Synthesis and cytotoxic analysis of some disodium 3β,6β-dihydroxysterol disulfates

Article abstract of DOI:10.1016/j.steroids.2009.08.006

Disodium 3β,6β-dihydroxy-5α-cholestane disulfate (1) was synthesized in 4 steps with a high overall yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH₄ in the presence of NiCl₂ to produce cholest-3β,6β-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated diammonium 3β,6β-dihydroxy-5α-cholestane disulfate (7a) and the treatment of 7a by cation exchange resin 732 (sodium form)(Na⁺) yielded the target steroid 1. Disodium 24-ethyl-3β,6β-dihydroxycholest-22-ene disulfate (2) and disodium 24-ethyl-3β,6β-dihydroxycholestane disulfate (3) were synthesized using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a cholesterol-type side chain at position 17 is necessary for their biological activity.

Full text of DOI:10.1016/j.steroids.2009.08.006

Steroids 74 (2009) 1057–1060
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and cytotoxic analysis of some disodium
3␤,6␤-dihydroxysterol disulfates
Jianguo Cuia,∗, Hui Wanga, Yanmin Huanga, Yi Xinb, Aimin Zhoub,∗∗
a College of Chemistry and Life Science, Guangxi Teachers Education University, Nanning, 530001, China
b Department of Chemistry, Cleveland State University, Cleveland, OH 44115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Disodium 3␤,6␤-dihydroxy-5␣-cholestane disulfate (1) was synthesized in 4 steps with a high overall
yield from cholesterol. First, cholesterol (4a) was converted to cholest-4-en-3,6-dione (5a) via oxidation
with pyridinium chlorochromate (PCC) and then 5a was reduced by NaBH4 in the presence of NiCl2 to pro-
duce cholest-3␤,6␤-diol (6a). The reaction of 6a with the triethylamine-sulfur trioxide complex generated
diammonium 3␤,6␤-dihydroxy-5␣-cholestane disulfate (7a) and the treatment of 7a by cation exchange
resin 732 (sodium form)(Na+) yielded the target steroid 1. Disodium 24-ethyl-3␤,6␤-dihydroxycholest-
22-ene disulfate (2) and disodium 24-ethyl-3␤,6␤-dihydroxycholestane disulfate (3) were synthesized
using a similar method. The cytotoxicity of these compounds against Sk-Hep-1 (human liver carcinoma
cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-
1B (human ovarian carcinoma cell line) cells was investigated. Our results indicate that presence of a
cholesterol-type side chain at position 17 is necessary for their biological activity.
Received 21 February 2009
Received in revised form 7 August 2009
Accepted 13 August 2009
Available online 22 August 2009
Keywords:
Disodium 3␤,6␤-dihydroxysterol disulfates
Synthesis
Biological activity
Antineoplastic activity
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
2. Results and discussion
In recent years, a variety of steroids with unusual and inter-
esting structures have been isolated from a wide range of marine
organisms, particularly from sponges and echinoderms [1–3].
Among these steroidal compounds, many are sulfated polyhy-
droxysterols in sodium salt forms [4,5]. These compounds have
exhibited a broad spectrum of biologic activities, such as sup-
pression of HIV replication [6–9], inhibition of protein tyrosine
kinases [10] and antitumor activities [11–13]. Therefore they
greatly attract the attention of organic chemists and biomedical
scientists [14–16]. Most of the sulfated polyhydroxysteroids iso-
lated from these echinoderms or ophiuroids are found that the
disulfation occurs on two hydroxyls located at C-2 and C-3 or
C-3 and C-7, or C-3 and C-21 [17]. In this report, we synthe-
sized three disulfated steroids with sulfate groups located at C-3
and C-6 on the ring A and B as shown in Fig. 1 and determined
their cytotoxicity against cancer cells. Interestingly we found that
the cytotoxicity of these compounds was differentially changed
along with the structure of the R group. Our finding provides
new evidence for the relationship between chemical structure and
biofunctions.
2.1. Chemistry
The synthesis of sulfated 3,6-dihydroxysterol was based on
the methodology developed by Arnostova et al. [18]. Here
we present a more efficient method for the synthesis of dis-
odium 3␤,6␤-dihydroxysterol disulfates with a cholesterol-like or
stigmasterol-like or sitosterol-like side chain at position 17 [19,20].
As shown in Scheme 1, cholesterol, stigmasterol, and ␤-sitosterol
were used as raw materials.
