Syntheses and antiproliferative activity of some sulfated hydroximinosterols

Article abstract of DOI:10.1007/s00044-012-0047-5

Four new sulfated steroidal compounds, sodium 3-hydroxy-6- hydroximinocholestane 3-sulfate (7a), sodium 3-hydroxy-6-hydroximinostigmastane 6-sulfate (7b), sodium 3-hydroxy-6-hydroximinocholest-4-ene 3-sulfate (10a), and sodium 6-hydroxy-3-hydroximinocholestane 6-sulfate (16) had been synthesized using cholesterol or stigmasterol as starting materials by different synthetic methods. The synthetic compounds were characterized by their analytical and spectral data, and their antiproliferative activity against MGC 7901 (human gastric carcinoma cell line), HeLa (human cervical carcinoma cell line), and SMMC 7404 (human liver carcinoma cell line) were investigated. The results showed that the compounds exhibited a remarkable cytotoxicity against HeLa tumor cells in vitro and 7a displayed a better cytotoxicity than cisplatin (a positive contrast).

Full text of DOI:10.1007/s00044-012-0047-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:409–414
DOI 10.1007/s00044-012-0047-5
ORIGINAL RESEARCH
Syntheses and antiproliferative activity of some sulfated
hydroximinosterols
•
•
•
•
•
Yanmin Huang Jianguo Cui Ying Li Lei Fan Yanxiao Jiao
Shaoyang Su
Received: 8 September 2011 / Accepted: 27 March 2012 / Published online: 7 April 2012
Ó Springer Science+Business Media, LLC 2012
´
evaluated for their anti-tumor activity (Krstica et al., 2007;
´
Poza et al., 2007; Bansal and Guleria, 2008; Djurendıc
Abstract Four new sulfated steroidal compounds, sodium
3-hydroxy-6-hydroximinocholestane 3-sulfate (7a), sodium
3-hydroxy-6-hydroximinostigmastane 6-sulfate (7b), sod-
ium 3-hydroxy-6-hydroximinocholest-4-ene 3-sulfate (10a),
and sodium 6-hydroxy-3-hydroximinocholestane 6-sulfate
(16) had been synthesized using cholesterol or stigmasterol as
starting materials by different synthetic methods. The syn-
thetic compounds were characterized by their analytical and
spectral data, and their antiproliferative activity against MGC
7901 (human gastric carcinoma cell line), HeLa (human
cervical carcinoma cell line), and SMMC 7404 (human liver
carcinoma cell line) were investigated. The results showed
that the compounds exhibited a remarkable cytotoxicity
against HeLa tumor cells in vitro and 7a displayed a better
cytotoxicity than cisplatin (a positive contrast).
et al., 2008; Koutsourea et al., 2008; Thibeault et al., 2008;
Mohamed et al., 2009; Stephen, 2009). Among these ste-
roidal compounds, many are sulfated polyhydroxysterols in
sodium salt forms (Han et al., 2003; Huang et al., 2006).
These compounds have exhibited a broad spectrum of
biologic activities, such as suppression of HIV replication
(Rudi et al., 2001), inhibition of protein tyrosine kinases
(Fu et al., 1994), and anti-tumor activities (Ivanchina et al.,
2000; Yang et al., 2003). Recently, we had described the
synthesis of some disodium 3b,6b-dihydroxysterol disul-
fates and their antiproliferative analysis (Cui et al., 2009a).
The results showed that disodium 3b,6b-dihydroxycholes-
tane disulfate (1) displayed a remarkable cytotoxicity
against Sk-Hep-1 (human liver carcinoma), H-292 (human
lung carcinoma), PC-3 (human prostate carcinoma), and
Hey-1B (human ovarian carcinoma) tumor cells in vitro.
Steroidal oximes have interesting bioactivity also. In our
previous work (Cui et al., 2009b, c), we found that a ste-
roidal oxime had a distinct cytotoxicity when it had a
cholesteric side chain and the 3- or 6-position on steroidal
nucleus was substituted by a hydroxyl or hydroximino. For
example, the IC50 values of the (3E,6E)-dihydroximino-
cholestane (2) (Fig. 1) against PC-3 and Hey-1B cells are
63 and 35 lmol/L, respectively.
