Synthesis of 6-thia analogs of the natural neurosteroid allopregnanolone

Article abstract of DOI:10.1016/j.tet.2006.03.025

A procedure is described for the preparation of 6-thiapregnanes in five steps from pregnenolone via a 5-oxo-7-iodo-secopregnane intermediate. The 6-thiasteroid obtained was converted into 6-thia-allopregnanolone and its sulfoxide and sulfone derivatives.

Full text of DOI:10.1016/j.tet.2006.03.025

Tetrahedron 62 (2006) 4762–4768
Synthesis of 6-thia analogs of the natural neurosteroid
allopregnanolone
a
Fernando J. Duran, Alberto A. Ghini, Hector Coirini and Gerardo Burton
a
b
a,
*
´
a
´
Departamento de Quımica Organica and UMYMFOR (CONICET-FCEN), Facultad de Ciencias Exactas y Naturales, Universidad de
´
´
Buenos Aires, Pabellon 2, Ciudad Universitaria, C1428EGA Buenos Aires, Argentina
b
´
Instituto de Biologıa y Medicina Experimental (CONICET), Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina
Received 16 January 2006; revised 4 March 2006; accepted 8 March 2006
Available online 3 April 2006
Abstract—A procedure is described for the preparation of 6-thiapregnanes in five steps from pregnenolone via a 5-oxo-7-iodo-secopregnane
intermediate. The 6-thiasteroid obtained was converted into 6-thia-allopregnanolone and its sulfoxide and sulfone derivatives. The trans stereo-
chemistryattheA/Bringjunction wasaccomplishedbystereoselectivereductionofanintermediatehemithioketalwithtriethylsilane/BF3$Et2O.
The compounds synthesized are analogs of natural neurosteroids, and exhibited GABAA receptor activity comparable to allopregnanolone.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
than 1 in their in vitro activity on the GABAA receptor, sug-
gesting a significant involvement of the lipophilic character
of ring B in modulating ligand binding to neighboring sites.
A recent report on the activity of 6-aza-allopregnanolone
points to the same conclusion.7 The replacement of oxygen
by sulfur (other characteristics being equal) often generates
marked changes in the biological activity of a compound.8
These biological and physicochemical properties are mostly
attributed to the different electronic properties (electronega-
tivity) of the sulfur and oxygen atoms. The larger size of the
sulfur atom, and its more disperse electron density, result in
a higher lipophilicity and diminished hydrogen bond accep-
Neuroactive steroids are positive allosteric modulators of the
GABAA receptor that interact with a specific steroid recog-
nition site on the receptor–ion channel complex. They have
potential therapeutic interest as anticonvulsants, anesthetics
and for the treatment of several neurological and psychiatric
disorders.1 Endogenous neuroactive steroids (neurosteroids)
such as 3a-hydroxy-5a-pregnan-20-one (allopregnanolone,
1) and its 5b isomer (pregnanolone, 2), are rapidly biotrans-
formed when administered exogenously and several syn-
thetic analogs of these compounds with improved
bioavailability have been developed.2 An important advance
in this search was the finding that substituents conferring
water-solubility to these lipophilic steroids could be incor-
porated at a number of different positions of the steroid nu-
cleus, without loss of anesthetic activity.3 Analogs obtained
by isosteric replacement of carbon atoms of the steroid nu-
cleus by heteroatoms (e.g., O) exhibit localized changes in
hydrophobicity and hydrogen bonding acceptor capacity;
however, at variance with the presence of additional polar
substituents, these modifications do not produce major
changes in the overall conformation of the steroid nucleus
or increased steric bulk.
˚
tor capacity. Also, the C–S bond is longer (ca. 1.8 A) and the
C–S–C angle (ca. 95–100ꢀ) is smaller than the correspond-
ing oxygen counterparts, thus some changes in flexibility
and conformation may be expected. Additionally, the soft
sulfur atom can be selectively oxidized to the sulfoxide or
sulfone moieties, giving rise to new derivatives with differ-
ent steric bulk and dipolar moments stereospecifically di-
rected towards the a- and/or b-faces. With these guidelines
in mind, we decided to synthesize the 6-thia analog 6. To
our knowledge, only a few procedures for the synthesis of
6-thiasteroids have been reported,9 none of which corre-
spond to analogs of neuroactive steroids.
We have described a synthetic approach to 5a-H and 5b-H
6-oxapregnanes (3 and 4),4 analogs of the endogenous neu-
roactive steroids 1 and 2, via stereospecific cyclizations of
secosteroid 5.5,6 These compounds were 100-fold less active
Keywords: 6-Thiasteroids; S-Oxo-thiasteroids; S,S-Dioxo-thiasteroids;
Neurosteroid analogs; Allopregnanolone; GABAA receptor.
* Corresponding author. Tel./fax: +54 11 4576 3385; e-mail: burton@qo.
fcen.uba.ar
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.03.025

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4763
Stereospecific reduction of the hemithioketal moiety in 11 to
the 5a-H-cyclic thioether was accomplished in two steps by
protection of the hydroxyl group at C-19 as a formate, fol-
lowed by selective reduction of the hemithioketal moiety
with Et3SiH and BF3$Et2O in Cl2CH2. The attack of the hy-
dride on the thiocarbenium intermediate occurred, as ex-
pected, from the less hindered a-face with stereoselective
formation of the 5a-H-6-thiasteroid 12. The pyranosic thio-
carbenium ion preferently accepts nucleophiles from the
a (axial) side due to the anomeric effect of the sulfur
atom; this preference is magnified, relative to the tetrahydro-
pyran system, because 1,3-diaxial repulsions are smaller as
a result of the longer C–S bond.12 The silyl protecting group
was also cleaved in this step. The coupling pattern of the
3a-H resonance that appeared as a broad multiplet
(W1/2¼15.3 Hz) typical of an axial hydrogen, was consistent
with the a orientation of 5-H. Confirmation of that stereo-
chemistry came from the observation of a strong NOE corre-
lation between H-5 and H-3 in the NOESY spectrum of 12,
thus indicating the a orientation for H-5.
2. Results and discussion
2.1. Chemistry
By analogy with the approach used in the synthesis of the
oxa-analogs 3 and 4,5,6 we focused our attention on the
iodo substituent present in secosteroid 5. This could be re-
placed by sulfur nucleophiles, giving rise to thioketals that
may be transformed into the desired cyclic thioether. In
this case however, the acetate group at C-3 in the 5-oxo
secosteroid 5 proved to be too labile, giving elimination by-
products throughout the synthesis; thus a silylated derivative
was used.10 Secosteroid 9 was synthesized from commer-
cially available pregnenolone (7) in four steps (Scheme 1).
