Synthesis and anti-glioma activity of 25(R)-spirostan-3β,5α,6β,19-tetrol

Article abstract of DOI:10.1016/j.steroids.2009.12.005

Malignant gliomas are common and aggressive brain tumours in adults. The rapid proliferation and diffuse brain migration are the main obstacles to successful treatment. Here, we show 25(R)-spirostan-3β,5α,6β,19-tetrol, a polyhydroxy steroid, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. The compound 25(R)-spirostan-3β,5α,6β,19-tetrol was synthesised by seven steps starting from diosgenin in 8.55% overall yield. The structures of the synthetic compounds were characterised by infrared (IR), ¹H nuclear magnetic resonance (NMR), ¹³C NMR spectra and EA.

Full text of DOI:10.1016/j.steroids.2009.12.005

Steroids 75 (2010) 224–229
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Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and anti-glioma activity of 25(R)-spirostan-3␤,5␣,6␤,19-tetrol
TianDong Lenga,1, JingXia Zhangb,1, Jun Xiea,1, ShuJia Zhoub, YiJun Huanga, YueHan Zhoua,
WenBo Zhua, GuangMei Yana,∗
a Department of Pharmacology, Zhong-Shan Medical College, Sun Yat-Sen University, Guangzhou, 74 Zhongshan Road II, Guangzhou 510080, China
b School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, 74 Zhongshan Road II, Guangzhou 510080, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Malignant gliomas are common and aggressive brain tumours in adults. The rapid proliferation and
diffuse brain migration are the main obstacles to successful treatment. Here, we show 25(R)-spirostan-
3␤,5␣,6␤,19-tetrol, a polyhydroxy steroid, is capable of suppressing proliferation and migration of
C6 malignant glioma cells in a concentration-dependent manner. The compound 25(R)-spirostan-
3␤,5␣,6␤,19-tetrol was synthesised by seven steps starting from diosgenin in 8.55% overall yield. The
structures of the synthetic compounds were characterised by infrared (IR), 1H nuclear magnetic resonance
(NMR), 13C NMR spectra and EA.
Received 25 September 2009
Received in revised form 7 December 2009
Accepted 8 December 2009
Available online 21 December 2009
Keywords:
Polyhydroxy steroid
25(R)-Spirostan-3␤,5␣,6␤,19-tetrol
Glioma
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
8.55% overall yield. We found 25(R)-spirostan-3␤,5␣,6␤,19-tetrol
could significantly inhibit the proliferation and migration of glioma
In the central nervous system (CNS), gliomas are the most com-
mon primary tumours with poor prognosis, which account for 60%
of primary brain tumours [1,2]. Although systemic metastases are
relatively rare, the highly infiltrative nature of glioma cells that
migrate into surrounding brain parenchyma makes total surgical
resection impossible [3,4]. The residual cancer cells peripheral to
the excised lesion after surgery give rise to a recurrent tumour,
which, in more than 90% of cases, develops immediately adjacent to
the resection margin [4,5]. Therefore, it is needed to develop novel
pharmacological agents to inhibit the proliferation and migration
of the malignant gliomas, which could be used in combination with
conventional systemic therapies.
Steroids are one of the most ancient molecules [6], which exhibit
diverse biological activities, and are regarded as the key to life. In
the past, much attention has been paid to the use of steroids in the
treatment of some neurodegenerative diseases such as Parkinson’s
disease, Alzheimer’s disease, and cerebral ischaemia [7,8], partly
due to their relatively high permeability across the blood–brain
barrier. However, little attention has been paid to the use of
steroids in the treatment of brain tumours, especially the malignant
gliomas.
cells, implying the potential use of 25(R)-spirostan-3␤,5␣,6␤,19-
tetrol as a drug candidate for the treatment of malignant gliomas.
2. Experimental
2.1. Chemistry
All melting points were determined on an X6 melting point
apparatus and were uncorrected. IR spectra were recorded as
KBr pellets on an EQUINOX 55 FT spectrophotometer. 1H NMR
and 13C NMR spectra were recorded on a Varian Unity INOVA
500 NB (500 MHz) spectrometer. Chemical shifts were reported in
values relative to TMS as the internal standard. Flash column chro-
matography was carried out with silica gel (200–300 mesh). All
chemicals and solvents were analytical grade and purchased from
the Guangzhou Chemical Reagents Company China.
