. Anal.
C H O ) C, H.
−1 1
3
350, 2968, 2920, 2873, 1595, 1454, 1374, 1308, 1274, 1204 cm ; H
3
(
NMR δ 5.35 (m, 1H), 4.04 (br s, 1H), 4.00−3.80 (m, 4H), 2.38 (d,
2
3
38
5
13
1
1
3
H, J = 16.5 Hz), 0.92 (s, 3H), 0.78 (s, 3H); C NMR δ 147.0, 119.2,
15.4, 66.4, 65.1, 64.5, 48.1, 44.2, 38.4, 37.9, 37.2, 36.2, 34.0, 32.6,
2.2, 30.7, 29.1, 28.3, 23.6, 16.9, 14.1; HRMS (EI) m/z calcd for
(
3α,5α,11β)-11-(Benzyloxy)-3-[[(methyloxy)methyl]oxy]-
androstan-17-one, Cyclic 17-(1,2-Ethanediyl acetal) (10b). KH
400 mg, 40% in mineral oil, 4 mmol) and then benzyl bromide (0.31
(
C H O 332.2351, found 332.2352.
mL, 2.5 mmol) were added to steroid 10a dissolved in THF (10 mL),
and the mixture was refluxed for 2 h. The reaction was cooled to 0 °C,
and the excess KH was carefully destroyed by MeOH (5 mL) addition.
Water (20 mL) was then added, and the product was extracted with
EtOAc. The EtOAc was washed with brine, dried, and concentrated to
give a residue which was purified by flash column chromatography
21 32
3
(
3β,5β,8α,10α,13α,14β)-3-[[(Methyloxy)methyl]oxy]androst-
9
(11)-en-17-one, Cyclic 17-(1,2-Ethanediyl acetal) (13b). Com-
pound 13a (1 g, 3 mmol) was converted into compound 13b using the
procedure reported for the preparation of steroid 9b. Flash column
chromatography (silica gel eluted with 15−25% EtOAc in hexanes)
gave product 13b (1.1 g, 97%): mp 73−75 °C; IR ν 2968, 2923,
max
(
silica gel eluted with 20−35% EtOAc in hexanes) to give product 10b
as a liquid (294 mg, 75%): IR ν 2926, 1454, 1279, 1210 cm ; H
−
1 1
1
6
3
1
3
595, 1454, 1374, 1308, 1275, 1207 cm ; H NMR δ 5.33 (d, 1 H, J =
−1 1
max
.1 Hz), 4.65 (dd, 2H, J = 9.1, 6.9 Hz), 3.96−3.80 (m, 4H), 3.36 (s,
NMR δ 7.40−7.18 (m, 5H), 4.70−4.52 (m, 3H), 4.20 (d, 1H, J = 11.3
13
H), 2.37 (d, 1H, J = 16.2 Hz), 0.93 (s, 3H), 0.79 (s, 3H); C NMR δ
13
Hz), 4.00−3.70 (m, 5H), 3.35 (s, 3H), 1.07 (s, 3H), 0.98 (s, 3H);
C
47.1, 119.2, 115.3, 94.5, 71.5, 65.1, 64.5, 55.1, 48.1, 44.3, 28.4, 38.2,
7.2, 34.0, 33.9, 32.56, 32.2, 31.4, 28.4, 26.5, 23.6, 17.2, 14.1. Anal.
NMR δ 139.0, 128.0 (2C), 127.5 (2C), 126.9, 119.4, 94.4, 75.1, 71.5,
7
3
0.1, 65.0, 64.3, 58.2, 55.0, 52.1, 45.3, 40.5, 36.0, 33.9, 33.0, 32.6, 31.9,
1.8, 31.6, 27.9, 26.0, 22.5, 15.7, 14.4; HRMS (FAB) m/z calcd for
(
C H O ) C, H.
23 36 4
(
3β,5β,8α,9β,10α,11β,13α,14β)-3-[[(Methyloxy)methyl]oxy]-
C H O Na 507.3086, found 507.3083.