Thus, pregnenolone was transformed into the 3b-tert-butyl-
dimethylsilyloxy-5a,6b-diol 8 by epoxidation and cleavage
(hydrogen peroxide, formic acid) followed by deformylation
(methanolic base) to the 3b,5a,6b-trihydroxypregnane (one-
pot)11 and subsequent regioselective silylation of the less
hindered equatorial 3b-hydroxy moiety. Treatment of 8
with HgO/I2 (CCl4, hn) gave the precursor secosteroid 9 in
42% yield. The structure of 9 was confirmed by comparison
of its 1H and 13C NMR spectra with those of the acetylated
analog 5 previously synthesized by us.5
Inversion of the configuration at C-3, a requisite for agonistic
GABAA receptor activity,13 was accomplished in a straight-
forward way using the Mitsunobu reaction. Adequate choice
Scheme 1. Reagents and conditions: (a) i. HCO2H, then 30% H2O2; ii. 40% NaOH, MeOH; iii. TBDMSCl, imidazole, DMF; (b) HgO, I2, Cl2CH2–Cl4C, hn,
25 ꢀC, 4.5 h.
Reaction of 9 with potasium thioacetate gave the sulfur
substituted secosteroid 10 in 70% yield (Scheme 2). The
presence of the sulfur moiety was evident in the NMR and
mass spectra. Particularly diagnostic was the shift of the
7-CH2 hydrogen and carbon resonances (3.14/3.45 and
17.4 ppm for 9; 2.95/3.05 and 31.5 ppm for 10), consistent
with the change of iodine by thioacetyl at that position.
Treatment of 10 with base, simultaneously cleaved the
C-19 formate and the C-7 thioacetate, with concomitant cy-
clization of the intermediate 7-sulfanyl anion to give the
hemithioketal 11 as a 9:1 5a/5b mixture (78% yield).y Ab-
sence of the characteristic signal of a carbonyl group at
C-5 in the 13C NMR spectrum of 11 and the presence of a res-
onance at 81.9 ppm assigned to the hemithioketalic carbon
of the 5-a isomer, indicated the success of this transforma-
tion. Attempts to purify the diastereomeric mixture on silica
gel resulted in partial decomposition and isomerization of
the a-epimer into the b-epimer giving rise to a new diaste-
reomeric ratio of ca. 1:1.
of the acid for this reaction was mandatory, taking into ac-
count that the following step involved the regioselective de-
protection of the formate group at C-19. The 3a-benzoate in
13 proved to be adequate for this purpose. The success of the
Mitsunobu reaction was evident in the 1H NMR spectrum of
the reaction product that showed the equatorial orientation
of H-3 (W1/2¼7.5 Hz, at variance with the same hydrogen
in 12, vide supra). Aromatic hydrogens, belonging to the
incorporated benzoyl moiety, were also observed. The 19-
hydroxy derivative 13 was obtained by regioselective
deprotection of the 19-ester moiety with hydrochloric acid
in methanol; under these mild and controlled conditions,
the 3a-benzoate was not affected.
Deoxygenation of the primary hydroxyl group attached at
C-19 was then carried out using the Barton–Mc Combie pro-
cedure, by formation of the 19-imidazoylthionocarbonate
derivative and reduction with diphenylsilane to give 14;
attempts to obtain this compound free of unreacted diphenyl-
silane were unsuccessful and so it was used directly in the
next step. The low reactivity of the axial benzoate at C-3 re-
quired the use of strong base for its removal, which led to
partial isomerization of the side chain at C-17. Smooth de-
benzoylation was achieved under nucleophilic conditions
with sodium methylthiolate in DMF, to give 6-thia-allopreg-
nanolone 6 as single product (61% yield from 13).
1
y
Determined in the H NMR spectrum of the mixture, from the intensity
ratio of the 13-CH3 resonances for the a (0.68 ppm) and b (0.62 ppm) iso-
mers. The coupling pattern of the 3a-H resonance of the major isomer,
that appeared as a broad multiplet (W1/2¼22.3 Hz) typical of an axial
hydrogen, was consistent with the a orientation of the 5-hydroxyl.

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F. J. Duran et al. / Tetrahedron 62 (2006) 4762–4768
4764
Scheme 2. Reagents and conditions: (a) KSAc, acetone, 3 h, 25 ꢀC; (b) NaOH, MeOH, 3 h, 25 ꢀC; (c) i. formic acetic anhydride, py, 2 h, 25 ꢀC; ii. Et3SiH,
BF3$Et2O, Cl2CH2, 1 h, ꢁ15 ꢀC; (d) i. benzoic acid, Ph3P, DEAD, THF, 18 h, 25 ꢀC; ii. 6 N HCl, MeOH, 1 h, 25 ꢀC; (e) i. thiocarbonyldiimidazole,
DMAP, Cl2CH2, 5 h, reflux; ii. Ph2SiH2, AIBN, toluene, 4.5 h, 115 ꢀC; (f) NaSCH3, DMF, 2 h, 100 ꢀC.
Selective oxidation of 6 with potassium peroxymonosulfate
(OxoneÒ) gave either sulfoxide 15 or sulfone 16, depending
on the product–reagent ratio and temperature.14 Thus, treat-
ment with OxoneÒ (1.3 equiv) in methanol at 0 ꢀC gave sulf-
oxide derivative 15 as single product (87% yield) whereas
excess of the same reagent (3 equiv) at room temperature
gave sulfone 16 in 90% yield. Configuration of the sulfur
1
atom in the sulfoxide 15 was inferred from the H NMR
spectrum, considering the shift in the 10-CH3 resonance in
sulfone 16 compared to 6 (+0.2 ppm) due to the axial oxygen
on the sulfur. A negligible shift was observed in the transfor-
mation of 6 to the sulfoxide 15 (ca. +0.05 ppm) indicating
that the oxygen was equatorially oriented. This is the ex-
pected stereochemistry for 15 considering attack of the re-
agent from the sterically less demanding a-face.
Figure 1. Inhibition of binding of [35S]-tert-butylbicyclo-phosporothionate
([35S]-TBPS) to membranes from rat cerebellum by allopregnanolone
(1, B), 3a-hydroxy-6-thia-5a-pregnan-20-one (6-thia-allopregnanolone,
6, C), S-oxo-3a-hydroxy-6-thia-5a-pregnan-20-one (15, ,), and S,S-di-
oxo-3a-hydroxy-6-thia-5a-pregnan-20-one (16, -). Cerebellum mem-
brane preparations were incubated with 10 nM [35S]-TBPS in the absence
(100% binding) or presence of increasing concentrations of the steroids
(50–600 nM). Picrotoxin (2 mM) was used to determine non-specific bind-
ing. Assays were carried out at 22 ꢀC for 2 h in the presence of 5 mM GABA.
2.2. GABAA receptor activity
Calculated IC50 values are: 1, 92.8ꢂ14.1 nM; 6, 171.2ꢂ39.2 nM; 15,
241.1ꢂ97.0 nM; 16, 200.3ꢂ37.1 nM.