2.1.1. 25(R)-3ˇ-Acetoxy-spirost-5-ene (2)
Diosgenin 1 (20 g, 0.48 mol) and pyridine (1 ml) were added to
150 ml acetic anhydride, and the mixture was heated and stirred
at 90 ◦C for 1 h [9]. The solution was poured into ice water, The
precipitate was filtered, washed with water and dried under vac-
uum to yield a white solid, which was recrystallised with CH3OH
to give compound 2 (20.9 g, 95%) as colourless needle crystals.
mp 193.6–194.5 ◦C. IR (KBr) ꢀ: 3047, 1723, 1451, 1378, 1051, 982,
In this study, we synthesised the polyhydorxy steroid, 25(R)-
spirostan-3␤,5␣,6␤,19-tetrol starting from diosgenin (Scheme 1) in
901 cm−1 1H NMR (CDCl3) ı: 0.78 (d, J = 5.2, 3H, 27-CH3), 0.79 (s,
.
3H, 18-CH3), 0.98 (d, 3H, J = 7.6, 21-CH3), 1.04 (s, 3H, 19-CH3), 2.03
(s, 3H, CH3CO), 3.37 (t, 1H, J = 10.8 Hz, 26-Ha), 3.46 (m, 1H, 26-Hb),
∗
Corresponding author. Tel.: +86 20 87333762; fax: +86 20 87330578.
E-mail address: yguangmei2009@yahoo.cn (G. Yan).
These authors contributed equally to this work.
1
0039-128X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.12.005

T. Leng et al. / Steroids 75 (2010) 224–229
225
Scheme 1.
4.41 (q, 1H, 16-H), 4.60 (m, 1H, 3-H), 5.37 (d, 1H, 6-H). 13C NMR
(CDCl3) ı: 14.51 (CH3), 16.27 (CH3), 17.13 (CH3), 19.92 (CH3), 20.83
(CH2), 21.39 (CH3), 27.76 (CH2), 28.83 (CH2), 30.31 (CH), 31.43 (CH),
31.45 (CH2), 31.45 (CH2), 31.86 (CH), 32.06 (CH2), 36.75 (CH2), 36.98
(CH2), 38.11 (CH2), 39.75 (CH2), 40.28 (C), 41.46 (CH), 49.99 (CH),
56.46 (CH), 62.15 (CH), 66.84 (CH2), 73.89 (CH), 80.80 (CH), 109.25
(C), 122.36 (CH), 139.72 (C) and 170.46 (C). Analysis calculated for
dissolved with ethyl acetate, and then the solution was sequen-
tially washed with 10% solution of sodium sulphite and 5% NaHCO3
and water, and then dried with Na2SO4. Evaporation of the solvent
gave an oil which was purified by flash column chromatogra-
phy (petroleum ether/EtOAc 9:1) and recrystallised from MeOH
to afford colourless needles 4 (8.2 g, 70.18%). mp 196.6–197.9 ◦C.
IR (KBr) ꢀ: 1740, 1452, 1368, 1053, 694, 606, 544 cm−1 1H NMR
.
(CDCl3) ı: 0.74 (d, J = 6.4, 3H, 27-CH3), 0.78 (s, 3H, 18-CH3), 0.91 (d,
J = 6.8, 21-CH3), 1.99 (s, 3H, CH3CO), 3.32 (t, 1H, J = 10.8Hz, 26-Ha),
3.35 (q, 1H, 26-Hb), 3.71 (d, J = 8.4, 1H, 19-Ha), 3.89 (d, J = 8.4, 1H,
19-Hb), 4.01 (d, J = 4.8, 1H, 16-H), 4.38 (m, 1H, 6-H), 5.15 (m, 1H, 3-
H). 13C NMR (CDCl3) ı: 13.68 (CH3), 15.99 (CH3), 16.35 (CH3), 20.48
(CH2), 21.73 (CH3), 22.52 (CH2), 26.11 (CH2), 28.03 (CH2), 29.51
(CH2), 30.54 (CH), 30.63 (CH2), 32.23 (CH), 32.28 (CH2), 38.95 (CH2),
40.45 (CH2), 40.57 (CH2), 40.78 (C), 45.17 (C), 48.06 (CH), 53.42 (CH),
61.34 (CH), 66.07 (CH), 66.81 (CH2), 69.12 (CH2), 73.61 (CH), 80.04
(C), 81.44 (CH), 108.45 (C) and 169.44 (C). Analysis calculated for
C
29H44O4: C, 76.27; H, 9.71; found: C, 76.53; H, 9.70.