Compounds (4a–4c) were converted to the corresponding 4-en-
3, 6-dioxysteroids (5a–5c) via oxidation with PCC in CH2Cl2. The
reduction of 5a–5c by NaBH4 in the presence of NiCl2 gave 3␤,6␤-
dihydroxysteroids (6a–6c) according to the synthetic method we
previously developed [21]. The compounds 6a–6c presented a typ-
ical difference in their spectrum from their parent compounds
5a–5c. In the IR spectra the absorption at 1714 and 1686 cm−1 for
the original diketone carbonyl groups (CO) of 5b, was replaced by a
new broad absorption at 3427 cm−1 for 6b, indicating that the car-
bonyl groups in 5b had been changed to the hydroxy groups in 6b. In
addition, the double bond of 5b at 1609 cm−1 had been eliminated
in 6b. In the 1H NMR spectrum the chemical shifts at 3.650 ppm (1H,
tt, J = 11.0, 5.0, C3-␣H) and 3.803 ppm (1H, dd, J = 5.5, 2.5, C6-␣H) for
6b showed a ␣-configuration of 3-H and 6-H.
∗
Corresponding author. Tel.: +86 771 3908308; fax: +86 771 3908308.
Corresponding author. Tel.: +1 216 687 2416; fax: +1 216 687 9298.
Based on the synthetic method developed by Comin et al.
[14], the treatment of 6a–6c with triethylammonium-sulfur tri-
∗∗
E-mail addresses: cuijg1954@126.com (J. Cui), a.zhou@csuohio.edu (A. Zhou).
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.08.006

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J. Cui et al. / Steroids 74 (2009) 1057–1060
Table 1
In vitro antitumor activities (IC50 in nmol/mL) of compounds 1, 2 and 3.a.
Compound
Sk-Hep-1
H-292
PC-3
Hey-1B
1
2
3
21
160
>200
63
120
>200
35
121
>200
33
170
>200
a
The MTS method was used to analyze the antiproliferative activity.
(2), and sitosterol-like side chain (3). Obviously the presence of a
cholesterol-type side chain at 17-C is necessary for the best biolog-
ical activity. The morphological change of the cancer cells induced
by compound 1 was shown in Fig. 1A and B.
3. Experiments
The sterol and NaBH4 were purchased from Merck Co. All chem-
icals and solvents were analytical grade and solvents were purified
by general methods before being used. Melting points were deter-
mined on an X4 apparatus and were uncorrected. Infrared spectra
were measured with a Nicolet FT-360 Spectrophotometer. The 1
H
and 13C NMR spectra were recorded in CDCl3 or DMSO on a Bruker
AV-500 spectrometer at working frequencies 500 and 125 MHz,
respectively. Chemical shifts are expressed in ppm (ı) values and
coupling constants (J) in Hz. Mass spectra (ESI) were recorded on
an LCMS-2010A instrument. The cell viability was determined by
the MTS method using 96-well plates in a LD400 AD/LD analysis
spectrometer (Beckman Coulter).
Scheme 1. Reagents and conditions: (a) PCC/CH2Cl2, (b) NaBH4/NiCl2·6H2O, (c)
Et3N-SO3/DMF, and (d) cation exchange resin 732 (sodium form)(Na+)/MeOH.
oxide complex gave the ammonium sulfate of compounds 1–3,
which was converted to their disodium salt via ion exchange
(Scheme 1). The presence of the sulfate group in 1–3 was con-
firmed by the downfield shift of 3-H and 6-H to 3.961–3.966 ppm
and 4.140–4.152 ppm, and by comparing to the 1H NMR spectra of
6a and 6c (3.65–3.70 ppm and 3.80–3.82 ppm, respectively).
3.1. The synthesis of compounds 5a–5c
Cholest-4-en-3,6-dione (5a): Pyridinium chlorochromate (PCC)
(2.564 g, 11.87 mmol) was added to a solution of cholesterol (4a)
(0.924 g, 2.2 mmol) in dried CH2Cl2 (40 mL) in one portion at room
temperature. The reaction was completed in 28 h. To the mixture
was then added 30 mL of CH2Cl2, and the suspension was poured
over a silica gel column and eluted with CH2Cl2. The eluate was
washed with cold water and saturated brines. After drying over
anhydrous sodium sulfate, the solvent was removed under reduced
pressure, and the crude product was purified by chromatography
on silica gel using petroleum ether (60–90 ◦C)/EtOAc (5: 1) as the
eluent to give 0.795 g (84%) of 5a as pale yellow crystals, m.p.
90–91 ◦C; IR (KBr) ꢀ 2953, 2865, 1693, 1600, 1486, 1249, 1221,
2.2. Antitumor activities
The antitumor activities of all these sulfated 3␤,6␤-
dihydroxysterols were determined in vitro on Sk-Hep-1 (human
liver carcinoma), H-292 (human lung carcinoma), PC-3 (human
prostate carcinoma) and Hey-1B (human ovarian carcinoma)
tumor cells. The results were summarized as IC50 values in
nmol/mL in Table 1.
