An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system

Article abstract of DOI:10.1016/j.steroids.2015.01.005

Abstract Spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, were synthesized from commercially available stigmasterol by a four steps reactions. In addition, the effects of these phytosterols on cell growth and

Full text of DOI:10.1016/j.steroids.2015.01.005

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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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An expeditious synthesis of spinasterol and schottenol, two phytosterols
present in argan oil and in cactus pear seed oil, and evaluation of their
biological activities on cells of the central nervous system
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Asmaa Badreddine a,b, Karym El Mostafa a,b, Amira Zarrouk a,c, Thomas Nury a, Youssef El Kharrassi a,b
,
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Boubker Nasser b, Mustapha Cherkaoui Malki a, Gérard Lizard a, ,1, Mohammad Samadi d,
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,1
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a Université de Bourgogne, Equipe ‘Biochimie du peroxysome, inflammation et métabolisme lipidique’ EA 7270/INSERM, Dijon, France
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b Laboratory of ‘Biochemistry of Neuroscience’, University Hassan I, Settat, Morocco
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c University of Monastir, LR12ES05, Lab-NAFS ‘Nutrition – Functional Food & Vascular Health’, Monastir, Tunisia
d LCPMC-A2, ICPM, Département de Chimie, Université de Lorraine, 1 Bd Arago, Metz Technopôle, Metz, France
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Spinasterol and schottenol, two phytosterols present in argan oil and in cactus pear seed oil, were syn-
thesized from commercially available stigmasterol by a four steps reactions. In addition, the effects of
these phytosterols on cell growth and mitochondrial activity were evaluated on 158N murine oligoden-
drocytes, C6 rat glioma cells, and SK-N-BE human neuronal cells with the crystal violet test and the MTT
test, respectively. The effects of spinasterol and schottenol were compared with 7-ketocholesterol (7KC)
and ferulic acid, which is also present in argan and cactus pear seed oil. Whatever the cells considered,
dose dependent cytotoxic effects of 7KC were observed whereas no or slight effects of ferulic acid were
found. With spinasterol and schottenol, no or slight effects on cell growth were detected. With spinaster-
ol, reduced mitochondrial activities (30–50%) were found on 158N and C6 cells; no effect was found on
SK-N-BE. With schottenol, reduced mitochondrial activity were revealed on 158N (50%) and C6 (10–20%)
cells; no effect was found on SK-N-BE. Altogether, these data suggest that spinasterol and schottenol can
modulate mitochondrial activity and might therefore influence cell metabolism.
Received 4 November 2014
Received in revised form 15 December 2014
Accepted 6 January 2015
Available online xxxx
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Keywords:
Spinasterol
Schottenol
Organic synthesis
Crystal violet test
MTT test
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Nervous cells
Ó 2015 Elsevier Inc. All rights reserved.
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1. Introduction
seed oil [5]. In order to evaluate their biological activities in vitro
and in vivo, high amounts of these compounds are required. Due
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Phytosterols are structurally related to cholesterol and are
mainly C-28 and C-29 carbon steroid alcohols [1]. Phytosterols
might have some benefits in preventing cardiovascular diseases,
and they could also contribute to preventing cancer and inflamma-
tory diseases [2–4]. These benefits have led to using phytosterols
as nutraceuticals. As the functional food market has grown expo-
nentially in recent years, the understanding of the potential health
benefits of phytosterol–enriched foods and nutrients is continually
evolving, and it is therefore necessary to have a better knowledge
of these molecules. However, only few data currently exist on the
biological activities of most phytosterols, mainly on spinasterol
and schottenol, which are present in argan oil and in cactus pear
to very low natural abundance of spinasterol and schottenol from
natural sources and despite the potential of these sterols in biolog-
ical studies, only three syntheses of spinasterol and schottenol are
available but they require 5–10 reaction steps with low overall
yield [6–8]. As part of our program directed toward the synthesis
of bioactive natural products an expeditious synthesis of spinaster-
ol and schottenol was developed. As some phytosterols have the
ability to cross the blood brain barrier [9] and to modulate amylo-
idogenesis in mice in vivo [10], we evaluated the biological activi-
ties of these synthetic phytosterols (spinasterol and schottenol) on
cells of the central nervous systems (158N murine oligodendro-
cytes, C6 rat glioma cells, and SK-N-BE human neuronal cells) with
the crystal violet test and the MTT test allowing to evaluate cell
growth and mitochondrial activity, respectively. Their effects were
compared with 7-ketocholesterol (used as positive control, known
to inhibit cell growth and to trigger mitochondrial dysfunction)
[11], and with ferulic acid (a potent anti-oxidant) also present in
argan and cactus pear seed oil [12]. Altogether, our data report
⇑
Corresponding authors. Tel.: +33 380 39 62 56 (G. Lizard), +33 387 54 77 93 (M.