2.1.2. 25(R)-3ˇ-Acetoxy-5˛-bromo-6ˇ-hydroxy-spirostane (3)
Compound 3 was prepared as described previously with some
modification [9]. A solution of compound 2 (19 g, 41.6 mmol) in
dioxane (180 ml) containing 70% HClO4 (1 ml) and water (10 ml)
was stirred in the dark at 10 ◦C. Immediately, four portions of NBS
(8 g, 45 mmol) were added stepwise at 10-min intervals to the solu-
tion. The mixture was stirred for 1 h, and then was poured into the
cool saturated solution of sodium sulphite. The mixture was fil-
tered and the residue was purified by flash column chromatography
(petroleum ether/EtOAc 4:1) to give 3 as needle crystals (16.5 g,
72.10%). mp 135.5–143.1 ◦C. IR (KBr) ꢀ: 3437, 1738, 1456, 1377,
C29H43BrO4: C, 63.15; H, 7.86; found: C, 63.14; H, 8.011.
2.1.4. 25(R)-3ˇ-Acetoxy-19-hydroxy-spirost-5-ene (5)
1055, 690, 521 cm−1 1H NMR (CDCl3) ı: 0.76 (d, J = 6.0, 3H, 27-
.
Compound 5 was prepared according to the reported method
[9], with some modification. Briefly, compound 4 (6 g, 10.88 mmol)
in ethanol (500 ml) was treated with zinc dust (18 g, 276.92 mmol)
under reflux with stirring for 5 h. The suspension was filtered, and
the filtrate was evaporated under reduced pressure. The residue
was dissolved in EtoAc, and washed with water and dried with
Na2SO4. The solvent was removed under reduced pressure, and
the resulting white solid was recrystallised from MeOH-H2O to
afford compound 5 (4.2 g, 78%) as needles; mp 223–225 ◦C. IR (KBr)
CH3), 0.77 (s, 3H, 18-CH3), 0.94 (d, J = 7.2, 3H, 21-CH3), 1.33 (s, 3H,
19-CH3), 2.03 (s, 3H, CH3CO), 3.35(t, 1H, J = 10.8 Hz, 26-Ha), 3.47
(m, 1H 26-Hb), 4.11 (m, 1H, 6-H), 4.17 (q, 1H, 16-H), 4.36 (m, 1H, 3-
H). 13C NMR (CDCl3) ı:14.48 (CH3), 16.59 (CH3), 17.12 (CH3), 17.98
(CH3), 21.15 (CH2), 21.32 (CH3), 26.36 (CH2), 28.83 (CH2), 30.27
(CH2), 30.30 (CH), 31.41 (CH2), 31.66 (CH), 34.84 (CH2), 35.10 (CH2),
38.44 (CH2), 39.72 (CH2), 40.50 (C), 40.69 (C), 41.67 (CH), 47.54 (CH),
55.60 (CH), 62.13 (CH), 66.87 (CH2), 72.04 (CH), 75.69 (CH), 80.73
(CH), 86.42 (C), 109.26 (C) and 170.36 (C). Analysis calculated for
C29H45BrO4: C, 62.92; H, 8.19; found: C, 62.58; H, 8.180.
v: 3474, 3040, 2957, 1732, 1450, 1377, 1244, 1045 cm−1 1H NMR
.
(CDCl3) ı: 0.75 (d, J = 6.0, 3H, 27-CH3), 0.82 (s, 3H, 18-CH3), 0.93 (d,
J = 6.8, 3H, 21-CH3), 2.00 (s, 3H, CH3CO), 3.34(t, 1H, J = 10.8 Hz, 26-
Ha), 3.42–3.43 (m, 1H, 26-Hb), 3.59 (d, J = 11.2, 1H, 19-Ha), 3.89 (d,
J = 11.6, 1H, 19-Hb), 4.37 (m, 1H, 16-H), 4.61 (m, 1H, 6-H) and 5.75
(m, 1H, 3-H). 13C NMR (CDCl3) ı: 14.48(CH3), 16.62 (CH3), 17.11
(CH3), 21.35 (CH2), 21.52 (CH3), 28.08 (CH2), 28.81 (CH2), 30.30
(CH), 31.38 (CH2), 31.72 (CH2), 32.88 (CH), 33.16 (CH2), 38.20 (C),
40.00 (CH2), 40.50 (C), 41.62 (CH2), 41.74 (CH), 50.26 (CH), 57.31
(CH), 62.06 (CH), 62.80 (CH2), 66.85 (CH2), 73.36 (CH), 80.84 (CH),
109.30 (CH), 127.99 (CH), 134.62 (C) and 170.46 (C). Analysis cal-
culated for C29H44O5: C, 73.69; H, 9.38; found: C, 73.94; H, 9.30.
