Synthesis and antiviral activity of sulfated and acetylated derivatives of 2β,3α-dihydroxy-5α-cholestane

Article abstract of DOI:10.1016/S0039-128X(02)00166-6

Five new steroid sulfates, sodium 2β,3α-dihydroxy-5α-cholestane 3-sulfate (6), sodium 2β,3α-dihydroxy-5α-cholestane 2-sulfate (7), disodium 2β,3α-dihydroxy-5α-cholestane disulfate (8), sodium 3α-acetoxy-2β-hydroxy-5α-cholestane 2-sulfate (12), and sodium

Full text of DOI:10.1016/S0039-128X(02)00166-6

Steroids 68 (2003) 125–132
Synthesis and antiviral activity of sulfated and acetylated
derivatives of 2␤,3␣-dihydroxy-5␣-cholestane
Gustavo A. Garrido Santosa, Ana P. Murraya,b, Carlos A. Pujolc,
Elsa B. Damontec, Marta S. Maiera,∗
Departamento de Qu´ımica Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
a
Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina
Departamento de Qu´ımica, Universidad Nacional del Sur, Avda. Alem 1253, 8000 Bah´ıa Blanca, Buenos Aires, Argentina
b
c
Laboratorio de Virolog´ıa, Departamento de Qu´ımica Biológica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428 Buenos Aires, Argentina
Received 2 July 2002; received in revised form 13 September 2002; accepted 1 October 2002
Abstract
Five new steroid sulfates, sodium 2␤,3␣-dihydroxy-5␣-cholestane 3-sulfate (6), sodium 2␤,3␣-dihydroxy-5␣-cholestane 2-sulfate
(7), disodium 2␤,3␣-dihydroxy-5␣-cholestane disulfate (8), sodium 3␣-acetoxy-2␤-hydroxy-5␣-cholestane 2-sulfate (12), and sodium
2␤-acetoxy-3␣-hydroxy-5␣-cholestane 3-sulfate (13), have been synthesized starting from 3␤-hydroxy-5␣-cholestane (1). The synthetic
steroids were completely characterized by one-dimensional and two-dimensional NMR and FABMS spectra. Sulfation was performed
using triethylamine–sulfur trioxide complex in dimethylformamide as the sulfating agent. The sulfated steroids were comparatively eval-
uated for their inhibitory effect on the replication of herpes simplex virus type 2 (HSV-2). Compounds 7 and 8 were the most effective in
their inhibitory action against HSV-2. The disulfated steroid 8 also proved to be active against DEN-2 and JV.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Sulfated steroids; Synthesis; Antiviral activity
1. Introduction
synthesize disodium 2␤,3␣-dihydroxy-5␣-cholestane (8)
disulfate as well as monosulfated and acetylated derivatives
of 2␤,3␣-dihydroxy-5␣-cholestane (6, 7, 12, 13) (Fig. 1) in
order to evaluate the antiviral activity of these compounds
and gain insight into structure–activity correlations.
Sulfated polyhydroxysterols are naturally occurring
metabolites in sponges and echinoderms [1,2]. These com-
pounds have exhibited a broad spectrum of biologic activ-
ities, such as anti-HIV effects [3] and inhibition of protein
tyrosine kinases [4]. Among echinoderms, ophiuroids are
characterized by their content of sulfated steroids with
sulfate groups located at C-3 and C-21 and additional hy-
droxy groups located in rings A and C. Recently, we have
demonstrated the antiviral activity of sulfated steroids iso-
lated from the cold water ophiuroids, Ophioplocus januarii
[5] and Astrotoma agassizii [6], and synthetic derivatives
and analogs against pathogenic viruses of humans [7].
Steroids sulfated at C-21 and C-2 (␤) or C-3 (␣) with an
additional hydroxyl function at C-2 (␤) or C-3 (␣) were
the most effective in their inhibitory action against her-
pes simplex virus (HSV-2). These results prompted us to
2. Experimental
Melting points (m.p.) were determined on a Fisher Johns
1
apparatus and are uncorrected. H and 13C NMR spectra
were recorded on a Bruker AM 500 spectrometer. Chemi-
cal shifts (δ) are given in ppm downfield from TMS as the
internal standard. Two-dimensional NMR spectra were ob-
tained using standard Bruker software. Mass spectra were
collected on a VG-ZAB (HREIMS and FABMS) and on
a SHIMADZU QP-5000 (EIMS) mass spectrometer. The
FABMS (negative ion mode) were obtained on a glycerol
matrix. Reversed-phase chromatography was carried out
on octadecyl-functionalized silica gel (Aldrich). Prepara-
tive HPLC was carried out on an SP liquid chromatograph
equipped with a Spectra Series P100 solvent delivery sys-
∗
Corresponding author. Tel.: +54-11-4576-3346;
fax: +54-11-4576-3346.