Keywords Synthesis Á
Sodium hydroximinosterols sulfate Á
Antiproliferative activity Á Cytotoxicity
Introduction
In recent years, a variety of steroids with unusual and
interesting structures have been isolated, synthesized, and
Y. Huang Á J. Cui (&) Á Y. Jiao Á S. Su
College of Chemistry and Life Science, Guangxi Teachers
Education University, Nanning 530001, China
e-mail: cuijg1954@126.com
In order to evaluate the anti-tumor activity of new ste-
roidal derivatives and to compare the biological activity of
hydroximino and sodium sulfate groups to tumor cells, we
designed and synthesized four new sulfated steroids with
the hydroximino and sodium sulfate groups located at C-3
or C-6 on the steroidal nucleus, and investigated their
antiproliferative against HeLa (human cervical carcinoma),
SMMC 7404 (human liver carcinoma), and MGC 7901
(human gastric carcinoma) tumor cell lines.
Y. Li
Guangxi Institute of Special Equipment Supervision
and Inspection, Nanning 530219, China
L. Fan
Nanning Environmental Protection Monitoring Station,
Nanning 530012, China
123

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Med Chem Res (2013) 22:409–414
via oxidation from the 11 with Jones’ reagent. The oxima-
tion of compound 12 with NH2OHÁHCl (1:1.2) in ethanol in
the presence of NaOAc gave 3-hydroximinocholest-6-one
(13), and 13 was reduced by NaBH4 to produce 3-hydroxi-
minocholest-6-ol (14). The compound (15) was yielded by
the reaction of 14 with the triethylamine-sulfur trioxide
complex and the treatment of cation exchange resin 732
(sodium form, Na?). In the reaction, the 3-hydroximino of
14 was converted into the 3-carbonyl group of 15 at acidic
condition again. Lastly, the target product (16) was obtained
by the oximation of compound 15.
C
H3
H3C
H3C
H3C
HO
N
NaO3SO
N
OSO3Na
OH
1
2
Fig. 1 Chemical structures of compound 1 and 2
Results and discussion
Chemistry
The structures of all synthesized compounds had been
proved by their IR, NMR, and MS data.
The synthetic route of target compounds (7a, 7b, and 10a)
is outlined below in Scheme 1. First, sterols (3a, b) were
converted to 3,6-dione (4a, b) via oxidation with pyridi-
nium chlorochromate (PCC). Then the compounds 4a,
b were transformed into (5a, b) by selective reduction
using NaBH4 in the presence of NiCl2, and 4a was trans-
formed into (8a) in the presence of CoCl2, according to the
synthetic method we developed (Cui et al., 2000). The
compounds (6a, b, and 9a) were produced by the reaction
of 5a, b and 8a with the triethylamine-sulfur trioxide
complex and the treatment of cation exchange resin 732
(sodium form, Na?). Finally, the compounds (7a, b, and
10a) were obtained by the reaction of 6a, b and 9a with
hydroxylamine hydrochloride in ethanol in the presence of
NaOAc.
Biological evaluation
The cytotoxicity of compounds 7a, b, 10a, and 16 was
determined in vitro on HeLa (human cervical carcinoma),
SMMC 7404 (human liver carcinoma), and MGC 7901
(human gastric carcinoma) tumor cell lines. The MTT
method was used to analyze the antiproliferative activity.
The results are summarized as IC50 values in lmol/L in
Table 1.
As shown in Table 1, the compounds 7a and 16 dis-
played a distinct cytotoxicity against HeLa, SMMC 7404,
and MGC 7901 tumor cell lines, and 7a showed a better
cytotoxicity than cisplatin (a positive contrast) especially.
However, 7b against SMMC 7404 and 10a against MGC
7901 were almost inactive. We previously reported that the
disodium dihydroxysterol disulfates with a cholesterol-type
side chain at 17-C displayed a better cytotoxicity than the
compounds with a stigmasterol-type side chain (Cui et al.,
The synthetic route of sodium 6-hydroxy-3-hydro-
ximinocholestane 6-sulfate (16) is outlined in Scheme 2.