Biological activity of the three synthetic steroids was
assayed by in vitro tests using [35S]-tert-butylbicyclo[2,2,2]-
phosporothionate (TBPS), [3H]-flunitrazepam, and [3H]-
muscimol as radiolabelled ligands to g-aminobutyric acid
receptors (GABAA). Crude membrane receptors from male
rats’ cerebellum were used to test the capacity of the steroids
to displace the specific binding of the radioactive ligands.13
Allopregnanolone was used as a positive control to check the
viability of the methods. Preliminary results revealed that
the synthetic steroids show a displacement pattern similar
to that of allopregnanolone with an IC50 for [35S]-TBPS
binding in the 10ꢁ7 M range (Fig. 1). All three steroids
stimulated in a similar way the binding of [3H]-flunitraze-
pam and affected [3H]-muscimol binding as well, rendering
comparable results to allopregnanolone. Complete biologi-
cal results will be published in a separate paper.
3. Conclusions
The first 6-thia analog of a neurosteroid and its oxidized de-
rivatives 6-sulfoxide and 6-sulfone, were synthesized from

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F. J. Duran et al. / Tetrahedron 62 (2006) 4762–4768
4765
pregnenolone in 8.1, 7.0, and 7.3% yield, respectively. The
key reduction of the intermediate hemithioketal to give the
trans-fused A/B rings in the steroidal framework was accom-
plished stereospecifically, its stereochemical outcome being
consistent with the intermediacy of a pyranosic thiocarbe-
nium ion. Furthermore compound 13 is a synthetic precursor
of C-19 substituted analogs. GABAA receptor activity for 6-
thia-allopregnanolone (6) was similar to that of allopregna-
nolone (1) giving further support to our assumption that the
decrease in activity observed for the 6-oxa and 6-aza analogs
is related to their hydrogen bonding acceptor and/or donor
properties at position 6. The small change in activity ob-
served upon oxidation to the sulfoxide and sulfone analogs
(15 and 16) indicates that lipophylicity and electrostatic po-
tential changes in the vicinity of position 6 are not critical for
GABAA receptor activity.
and diluted with cold water (50 mL). 3b,5a,6b-Trihydroxy-
pregnan-20-one precipitated as a white solid after cooling; it
was collected, washed with cold water and dried under vac-
uum (3.34 g). The triol was dissolved in anhydrous DMF
(45 mL) under nitrogen, imidazole (1.94 g, 28.50 mmol)
and tert-butyldimethylsilyl chloride (2.87 g, 19.03 mmol)
were added at 0 ꢀC and the mixture was allowed to reach
25 ꢀC. After 2 h the solution was poured into saturated aque-
ous NaCl (60 mL), extracted with diethyl ether (3ꢃ20 mL),
dried with sodium sulfate and evaporated under vacuum.
The white solid obtained was purified by flash chromatogra-
phy on silica gel using hexane/ethyl acetate (90:10), to give
the title compound 8 (3.17 g, 72% from 7). Mp 202–203 ꢀC
(hexane/ethyl acetate) [found: C, 69.9, H, 10.5. C27H48O4Si
requires: C, 69.78, H, 10.41]; nmax 3479, 2937, 2860, 1697,
1466, 1359, 1251, 1077, 871, 834, 776; dH (500 MHz) 0.05
(6H, s, TBDMS–H), 0.63 (3H, s, 18-H), 0.87 (9H, s,
TBDMS–H), 1.18 (3H, s, 19-H), 2.11 (3H, s, 21-H), 2.53
(1H, t, J¼8.9 Hz, 17-H), 3.52 (1H, br s, 6-H), 4.07 (1H, m,
3-H); dC (50 MHz) ꢁ4.6 (CH3Si), ꢁ4.5 (CH3Si), 13.5 (C-
18), 16.8 (C-19), 18.1 (SiC(CH3)3), 21.1 (C-11), 22.8 (C-
16), 24.2 (C-15), 25.9 (C(CH3)3), 30.3 (C-8), 31.1 (C-2),
31.5 (C-21), 32.4 (C-1), 34.3 (C-7), 38.3 (C-10), 39.0 (C-
12), 41.3 (C-4), 44.3 (C-13), 45.7 (C-9), 56.1 (C-14), 63.7
(C-17), 68.1 (C-3), 75.9 (C-6), 76.1 (C-5), 209.6 (C-20);
m/z (EI) 465 (M+H, 0.1), 447 (M+HꢁH2O, 0.2), 389 (2),
315 (24), 297 (30), 279 (8).
4. Experimental
4.1. General experimental procedures
Melting points were determined on a Fisher Johns apparatus
and are uncorrected. 1H and 13C NMR spectra were recorded
in CDCl3 solutions in a Bruker AC-200 NMR spectrometer at
200.13 (1H) and 50.32 (13C) MHz or a Bruker AM-500 at
500.13 (1H) and 125.77 (13C) MHz. NMR assignments are
based on multiplicity determinations (DEPT) and 2D spectra
(COSY, HETCOR, HMBC, HSQC, and NOESY) are ob-
tained using standard Bruker software. Chemical shifts are
given in parts per million (d) downfield from TMS internal
standard. IR spectra were measured as thin films (from di-
chloromethane solution) on KBr disks, using a FT-IR Nicolet
Magna 550 spectrophotometer, wave-numbers are given in
cmꢁ1. The electron impact mass spectra were obtained at
70 eV by direct inlet in a Shimadzu QP 5000 mass spectro-
meter. Thin layer chromatography was performed on silica
gel 60 plates with fluorescent indicator. The plates were visu-
alized with a 3.5% solution of phosphomolybdic acid in etha-
nol. Column chromatography was conducted on silica gel
(230–400 mesh) or octadecyl-functionalized silica gel. All
4.1.2. 3b-tert-Butyldimethylsilyloxy-19-formyloxy-7-
iodo-6-nor-5,7-secopregnane-5,20-dione (9). To a solution
of diol 8 (1.00 g, 2.15 mmol) in dry dichloromethane
(85.0 mL) in a water-jacketed vessel, were added recently
distilled carbon tetrachloride (85.0 mL), mercury(II) oxide
(1.22 g, 4.73 mmol), and iodine (1.77 g, 7.00 mmol) under
nitrogen. The solution was vigorously stirred and irradiated
with two 300 W tungsten lamps (5000 lm each) at 25 ꢀC.
