Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues of allopregnanolone

Article abstract of DOI:10.1016/j.bmc.2009.08.008

The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the β-face of ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy. The methodology also allows the synthesis of 3

Full text of DOI:10.1016/j.bmc.2009.08.008

Bioorganic & Medicinal Chemistry 17 (2009) 6526–6533
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and GABAA receptor activity of 2,19-sulfamoyl analogues
of allopregnanolone
Fernando J. Durán a, Valeria C. Edelsztein a, Alberto A. Ghini a, Mariana Rey b, Héctor Coirini b,
Philippe Dauban c, Robert H. Dodd c, Gerardo Burton a,
*
a Departamento de Química Orgánica and UMYMFOR (CONICET-UBA), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2,
Ciudad Universitaria, C1428EHA Buenos Aires, Argentina
b Instituto de Biología y Medicina Experimental (CONICET), Vuelta de Obligado 2490, C1428ADN Buenos Aires, Argentina
c Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, 91198 Gif-sur-Yvette, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of new analogues of allopregnanolone with a bridged sulfamidate ring over the b-face of
Received 23 May 2009
Revised 1 August 2009
Accepted 5 August 2009
Available online 9 August 2009
ring A has been achieved from easily available precursors, using an intramolecular aziridination strategy.
The methodology also allows the synthesis of 3
a-substituted analogues such as the 3a-fluoro derivative.
GABAA receptor activity of the synthetic analogues was evaluated by assaying their effect on the binding
of [3H]flunitrazepam and [3H]muscimol. The 3
a-hydroxy-2,19-sulfamoyl analogue and its N-benzyl
derivative were more active than allopregnanolone for stimulating binding of [3H]flunitrazepam. For
the binding of [3H]muscimol, both synthetic analogues and allopregnanolone stimulated binding to a
Keywords:
Steroids
Neurosteroids
similar extent, with the N-benzyl derivative exhibiting a higher EC50. The 3a-fluoro derivative was inac-
tive in both assays.
c
-Aminobutyric acid
Ó 2009 Elsevier Ltd. All rights reserved.
GABAA
1. Introduction
Neuroactive steroids are positive allosteric modulators of the
GABAA receptor that interact with a specific steroid recognition site
on the receptor–ion channel complex.1 The current interest in the
therapeutic properties of these compounds results from their po-
tential activity as anticonvulsants,2–4 anaesthetics5 and for the
treatment of several neurological and psychiatric disorders.6–9
Endogenous neuroactive steroids (neurosteroids) such as 3
a-hy-
droxy-5 -pregnan-20-one (1) and its 5b isomer (2), are rapidly
a
biotransformed when administered exogenously and several syn-
thetic analogues of these compounds with improved bioavailabil-
ity have been developed.10 Structure–activity relationship studies
have demonstrated that a saturated pregnane nucleus with either
The reported synthesis of 2b-aminopregnanes (such as 4) starts
5a- or 5b-stereochemistry, a 3a-hydroxyl group and a 20-ketone
from 2a,3a
-epoxysteroids.14 Opening of these conformationally ri-
form the basic pharmacophore.11 However, some variations to this
core unit are well tolerated, such as relatively large, polar (for
water solubility) substituents at the 2-position on the b-face that
can include ethers, for example, minaxolone 312,13 and amines,
for example, Org 21465 4.14
gid unsymmetrical epoxides by nucleophiles follows the Fürst-
Plattner rule with exclusive formation of the diaxial product under
kinetically controlled conditions,15,16 that is, attack of the nucleo-
phile on the b-face at C-2, giving rise simultaneously to 3a-hydro-
xy derivatives (a condition for central nervous system activity)
having a C-2b substituent. We envisaged another approach to such
analogues and focused again our attention on the intramolecular
aziridination reaction of olefinic sulfamates.17 We have previously
shown18 that treatment of a pregn-5-en-19-yl sulfamate (i.e., com-
pound 5) with iodosylbenzene and a copper salt catalyst, leads to
* Corresponding author. Tel./fax: +54 11 4576 3385.
E-mail address: burton@qo.fcen.uba.ar (G. Burton).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.008

F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
6527
2. Chemistry
With these considerations in mind, we chose 19-hydroxypregn-
2-en-20-one (10) as an appropriate precursor for our study. The
published synthesis of such unsaturated pregnenes starts from
pregnanolone acetate but gives only modest yields owing mainly
to the poor regioselectivity of 3b-tosylate elimination in the
Scheme 1. Reagents and conditions: (a) PhI@O, Cu(CH3CN)4PF6, CH3CN, N2, 25 °C,
20 h.
5a
H-reduced pregnane.22 We thus envisaged a different strategy
starting from the 6-keto-derivative 11 (Scheme 2). This compound
can be synthesized from cheap, commercially available pregneno-
lone acetate in seven steps following a procedure described in part
by Mori and co-workers for the synthesis of brassinolides.23 The
keto group of compound 11 was first reduced stereoselectively to
6b-hydroxypregnane 12 using the bulky lithium tri-tert-butoxy-
aluminum hydride, which approaches from the less sterically hin-
formation of the corresponding nitrene and consequent aziridin-
ation of the 5,6 double bond with total facial stereoselectivity on
the b-face (compound 6; Scheme 1). By analogy, in the case of a
2,3-pregnene C-19 sulfamoyl derivative, a 2b,3b,19-sulfamoyl azi-
ridine derivative should be obtained. Subsequent regioselective
opening of the aziridine ring at C-3 by nucleophiles (vide supra)
would give access to analogues of allopregnanolone (1) having a
2b amino group as part of a cyclic sulfamidate moiety in their
structure. These types of compounds are of interest per se, since
the sulfamidate moiety may be considered as part of the pharma-
cophore which provides a certain degree of conformational rigidity
to the molecules that could favor their eventual binding to the
receptor. Alternatively, the sulfamidate group, after activation,
serves as a point of entry for a variety of nucleophiles at C-19
thereby allowing further structural modifications.17,19 Finally, this
dered
the 6
indicative of the correct configuration at this position (6
a
a
face. The characteristic resonance and coupling pattern for
-H in the 1H NMR spectra of 12 (d 3.85 ppm, J = 2.8 Hz) was
-H in an
a
equatorial orientation). Treatment of compound 12 with (diacet-
oxyiodo)benzene (DIB) and I2 in the presence of light, generated
the 6b-alkoxy radical resulting in remote functionalization of the
19-CH3 group24 to epoxysteroid 13 (75% yield). Regeneration of
the carbonyl group at C-20 was accomplished using standard pro-
cedures. Thus, removal of the acetate group with LAH in THF affor-
ded the 20b-hydroxy derivative which upon further oxidation with
PCC provided compound 14 in good yield (73% over two steps).