First, 4a was reduced by NaBH4 in the presence of NiCl2 to
produce cholest-3,6-diol (11). Compound (12) was obtained
R
R
R
b
c
d
R
R
HO
NaO SO
3
NaO SO
3
O
O
N
OH
5
a
6
7
R
R
R
O
HO
O
4
e
3
g
f
HO
NaO SO
3
NaO SO
3
O
O
N
OH
10a
9a
8a
b:
a:
R =
Scheme 1 Synthesis of sodium 3-hydroxy-6-hydroximinosterols
3-sulfates compounds. Reagents and conditions: (a) PCC/CH2Cl2,
48 h; (b) NaBH4/CH3OH, NiCl2Á6H2O, 5 min; (c) Et3N–SO3/DMF,
60 °C, 3 h; cation exchange resin 732 (sodium form)/(Na?)/MeOH, 5 h;
(d) NH2OHÁHCl, NaOAcÁ3H2O/95 % C2H5OH, 1 h; (e) NaBH4/
CH3OH, CoCl2Á6H2O; (f) Et3N–SO3/DMF, 60 °C, 3 h; cation exchange
resin 732 (sodium form)/MeOH, 5 h; and (g) NH2OHÁHCl,
NaOAcÁ3H2O/95 %C2H5OH, 1 h
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Med Chem Res (2013) 22:409–414
411
Scheme 2 Synthesis of sodium
6-hydroxy-3-
hydroximinocholestane
6-sulfate. Reagents and
conditions: (a) NaBH4/
NiCl2Á6H2O, 30 min; (b) Jones/
Me2CO, 1 h; (c) NH2OHÁHCl,
NaOAcÁ3H2O/95 %C2H5OH;
1 h; (d) NaBH4/MeOH, 30 min;
(e) Et3N–SO3/DMF, 60 °C, 3 h;
cation exchange resin 732
(sodium form)/MeOH, 5 h; and
(f) NH2OHÁHCl, NaOAcÁ3H2O/
95 %C2H5OH, 1 h
a
b
O
HO
O
O
OH
O
4
12
11
c
e
d
N
HO
HO
N
O
OH
13
14
f
O
HO N
OSO3Na
OSO3Na
15
16
Table 1 In vitro antiproliferative activities (IC50 in lmol/L) of the synthetic hydroximinosteroid analogues
Compounds
7a
7b
10a
16
Cisplatin
HeLa
9.6 1.0
14.3 1.2
[100
27.7 1.9
25.3 2.1
62.2 4.8
[100
12.5 1.0
10.2 0.8
33.3 2.7
10.1 0.9
23.2 1.8
6.7 0.4
SMMC 7404
MGC 7901
12.9 0.8
6.5 0.4
Data represent the mean values of three independent determinations
Cytotoxicity, as IC50 for each cell line, is the concentration of compound which reduced by 50 % the optical density of treated cells with respect
to untreated cells using the MTT assay
2009b. Here, the presence of a cholesterol-type side chain
appearing to be necessary for achieving a higher cytotox-
icity was confirmed again in our present work (comparing
the IC50 values of 7a with 7b). At the same time, we
previously indicated that the elimination of the 4, 5-double
bond for the steroidal oximes augmented the cytotoxic
activity against some tumor cancer cells (Cui et al., 2009c).
Similarly, the cytotoxic activity of compound 7a without
the 4,5-double bond was obviously higher than 10a with
the 4,5-double bond against these tumor cells, especially to
MGC 7901 and SMMC 7404 tumor cell line.
activity study in vivo should continue with more intensity.
Our findings could provide new evidence showing the
relationship between the chemical structure and biological
activity and may be useful for the design of new chemo-
therapeutic drugs.
Experimental
Chemistry
The sterols and NaBH4 were purchased from the Merck
Co. All chemicals and solvents were analytical grade, and
the solvents were purified by general methods before being
used. Melting points were determined on an X4 apparatus
and were uncorrected. Infrared spectra were measured with
a Nicolet FT-360 Spectrophotometer. The 1H and 13C
NMR spectra were recorded on a Bruker AV-500 spec-
trometer at working frequencies 500 and 125 MHz and on
a Bruker AV-300 spectrometer at working frequencies 300
and 75 MHz, respectively. Chemical shifts are expressed in
ppm (d) values and coupling constants (J) in Hz. LREIMS
Conclusions
In conclusion, we have prepared some sulfated hydroxi-
minosterols which were proven to be potent antitumor
activities against HeLa (human cervical carcinoma),
SMMC 7404 (human liver carcinoma), and MGC 7901
(human ventriculi carcinoma) tumor cell lines. The com-
pound 7a was found to be a more potent compound,
therefore, the further structural modification and antitumor
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Med Chem Res (2013) 22:409–414
were recorded on a Thermo-DSQ instrument. The cell
proliferation assay was performed by a MTT method using
96-well plates in Biocell ELISA analysis spectrometer.