Compound 1 displayed significantly higher cytotoxicity against
these cancer cells when compared to compounds 2 and 3. Inter-
estingly, the cytotoxicity of the compounds against these cancer
cells was increased along with the order of the side chain at
17-C: cholesterol-like side chain (1), stigmasterol-like side chain
1117, 942 cm−1 1H NMR(CDCl3, 500 MHz): 0.746(3H, s, 18-CH3),
;
0.886(3H, d, J = 6.4, 26 or 27-CH3), 0.899(3H, d, J = 6.4, 26 or 27-
CH3), 0.952(3H, d, J = 6.5, 21-CH3), 1.172(3H, s, 19-CH3), 2.546(1H,
dd, J = 14.6, 5.2, C2-␤H), 2.706(1H, dd, J = 16.0, 4.0, C7-␣H), 6.196(1H,
s, C4-H).
Fig. 1. The morphology of cancer cells. Hey 1B (A) and Sk-Hep-1 (B) cells were treated with compound 1 at various doses for 24 h and 48 h, respectively. The photos of cell
morphology were taken under an Olympus CKX31 microscope at 100× magnification.

J. Cui et al. / Steroids 74 (2009) 1057–1060
1059
24-Ethylcholest-4,22-dien-3,6-dione (5b): Yield: 83%, m.p.
134–135 ◦C; IR (KBr) ꢀ: 2959, 1714, 1686, 1609, 969, 864 cm−1 1H
trated. This process was repeated one more time with 15 g of the
resin. Finally compound 1 was obtained as a colorless solid (45 mg),
yield: 60%, m.p. 173–174 ◦C; IR(KBr) ꢀ: 3464, 2937, 2868, 1470,
;
NMR(CDCl3, 500 MHz): 0.743(3H, s, 18-CH3), 0.805(3H, t, J = 7.0,
29-CH3), 0.798(3H, d, J = 6.5, 26- or 27-CH3), 0.849(3H, d, J = 6.5,
26- or 27-CH3), 1.036(3H, d, J = 7.0, 21-CH3), 1.169(3H, s, 19-CH3),
5.040(1H, dd, J = 15.2, 9.0, C22-H), 5.150(1H, dd, J = 15.2, 8.5, C23-H),
6.171(1H, s, C4-H).
1383, 1226, 1066, 980, 947, 853, 820, 624 cm−1 1H NMR(DMSO,
;
500 MHz): 0.657(3H, s, 18-CH3), 0.851(3H, d, J = 6.5, 26- or 27-CH3),
0.855(3H, d, J = 6.5, 26- or 27-CH3), 0.896(3H, d, J = 6.5, 21-CH3),
0.932(3H, s, 19-CH3), 1.935(1H, brd, J = 12.5, C2-␤H), 3.966(1H, m,
C3-␣H), 4.140(1H, s, C6-␣H); 13C NMR(DMSO, 125 MHz): 36.1(1-
C), 28.2(2-C), 70.1(3-C), 33.4(4-C), 35.4(5-C), 76.1(6-C), 42.8(7-C),
30.5(8-C), 47.7(9-C), 38.6(10-C), 21.1(11-C), 39.4(12-C), 40.6(13-C),
56.2(14-C), 24.4(15-C), 27.8(16-C), 54.2(17-C), 12.4(18-C), 15.8(19-
C), 35.6(20-C), 19.0(21-C), 36.1(22-C), 23.7(23-C), 40.5(24-C),
24-Ethylcholest-4-en-3,6-dione (5c): Yield: 86%, m.p. 172–174 ◦C.
IR (KBr) ꢀ: 2959, 1683, 1601, 1581, 1461, 1377, 1246, 1124,
948, 871 cm−1 1H NMR(CDCl3, 500 MHz): 0.724(3H, s, 18-CH3),
;
0.816(3H, d, J = 7.0, 26- or 27-CH3), 0.841(3H, d, J = 7.0, 26- or 27-
CH3), 0.848(3H, t, J = 8.0, 29-CH3), 0.935(3H, d, J = 6.5, 21-CH3),
1.167(3H, s, 19-CH3), 2.13–2.17(1H, m, C2-␣H), 2.44–2.58 (2H, m,
C7-␤H and C2-␤H), 2.682(1H, dd, J = 15.5, 4.5, C7-␣H), 6.170(1H, s,
C4-H).
29.2(25-C), 22.8(26-C), 23.1(27-C); (-)LRESIMS m/z: 585[M−Na]−1
,
563[M−2Na+H]−1
.