Opening of the 6,19-epoxy moiety in compound 14 was achieved
using a reaction previously developed by us for the cleavage of
ethers.25 Thus, treatment of 14 with ZnI2 in acetic anhydride gave
methodology gives easy access to modified 3a-fluoro derivatives
(via nucleophilic opening of the aziridine ring with fluoride anion),
recently shown to be an interesting new family of potentially ac-
tive analogues of neurosteroids.20,21 We report herein the synthesis
and GABAA receptor activity of rigid analogues of allopregnanolone
having a sulfamoyl moiety on the b-face at the C-2 position and
either a pharmacologically relevant hydroxy group or fluoride
6a
-iodo-19-acetyloxypregnane 15 in 74% yield. Removal of iodine
in compound 15 was easily performed by a classical radical dehalo-
genation (tributyltin hydride, h
, toluene)26 and further treatment
atom at the 3a position (compounds 7, 8 and 9).
m
of the product with potassium hydroxide in methanol afforded
compound 10 in excellent yield (92% from 15). Absence of the typ-
ical signal for CHI in the 1H NMR spectrum of compound 10 con-
firmed the success of the radical reaction.
The synthesis of sulfamidates 7, 8 and 9 is shown in Scheme 3.
Reaction of 19-hydroxypregnane 10 with sulfamoyl chloride gen-
erated in situ,27 then gave the polar, unsaturated steroidal sulfa-
mate 16 as the sole isolable product in 66% yield. The presence
of the sulfamidate group in 16 was confirmed by NMR and mass
spectrometry. Compound 16 was then used for the direct intramo-
lecular aziridination of the 2,3-double bond.18 Thus, reaction of 16
with 1.5 equiv of iodosylbenzene and 10% Cu(CH3CN)4PF6 in etha-
nol at room temperature for 18 h led stereospecifically to the sul-
famoylaziridine 17 in 75% yield. No other products could be
Scheme 2. Reagents and conditions: (a) LiAl(O-tBu)3H, THF, 0–25 °C, 1 h; (b) DIB, I2, h
m
, 25 °C, 2 h; (c) (i) LAH, THF, 0–25 °C, 1 h; (ii) PCC, BaCO3, MS 3 Å, Cl2CH2, 25 °C, 4 h; (d)
ZnI2/Ac2O, N2, 25 °C, 1 h; (e) (i) n-Bu3SnH, N2, 25 °C, 5 h; (ii) KOH, MeOH, N2, 20 °C, 5 h.

6528
F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
Table 2
Effects of allopregnanolone (1) and sulfamidates 7, 8 and 9 on binding of [3H]mus-
cimol to membranes from rat cerebelluma
Concentration (nM)
% Stimulation
1
7
8
9
20
50
95 19
100 22
108 25
106 24
116 26
165 13c
173 25c
100 11
105 18
108 20
106 19
118 17
170 30c
176 31b
119 15
121 5c
121 8c
117 10b
124 13b
124 5b
141 14b
n.d.
n.d.
n.d.
n.d.
n.d.
96
100
200
400
800
1000
2
n.d.
EC50 (nM)
658 259
542 145
1890 617
—
a
Membranes were incubated with 10 nM [3H]muscimol in the absence or
presence of 1, 7, 8 or 9. The samples were filtered under vacuum through glass
microfiber filters, and specific radioactivity bound to membranes was determined
by liquid scintillation counting. Data shown represent mean values from four
separate experiments performed in duplicate.
b
p <0.05 versus controls with no steroid added.
p <0.01 versus controls with no steroid added. n.d.: not determined.
c
Scheme 3. Reagents and conditions: (a) HCO2H/ClSO2NCO, DMA, 0–25 °C, 4 h; (b)
PhI@O, Cu(CH3CN)4PF6, CH3CN, N2, 25 °C, 18 h; (c) TBAA, THF, N2, 0–25 °C, 1 h; (d)
KOH, MeOH, N2, 25 °C, 2 h; (e) (i) PhCH2Br, 40% NaOH, Et3PhCH2NCl, Cl2CH2, 25 °C,
45 min; (ii) KOH, MeOH, N2, 25 °C, 2 h; (f) TBAF, THF, N2, 25 °C, 1 h.
With the aim of extending the scope of this methodology to the
synthesis of 3
with TBAF in THF at room temperature (Scheme 3, step f). As ex-
pected, regioselective opening of the aziridine ring gave the 3 -flu-
a
-fluoro derivatives,20,21 we treated compound 17
a
detected by TLC and 1H NMR analysis of the crude reaction mix-
ture. The structure of compound 17 was confirmed by 1H and 13C
NMR (proton decoupled and DEPT) spectroscopy. Treatment of
compound 17 with tetrabutylammonium acetate afforded the cyc-
lic sulfamate 18 resulting from regioselective opening of the aziri-
oro-2b,19-sulfamidate 9 in 7.7% overall yield (from pregnenolone
acetate). The structure of 9 was confirmed by NMR and mass spec-
trometry. In the former, signals at dH 4.53 and dC 88.9 correspond-
ing to H-3b and C-3, respectively, together with the values of the
coupling constants of these atoms with the neighboring fluorine
1
atom (2JH–F 44.2 Hz and JC–F 175.0 Hz), provided adequate proof
dine at C-3 (79% yield), thereby providing the 3a-oxygenated
moiety required for central nervous system (CNS) activity. The
same reaction using cesium acetate as nucleophile resulted in a
lower yield (71%). Smooth deacetylation of 18 with potassium
of structure.
3. GABAA receptor activity
hydroxide furnished the 3a-hydroxy derivative 7 in almost quanti-
tative yield. The equatorial orientation of 3b-H in this compound
was clearly established by its narrow resonance (W1/2 ꢀ 9 Hz) in
the 1H NMR spectrum. The overall yield of 7 starting from pregnen-
olone acetate (18 steps) was 7.2%.
To test the effect of substituents on the sulfamidate nitrogen on
the biological activity, we synthesized the benzyl derivative 8.
Thus, treatment of 18 under the usual conditions of benzyla-
tion28,29 (Scheme 3, step e), gave N-benzyl derivative in 96% yield.
Smooth hydrolysis of the acetate group in C-3 with KOH in meth-
anol at room temperature gave the analogue 8 in 7.3% yield from
pregnenolone acetate.
GABAA receptor affinity was evaluated by assaying the effect of
the synthetic analogues 7, 8 and 9 on the binding of [3H]flunitraz-
epam (a specific ligand for the benzodiazepine binding site of GA-
BAA receptors) and [3H]muscimol (a specific ligand for the GABA
binding site) as compared with the effects of the typical neuroster-
oid allopregnanolone.11 This compound is known to stimulate
binding of [3H]flunitrazepam and [3H]muscimol by noncompeti-
tive interactions of the steroid with these binding sites. Thus, as
shown in Table 1 allopregnanolone (1) stimulated [3H]flunitraze-
pam with
a maximum binding in excess of 140% (EC50
265 117 nM). Compound 7 and its N-benzyl derivative 8, were
significantly more active in the 25–100 nM range, with a smaller
EC50 (63 45 nM and 81 35 nM). For the stimulation of [3H]mus-
cimol binding (Table 2), allopregnanolone and the sulfamidates 7
Table 1
Effects of allopregnanolone (1) and sulfamidates 7, 8 and 9 on binding of [3H]flu-
nitrazepam to membranes from rat cerebelluma
Concentration (nM)
% Stimulation
1
7
8
9
10
25
50
100
250
500
800
93 10
93
104 11
113 10
122 9b
122 16b
126 9c
126 11b
170 7c
108
2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
98
7
113 6b
118 2c
117 3c
122 2b
125 5c
134 8c
92 12
97 11
122 10c
132 14c
140 19b
8
EC50 (nM)
265 117
63 45
81 35
—
a
Membranes were incubated with 1 nM [3H]flunitrazepam in the absence or
presence of 1, 7, 8 or 9. The samples were filtered under vacuum through glass
microfiber filters, and specific radioactivity bound to membranes was determined
by liquid scintillation counting. Data shown represent mean values from four
separate experiments performed in duplicate.