The compounds 5a, b and 14 were prepared according
to procedures in the literature (Cui et al., 2009c), and
compound 8a was prepared according to procedures in the
literature (Cui et al., 2009b).
53.5(5-C), 51.4(24-C), 46.0(7-C), 42.7(13-C), 40.6(20-C),
40.4(12-C), 39.3(10-C), 38.0(8-C), 36.3(1-C), 31.8(4-C),
28.6(25-C), 27.8(16-C), 27.0(2-C), 25.1(28-C), 23.6(15-C),
23.5(11-C), 21.2(21-C), 20.3(26-C), 20.1(27-C), 18.1(19-
C), 12.0(18-C), 11.2(29-C).
Sodium 3-hydroxy-6-hydroximinocholestane 3-sulfate (7a)
Sodium 3-hydroxycholest-6-one 3-sulfate (6a)
CH3COONaÁ3H2O (55 mg, 0.4 mmol) and NH2OHÁHCl
(42 mg, 0.6 mmol) were added to the solution of 6a
(101 mg, 0.2 mmol) in 25 mL 95 % ethanol. The mixture
was stirred at the room temperature for 1 h. Then the
reaction was terminated and the solution was neutralized
with saturated NaHCO3. The majority of solvent was
evaporated under reduced pressure. The residue was sub-
jected to chromatography using CH3OH/CHCl3 (1:4) as the
Triethylamine-sulfur trioxide complex (180 mg, 1 mmol)
was added to a solution of 3b-hydroxycholest-6-one (5a)
(100 mg, 0.25 mmol) in DMF (1.5 mL) under an argon
atmosphere, and the mixture was stirred at 30 °C for 1 h.
Then the reaction mixture was quenched with water
(0.2 mL). The solution was poured over a silica gel column
to remove excess SO3ÁNEt3 using petroleum ether/CH2Cl2
(1:1) as the eluent, continued to elute with MeOH/CHCl3
(1:12) and followed by evaporation of the solvent obtaining
a white solid. 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 tempera-
ture. The resin was removed by filtration, and the filtrate
was concentrated. This process was repeated one more time
with 15 g of the resin. Finally, compound 6a was obtained
as colorless solid (112 mg), yield: 89 %. hmp 192–194 °C;
IR(KBr) m/cm-1: 1699, 1466, 1372, 1258, 1209, 1066,
1004, 967; 1H NMR(CD3OD, 500 MHz) d: 0.71(3H, s,
18-CH3), 0.75(3H, s, 19-CH3), 0.88(6H, d, J = 6.5, 26-
and 27-CH3), 0.95(3H, d, J = 6.0, 21-CH3), 2.21(1H, dd,
J = 13.0, 4.0, C7-aH), 2.41(1H, d, J = 12.0, C5-aH),
4.26–4.22(1H, m, C3-aH); 13C NMR(CD3OD, 125 MHz)
d: 211.8(6-C), 77.6(3-C), 56.5(14-C), 56.2(17-C), 56.1(9-
C), 53.5(5-C), 46.1(7-C), 42.8(13-C), 40.6(10-C), 39.5
(12-C), 39.3(24-C), 38.1(8-C), 36.3(22-C), 36.0(20-C),
35.7(1-C), 27.9(4-C), 27.8(25-C), 27.7(16-C), 27.1(2-C),
23.7(15-C), 23.6(23-C), 21.8(26-C), 21.7(27-C), 21.2(11-
C), 17.8(21-C), 12.0(19-C), 11.1(18-C).