E-mail address: maier@qo.fcen.uba.ar (M.S. Maier).
0039-128X/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
doi:10.1016/S0039-128X(02)00166-6

126
G.A.G. Santos et al. / Steroids 68 (2003) 125–132
poured into 10% HCl solution and extracted with CH2Cl2
(3 × 15 ml). The combined organic layer was washed suc-
cessively with water, saturated NaHCO3 solution, and water,
and then dried over anhydrous MgSO4 and evaporated to
dryness to give 537 mg (85%) of 3 as white solid, m.p. 66 ◦C
1
(acetone–H2O). H NMR δ (CDCl3): 0.67 (s, 3H, H-18),
0.76 (s, 3H, H-19), 0.87 (d, J = 6.6 Hz, 6H, H-26, H-27),
0.91 (d, J = 8 Hz, 3H, H-21), 5.58 (m, 2H, H-2, H-3).
HREIMS m/z = 370.3600 [M]+ (C27H46, ꢀ = 3.0 mmu).
2.3. 2,3α-Epoxy-5α-cholestane (4)
To a solution of 5␣-cholest-2-ene (3) (481 mg, 1.30 mmol)
in CH2Cl2 (33 ml) were added water (22.6 ml) and Na2CO3
(566 mg, 4.77 mmol). The reaction mixture was stirred vig-
orously and m-chloroperbenzoic acid (310 mg, 1.80 mmol)
was added slowly. The mixture was stirred for 4 h at room
temperature, and then the aqueous layer was extracted with
CH2Cl2 (3 × 25 ml). The combined CH2Cl2 extracts were
washed successively with 5% Na2SO3 solution, saturated
NaHCO3 solution, and water, dried over anhydrous MgSO4
and evaporated to dryness to give 497 mg (99%) of 4 as
a white powder, m.p. 105–106 ◦C (acetone). 1H NMR δ
(CDCl3): 0.64 (s, 3H, H-18), 0.75 (s, 3H, H-19), 0.86 (d,
J = 6.6 Hz, 6H, H-26, H-27), 0.90 (d, J = 8 Hz, 3H, H-21),
3.13 (m, 2H, H-2, H-3). HREIMS m/z = 386.3549 [M]+
(C27H46O, ꢀ = 5.0 mmu).
Fig. 1. Synthetic intermediates (5, 9–11) and new sulfated steroids (6–8,
12–13).
tem, a Rheodyne manual injector and a refractive index
detector using a Phenomenex AQUA 5 ␮ C18 125A column.
TLC of sulfated steroids was performed on silica gel F254
(n-BuOH/AcOH/H2O (12:3:5)) and C18 reversed-phase
plates (MeOH/H2O (80:20 v/v)) and detected by spraying
with sulfuric acid (10% EtOH).
Triethylamine–sulfur trioxide complex was prepared by
treatment of triethylamine with chlorosulfonic acid follow-
ing the procedure of Nair and Bernstein [8]. Commercially
available 3␤-hydroxy-5␣-cholestane (1) was obtained from
Aldrich and used as starting material for the synthesis of
1
compounds 5–13. Compound 1: H NMR δ (CDCl3): 0.65
(s, 3H, H-18), 0.80 (s, 3H, H-19), 0.87 (d, J = 6.9 Hz, 6H,
H-26, H-27), 0.90 (d, J = 6.9 Hz, 3H, H-21), 3.58 (m, 1H,
H-3).
2.4. 2β,3α-Dihydroxy-5α-cholestane (5)
A solution of epoxide 4 (475 mg, 1.23 mmol) in THF
(23 ml) was treated with 1N H2SO4 (1.53 ml, 3.1 mmol) and
stirred for 24 h at room temperature. After neutralization
with saturated NaHCO3 solution the mixture was evap-
orated to fifth initial volume, diluted with water (18 ml),
and extracted with ethyl acetate (3 × 15 ml). The combined
organic extracts were washed with water, dried over an-
hydrous MgSO4, filtered and evaporated to dryness. The
crude diol 5 (477 mg, 96%) was submitted to vacuum-dry
column chromatography on silica gel (32–63 ␮m), eluted
with cyclohexane/acetone (50:50 v/v), evaporated under
reduced pressure, and subjected to vacuum-dry column
chromatography on silica gel C18 (35–75 ␮m). Frac-
tions eluted with methanol/water (98:2 v/v) afforded
pure 2␤,3␣-dihydroxy-5␣-cholestane (5) (305.1 mg, 62%,
m.p. 178–180 ◦C (acetone–H2O)). 1H NMR δ (CDCl3):
0.66 (s, 3H, H-18), 0.87 (d, J = 6.6 Hz, 6H, H-26, H-27),
0.90 (d, J = 7.3 Hz, 3H, H-21), 0.99 (s, 3H, H-19), 3.88
(m, 2H, H-2, H-3). EIMS m/z = 404 (14%, M+), 386 (2%,
M+·H2O), 368 (1%, M+·2H2O).