3
0
44
5
11-hydroxyandrostan-17-one, Cyclic 17-(1,2-Ethanediyl ace-
tal) (14a). Compound 13b was dissolved in THF (10 mL) and stirred
at 0 °C. A 1 M borane−THF complex in THF (3 mL, 3 mmol) was
added, and the reaction was stirred at room temperature for 8 h. The
reaction was cooled to 0 °C, and excess borane complex was carefully
quenched with a few drops of water. Aqueous NaOH (3 N, 10 mL)
followed by 30% H O (10 mL) was added, and the reaction was
(
5β,8α,10α,13α,14β,17α)-17-Hydroxyandrost-9(11)-en-3-one
(
12a). NH (150 mL) was condensed into a cold (−78 °C) two-
3
necked flask (250 mL) fitted with a Dewar condenser. Toluene (15
mL) and THF (20 mL) were added. Lithium (294 mg, 42.3 mmol), in
small pieces, was added to the solution, and the mixture was stirred for
1
0 min, resulting in the formation of a deep blue colored solution.
2
2
17
Compound 11 (2.4 g, 8.45 mmol) in THF (20 mL) was added, and
the mixture was stirred at −78 °C for 45 min. Solid ammonium
chloride (6 g) was added, and the ammonia was allowed to evaporate.
Water (200 mL) was added, and the reaction mixture was extracted
with EtOAc (100 mL × 3). The combined EtOAc extracts were
washed with brine, dried, and concentrated to give a white solid which
was purified by flash column chromatography (silica gel eluted with
stirred at room temperature for 16 h. The mixture was extracted with
Et O (3 × 75 mL) followed by EtOAc (2 × 50 mL). The combined
2
extracts were dried and concentrated to give an oil which was purified
by flash column chromatography (silica gel eluted with 20−40%
EtOAc in hexanes) to give product 14a as a colorless liquid (351 mg,
−
1
6
7%): IR νmax 3435, 2923, 1595, 1457, 1380, 1312, 1282, 1212 cm ;
1
H NMR δ 4.59 (dd, 2H, J = 10.7, 6.6 Hz), 3.93−3.68 (m, 5H), 3.30
s, 3H), 0.88 (s, 3H), 0.78 (s, 3H); C NMR δ 118.7, 94.2, 71.1, 69.2,
2
0−35% EtOAc in hexanes) to give product 12a (1.97 g, 81%): mp
13
(
1
66−168 °C; IR ν 3391, 2921, 2869, 1713, 1596, 1445, 1374, 1271
max
65.0, 64.4, 60.4, 54.9, 49.3, 46.2, 42.5, 39.5, 37.6, 34.9, 34.5, 34.1, 34.1,
−1 1
cm ; H NMR δ 5.41 (m, 1H), 3.74 (t, 1H, J = 9.1 Hz), 1.16 (s, 3H),
3
1.1, 28.9, 26.2, 22.5, 15.0, 11.7. Anal. (C H O ) C, H.
23 38 5
0
4
.72 (s, 3H); 13C NMR δ 211.6, 146.1, 116.7, 81.9, 48.4, 45.0, 44.6,
1.4, 38.3, 38.1, 37.8, 37.0, 36.9, 32.2, 30.7, 28.7, 24.2, 17.3, 10.5. Anal.
(
3β,5β,8α,9β,10α,13α,14β)-3-[[(Methyloxy)methyl]oxy]-
androstane-11,17-dione, Cyclic 17-(1,2-Ethanediyl acetal)
(
C H O C, H.
