Naturally occurring marine steroid 24-methylenecholestane-3β,5α,6β,19-tetraol functions as a novel neuroprotectant

Article abstract of DOI:10.1016/j.steroids.2015.11.005

Steroids have been shown to have multiple effects on the nervous system including neuroprotective activities, and they have the potential to be used for the treatment of neurodegenerative diseases. In this current study, we tested the hypothesis that the marine steroid 24-methylenecholestane-3β,5α,6β,19-tetraol (Tetrol) has a neuroprotective effect. (1) We synthesized Tetrol through a multiple step reaction starting from hyodeoxycholic acid (HDCA). (2) We then evaluated the neuroprotective effect of Tetrol with a glutamate-induced neuronal injury model in vitro. Tetrol concentration dependently increased the survival rate of cerebellar granule neurons challenged with toxic concentration of glutamate. Consistently, Tetrol significantly decreased glutamate-induced lactate dehydrogenase (LDH) release with a threshold concentration of 2.5 μM. (3) We further evaluated the neuroprotective effect of Tetrol in a middle cerebral artery occlusion (MCAO)-induced cerebral ischemia model in rat. Tetrol, at a dose of 12 mg/kg, significantly decreased MCAO-induced infarction volume by ～50%. (4) Finally, we probed the mechanism and found that Tetrol concentration dependently attenuated N-methyl-d-aspartate (NMDA)-induced intracellular calcium ([Ca²⁺]_i) increase with an IC₅₀ of 7.8 ± 0.62 μM, and inhibited NMDA currents in cortical neurons with an IC₅₀ of 10.28 ± 0.71 μM. Taken together, we have synthesized and characterized Tetrol as a novel neuroprotectant through negative modulation of NMDA receptors.

Full text of DOI:10.1016/j.steroids.2015.11.005

Steroids 105 (2016) 96–105
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Naturally occurring marine steroid 24-methylenecholestane-
3b,5a,6b,19-tetraol functions as a novel neuroprotectant
Tiandong Leng a,b,1, Ailing Liu a,1, Youqiong Wang a,1, Xinying Chen c,1, Shujia Zhou c, Qun Li a,
Wenbo Zhu a, Yuehan Zhou a, Xingwen Su a, Yijun Huang a, Wei Yin a, Pengxin Qiu a, Haiyan Hu c,
a,
Zhi-gang Xiong b, Jingxia Zhang c, , Guangmei Yan
⇑
⇑
a Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, 74 Zhongshan Road II, Guangzhou, GD 510080, China
b Neuroscience Institute, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA 30329, USA
c School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, GD 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Steroids have been shown to have multiple effects on the nervous system including neuroprotective
activities, and they have the potential to be used for the treatment of neurodegenerative diseases. In this
current study, we tested the hypothesis that the marine steroid 24-methylenecholestane-3b,5a,6b,19-
tetraol (Tetrol) has a neuroprotective effect. (1) We synthesized Tetrol through a multiple step reaction
starting from hyodeoxycholic acid (HDCA). (2) We then evaluated the neuroprotective effect of Tetrol
with a glutamate-induced neuronal injury model in vitro. Tetrol concentration dependently increased
the survival rate of cerebellar granule neurons challenged with toxic concentration of glutamate.
Consistently, Tetrol significantly decreased glutamate-induced lactate dehydrogenase (LDH) release with
Received 5 September 2015
Received in revised form 2 November 2015
Accepted 19 November 2015
Available online 26 November 2015
Keywords:
Marine steroid
Neuroprotection
Cerebral ischemia
NMDA
a threshold concentration of 2.5 lM. (3) We further evaluated the neuroprotective effect of Tetrol in a
middle cerebral artery occlusion (MCAO)-induced cerebral ischemia model in rat. Tetrol, at a dose of
12 mg/kg, significantly decreased MCAO-induced infarction volume by ꢀ50%. (4) Finally, we probed
Glutamate toxicity
the mechanism and found that Tetrol concentration dependently attenuated N-methyl-
D
-aspartate
M, and inhibited
M. Taken together, we have synthesized
(NMDA)-induced intracellular calcium ([Ca2+]i) increase with an IC50 of 7.8 0.62
NMDA currents in cortical neurons with an IC50 of 10.28 0.71
l
l
and characterized Tetrol as a novel neuroprotectant through negative modulation of NMDA receptors.
Ó 2015 Published by Elsevier Inc.
1. Introduction
sulfate is a use-dependent NMDA receptor inhibitor [7,8], and
dehydroepiandrosterone (DHEA), allotetrahydrodeoxycorticos-
Over the past few decades, neurosteroids or neuroactive ster-
oids have attracted much interest from an academic perspective
as well as clinical and therapeutic aspects [1–3]. Intensive studies
carried out recently have demonstrated that some endogenous
steroids including estrogens, progestins and allopregnanolone, dis-
play beneficial neuroprotective properties, implying steroids may
represent novel and promising neuroprotectants for the treatment
of neurodegenerative diseases [4–6]. In addition to the classic
genomic actions, increasing evidence shows that steroids exert
nongenomic actions including rapid modulation of membrane
associated receptors and ion channels. For example, pregnanolone
terone (THDOC) and allopregnanolone are positive allosteric
GABAA receptor modulators [9,10]. These nongenomic actions
endow steroids with therapeutic potential to treat neurological
diseases including cerebral ischemia, Alzheimer disease, traumatic
brain injury, and epilepsy [11–16].
Excessive glutamate release and glutamate receptor activation
mediated excitotoxicity is observed in numerous neurological
disorders including stroke, amyotrophic lateral sclerosis (ALS), epi-
lepsy, Parkinson’s, Huntington’s and Alzheimer’s diseases [17–20].
Particularly, NMDA receptor mediated calcium overload is consid-
ered as a primary mediator of the central neuronal injury/death
consequent to hypoxic-ischemic insults [17,21]. Inhibition of
excessive NMDA receptor activation would provide therapeutic
benefits for these neurological disorders implicated with NMDA
receptor over-stimulation [22,23]. For example, memantine, as a
NMDA receptor antagonist has been successfully used in clinical
⇑
Corresponding authors at: Department of Pharmacology, Zhongshan School of
Medicine, Sun Yat-Sen University, 74 Zhongshan Road II, Guangzhou 510080, China
(G. Yan).