2.1.3. 25(R)-3ˇ-Acetoxy-5˛-bromo-6ˇ,19-epoxy-spirostane (4)
The synthesis of compound 4 was performed as described
previously with some modification [9].
drous cyclohexane (1420 ml) and benzene (480 ml) containing
lead tetraacetate (50.9 g, 112.76 mmol), calcium carbonate (20 g,
200 mmol) was stirred, then compound 3 (12 g, 21.68 mmol) and
iodine (12 g, 47.28 mmol) were added. The solution was irradiated
by a 250 W tungsten lamp for 6 h. The mixture was filtered, and
the solvent was removed under reduced pressure. The residue was
A mixture of anhy-

226
T. Leng et al. / Steroids 75 (2010) 224–229
2.1.5. 25(R)-3ˇ,19-Diacetoxy-spirost-5-ene (6)
108.30 (C). Analysis calculated for. C27H44O6: C, 69.79; H, 9.54;
found: C, 70.19; H, 9.80.
Compound 5 (4 g, 8.46 mmol) and pyridine (1 ml) were added to
acetic anhydride (20 ml), and the mixture was heated and stirred
at 90 ◦C for 1 h. The solution was poured into ice water, The pre-
cipitate was filtered, washed with water, dried under vacuum to
yield compound 6 as colourless needle crystals (4.2 g, 95%). mp
2.2. Biological assays
25(R)-Spirostan-3␤,5␣,6␤,19-tetrol was prepared by the above
route described in Scheme 1. Dulbecco’s modified eagle medium
(DMEM) and foetal bovine serum (FBS) were purchased from
Gibco BRL (Grand Island, NY, USA). Hoechst 33258 was obtained
from Molecular Probes, Inc. (Eugene, OR, USA). Antibodies against
cyclin D1, cyclin D3 and glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) were obtained from Cell Signaling Technology
(Beverly, MA, USA), and cyclin-dependent kinases CDK2 and CDK4
antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All
other reagents were of analytical grade unless otherwise indicated.
156.8–157.3 ◦C. IR (KBr) ꢀ: 3047, 1747, 1449, 1371, 1050 cm−1 1H
.
NMR (CDCl3) ı: 0.78 (d, J = 4.8, 3H, 27-CH3), 0.81 (s, 3H, 18-CH3),
0.96 (d, J = 5.6, 3H, 21-CH3), 2.00 (s, 3H, 3-CH3CO), 2.02 (s, 3H, 19-
CH3CO), 3.35 (t, 1H, J = 8.8 Hz, 26-Ha), 3.46–3.47 (m, 1H, 26-Hb),
3.96 (d, J = 9.2, 1H, 19-Ha), 4.39 (m, 1H, 16-H), 4.49 (d, J = 9.6, 1H,
19-Hb), 4.62 (m, 1H, 6-H), 5.63 (m, 1H, 3-H). 13C NMR (CDCl3) ı:
14.40 (CH3), 16.27 (CH3), 17.02 (CH3), 20.92 (CH3), 21.21 (CH3),
21.29 (CH2), 27.73 (CH2), 28.67 (CH2), 30.15 (CH), 31.29 (CH2),
31.36 (CH2), 31.66 (CH), 32.38 (CH), 33.41 (CH2), 37.98 (CH2), 39.71
(CH2), 40.29 (C), 41.51 (CH), 49.90 (CH), 56.85 (CH), 61.94 (CH),
64.43 (CH2), 66.70 (CH2), 73.20 (CH), 80.66 (CH), 109.14 (C), 126.40
(CH), 134.47 (C), 170.42 (C) and 170.60 (C). Analysis calculated for
2.2.1. Cell culture and drug treatment
Rat C6 glioma cells were obtained from the American Type
Culture Collection (Manassas, VA, USA) and maintained in DMEM
(Invitrogen, Grand Island, NY, USA) supplemented with 10%
FBS (PAA Laboratories Inc., Etobicoke, ON, USA), 100 units ml−1
C31H46O6: C, 73.69; H, 9.38; found: C, 73.50; H, 9.60.
2.1.6. 25(R)-3ˇ,19-Diacetoxy-5˛,6˛-epoxy-spirostane (7)
H2O (80 ml), Na2CO3 (1.8 g, 21.68 mmol) and m-CPBA (2.2 g,
12.75 mmol) were added to the solution of compound 6 (2 g,
3.77 mmol) in 120 ml of CH2Cl2, and the mixture was stirred at
room temperature for 6 h. The solvent was removed under reduced
pressure, and extracted with ethyl acetate. The organic layer was
washed with Na2SO3, NaHCO3 and brine, and then dried with anhy-
drous Na2SO4. Evaporation of the solvent gave the crude product
which was purified by flash column chromatography to give a white
solid. The resulting product was recrystallised from MeOH to afford
colourless needle crystals 7 (1 g, 55.4%). mp 214–216 ◦C. IR (KBr) ꢀ:
penicillin (PAA), and 100 ␮g ml−1 streptomycin (PAA) in
a
humidified atmosphere of 5% CO2 at 37 ◦C. Before adminis-
tration, the medium was changed to DMEM with 1% FBS,
100 units ml−1 penicillin, and 100 ␮g ml−1 streptomycin. 25(R)-
Spirostan-3␤,5␣,6␤,19-tetrol was dissolved in DMSO and added to
the cultures at indicated concentrations for 48 h. DMSO was used
at a final concentration below 0.1% and the same volume of DMSO
was added to the control group.