(14b). Compound 14a (250 mg, 0.63 mmol) was dissolved in stirred
1
9
28 2)
1
342
dx.doi.org/10.1021/jm2014925 | J. Med. Chem. 2012, 55, 1334−1345

Journal of Medicinal Chemistry
Article
CH Cl (8 mL), solid NaOAc (154 mg, 1.89 mmol) and pyridinium
filtrate and washings were concentrated to give an off-white solid
which was purified by flash column chromatography (silica gel eluted
with 15−25% ethyl acetate in hexanes) to give product 18a (471 mg,
2
2
chlorochromate (PCC; 407.4 mg, 1.89 mmol) were added, and the
reaction was stirred at room temperature for 3 h. The CH Cl solution
2
2
was directly purified by flash column chromatography (silica gel eluted
80%): mp 117−119 °C; IR ν 2944, 2882, 1708, 1448, 1379, 1292,
max
−1 1
with 10−35% EtOAc in hexanes) to give product 14b (237 mg, 96%)
1223 cm ; H NMR δ 4.67 (s, 2H), 4.00−3.76 (m, 4H), 3.50 (m,
−1
1
13
as an oil: IR ν 2924, 1705, 1594, 1456, 1385, 1204 cm ; H NMR δ
1H), 3.36 (s, 3H), 1.08 (s, 3H), 0.84 (s, 3H); C NMR δ 210.9,
max
4
(
.63 (dd, 2H, J = 8.2, 6.9 Hz), 3.94−3.70 (m, 5H), 3.32 (s, 3H), 2.57
118.6, 94.7, 75.7, 65.2, 64.4, 55.1, 50.0, 46.4, 45.9, 45.7, 43.4, 36.2,
36.0, 35.2, 34.1, 29.7, 28.4, 23.8, 21.3, 14.4, 11.7. Anal. (C H O ) C,
d, 1H, J = 12 Hz), 0.98 (s, 3H), 0.75 (s, 3H); 13C NMR δ 211.4,
2
3
36
5
1
3
17.7, 94.4, 71.4, 65.2, 64.5, 64.2, 55.0, 50.1, 49.6, 49.5, 39.6, 37.0,
5.5, 34.3, 33.2, 31.8, 31.5, 27.8, 25.9, 22.1, 14.8, 11.1. Anal.
H.
(3β,5α)-7-Hydroxy-3-[[(methyloxy)methyl]oxy]androstan-
(
C H O ) C, H.
17-one, Cyclic 17-(1,2-Ethanediyl acetal) (18b). Steroid 18a (498
mg, 1.27 mmol) was converted into an inseparable mixture of the 7α-
and 7β-hydroxysteroids 18b using the procedure reported for the
preparation of steroid 10a. Filtration through a short column of silica
gel eluted with 40% EtOAc in hexanes gave product 18b (431 mg,
86%), which was then converted without characterization into the
separable steroids 18c and 18d.
23
36
5
(
3β,5β,8α,9β,10α,11α,13α,14β)-3-[[(Methyloxy)methyl]oxy]-
1
1-hydroxyandrostan-17-one, Cyclic 17-(1,2-Ethanediyl ace-
tal) (14c). Compound 14b (208 mg, 0.53 mmol) was converted into
compound 14c using the procedure reported for the preparation of
steroid 10a. The reaction time was 3 h. Flash column chromatography
(
silica gel eluted with 35% EtOAc in hexanes) gave product 14c (199
−
1 1
mg, 95%) as an oil: IR νmax 3504, 2925, 1455, 1279, 1212 cm ; H
NMR δ 4.58 (dd, 2H, J = 8.0, 6.6 Hz), 4.31 (br s), 3.90−3.70 (m, 4H),
3
6
3
(3β,5α,7β)-7-(Benzyloxy)-3-[[(methyloxy)methyl]oxy]-
androstan-17-one, Cyclic 17-(1,2-Ethanediyl acetal) (18c) and
(3β,5α,7α)-7-(Benzyloxy)-3-[[(methyloxy)methyl]oxy]-
androstan-17-one, Cyclic 17-(1,2-Ethanediyl acetal) (18d).
Steroid 18b (431 mg, 1.09 mmol) was converted into products 18c
and 18d using the procedure reported for the benzylation of steroid
13
.29 (s, 3H), 1.01 (s, 3H), 0.96 (s, 3H); C NMR δ 119.4, 94.4, 71.3,
7.9, 65.0, 64.4, 57.8, 55.0, 51.8, 45.1, 40.4, 39.6, 35.9, 33.9, 32.9, 32.4,
1.6, 31.3, 27.8, 25.9, 22.6, 16.8, 14.3; HRMS (FAB) m/z calcd for
C H O Na 417.2617, found 417.2617.