Disodium 24-ethyl-3ˇ,6ˇ-dihydroxycholest-22-ene disulfate (2):
Yield: 66%; m.p. 174–175 ◦C; IR(KBr) v: 2937, 2864, 1646, 1466,
3.2. The synthesis of compounds 6a–6c
1368, 1209, 1066, 967, 853, 812 cm−1 1H NMR(DMSO, 500 Hz):
;
0.679(3H, s, 18-CH3), 0.778 (3H, t, J = 4.4, 29-CH3), 0.795(3H,
d, J = 6.1, 26- or 27-CH3), 0.835(3H, d, J = 6.1, 26- or 27-CH3),
0.935(3H, s, 19-CH3), 0.998(3H, d, J = 6.5, 21-CH3), 1.917(1H,
d, J = 12.5, C2-␤H), 2.031(1H, m, C20-H), 3.961(1H, m, C3-␣H),
4.152(1H, d, J = 3.4, C6-␣H), 5.031(1H, dd, J = 15.1, 8.6, C22-H),
5.161(1H, dd, J = 15.1, 8.8, C23-H); 13C NMR(DMSO, 125 MHz):
35.5(1-C), 27.4(2-C), 70.1(3-C), 33.4(4-C), 31.8(5-C), 76.1(6-C),
45.7(7-C), 29.2(8-C), 54.2(9-C), 38.6(10-C), 21.4(11-C), 40.6(12-C),
44.2(13-C), 56.4(14-C), 25.3(15-C), 29.0(16-C), 56.0(17-C), 12.7(18-
C), 15.9(19-C), 42.6(20-C), 21.6(21-C), 138.6(22-C), 129.3(23-C),
47.8(24-C), 30.5(25-C), 21.1(26-C), 19.4(27-C), 24.4(28-C), 12.6(29-
Cholest-3ˇ,6ˇ-diol (6a): NaBH4 (90 mg, 2.38 mmol) was added
to a solution of 5a (200 mg, 0.50 mmol) and NiCl2.6H2O (120 mg,
0.50 mmol) in CH3OH (20 mL) in 1 min at room temperature. After
20 min, the reaction was stopped. The solution was neutralized
with 1 M HCl. After evaporating the majority of the MeOH under
reduced pressure, ethyl acetate (40 mL) was added to the residue.
The resulting solution was washed with cold water and saturated
brines. After drying over anhydrous sodium sulfate, the solvent
was removed under reduced pressure, and the crude product was
then purified by flash chromatography on silica gel using petroleum
ether /ethyl acetate (1:1) as the eluent, to give 6a as a white solid
(164 mg, 81%), m.p. 161–162 ◦C; IR(KBr) ꢀ: 3399, 2925, 2868, 1470,
1360, 1319, 1172, 1078, 1041, 1021, 955, 767 cm−1; 1H NMR(CDCl3,
500 MHz): 0.709(3H, S, 18-CH3), 0.878(3H, d, J = 2.2, 26 or 27-CH3),
0.891(3H, d, J = 2.2, 26 or 27-CH3), 0.927(3H, d, J = 6.4, 21-CH3),
1.052(3H, s, 19-CH3), 3.64–3.82(1H, m, C3-␣H), 3.825(1H, d, J = 2.4,
C6-␣H).
C); (-)LRESIMS m/z: 611[M−Na]−1, 588[M−2Na]−2
.
Disodium 24-ethyl-3ˇ,6ˇ-dihydroxycholestane disulfate (3):
Yield: 65%; m.p. 242–249 ◦C; IR(KBr) v: 3427, 2925, 1634, 1401,
1209, 815 cm−1 1H NMR(DMSO, 500 MHz): 0.650(3H, s, 18-CH3),
;
0.798(3H, d, J = 7.0, 26- or 27-CH3), 0.818(3H, d, J = 7.0, 26- or
27-CH3), 0.825(3H, t, J = 6.5, 29-CH3), 0.896(3H, d, J = 6.3, 21-CH3),
0.923(3H, s, 19-CH3), 1.924(1H, brad, J = 12.5, C2-␤H), 3.961(1H, m,
24-Ethylcholest-22-en-3ˇ,6ˇ-diol (6b): Yield: 72%; m.p.
C3-H), 4.181(1H, d, J = 3.5, C6-H); (-)LRESIMS m/z: 613[M−Na]−1
,
207–209 ◦C; IR(KBr) ꢀ: 3427, 2929, 2864, 1581, 1462, 1380,
590[M−2Na]−2
.