E-mail addresses: jingxiaz@163.com (J. Zhang), ygm@mail.sysu.edu.cn (G. Yan).
These authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.steroids.2015.11.005
0039-128X/Ó 2015 Published by Elsevier Inc.

T. Leng et al. / Steroids 105 (2016) 96–105
97
for Alzheimer’s disease [24]. Thus, NMDA receptor inhibitors may
serve as promising candidates to treat neurological disorders
including stroke.
2.1.3. 3b-Acetoxychol-5-en-24-oyl chloride
32 ml thionyl chloride and 1 ml pyridine were added to a solu-
tion of compound 2 (100 g) in 400 ml dry benzene, which mixture
was stirred for 4 h, at room temperature, and evaporated. The
resulting crude product (3b-acetoxychol-5-en-24-oyl chloride)
was dissolved in 200 ml dry benzene for the following Grignard
reaction.
Tetrol is
a steroidal metabolite isolated from soft corals
Nephthea albida and N. tiexieral verseveldt, and was first synthesized
from stigmasterol [25]. It has been shown to exert significant anti-
cancer activity [26]. In the current study, we examined whether
Tetrol has neuroprotective effect. Our data showed that Tetrol
exerted potent neuroprotective activity in both glutamate-medi-
ated neurotoxicity model and MCAO induced cerebral ischemia
model. We also demonstrated that negative modulation of NMDA
receptors might be an underlying mechanism for its neuroprotec-
tive effect. Taken together, our findings demonstrated that the
marine steroid Tetrol functions as a promising therapeutic candi-
date for stroke treatment, which may also be extended to a range
of neurological disorders with NMDA receptors overstimulation.
Additionally, Tetrol would serve as a novel pharmacological tool
for the investigation of NMDA receptor functions.
2.1.4. 3b-Acetoxycholest-5-en-24-one (3)
The isopropyl group was introduced to the side chain through
Grignard reaction. Briefly, dry Mg powder (20 g) was added to
anhydrous ethyl ether (60 ml), and then 73 ml isopropyl bromide
and 100 ml anhydrous ethyl ether were added in 8 portions at
intervals of 5 min, under ultrasound conditions. After this, 78 g
cadmium chloride and 100 ml anhydrous ethyl ether were added
in four portions under ultrasound conditions. Then, the reaction
was extended for 30 min at 40 °C with ultrasound heating. The
reaction mixture was cooled by ice-cold water bath, and then 3b-
acetoxychol-5-en-24-oyl chloride in 200 ml benzene was added
dropwise and stirred for 2 h with ultrasound applied. The reaction
was then cooled again by ice-cold water bath, and quenched by
dropwise addition of 150 ml HCl/H2O mixture (1:1). The resulting
crude product was extracted with benzene, washed with water
to neutral pH, dried over anhydrous Na2SO4, evaporated by vacuum
and recrystallized with 50 ml methanol to afford 71 g colorless
needles (77%). m.p. 125–127 °C. IR (KBr, cmÀ1): 1725, 1706,
1468, 1373, 1255, 1036, 960. 1H NMR (CDCl3, 400 MHz) d: 0.68
(s, 3H, 18-CH3), 0.91 (d, 3H, J = 6.5 Hz 21-CH3), 1.01 (s, 3H, 19-
CH3), 1.10 (6H, J = 7.4 Hz, d, 26-CH3, 27-CH3), 2.03 (s, 3H, CH3CO),
2.60 (m, 1H, 25-CH), 4.59 (m, 1H, 3-CH), 5.37 (d, 1H, J = 4.5 Hz, 6-
CH). 13C NMR (CDCl3, 400 MHz): 12.24 (CH3), 18.68 (CH3), 18.75
(CH3), 18.89 (CH3), 19.67 (CH3), 21.36 (CH2), 21.76 (CH3), 24.31
(CH2), 28.10 (CH2), 28.46 (CH2), 30.12 (CH2), 31.20 (CH2), 31.98
(CH), 35.72 (CH), 36.90 (C), 37.34 (CH2), 37.54 (CH2), 38.45 (CH2),
40.04 (CH2), 41.15 (CH), 42.68 (C), 50.35 (CH), 56.47 (CH), 57.00
(CH), 74.27 (CH), 122.93 (CH), 139.93 (C), 170.82(C), 215.69 (C);
EIMS m/z: 399(MÀC3H7)+, 382 (MÀAcOH)+; Analysis calculated
for C29H46O3: C 78.73, H 10.43; found C 78.25, H 10.25.
2. Experimental
2.1. Synthesis of Tetrol
General procedures
The analytical grade chemicals and solvents were purchased
from the Guangzhou Chemical Reagents Company (Guangzhou,
Guangdong, China). The melting points, IR, 1H NMR and 13C NMR
spectra were recorded as described previously [27]. Chemical shifts
were reported in values relative to TMS as the internal standard.
MS data were measured on a DSQ and LCQ mass spectrometer
(Suwanee, GA, USA). Flash column chromatography was carried
out with silica gel (200–300 mesh).
2.1.1. 3b-Hydroxychol-5-en-24-oic acid (1)
Compound 1 was synthesized starting from HDCA as described
previously [28]. m.p. 220–221 °C; IR:(KBr)
m: 3359, 2939, 2667,
1698, 1436, 1377, 1195, 1049, 957; 1H NMR (CDCl3) d: 5.32(t, 1H,
6b-H), 4.41(m, 1H, 3-CH), 3.75 (s,1H, 3-OH), 0.98 (s, 3H, 19-CH3),
0.94 (d, 3H, 21-CH3), 0.68 (s, 3H, 18-CH3); 13C NMR(CDCl3) d:
175.71 (C), 140.96 (C), 120.69 (CH), 70.55 (CH), 56.38 (CH), 55.49
(CH), 49.74 (CH), 41.99 (C), 41.93 (CH2), 39.37 (CH2), 36.98 (CH2),
36.15 (C), 34.94 (CH), 31.54 (CH), 31.49 (CH2), 31.18 (CH2), 30.84
(CH2), 30.70 (CH2), 27.68 (CH2), 23.88 (CH2), 20.68 (CH2), 19.07
(CH3), 18.03 (CH3), 11.56 (CH3); MS (EI) m/z: 375 [M+1]+, 374
[M]+, 356 [MÀH2O]+; Analysis calculated for C24H38O3: C 77.01, H
10.16; found C 77.09, H 9.87.