23
38
5
1
0b. Flash column chromatography (silica gel eluted with 10−35%
(
3β,5β,8α,9β,10α,11α,13α,14β)-11-(Benzyloxy)-3-
[
[(Methyloxy)methyl]oxy]androstan-17-one, Cyclic 17-(1,2-
EtOAc in hexanes) gave product 18c (120 mg, 23%), product 18d
(240 mg, 45%), and recovered steroid 18b (120 mg, 23%). Product
18d eluted from the column before product 18c. Data for 18c: oil; IR
Ethanediyl acetal) (14d). Compound 14c (200 mg, 0.51 mmol)
was converted into compound 14d using the procedure reported for
the preparation of steroid 10b. Flash column chromatography (silica
gel eluted with 20−35% EtOAc in hexanes) gave product 14d (170
−1 1
ν
max 2936, 1454, 1307 cm ; H NMR δ 7.40−7.19 (m, 5H), 4.68 (s,
2H), 4.55 (d, 1H, J = 11 Hz), 4.34 (d, 1H, J = 11 Hz), 4.00−3.79 (m,
4H), 3.49 (m, 1H), 3.37 (s, 3H), 3.13 (m, 1H), 0.86 (s, 3H), 0.84 (s,
3H); 13C NMR δ 138.9, 128.3 (2C), 127.9 (2C), 127.3, 119.0, 94.6,
82.7, 76.1, 70.4, 65.2, 64.5, 55.1, 52.9, 50.2, 46.7, 42.0, 41.5, 37.0, 35.2,
−
1 1
mg, 69%) as an oil: IR ν 2924, 1454, 1278 cm ; H NMR δ 7.40−
max
7
3
1
6
3
.18 (m, 5H), 4.70−4.52 (m, 3H), 4.20 (d, 1H, J = 11.2 Hz), 4.02−
.69 (m, 5H), 3.36 (s, 3H), 1.06 (s, 3H), 0.98 (s, 3H); 13C NMR δ
39.0, 128.0 (2C), 127.6 (2C), 127.0, 119.5, 94.5, 75.2, 71.5, 70.1,
5.1, 64.4, 58.2, 55.1, 52.1, 45.3, 40.6, 36.0, 34.0, 33.1, 32.7, 31.9, 31.9,
1.7, 27.9, 26.0, 22.6, 15.8, 14.4; HRMS (FAB) m/z calcd for
35.1, 34.3, 33.8, 30.4, 28.7, 25.4, 20.9, 14.7, 12.4. Anal. (C30H O ) C,
44 5
−1 1
H. Data for 18d: oil; IR νmax 2935, 1453, 1306 cm ; H NMR δ 7.43−
7.20 (m, 5H), 4.68 (s, 2H), 4.57 (d, 1H, J = 12 Hz), 4.32 (d, 1H, J =
11.8 Hz), 3.97−3.76 (m, 4H), 3.48 (m, 1H), 3.46 (br s, 1H), 3.37 (s,
3H), 0.81 (s, 3H), 0.81 (s, 3H); 13C NMR δ 139.5, 128.2 (2C), 127.4
(2C), 127.2, 119.4, 94.6, 76.4, 74.5, 70.4, 65.1, 64.6, 55.1, 46.3, 46.0,
44.2, 40.4, 37.2, 36.8, 35.7, 35.0, 34.3, 31.6, 30.4, 28.7, 22.4, 20.6, 14.1,
11.4. Anal. (C H O ) C, H.
C H O Na 507.3087, found 507.3091.
30
44
5
(
3β)-3-[[(Methyloxy)methyl]oxy]androst-5-en-17-one, Cyclic
26
1
7-(1,2-Ethanediyl acetal) (16). Steroid 15 (3 g, 9 mmol) was
converted into steroid 16 using the procedure reported for the
preparation of steroid 9b. Flash column chromatography (silica gel
eluted with 15−20% EtOAc in hexanes) gave product 16 (3.1 g, 91%):
30
44
5
(3β,5α,7β)-7-(Benzyloxy)-3-hydroxyandrostan-17-one (19a).