Three additional portions of mercury(II) oxide (1.22 g,
4.73 mmol) and iodine (1.77 g, 7.00 mmol) were added at
intervals of 1 h. After 4.5 h the solution was diluted with di-
chloromethane (85.0 mL) and filtered. The organic layer
was washed with a saturated solution of sodium thiosulfate
(60 mL) and water (60 mL), dried, and evaporated under re-
duced pressure. The oily residue was purified by flash chro-
matography on octadecyl-functionalized silica gel using
methanol/water (70:30) to give the title compound as
a foamy yellow solid (0.85 g, 65%). nmax 3488, 2950,
2887, 2858, 1726, 1700, 1467, 1360, 1179, 1052, 837,
778, 738, 700; dH (500 MHz) 0.03 (3H, s, TBDMS–H),
0.06 (3H, s, TBDMS–H), 0.69 (3H, s, 18-H), 0.86 (9H, s,
TBDMS–H), 0.90 (1H, m, 8-H), 1.13 (1H, m, 15b-H),
1.39 (1H, m, 14-H), 1.48 (1H, m, 12a-H), 1.69 (1H, m,
2b-H), 1.75 (2H, m, 15a-H and 16a-H), 1.81–1.88 (4H, m,
11b-H, 2a-H, 9-H and 11a-H), 2.04 (1H, m, 1a-H), 2.09
(1H, m, 12b-H), 2.11 (1H, m, 1b-H), 2.12 (3H, s, 21-H),
2.15 (1H, m, 16b-H), 2.41 (1H, dt, J¼13.9, 2.8 Hz, 4a-H),
2.57 (1H, t, J¼9.2 Hz, 17-H), 3.14 (1H, dd, J¼10.8,
2.8 Hz, 7a-H), 3.45 (1H, dd, J¼10.8, 1.8 Hz, 7b-H), 3.49
(1H, dd, J¼13.9, 3.5 Hz, 4b-H), 4.32 (1H, d, J¼11.8 Hz,
19a-H), 4.45 (1H, m, 3a-H), 4.45 (1H, d, J¼11.8 Hz, 19b-
H), 8.09 (1H, s, formate); 13C NMR (125 MHz) d: ꢁ5.0
(C–CH3Si), ꢁ4.9 (C–CH3Si), 14.0 (C-18), 17.4 (C-7), 17.9
(C–CSi), 22.4 (C-16), 23.3 (C-11), 23.6 (C-15), 25.6 (C–
CH3), 27.4 (C-1), 28.8 (C-2), 31.3 (C-21), 37.6 (C-8), 38.6
˚
solvents were distilled and stored over 4 A molecular sieves
before use. Solvents were evaporated at 45 ꢀC under vacuum.
High-resolution mass spectra were measured at the Mass
Spectrometry Facility of the Chemistry Department, Univer-
sity of California at Riverside. Pregnenolone was purchased
from Steraloids Inc. (USA). All new compounds were deter-
mined to be >95% pure by 1H NMR spectroscopy.
4.1.1. 3b-tert-Butyldimethylsilyloxy-5a,6b-dihydroxy-
pregnan-20-one (8). A suspension of pregnenolone (7,
3.0 g, 9.48 mmol) in 88% formic acid (33 mL) was heated
to 70–80 ꢀC with stirring for 5 min and then cooled to
25 ꢀC. The resulting thick slurry containing the 3-formate,
was treated with 30% hydrogen peroxide (3.4 mL) keeping
the temperature at 40 ꢀC by cooling. After 20 min a pink so-
lution resulted and stirring was continued at ca. 20 ꢀC for
20 h. The reaction mixture was treated with boiling water
(50 mL) with stirring, allowed to cool and the white solid
was collected and dried. The solid was dissolved in methanol
(100 mL) and the solution was treated with 40% sodium hy-
droxide (5.5 mL) at 0 ꢀC and allowed to reach 25 ꢀC. After
45 min the solution was neutralized with 2 N HCl (27 mL)

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F. J. Duran et al. / Tetrahedron 62 (2006) 4762–4768
4766
(C-12), 39.6 (C-9), 43.1 (C-13), 49.1 (C-4), 53.7 (C-10), 55.4
(C-14), 63.2 (C-17), 65.2 (C-19), 70.4 (C-3), 160.8 (C-for-
mate), 208.8 (C-20), 212.9 (C-5); m/z (FAB) 627 (M+Na+,
11), 477 (9), 461 (100), 329 (40); HRMS (FAB) found
627.1980, C27H45IO5SiNa requires 627.1979.
To a solution of the hemithioketal 11 obtained above
(0.318 g, 0.659 mmol) in dry pyridine (14.2 mL) was added
recently prepared formic acetic anhydride15 (8.25 mL) at
25 ꢀC under nitrogen. After 2 h the solution was poured
into cold 2 N HCl (50 mL), extracted with dichloromethane
(3ꢃ15 mL), dried with sodium sulfate, and the solvent was
evaporated. The residue was dissolved in dry dichloro-
methane (50 mL) and triethylsilane (1.05 mL, 6.6 mmol)
and BF3$Et2O (0.85 mL, 6.6 mmol) were added at ꢁ15 ꢀC
under nitrogen. After 1 h, cold water (20 mL) was added fol-
lowed by solid sodium bicarbonate until neutral. The solu-
tion was washed with saturated sodium bicarbonate
(20 mL) and brine (20 mL), dried with sodium sulfate, and
the solvent was evaporated. Purification by column chroma-
tography on silica gel with a gradient of hexane/ethyl acetate
gave the 6-thiapregnane 12 (0.157 g, 64% from 10). Mp
161–162 ꢀC (hexane/ethyl acetate); nmax 3407, 2937, 2869,
1715, 1360, 1174, 1060; dH (500 MHz) 0.64 (3H, s, 18-H),
0.89 (1H, td, J¼11.4, 3.7 Hz, 9-H), 0.98 (1H, td, J¼13.9,
3.6 Hz, 1a-H), 1.18 (1H, m, 14-H), 1.26–1.33 (2H, m,
15b-H and 12a-H), 1.38 (1H, m, 2b-H), 1.50 (1H, m, 11b-
H), 1.55 (1H, m, 4a-H), 1.67–1.70 (2H, m, 16a-H and
15a-H), 1.77 (1H, ddd, J¼13.9, 7.0, 3.7 Hz, 11a-H), 1.86–
1.88 (1H, m, 8-H and 2a-H), 2.00–2.02 (1H, m, 4b-H and
12b-H), 2.10 (3H, s, 21-H), 2.19 (1H, m, 16b-H), 2.33
(1H, dt, J¼14.0, 3.5 Hz, 1b-H), 2.41 (1H, dd, J¼13.2,
11.3 Hz, 7a-H), 2.49 (1H, t, J¼9.0 Hz, 17-H), 2.55 (1H,
dd, J¼13.2, 3.6 Hz, 7b-H), 2.72 (1H, dd, J¼13.4, 3.6 Hz,
5a-H), 3.69 (1H, m, 3a-H), 4.48 (1H, d, J¼12.8 Hz, 19a-
H), 4.75 (1H, d, J¼12.8 Hz, 19b-H), 8.12 (1H, s, formate);
dC (125 MHz) 13.4 (C-18), 22.3 (C-11), 22.7 (C-16), 24.3
(C-15), 31.2 (C-1), 31.3 (C-2), 31.3 (C-21), 34.5 (C-7),
37.3 (C-4), 37.3 (C-8), 38.5 (C-10), 39.1 (C-12), 44.0 (C-
13), 48.8 (C-5), 54.5 (C-9), 55.8 (C-14), 62.4 (C-19), 63.5
(C-17), 69.9 (C-3), 160.9 (formate), 208.9 (C-20); m/z (EI)
380 (M+, 33), 335 (MꢁHCOOH, 24), 321 (4), 251 (8), 93
(55), 79 (82); HRMS (EI) found 380.2035, C21H32O4S re-
quires 380.2021.