2.1.5. 3b-Acetoxy-5a-bromocholestan-6b-ol-24-one (4)
1.5 ml 70% HClO4 and 5.8 ml H2O was added to a solution of
compound 3 (20 g) in 200 ml THF, which was followed by drop-
wise addition of 20 g NBS (N-bromosuccinimide) dissolved in
100 ml THF at a temperature maintained <10 °C. The mixture
was stirred vigorously for 1.5 h in the dark with an ice-cold water
bath, and then 200 ml ice-cold 10% Na2S2O3 was added dropwise to
quench the reaction. The upper layer was evaporated and the lower
layer was extracted twice with 100 ml of dichloromethane. These
two portions were collected and neutralized with saturated
NaHCO3, washed with brine, dried over Na2SO4. The crude product
was obtained by vacuum evaporation, which was further purified
by flash column chromatography to afford 22 g white solid (90%).
m.p. 137–139 °C. IR (KBr, cmÀ1): 3451, 2943, 2872, 1735, 1709,
1467, 1378, 1273, 1244, 1028, 757, 669. 1H NMR (CDCl3,
400 MHz) d: 0.69 (s, 3H, 18-CH3), 0.89 (d, 3H, J = 6.4 Hz, 21-CH3),
1.08 (d, 6H, J = 7.2 Hz, 26-CH3, 27-CH3), 1.26 (s, 3H, 19-CH3), 2.02
(s, 3H, 3-CH3COO), 2.60 (m, 1H, 25-CH), 4.05 (brt, 1H, J = 3.0 Hz,
6-CH), 5.20 (m, 1H, 3-CH); 13C NMR (CDCl3, 400 MHz):12.41
(CH3), 17.99 (CH3), 18.29 (CH3), 18.36 (CH3), 18.47 (CH3), 21.33
(CH3), 21.29(CH2), 24.03 (CH2), 26.36 (CH2), 28.07 (CH2), 29.79
(CH2), 30.59 (CH), 34.59 (CH2), 35.12 (CH2), 35.31 (CH), 37.15
(CH2), 38.45 (CH2), 39.66 (CH2), 40.35(C), 40.85 (CH), 42.74(C),
47.42 (CH), 55.72 (CH), 55.88 (CH), 72.12 (CH), 75.74 (CH), 86.74
(C), 170.40 (C), 215.37 (C); FABMS m/z:520 (MÀH2O)+, 441 (MÀH2-
OÀBr)+; Analysis calculated for C29H47O4Br: C, 64.68; H, 8.74.
Found: C, 64.30; H, 8.77.
2.1.2. 3b-Acetoxychol-5-en-24-oic acid (2)
Pyridine (2.5 ml) was added to a solution of compound 1 (100 g)
in 500 ml acetic anhydride, and then the mixture was heated and
stirred at 100 °C for 2 h. After cooling, 2500 ml of ice-cold water
was poured into the mixture to quench the reaction. After 24 h,
the reaction mixture was filtered, washed with water and dried.
m.p. 178–179 °C. IR (KBr, cmÀ1): 2939, 1733, 1442, 1373, 1247,
1038, 959. 1H NMR (CDCl3, 400 MHz) d: 0.69 (s, 3H, 18-CH3),
0.93 (d, 3H, J = 6.5 Hz 21-CH3), 1.02 (s, 3H, 19-CH3), 2.02 (s, 3H,
CH3CO), 4.61 (m, 1H, 3-CH), 5.38 (t, 1H, J = 4.40 Hz 6-CH). 13C
NMR (CDCl3, 400 MHz), d: 11.87 (CH3), 18.29 (CH3), 19.28 (CH3),
21.03 (CH2), 21.36 (CH3), 24.24 (CH2), 27.77 (CH2), 28.06 (CH2),
29.99 (CH2), 30.80 (CH2), 31.86 (CH2), 31.89 (CH), 35.31 (CH),
36.59 (C), 37.01 (CH2), 38.12 (CH2), 39.73 (CH2), 42.40 (C), 50.03
(CH), 55.81 (CH), 56.68 (CH), 73.99 (CH), 122.56 (CH), 139.67 (C),
170.51 (C), 179.94 (C); EIMS m/z: 356 (MÀAcOH)+; Analysis calcu-
lated for C26H40O4: C 75.00, H 9.62; found C 75.40, H 9.38.

98
T. Leng et al. / Steroids 105 (2016) 96–105
2.1.6. 3b-Acetoxy-5
a
-bromo-6b,19-epoxy-cholest-24-one (5)