Steroid 18c (120 mg, 0.25 mmol) was converted into steroid 19a
using the procedure reported for the preparation of compound ent-4.
Flash column chromatography (silica gel eluted with 30−40% EtOAc
mp 94−96 °C; IR ν 2926, 2893, 2823, 1667, 1466, 1437, 1381,
max
−1 1
1
4
3
4
1
305, 1277 cm ; H NMR δ 5.33 (d, 1H, J = 5.2 Hz), 4.66 (s, 2H),
.00−3.78 (m, 4H), 3.36 (m, 1H), 3.34 (s, 3H), 0.99 (s, 3H), 0.83 (s,
H); 13C NMR δ 140.6, 121.4, 119.4, 94.6, 76.8, 65.1, 64.5, 55.1, 50.5,
9.9, 45.6, 39.5, 37.2, 36.7, 34.1, 32.1, 31.2, 30.5, 28.8, 22.7, 20.4, 19.3,
4.1. Anal. (C H O ) C, H.
in hexanes) gave product 19a (90 mg, 91%): mp 175−177 °C; IR ν
max
−1 1
3447, 2930, 2855, 1735, 1497, 1453, 1405, 1362, 1202 cm ; H NMR
δ 7.40−7.20 (m, 5H), 4.64 (d, 1H, J = 11 Hz), 4.38 (d, 1H, J = 11 Hz),
23
36
4
1
3
(
3β)-3-[[(Methyloxy)methyl]oxy]androst-5-ene-7,17-dione,
Cyclic 17-(1,2-Ethanediyl acetal) (17). A mixture of steroid 16
2.26 g, 6 mmol), 70% aqueous t-BuOOH (4.32 mL, 30 mmol), and
3.60 (m, 1H), 3.23 (m, 1H), 0.87 (s, 3H), 0.86 (s, 3H); C NMR δ
221.6, 138.5, 128.4 (2C), 127.7 (2C), 127.5, 81.9, 70.9, 70.2, 53.0,
51.3, 48.4, 41.8, 40.9, 37.9, 36.9, 36.1, 35.2, 33.6, 31.6, 31.4, 24.9, 20.8,
14.1, 12.4. Anal. (C H O ) C, H
(
NaOCl (813 mg, 7.2 mmol) in premixed acetonitrile (60 mL) and
water (20 mL) was heated at 50 °C for 36 h. The reaction was cooled,
poured into aqueous 10% Na SO (100 mL), and stirred for 0.5 h. The
product was extracted into Et O (100 mL × 4), and the combined
extracts were washed with brine, dried, and concentrated to give a
viscous liquid which was purified by flash column chromatography
(
2
26 36
3
(5α,7β)-7-(Benzyloxy)androstane-3,17-dione (19b). Steroid
19a (71 mg, 0.18 mmol) was converted into compound 19b using
the procedure reported for the preparation of steroid 14b. Flash
column chromatography (silica gel eluted with 25% EtOAc in
2
3
2
hexanes) gave product 19b (61 mg, 86%): mp 181−183 °C; IR ν
max
−1 1
silica gel eluted with 15−25% EtOAc in hexanes) to yield recovered
2945, 2921, 2856, 1733, 1716, 1454, 1367, 1289, 1257, 1227 cm ; H
steroid 16 (450 mg, 20%) and product 17 (800 mg, 34%): mp 136−
NMR δ 7.40−7.24 (m, 5H), 4.63 (d, 1H, J = 11 Hz), 4.40 (d, 1H, J =
−
1
13
1
38 °C; IR ν 2946, 2880, 1668, 1629, 1461, 1382, 1295, 1215 cm ;