Figure 1. Superposition of calculated structures (HF/6-31G**) of sulfamidate 7
(blue) and allopregnanolone (1) (white). Overlay corresponds to best fit for O(3),
C(3), C(17) and C(20) (RMS error 0.051 Å); O(3)–C(20) distances are 1: 10.04 Å; 7:
10.15 Å.
b
p <0.05 versus controls with no steroid added.
c
p <0.01 versus controls with no steroid added. n.d.: not determined.

F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
6529
and 8, had similar dose–response curves, with the N-benzyl deriv-
ative 8 exhibiting a higher EC50 (1890 617 nM). On the other
hand, the 3a-fluoro analogue 9 differing from 7 only in the substi-
tuent at C-3, showed no significant effects (compared to controls)
on the binding of [3H]flunitrazepam and [3H]muscimol, when as-
sayed at the highest concentration allowed by its solubility in the
biological medium (800 nM).
Ab-initio calculations (HF/6-31G**) showed that introduction of
the sulfamidate bridge in allopregnanolone does not affect signifi-
cantly the steroid backbone conformation (Fig. 1). Thus, the relative
tert-butoxyaluminum hydride (1.94 g, 7.63 mmol) under N2 at
0 °C. After 1 h, acetone (10 mL) was added and when gas evolution
ceased the reaction mixture was neutralized with 1 N HCl. The sol-
vent was evaporated to one fifth of its original volume and the res-
idue was diluted with dichloromethane (60 mL) and successively
washed with a saturated solution of NaHCO3 (20 mL), brine
(20 mL) and water (20 mL). The organic layer was dried with so-
dium sulfate and evaporated in vacuo. Purification by flash chro-
matography gave 6b-hydroxy steroid 12 (1.38 g, 95%) as a white
solid: mp 150–152 °C (from AcOEt–hexane); IR (KBr) 3502, 2937,
positions and orientations of the C-17 side chain and the 3
a
-hydro-
2872, 1724, 1454, 1375, 1075, 1047, 1022 cmꢁ1
(200.13 MHz): 5.68 (1H, m, 3-H), 5.57 (1H, m, 2-H), 4.85 (1H, m,
20-H), 3.85 (1H, t, J = 2.8 Hz, 6 -H), 2.31 (1H, m, 4 -H), 2.02 (3H,
s, acetate), 1.84–1.86 (4H, m, 4b-H, 7b-H, 12b-H and 1b-H), 1.78
(1H, m, 1 -H), 1.67–1.72 (3H, m, 15 -H, 8-H and 16b-H), 1.60
(1H, m, 17-H), 1.41–1.46 (3H, m, 11b-H, 5 -H and 11 -H), 1.17–
1.25 (4H, 7 -H, 15b-H, 12 -H and 16 -H), 1.15 (3H, d, J = 6.2 Hz,
;
1H NMR
xyl are maintained. The enhanced biological activity displayed by
analogues 7 and 8 may then be related to favorable interactions of
the sulfamidate moiety with the receptor, in agreement with obser-
vations that H-bond acceptors in the proximity of positions 2 and 3
increase activity.30 Most interesting is the fact that introduction of
the bulky benzyl substituent (8) is well tolerated; this is in line with
results on 2- and 3-substituted analogues of allopregnanolone, that
suggested the presence of a hydrophobic pocket in the receptor that
could accommodate bulky groups in the neighborhood of ring A.30,31
The lack of activity of the 3-fluoro derivative 9 was somewhat
disappointing, considering previous reports on activity of other
a
a
a
a
a
a
a
a
a
21-H), 1.08 (1H, m, 14-H), 0.77 (1H, m, 9-H), 0.76 (3H, s, 19-H),
0.67 (3H, s, 18-H); 13C NMR (50.32 MHz): 170.4 (acetate), 125.6
(C-2 and C-3), 72.8 (C-20), 70.3 (C-6). 55.5 (C-14), 55.1 (C-17),
54.2 (C-9), 44.5 (C-5), 42.4 (C-13), 41.7 (C-1), 39.7 (C-7), 39.3 (C-
12), 34.6 (C-10), 30.0 (C-8), 26.4 (C-4), 25.4 (C-16), 24.2 (C-15),
21.5 (acetate), 20.7 (C-11), 19.9 (C-21), 15.1 (C-19), 12.5 (C-18);
MS (ESI): m/z 401 (M+H2O+Na, 12), 383 (M+Na, 100), 283 (40),
243 (20); Anal. Calcd for C23H36O3: C, 76.62; H, 10.06. Found: C,
76.22; H, 10.04.
3a a-hydro-
-fluoro steroids.20,21 In this case, replacement of the 3
xyl by fluorine was highly deleterious to GABAA receptor activity,
indicating that further work is needed to ascertain the effect of
alternate substituents at position 3a of neuroactive steroids.
4.2.2. 20b-Acetyloxy-6,19-epoxypregn-2-ene (13)
4. Experimental
4.1. General
To a solution of alcohol 12 (1.00 g, 2.77 mmol) in dry dichloro-
methane (330 mL) in a water jacketed flask were added iodoben-
zene diacetate (0.45 g, 1.39 mmol) and iodine (0.70 g, 2.77 mmol)
under N2. The solution was irradiated with a 300 W tungsten lamp
(5000 lm) while being stirred at 25 °C. Two aliquots of iodoben-
zene diacetate (0.45 g, 1.39 mmol) and iodine (0.70 g, 2.77 mmol)
were added each 30 min. After 1 h, the reaction was finished (tlc)
and a solution of saturated sodium thiosulfate (60 mL) was added.
The organic layer was washed with brine (60 mL) and water
(60 mL), dried with sodium sulfate and evaporated in vacuo. The
oily mixture was chromatographed on neutral alumina with cyclo-
hexane/AcOEt (95:5–8:2), to give epoxypregnene 13 (0.74 g, 75%)
as a white solid: mp 147–149 °C (from AcOEt–hexane); IR (KBr)
Melting points (mp) were taken on a Fisher-Johns apparatus and
are uncorrected. IRspectrawererecordedinthinfilmsusingKBrdisks
on a Nicolet Magna 550 FT-IR spectrophotometer. 1H and 13C NMR
spectra were measured in a Bruker AC-200 (200.13 and 50.32 MHz)
or a Bruker Avance II 500 (500.13 and 125.72 MHz) NMR spectrome-
ter indeuteriochloroform unlessindicated otherwise. Chemicalshifts
are given in ppm downfield from internal TMS; J values are given in
hertz. Spectra were assigned by analysis of the DEPT, COSY 45, HET-
COSY and HMBC obtained using standard Bruker software. The
electron impact mass spectra (MS) were collected on a Shimadzu
QP-5000 mass spectrometer at 70 eV by direct inlet. Vacuum liquid
chromatography (VLC) was carried out on Kieselgel 60-G (Merck);
column flash chromatography was carried out on Kieselgel S 0.040–
0.063 mm or Florisil. Thin layer chromatography (TLC) analysis was
performed on Silica Gel 60 F254 (0.2 mm thick). The plates were visu-
alized by spraying with a 3.5% solution of phosphomolybdic acid in
ethanol. All solvents were distilled and stored over 4 Å molecular
sieves before use. The homogeneity of all compounds was confirmed
by TLC. Solvents were evaporated at 45 °C under vacuum. High-reso-
lution mass spectrometry and elemental analyses were performed at
the ICSN, CNRS, Gif-sur-Yvette, France.