1037, 967 cm−1 1H NMR(CDCl3, 500 MHz): 0.708(3H, s, 18-CH3),
;
0.795(3H, d, J = 6.5, 26 or 27-CH3), 0.845(3H, d, J = 6.5, 26 or
27-CH3), 0.802(3H, t, J = 7.5, 29-CH3), 1.013(3H, d, J = 7.0, 21-CH3),
1.033(3H, s, 19-CH3), 3.650(1H, tt, J = 11.0, 5.0, C3-␣H), 3.803(1H,
dd, J = 5.5, 2.5, C6-␣H), 5.022(1H, dd, J = 15.0, 8.5, C22-H), 5.140(1H,
dd, J = 15.0, 8.5, C23-H).
3.4. Assay for cell viability
3.4.1. Materials and methods
Stock solutions of compounds 1, 2 and 3, were prepared in sterile
dimethyl sulfoxide (DMSO) (Sigma) at a concentration of 10 mg/mL,
and diluted to proper concentrations according to the experimental
design with the cell culture medium.
24-Ethylcholest-3ˇ,6ˇ-diol (6c): Yield: 72%; m.p. 172–173 ◦C;
IR(KBr) ꢀ: 3419, 3125, 2941, 2872, 1397, 1172, 1033 cm−1 1H
;
NMR(CDCl3, 500 MHz): 0.728(3H, s, 18-CH3), 0.831(6H, d, J = 7.6 Hz,
26 and 27-CH3), 0.866(3H, d, J = 6.2 Hz, 29-CH3), 0.896(3H, s, 21-
CH3), 1.027(3H, s, 19-CH3), 3.667(1H, t, J = 5.5, C3-H), 3.821(1H, s,
C6-H).
3.4.2. Cell culture
Sk-Hep-1, H-292, PC-3 (ATCC) and Hey-1B (a gift from Dr. Yan
Xu, University of Indiana) cells were cultured in a proper medium
supplemented with 10% fetal bovine serum and 0.1 g/L penicillin
G + 0.1 g/L streptomycin sulfate in a humidified atmosphere of 5%
CO2 at 37 ◦C.
3.3. General procedure for the synthesis of compounds 1–3
Disodium 3ˇ,6ˇ-dihydroxy-5˛-cholestane disulfate (1): The
triethylamine-sulfur trioxide complex (173 mg, 0.96 mmol) was
added to a solution of cholest-3␤,6␤-diol (6a) (50 mg, 0.12 mmol)
in DMF (0.6 mL) under an argon atmosphere, and the mixture was
stirred at 95 ◦C for 3 h. Then the reaction mixture was quenched
with water (0.2 mL). The solution was poured over a silica gel
column to remove excess SO3·NEt3. The product was eluted by
using petroleum ether /CH2Cl2 (1:1) as the eluent, and then con-
tinued to be eluted with MeOH/CHCl3(1:12), and followed by
evaporation of the solvent to yield a white solid (diammonium
3␤,6␤-dihydroxy-5␣-cholestane disulfate). To the solution of the
solid in methanol (15 mL) was added Cation exchange resin 732
(sodium form) (Na+) (10 g), and stirred for 5 h at room temperature.
The resin was removed by filtration, and the filtrate was concen-
3.4.3. Treatment of cancer cells
Cancer cells (4 × 104 cells/mL, 200 ␮L) were seeded into each
well of a 96-well microtiter plate. After incubation for 24 h, the
compounds with a series of concentrations (range 5–100 ␮g/mL)
were added to the cells. An equal amount of DMSO was added to
the cells as negative controls. All were treated in triplicate.
3.4.4. Determination of cell viability
MT Stetrazolium salt ((3-4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
(CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay,
Cat #G5421, Promega Corporation) dye reduction assay was used
to determine the cell viability. The assay is dependent on the

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J. Cui et al. / Steroids 74 (2009) 1057–1060
reduction of MTS by mitochondrial dehydrogenases of viable cells
to a blue water soluble formazan product, which can be measured
spectrophotometrically. The absorbance of the formazan product
at 490 nm is in linear proportion to cell numbers. Briefly, after
treatment (see 3.4.3) with the compounds for 48 h, the medium
was removed and the cells were incubated with 100 ␮L of fresh
medium plus 20 ␮L of MTS solution for additional 4 h according
to the instruction provided by the manufacturer. The absorbance
(A) at 490 nm was measured using a LD400 AD/LD analysis spec-
trometer (Beckman coulter). The IC50 value was calculated as the
concentration of drug yielding 50% cell survival.
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The authors acknowledge the financial support of the National
Natural Science Foundation of China (No.: 20562001), the Natural
Science Foundation of Guangxi Province (No.: 0575054) of China.
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