Dual bioactivity of resveratrol fatty alcohols: Differentiation of neural stem cells and modulation of neuroinflammation

Article abstract of DOI:10.1016/j.bmcl.2007.05.037

The synthesis of resveratrol fatty alcohols (RFAs), a new class of small molecules presenting strong potential for the treatment of neurological diseases, is described. RFAs, hybrid compounds combining the resveratrol nucleus and ω-alkanol side chains, are able to modulate neuroinflammation and to induce differentiation of neural stem cells into mature neurons. Acting on neuroprotection and neuroregeneration, RFAs represent an innovative approach for the treatment or cure of neuropathies.

Full text of DOI:10.1016/j.bmcl.2007.05.037

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4218–4222
Dual bioactivity of resveratrol fatty alcohols: Differentiation
of neural stem cells and modulation of neuroinflammation
a,b
Frederique Hauss, Jiawei Liu,a Alessandro Michelucci,b Djalil Coowar,a
´ ´
Eleonora Morga,b Paul Heuschlingb and Bang Luua,*
aLaboratoire de Chimie Organique des Substances Naturelles, UMR 7177 CNRS, Universite´ Louis Pasteur,
67084 Strasbourg Cedex, France
bLaboratoire de NeuroBiologie, Unite´ de Recherche Sciences de la Vie, Faculte´ des Sciences,
de la Technologie et de la Communication,
Universite´ du Luxembourg, 1511 Luxembourg, Luxembourg
Received 5 April 2007; revised 10 May 2007; accepted 11 May 2007
Available online 17 May 2007
Abstract—The synthesis of resveratrol fatty alcohols (RFAs), a new class of small molecules presenting strong potential for the
treatment of neurological diseases, is described. RFAs, hybrid compounds combining the resveratrol nucleus and x-alkanol side
chains, are able to modulate neuroinflammation and to induce differentiation of neural stem cells into mature neurons. Acting
on neuroprotection and neuroregeneration, RFAs represent an innovative approach for the treatment or cure of neuropathies.
Ó 2007 Elsevier Ltd. All rights reserved.
Neural cell death is one of the major characteristics of
degenerative neuropathologies like Alzheimer’s disease
(AD) or Multiple sclerosis (MS), although loss of cell
functions of neurons and/or glial cells is also known
to contribute to these degenerative diseases.
into mature neural cells.3 However, few non-proteic syn-
thetic molecules have been shown to confer such effect.4
Recently, Schultz et al. showed that a class of 4-amino-
thiazoles can induce, in a dose-dependent manner, neu-
ronal differentiation of adult hippocampal neural
progenitor cells.5
At present, most treatments of these incurable neurolog-
ical disorders are symptomatic. The discovery of the
presence of neural stem/progenitor cells (NSCs) in the
adult brain has opened new approaches for therapeutic
interventions.1 NSCs are multipotent progenitor cells
which can differentiate into all different nervous cell
types (e.g., neurons, astrocytes and oligodendrocytes)
and can contribute to neurogenesis in adulthood.2 The
differentiation of these NSCs is regulated by key factors
involved in neurogenesis and stem cell development.
Hence, potent inducing agents for NSC differentiation
represent an innovative approach with good potential
to treat or even cure neurological diseases by resupply-
ing the central nervous system (CNS) with new mature
nerve cells. Some proteic growth factors [e.g., Fibroblast
growth factor (FGF) and Epidermal growth factor
(EGF)] are able to induce the differentiation of NSCs
A second factor contributing significantly to the cell
death in patient’s brain tissues is the inflammatory pro-
cesses accompanying neurodegenerative diseases.6 Dur-
ing the development of neurodegenerative or
demyelinating diseases, microglial cells, the brain resi-
dent monocyte-macrophage cell population, become
highly activated.7 This activation produces large
amounts of devastating pro-inflammatory cytokines,
like tumour necrosis factor-a (TNF-a), interleukin-1b
(IL-1b) as well as free radicals like nitric oxide (NO)
and superoxide anion.8,9 In AD, the extracellular depo-
sitions of amyloid beta (Ab) represent the major histo-
logical lesion and are responsible for the death of
neurons by a currently unclear mechanism. One theory
involves neuroinflammation which is supported by stud-
ies showing clustering of microglial cells within Ab
depositions in human brain tissues.10
Previous studies have shown that n-hexacosanol, a
long chain primary alcohol containing 26 carbon
atoms, confers neurotrophic activity. It has been
Keywords: Resveratrol fatty alcohols; Neuroinflammation; Neural
stem cells; Microglia; Neurodegenerative diseases.
*
Corresponding author. E-mail: luu@chimie.u-strasbg.fr
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.037