3448, 2931, 2872, 1731, 1445, 1372, 1033, 1019, 943, 852 cmꢁ1
1H NMR (200.13 MHz): 5.67 (1H, m, 3-H), 5.57 (1H, m, 2-H), 4.85
(1H, m, 20-H), 3.97 (1H, d, J = 4.8 Hz, -H), 3.78 (1H, d,
.
6a
J = 8.0 Hz, 19b-H), 3.53 (1H, d, J = 8.0 Hz, 19a-H), 2.01 (3H, s, ace-
tate), 1.15 (3H, d, J = 6.1 Hz, 21-H), 0.70 (3H, s, 18-H); 13C NMR
(50.32 MHz): 170.3 (acetate), 124.9 (C-3), 123.8 (C-2), 81.5 (C-6),
72.8 (C-20), 70.9 (C-19), 54.9 (C-14), 54.8 (C-17), 53.9 (C-9), 46.1
(C-5), 42.9 (C-10 and C-13), 39.3 (C-12), 36.9 (C-7), 34.6 (C-8),
27.5 (C-1), 26.6 (C-4), 25.4 (C-16), 23.5 (C-15), 22.2 (C-11), 21.5
(acetate), 19.9 (C-21), 12.9 (C-18); MS (ESI): m/z 399 (M+H2O+Na,
25), 381 (M+Na, 100), 299 (55); Anal. Calcd for C23H34O3: C,
77.05; H, 9.56. Found: C, 77.08; H, 9.39.
20b-Acetyloxypregn-2-en-6-one (11) was obtained from 20b-
acetyloxypregn-5-en-3b-ol following essentially the procedure de-
scribed by Mori and co-workers.23 The latter compound was ob-
tained from pregnenolone acetate following the procedure
described previously by us.32 The geometries of compounds 1
and 7 were optimized using the ab-initio quantum chemistry pro-
gram GAUSSIAN 0333 and the HF/6-31G** basis set.
4.2.3. 6,19-Epoxypregn-2-en-20-one (14)
To a stirred solution of compound 13 (1.00 g, 2.79 mmol) in dry
THF (23 mL) was added lithium aluminum hydride (0.52 g,
13.84 mmol) under N2 at 0 °C. After 1 h, acetone (5 mL) was added,
and when gas evolution ceased the reaction mixture was neutral-
ized with 1 N HCl. The solvent was evaporated to one third of its
original volume, a solution of saturated sodium potassium tartrate
(40 mL) was added and the mixture extracted with dichlorometh-
ane (2 ꢂ 20 mL); the organic layer was successively washed with a
saturated solution of NaHCO3 (15 mL), brine (15 mL) and water
(15 mL), dried with sodium sulfate and evaporated under reduced
pressure. Chromatography on silica gel using a gradient of cyclo-
4.2. Syntheses
4.2.1. 20b-Acetyloxypregn-2-en-6b-ol (12)
To a stirred solution of 20b-acetyloxy-pregn-2-en-6-one 11
(1.44 g, 4.02 mmol) in dry THF (75 mL) was added lithium tri-

6530
F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
hexane/AcOEt (9:1–6:4) gave the 20-alcohol (0.77 g, 87%): mp
4.2.5. 19-Hydroxypregn-2-en-20-one (10)
155–157 °C (from AcOEt–hexane); IR (KBr) 3427, 2929, 2869,
To a stirred solution of compound 15 (0.60 g, 1.44 mmol) in dry
toluene (12.5 mL) was added tri-n-butyltin hydride (0.5 mL,
1.88 mmol) dropwise under N2 over a 5 min period. The resulting
solution was stirred at 25 °C for 5 h and then concentrated in va-
cuo. The residue was dissolved in acetonitrile (40 mL) and parti-
tioned with hexane (3 ꢂ 10 mL). The acetonitrile layer was
evaporated in vacuo and the residue was purified by flash chroma-
tography on silica gel (gradient of hexane–AcOEt) to give 19-acet-
yloxypregn-2-en-20-one (0.48 g, 1.33 mmol, 92%). To a solution of
the latter compound (312 mg, 0.87 mmol) in methanol (55 mL)
was added KOH 10% (10 mL) under N2 at 20 °C. After 5 h the reac-
tion mixture was neutralized with 2 N HCl and evaporated to a fifth
of its original volume. The resulting mixture was diluted with
dichloromethane (50 mL) washed with brine, dried with sodium
sulfate and the solvent evaporated. Column chromatography on
silica gel using cyclohexane/AcOEt (8:2) as eluent gave compound
10 (265 mg, 0.84 mmol, 97%) as a white solid: mp 143–144 °C
(from AcOEt–hexane); IR (KBr) 3477, 2911, 2845, 1701, 1447,
1453, 1374, 1494, 1033, 737, 656 cmꢁ1 1H NMR (200.13 MHz):
;
5.68 (1H, m, 3-H), 3.98 (1H, d, J = 4.6 Hz, 6a-H), 3.80 (1H, d,
J = 7.9 Hz, 19b-H), 3.74 (1H, m, 20-H), 3.54 (1H, d, J = 7.9 Hz, 19a-
H), 2.56 (1H, m, 2-H), 1.14 (3H, d, J = 6.1 Hz, 21-H), 0.82 (3H, s,
18-H); 13C NMR (50.32 MHz): 124.9 (C-3), 123.9 (C-2). 81.6 (C-6),
70.9 (C-19), 70.4 (C-20), 58.4 (C-17), 54.9 (C-14), 53.9 (C-9), 46.1
(C-5), 43.1 (C-13), 42.9 (C-10), 40.1 (C-12), 36.8 (C-7), 34.6 (C-8),
27.5 (C-1), 26.6 (C-4), 25.7 (C-16), 23.8 (C-15), 23.6 (C-21), 22.2
(C-11), 12.9 (C-18); MS (ESI) m/z 633 (2 M+Na+, 100); Anal. Calcd
for C21H32O2: C, 79.70; H, 10.19. Found: C, 79.68; H, 10.09.
Pyridinium chlorochromate (0.95 g, 4.42 mmol), barium car-
bonate (0.65 g, 3.32 mmol) and 4 Å molecular sieves (0.30 g) in
dry dichloromethane (25 mL) were vigorously stirred at 25 °C un-
der N2 for 15 min and then a solution of the alcohol obtained above
(0.70 g, 2.21 mmol) in dry dichloromethane (25 mL) was added.