56.27 (CH), 56.75 (CH), 60.93 (CH), 61.53 (C), 64.63 (CH2), 70.80
(CH), 170.99 (C), 171.36 (C), 215.80 (C); EIMS m/z: 517 (M+1)+,
473(MÀC3H7)+. Analysis calculated for C31H48O6: C, 72.06; H,
9.36. Found: C, 71.91; H, 9.42.
Compound 4 (5 g) was dissolved in an anhydrous mixture of
800 ml cyclohexane and 200 ml benzene, followed by addition of
(Diacetoxyiodo) benzene (DIB) (4.14 g) and iodine (2.80 g). The
reaction mixture was maintained at 45 °C with a 50 min ultra-
sound (35 kHz), and then washed with 10% Na2SO3, H2O, brine,
dried over anhydrous Na2SO4, and evaporated by vacuum. The pro-
duct was purified by flash column chromatography (petroleum
ether/EtOAc (8:2)) and recrystallized from methanol to afford 5
as colorless needles (4.23 g, 85%). m.p. 141–143 °C. IR (KBr,
cmÀ1): 2934, 2865, 1734, 1705, 1467, 1445, 1369, 1243, 1033,
917, 791, 698, 606. 1H NMR (CDCl3, 400 MHz) d: 0.68 (s, 3H, 18-
CH3), 0.90 (d, 3H, J = 6.4 Hz, 21-CH3), 1.09 (d, 6H, J = 6.8 Hz, 26-
CH3, 27-CH3), 2.03 (s, 3H, 3-CH3COO), 2.61 (m, 1H, 25-CH), 3.74
(d, 1H, J = 8.0 Hz, 19-CHa), 3.92 (d, 1H, J = 8.0 Hz, 19-CHb), 4.10
(brd, 1H, J = 4.5 Hz, 6-CH), 5.40 (m, 1H, 3-CH); 13C NMR (CDCl3,
400 MHz): 12.40 (CH3), 18.20 (CH3), 18.34 (CH3), 18.39 (CH3),
21.83 (CH3), 22.64 (CH2), 23.25 (CH2), 23.42 (CH2), 26.86 (CH2),
28.13 (CH2), 29.75 (CH2), 32.79 (CH2), 33.27 (CH), 35.28 (CH),
37.11 (CH2), 39.72 (CH2), 40.82 (CH), 41.32 (CH2), 43.81 (C),
45.81 (C) 48.64 (CH), 54.33 (CH), 55.78 (CH), 67.43 (CH2), 69.97
(CH), 74.52 (C), 82.28 (CH), 170.25 (C), 215.22 (C); FABMS m/z:
457 (MÀBr)+; Analysis calculated for C29H45O4Br: C, 64.93; H,
8.40. Found: C, 64.70; H, 8.51.
2.1.9. 3b,5a,6b,19-tetrahydroxy-cholest-24-one (8)
At room temperature, 1 N H2SO4 (11.98 ml) was mixed with
compound 7 (4.56 g) in 225 ml acetone and stirred for 24 h. The
reaction mixture was neutralized with NaHCO3 followed by evap-
oration to remove acetone. Then the reaction mixture was
extracted using ethyl acetate, washed with water and saturated
brine solution, dried over anhydrous Na2SO4, evaporated by vac-
uum and purified by flash column chromatography (petroleum
ether/acetone (1:1)) to afford 2.87 g white solid (65%). m.p. 214–
216 °C. IR (KBr, cmÀ1): 3323, 2941, 2868, 1707, 1464, 1380, 1344,
1048, 975. 1H NMR (DMSO-d6, 400 MHz) d: 0.68 (s, 3H, 18-CH3),
0.87 (d, 3H, J = 6.5 Hz, 21-CH3), 0.99 (d, 6H, J = 7.0 Hz, 26-CH3 and
27-CH3), 2.61 (m, 1H, 25-CH), 3.27 (m, 1H, 6-CH), 3.50 (dd, 1H,
J = 12.1, 6.0 Hz, 19-CHa), 3.67 (s, 1H, 5-OH), 3.85 (m, 1H, 3-CH),
3.99 (dd, 1H, J = 12.0, 3.5 Hz, 19-CHb), 4.16 (d, 1H, J = 5.5 Hz, 3-
OH), 4.58 (m, 1H, 19-OH), 5.09 (dd, 1H, J = 5.0, 1.5 Hz, 6-OH). 13C
NMR (DMSO-d6, 400 MHz) d: 12.62 (CH3), 18.48 (CH3), 18.54
(CH3), 18.71 (CH3), 22.58 (CH2), 24.71 (CH2), 27.76 (CH2), 28.60
(CH2), 30.25(CH), 31.71 (CH), 32.08 (CH2), 34.78 (CH2), 35.78
(CH), 37.34 (CH2), 40.74 (CH), 41.21 (CH2), 41.87 (CH2), 42.79 (C),
43.06 (C), 45.84 (CH), 56.39 (CH), 57.40 (CH), 62.92 (CH2), 66.71
(CH), 74.67 (CH), 75.02 (C), 214.76 (C); EIMS m/z: 432 (MÀH2O)+.
Analysis calculated for C27H46O5: C, 71.96; H, 10.29. Found: C,
71.57; H, 9.89.
2.1.7. 3b,19-Diacetoxycholest-5-en-24-one (6)
Compound 5 (4 g) was added to 80 ml CH3COOH, and then 10 g
zinc powder was added, and stirred and heated under reflux for
20 h. The crude product was filtered and extracted with 150 ml
CH2Cl2, neutralized with 40 ml saturated NaHCO3, washed with
water and then dried over anhydrous Na2SO4. The final product
was evaporated and further purified by flash column chromatogra-
phy to afford 3.32 g white solid (90%). IR (KBr, cmÀ1): 2941, 1739,
1711, 1467, 1371, 1240, 1091, 979, 981. 1H NMR (CDCl3, 400 MHz)
d: 0.69 (s, 3H, 18-CH3), 0.91 (d, 3H, J = 6.4 Hz, 21-CH3), 1.09 (d, 6H,
J = 6.8 Hz, 26-CH3, 27-CH3), 2.02 (s, 3H, 3-CH3COO), 2.04 (s, 3H, 19-
CH3COO), 2.61 (m, 1H, 25-CH), 3.98 (d, 1H, J = 12.0 Hz, 19-CHa),
4.45(d, 1H, J = 12.0 Hz, 19-CHb), 4.64 (1H, m, 3-CH), 5.62 (1H, t,
J = 3.2 Hz, 6-CH); 13C NMR (CDCl3, 400 MHz): 12.23 (CH3), 18.59
(CH3), 18.65 (CH3), 18.78 (CH3), 21.32 (CH3), 21.58 (CH3), 21.93
(CH2), 24.46 (CH2), 28.16 (CH2), 28.38 (CH2), 30.06 (CH2), 33.15
(CH), 33.68 (CH2), 35.63 (CH), 38.47 (CH2), 39.98 (CH2), 39.72
(CH2), 39.98 (C), 40.13 (CH2), 41.04 (CH), 42.73 (C), 50.36 (CH),
56.14 (CH), 57.46 (CH), 64.63 (CH2), 73.55 (CH), 126.96 (CH),
134.87 (C), 170.58 (C), 170.81 (C), 215.27 (C); EIMS m/z: 457
(MÀC3H7)+; Analysis calculated for C31H48O5: C, 74.36; H, 9.66.