11 Hz), 3.26 (m, 1H), 1.07 (s, 3H), 0.90 (s, 3H); C NMR δ 221.2,
max
1
H NMR δ 5.70 (s, 1H), 4.69 (dd, 2H, J = 8.0, 6.9 Hz), 4.00−3.80 (m,
210.9, 138.3, 128.4 (2C), 127.8 (2C), 127.6, 81.5, 70.5, 52.2, 51.2,
48.3, 44.2, 43.7, 40.8, 38.2, 37.9, 36.0, 35.3, 33.8, 31.5, 24.9, 21.2, 14.1,
11.6. Anal. (C H O ) C, H.
4
H), 3.56 (m, 1H), 3.38 (s, 3H), 1.20 (s, 3H), 0.87 (s, 3H); 13C NMR
δ 201.2, 165.3, 125.9, 118.5, 94.9, 75.5, 65.1, 64.3, 55.2, 49.8, 46.0,
26
34
3
4
5.2, 44.3, 39.3, 38.3, 36.2, 34.0, 29.5, 28.5, 25.0, 20.5, 17.2, 14.3. Anal.
(8α,9β,10α,13α,14β)-Androst-5-ene-3,17-dione, Cyclic 3,17-
18
(
C H O ) C, H.
Bis(1,2-Ethanediyl acetal) (21a). Compound 20 (1.6 g, 5.6
mmol) was converted into compound 21a using the procedure
reported for the preparation of compound 9a. Flash column
chromatography (silica gel eluted with 15−25% EtOAc in hexanes)
23
34
5
(
3β,5α)-3-[[(Methyloxy)methyl]oxy]androstane-7,17-dione,
Cyclic 17-(1,2-Ethanediyl acetal) (18a). Steroid 17 (586 mg, 1.5
mmol), 10% Pd/C (300 mg), and EtOAc (80 mL) were placed in a
Parr hydrogenation flask and hydrogenated (50 psi) for 18 h. The Pd/
C was filtered under vacuum through a pad of Celite 454 on a sintered
glass funnel. The filter cake was washed with EtOAc. The combined
gave product 21a (1.74 g, 83%): mp 158−160 °C; IR ν 2943, 2885,
max
−1 1
1431, 1381, 1305 cm ; H NMR δ 5.34 (m, 1H), 4.00−3.80 (m, 8H),
1.04 (s, 3H), 0.86 (s, 3H); 13C NMR δ 140.0, 121.8, 119.3, 109.3, 65.0,
1
343
dx.doi.org/10.1021/jm2014925 | J. Med. Chem. 2012, 55, 1334−1345

Journal of Medicinal Chemistry
Article
6
3
4.4, 64.3, 64.1, 50.4, 49.4, 45.6, 41.7, 36.6, 36.2, 34.1, 32.1, 31.0, 30.9,
0.5, 22.7, 20.3, 18.7, 14.1. Anal. (C H O ) C, H.
harvested from sexually mature female X. laevis frogs (Xenopus 1,
Northland, MI). The frogs were anesthetized with 0.1% tricane (3-
aminobenzoic acid ethyl ester) and oocytes obtained by partial
ovariectomy. The follicular layer was removed from the oocytes by
shaking for 20 min at 37 °C in 2 mg/mL collagenase dissolved in a
calcium-free solution containing 96 mM NaCl, 2 mM KCl, 1 mM
23
34
4
(
8α,9β,10α,13α,14β)-Androst-5-ene-3,7,17-trione, Cyclic
3
,17-Bis(1,2-Ethanediyl acetal) (21b). Compound 21a (1.73 g,
4.62 mmol) was converted into compound 21b using the procedure
reported for the preparation of steroid 17. Flash column
chromatography (silica gel eluted with 15−25% EtOAc in hexanes)
gave recovered compound 21a (500 mg, 28%) and product 21b (560
MgCl , and 5 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
2
acid (HEPES), pH 7.4. Capped RNA for α , β , and γ subunits was
1
2
2L
mg, 31%): mp 198−200 °C; IR ν 2953, 2882, 1672, 1634, 1457,
prepared in vitro (mMESSAGE mMachine kit, Ambion, Austin, TX)
max
−1 1
1
(
383, 1295 cm ; H NMR δ 5.66 (s, 1H), 4.03−3.78 (m, 8H), 2.69
from linearized pBluescript vectors containing subunit coding regions.
d, 1H, J = 14.8 Hz), 1.22 (s, 3H), 0.88 (s, 3H); 13C NMR δ 201.2,
20
The α Q241L mutation was introduced as described previously.