After 4 h, the reaction mixture was diluted with diethyl ether
(45 mL), and percolated through a silica gel pad with hexane/AcOEt
8:2. Evaporation of the solvent gave 20-ketosteroid 14 (0.58 g, 83%)
as a white solid: mp 105–107 °C (from AcOEt–hexane); IR (KBr)
1357, 1216, 1043, 748, 668, 594 cmꢁ1 1H NMR (500.13 MHz):
;
5.60 (2H, m, 2-H and 3-H), 3.81 (1H, dd, J = 11.5, 5.4 Hz, 19b-H),
3.73 (1H, dd, J = 11.5, 3.5 Hz, 19a-H), 2.52 (1H, t, J = 9.1 Hz, 17-H),
2.40 (1H, br d, J = 16.4 Hz, 1b-H), 2.17 (1H, m, 16b-H), 2.11 (3H, s,
2927, 2873, 1704, 1437, 1356, 1222, 1167, 1017, 850, 758 cmꢁ1
1H NMR (500.13 MHz): 5.68 (1H, m, 3-H), 5.57 (1H, m, 2-H), 3.98
(1H, d, J = 4.8 Hz, 6 -H), 3.78 (1H, d, J = 8.0 Hz, 19bH), 3.55 (1H,
d, J = 8.0 Hz, 19a-H), 2.52 (1H, t, J = 9.0 Hz, 17-H), 2.16–2.17 (2H,
m, 4 -H and 16b-H), 2.11 (3H, s, 21-H), 2.05 (1H, dt, J = 12.4,
3.2 Hz, 12b-H), 1.95–1.99 (3H, m, 1 -H, 1b-H and 4b-H), 1.80
(1H, dt, J = 12.7, 5.2 Hz, 7b-H), 1.74 (1H, m, 8-H), 1.60–1.67 (4H,
m, 5-H, 11 -H, 15 -H and 16 -H), 1.44 (1H, td, J = 12.7, 3.8 Hz,
12 -H), 1.27–1.30 (3H, m, 11b-H, 14-H and 15b-H), 1.12 (1H, t,
J = 12.0 Hz, 7 -H), 1.05 (1H, td, J = 11.4, 2.7 Hz, 9-H), 0.69 (3H, s,
;
a
21-H), 2.00–2.03 (2H, m, 4b-H and 12b-H), 1.88 (1H, m, 4
1.62–1.69 (6H, m, 1 -H, 7b-H, 11 -H, 11b-H, 15 -H and 16
1.46–1.55 (3H, m, 6
5.1 Hz, 12 -H), 1.32 (1H, dd, J = 13.1, 3.9 Hz, 6b-H), 1.21 (1H, m,
15b-H), 1.12 (1H, m, 14-H), 0.95 (1H, m, 7 -H), 0.86 (1H, td,
a
-H),
a
a
a
a
-H),
a
a-H, 8-H, and 5-H), 1.37 (1H, dd, J = 12.3,
a
a
a
a
a
a
J = 11.3, 5.0 Hz, 9-H), 0.65 (3H, s, 18-H); 13C NMR (50.32 MHz):
209.6 (C-20), 127.0 (C-3), 126.3 (C-2), 63.8 (C-17), 61.8 (C-19),
57.1 (C-14), 54.1 (C-9), 44.2 (C-13), 41.4 (C-5), 39.3 (C-12), 38.8
(C-10), 36.0 (C-8), 34.0 (C-1), 31.7 (C-7), 31.5 (C-21), 30.7 (C-4),
28.4 (C-6), 24.3 (C-15), 22.6 (C-16), 21.9 (C-11), 13.3 (C-18); MS
(ESI): m/z 339 (M+Na+, 100), 295 (5); Anal. Calcd for C21H32O2: C,
79.70; H, 10.19. Found: C, 80.09; H, 10.02.
a
a
18-H); 13C NMR (125.77 MHz): 209.4 (C-20), 125.0 (C-3), 123.7
(C-2), 81.5 (C-6), 70.9 (C-19), 63.6 (C-17), 55.7 (C-14), 53.8 (C-9),
46.1 (C-5), 44.7 (C-13), 42.9 (C-10), 39.1 (C-12), 36.7 (C-7), 34.8
(C-8), 31.4 (C-21), 27.5 (C-1), 26.6 (C-4), 23.7 (C-15), 22.9 (C-16),
22.3 (C-11), 13.8 (C-18); MS (ESI) m/z 369 (M+MeOH+Na+, 100);
Anal. Calcd for C21H30O2: C, 80.21; H, 9.62. Found: C, 80.00; H, 9.36.
4.2.6. 19-Sulfamoyloxypregn-2-en-20-one (16)
Formic acid (0.143 mL, 3.77 mmol) was added dropwise to neat
chlorosulfonyl isocyanate (0.33 mL, 3.79 mmol) at 0 °C with rapid
stirring. Gas evolution was observed during the addition process.
The resulting viscous suspension was stirred for 18 h at 25 °C.
The reaction mixture was cooled to 0 °C, DMA (0.8 mL) was added
and the solution was stirred for 5 min. A solution of compound 10
(200 mg, 0.63 mmol) in DMA (2.2 mL) was added dropwise, the
resulting solution was allowed to warm to 25 °C and stirring con-
tinued until the reaction was complete, as determined by TLC (ca.