F. Hauss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4218–4222
4219
shown that the length of the chain and the x-hydroxyl
function are essential for the biological activity.11 Be-
sides, in order to take into account the presumed con-
tribution of inflammatory and oxidative phenomena in
neurodegenerative pathologies, we decided to synthe-
size small molecules bearing a x-alkanol side chain
combined with a neuroprotective moiety. Structure–
activity relationship studies led to the identification
of compounds, presenting even better neurotrophic
activities with a shorter chain length. Moreover, some
of these fatty alcohol derivatives were able to induce
differentiation of neural stem cells into mature neu-
rons12,13 or to induce the differentiation of oligoden-
drocyte precursors into mature oligodendrocytes.14
The synthesis of RFAs was based on two key steps. On
one hand, a Sonogashira cross-coupling16 between
methyl 4-iodo-3,5-dimethoxybenzoate (2) and differ-
ently protected x-alkynols 3 allowed us to couple the
x-alkanol side chains. On the other hand, a Wads-
worth–Emmons17 reaction between 3,5-dimethoxy-
benzaldehyde derivatives 5 bearing various x-alkanol
side chains and methoxybenzylphosphonate 6 gave
methylated RFAs 7 which, after deprotection, yielded
RFAs 1 (Scheme 1).
Our procedure started with commercially available res-
orcylic acid which was iodinated in presence of N-iodo-
succinimide and then methylated with potassium
carbonate and dimethylsulfate in refluxing acetone to
give methyl 4-iodo-3,5-dimethoxybenzoate (2). The
differently protected x-alkynols 3 were prepared from
O-protected bromo alcohols obtained by mono-bromin-
ation of the corresponding diols. Finally, the O-pro-
tected bromo alcohols in the presence of a lithium
acetylide-ethylenediamine complex gave the desired
x-alkynols 3 in good overall yields (53–76%).
In view of these encouraging findings, we focused on the
elaboration of new hybrid compounds. These molecules
combine the neuroregenerative activities of the x-alka-
nol structure and the neuroprotective effects of an
anti-inflammatory and anti-oxidative core, trans-resve-
ratrol (trans-3,40,5-trihydroxystilbene). Thus, long
chain fatty alcohols bearing the trans-resveratrol as a
neuroprotective core, the resveratrol fatty alcohols
(RFAs), were prepared (Fig. 1). Resveratrol is a phyto-
alexin mainly found in plants, grapes and peanuts which
has been shown to display a wide range of interesting
and useful bioactivities (e.g., cancer chemoprotective,
anti-inflammatory, anti-platelet aggregation, anti-oxi-
dative and anti-bacterial activities).15
The Sonogashira cross-coupling between the protected
x-alkynols 3 and the methyl 4-iodo-3,5-dimethoxybenzo-
ate (2) was realized in classical conditions and allowed us
to obtain the different coupling products in good to
excellent yields (Scheme 1). Then, catalytic hydrogena-
tion with palladium on charcoal 5% followed by reduc-
tion with lithium and aluminium hydride yielded the
corresponding hydrogenated benzyl alcohols. A Ley oxi-
dation18 gave then the desired benzaldehydes 5, needed
for the Wadsworth–Emmons coupling.
OR1
OH
(CH2)nOH
OR1
R1 = H, Me
The final key step in the synthesis was the Wadsworth–
Emmons reaction17 between the different benzaldehydes
5 and the phosphonate 6 in the presence of sodium meth-
ylate which afforded the different series of methylated
RFAs 7. RFAs 1 were obtained after deprotection using
boron tribromide. Phosphonate 6 was obtained in 80% in
OH
R1O
HO
n = 10, 12, 14, 16, 18
Resveratrol
Resveratrol Fatty Alcohols (RFAs)
Figure 1. Structures of resveratrol and RFAs.
OMe
OMe
(CH2)n-2OTBS
I
a
(CH2)n-2OTBS
MeO2C
OMe
MeO2C
OMe
2
3
4
OMe
b, c, d
(CH2)n-1OTBS
OHC
OMe
5
P(O)(OEt)2
e
OMe
OH
(CH2)nOH
(CH2)nOH
OH
f
OMe
OMe
MeO
HO
7
1
6
Scheme 1. Synthesis of RFA analogues. Reagents and conditions: (a) Alkynols 3, PdCl2(PPh3)2, CuI, Et3N, 80 °C, 24–48 h, 65–86%; (b) H2, Pd/C
5%, EtOH, rt, 24 h, 90–98%; (c) LiAlH4, THF, 0 °C to rt, 2 h, 92–99%; (d) TPAP, NMO, CH2Cl2, 0 °C to rt, 1 h, 81–94%; (e) i—NaOMe, DMF,
0 °C, 1 h; ii—5, DMF, 0 °C to rt, 1 h; iii—100 °C, 1 h; iv—HCl 2 N, rt, 17 h, 60–82%; (f) BBr3, CH2Cl2, ꢀ78 °C to rt, 2 h, 46–76%.