Samadi).
E-mail addresses: gerard.lizard@u-bourgogne.fr (G. Lizard), mohammad.
samadi@univ-lorrraine.fr (M. Samadi).
1
Prof. Mohammad Samadi and Dr. Gérard Lizard equally contributed.
http://dx.doi.org/10.1016/j.steroids.2015.01.005
0039-128X/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: Badreddine A et al. An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in
cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system. Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.01.005
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an expeditious synthesis of spinasterol and schottenol which are
able to modulate mitochondrial activity on various cell types of
the central nervous system.
2.1.1. -Ethylcholest-5, 22-dien-3-hydroxy-7-one (compound 5; Fig. 1)
To a solution of stigmasterol 3 (1 mmol) and CuCl(OH)ÁTMEDA
(23.2 mg, 0.05 mmol) in CH2Cl2 (8 mL) and MeOH (2 mL) was
added t-BuOOH (2 mL, 10 mmol) [13]. After, the reaction was stir-
red at room temperature for 40 h, solvent was removed under
reduced pressure. The residue was purified by flash column chro-
matography with EtOAc–petroleum ether (40–60) as eluent to
yield 5 (247 mg, 58%) as a white solid. mp 150–152 °C; IR (neat):
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2. Materials and methods
2.1. Chemistry
m
: 3349, 2958, 2938, 2865, 1677, 1633, 1042 cmÀ1. 1H NMR (CDCl3)
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All of the reactions were carried out under an argon atmo-
sphere. All reagents were obtained from commercial suppliers
and used without further purification. Stigmasterol 90% (TCI)
and Sitosterol 70% (Sigma–Aldrich) were used as such without
5.71 (s, 1H, 6-CH), 5.19 (dd, 1H, J = 15.16, 8.56 Hz, 23-CH), 5.043
(dd, 1H, J = 15.16, 8.64 Hz, 22-CH), 3.731–3.677 (m, 1H, 3-CH),
1.21 (s, 3H, 19-CH3), 1.04 (d, 3H, J = 6.5 Hz, 21-CH3), 0.86 (d, 3H,
J = 6.5 Hz, 26-CH3 or 27-CH3), 0.82 (t, 3H, J = 7.0 Hz, 29-CH3), 0.81
(d, 3H, J = 6.5 Hz, 26-CH3 or 27-CH3), 0.71 (s, 3H, 18-CH3), 13C
NMR (CDCl3) d = 202.29, 165.24, 138.07, 129.5, 126.06, 70.49,
54.7, 51.21, 50.04, 49.95, 45.39, 42.99, 41.83, 40.23, 38.58, 38.3,
36.36, 31.87, 31.17, 29.03, 26.4, 25.36, 21.41, 21.2, 21.04, 19.00,
17.31, 12.24, 12.19. HRMS: m/z calcd for C29H46O2 [M] 426.3498,
found 402.3489. 1H, 13C NMR, and IR data were in accordance with
literature values [14] (Suppl. Fig. 1).
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further purification. Cu(OH)ClÁTMEDA dimmer (di-
l-hydroxo-
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bis[(N,N,N’,N’-tetramethylethylenediamine) copper(II)] chloride)
was prepared according to literature [13]. Flash chromatography
was carried out using silica gel (Merck Kieselgel 60, 230–400
mesh) with mixtures of ethyl acetate and petroleum ether as elu-
ent unless specified otherwise. TLC analyses were performed on
thin-layer analytical plates 60 F254 (Merck). The tert-butyl hydro-
peroxide (TBHP) 5–6 M solution in Decane was purchased from
Sigma–Aldrich. TLC analyses were performed on thin layer analyt-
ical Plates 60 F254 (Merck). Melting points were measured on a
Kofler Heizbank apparatus and are uncorrected. IR spectra were
recorded on a PerkinElmer-Spectrum One FTIR spectrometer. 1H
and 13C NMR spectra were recorded with a Bruker Advance 400
spectrometer. High-resolution mass spectra (HRMS) were taken
in electron ionization (EI) mode on Jeol GCmate.