2.1. 3β-Hydroxy-5α-cholestane tosylate (2)
To a solution of 3␤-hydroxy-5␣-cholestane (1) (2 g,
5.15 mmol) in dry pyridine (6.8 ml) p-toluenesulfonyl chlo-
ride (2.22 g, 11.65 mmol) was added. The solution was
stirred at room temperature for 24 h and then poured into
cold water (20 ml) and extracted with CH2Cl2 (3 × 15 ml).
The combined organic layer was washed successively with
2N HCl solution, water, saturated NaHCO3 solution, and
water, and then dried over anhydrous MgSO4 and evapo-
rated in vacuo to give 2.49 g (89%) of 2, m.p. 177–178 ◦C
1
(acetone–H2O). H NMR δ (CDCl3): 0.63 (s, 3H, H-18),
0.77 (s, 3H, H-19), 0.86 (d, J = 6.6 Hz, 6H, H-26, H-27),
0.89 (d, J = 6 Hz, 3H, H-21), 2.44 (s, 3H, CH3-Ph), 4.42
(m, 1H, H-3), 7.32 (d, J = 8 Hz, 2H, H-3ꢀ, H-5ꢀ), 7.79 (d,
J = 8.4 Hz, 2H, H-2ꢀ, H-6ꢀ). HREIMS m/z = 542.3793
[M]+ (C34H54O3S, ꢀ = 2.0 mmu).
2.2. 5α-Cholest-2-ene (3)
2.5. Sodium 2β,3α-dihydroxy-5α-cholestane 3-sulfate (6)
and sodium 2β,3α-dihydroxy-5α-cholestane 2-sulfate (7)
Lithium bromide (1.5 g, 17.27 mmol) and lithium car-
bonate (1.29 g, 17.27 mmol) were added to a solution of
3␤-hydroxy-5␣-cholestane tosylate (2) (0.93 g, 1.71 mmol)
in dry DMF (15 ml), and the mixture was refluxed for 1.5 h.
After cooling to room temperature the mixture was slowly
Triethylamine–sulfur trioxide complex(27mg, 0.12mmol)
was added to a solution of 2␤,3␣-dihydroxy-5␣-cholestane

G.A.G. Santos et al. / Steroids 68 (2003) 125–132
127
Table 1
1H NMR data for compounds 6–8, 12, and 13a,b
Proton
δH multiplet (J)
6
7
8
12
13
H-2
H-3
4.08 d (2.7)
4.40 d (2.7)
0.68 s
4.42 d (2.3)
4.02 d (2.3)
0.68 s
4.73 d (2.25)
4.70 d (2.25)
0.69 s
4.44 d (2)
5.11 d (2)
0.69 s
5.14 d (2.5)
4.40 d (2.5)
0.68 s
H-18
H-19
H-21
H-26
H-27
CH3CO
0.99 s
0.99 s
0.99 s
1.01 s
0.93 s
0.93 d (6.6)
0.88 d (6.6)
0.88 d (6.6)
0.93 d (6.6)
0.88 d (6.6)
0.88 d (6.6)
0.93 d (6.6)
0.88 d (6.6)
0.88 d (6.6)
0.93 d (6.6)
0.88 d (6.6)
0.88 d (6.6)
2.05 s
0.93 d (6.6)
0.88 d (6.6)
0.88 d (6.6)
2.03 s
a Recorded at 500 MHz in methanol-d4.
b J in Hz.
(5) (24.2 mg, 0.06 mmol) in DMF (0.5 ml), and the mix-
ture was stirred at room temperature for 3 h. Then, the reac-
tion mixture was quenched with water (0.5 ml), evaporated
to dryness under reduced pressure, and the residue eluted
through Amberlite CG-120 (sodium form) with methanol
to afford a mixture of compounds 6 and 7. Preparative
HPLC (methanol/water (80:20 v/v)) of the mixture afforded
pure 6 (11.84 mg, 39%) and 7 (6.38 mg, 21%) together
with the disulfated analog 8 (11.3 mg, 31%). Compound
(3 × 30 ml). The combined organic layer was washed suc-
cessively with water, saturated NaHCO3 solution, and water,
dried over anhydrous MgSO4, and evaporated to dryness.