4220
F. Hauss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4218–4222
a single step from the corresponding benzyl alcohols in
presence of iodide in refluxing triethylphosphite.19
In order to evaluate the potential neuroregenerative
activity of RFAs, their ability to influence the cell fate
decision and the differentiation of neural stem cell de-
rived neurospheres into neurons or glial cells was inves-
tigated. Neurospheres are undifferentiated cell
aggregates floating in the culture medium which contain
5–10% neural stem cells (multipotent progenitors).20
They arise from the cultivated NSCs due to selective ac-
tion of growth factors like EGF and bFGF (brain
FGF).20
Figure 3. RFA12 induced differentiation of neural stem cell derived
neurospheres into neurons. Neurospheres were allowed to differentiate
for 3 days without (a) or with (b) RFA12 (0.1 lM), and immuno-
stained with anti-MAP(2a+2b) and Tuj1 coupled to Alexa 555 for
neurons (red), anti-GFAP coupled to Alexa 488 for astrocytes (green)
and TO-PRO, a nuclei specific marker (blue).
After dissociation, neurospheres were grown up for
three days (proliferation phase) and allowed to differen-
tiate on a polyornithine support (differentiation phase)
for a subsequent three days. RFAs were added at
T = 0 (Fig. 2).21 The differentiation was then analyzed
by immunocytochemistry, using specific antibodies for,
respectively, mitotic neurons [microtubule associated
protein 2, MAP(2a+2b)], postmitotic neurons (mono-
clonal antibody against neuronal class III tubulin,
TuJ1) and an astrocyte-specific intermediate filament
protein (glial fibrillary acidic protein, GFAP). This
study does not take into account oligodendrocytes.
The total number of cells was quantified using TO-
PRO-3-iodide, a nuclear marker (Fig. 3).
(Table 1). Resveratrol alone does not enhance the neu-
ronal production. RFA12 at 0.1 lM was more active
than retinoic acid (RA) at 5 lM, known for its strong
neurogenic capacity.22 Finally, we confirmed that the
x-hydroxyl function was necessary for the activity as
far as compound 8 has no effect on the number of
neurons. These results comfort our starting hypothesis
that the presence of a precise side chain length and the
x-alkanol function are necessary for the neurotrophic
activity.
First of all, we determined that quantitatively the stron-
gest effect on the neuronal population was observed at
concentrations of 0.1 lM or 1 lM for most of the com-
pounds. Higher concentrations (5 lM, 10 lM) were
toxic and lower ones (1 nM) inefficient.
Besides, RFA12 increased in a concentration-dependent
manner the number of neurons accompanied by a de-
crease of the number of astrocytes. In addition, in the
The efficiency to induce the differentiation of neuro-
spheres into mature neurons of the most promising can-
didate RFA12 1b (R1 = H, n = 12) was estimated
quantitatively (Table 1). The qualitative aspect was esti-
mated by the morphology of the neurons, including
their size and the length of their extensions (Fig. 3).
Table 1. Neuronal differentiation activity of RFAsa
Compound
(0.1 lM)
R1
n
% neurons
versus
control
% neurons
versus RAb
Diffc
Ctrl
RA 5 lM
Resveratrol
100
203
105
147
188
135
107
108
263
160
106
0.50
1
+
++
+
Immunostaining showed that the length of the side
chain and the presence of the hydroxyl group on the
aromatic ring system were essential for a better biologi-
cal activity. In fact, the most active compounds 7b and
1b are those bearing a x-alkanol chain having 12 carbon
atoms. Moreover, compound 1b, RFA12 (R1 = H,
n = 12), is able to increase by 160% the number of neu-
rons from the neurospheres compared to untreated cells
0.52
0.72
0.93
0.67
0.53
0.53
1.30
0.79
0.52
7a
7b
7c
7d
1a
1b
1c
8
Me
Me
Me
Me
H
10
12
14
16
10
12
14
12d
+
++
+
+
+
H
++
+
H
H
+
+ indicates that neurospheres differentiate into neurons with a mor-
phology similar to control conditions. ++ neurons are more differen-
tiated, they elicit larger morphologies with longer process than in the
control.
a Quantitative effect of RFAs, resveratrol and retinoic acid (RA) on
neurosphere differentiation into neurons. Control (Ctrl) is EtOH.
Each compound was evaluated in at least three independent experi-
ments; variation was generally 20%.
b % of neurons divided by % of neurons when cells were treated with
RA 5 lM.
c Diff stands for qualitative result on the neuronal differentiation of
neurospheres.
d RFA without x-OH.
Figure 2. Experimental protocol for the neurosphere assay.