4.1.3. 3b-tert-Butyldimethylsilyloxy-19-formyloxy-7-
acetylsulfanyl-6-nor-5,7-seco-pregnane-5,20-dione (10).
A mixture of secosteroid 7 (0.65 g, 1.08 mmol) and potas-
sium thioacetate (0.37 g, 3.23 mmol) in dry acetone
(33 mL) was stirred at room temperature for 3 h under nitro-
gen. The reaction mixture was diluted with dichloromethane
(70 mL), filtered and the solvent was evaporated. The residue
was purified by flash chromatography on silica gel with an
hexane/ethyl acetate gradient to give thioacetate 10 (0.42 g,
70%) as a vitreous solid; nmax 2955, 2932, 2894, 2857,
1725, 1700, 1464, 1357, 1174, 1128, 1041, 835; dH
(500 MHz) 0.03 (3H, s, TBDMS–H), 0.05 (3H, s,
TBDMS–H), 0.65 (3H, s, 18-H), 0.85 (9H, s, TBDMS–H),
1.23 (1H, m, 15b-H), 1.41-1.43 (2H, m, 14-H and 12a-H),
1.62 (1H, m, 15a-H), 1.67–1.69 (3H, m, 2b-H, 16a-H and
11b-H), 1.81 (1H, m, 11a-H), 1.88–1.91 (2H, m, 9-H and
2a-H), 1.98–1.99 (2H, m, 8-H and 1a-H), 2.08 (2H, m, 1b-
H and 12b-H), 2.11 (3H, s, 21-H), 2.12 (1H, m, 16b-H),
2.30 (3H, s, CH3COS), 2.36 (1H, dt, J¼13.8, 2.8 Hz, 4a-
H), 2.50 (1H, t, J¼9.2 Hz, 17-H), 2.95 (1H, dd, J¼13.9,
2.8 Hz, 7a-H), 3.05 (1H, dd, J¼13.9, 3.0 Hz, 7b-H), 3.14
(1H, dd, J¼13.8, 3.5 Hz, 4b-H), 4.36 (1H, d, J¼11.8 Hz,
19a-H), 4.43 (1H, m, 3a-H), 4.48 (1H, d, J¼11.8 Hz, 19b-
H), 8.08 (1H, s, formate). dC (125 MHz) ꢁ5.0 (CH3Si),
ꢁ4.9 (CH3Si), 13.1 (C-18), 17.9 (SiC(CH3)3), 22.6 (C-16),
23.7 (C-11), 24.4 (C-15), 25.6 (C(CH3)3), 27.5 (C-1), 28.8
(C-2), 30.9 (CH3COS), 31.2 (C-21), 31.5 (C-7), 36.7 (C-8),
38.8 (C-12), 41.1 (C-9), 43.5 (C-13), 48.0 (C-4), 53.5
(C-14), 54.0 (C-10), 63.4 (C-17), 65.6 (C-19), 70.4 (C-3),
160.8 (formate), 193.8 (CH3COS), 208.8 (C-20), 211.9 (C-
5); m/z (FAB) 575 (M+Na+, 100), 494 (30), 493 (83), 441
(9), 419 (35), 361 (26); HRMS (FAB) found 575.2810,
C29H48O6SSiNa requires 575.2839.
4.1.5. 3a-Benzoyloxy-19-hydroxy-6-thia-5a-pregnan-20-
one (13). To a solution of 6-thiapregnane 12 (0.125 g,
0.328 mmol) in dry THF (5.0 mL), were added triphenyl-
phosphine (0.258 g, 0.984 mmol), benzoic acid (0.094 g,
0.770 mmol), and DEAD (0.090 mL, 0.659 mmol) at
25 ꢀC under nitrogen. The mixture was stirred for 18 h and
the THF was evaporated under vacuum. The residue was pu-
rified by column chromatography on silica gel (cyclohexane/
ethyl acetate), dissolved in methanol (26 mL) and 6 N HCl
(5.9 mL, 35.8 mmol) added at 0 ꢀC. The solution was al-
lowed to reach 25 ꢀC under nitrogen, after 1 h a saturated so-
lution of potassium bicarbonate was added until neutral, the
methanol was evaporated to a fifth of the original volume
and the mixture was poured into dichloromethane (30 mL),
washed with brine (1 mL), dried with sodium sulfate, and
the solvent was evaporated. The light yellow solid was puri-
fied by column chromatography on silica gel using a gradient
of hexane/ethyl acetate, to give the title compound 13
(0.140 g, 93%) as a white solid. Mp 86–88 ꢀC (hexane/ethyl
acetate) [found: C, 70.8, H, 8.2. C27H36O4S requires: C,
71.02, H, 7.95]; nmax 3496, 2945, 2873, 1707, 1449, 1353,
1112, 715; dH (500 MHz) 0.72 (3H, s, 18-H), 0.99 (1H,
td, J¼11.4, 3.7 Hz, 9-H), 1.21 (1H, m, 14-H), 1.28–1.35
(2H, m, 15b-H and 1a-H), 1.43 (1H, td, J¼12.8, 4.1 Hz,
4.1.4. 3b-Hydroxy-19-formyloxy-6-thia-5a-pregnan-
20-one (12). To a solution of thioacetate 10 (0.40 g,
0.72 mmol) in methanol (40.0 mL) was added 10% aqueous
sodium hydroxide (10 mL) at 0 ꢀC under nitrogen. The reac-
tion mixture was warmed to 25 ꢀC; after 3 h the solution was
neutralized with 1 N hydrochloric acid (20 mL) and evapo-
rated under vacuum to a fifth of its original volume. The
remaining solution was diluted with dichloromethane
(50 mL), washed with brine (20 mL), and evaporated to dry-
ness. Purification on a silica gel column using a gradient of
hexane/ethyl acetate gave hemithioketal 11 (5a/5b 9:1;
0.318 g, 91%). Data for the 5a-hydroxy isomer: dH
(200 MHz) 0.05 (6H, s, TBDMS–H), 0.68 (3H, s, 18-H),
0.87 (9H, s, TBDMS–H), 2.11 (3H, s, 21-H), 2.35 (1H, dd,
J¼12.8, 4.0 Hz, 7a-H), 2.53 (1H, t, J¼8.8 Hz, 17-H), 2.84
(1H, t, J¼12.8 Hz, 7b-H), 3.71 (1H, d, J¼12.6 Hz, 19a-H),
4.10 (1H, m, 3a-H), 4.25 (1H, d, J¼12.6 Hz, 19b-H); dC
(50 MHz) ꢁ4.7 and ꢁ4.6 (C–CH3Si), 13.8 (C-18), 18.1
(SiC(CH3)3), 22.1 (C-16), 22.1 (C-11), 22.4 (C-15), 25.8
(C(CH3)3), 28.1 (C-2), 29.1 (C-1), 31.4 (C-21), 31.6 (C-7),
37.1 (C-8), 39.4 (C-12), 43.5 (C-13), 44.5 (C-10), 45.4 (C-
9), 45.8 (C-4), 56.2 (C-14), 63.6 (C-17), 64.1 (C-19), 67.0
(C-3), 81.9 (C-5), 209.2 (C-20).