The residue was purified by column chromatography on sil-
ica gel using cyclohexane/CH2Cl2 (50:50 v/v) as eluent to
give 133.1 mg (92%) of 9 as white solid, m.p. 110–111 ◦C
(acetone). 1H NMR δ (CDCl3): 0.64 (s, 3H, H-18), 0.86 (d,
J = 6.6 Hz, 6H, H-26, H-27), 0.89 (d, J = 6.2 Hz, 3H,
H-21), 0.90 (s, 3H, H-19), 2.03 (s, 3H, CH3CO), 2.06 (s,
6: m.p. 124–125 ◦C. For H and 13C NMR see Tables 1
1
and 2. FAB− m/z = 483 [M–Na]−. HRFABMS (negative
Table 2
13C NMR data for compounds 6–8, 12, and 13
ion mode) m/z = 483.3164 [M–Na]− (C27H47O5S, ꢀ =
a
1
2.0 mmu). Compound 7: m.p. 151–152 ◦C. For H and 13C
Carbon
δC
NMR see Tables 1 and 2. FAB− m/z = 483 [M–Na]−.
HRFABMS (negative ion mode) m/z = 483.3164 [M–Na]−
(C27H47O5S, ꢀ = 2.0 mmu).
6
7
8
12
13
1
2
3
4
5
6
7
8
41.0 t
70.0 d
78.5 d
30.3 t
40.6 d
29.3 t
33.3 t
36.3 d
56.6 d
36.4 s
22.0 t
41.5 t
43.8 s
57.9 d
25.2 t
29.3 t
57.7 d
12.6 q
14.6 q
37.1 d
19.2 q
37.4 t
24.9 t
40.7 t
29.1 d
38.8 t
78.8 d
69.0 d
32.5 t
39.7 d
39.2 t
76.5 d
76.1 d
30.5 t
40.2 d
40.9 t
76.0 d
72.2 d
29.9 t
39.6 d
38.3 t
72.6 d
75.5 d
30.9 t
40.3 d
2.6. Disodium 2β,3α-dihydroxy-5α-cholestane disulfate (8)
29.3b
t
29.2b
33.2 t
36.4b
t
29.2b
t
29.4b
t
Triethylamine–sulfur trioxide complex (48 mg, 0.265
mmol) was added to a solution of 2␤,3␣-dihydroxy-5␣-
cholestane (5) (17 mg, 0.042 mmol) in DMF (0.5 ml). The
reaction mixture was stirred at 95 ◦C for 13 h and then
quenched with water (0.5 ml). After evaporation to dryness
the residue was eluted through Amberlite CG-120 (sodium
form) with methanol. Further purification by preparative
HPLC (methanol/water (80:20 v/v)) afforded pure disodium
2␤,3␣-dihydroxy-5␣-cholestane disulfate (8) (23 mg, 93%),
m.p. 147−148 ◦C. For 1H and 13C NMR see Tables 1
and 2. FAB− m/z = 585 [M–Na]−. HRFABMS (negative
ion mode) m/z = 585.2534 [M–Na]− (C27H46O8S2Na,
ꢀ = 0.2 mmu).
33.3 t
36.4 d
56.7 d
36.8 s
22.0 t
41.5 t
43.8 s
57.9 d
25.2 t
33.2 t
36.4 d
56.7 d
36.3 s
22.0 t
41.4 t
43.8 s
57.9 d
25.2 t
33.1 t
36.4 d
56.4 d
36.3 s
21.9 t
41.4 t
43.8 s
57.8 d
25.2 t
d
9
56.6 d
36.4b
s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
27
26
CH3
CO
22.0 t
41.4 t
43.8 s
57.9 d
25.2 t
29.4b
t
29.3b
t
29.3b
t
29.3b
t
57.7 d
12.6 q
14.2 q
37.1 d
19.2 q
37.4 t
24.9 t
40.7 t
29.1 d
22.9bq
57.7 d
12.5 q
14.3 q
37.1 d
19.2 q
37.4 t
24.9 t
40.7 t
29.1 d
57.7 d
12.5 q
14.2 q
37.1 d
19.2 q
37.4 t
24.9 t
40.7 t
29.1 d
57.7 d
12.6 q
14.4 q
37.1 d
19.2 q
37.4 t
24.9 t
40.7 t
29.1 d
2.7. 2β,3α-Diacetoxy-5α-cholestane (9)
22.9b
23.2b
q
q
22.9b
23.2b
q
q
22.9b
23.2b
q
q
22.9b
23.2b
q
q
23.2b
q
Acetic anhydride (10.5 ml) was added to a solution of
2␤,3␣-dihydroxy-5␣-cholestane (5) (120 mg, 0.297 mmol)
in 5 ml of pyridine, and the mixture was stirred at room
temperature for 24 h. The mixture was then poured onto ice
and 2N HCl solution (20 ml), and extracted with CH2Cl2
21.1 q
171.6 s
21.3 q
171.6 s
a Recorded at 125 MHz in methanol-d4; multiplicity by DEPT.
b Chemical shifts are interchangeable.