2.2.2. Proliferation and cytotoxicity assay
2925, 2874, 1740, 1454, 1370, 1240, 1037 cm−1
.
1H NMR (CDCl3)
Cell proliferation was evaluated by MTT assay as we described
previously [10]. Briefly, C6 cells were plated in 96-well plates and
treated with 25(R)-spirostan-3␤,5␣,6␤,19-tetrol for the 48 h, fol-
lowing which 10 ␮l MTT (5 mg ml−1) was added and incubated for
another 4 h at 37 ◦C. Finally, the MTT solution was removed and
100 ␮l DMSO was added to dissolve the crystal. Absorbance was
read at 570 nm with an EXL800 microimmunoanalyser (BioTek,
Burlington, VT, USA).
Cytotoxicity of 25(R)-spirostan-3␤,5␣,6␤,19-tetrol was evalu-
ated by lactate dehydrogenase (LDH) release. LDH release was
quantified with a CytoTox 96 nonradioactive cytotoxicity assay
kit (Promega, Madison, WI, USA) according to the manufacturer’s
instructions. Absorbance was measured at 490 nm with an EXL800
microimmunoanalyser.
ı: 0.65 (s, 3H, 18-CH3), 0.705 (d, J = 5.6 3H, 27-CH3), 0.84 (m, 3H,
21-CH3), 2.02 (s, 3H, 3-CH3CO), 2.05 (s, 3H, 19-CH3CO), 3.26–3.31
(m, 1H, 26-Ha), 3.38–3.40 (m, 1H, 26-Hb), 3.99 (d, J = 9.2, 1H, 19-
Ha), 4.25–4.38 (m, 1H, 16-H), 4.41 (d, J = 9.2, 1H, 19-Hb), 4.74–4.78
(m, 1H, 6-H), 4.87–4.94 (m, 1H, 3-H). 13C NMR (CDCl3) ı: 14.38
(CH3), 17.02 (CH3), 20.89 (CH2), 21.45 (CH3), 27.31 (CH2), 29.64
(CH2), 30.57 (CH2), 31.34 (CH), 31.64 (CH2), 37.30 (CH2), 38.34 (C),
39.78 (CH2), 40.38 (C), 41.58 (CH), 42.63 (CH), 43.11 (CH), 56.98
(CH), 59.61 (CH), 61.82 (CH), 62.44 (C), 63.46 (CH2), 66.78 (CH2),
70.46 (CH), 80.49 (CH), 109.21 (C), 170.12 (C) and 170.67 (C). Anal-
ysis calculated for C31H46O7: C, 70.16; H, 8.74; found: C, 70.35;
H, 8.96.
2.1.7. 25(R)-Spirostan-3ˇ,5˛,6ˇ,19-tetrol (8)
H2SO4 (1N, 5 ml) was added to the solution of compound 7 (0.5 g,
0.94 mmol) in THF (25 ml), and stirred at room temperature for 24 h.
The mixture was neutralised with Na2CO3 and then evaporated. The
residue was dissolved in methanol (100 ml), Na2CO3 (2 g, 24 mmol)
was added and then refluxed for 2 h. The solution was poured into
200 ml ice water, and the precipitate was filtered and washed with
water and purified by flash column chromatography to give white
solid. The resulting solid was further purified by recrystallisation
from MeOH-H2O to afford colourless needles of 25(R)-spirostan-
3␤,5␣,6␤,19-tetrol (0.2 g, 43.31%). mp 153–155 ◦C. IR (KBr) ꢀ: 3408,
2.2.3. Migration assay
Cell migration was evaluated by a wound healing assay as
described previously with some modification [11]. Briefly, the
cells were plated on a 6-well plate. The next day, scratches were
made with a 200 ␮l sterile pipette tip, and then the cells were
washed with phosphate-buffered saline (PBS) three times and incu-
bated with 25(R)-spirostan-3␤,5␣,6␤,19-tetrol for 48 h. The cells
were washed twice with PBS and fixed with 4% paraformaldehyde
for 30 min. To make cell counting easier, the nucleus of C6 cells
was stained with Hoechst 33258 (Invitrogen, 5 ␮g ml−1, diluted in
PBS) for 5 min after fixation. Finally, the cells were washed three
times with PBS and photographed with an Olympus fluorescence
microscope (Melville, NY, USA). The percentage of migration was
calculated by cells that migrated into the scratched areas compared
with cells that stayed in the peripheral areas.