1
1
64.7, 126.5, 118.6, 108.8, 65.1, 64.5, 64.5, 49.5, 46.2, 45.3, 44.4, 41.7,
Subunit RNA was injected in equal parts (20−40 ng of total RNA),
after which the oocytes were incubated at 18 °C in ND96 solution
containing 96 mM NaCl, 2 mM KCl, 2 mM CaCl , 1 mM MgCl , and
38.2, 35.5, 34.1, 31.0, 29.6, 25.1, 20.6, 17.0, 14.4. Anal. (C H O ) C,
23
32
5
H.
2
2
(
5β,8α,9β,10α,13α,14β)-Androstane-3,7,17-trione, Cyclic
,17-Bis(1,2-Ethanediyl acetal) (22a). Compound 21b (500 mg,
.28 mmol) was converted into compound 22a using the procedure
5 mM HEPES, pH 7.4. ND96 was supplemented with pyruvate (5
mM), penicillin (100 U/mL), streptomycin (100 μg/mL), and
gentamycin (50 μg/mL).
Oocyte responses were recorded 2−5 days following RNA injection.
GABA currents were recorded using a two-electrode voltage clamp
(OC725C amplifier, Warner Instruments) at a membrane potential of
3
1
reported for the preparation of steroid 18a. Flash column
chromatography (silica gel eluted with 20−30% EtOAc in hexanes)
gave product 22a (380 mg, 76%): mp 208−210 °C; IR ν 2952,
2
1
6
2
max
−1 1
880, 1705, 1456, 1386, 1293 cm ; H NMR δ 4.00−3.70 (m, 8H),
.08 (s, 3H), 0.84 (s, 3H); 13C NMR δ 210.8, 118.6, 108.6, 65.1, 64.4,
4.3, 64.1, 54.7, 50.0, 45.7, 45.6, 45.2, 43.4, 37.7, 35.9, 35.2, 34.1, 31.1,
−
70 mV. The bath solution was ND96 solution, and glass recording
pipets (∼1 MΩ resistance) were filled with 3 M KCl. Compounds
were applied to the oocytes using a multibarrel pipet with a common
output tip. Data acquisition and analysis were performed with
pCLAMP software (Molecular Devices, Sunnyvale, CA). Statistical
differences were assessed with one-tailed or two-tailed paired or
unpaired t tests as indicated.
9.7, 23.8, 21.3, 14.4, 10.9. Anal. (C H O ) C, H.
23
34
5
(
5β,8α,9β,10α,13α,14β)-7-Hydroxyandrostane-3,17-dione,
Cyclic 3,17-Bis(1,2-Ethanediyl acetal) (22b). Compound 22a (140
mg, 0.36 mmol) dissolved in 2-propanol (30 mL) and Raney nickel
were placed in a Parr hydrogenation flask and hydrogenated (H , 60
psi) for 18 h. The Raney nickel was removed by filtration under
vacuum through Celite 454 in a sintered glass funnel. The Celite 454
was washed with methanol, and the filtrate was evaporated to yield a
solid product which was purified by flash column chromatography
2
Tadpole Behavioral Methods. The methods used were described
27
previously.
ASSOCIATED CONTENT
■
(
silica gel eluted with 40% EtOAc in hexanes) to give the inseparable
*
S
Supporting Information
mixture of 7α- and 7β-alcohol products 22b (136 mg, 96%). This
Table of elemental analysis results. This material is available
free of charge via the Internet at http://pubs.acs.org.
product was not characterized.