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F. J. Duran et al. / Tetrahedron 62 (2006) 4762–4768
4767
12a-H), 1.67–1.72 (3H, m, 16a-H, 15a-H and 11b-H), 1.79–
1.82 (2H, m, 2b-H and 11a-H), 1.93 (2H, m, 1b-H and
2a-H), 2.06–2.07 (2H, m, 4b-H and 12b-H), 2.11 (1H, m,
4a-H), 2.12 (3H, s, 21-H), 2.20 (1H, m, 16b-H), 2.28 (1H,
qd, J¼10.9, 3.8 Hz, 8-H), 2.50 (1H, dd, J¼13.1, 11.4 Hz,
7a-H), 2.52 (1H, t, J¼9.2 Hz, 17-H), 2.61 (1H, dd,
J¼13.1, 3.8 Hz, 7b-H), 3.16 (1H, dd, J¼13.1, 3.8 Hz, 5a-
H), 3.83 (1H, dd, J¼12.2, 7.0 Hz, 19a-H), 4.34 (1H, d,
J¼12.2 Hz, 19b-H), 5.35 (1H, m, 3b-H), 7.46 (2H, m,
meta-ArH), 7.59 (1H, m, para-ArH), 8.06 (2H, m, ortho-
ArH); dC (50 MHz) 13.6 (C-18), 21.6 (C-11), 22.5 (C-16),
24.0 (C-15), 26.3 (C-2), 29.3 (C-1), 31.3 (C-21), 32.1
(C-4), 34.2 (C-7), 38.1 (C-8), 39.0 (C-12), 39.5 (C-10),
44.2 (C-13), 45.8 (C-5), 54.7 (C-9), 56.3 (C-14), 62.5
(C-19), 63.4 (C-17), 69.5 (C-3), 128.3 (meta-phenyl), 129.4
(ortho-phenyl), 130.5 (ipso-phenyl), 132.9 (para-phenyl),
165.5 (PhCOO), 209.2 (C-20); m/z (EI) 456 (M+, 10), 334
(61), 316 (5), 316 (5), 304 (25), 303 (24), 105 (41).
19-H), 1.17 (1H, m, 14-H), 1.25 (1H, m, 15b-H), 1.33 (1H, m,
11b-H), 1.40 (2H, m, 1a-H and 12a-H), 1.64–1.65 (3H, m,
1b-H, 4a-H and 4b-H), 1.66–1.67 (1H, m, 16a-H and 2a-
H), 1.70–1.74 (4H, m, 15a-H, 2b-H, 11a-H and 8-H), 2.06
(1H, br d, J¼11.6 Hz, 12b-H), 2.11 (3H, s, 21-H), 2.17
(1H, m, 16b-H), 2.45 (1H, dd, J¼13.1, 11.2 Hz, 7a-H),
2.55 (1H, dd, J¼13.1, 3.8 Hz, 7b-H), 2.56 (1H, t,
J¼9.3 Hz, 17-H), 3.12 (1H, dd, J¼10.4, 6.7 Hz, 5a-H),
4.08 (1H, m, 3b-H); dC (125 MHz) 11.4 (C-19), 13.2 (C-
18), 21.0 (C-11), 22.7 (C-16), 24.3 (C-15), 28.5 (C-2), 31.0
(C-1), 31.4 (C-21), 34.3 (C-7), 34.9 (C-4), 36.9 (C-8), 37.1
(C-10), 38.8 (C-12), 44.0 (C-13), 45.5 (C-5), 54.1 (C-9),
56.7 (C-14), 63.6 (C-17), 65.8 (C-3), 209.2 (C-20); m/z (EI)
336 (M+, 5), 318 (MꢁH2O, 10), 303 (2), 251 (2), 207 (1);
HRMS (EI) found 336.2131, C20H32O2S requires 336.2123.
4.1.7. S-Oxo-3a-hydroxy-6-thia-5a-pregnan-20-one (15).
To a solution of 6-thiapregnane 6 (0.009 g, 0.027 mmol) in
methanol (1.0 mL) cooled to 0 ꢀC, was added a suspension
of OxoneÒ (0.011 g, 0.018 mmol) in water (0.8 mL). After
5 min, a saturated solution of sodium bisulfite (1.0 mL)
was added, the methanol was evaporated and the resulting
mixture was extracted with ethyl ether (10 mL). The residue
obtained after evaporation of the solvent was purified by pre-
parative TLC (dichloromethane/methanol 20:1) to give sulf-
oxide 15 (0.0082 g, 87%). Mp 182–183 ꢀC (hexane/ethyl
acetate); nmax 3382, 2940, 2863, 1701, 1429, 1359, 1014,
756; dH (500 MHz) 0.64 (3H, s, 18-H), 0.95 (3H, s, 19-H),
1.15 (1H, td, J¼11.4, 4.1 Hz, 9-H), 1.30 (1H, m, 11b-H),
1.31 (1H, m, 15b-H), 1.39 (1H, m, 14-H), 1.42 (1H, m,
12a-H), 1.50 (1H, m, 1a-H), 1.57 (1H, dd, J¼13.1, 4.0 Hz,
1b-H), 1.64 (1H, m, 2b-H), 1.72–1.74 (4H, m, 2a-H, 11a-
H, 16a-H and 15a-H), 1.78–1.79 (1H, m, 4b-H and 8-H),
2.05 (1H, br d, J¼12.2 Hz, 12b-H), 2.12 (3H, s, 21-H),
2.19 (1H, m, 16b-H), 2.35 (1H, t, J¼12.2 Hz, 7a-H), 2.36
(1H, m, 4a-H), 2.55 (1H, t, J¼8.9 Hz, 17-H), 2.89 (1H,
dd, J¼13.2, 3.7 Hz, 5a-H), 3.41 (1H, dd, J¼11.6, 2.8 Hz,
7b-H), 4.23 (1H, m, 3b-H); dC (125 MHz) 13.1 (C-19),
13.2 (C-18), 20.7 (C-11), 22.7 (C-16), 24.3 (C-15), 27.6
(C-2), 29.1 (C-4), 31.4 (C-21), 32.0 (C-1), 33.5 (C-8), 38.3
(C-12), 38.9 (C-10), 43.9 (C-13), 53.4 (C-9), 55.4 (C-14),
56.4 (C-7), 63.3 (C-17), 64.3 (C-3), 64.7 (C-5), 208.7
(C-20); MS (EI) m/z (%): 352 (M+, 0.5), 318 (1), 298 (1),
173 (4), 121 (13); HRMS (EI) found 352.2070, C20H32O3S
requires 352.2072.