2927, 2856, 1456, 1378, 1054 cm−1 1H NMR (CDCl3) ı: 0.76 (s, 3H,
.
18-CH3), 0.91(d, J = 5.63H, 27-CH3), 1.02(m, 3H, 21-CH3), 3.16–3.25
(m, 1H, 26-Ha), 3.32–3.42 (m, 1H, 26-Hb), 3.61–3.68 (m, 1H, 6-CH),
3.75–3.85 (m, 1H, 3-CH), 4.19 (d, J = 3.2, 1H, 19-Ha), 4.26–4.30 (m,
1H, 16-H) and 4.46 (d, J = 4.4, 1H, 19-Hb). 13C NMR (CDCl3) ı:14.52
(CH3), 16.16 (CH3), 16.99 (CH3), 20.46 (CH2), 21.62 (CH2), 28.44
(CH2), 28.92 (CH2), 29.74 (CH), 30.82 (CH), 31.38 (CH2), 32.92 (CH),
34.60 (CH), 39.40 (CH2), 39.66 (CH2), 39.92 (CH), 40.81 (C), 41.04
(CH2), 43.77 (CH), 44.57 (C), 55.44 (CH), 61.85 (CH), 65.62 (CH),
65.82 (CH2), 65.83 (CH2), 73.98 (CH), 79.19 (C), 80.16 (CH) and
2.2.4. Cell cycle analysis
A flow cytometry analysis of DNA content of cells was performed
to assess the cell cycle phase distributions as described [2]. In brief,
after treatment, the cells were collected by trypsinisation, washed

T. Leng et al. / Steroids 75 (2010) 224–229
227
in PBSand fixed in 70% ethanol for 30 min at 4 ◦C. After washing with
3. Results and discussion
PBS, cells were incubated with the DNA-binding dye propidium
iodide (50 ␮g ml−1) and RNase (1.0 mg ml−1) for 30 min at 37 ◦C in
the dark. Finally, cells were washed and red fluorescence was anal-
ysed by a FACSCalibur flow cytometer (BD, Heidelberg, Germany)
using a peak fluorescence gate to discriminate aggregates.
3.1. Chemistry
The planned synthetic route from diosgenin (1) to the tar-
get compound 25(R)-spirostan-3␤,5␣,6␤,19-tetrol (8) is shown in
Scheme 1. The 3␤-hydroxy group of 1 was protected by acetylation
to give 3␤-acetoxydiosgenin (2). Compound 2 was converted to
compound 3 with NBS containing a catalytic amount of 70% HClO4.
When the acidity was not suitable, it yielded a large amount of
byproduct 25(R)-3␤-acetoxy-5␣-hydroxy-spirostan-6-one which
was difficult to be separated from compound 3. The 6␤, 19-epoxide
(4) was the product of remote oxidation when bromohydrin (3)
was treated with Pb (OAc)4 and I2 under the irradiation of a tung-
2.2.5. Western blot analysis
C6 cells were treated with 25(R)-spirostan-3␤,5␣,6␤,19-tetrol
for the indicated time and the total protein was extracted with
M-PER (Mammalian Protein Extraction Reagent, Pierce, Rockford,
IL, USA) according to the manufacturer’s instructions. After mea-
surement of the protein concentration with BCA Protein Assay
Kit (Pierce, Rockford, IL, USA), equal amount of protein sam-
ples were combined with concentrated sodium dodecyl sulphate
(SDS) loading buffer and heated to 95 ◦C for 5 min, separated
by sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) and Western blots were performed. After incubation
with horseradish peroxidase labelled secondary antibody (1:1000,
Cell Signaling Technology, Beverly, MA, USA), the visualisation of
protein was done with enhanced chemiluminescence detection
system (Pierce, Rockford, IL, USA) and a GeneGnome chemilu-
minescence imaging and analysis system (Syngene Bio Imaging,
Cambridge, UK).
2.2.6. Data analysis
Data are expressed as mean SD from three independent exper-
iments. Statistical evaluations were done by one-way analysis of
variance (ANOVA). p < 0.05 was considered statistically significant.
Fig. 2. 25(R)-Spirostan-3␤,5␣,6␤,19-tetrol (Tetrol) inhibited migration of C6 glioma
cells. C6 cells were plated on a 6-well plate and scraped with a 200 ␮l sterile pipette
tip and then incubated with different concentrations of Tetrol for 48 h. (A) Phase
contrast of migrated cells. Representatives of three independent experiments were
shown. (B) Hochest 33258 staining of nucleus. (C) Statistical analysis of the percent-
age of migrated cells. *p < 0.05, **p < 0.01 compared with control.