4 h). The reaction was quenched by the successive addition of
AcOEt (15 mL) and brine (5 mL) and the mixture was poured into
AcOEt (20 mL) and water (10 mL). The organic layer was collected
and the aqueous layer was extracted with AcOEt (3 ꢂ 20 mL). The
organic extracts were washed with brine (2 ꢂ 10 mL), dried over
sodium sulfate and concentrated in vacuo. Purification of the resi-
due by flash chromatography on Florisil (gradient of hexane–
AcOEt) gave 19-sulfamoyloxypregn-2-en-20-one 16 (165.3 mg,
0.42 mmol, 66%) as a white solid: mp 163–165 °C (from AcOEt–
hexane); IR (KBr) 3348, 3281, 2918, 2839, 1703, 1448, 1356,
4.2.4. 6a-Iodo-19-acetyloxypregn-2-en-20-one (15)
6,19-Epoxypregn-2-en-20-one (14) (0.70 g, 2.23 mmol) was
added to freshly prepared zinc iodide (2.80 g, 17.8 mmol) and acetic
anhydride(21.5 mL, 228.0 mmol) at 25 °C under N2. After 10 min the
reaction mixture was diluted with dichloromethane (70 mL) and a
saturated solution of NaHCO3 (30 mL) was added. The organic layer
was separated and washed with aqueous sodium thiosulfate
(30 mL), brine (30 mL) and water (30 mL), dried with sodium sulfate
and the solvent evaporated in vacuo. The residue was chromato-
graphed on Florisil with cyclohexane/AcOEt (95:5–8:2), to give
iodosteroid 15 (0.69 g, 74%) as an amorphous solid; attempts to
recrystallize this compound were unsuccessful, resulting in exten-
sive decomposition. IR (KBr) 2940, 2880, 1742, 1703, 1439, 1364,
1039, 738, 672 cmꢁ1 1H NMR (500.13 MHz): 5.67 (1H, m, 3-H),
;
5.55 (1H, m, 2-H), 4.49 (1H, td, J = 12.0, 4.3 Hz, 6b-H), 4.23 (1H, d,
J = 12.2 Hz, 19b-H), 4.15 (1H, d, J = 12.2 Hz, 19a-H), 2.67 (1H, br d,
J = 16.2 Hz, 4a-H), 2.60 (1H, dt, J = 12.8, 3.9 Hz, 7b-H), 2.52 (1H, t,
J = 9.0 Hz, 17-H), 2.17–2.21 (2H, m, 16b-H and 1b-H), 2.11 (3H, s,
21-H), 2.05 (3H, s, acetate), 2.04 (1H, m, 12b-H), 1.92 (2H, m, 5-H
1266, 1204, 985, 921, 744 cmꢁ1 1H NMR (500.13 MHz): 5.69
;
and 7
a
-H), 1.80–1.81 (2H, m, 1
a
-H and 4b-H), 1.64–1.74 (4H, m,
-H and 11b-
(1H, m, 3-H), 5.61 (1H, m, 2-H), 4.66 (2H, br s, SO2NH2), 4.41
(1H, d, J = 10.0 Hz, 19b-H), 4.18 (1H, d, J = 10.0 Hz, 19a-H), 2.51
(1H, t, J = 9.0 Hz, 17-H), 2.35 (1H, dd, J = 17.3, 5.0 Hz, 1b-H), 2.11
(3H, s, 21-H), 0.66 (3H, s, 18-H); 13C NMR (125.77 MHz): 210.7
(C-20), 125.9 (C-3), 124.9 (C-2). 69.3 (C-19), 63.6 (C-17), 57.0 (C-
14), 53.6 (C-9), 44.1 (C-13), 41.2 (C-5), 39.1 (C-12), 37.6 (C-10),
35.8 (C-8), 34.0 (C-1), 31.4 (C-7), 31.1 (C-21), 30.0 (C-4), 28.3 (C-
6), 24.1 (C-15), 22.5 (C-16), 21.5 (C-11), 13.1 (C-18); MS (ESI): m/
z 813 (M2+Na, 60), 450 (M+MeOH+Na, 53), 353 (100); Anal. Calcd
11 -H, 16
a
a
-H, 15 -H and 8-H), 1.39–1.47 (2H, m, 12a
a
H), 1.21 (1H, m, 15b-H), 1.15 (1H, m, 14-H), 1.07 (H, td, J = 11.6,
3.7 Hz, 9-H), 0.59 (3H, s, 18-H); 13C NMR (50.32 MHz): 209.2 (C-
20), 170.6 (acetate), 126.5 (C-3), 124.7 (C-2), 63.4 (C-19 and C-17),
56.5 (C-14), 53.2 (C-9), 50.3 (C-5), 47.2 (C-7), 44.1 (C-13), 41.3 (C-
10), 39.5 (C-8), 38.8 (C-6), 38.7 (C-12), 36.2 (C-1), 32.9 (C-4), 31.4
(C-21), 24.2 (C-15), 22.8 (C-16), 21.5 (C-11), 21.2 (acetate), 13.3 (C-
18); MS (ESI): m/z 485 (M+H, 5), 357 (M-I, 20), 297 (65).

F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
6531
for C21H33NO4S: C, 63.77; H, 8.41; N, 3.54; S, 8.11. Found: C, 63.52;
H, 8.23; N, 3.41; S, 7.96.
m, 5-H and 11
16
and 12
m, 14-H and 15b-H), 1.08 (1H, m, 6
a
-H), 1.63–1.71 (4H, m, 1
a
-H, 7b-H, 15
a
-H and
a
-H), 1.51 (1H, d, J = 14.7 Hz, 4b-H), 1.36–1.40 (2H, m, 6b-H
-H), 1.26–1.31 (2H, m, 8-H and 11b-H), 1.17–1.22 (2H,
-H), 0.97–0.99 (2H, m, 7 -H
a
4.2.7. 2b,3b -Iminopregnan-20-one N,19-sultone (17)
a
a
To a solution of 19-sulfamate 16 (55.0 mg, 0.139 mmol) in
anhydrous acetonitrile (2.0 mL) containing activated 3 Å molecular
sieves (0.1 g), were added at 25 °C under N2, iodosylbenzene
and 9-H), 0.62 (3H, s, 18-H); 13C NMR (125.77 MHz): 209.5 (C-
20), 71.7 (C-19), 68.5 (C-3), 63.6 (C-17), 56.3 (C-14), 52.8 (C-9),
52.3 (C-2), 44.1 (C-13), 40.3 (C-10), 38.8 (C-5), 38.7 (C-12), 36.1
(C-8), 31.6 (C-4), 31.5 (C-21), 31.1 (C-7), 28.9 (C-1), 27.9 (C-6),
24.2 (C-15), 22.7 (C-16), 21.4 (C-11), 13.5 (C-18); HRMS (MALDI)
m/z [M+Na+] 434.1958 (calcd for C21H33NO5SNa 434.1977).
(122.4 mg,
0.556 mmol)
and
Cu(CH3CN)4PF6
(15.5 mg,
0.042 mmol). The reaction mixture was stirred at room tempera-
ture for 18 h, molecular sieves were removed by filtration and
the filtrate was evaporated to dryness in vacuo. The residue was
purified by flash chromatography on silica gel (hexane/AcOEt
6:4–3:7) to give aziridine 17 (41.0 mg, 75%) as a white solid: mp
141–143 °C (from AcOEt–hexane); IR (KBr) 3393, 2937, 2874,
4.2.9. N-Benzyl-3a-hydroxy-S,S-dioxo-19,2-(epoxythioimino)-
pregnan-20-one (8)
To a solution of sulfamidate 18 (13.0 mg, 0.029 mmol) in dichlo-
romethane (0.5 mL) were added NaOH 40% (0.2 mL), triethylbenzy-
lammonium chloride (1.0 mg, 0.004 mmol) and benzyl bromide
1699, 1366, 1174, 1003, 975, 837, 794 cmꢁ1
;
1H NMR
(500.13 MHz): 4.42 (1H, dd, J = 12.3, 1.7 Hz, 19b-H), 4.19 (1H, d,
J = 12.3 Hz, 19a-H), 3.08 (1H, dd, J = 7.5, 5.5 Hz, 3b-H), 2.97 (1H,
(17 lL, 0.143 mmol) at 25 °C with vigorous stirring. After 45 min
m, 2
J = 8.8 Hz, 17-H), 2.28 (1H, dd, J = 16.3, 11.8 Hz, 4
m, 16b-H), 2.10 (3H, s, 21-H), 2.04–2.07 (2H, m, 4b-H and 12
H), 1.87 (1H, ddd, J = 15.3, 3.2, 1.7 Hz, 1 -H), 1.80 (1H, dd,
J = 12.6, 3.5 Hz, 7b-H), 1.66–1.73 (3H, m, 11 -H, 15 -H and 16
H), 1.60 (1H, m, 6 -H), 1.37–1.40 (4H, m, 5-H, 6b-H, 8-H and
12 -H), 1.18–1.22 (2H, m, 11b-H and 15b-H), 1.13 (1H, m, 14-H),
0.98 (1H, m, 7
-H), 0.87 (1H, m, 9-H), 0.60 (3H, s, 18-H); 13C
a
-H), 2.80 (1H, dd, J = 15.3, 0.9 Hz, 1b-H), 2.48 (1H, t,
dichloromethane (10.0 mL) and water (3.0 mL) were added. The or-
ganic layer was separated, washed with brine (3.0 mL), dried with
sodium sulfate and evaporated in vacuo. The residue was purified
by preparative TLC (hexane/AcOEt 7:3) to give the N-benzyl preg-
nane (15.0 mg, 0.027 mmol, 96%): 1H NMR (200.13 MHz): 7.32–
7.44 (5H, m, Ph-H), 4.97 (1H, m, 3b-H), 4.83 (1H, d, J = 16.1 Hz,
CHH-Ph), 4.61 (1H, d, J = 12.5 Hz, 19b-H), 4.46 (1H, d, J = 12.5 Hz,
a-H), 2.14 (1H,
a
-
a
a
a
a-
a
a
a
19a-H), 4.44 (1H, d, J = 16.1 Hz, CHH-Ph), 3.27 (1H, m, 2a-H),
NMR (125.77 MHz, CDCl3 + 5% CD3OD): 209.9 (C-20), 72.3 (C-19),
63.2 (C-17), 56.4 (C-14), 53.9 (C-9), 43.7 (C-13), 40.4 (C-5), 40.2
(C-12), 39.6 (C-10), 38.7 (C-3), 37.9 (C-2), 35.9 (C-8), 35.5 (C-1),
31.1 (C-21), 30.9 (C-7), 26.9 (C-6), 24.7 (C-4), 23.9 (C-15), 22.5
(C-16), 20.7 (C-11), 13.1 (C-18); HRMS (MALDI) m/z [M+Na+]
416.1854 (calcd for C21H31NO4SNa 416.1872).