128
G.A.G. Santos et al. / Steroids 68 (2003) 125–132
3H, CH3CO), 4.88 (m, 2H, H-2, H-3). EIMS m/z = 488
2.11. Acetylation of compound 7
(1%, M+), 426 (4%, M+-AcOH-CH2CO).
Acetic anhydride (1 ml) was added to a solution of
sodium 2␤,3␣-dihydroxy-5␣-cholestane 2-sulfate (7) (4 mg,
0.0079 mmol) in 1 ml of pyridine, and the mixture was
stirred at room temperature for 24 h. The mixture was then
poured onto ice and 2N HCl solution (2 ml), and extracted
with CH2Cl2 (3 × 2 ml). The combined organic layer was
washed successively with water, saturated NaHCO3 solu-
tion, and water, dried over anhydrous MgSO4, and evapo-
rated to dryness. The residue was purified by preparative
HPLC to afford 3.85 mg (89%) of pure 12.
2.8. 3α-Acetoxy-2β-hydroxy-5α-cholestane (10) and
2β-acetoxy-3α-hydroxy-5α-cholestane (11)
bis(Tributyltin)oxide (BBTO) (0.27 ml, 0.545 mmol) was
added to a solution of 2␤,3␣-diacetoxy-5␣-cholestane (9)
(133 mg, 0.273 mmol) in toluene (2.7 ml), and the mixture
was refluxed for 5 h. The reaction mixture was cooled to
room temperature and purified by vacuum-dry column chro-
matography on silica gel using CH2Cl2/methanol (80:20
v/v) as eluent to give 108.3 mg (89%) of a mixture of
monoacetylated compounds 10 and 11. Further purification
of this mixture by column chromatography on silica gel
using cyclohexane/acetone (90:10 v/v) as eluent afforded
pure 10 (29.2 mg, 27%) and 11 (79.1 mg, 73%). Compound
2.12. Cells and viruses
Vero (African green monkey kidney) cells were grown
in 24-cell culture plates with Eagle’s Minimum Essential
Medium (MEM) containing 5% of bovine serum. Main-
tenance medium (MM), consisted of MEM with 1.5% of
bovine serum. HSV-2 strain G was obtained from the Ameri-
can Type Culture Collection (Rockville, USA). Dengue virus
type 2 (DEN-2) strain NG was obtained from the Instituto
Nacional de Enfermedades Virales Humanas (INEVH), Dr.
J. Maiztegui, Pergamino, Argentina. Junin virus (JV) strain
IV 4454 was provided by Dr. Marta S. Contigiani from the
1
10: m.p. 106–107 ◦C (acetone). H NMR δ (CDCl3): 0.65
(s, 3H, H-18), 0.86 (d, J = 6.6 Hz, 6H, H-26, H-27), 0.90
(d, J = 7 Hz, 3H, H-21), 0.99 (s, 3H, H-19), 2.06 (s, 3H,
CH3CO), 3.91 (s, 1H, H-2), 4.84 (d, J = 2.2 Hz, 1H, H-3).
EIMS m/z = 446 (1%, M+), 386 (31%, M+-AcOH), 368
(3%, M+-AcOH-H2O). Compound 11: m.p. 85–86 ◦C (ace-
1
tone). H NMR δ (CDCl3): 0.64 (s, 3H, H-18), 0.86 (d,
J = 6.8 Hz, 6H, H-26, H-27), 0.89 (d, J = 7 Hz, 3H, H-21),
0.90 (s, 3H, H-19), 2.04 (s, 3H, CH3CO), 3.84 (s, 1H, H-3),
4.87 (d, 1H, J = 2.6 Hz, H-2). EIMS m/z = 446 (1%, M+),
386 (31%, M+-AcOH), 368 (3%, M+-AcOH-H2O).
´
Instituto de Virologıa, Córdoba University, Argentina.
2.13. Compounds solutions
Stock solutions of the compounds at a concentration of
10 mg/ml were prepared in DMSO and sterilized by filtra-
tion. The solutions were kept at 4 ◦C until use.
2.9. Sodium 3α-acetoxy-2β-hydroxy-5α-cholestane
2-sulfate (12)
Triethylamine–sulfur trioxide complex (8.0 mg, 0.044
mmol) was added to a solution of 3␣-acetoxy-2␤-hydroxy-
5␣-cholestane (10) (9.9 mg, 0.022 mmol) in DMF (0.4 ml).
The reaction mixture was stirred at room temperature for
13 h, and then quenched with water (0.5 ml). After evapo-
ration to dryness the residue was eluted through Amberlite
CG-120 (sodium form) with methanol. Further purification
by preparative HPLC (methanol/water (85:15 v/v)) afforded
4.6 mg (38%) of pure 12, m.p. 120–121 ◦C together with
4 mg of compound 7 (36%). For 1H and 13C NMR see
Tables 1 and 2. FAB− m/z = 525 [M–Na]−. HRFABMS
(negative ion mode) mz = 525.3253 [M–Na]− (C29H49O6S,
ꢀ = 0.3 mmu).