Found: C, 74.16; H, 9.53.
2.1.10. 24-Methylenecholestane-3b,5a,6b,19-tetraol (Tetrol)
n-Butyllithium (2.8 ml) was added to a solution of methyltriph-
enylphosphonium bromide (500 mg, 1.90 mmol) in THF (35 ml).
The mixture was stirred at room temperature for 3.5 h under nitro-
gen atmosphere. When the reaction mixture turned red, compound
8 (420 mg) was added and stirred for 1 h. The product was poured
to NH4Cl and evaporated to remove THF. The residue was extracted
with ethyl acetate (100 ml  2), washed with water, dried with
Na2SO4. The final product was evaporated under vacuum and puri-
fied with flash column chromatography (petroleum ether/acetone
(1:1)) and was further purified by recrystallization from MeOH–
H2O to afford colorless needles 0.337 g (81%). m.p. 223–225 °C. IR
(KBr, cmÀ1): 3416, 2942, 2868, 1642, 1464, 1377, 1343, 1080,
1057, 1030, 879; 1H NMR (DMSO-d6, 400 MHz) d: 0.67 (s, 3H, 18-
CH3), 0.87 (d, 3H, J = 6.5 Hz, 21-CH3), 0.99 (d, 3H, J = 3.5 Hz, 27-
CH3), 1.01 (d, 3H, J = 3.5 Hz, 26-CH3), 2.20 (m, 1H, 25-CH), 3.27
(m, 1H, 6-CH), 3.52 (dd, 1H, J = 12.0, 6.0 Hz, 19-CHa), 3.73 (s, 1H,
5-OH), 3.84 (m, 1H, 3-CH), 4.01 (dd, 1H, J = 12.0, 3.5 Hz, 19-CHb),
4.16 (d, 1H, J = 5.5 Hz, 3-OH), 4.51 (dd, 1H, J = 6.0, 3.5 Hz, 19-OH),
4.65 (brs, 1H, 28-CHa), 4.71 (brs, 1H, 28-CHb), 5.05 (d, 1H,
J = 5.14 Hz, 6-OH); 13C NMR (DMSO-d6, 400 MHz) d: 12.74 (CH3),
19.37 (CH3), 22.50 (CH3), 22.58 (CH2), 22.64 (CH3), 24.70 (CH2),
27.89 (CH2), 28.75 (CH2), 30.18 (CH2), 31.76 (CH), 32.07 (CH2),
33.95 (CH), 34.76 (CH2), 35.09 (CH2), 36.13 (CH), 41.26 (CH2),
41.84 (CH2), 43.34 (C), 43.49(C), 45.87 (CH), 57.45 (CH), 56.68
(CH), 63.02 (CH2), 66.74 (CH), 74.70 (CH), 75.37 (C), 107.25 (CH2),
156.81 (C); FABMS m/z: 449 (M+1)+. Analysis calculated for
2.1.8. 3b,19-Diacetoxy-5a,6a-epoxy-cholest-24-one (7)
4.4 g Na2CO3 and 170 ml H2O was mixed with compound 6
(4.5 g) in CH2Cl2 (250 ml) and stirred, and then MCPBA (2.8 g)
was added to the reaction mixture and stirred for 4 h. The product
was extracted with CH2Cl2 and washed with 5% Na2SO3, saturated
NaHCO3 and H2O, dried with anhydrous Na2SO4, extracted with
CH2Cl2 and evaporated with vacuum to afford 4.56 g white solid
(99%). m.p. 178–180 °C. IR (KBr, cmÀ1): 2941, 1734, 1708, 1467,
1446, 1369, 1232, 1036, 964. 1H NMR (CDCl3, 400 MHz) d: 0.68
(s, 3H, 18-CH3), 0.88 (d, 3H, J = 6.5 Hz, 21-CH3), 1.10 (d, 6H,
J = 7.1 Hz, 26-CH3, 27-CH3), 2.01 (s, 3H, 3-CH3COO), 2.04 (s, 3H,
19-CH3COO), 2.60 (m, 1H, 25-CH), 4.06 (d, 1H, J = 11.6 Hz, 19-
CHa), 4.20 (d, 1H, J = 11.6 Hz, 19-CHb), 4.80 (1H, m, 3-CH), 4.97
(1H, t, J = 3.3 Hz, 6-CH); 13C NMR (CDCl3, 400 MHz): 12.16 (CH3),
18.71 (CH3), 18.77 (CH3), 18.86 (CH3), 21.65 (CH3), 21.68 (CH3),
22.29 (CH2), 24.49 (CH2), 27.75 (CH2), 28.68 (CH2), 30.12 (CH2),
32.38 (CH), 32.54 (CH2), 38.40 (C), 37.51 (CH2), 35.76 (CH), 37.62
(CH2), 38.72 (CH2), 66.19 (CH2), 41.23 (CH), 42.67(C), 49.85 (CH),
C28H48O4: C, 75.06; H, 10.71. Found: C, 74.76; H, 10.74.
2.2. Neuronal cultures and glutamate treatment
The experimental procedures on live animals were performed
following the guidelines for the care and use of laboratory animals
as published by National Institutes of Health (NIH Publications No.