eluent to give 94 mg of 7a (90 %) as a white solid, hmp
:
192–194 °C; IR(KBr) m/cm-1: 3470, 2933, 2868, 2843,
1642, 1552, 1470, 1442, 1384, 1335, 1217, 1147, 1070,
1012, 967, 890, 865, 829, 702, 628, 587; 1H NMR(CD3OD,
500 MHz) d: 0.72(3H, s, 18-CH3), 0.77(3H, s, 19-CH3),
0.90(3H, d, J = 6.5, 26-CH3 or 27-CH3), 0.91(3H, d, J =
6.5, 26-CH3 or 27-CH3), 0.96(3H, d, J = 6.5, 21-CH3),
2.17(1H, brd, J = 13.0, C5-H), 3.34(1H, dd, J = 14, 4.5,
C7-aH), 4.27(ddd, 1H, J = 16.5, 11.0, 4.5, C3-H), 4.58 (s,
1H, =N–OH); 13C NMR(CD3OD, 125 MHz) d: 158.7(6-C),
78.4(3-C), 56.6(14-C), 56.2(17-C), 54.2(9-C), 49.4(13-C),
42.7(5-C), 39.7(10-C), 39.3(24-C), 38.4(12-C), 36.05(4-C),
36.00(22-C), 35.7(20-C), 35.7(1-C), 29.1(8-C), 28.7(16-C),
28.1(25-C), 27.9(2-C), 27.8(7-C), 23.8(15-C), 23.6(23-C),
21.9(27-C), 21.6(26-C), 21.2(11-C), 17.9(21-C), 11.5(19-
C), 11.2(18-C). ESI–MS m/z: 496 (M-23).
Sodium 3-hydroxy-6-hydroximinostigmastane 3-sulfate
(7b)
Compound 7b was prepared similarly according to the
procedure of 7a. Yield 93 %, hmp 190–192 °C; IR(KBr) m/
cm-1: 3398, 2957, 2859, 1655, 1454, 1380, 1247, 1208,
1
Sodium 3-hydroxystigmast-6-one 3-sulfate (6b)
1073, 967, 828, 632; H NMR(CD3OD, 500 MHz) d: 0.74
(3H, s, 18-CH3), 0.78(3H, s, 19-CH3), 0.84(3H, d, J = 4.5,
26 or 27-CH3), 0.85(3H, t, J = 7.0, 29-CH3), 0.89(3H, d,
J = 4.5, 26 or 27-CH3), 1.06(3H, d, J = 5.0, 21-CH3), 2.17
(1H, brd, J = 12.5, C5-H), 3.37–3.32(2H, m, C7-H), 4.29
(1H, ddd, J = 17.0, 12.5, 5.5, C3-H), 5.07(1H, dd, J = 14.5,
9.0, C22-H), 5.20(1H, dd, J = 14.5, 8.5, C23-H); 13C
NMR(CD3OD, 125 MHz) d: 157.2(6-C), 138.4(22-C), 129.2
(23-C), 76.3(3-C), 56.4(14-C), 55.8(17-C), 53.7(9-C), 53.5(24-
C), 51.4(13-C), 46.0(5-C), 42.7(10-C), 40.6(12-C), 40.4(20-
C), 39.3(4-C), 38.0(1-C), 36.3(8-C), 31.8(25-C), 28.6(2-C),
27.8(7-C), 27.0(28-C), 25.1(16-C), 23.6(19-C), 23.5(15-C),
21.2(11-C), 20.3(27-C), 20.1(26-C), 18.1(21-C), 12.4(18-C),
12.3 (29-C). ESI–MS m/z: 522 (M-23) .
Compound 6b was prepared similarly according to the
procedure of 6a. Yield 96 %, hmp 169–171 °C; IR(KBr) m/
cm-1: 1711, 1650, 1458, 1384, 1253, 1221, 1066, 1004,
1
959; H NMR(CD3OD, 500 MHz) d: 0.75(3H, s, 18-CH3),
0.78(3H, s, 19-CH3), 0.84(3H, d, J = 6.0, 26- or 27-CH3),
0.85(3H, t, J = 7.5, 29-CH3), 0.89(3H, d, J = 6.0, 26- or
27-CH3), 1.06(3H, d, J = 7.0, 21-CH3), 2.22(1H, dd,
J = 13.0, 4.5, C7-aH), 2.43(1H, dd, J = 12.5, 2.5, C5-aH),
4.26(1H, tdd, J = 11.0, 5.5, 4.5, C3-aH), 5.08(1H, dd,
J = 15.0, 8.0, C22-H), 5.21(1H, dd, J = 15.0, 9.0, C23-H);
13C NMR(CD3OD, 125 MHz) d: 211.8(6-C), 138.2(22-C),
129.4(23-C), 77.5(3-C), 56.6(14-C), 56.2(17-C), 56.0(9-C),
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Med Chem Res (2013) 22:409–414
413
Sodium 3b-hydroxycholest-4-en-6-one 3-sulfate (9a)
was obtained as a colorless solid (85 mg), yield: 82 %, hmp
186–188 °C. IR (KBr) m/cm-1: 2945, 1703, 1466, 1393,
1245, 1074, 1008, 914, 780; 1H NMR (DMSO-d6,
500 MHz) d: 0.67(3H, s, 18-CH3), 0.83(3H, d, J = 6.6, 26
or 27-CH3), 0.83(3H, d, J = 6.6, 26 or 27-CH3), 0.89(d,
3H, J = 6.6, 21-CH3), 1.03(3H, s, 19-CH3), 4.06(1H, d,
J = 2.6, C6-H); 13C NMR (DMSO-d6, 125 MHz) d: 211.9,
75.1, 56.3, 56.2, 53.4, 47.9, 46.3, 42.7, 40.6, 39.4, 39.1,
38.0, 36.8, 36.1, 35.6, 35.6, 30.0, 28.3, 27.8, 24.4, 23.7,
23.1, 22.9, 21.3, 19.1, 14.6, 12.4.