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2.1.2. -Ethylcholest-5-en-3-hydroxy-7-one (compound 8; Fig. 1)
Sitosterol (414 mg, 1 mmol) was oxidized by same procedure
described for 3 and purified over silica gel using EtOAc–petroleum
ether (40–60) as eluent to give compound 8 (223 mg, 52%) as a
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white solid. mp 122–124 °C; IR (neat):
m: 3524, 2938, 2861, 1660,
1627, 1062 cmÀ1 1H NMR (CDCl3) 5.7 (d, J = 0.84 Hz, 1H, 6-CH),
.
Fig. 1. Synthesis scheme of spinasterol and schottenol.
Please cite this article in press as: Badreddine A et al. An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in
cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system. Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.01.005
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3.731–3.677 (m, 1H, 3-CH), 1.21 (s, 3H, 19-CH3), 0.94 (d, 3H,
J = 6.5 Hz, 21-CH3), 0.86 (d, 3H, J = 6.5 Hz, 26-CH3 or 27-CH3),
0.82 (t, 3H, J = 7.0 Hz, 29-CH3), 0.81 (d, 3H, J = 6.5 Hz, 26-CH3 or
27-CH3), 0.7 (s, 3H, 18-CH3) ppm. 13C NMR (CDCl3) d = 202.40,
165.28, 126.07, 70.48, 54.71, 49.96, 49.94, 45.82, 45.41, 43.1,
41.82, 38.7, 38.29, 36.36, 36.07, 33.94, 31.16, 29.14, 28.54, 26.32,
26.1, 23.05, 21.21, 19.79, 19.04, 18.92, 17.3, 11.97 (2 Â CH3) ppm.
HRMS: m/z calcd for C29H48O2 [M] 428.3654, found 428.3658. 1H,
13C NMR, and IR data were in accordance with literature values
[15] (Suppl. Fig. 1).
18-CH3) ppm. 13C NMR (CDCl3) d = 139.56, 138.17, 129.45, 117.46,
71.06, 55.91, 55.13, 51.25, 49.46, 43.29, 40.83, 40.27, 39.47, 37.99,
37.15, 34.22, 31.88, 31.47, 29.65, 28.51, 25.40, 23.02, 21.55, 21.38,
21.09, 19.00, 13.04, 12.24, 12.04 ppm. HRMS: m/z calcd for
C29H48O [M] 412.3705, found 412.3716. 1H and 13C NMR data and
were in accordance with literature values [16] (Suppl. Fig. 1).
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2.1.6. -Schottenol (compound 2; Fig. 1)
Schottenol was obtained from ketone 9 (215 mg, 0.5 mmol) by
the same procedure described for spinasterol. Yield (156 mg.
75%) as a white solid. mp: 150–152 °C; IR (neat): m: 3538, 2930,
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2.1.3. -Ethylcholesta-3-hydroxy-22-en-7-one (compound 6; Fig. 1)
Ammonium formate (252 mg, 4 mmol) was added to a mixture
of the enone 5 (426 mg, 1 mmol) and Pd/C (10%, 40 mg) in metha-
nol (5 mL) and ethyl acetate (5 mL). The mixture was heated at
70 °C for 1 h and then cooled to room temperature. The solvent
was evaporated under reduced pressure and the residue was puri-
fied by flash chromatography on silica gel using EtOAc–petroleum
ether (40–60) as eluent to yield product 6 (390 mg, 91%) as white
291, 2863, 2850, 1446, 1038 cmÀ. 1 1H NMR (CDCl3) 5.18 (dd, 1H,
J = 4.36, 2.33 Hz, 7-CH), 3.61 (m, 1H, 3-CH), 094 (d, 3H, J = 6.4 Hz,
21-CH3), 0.86 (t, 3H, J = 7.6 Hz, 29-CH3), 0.85 (d, 3H, J = 7.2 Hz, 26-
CH3 or 27-CH3), 0.83 (d, 3H, J = 6.8 Hz, 26-CH3 or 27-CH3), 0.81 (s,
3H, 19-CH3) 0.55 (s, 3H, 18-CH3) ppm. 13C NMR (CDCl3) d = 139.62,
117.43, 71.06, 56.10, 55.05, 49.46, 45.85, 43.39, 40.27, 39.57, 37.99,
37.15, 36.59, 34.21, 33.91, 31.49, 29.66, 29.17, 27.97, 26.21, 23.08,
22.98, 21.56, 19.83, 19.05, 18.91, 13.04, 11.98, 11.85 ppm. HRMS:
m/z calcd for C29H50O [M] 414.3862, found 414.3853. 1H and 13C
NMR data were in accordance with literature values [16] (Suppl.