8023, revised 1978). Rat cerebellar granule neurons (CGNs) were
cultured from 8 day old Sprague–Dawley rat pups as described

T. Leng et al. / Steroids 105 (2016) 96–105
99
previously [29]. The neurons were cultured and seeded at a density
of 2.0 Â 106 cells/ml in basal modified Eagle’s medium (Invitrogen)
containing 10% fetal bovine serum (FBS), 25 mM KCl, 2 mM glu-
surgical operation, the right femoral artery was cannulated for
sampling blood to measure the physiological variables including
pH, PaO2, PaCO2 and glucose. Rectal temperature was maintained
at 37 0.5 °C with a thermostatically controlled heating pad and
lamp. Animals were sacrificed with chloral hydrate at 7 days after
ischemia. Brains were rapidly removed, sectioned coronally at
2 mm intervals, and stained with 2,3,5-triphenyltetrazolium
hydrochloride (TTC). The corrected infarct volume percent-
age = 100 Â (contralateral hemisphere volume À non-infarct ipsi-
lateral hemisphere volume)/contralateral hemisphere volume. All
manipulations and analysis were performed by individuals blinded
to the treatment groups.
tamine, and penicillin (100 units/ml)/streptomycin (100
To suppress the growth of non-neuronal cells, cytosine arabinoside
(10 M) was added 24 h after plating. For glutamate treatment,
lg/ml).
l
CGN were used at 8–10 days. Briefly, CGNs were washed two times
with Locke’s buffer (134 mM NaCl, 25 mM KCl, 4 mM NaHCO3,
2.3 mM CaCl2, 5 mM D-glucose, 5 mM HEPES, pH 7.4). Then DMSO
vehicle (<0.1%) and different concentrations of Tetrol were added
to the culture in Locke’s buffer, followed by addition of 200
lM
glutamate and 10 M glycine and incubated for 45 min. After that,
l
the cultures were changed back to the normal culture medium for
24 h, until the viability and LDH measurement were performed.
Mouse cortical neurons were prepared from the brains of
fetuses (embryonic day 16) of Swiss Webster mice as described
previously [30]. Cells were cultured and seeded in poly-L-
ornithine-coated culture dishes at a density of 1 Â 106 cells/dish.
Cells were initially maintained in minimal essential medium con-
taining 10% FBS, 10% horse serum at 37 °C in a humidified 5%
CO2 atmosphere incubator for 24 h. Culture medium was changed
to Neurobasal medium supplemented with B-27 (Invitrogen) at
24 h. The culture medium was changed twice a week. Neurons
were used for the experiments at 10–14 days in vitro.
2.5. Measurement of intracellular calcium
Fluorescence dye fura-2-acetoxymethyl ester was used for cal-
cium imaging. Briefly, cortical neurons grown in 6-well plates with
coverslips were washed three times with normal extracellular fluid
(ECF) (ECF composition, in mM: NaCl, 140; KCl, 5.4; MgCl2, 1;
CaCl2, 2; HEPES, 20; glucose, 10, pH 7.4), and then were incubated
in ECF with 5 lM fura-2-acetoxymethyl ester for 30 min in the
dark. After that, the cells were washed three times with ECF and
kept in the dark for additional 30 min until Ca2+ measurement.
Ca2+ measurement was conducted in the presence of Nimodipine
(5
2,3-dione (CNQX) (10
(0.3
M; Alomone labs). Mg2+-free ECF containing 200
and 3
M glycine was applied to induce the increase of [Ca2+]i. Dig-
ital images were acquired and analyzed by Axon Imaging Work-
bench software 6.0 (Axon Instruments). Ratio images (340/380)
were analyzed by averaging pixel ratio values in circumscribed
regions of cells in the field of view. Data were finally presented
by (F À F0)/F0 for the dose response analysis. F indicates the
340/380 values over time and F0 indicates the 340/380 value at
0 S time point. Sigmaplot 8.0 was used for further analysis.
l
M; Sigma, St. Louis, MO, USA), 6-cyano-7-nitroquinoxaline-
M; Sigma) and Tetrodotoxin (TTX)
M NMDA
2.3. Neuronal viability and injury assays
l
l
l
The viability of CGNs was assessed by fluorescein diacetate
(FDA) alive staining, at 24 h after glutamate treatment. FDA is
taken up by cells which convert the non-fluorescent FDA into the
green fluorescent metabolite fluorescein. As the conversion is
esterase dependent, the measured signal serves as indicator for
viable cells [31]. Briefly, CGNs were washed twice with PBS then
l
incubated in PBS containing FDA (10 lg/ml) for 5 min in the dark.
The cultures were then washed twice with PBS and examined by
random photography under UV light microscopy. FDA positive
neurons from 4 to 5 random fields were counted by a blinded
observer, and normalized to the normal control.
2.6. Electrophysiology
Cell injury was quantified by LDH assay using a Cytotox 96 non-
radioactive cytotoxicity assay kit (Promega, Madison, WI, USA). At
the end of the experiments, 50 ll medium was transferred from
the culture wells to 96-well plates for LDH release measurement.
For the final sample, cells were incubated with 1% Triton X-100
Whole-cell patch-clamp recordings were performed under volt-
age-clamp mode at room temperature (22–25 °C), with Multiclam-
p200B amplifier and Digidata1322 series interface (AXON
Instrument, Foster City, CA). Data analysis was performed using
pCLAMP 9.0 software (AXON Instrument). Recording pipettes were
made from glass pipettes of 1.2 mm outside diameter and 0.5 mm
inside diameter, through a horizontal puller P-97 (Sutter Instru-
ment Co., Novato, CA, USA). The pipettes were filled with internal
solution containing (in mM): 140 CsF, 1 CaCl2, 10 HEPES, 11 ethy-
lene glycol tetraacetic acid (EGTA), 2 Tetraethylammonium (TEA),
4 MgCl2, pH 7.3, adjusted with CsOH, 290–300 mOsm. The perfu-
sion solution contained (in mM): 140 NaCl, 5.4 KCl, 20 HEPES, 10
Glucose, 2 CaCl2, pH 7.4, adjusted with NaOH and HCl, 320–
330 mOsm. NMDA current was induced by co-application of
for 30 min, and then 50
well to 96-well plates for maximal LDH release measurement.
50 l of the reconstituted Substrate Mix was added to each well
of the plate and incubated for 30 min in dark at room temperature.
Then, 50 l stop solution was added and then the absorbance at
lL supernatant was withdrawn from each
l
l
490 nm was measured within one hour.