Compound 9a was prepared similarly according to the
procedure of 6a. Yield 85 %. hmp : 141–143 °C; IR (KBr)
m/cm-1: 1658, 1466, 1376, 1229, 1045, 972; 1H NMR
(DMSO-d6, 300 MHz) d: 0.69(3H, s, 18-CH3), 0.84(3H, d,
J = 6.6, 26- or 27-CH3), 0.85(3H, d, J = 6.6, 26- or
27-CH3), 0.89(3H, d, J = 6.3, 21-CH3), 1.14(3H, s,
19-CH3), 3.10(1H, dd, J = 7.2, 4.8, C7-aH), 4.68(1H, ddd,
J = 12.3, 5.4, 1.8, C3-aH), 5.97(1H, s, C6-H); 13C NMR
(DMSO-d6, 75 MHz) d: 198.6(6-C), 169.1(5-C), 120.4(4-C),
72.6(3-C), 56.0(14-C), 55.6(17-C), 53.3(9-C), 46.3(13-C),
42.5(7-C), 36.2(12-C), 36.1(24-C), 35.6(10-C), 33.9(22-C),
33.8(20-C), 28.2(8-C), 27.9(1-C), 24.2(2-C), 23.6(15-C),
23.1(16-C), 23.0(25-C), 22.9(26-C), 22.8(27-C), 21.0(23-C),
19.0(11-C), 18.2(19-C), 12.2(21-C), 9.2(18-C).
Sodium 6-hydroxy-3-hydroximinocholestane 6-sulfate (16)
NH2OHÁHCl (21 mg, 0.3 mmol) was added to the solution
of 15 (76 mg, 0.15 mmol) and CH3COONaÁ3H2O (41 mg,
0.3 mmol) in 10 mL 95 % ethanol in the interval of
10 min. The mixture was stirred at the room temperature
for 1 h. Then the reaction was terminated and the solution
was neutralized with saturated NaHCO3. The majority of
solvent was evaporated under reduced pressure. The resi-
due was subjected to chromatography using CH3OH/
CHCl3 (1:4) as the eluent to give 60 mg of 16 (90 %) as a
white solid, hmp 197–199 °C; IR (KBr) m/cm-1: 3440,
2941, 2872, 1638, 1544, 1470, 1380, 1213, 1066, 1008,
Sodium 3b-hydroxy-6-hydroximinocholest-4-ene 3-sulfate
(10a)
Compound 10a was prepared similarly according to the
procedure of 7a. Yield 72 %. hmp 98–103 °C; IR (KBr) m/
cm-1: 3440, 2942, 2860, 1617, 1478, 1380, 1106, 800; H
1
NMR (DMSO-d6, 300 MHz) d: 0.67(3H, s, 18-CH3),
0.838(3H, d, J = 6.6, 26- or 27-CH3), 0.842(3H, d,
J = 6.6, 26- or 27-CH3), 0.88(3H, d, J = 6.0, 21-CH3),
1.03(3H, s, 19-CH3), 3.14(1H, m, C7-aH), 4.65–4.55 (1H,
m, C3-aH), 6.08(1H, d, J = 1.8, C4-H); 13C NMR(CD3OD,
125 MHz) d: 175.7(6-C), 151.4(5-C), 108.4(4-C), 73.1(3-
C), 56.0(14-C), 55.8(17-C), 53.7(9-C), 49.1(13-C),
42.4(10-C), 38.5(12-C), 37.2(24-C), 36.1(22-C), 35.6(20-
C), 34.2(1-C), 34.0(8-C), 28.2(7-C), 25.6(2-C), 24.6(16-C),
24.3(25-C), 23.6(15-C), 23.1(23-C), 22.9(11-C), 21.5(26-
C), 21.3(27-C), 19.0(19-C), 18.8(21-C), 12.2(18-C); ESI–
MS m/z: 498 (M-23).