Fig. 1).
solid. mp: 156–158 °C; IR (neat): m: 3398, 2965, 2937, 2868, 1705,
1633, 1029 cmÀ1 1H NMR (CDCl3) 5.17 (dd, 1H, J = 15.16, 8.56 Hz,
23-CH), 5.02 (dd, 1H, J = 15.16, 8.68 Hz, 22-CH), 3.62 (m, 1H,
3-CH), 1.09 (s, 3H, 19-CH3), 1.02 (d, 3H, J = 6.4 Hz, 21-CH3), 0.85
(d, 3H, J = 6.4 Hz, 26-CH3 or 27-CH3), 0.82 (t, 3H, J = 7.0 Hz,
29-CH3), 0.81 (d, 3H, J = 6.4 Hz, 26-CH3 or 27-CH3), 0.68 (s, 3H,
18-CH3) ppm. 13C NMR (CDCl3) d = 212.03, 138.08, 129.45, 70.69,
55.26, 54.94, 51.19, 49.96, 48.98, 46.83, 46.08, 42.39, 40.23,
38.63, 37.89, 36.31, 35.99, 31.86, 31.04, 28.95, 25.36, 25.06,
21.83, 21.3, 21.04, 18.98, 12.27, 12.23, 11.81 ppm. HRMS: m/z calcd
for C29H48O2 [M] 428.3654, found 428.3661 (Suppl. Fig. 1).
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2.2. Cell culture and treatments
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Murine oligodendrocytes (158N), with some characteristics of
differentiated oligodendrocytes were seeded at 120,000 cells per
well in 12 wells microplates containing 1 mL of culture medium,
constituted by Dulbecco’s modified Eagle medium (DMEM) (Lonza)
supplemented with 5% (v/v) heat-inactivated fetal bovine serum
(FBS) (Pan Biotech) and 1% antibiotics (100 U/mL penicillin,
100 mg/mL streptomycin) (Pan Biotech) [17,18].
Rat glioma cells (C6) were seeded at 250,000 cells per well in 12
wells microplates containing 1 mL of culture medium, constituted
by Dulbecco’s modified Eagle medium F12 (DMEM F12) (Lonza)
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(FBS) (Pan Biotech) and 1% antibiotics (100 U/mL penicillin,
100 mg/mL streptomycin) (Pan Biotech) [18,19].
Human neuroblastoma cells (SK-N-BE) were seeded at 400,000
cells per well in 12 wells microplates containing 1 mL of culture
medium, constituted by Dulbecco’s modified Eagle medium
(DMEM) (Lonza) supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (FBS) (Pan Biotech) and 1% antibiotics (100 U/
mL penicillin, 100 mg/mL streptomycin) (Pan Biotech) [18,20].
158N, C6, and SK-N-BE cells were incubated at 37 °C in a
humidified atmosphere containing 5% CO2, and passaged twice a
week. At each passage, cells were trypsinized with a (0.05% tryp-
sin–0.02% EDTA) solution (Pan Biotech).
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2.1.4. -Ethylcholesta-3-hydroxy-7-one (compound 9; Fig. 1)
Enone 8 (428 mg, 1 mmol) was hydrogenated by same proce-
dure described for 6 and purified over silica gel using EtOAc–petro-
leum ether (40–60) as eluent to give compound 9 (405 mg, 94%) as
a white solid. mp: 150–152 °C; IR (neat):
m: 3364, 2954, 2868,
1704, 1025 cmÀ1 1H NMR (CDCl3) 3.62 (m, 1H, 3-CH), 1.09 (s,
.