2.60 (1H, d, J = 15.4 Hz, 1b-H), 2.47 (1H, t, J = 9.2 Hz, 17-H), 2.10
(3H, s, 21-H), 2.04 (3H, s, acetate), 0.59 (3H, s, 18-H). KOH 2.5 N
in methanol (1.1 mL) was added at 25 °C under N2 to a solution
of the above compound (14.0 mg, 0.026 mmol) in methanol
(6.4 mL). After 2 h the mixture was neutralized with HCl 2 N
(1.3 mL) and concentrated in vacuo to a fifth of its volume. The
resulting mixture was diluted with dichloromethane (15.0 mL)
washed with brine (5.0 mL) and water (5.0 mL), dried with sodium
sulfate and the solvent evaporated in vacuo. The oily residue was
purified by preparative TLC (hexane/AcOEt 4:6) to give the N-ben-
zyl sulfamidate 8 (12.6 mg, 0.025 mmol, 96%) as a white solid: mp
147–149 °C (from AcOEt–hexane); IR (KBr); 3500, 3290, 2929,
4.2.8. 3
20-one (7)
To a solution of aziridine 17 (17.0 mg, 0.043 mmol) in anhy-
a-Hydroxy-S,S-dioxo-19,2-(epoxythioimino)pregnan-
drous THF (1.0 mL) was added TBAA (65.0 mg, 0.216 mmol) at
0 °C and the mixture was allowed to reach 25 °C under N2. After
1 h, the THF was removed in vacuo and the oily residue was puri-
fied by preparative TLC (hexane/AcOEt 1:1) to give sulfamidate 18
(15.5 mg, 0.034 mmol, 79%) as a white solid: mp 138–140 °C (from
AcOEt–hexane); IR (KBr) 3357, 2945, 2878, 1742, 1374, 1240, 1180,
2854, 1697, 1448, 1362, 1173, 1077, 963, 738 cmꢁ1 1H NMR
;
(500.13 MHz): 7.31–7.39 (5H, m, Ph-H), 4.63 (1H, d, J = 15.9 Hz,
CHH-Ph), 4.60 (1H, d, J = 12.1 Hz, 19b-H), 4.46 (1H, d, J = 12.1 Hz,
19a-H), 4.45 (1H, d, J = 15.9 Hz, CHH-Ph), 3.83 (1H, m, 3b-H), 3.22
1033, 980, 780 cmꢁ1
4.59 (1H, br s, SO2NH), 4.51 (1H, d, J = 12.6 Hz, 19b-H), 4.30 (1H,
d, J = 12.6 Hz, 19a-H), 3.55 (1H, m, 2 -H), 2.71 (1H, d, J = 14.8 Hz,
;
1H NMR (200.13 MHz): 4.78 (1H, m, 3b-H),
(1H, m,
J = 9.0 Hz, 17-H), 2.11–2.15 (2H, m, 4
21-H), 2.03 (1H, m, 12b-H), 1.73–1.77 (4H, m, 1
and 7b-H), 1.65–1.67 (2H, m, 15 -H and 16 -H), 1.35–1.40 (5H,
m, 4b-H, 6 -H, 6b-H, 11b-H and 12 -H), 1.19–1.23 (2H, m, 8-H
-H and 9-
2a
-H), 2.60 (1H, d, J = 15.1 Hz, 1b-H), 2.48 (1H, t,
-H and 16b-H), 2.10 (3H, s,
-H, 5-H, 11 -H,
a
a
a
a
1b-H), 2.52 (1H, t, J = 9.0 Hz, 17-H), 2.12 (3H, s, acetate), 2.11
(1H, s, 21-H), 0.63 (3H, s, 18-H); 13C NMR (50.32 MHz): 209.3 (C-
20), 170.2 (acetate), 71.6 (C-19), 70.2 (C-3), 63.6 (C-17), 56.3 (C-
14), 52.8 (C-9), 49.5 (C-2), 44.0 (C-13), 40.2 (C-10), 39.9 (C-5),
38.6 (C-12), 36.0 (C-8), 31.5 (C-21), 31.1 (C-7), 29.8 (C-4), 28.9
(C-1), 27.6 (C-6), 24.2 (C-15), 22.7 (C-16), 21.3 (C-11), 21.2 (ace-
tate), 13.5 (C-18). Compound 18 (21.0 mg, 0.046 mmol) was dis-
solved in methanol and KOH 2.5 N in methanol (3.7 mL) added
under N2 at 0 °C. The reaction mixture was warmed to 25 °C and
after 2 h the solution was neutralized with 2 N HCl (4.6 mL) and
concentrated in vacuo to a fifth of its volume. The resulting mix-
ture was diluted with AcOEt (15.0 mL) washed with brine
(5.0 mL) and water (5.0 mL), dried with sodium sulfate and evapo-
rated to dryness. The residue was purified by preparative TLC (hex-
ane/AcOEt 1:1) to give sulfamidate 7 (19.0 mg, 99%) as a white
solid: mp 195–197 °C (from AcOEt–hexane); IR (KBr); 3500,
a
a
a
a
and 15b-H), 1.07 (1H, m, 14-H), 0.94–0.99 (2H, m, 7
a
H), 0.59 (3H, s, 18-H); 13C NMR (125.77 MHz): 209.2 (C-20),
136.3 (Ph C-10), 128.8 (Ph C-30,50), 128.3 (Ph C 40), 128.0 (Ph C-
0
´
2,6 ), 72.1 (C-19), 66.9 (C-3), 63.5 (C-17), 57.8 (C-2), 57.0 (C-14),
53.5 (CH2-Ph), 52.8 (C-9), 43.8 (C-13), 40.0 (C-10), 38.7 (C-12),
38.5 (C-5), 35.9 (C-8), 32.9 (C-4), 31.6 (C-7), 31.4 (C-21), 31.0 (C-
1), 27.4 (C-6), 24.2 (C-15), 22.7 (C-16), 21.0 (C-11), 13.4 (C-18);
MS (ESI): m/z 524 (M+Na, 100), 363 (10); HRMS (MALDI) m/z
[M+Na+] 524.2451 (calcd for C28H39NO5SNa 524.2447).