(
5β,7β,8α,9β,10α,13α,14β)-7-(Benzyloxy)androstane-3,17-
dione, Cyclic 3,17-Bis(1,2-Ethanediyl acetal) (22c) and
5β,7α,8α,9β,10α,13α,14β)-7-(Benzyloxy)androstane-3,17-
AUTHOR INFORMATION
(
■
*
dione, Cyclic 3,17-Bis(1,2-Ethanediyl acetal) (22d). The insep-
arable epimeric products 22b (130 mg, 0.33 mmol) were converted
into separable compounds 22c (35 mg, 22%) and 22d (80 mg, 50%)
using the procedure reported for the preparation of steroids 18c and
Corresponding Author
Phone: (314) 362-1726. Fax: (314) 362-7058. E-mail:
dcovey@wustl.edu.
1
8d from the inseparable 7α- and 7β-alcohols 18b. Data for 22c: mp
ACKNOWLEDGMENTS
1
41−143 °C; IR ν 2943, 2872, 1496, 1456, 1362, 1306, 1286, 1245
max
■
−1 1
cm ; H NMR δ 7.40−7.20 (m, 5H), 4.57 (d, J = 12.1 Hz), 4.33 (d, J
=
This work was supported by NIH Grant GM47969 (D.F.C.,
A.S.E., C.F.Z., J.H.S.) and the Bantly Foundation (C.F.Z).
A.S.E. is a consultant for Sage Therapeutics. D.F.C. and C.F.Z.
are members of the Scientific Advisory Board of Sage
Therapeutics and may receive income based on equity holdings
in Sage Therapeutics. Washington University may receive
income based on a license of related technology by the
University to Sage Therapeutics. Sage Therapeutics did not
support this work.
13
12.4 Hz), 3.96−3.78 (m, 8H), 3.45 (br s, 1H), 0.81 (s, 6H);
C
NMR δ 139.7, 128.1 (2C), 127.4 (2C), 127.0, 119.4, 109.2, 74.5, 70.4,
6
3
2
1
4
3
1
4
5.0, 64.5, 64.0 (2C), 45.9, 45.9, 44.1, 40.3, 37.6, 36.1, 35.7, 35.5, 34.2,
1.4, 31.2, 30.4, 22.3, 20.5, 14.0, 10.5. Anal. (C H O ) C, H. Data for
30
42
5
2d: mp 145−147 °C; IR ν 2943, 2871, 1586, 1454, 1361, 1307,
max
−1 1
205 cm ; H NMR δ 7.40−7.20 (m, 5H), 4.54 (d, 1H, J = 11 Hz),
.34 (d, 1H, J = 11 Hz), 3.96−3.80 (m, 8H), 3.15 (m, 1H), 0.86 (s,
H), 0.84 (s, 3H); 13C NMR δ 138.8, 128.3 (2C), 127.8 (2C), 127.7,
18.9, 109.1, 82.7, 70.4, 65.1, 64.5, 64.1 (2C), 52.4, 50.4, 46.6, 41.4,
0.8, 37.8, 35.9, 35.1, 34.3, 33.6, 31.1, 30.4, 25.4, 20.9, 14.7, 11.5. Anal.
(
C H O ) C, H.
30 42 5
ABBREVIATIONS USED
■
(
5β,7α,8α,9β,10α,13α,14β)-7-(Benzyloxy)androstane-3,17-
dione (23). Compound 22d (193 mg, 0.4 mmol) was converted into
compound 23 using the procedure reported for the preparation of
steroid 4. Flash column chromatography (silica gel eluted with 30−
4
1
7
3
1
4
GABA, γ-aminobutyric acid; GABA , γ-aminobutyric acid type
A
35
35
A; [ S]TBPS, [ S]-tert-butylbicyclophosphorothionate; SAR,
structure−activity relationship; LRR, loss of righting reflex;
LSR, loss of swimming reflex
0% EtOAc in hexanes) gave product 23 (140 mg, 89%): mp 183−
−1 1
85 °C; IR 2921, 2855, 1733, 1716, 1453, 1367 cm ; H NMR δ
.40−7.20 (m, 5H), 4.63 (d, 1H, J = 10.7 Hz), 4.40 (d, 1H, J = 11 Hz),
13
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3.7, 40.8, 38.2, 37.9, 36.0, 35.3, 33.8, 31.5, 24.9, 21.2, 14.1, 11.6. Anal.
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