2.14. Cytotoxicity test
Cellular viability was measured with 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-
Aldrich) method in Vero cells. Confluent cultures in 96-well
plates were exposed to different concentrations of the
steroids, with three wells for each concentration, and incu-
bated for 48 h at 37 ◦C. Then 10 ␮l of MM containing MTT
(5 mg/ml) was added to each well. After 2 h of incubation at
37 ◦C, the supernatant was removed and 200 ␮l of ethanol
was added to each well to solubilize the formazan crys-
tals. After vigorous shaking, absorbance was measured in
a microplate reader at 595 nm. The cytotoxic concentration
50% (CC50) was calculated as the compound concentration
required to reduce cell viability by 50%.
2.10. Sodium 2β-acetoxy-3α-hydroxy-5α-cholestane
3-sulfate (13)
2.15. Antiviral assays
By the same procedure used for the preparation of 12,
compound 11 was converted into 2␤-acetoxy-3␣-hydroxy-
5␣-cholestane 3-sulfate (13) in 75% yield, m.p. 146–148 ◦C.
For 1H and 13C NMR see Tables 1 and 2. FAB− m/z = 525
[M–Na]−. HRFABMS (negative ion mode) m/z = 525.3253
[M–Na]− (C29H49O6S, ꢀ = 0.3 mmu).
Antiviral activity was evaluated by reduction of virus
plaque formation. Vero cell monolayers grown in 24-well
plates were infected with about 50 plaque forming units
(PFU) of virus per well in the absence or presence of various

G.A.G. Santos et al. / Steroids 68 (2003) 125–132
129
concentrations of the compounds. After 1 h of adsorption,
residual inoculum was replaced by MM containing 0.7%
methylcellulose and the corresponding dose of each com-
pound. Plaques were counted after 2 days of incubation
at 37 ◦C for HSV-2 and 7 days post-infection for JV and
DEN-2. The 50% inhibitory concentration (IC50) was cal-
culated as the compound concentration required to reduce
virus plaque by 50%. All determinations were performed
twice and each in duplicate.
hand, the reaction of compound 5 with 2 equivalents of the
sulfating reagent at room temperature rendered after ion
exchange sodium 2␤,3␣-dihydroxy-5␣-cholestane 3-sulfate
(6) and sodium 2␤,3␣-dihydroxy-5␣-cholestane 2-sulfate
(7) in a 6.5:3.5 ratio, together with minor amounts of the
disulfated derivative 8 (Scheme 2). Compounds 6, 7, and
8 were purified by preparative reversed-phase HPLC and
1
characterized by HRFABMS and H and 13C NMR spec-
troscopy (Tables 1 and 2). The assignments of the NMR
signals were derived from 1H–1H COSY and HETCOR
experiments. Disodium 2␤,3␣-dihydroxy-5␣-cholestane
disulfate (8) showed two methine signals at δ 4.73 (H-2)
and 4.70 (H-3), characteristic of the presence of two sulfate
groups at C-2 and C-3 [13]. This is in accordance with the
chemical shifts observed for C-2 (76.1) and C-3 (76.1) in
the 13C NMR spectrum. The 13C NMR spectra of mono-
sulfated compounds 6 and 7 differed in the chemical shifts
of C-1 (δ 41.0 in 6, 38.8 in 7), C-2 (δ 70.0 in 6, 78.8 in 7),
C-3 (δ 78.5 in 6, 69.0 in 7), and C-4 (δ 30.3 in 6, 32.5 in
7). The downfield shift of 9.5 ppm for C-3 and the upfield
shift of 2.2 ppm for C-4 in compound 6 indicated that the
sulfate group was located at C-3. These values confirm the
assignment of the position of the sulfate group in ring A for
disodium (20R)-2␤,3␣,21-trihydroxy-5␣-cholest-24-ene-
3,21-disulfate, isolated previously by us from the antarctic
ophiuroid A. agassizii [6]. The presence of the sulfate group
at C-2 in 7 was confirmed by the downfield shift of 8.8 ppm
2.16. Virucidal assay
A virus suspension of HSV-2 containing 4 × 105 PFU
was incubated with an equal volume of MM with or without
various concentrations of the compounds for 1 h at 37 ◦C.
The samples were then diluted in cold MM to determine
residual infectivity by plaque formation. The sample dilution
effectively reduced the drug concentration to be incubated
with the cells at least 100-fold to assess that titer reduction
was only due to cell-free virion inactivation. The virucidal
concentration 50% (VC50), defined as the concentration re-
quired to inactivate virions by 50% was then calculated.