4.2.10. 3a-Fluoro-S,S-dioxo-19,2-(epoxythioimino)pregnan-20-
one (9)
To a solution of aziridine 17 (20.0 mg, 0.051 mmol) in anhy-
drous THF (1.0 mL) was added TBAF (29.0 mg, 0.127 mmol) at
0 °C and the mixture was allowed to reach 25 °C under N2. After
1 h the THF was removed in vacuo and the oily residue was puri-
fied by preparative TLC on silica gel (hexane/AcOEt 4:6), to give
3290, 2929, 2854, 1697, 1448, 1362, 1173, 1077, 963, 738 cmꢁ1
;
1H NMR (500.13 MHz): 4.85 (1H, br s, SO2NH); 4.46 (1H, d,
J = 12.7 Hz, 19b-H), 4.26 (1H, d, J = 12.7 Hz, 19a-H), 3.83 (1H, br s,
W1/2 ꢀ 9 Hz, 3b-H), 3.50 (1H, m, 2
2.2 Hz, 1b-H), 2.52 (1H, t, J = 8.9 Hz, 17-H), 2.16 (1H, m, 16b-H),
2.12 (3H, s, 21-H), 2.02–2.08 (2H, m, 4 -H and 12b-H), 1.80 (2H,
a
-H), 2.60 (1H, dd, J = 15.0,
the 3
solid: mp 188–190 °C (from AcOEt–hexane); IR (KBr); 3553,
3276, 2934, 2877, 1697, 1448, 1362, 1176, 1059, 967, 738 cmꢁ1
a-fluoro derivative 9 (17.7 mg, 0.043 mmol, 84%) as a white
a
;

6532
F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
1H NMR (500.13 MHz): 4,75 (1H, br s, SO2NH), 4.53 (1H, br d, JH–F
44.2 Hz, 3b-H), 4.50 (1H, d, J = 12.5 Hz, 19b-H), 4.33 (1H, d,
J = 12.5 Hz, 19a-H), 3.70 (1H, m, 2 -H), 2.71 (1H, br d, J = 15.7 Hz,
1b-H), 2.52 (1H, t, J = 8.9 Hz, 17-H), 2.17 (1H, m, 16b-H), 2.11
(3H, s, 21-H), 2.06–2.08 (2H, m, 4 -H and 12b-H), 1.74–1.79 (4H,
m, 4b-H, 5-H, 7b-H and 11 -H), 1.65–1.69 (3H, m, 1
and 16 -H), 1.35–1.43 (3H, m, 6b-H, 11b-H and 12
1.27 (4H, m, 6
=
Acknowledgments
a
We thank Agencia Nacional de Promoción Científica y Tecnológ-
ica (PICT 00727), Universidad de Buenos Aires and CONICET
(Argentina) for financial support.
a
a
a
a
-H, 15
a-H
-H), 1.16–
a
Supplementary data
a
-H, 8-H, 14-H and 15b-H), 0.97–1.03 (2H, m, 7a-
H and 9-H), 0.62 (3H, s, 18-H); 13C NMR (125.77 MHz): 209.2
(C-20), 88.9 (d, JC–F = 175.0 Hz, C-3), 71.8 (C-19), 63.5 (C-17), 56.5
(C-14), 52.9 (C-9), 49.8 (d, JC–F = 31.4 Hz, C-2), 44.0 (C-13),
40.4 (C-10), 39.2 (C-5), 38.7 (C-12), 36.0 (C-8), 31.5 (C-21), 31.2
(C-7), 30.0 (d, JC–F = 19.7 Hz, C-4), 29.9 (C-1), 27.5 (C-6), 24.2
(C-15), 22.8 (C-16), 21.3 (C-11), 13.5 (C-18); HRMS (MALDI) m/z
[M+Na+] 436.1956 (calcd for C21H32FNO4SNa 436.1934).
Supplementary data (1H and 13C NMR spectra of compounds 7,
8, 9, 15, 17 and 18) associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2009.08.008.
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4.3.2. [3H]Flunitrazepam binding assays
The effects of allopregnanolone (1) and sulfamidates 7, 8 and 9
on [3H]flunitrazepam binding were evaluated in cerebellum P2
homogenates using essentially the protocol described previously.34
Briefly, 100 lL aliquots of cerebellum membrane preparation P2
were incubated with 1 nM [3H]flunitrazepam (85.2 Ci/mmol) in
the presence or absence of different concentrations of the steroids
(10–800 nM). All steroids were dissolved in DMSO (Sigma–Aldrich
Corp., St. Louis, MO) and diluted with the incubation buffer
(1:1000) immediately before use. Diazepam (1 lM) was used to
determine non-specific binding. Assays were carried out at 4 °C
for 90 min and terminated by rapid filtration through glass micro-
fiber filters (Micro 96 Harvester, Molecular Devices). Filter
bound radioactivity was quantified by liquid scintillation
spectrophotometry.
4.3.3. [3H]Muscimol binding assays
The effects of allopregnanolone (1) and sulfamidates 7, 8 and 9
on [3H]muscimol binding were evaluated in cerebellum P2 homog-
enates using essentially the protocol described previously.34
Briefly, 100 lL aliquots of cerebellum membrane preparation P2
were incubated with 10 nM [3H]muscimol (18.0 Ci/mmol) in the
presence or absence of different concentrations of the steroids
(20–1000 nM). All steroids were dissolved in DMSO (Sigma–
Aldrich Corp., St. Louis, MO) and diluted with the incubation buffer
(1:1000) immediately before use. GABA (10 lM) was used to deter-
mine non-specific binding. Assays were carried out at 4 °C for
60 min and terminated by rapid filtration through glass microfiber
filters (Micro 96 Harvester, Molecular Devices). Filter bound radio-
activity was quantified by liquid scintillation spectrophotometry.

F. J. Durán et al. / Bioorg. Med. Chem. 17 (2009) 6526–6533
6533
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.;
GAUSSIAN 03, Revision B.05, Gaussian, Inc.: Pittsburgh, PA, 2003.
34. Nicoletti, D.; Ghini, A. A.; Furtmüller, R.; Sieghart, W.; Dodd, R. H.; Burton, G.
Steroids 2000, 65, 349.