4.1.6. 3a-Hydroxy-6-thia-5a-pregnan-20-one (6). To a
solution of 3a-benzoate 13 (0.127 g, 0.279 mmol) in dry
dichloromethane (6.4 mL), were added thiocarbonyldi-
imidazole (0.253 g, 1.40 mmol) and 4-dimethylaminopyri-
dine (0.002 g, 0.015 mmol) and the solution was refluxed
under nitrogen for 5 h. The reaction mixture was evaporated
to dryness and purified by column chromatography on silica
gel with hexane/ethyl acetate (8:2) to give the intermediate
19-imidazoylthionocarbonate as a yellow solid (0.136 g).
The solid was dissolved in anhydrous toluene (6.6 mL)
and heated to 115 ꢀC under nitrogen, diphenylsilane
(0.265 mL, 1.450 mmol) was added followed by 18 aliquots
(0.05 mL each) of a solution of AIBN in anhydrous toluene
(0.158 g/mL, 1.8 equiv) at 15 min intervals. The solvent was
evaporated and the residue was purified by column chroma-
tography on silica gel with hexane/ethyl acetate as eluent to
give an oily fraction of 14 containing residual diphenylsilane
that could not be separated; dH (500 MHz) 0.65 (3H, s, 18-
H), 1.09 (3H, s, 19-H), 2.11 (3H, s, 21-H), 2.44 (1H, dd,
J¼13.2, 12.2 Hz, 7a-H), 2.51 (1H, t, J¼9.3 Hz, 17-H),
2.53 (1H, dd, J¼13.2, 3.8 Hz, 7b-H), 3.09 (1H, dd,
J¼13.4, 3.5 Hz, 5a-H), 5.29 (1H, m, 3b-H), 7.47 (2H, m,
meta-ArH), 7.58 (1H, m, para-ArH), 8.06 (2H, m, ortho-
ArH); dC (125 MHz) 11.5 (C-19), 13.2 (C-18), 21.0 (C-
11), 22.7 (C-16), 24.2 (C-15), 25.9 (C-2), 31.3 (C-21),
31.9 (C-1), 31.9 (C-4), 34.3 (C-7), 36.7 (C-8), 36.9 (C-10),
38.8 (C-12), 43.9 (C-13), 46.6 (C-5), 54.1 (C-9), 55.5
(C-14), 63.5 (C-17), 69.7 (C-3), 128.3 (meta-phenyl),
129.5 (ortho-phenyl), 130.7 (ipso-phenyl), 132.8 (para-phe-
nyl), 165.5 (PhCOO), 209.0 (C-20).
4.1.8. S,S-Dioxo-3a-hydroxy-6-thia-5a-pregnan-20-one
(16). To a solution of 6-thiapregnane 6 (0.0116 g,
0.0416 mmol) in methanol (1.3 mL) cooled to 0 ꢀC, was
added a suspension of OxoneÒ (0.038 g, 0.062 mmol) in
water (1.0 mL). The reaction mixture was allowed to reach
25 ꢀC and after 5 h a saturated solution of sodium bisulfite
(1.3 mL) was added, the methanol was evaporated, and the
residue was extracted with ethyl ether (10 mL). The residue
obtained after evaporation of the solvent was purified by pre-
parative TLC (dichloromethane/methanol 20:1) to give the
sulfone 16 (0.0114 g, 90%). Mp 187–188 ꢀC (hexane/ethyl
acetate); nmax 3491, 2947, 2865, 1699, 1289, 1131, 1012,
903; dH (500 MHz) 0.67 (3H, s, 18-H), 1.15 (1H, m, 9-H),
1.16 (3H, s, 19-H), 1.31–1.32 (2H, m, 14-H and 15b-H),
1.43 (2H, m, 11b-H and 12a-H), 1.53 (1H, m, 1a-H), 1.59
(1H, m, 1b-H), 1.67 (1H, m, 15b-H), 1.70 (2H, m, 2a-H
and 2b-H), 1.73 (1H, m, 16a-H), 1.77 (1H, m, 11a-H),
To a solution of the crude fraction obtained above containing
14, in dry DMF (5.0 mL), was added sodium methanethiolate
(0.254 g, 3.63 mmol) and the mixture was heated for 2 h at
100 ꢀC under nitrogen. The resulting solution was cooled,
poured into brine (10 mL), and extracted with ethyl ether
(30 mL). The organic layer was washed with brine
(2ꢃ10 mL), dried with sodium sulfate, and the solvent was
evaporated. The yellowish solid was purified by column
chromatography on silica gel using hexane/ethyl acetate
(8:2 to 1:1) to give the title compound 6 as a white solid
(0.057 g, 61% from 13). Mp 174–175 ꢀC (hexane/ethyl ace-
tate); nmax 3430, 2939, 2875, 1696, 1426, 1359, 1010, 752; dH
(500 MHz) 0.63 (3H, s, 18-H), 0.90 (1H, m, 9-H), 1.03 (3H, s,

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F. J. Duran et al. / Tetrahedron 62 (2006) 4762–4768
4768
1.93 (1H, m, 4b-H), 2.07 (1H, m, 12b-H), 2.12 (3H, s, 21-H),
2.20 (2H, m, 8-H and 16b-H), 2.24 (1H, m, 4a-H), 2.54 (1H,
t, J¼9.2 Hz, 17-H), 2.66 (1H, t, J¼13.9 Hz, 7a-H), 3.08 (1H,
dd, J¼13.9, 3.4 Hz, 7b-H), 3.24 (1H, dd, J¼12.9, 2.6 Hz,
5a-H), 4.28 (1H, m, 3b-H); dC (125 MHz) 12.2 (C-19),
13.1 (C-18), 20.8 (C-11), 22.6 (C-16), 23.9 (C-4), 24.1 (C-
15), 27.7 (C-2), 31.4 (C-21), 32.9 (C-1), 34.2 (C-8), 38.2
(C-12), 39.4 (C-10), 43.8 (C-13), 52.5 (C-9), 54.8 (C-14),
56.2 (C-7), 61.2 (C-5), 63.2 (C-17), 64.2 (C-3), 208.5 (C-
20); m/z (EI) 368 (M+, 0.3), 350 (MꢁH2O, 0.3), 298 (1),
207 (8), 121 (4), 44 (100); HRMS (EI) found 368.2016,
C20H32O4S requires 368.2021.