1
967, 829, 624, 583; H NMR (DMSO-d6, 500 MHz) d:
0.66(3H, s, 18-CH3), 0.83(3H, d, J = 3.0, 26-CH3 or
27-CH3), 0.85(3H, d, J = 3.0, 26-CH3 or 27-CH3),
0.89(3H, d, J = 6.6, 21-CH3), 0.94(3H, s, 19-CH3),
3.04–3.01(1H, m, C2-bH), 4.12 (1H, ddd, J = 12.0, 5.4,
3.0, C6-bH), 10.08(brs, 1H, =NOH); 13C NMR (DMSO-d6,
125 MHz) d: 158.4, 76.0, 56.7, 54.1, 53.9, 49.0, 47.8, 43.1,
39.0, 37.2, 36.6, 36.2, 32.2, 31.1, 28.7, 28.3, 25.4, 24.8,
24.2, 23.6, 23.3, 21.5, 20.8, 19.5, 15.2, 14.9, 12.9; ESI–MS
m/z: 496 (M-23).
Cell culture and assay for cell viability
Sodium 6-hydroxycholest-3-one 6-sulfate (15)
SMMC 7404 (human liver carcinoma), MGC 7901 (gastric
carcinoma), and HeLa (cervical carcinoma) cancer cells
were grown in RPMI-1640 supplemented with 10 % cos-
mic calf serum (Hyclone) and antibiotics in a humidified
atmosphere of 5 % CO2 at 37 °C. The viability of these
cells was determined using the colorimetric Cell Titer 96
aqueous Cell Proliferation Assay (MTT) according to the
instructions provided by the manufacturer. In brief, cells
(1 9 104 cells per well) were seeded in 96-well plates. One
day after seeding, the cells were treated with or without
different concentration of each compound and reincubated
for 72 h. After the cells were washed with sterile phosphate
buffer saline (PBS), 190 lL of RPMI-1640 and 10 lL of
the tetrazolium dye (MTT) (5 mg/mL) solution were added
to each well, and the cells were incubated for an additional
4 h. The medium was discarded and 200 lL of DMSO was
Triethylamine-sulfur trioxide complex (290 mg, 1.6 mmol)
was added to a solution of (3E)-Hydroximinocholest-6-ol
(14) (104 mg, 0.2 mmol) in DMF (1 mL) under an argon
atmosphere, and the mixture was stirred at 60 °C for 2 h.
Then the reaction mixture was quenched with water
(0.2 mL). The solution was poured over a silica gel column
to remove excess SO3ÁNEt3 using petroleum ether/CH2Cl2
(1:1) as the eluent, continued to elute with MeOH/CHCl3
(1:12), and followed by evaporation of the solvent
obtaining a white solid. To the solution of the solid in
methanol (15 mL) was added Cation exchange resin 732
(sodium form) (Na?) (5 g), and stirred for 5 h at room
temperature. The resin was removed by filtration, and the
filtrate was concentrated. This process was repeated one
more time with 5 g of the resin. Finally, the compound 15
123

414
Med Chem Res (2013) 22:409–414
Huang SF, Cui JG, Liu ZP, Gan CF (2006) Recent advances of
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added to dissolve the purple formazan crystals formed. The
absorbance (A) at 492 nm was measured using a Biocell
ELISA analysis spectrometer.
Acknowledgments The authors acknowledge the financial support
of the Natural Science Foundation of Guangxi Province (No:
2010GXNSFD013019) and the Natural Science Foundation of
Guangxi Province (No: 2010GXNSFA013052).
´
Krstica NM, Bjelakovic MS, Zizakb Z, Pavlovic MD, Juranic ZD,
Pavlovic VD (2007) Synthesis of some steroidal oximes,
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