3. Results and discussion
Diol 5 was prepared from commercially available
3␤-hydroxy-5␣-cholestane (1) by the four step synthesis
shown in Scheme 1 [9–11]. Compound 5 was purified by
successive vacuum-dry column chromatography on normal
and C18 silica gel and used as starting material for the syn-
thesis of compounds 6–13. Triethylamine–sulfur trioxide
complex was selected as the sulfating reagent owing to its
facile preparation [8] and the stability of the triethylammo-
nium salts of sulfate esters [12].
Treatment of diol 5 with 6.3 equivalents of triethylamine–
sulfur trioxide complex at 95 ◦C afforded the ammonium
sulfate of compound 8, which was transformed via ion ex-
change into the disodium salt 8 (Scheme 2). On the other
1
for C-2 and the upfield shift of 2.2 ppm for C-1. H NMR
spectra showed signals at δ 4.08 (H-2) and 4.40 (H-3) for
compound 6 and δ 4.42 (H-2) and 4.02 (H-3) for compound
7, confirming the sulfate groups positions in monosulfated
derivatives 6 and 7.
Looking forward for a major selectivity in the obtention of
monosulfated analogs, an alternative synthetic strategy was
tried (Scheme 3). Diol 5 was treated with acetic anhydride
in pyridine rendering the diacetylated compound 9, which
was submitted to selective deprotection with BBTO. This
reagent has been utilized for the deprotection of a variety of
steroid esters in non-hydrolytic conditions, in particular in
Scheme 1. Conditions: (a) p-TsCl, pyridine; (b) LiCO3, LiBr, DMF; (c) m-ClPBA, H2O-CH2Cl2; (d) H2SO4, THF.

130
G.A.G. Santos et al. / Steroids 68 (2003) 125–132
Scheme 2. Conditions: (a) 2 equivalents Et3N–SO3, DMF, room temperature; (b) Amberlite CG-120 (MeOH); (c) 6.3 equivalents Et3N–SO3, DMF,
95 ◦C; (d) Amberlite CG-120 (MeOH).
the selective hydrolysis of the 3␤-acetyl group in the pres-
ence of the 6␣ one in 3␤,6␣-diacetoxy-5␣-pregnan-20-one
[14]. As shown in Scheme 2, when a solution of diacetate
9 in toluene and BBTO was refluxed for 5 h, a mixture of
monoacetylated compounds 10 and 11 was obtained in 89%
yield in a 2.7:7.3 ratio. Both monoacetylated derivatives
were separated and purified by chromatography and treated
separately with triethylamine–sulfur trioxide complex in the
same reaction conditions used for the monosulfation of diol
5 to give the sulfated analogs 12 and 13. It is to note that
while sulfation of compound 11 rendered only compound
13, compound 10 afforded the expected sulfated analog 12
together with compound 7 derived from the deacetylation of
12. As compounds 7 and 12 were easily separated by HPLC,
compound 7 was further transformed into 12 by a simple
acetylation reaction.
76.0 ppm, δH 4.44 ppm) and the acetoxy group is attached to
C-3 (δC 72.2 ppm, δH 5.11 ppm). The spectroscopic data of
13 revealed that the 2␤-hydroxy group remained protected
(δC 72.6 ppm, δH 5.14 ppm) while the sulfation took place
at the free 3␣-hydroxy group (δC 75.5 ppm, δH 4.40 ppm).
Again, assignment of proton and carbon resonances was
based on the analysis of 1H, 13C, DEPT, 1H–1H COSY, and
HETCOR NMR experiments.
A great selectivity between positions 2 and 3 was ob-
served for these reactions. Thus, in the monosulfation of diol
5, the analog sulfated at C-3 (6) was obtained as the major
product and in the deprotection reaction of the diacetylated
compound (9) with BBTO the 2␤-acetoxy-3␣-hydroxy
derivative 13 was the predominant product. These results
indicated that, as it was expected, groups attached to C-3
with ␣ configuration were more reactive than those attached
to C-2 (␤) because of the steric hindrance due to the axial
methyl group (C-19) at C-10 (Fig. 2).
1H and 13C NMR data of compound 12 (Tables 1 and
2) confirmed that the sulfate group is located at C-2 (δC
Scheme 3. Conditions: (a) Ac2O, pyridine; (b) BBTO, toluene; (c) 2 equivalents Et3N–SO3, DMF, room temperature; (d) Amberlite CG-120 (MeOH).