F. Hauss et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4218–4222
4221
Specifically, RFA12 can increase the number of neurons
by 160%. Hence, we succeed in combining two biological
properties in one molecule. A possible explanation of this
effect is that our compounds act on the cell fate of the
progenitors as the increase of neurons is accompanied
by a decrease of astrocytes. This supposition suggests
that RFA12 would act directly or indirectly on the Notch
pathway that mediates a large array of cellular processes
including the maintenance of stem cell self-renewal, pro-
liferation and differentiation.24,25 More systematic work
is needed to completely elucidate the mechanism of
action. Overall, this study indicates that this compound
has a better pharmacological profile than resveratrol
for the treatment of neurological diseases with degenera-
tive and/or inflammatory components.
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Ctrl
*
*
10-6M
5.10-6M
**
TNF-α RFA12 TNF-α Resv.
NO RFA12
NO Resv.
Figure 4. Effect of RFA12 and resveratrol (resv) on NO and TNF-a
expression in MMGT-12 cells in presence of LPS (0.01 lg/mL).
Culture supernatants were collected after, respectively, 48 h or 24 h
incubation. NO and TNF-a production data are means SEM (bars)
values obtained from three independent experiments (n = 3). Results
are expressed as percentage of control (0.01 lg/mL LPS; (a)
100% = 5.57 nmol/mL NO and (b) 100% = 3731 pg/mL TNF-a.
*p < 0.05, **p < 0.01, NO and TNF-a expression levels are significantly
different from the corresponding control levels.
Acknowledgment
This work was supported by a PhD and Post-doctoral
Fellowship Program from the Luxembourg Ministry of
Culture, Higher Education and Research (F.H., A.M.,
and D.C.). Special thanks to Prof. N.S. Yang (Taipei)
for critical reading of the manuscript.
presence of RFA12, the neurons were larger and pro-
cesses were longer which means that the neuronal pre-
cursors are more differentiated (Fig. 3). The fact that
the increase in the number of neurons takes place at
the expense of astrocytes suggests that RFA12 could
specifically act on the cell fate.
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