2.4. Focal ischemia
Male Sprague Dawley (SD) rats (weighing 250–300 g), under-
went focal cerebral ischemia, were randomly assigned to two
groups as follows: Tetrol group (n = 16), in which the animals were
administered with 12 mg/kg Tetrol intraperitoneally successively
at the time of 10 min, 6 h, 1 day and 4 days after middle cerebral
artery occlusion (MCAO); Vehicle group (n = 16) were adminis-
tered the same volume dimethyl sulfoxide (DMSO) intraperi-
toneally. Permanent focal ischemia was induced by suture
occlusion of the middle cerebral artery in male rats. A 3–0 nylon
monofilament suture (Ethicon Nylon Suture, Ethicon Inc., Osaka,
Japan) with blunt tip was inserted into the internal carotid artery
to a point approximately 17–18 mm distal to the carotid bifurca-
tion until a mild resistance was felt, thereby occluding the origins
of the anterior cerebral and middle cerebral arteries. During the
100 lM NMDA and 3 lM glycine, in the presence of 0.3 lM TTX.
The voltage was held at À60 mV. The peak currents were used
for data analysis. To avoid the possible interference from current
run-down, the formal recording wasn’t started until, at least, three
stable NMDA currents were achieved.
2.7. Statistical analysis
Results were expressed as mean S.E.M. Sigma Plot (San Jose,
CA, USA) and GraphPad Prism 4 (San Diego, CA, USA) were used
for statistical analysis. Student’s t-test and one-way ANOVA with
post hoc Dunnett’s test were used to examine the statistical signif-
icance where appropriate. Statistical significance was set at

100
T. Leng et al. / Steroids 105 (2016) 96–105
P < 0.05. The dose–response curve was fitted with 3 parameter
logistic nonlinear regression model.
Tetrol was established on the basis of spectroscopic analysis and
by comparing the physical and spectroscopic data with literature
values (Table 1).
3. Results
3.2. Neuroprotective effects of Tetrol against glutamate-induced
neuronal injury in vitro
3.1. Synthesis of 24-methylenecholestane-3b,5a,6b,19-tetraol
We tested the neuroprotective effect of the synthesized Tetrol
on glutamate (Glu)-induced neuronal injury in CGNs. Exposure of
In this study, we designed and synthesized Tetrol starting from
hyodeoxycholic acid (HDCA) via multiple steps of reactions as
described above (also see the scheme in Fig. 1). The final product
purification by flash column chromatography resulted in a white
solid, which was further purified by recrystallization from
MeOH–H2O to afford colorless needles. The structural identity of
the cultured cerebellar granule neurons to 200
nificantly reduced the number of surviving neurons compared with
normal control (Fig. 2A). Tetrol at 10 M effectively increased the
lM glutamate sig-
l
survival number of cerebellar granule neurons compared with
DMSO vehicle (Veh) (Fig. 2A). DMSO concentration was <0.1%,
Fig. 1. Synthesis of 24-methylenecholestane-3b,5
a,6b,19-tetraol (Tetrol). Tetrol was synthesized from hyodeoxycholic acid (HDCA) via several steps of reactions. Compound
1 was synthesized from HDCA as previously described [28].

T. Leng et al. / Steroids 105 (2016) 96–105
101
Table 1
Comparison of the 1H and 13C chemical shifts (ppm) of Tetrol with reference (Ref) values [25].
H
Synthetic product
Ref. value
C
Synthetic product
Ref. value
3
a
-H
-H
3.84
3.27
3.52
4.01
4.65
4.71
4.16
3.73
5.05
4.51
3.84
3.27
3.52
3.99
4.63
4.70
4.16
3.73
5.04
4.52
3-CH
5-C
6-CH
19-CH2
24-C
28-CH2
18-CH2
21-CH3
26-CH3
27-CH3
66.74
75.37
74.70
63.02
156.81
107.25
12.74
19.37
22.64
22.50
65.8
74.4
73.8
62.0
155.7
106.4
12.1
18.5
21.6
21.6
6a
19-Ha
19-Hb
28-Ha
28-Hb
3b-OH
5a-OH
6b-OH
19-OH
Fig. 2. Tetrol protects against glutamate toxicity in cerebellar granule neurons (CGNs). (A) Representative phase-contrast images show Tetrol at 10
l
M protects against
glutamate toxicity in cultured CGNs at 24 h after glutamate challenge. Normal control group (Control), neurons without glutamate treatment; Glu + Vehicle group, neurons
treated with 200 M glutamate, 10 M glycine and DMSO vehicle (<0.1%); Glu + Tetrol groups, neurons treated with 200 M glutamate, 10 M glycine and 10 M Tetrol. (B)
l
l
l
l
l
Representative FDA staining of alive neurons observed under UV light. (C) Summary data of FDA staining show Tetrol concentration-dependently increases the survival rate of
CGNs treated with glutamate. *P < 0.05 compared with vehicle (Veh) group, n = 4 independent experiments (one-way ANOVA, F = 17.46). 4–5 random fields were counted and
averaged for each coverslip. (D) Tetrol concentration-dependently inhibits glutamate-induced LDH release in CGNs at 24 h after glutamate treatment. Data were presented as
mean S.E.M, *P < 0.05 compared with vehicle (Veh) group, n = 4 independent experiments (one-way ANOVA, F = 27.69).
which has no significant neuroprotective effect (data not shown).
The survival rate was further evaluated by measurement of the
FDA (fluorescein diacetate) staining cell numbers (Fig. 2B). At
24 h following glutamate treatment, alive staining with FDA
showed that Tetrol significantly increased the viability (% FDA pos-
itive cells) in a concentration-dependent manner (Fig. 2C, n = 4,

102
T. Leng et al. / Steroids 105 (2016) 96–105
Fig. 3. Tetrol protects against MCAO induced cerebral ischemia injury in rats. (A)
Representative coronal brain sections stained with TTC showing the infarction area
(pale region). Tetrol (12 mg/kg, i.p.) significantly decreased the infarction area
compared with vehicle. (B) Summary data showing infarction volumes (%
contralateral structure) in male SD rats treated with DMSO vehicle (n = 16) or
12 mg/kg Tetrol (n = 16). All values are mean SEM. *P < 0.05 compared with
vehicle group (Student’s unpaired t test).