G.A.G. Santos et al. / Steroids 68 (2003) 125–132
131
Table 3
Antiviral and virucidal activities of sulfated steroids 6–8 and 12 and 13
Compound CC50
(␮g/ml)a
IC50
(␮g/ml)b
SI
VC50
(␮g/ml)d
c
(CC50/IC50
)
6
7
8
12
13
38
30
66
>100
39
>25
<1.5
1.6
2.8
29.0
20.4
19.4
>100
76.0
1.9
0.1
0.2
19.3
23.9
90.5
>25
2.9
1.5
13.0 >1.1
<1.6
0.9
Fig. 2. Spatial distribution of A-ring substituents in the synthetic inter-
mediates 5, 9, 10, and 11.
a Concentration required to reduce cell viability by 50%.
b Concentration that reduced the number of virus plaque by 50% S.D.
c Selectivity index.
d Concentration required to inactivate virus by 50% S.D.
The antiviral activity of the five compounds was tested
against HSV-2, the agent responsible for primary and re-
current infections of mucous membranes, genital lesions,
neonatal infections, and meningitis [15]. From the data
shown in Table 3, compounds 7 and 8 were the most active
against HSV-2 with IC50 values of 19.3 and 23.9 ␮g/ml,
respectively. Monosulfated steroid 6, differing from 7 in
the position of the sulfate group as well as acetylated and
sulfated compound 13 were not active against HSV-2 at a
concentration up to 25 ␮g/ml. The four steroids also showed
virucidal activity against HSV-2 with values of VC50 rang-
ing from 19.4 to 76.0 ␮g/ml. Due to its low cytotoxic effect
on Vero cells, the monosulfated steroid 12 containing an
acetyl group at C-2 was assayed for antiviral activity at a
higher concentration. Nevertheless, only a weak inhibitory
activity against HSV-2 was observed (IC50 = 90.5 ␮g/ml).
No virucidal effect of this compound was observed up to
100 ␮g/ml. Of all the synthetic compounds tested, disul-
fated steroid 8 showed the best selectivity index (SI = 2.8).
This result enabled us to test the antiviral activity of 8
against two pathogenic viruses in humans: JV and DEN-2.
JV causes a severe disease in humans known as Argen-
tine hemorrhagic fever [16] and DEN infection results in
acute febrile illness or a more severe manifestation known
as DEN hemorrhagic fever-shock syndrome [17]. Deriva-
tive 8 was not active against JV at a concentration up
to 25 ␮g/ml, while it elicited a marked DEN-2 inhibitory
activity (IC50 = 16.9 0.2 ␮g/ml).
Several sulfated polyhydroxysterols have been recently
isolated from sponges [18]. Most of these natural products
are characterized by the 2␤,3␣,6␣-tri-O-sulfate functions
but several bioactive sterols sulfated at C-2 (␤) and C-3
(␣) containing additional alkylation in the side chain have
been isolated from the sponges Petrosia weinbergi [13,19]
(14–18) and Pachastrella sp. [20] (19) (Fig. 3). Weinbes-
terols A (14) and B (15) and orthoesterol disulfates A (16), B
(17), and C (18) exhibited in vitro activity against the feline
leukemia virus (FeLV), mouse influenza virus (PR8), and
mouse corona virus. Compound 14 was also active in vitro
against HIV. Halistanol disulfate B (19) was active in the
Fig. 3. Disulfated steroids isolated from the sponges Petrosia weinbergi and Pachastrella sp.

132
G.A.G. Santos et al. / Steroids 68 (2003) 125–132
endothelin-converting enzyme assay while the correspond-
ing diol was totally inactive.
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Antiviral sulfated steroids from the ophiuroid Ophioplocus januarii.
J Nat Prod 1996;59:887–9.
In order to determine the role of the sulfate groups in
the antiviral activity of disulfated steroid 8, we evaluated
the in vitro activity of diol 5 against HSV-2. Although
compound 5 showed no cytotoxic effect on Vero cells
(CC50 > 200 ␮g/ml) it was not active against HSV-2 at
a concentration up to 50 ␮g/ml and showed no virucidal
effect (VC50 > 100 ␮g/ml). These results are indicative
of the role of the sulfate groups in the antiviral activity
of 8. Due to its antiviral activity, disulfated steroid 8 is a
good model compound for further introducing additional
functional groups at the steroidal skeleton or its side chain
in order to evaluate structure–activity correlations in the
inhibitory activity against pathogenic viruses in humans.
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2-Epirosten-12-ona. Qu´ımica & Industria 1995;2:19–22.
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Acknowledgments
We thank the International Foundation for Science, AN-
PCyT and the Universidad de Buenos Aires for financial
support of this work. We also wish to thank UMYMFOR
(CONICET-FCEN) for spectroscopic analysis. Mass spec-
trometry was provided by the Washington University Mass
Spectrometry Resource, an NIH Research Resource (Grant
No. PA1RR00954). M.S.M. and E.B.D. are research mem-
bers of the National Research Council of Argentina. C.A.P.
is a technical member of CONICET.
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disulfate B, a novel sulfated sterol from the sponge Pachastrella
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