GABA and terminated by rapid filtration through a glass
fiber filter as above.
4.2.4. [3H]-Muscimol ([3H]-Mus) binding. Aliquots
(100 mL) of washed cerebellum membrane preparation in
Tris–acetate 50 mM buffer pH 6.1 were incubated with
10 nM [3H]-Mus (18.0 Ci/mmol, Perkin Elmer Life Science
Inc., Boston, MA) in the absence or presence of increasing
concentrations of the steroids (50–600 nM); 1 mM GABA
(Sigma–Aldrich Corp.) was used to determine non-specific
binding. Incubations were carried out at 4 ꢀC for 60 min in
the absence of GABA and terminated by rapid filtration
through glass fiber filter as above.
4.2. Biological activity assays
Acknowledgments
4.2.1. Membrane preparation. Whole cerebellum from
male Sprague–Dawley rats (200–250 g) was rapidly re-
moved after sacrifice and stored at ꢁ80 ꢀC. The material
was thawed and homogenized in 5 vol (v/w) of ice-cold
0.32 M sucrose, using a Teflon-glass homogenizer at
1200 rpm. The homogenate was centrifuged at 1000g for
10 min at 4 ꢀC. The supernatant was carefully decanted
and centrifuged for 20 min at 15,000g at 4 ꢀC. The pellet
was washed twice with 50 mM Tris–HCl buffer (pH 7.4) fol-
lowed by centrifugation for 20 min at 15,000g at 4 ꢀC. The
final pellet was suspended in 1.2 mL of the same buffer
and frozen at ꢁ20 ꢀC. On the days of the assays, membranes
were thawed, centrifuged for 20 min at 15,000g at 4 ꢀC, and
the pellet was washed twice with 100 vol of the correspond-
ing ice-cold buffer by centrifugation (15,000g, 20 min). The
final pellet was suspended in the incubation buffer to a pro-
tein concentration of approximately 8 mg/mL.16
This work was supported by grants from Agencia Nacional
´
de Promocion Cientıfica y Tecnologica (PICT 10962),
CONICET (Argentina) and Universidad de Buenos Aires.
´
´
References and notes
1. Gasior, M.; Carter, R. B.; Witkin, J. M. Trends Pharmacol. Sci.
1999, 20, 107–112.
2. Hamilton, N. M. Curr. Top. Med. Chem. 2002, 2, 887–902.
3. Beekman, M.; Ungard, J. T.; Gasior, M.; Carter, R. B.; Dijkstra,
D.; Goldberg, S. R.; Witkin, J. M. J. Pharmacol. Exp. Ther.
1998, 284, 868–877.
4. Nicoletti, D.; Ghini, A. A.; Furtmuller, R.; Sieghart, W.; Dodd,
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R. H.; Burton, G. Steroids 2000, 65, 349–356 and references
cited therein.
5. Nicoletti, D.; Ghini, A. A.; Brachet-Cota, A. L.; Burton, G.
J. Chem. Soc., Perkin Trans. 1 1995, 1089–1093.
6. Nicoletti, D.; Ghini, A. A.; Burton, G. J. Org. Chem. 1996, 61,
6673–6677.
7. Kasal, A.; Matyas, L.; Budesinsky, M. Tetrahedron 2005, 61,
2269–2278.
8. See for example Witczak, Z. J. Curr. Med. Chem. 1999, 6, 165–
178.
9. Suginome, H.; Yamada, S.; Wang, J. B. J. Organomet. Chem.
1990, 55, 2170–2176; Speckamp, W. N.; Kesselaar, H. Tetra-
hedron Lett. 1974, 38, 3405–3408.
10. Back, T. G.; Baron, D. L.; Morzycki, J. W. Heterocycles 1994,
38, 1053–1060.
11. Fieser, L. F.; Rajagopalan, S. J. Organomet. Chem. 1949, 71,
3938–3941.
12. See for example Kirbi, A. J. The Anomeric Effect and Related
StereoelectronicEffectsatOxygen; Springer: Berlin, 1983; p 23.
13. Gee, K. W.; Bolger, M. B.; Brinton, R. E.; Coirini, H.;
McEwen, B. S. J. Pharmacol. Exp. Ther. 1988, 246, 803–812.
14. Trost, B. M.; Curran, D. P. Tetrahedron Lett. 1981, 22, 1287–
1291.
4.2.2. [35S]-tert-Butylbicyclo-phosporothionate ([35S]-
TBPS) binding. Binding assays were carried out using a
previously described protocol with some modifications.13
Aliquots (100 mL) of cerebellum membrane preparation
were incubated with 10 nM [35S]-TBPS (65.13 Ci/mmol,
Perking Elmer Life Science Inc., Boston, MA) in the ab-
sence or presence of increasing concentration of the steroids
(50–600 nM). The synthetic steroids and allopregnanolone,
used as control, were dissolved in DMSO and diluted with
the incubation buffer (1:1000; 50 mM Tris–HCl, 200 mM
NaCl, pH 7.4) immediately before use; 2 mM picrotoxin
was used to determine non-specific binding. Assays were
carried out at 22 ꢀC for 2 h in the presence of 5 mM GABA
(Sigma–Aldrich Corp.) and terminated by rapid filtration
through a glass fiber filter (Number 30, Schleicher & Schuell
Inc., Keene, NH). Filter bound radioactivity was quantified
by liquid scintillation spectrophotometry. IC50 (concentra-
tion at which half-maximal inhibition of control binding
occurs) values were determined by linear computerized
regression analysis after logit/log transformation.17
15. Zemlicka, J.; Beranek, J.; Smrt, J. Collect. Czech. Chem.
Commun. 1962, 27, 2784–2795.
4.2.3. [3H]-Flunitrazepam ([3H]-FLU) binding. Aliquots
(100 mL) of cerebellum membrane preparation were incu-
bated with 3 nM [3H]-FLU (85.2 Ci/mmol, Perking Elmer
Life Science Inc., Boston, MA) in the absence or presence
of increasing concentration of the steroids (50–600 nM). Al-
lopregnanolone, was used as a standard and 1 mM diazepam
(Roemmers Lab, Buenos Aires) was used as non-specific
binding.18 Incubations were carried out at 4 ꢀC for 90 min
in 50 mM Tris–HCl buffer (pH 7.4) in the absence of
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17. Rodbard, D.; Lewalds, J. E. Computer Analysis of Radioligand
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Research Methods in Reproductive Endocrinology. Karolinska
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18. Gonzalez, S. L.; Ferrini, M.; Coirini, H.; Gonzalez-Deniselle,
M. C.; De Nicola, A. F. Brain Res. 1992, 589, 97–101.