Fig. 1. 25(R)-Spirostan-3␤,5␣,6␤,19-tetrol (Tetrol) induced proliferation inhibition
but not cell death in C6 glioma cells. C6 cells were treated with different concen-
trations of Tetrol for 48 h. (A) MTT absorbance was measured for cell proliferation.
*p < 0.05, **p < 0.01 compared with control. (B) LDH absorbance was measured for
cytotoxicity.

228
T. Leng et al. / Steroids 75 (2010) 224–229
sten lamp. Compound 5 was an important intermediate, which was
synthesised via reduction of 4 with Zn powder.
scores of C6 cells (Fig. 1A). The reduction in MTT score in response to
25(R)-spirostan-3␤,5␣,6␤,19-tetrol treatment could be explained
either by cell injury or by reduced proliferation. LDH assay was
used to investigate whether 25(R)-spirostan-3␤,5␣,6␤,19-tetrol
could cause cell injury. We found there was no statistically sig-
nificant difference in the mean LDH absorbance between cells
treated with 25(R)-spirostan-3␤,5␣,6␤,19-tetrol and the control
(Fig. 1B). Therefore, cytotoxicity was not the possible contributor
to the reduction in MTT score. Taken together, the proliferation
inhibiting activity of 25(R)-spirostan-3␤,5␣,6␤,19-tetrol appears
to be responsible for the reduction in MTT score.
The most characteristic features of 19-hydroxy steroid (5)
involve the appearance of the 13C NMR peak at 62.80 (19-CH2OH),
134.62 (5-C), 127.99 (6-CH) and 1H NMR peak at 3.59 (d, J = 8.4,
1H, 19-Ha), 3.89 (d, J = 8.4, 1H, 19-Hb) and 5.75 (t, 1H, 6-H), which
are consistent with 19-OH and 5,6-double bonds. The C19-OH
of 5 was protected by acetylation to give 6. Compound 6 was
converted to 5,6-epoxide (7) with m-CPBA. The disappearance of
13C NMR peak at 134.47 (5-C), 126.40 (6-CH), and the appear-
ance of 13C NMR peak at 59.61 (6-CH) and 61.98 (5-C) implied
the formation of 5,6-epoxide (7). The target compound 25(R)-
spirostan-3␤,5␣,6␤,19-tetrol (8) was obtained when 7 was treated
with H2SO4 and then hydrolysed using Na2CO3 in methanol. The
appearance of 13C NMR peak at 73.98 (3-CH), 79.19 (5-C), 65.62 (6-
CH), 65.83 (19-CH2) proved the formation of 3␤, 5␣, 6␤, 19-tetrol
Except for the infinite proliferation ability, migration is
another characteristic feature of malignant gliomas. To deter-
mine the possible role of 25(R)-spirostan-3␤,5␣,6␤,19-tetrol in
migration, we performed a wound healing assay. As shown
in Fig. 2A and B, untreated C6 cells were able to invade the
scratched area that was fully re-colonised 48 h later. How-
ever, 25(R)-spirostan-3␤,5␣,6␤,19-tetrol significantly inhibited
this process in a concentration-dependent manner. In 25(R)-
spirostan-3␤,5␣,6␤,19-tetrol (40 ␮M) treated group, very few cells
were in the scratched area and the distance between the borders
of the wound was significantly different from that of the control
48 h after the scratch (Fig. 2A and B). Quantitative analysis clearly
indicated significant decreases of the cell migration rate following
25(R)-spirostan-3␤,5␣,6␤,19-tetrol treatment (Fig. 2C). These data
suggest that 25(R)-spirostan-3␤,5␣,6␤,19-tetrol has the ability to
suppress the cellular migration of C6 malignant glioma cells.
To investigate the chemical basis of 25(R)-spirostan-
3␤,5␣,6␤,19-tetrol (8) as an anti-glioma agent, the precursors of 8,
together with 8, were tested for their effect on the migration and
proliferation ability of C6 glioma at the concentration of 20 ␮M
(Table 1). We found, except for compound 8, only compounds 3
and 4 have the inhibitory effect on C6 glioma migration and prolif-
eration. However, compounds 3 and 4 could also cause significant
cytotoxicity at the effective concentration, which may account
for their inhibiting effect on the migration and proliferation of C6
glioma. By contrast, 25(R)-spirostan-3␤,5␣,6␤,19-tetrol did not
induce cytotoxicity as it significantly suppressed the C6 glioma
migration and proliferation. Furthermore, other structurally
related compounds (1–7) have no effect on C6 glioma migra-
tion and proliferation, which further confirm the polyhydroxy
(8). For compounds 2–7, the peak still existed at 108–110 in the 13
C
NMR spectrum. It was the chemical shift of spiral atom 22-C which
showed that the spiral ring structure was kept unchanged.