3H, 19-CH3), 0.93 (d, 3H, J = 6.4 Hz, 21-CH3), 0.85 (t, 3H,
J = 7.0 Hz, 29-CH3), 0.82 (d, 3H, J = 6.4 Hz, 26-CH3 or 27-CH3),
0.81 (d, 3H, J = 6.4 Hz, 26-CH3 or 27-CH3), 0.66 (s, 3H, 18-CH3)
ppm. 13C NMR (CDCl3) d = 212.15, 70.72, 55.26, 54.91, 50.00,
48.88, 46.86, 45.81, 43.1, 41.82, 38.7, 38.29, 36.36, 36.07, 33.94,
31.16, 29.14, 28.54, 26.015, 24.98, 23.04, 21.85, 19.78, 19.03,
18.84, 17.3, 12.06, 11.97, 11.82 ppm. HRMS: m/z calcd for
C29H50O2 [M] 430.3811, found 430.3829 (Suppl. Fig. 1).
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2.1.5. -Spinasterol (compound 1; Fig. 1)
In a 50-mL round-bottom flask, ketone 6 (214 mg, 0.5 mmol)
and p-tolylsulfonylhydrazine (112 mg, 0.6 mmol) were dissolved
in anhydrous methanol (10 mL). The mixture was stirred for 3 h
at reflux (75 °C). The reaction mixture was concentrated to dryness
under reduced pressure to give the crude tosylhydrazone 7 as white
solid. To this crude intermediate dissolved in dry THF (10 mL) and
anhydrous toluene (10 mL) was added LiH (40 mg, 5 mmol). The
mixture was heated at 110 °C for 5 h under argon. The solution
was cooled to 0 °C and water (2 mL) was slowly added and diluted
with EtOAc (50 mL). The organic phase was washed with saturated
NaCl, dried over Na2SO4, and evaporated. Flash chromatography
followed by elution with EtOAc–petroleum ether (20–80) gave
The conditions of treatment of murine oligodendrocytes 158N
cells, C6 rat glioma cells, and SK-N-BE human neuroblastoma cells
with 7-ketocholesterol (7KC), spinasterol, schottenol and ferulic
acid were realized in the following conditions. Initial concentra-
tions of oxysterols (800
1 mg (7KC, spinasterol, schottenol, ferulic acid) + 50
l
g/mL = 2 mM) were prepared as follows:
L absolute
l
ethanol + 1.2 mL culture medium. After plating 158N cells, C6 cells,
and SK-N-BE cells for 24 h, cells were further treated for 24 h with
various concentrations of these compounds (5, 10, 20, and 40 lM).
When compared to untreated cells; the maximal final concentra-
tion of absolute ethanol (0.08%) in the culture medium had no
effects on cell growth and viability [18].
151 mg (73%) as a white solid. mp: 171–173 °C; IR (neat):
m:
3424, 3314, 2960, 2937, 2868, 1447, 1039, 969 cmÀ1 1H NMR
.
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(CDCl3) 5.17 (dd, 1H, J = 15.2, 8.6 Hz, 23-CH), 5.18 (d, J = 3.8 Hz,
7-CH3) 5.04 (dd, 1H, J = 15.2, 8.6 Hz, 22-CH), 3.731–3.677 (m, 1H,
3-CH), 1.04 (d, 3H, J = 6.5 Hz, 21-CH3), 0.86 (d, 3H, J = 6.4 Hz,
26-CH3 or 27-CH3), 0.84 (d, 3H, J = 6.4 Hz, 26-CH3 or 27-CH3), 0.81
(t, 3H, J = 7.0 Hz, 29-CH3), 0.81 (s, 3H, 19-CH3), 0.57 (s, 3H,
2.3. Colorimetric MTT assay
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The MTT assay was used to evaluate the effects of treatments on
cell proliferation and/or mitochondrial activity. The MTT assay was
carried out as previously described [21] on 158N cells plated in
Please cite this article in press as: Badreddine A et al. An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in
cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system. Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.01.005
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6-well flat-bottom culture plates after 24 h of treatment with 7KC,
7b-OHC, or 24S-OHC (50 M) without or with -tocopherol
(400 M), DHA (50 M), or ( -tocopherol + DHA). MTT salt is
reduced to formazan in the metabolic active cells by mitochondrial
enzyme succinate dehydrogenase to form NADH and NADPH [22].