*P < 0.05, by one-way ANOVA). Consistent with FDA staining, the
LDH release assay showed that Tetrol concentration-dependently
decreased glutamate-induced neuronal injury (Fig. 2D, n = 4,
*P < 0.05, by one-way ANOVA).
3.3. Neuroprotective effects of Tetrol against cerebral ischemia injury
in vivo
Fig. 4. Inhibition of NMDA-induced [Ca2+
] increase by Tetrol in cortical neurons.
i
We then explored the neuroprotective effect of Tetrol in a rat
permanent focal cerebral ischemia model induced by MCAO. A sig-
nificant infarction area (white) was observed in the vehicle treated
group at 7 days after MCAO (Fig. 3A). Intraperitoneal administra-
tion of 12 mg/kg Tetrol significantly reduced the infarction volume
(A) Tetrol concentration dependently inhibited NMDA-induced [Ca2+
] increase in
i
cultured cortical neurons. 200
lM NMDA and 3 lM glycine were used to induce the
[Ca2+
]
i
elevation. DMSO (<0.1%) was added as a vehicle control. The data were
expressed as (F À F0)/F0. F indicates the 340/380 values over time, and F0 indicates
the basal 340/380 value at 0 S time point. (B) Dose–response curve was derived
from the data at the 200 S time point and fitted with a 3 parameters logistic
*
from 61 8.1% to 32 11% (Fig. 3B, n = 16, P < 0.05, by Student’s
nonlinear regression model, with an IC50 of 7.8 0.62 lM (n = 104–157 cells).
unpaired t test). The physiological variables during surgery in all
experimental animals were within normal range and no significant
differences were observed between groups (Table 2).
reduced the increased [Ca2+]i level (Fig. 4A). The dose–response
analysis of the values measured at the 200 S time point yielded a
half-maximal inhibitory concentration (IC50
) of 7.8 0.62 lM
3.4. Tetrol inhibits NMDA-induced [Ca2+]i increase in cortical neurons
(Fig. 4B, n = 104–157 cells).
NMDA receptor mediated intracellular calcium overload is an
important mechanism underlying glutamate mediated neuronal
toxicity. We then investigated whether Tetrol decreases NMDA
induced intracellular calcium increase. Bath application of
3.5. Tetrol inhibits NMDA currents in cortical neurons
The NMDA receptor channel is a major pathway conducting glu-
tamate-induced Ca2+ influx. Based on the above calcium imaging
findings, we hypothesized that NMDA receptors might be a target
of Tetrol. To test this hypothesis, we examined the effects of Tetrol
200 lM NMDA in cultures induced a remarkable increase of
[Ca2+]i. Co-application of Tetrol concentration-dependently
Table 2
Physiological variables during MCAO.
Group
pH
7.38 0.2
7.37 0.17
PaO2 (mmHg)
PaCO2 (mmHg)
Glucose (mol/L)
Rectal temperature (°C)
DMSO (n = 16)
Tetrol (n = 16)
124 2.45
129 3.74
41 2.45
39 1.73
5.78 0.91
5.63 0.62
37.2 0.77
37 1.09
Data are expressed as mean SEM. PaO2: arterial oxygen tension; PaCO2: arterial carbon dioxide tension. P > 0.05 for all parameters between two groups.

T. Leng et al. / Steroids 105 (2016) 96–105
103
Fig. 5. Inhibition of NMDA currents by Tetrol in cortical neurons. (A and B) Representative current traces were induced by 100
lM NMDA plus 3
lM glycine, with or without
various concentrations of Tetrol as indicated. (C) Summary data showed that Tetrol concentration-dependently inhibited NMDA currents. (D) The dose–response curve was
inferred from (C) and was fitted with 3 parameters logistic nonlinear regression model, with an IC50 of 10.28 0.71
currents was normalized to the peak amplitude of the control currents at 0 min.
lM (n = 5 cells). The inhibitory effect of Tetrol on NMDA
on NMDA receptor-mediated inward currents in cortical neurons.
The formal recording wasn’t performed until three stable current
traces were obtained. Vehicle has no significant effect on NMDA
currents (Fig. 5A and C, n = 6 cells), whereas Tetrol concentration-
dependently decreased NMDA currents (Fig. 5B and C, n = 5 cells).
The dose–response analysis yielded a half-maximal inhibitory con-
cal activities through classic steroid hormone nuclear receptors. In
the past few decades, increasing evidence shows that steroids have
non-genomic effects including the modulation of neuronal
excitability through interaction with cell surface receptors and li-
gand-gated ion channels such as GABAA and NMDA receptors
[32,33]. For example, the endogenous oxysterols including 24(S)-
centration (IC50) of 10.28 0.71
l
M (Fig. 5D, n = 5 cells).
hydroxycholesterol and cholestane-3b,5a,6b-triol have recently
been shown to be a positive allosteric modulator and use-depen-
dent inhibitor of NMDA receptors, respectively [34,35]. It is impor-
tant to study the effects of neurosteroids or neuroactive steroids in
view of their therapeutic potential in the treatment of neurological
diseases [36,37]. In this study, we demonstrated that the marine
steroid Tetrol protects neurons against glutamate toxicity and
4. Discussion
The steroids synthesized in the nervous system starting from
cholesterol or steroidal precursors, termed neurosteroids, have a
variety of diverse functions [1,3]. In general, they mediate biologi-

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T. Leng et al. / Steroids 105 (2016) 96–105
MCAO-induced neuronal injury partly via a mechanism of negative
modulation of NMDA receptors.
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Acknowledgements
Supported by the Project of Natural Science Foundation of
Investigation Team, Guangdong Province of China [No. 039191];
the National High Technology Research and Development Program
of China [No. 2006AA090502]; the National Natural Science Foun-
dation of China [81173045]; and the NIH R01NS066027.

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