3.2. Anti-glioma activity
Local migration and aggressive proliferation activity of the
remaining tumour cells following operation are inherent features
of malignant glioma s. Cell migration is a crucial event for tumour
spreading, metastasis and invasiveness. An invasive nature of
glioma cells leads to the local destruction of the adjacent tissue
with an extensive cellular infiltration into the normal brain [12].
The infiltration of tumour cells and their high proliferative activity
are key elements in turning gliomas into an unmanageable disease,
which prevents complete tumour removal and leads to therapeu-
tic failure and recurrence. Effective inhibition of the migration and
proliferation activity of the malignant gliomas may provide help
avoiding the spread and recurrence of the malignant gliomas, espe-
cially after surgical resection.
To evaluate the inhibiting activity of 25(R)-spirostan-
3␤,5␣,6␤,19-tetrol on glioma proliferation, we performed an
MTT score assay. The conversion of MTT requires active mitochon-
dria within cells; thus, MTT scores are used as a measure of cellular
activity and cell number [13]. Compared with the control, 25(R)-
spirostan-3␤,5␣,6␤,19-tetrol could dose-dependently inhibit MTT
Fig. 3. Effects of 25(R)-spirostan-3␤,5␣,6␤,19-tetrol (Tetrol) on the cell cycle and cell cycle regulatory proteins of C6 glioma cells. C6 cells were treated with 10, 20 and
40 ␮M of Tetrol for 48 h. (A) Cell cycle distributions. One of three independent experiments was shown. (B) Statistical analysis of three independent experiments. *p < 0.05,
**p < 0.01 compared with control. (C) Western blots analysis of cyclins and cyclin-dependent kinases.

T. Leng et al. / Steroids 75 (2010) 224–229
229
Table 1
In conclusion, we demonstrated here that 25(R)-spirostan-
3␤,5␣,6␤,19-tetrol was able to effectively inhibit proliferation and
migration of C6 glioma cells. Specially, it did not cause significant
tumour cell cytotoxicity at the effective concentrations used in this
study, which indicated 25(R)-spirostan-3␤,5␣,6␤,19-tetrol could
function as a novel and potential anti-glioma agent compared with
conventional cytotoxic chemotherapies. At the same time, it may
provide critical clues for the development of more potent anti-
tumour agents.
In Vitro anti-glioma activity of the tested compounds at 20 (M concentration.
Compound
% of Migration
MTT Absorbance
LDH Absorbance
1
2
3
4
5
6
7
8
69.32 4.42
71.31 5.34
58.35 2.68*
62.38 3.69*
65.44 6.56
69.79 5.76
65.97 7.29
48.35 3.29**
0.70 0.08
0.71 0.13
0.62 0.09*
0.64 0.06*
0.68 0.15
0.71 0.10
0.69 0.08
0.56 0.07**
0.19 0.02
0.20 0.03
0.41 0.05**
0.35 0.04*
0.18 0.02
0.21 0.03
0.20 0.02
0.18 0.02
Acknowledgements
Results are expressed as means SD (n = 3), *p < 0.05, **p < 0.01 compared with con-
trol.
This work was supported by the National Natural Sci-
ence Foundation of China (Project No. 30472089), the National
High Technology Development 863 Key Project (Project No.
2006AA090502).
structure is critical for the anti-glioma activity of this series of
compounds.
Dysregulation of cell cycle progression is a fundamental reason
that leads to aberrant proliferation of tumour cells. The G1/S-
phase checkpoint is the first critical restriction point in the cell
division cycle and the G1/S transition is closely linked to the acti-
vation of cell cycle regulatory proteins such as cyclins and CDKs
[14]. To gain insight into the mechanism of growth inhibitory
effects of 25(R)-spirostan-3␤,5␣,6␤,19-tetrol, we assessed cell
cycle distribution of C6 cells by flow cytometry. Analysis of
cell cycle showed that 25(R)-spirostan-3␤,5␣,6␤,19-tetrol led to
a dose-dependent C6 accumulation in the G1 phase to reach
approximately 75.4%, 82.0% and 87.7% of population at 10, 20
and 40 ␮M, respectively, while that in the control was 64.2%,
indicating cell cycle arrest at this phase. Concomitantly, there
was a striking decrease in the S and G2/M fractions over the
basal level. Cells in the S phase decreased from 25.8% of control
group to 7.3% of 40 ␮M 25(R)-spirostan-3␤,5␣,6␤,19-tetrol group
(p < 0.01) and G2/M phase cells from 10.1% to 3.7% (p < 0.01) (Fig. 3
A and B).
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