The plates were read at 570 nm with a microplate reader.
was read at 570 nm after extraction of the dye with 0.1 mol/L
sodium citrate in 50% ethanol.
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2.5. Statistical analysis
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Analyses were carried out with Excel software (version 2013)
with the ANOVA test. Data were considered statistically different
at a P-value of 0.05 or less.
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2.4. Staining with crystal violet
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Quantification of adherent cells was estimated by staining with
crystal violet [23]. Cells were seeded in triplicates in 6 well plates,
3. Results and discussion
and cultured without or with 7KC (5, 10, 20, and/or 40
lg/mL)
3.1. Synthesis
associated or not with -tocopherol (400 M) for 24 h. At the
a
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end of treatment, cells were washed with PBS, stained with crystal
violet (Sigma–Aldrich) (5 min), and rinsed with water. Absorbance
We have devised an improved rout to compound 1 (spinasterol)
and compound 2 (schottenol) starting from stigmasterol 3 and
Fig. 2. Effects of spinasterol and schottenol on cell growth and mitochondrial activity. 158N murine oligodendrocytes, C6 rat glioma cells, and SK-N-BE human neuronal cells
were treated for 24 h with 7-ketocholesterol (7KC) (used as positive control for induction of cell death and mitochondrial dysfunctions), spinasterol, schottenol and ferulic
acid (5, 10, 20, and 40
versus control (untreated cells). At each concentration of product corresponds one vehicle control (0%, 0.01%, 0.02%, 0.04%, and 0.08% absolute ethanol for the concentrations
0, 5, 10, 20 and 40
M, respectively) ANOVA test: ⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001.
lM). Cell growth and mitochondrial activity were measured with the crystal violet test and the MTT test, respectively. Values were expressed as %
l
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sitosterol 4 as a useful starting material both for its commercial
availability and for the presence of functional groups in positions
suitable for conversion to 1 and 2. The key step in the synthesis
of the two natural phytosterols 1 and 2 was the preparation of
7-keto steroids 6 and 9 and subsequent Bamford–Stevens reaction
to convert carbonyl function into corresponding olefin.
7KC important variations were observed from one cell type to
another. With this oxysterol used at 5 and 10 M, as cell growth
inhibition is usually observed on at these concentrations whatever
the cell line considered (excepted on SK-N-BE cells at 5 M), the
simultaneous increase of MTT values (Fig. 2D and F) suggests an
hyperactivity of mitochondria. At highest concentrations (10 and
l
l
According to literature, the allylic oxidation of
D
5-steroids to
20 lM), and whatever the cell line considered, the lowest MTT val-
corresponding 7-keto-
D
5-steroids have been carried out either
ues observed (Fig. 2B, D and F) are in agreement with cell growth
inhibition and also favor the hypothesis of a concomitant decrease
of mitochondrial activity. This impact of spinasterol and schottenol
on the mitochondrial activity is in agreement with previous data
obtained on murine microglial BV-2 cells reporting a loss of trans-
with very large excesses of chromiumVI [24] reagents or with
t-BuOOH in the presence of corresponding amounts of catalytic
complexes/salts of transition metals [25–27]. All these available
methods need the protection of the hydroxyl group at C-3 of ste-
roids, otherwise the allylic oxidation of the unprotected steroids
decreases the reaction efficiency. To avoid the protection and
deprotection steps, we have found that the use of CuCl(OH)ÁTMEDA
membrane mitochondrial potential (DWm) measured at the indi-
vidual cell level by flow cytometry with the fluorescent probe
DiOC6(3) [5]. These data also confirm a number of investigations
supporting that mitochondria constitute a potential direct or indi-
rect target of phytosterols [30]. It is therefore suggested that spin-
asterol and schottenol can modulate mitochondrial activity, and
might consequently influence cell metabolism.
[13] as catalyst allowed for the allylic oxidation of
D
5-steroids
under ambient temperature when tert-butyl hydroperoxide was
used as oxidant. Thus, treatment of the stigmasterol 3 with
t-BuOOH (10 equiv) in the presence of catalytic amount of
CuCl(OH)ÁTMEDA (5% mol) in CH2Cl2–MeOH afforded the 7-keto-
In conclusion, we have described an efficient and practical syn-
thesis of spinasterol and schottenol, from readily available stig-
masterol and sitosterol. A novel system for allylic oxidation of
D
5-sterols 5 [14] in 58% yield. In the same manner allylic oxidation
of sitosterol 4 produced the enone 8 [15] in 52% yield (Fig. 1).
Selective hydrogenation [28] of the enone 5 and 8 with Pd/C in
the presence of ammonium formate as hydrogen donor afforded
the saturated ketones 6 and 9 in 91% and 94% yield respectively.
To complete the synthesis the next two separate reaction steps
were performed in one reaction flask as follow: the ketone 6 was
reacted with p-toluenesulfonyl hydrazide in MeOH at 75 °C to give
the corresponding intermediate tosylhydrazone 7 followed by
reductive elimination [29] of the 7-hydrazone function with lith-
ium hydride in toluene–THF at 110 °C afforded the desired spinas-
terol 1 73% yield. Similarly in an identical manner the 7-keto
sitosterol 9 was converted to its tosylhydrazone 10 and subsequent
reductive elimination furnished schottenol 2 in 75%. The synthetic
compound 1 and 2 obtained here were identical by NMR with nat-
ural product spinasterol and schottenol [16].
unprotected
D D
5-steroids to 7-keto- 5-steroids by tert-butyl
hydroperoxide using CuCl(OH)ÁTMEDA as novel catalyst is note
worthy. Further study on the allylic oxidation of alkenes and others
D
5-steroids with CuCl(OH)ÁTMEDA is currently underway in our
laboratory and will be reported in due course. Our data also estab-
lished that spinasterol and schottenol have no effects on cell
growth of the cells of the central nervous system (158N, C6, and
SK-N-BE cells), whereas they decrease mitochondrial activity mea-
sured with the MTT test. Further investigations are required in
order to precise the effects of these phytosterols on normal cells
at the mitochondrial levels, and to identify which mechanisms
and metabolic pathways are involved.
376
Acknowledgements
314
315
3.2. Impact on schottenol and spinasterol on cell growth and
mitochondrial activity
377
378
379
380
381
382
383
384
This work was presented as a poster at the 4th ENOR sympo-
sium ‘Translational Research on Oxysterols’ September 18–19,
2014 – Coimbra, Portugal (ENOR’s website; http://oxysterols.
com/). This work was supported by the Action Intégrée of the
Comité Mixte Inter-universitaire Franco-Marocain (CMIFM, AIMA/
10/238, EGIDE) from the PHC Volubulis/Toubkal program, Ministè-
re des Affaires Etrangères, the Conseil Régional de Bourgogne, and
the Université de Bourgogne.
316
317
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321
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323
324
325
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328
329
330
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332
333
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337
338
339
340
341
The biological activities of schottenol and spinasterol on cell
growth and mitochondrial activity were evaluated with the crystal
violet and with the MTT tests on 158N, C6, and SK-N-BE cells cul-
tured in similar conditions to those previously used to evaluate the
effects of 4a- and 4b-hydroxycholesterol [18]. These phytosterols
were compared with 7KC (used as positive control) known to inhi-
bit cell growth and to induce mitochondrial dysfunctions at ele-
385
Appendix A. Supplementary data
vated concentrations [11] and with ferulic acid,
a potent
antioxidant, also found in argan oil and in cactus pear seed oil.
Whatever the cell considered and for the different concentrations
of schottenol, spinasterol, and ferulic acid used (5, 10, 20, and
386
387
388
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.steroids.2015.01.
005.
40
(Fig. 2A, C and E). However, significant inhibition of cell growth
were observed with 7KC at 5, 10, 20, and 40 M on 158N cells,
and at 10, 20, and 40 M on C6, and SK-N-BE cells (Fig. 2A, C and
lM), no or slight effects on cell growth were observed
389
References
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Please cite this article in press as: Badreddine A et al. An expeditious synthesis of spinasterol and schottenol, two phytosterols present in argan oil and in
cactus pear seed oil, and evaluation of their biological activities on cells of the central nervous system. Steroids (2015), http://dx.doi.org/10.1016/
j.steroids.2015.01.005
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