Tocopherol long chain fatty alcohols decrease the production of TNF-α and NO radicals by activated microglial cells

Article abstract of DOI:10.1016/j.bmcl.2004.09.078

Tocopherol derivatives were found to strongly modulate microglial activation induced by lipopolysaccharide. The synthesis of a series of Tocopherol long chain Fatty Alcohols (TFA) and their biological activities on the modulation of microglial activation are described. Specifically, the 2-(12-hydroxy-dodecyl)-2,5,7,8-tetramethyl-chroman-6-ol, the TFA bearing 12 carbon atoms on the side chain (n = 12), shows the most potent inhibition of secretion on nitric oxide (NO) and tumour necrosis factor-α (TNF-α) by lipopolysaccharide (LPS)-activated microglia.

Full text of DOI:10.1016/j.bmcl.2004.09.078

Bioorganic & Medicinal Chemistry Letters 14 (2004) 6023–6026
Tocopherol long chain fatty alcohols decrease the production
of TNF-a and NO radicals by activated microglial cells
a
Thierry Muller, Luc Grandbarbe, Eleonora Morga, Paul Heuschling and Bang Luu
b
b
b
a,
*
a
Laboratoire de chimie organique des substances naturelles, Centre de Neurochimie, UMR 7123 CNRS,
Universit e´ Louis Pasteur, 67084 Strasbourg cedex, France
b
Laboratoire de Neurobiologie, Facult e´ des Sciences, de la Technologie et de la Communication, Universit e´ du Luxembourg,
511 Luxembourg-Ville, Luxembourg
1
Received 14 May 2004;revised 27 September 2004;accepted 28 September 2004
Available online 18 October 2004
Abstract—The synthesis of a series of Tocopherol long chain Fatty Alcohols (TFA) and their biological activities on the modulation
of microglial activation are described. Specifically, the 2-(12-hydroxy-dodecyl)-2,5,7,8-tetramethyl-chroman-6-ol, the TFA bearing
1
2 carbon atoms on the side chain (n = 12), shows the most potent inhibition of secretion on nitric oxide (NO) and tumour necrosis
factor-a (TNF-a) by lipopolysaccharide (LPS)-activated microglia.
Ó 2004 Elsevier Ltd. All rights reserved.
5
1
. Introduction
to those observed in AlzheimerÕs disease. The appear-
ance of phagocytic vesicles finally completes the
microglial activation process.
3
Microglial cells, the brain resident monocyte–macroph-
age cell population, derive from the bone marrow mono-
nuclear phagocyte cell lineage and enter the brain
parenchyma during development. In the normal brain
tissue, microgliocytes are found as quiescent cells
throughout the brain parenchyma, where they represent
In an attempt to modulate these neurotoxic properties
of activated microglial cells, we have undertaken the
synthesis and the characterization of the pharmacologi-
cal properties of small molecules, which are based on a
chemical structure likely to present biological activities.
1
around 15% of the cell population. Upon appropriate
stimulation, these cells continue the previously halted
differentiation process to become immunocompetent
phagocytic cells. This type of activation is observed after
brain injury or infection, as well as during the develop-
ment of neuropathies like AlzheimerÕs disease, stroke or
demyelinating diseases such as multiple sclerosis. After a
rapid change in cell morphology, activated microglio-
cytes produce pro-inflammatory cytokine like tumour
Previous studies have shown the neurotrophic activity of
n-hexacosanol, a long chain primary alcohol containing
26 carbon atoms. Both, the length of the chain and the
x-hydroxyl function are crucial factors for the biological
6
activity. As we are dealing with neurodegeneration, we
are interested in compounds presenting a neuroprotec-
tive as well as a neuroregenerative activity. Our
approach is the investigation of hybrid compounds com-
bining a neurotrophic x-alkanol structure and an anti-
oxidant moiety. Former studies led to the synthesis of
3-(15-hydroxy-pentadecyl)-2,4,4-trimethyl-2-cyclohexen-
2
necrosis factor-a (TNF-a) as well as the free radicals ni-
Å 3
ÅÀ
4
tric oxide (NO ) and superoxide anion (O ). These
radicals combine to form the very reactive peroxynitrite
radical (ONOO ). We have previously shown that the in
2
À
7
vitro production of these radicals along with TNF-a
secretion induces neurodegenerative events comparable
1-one (tCFA-15). This trimethyl cyclohexenonic long
chain fatty alcohol induces the differentiation of foetal
8
neurons and neural stem cells.
Here we report the synthesis and biological activities of
long chain fatty alcohols bearing an a-tocopherol moi-
ety, the chroman ring as functionalized nucleus in addi-
tion to an x-alkanol side chain. a-Tocopherol, a
Keywords: Tocopherol;Long chain fatty alkanol;Neuroinflammation;
Microglia.
*
Corresponding author. Tel./fax: +33 (0)3 88 41 16 72;e-mail:
luu@chimie.u-strasbg.fr
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.078

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T. Muller et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6023–6026
member of the vitamin E family, is the major lipid solu-
9
3. Biological activities
ble antioxidant in vivo and has been shown to have
neuroprotective effects, which act, at least partially,
The mouse microglial cell line MMGT12 (a generous
15
gift of Dr. E. Vanmechelen, Gent) was activated by
1
0
through suppression of microglial activation.
exposure to lipopolysaccharide (LPS 0.01lg/mL) for
24 or 48h in the presence or absence of different concen-
trations of TFAs or a-tocopherol. The secretion of
TNF-a after 24h was measured using an ELISA assay
(R&D System), the production of nitric oxide after
Here, we report that the in vitro production of nitric
oxide and TNF-a by activated microglial cells is modu-
lated by a co-incubation with the tocopherol long-chain
fatty alcohols (TFAs).
4
8h was monitored by quantifying the nitrite concentra-
tion (the final degradation product of NO in buffered
solutions) using a microplate adapted assay of the Gri-
1
6
2. Chemistry
ess method. The expression levels of the NO-synthase
type-II, the enzyme responsible for the induced produc-
tion of NO, were examined using immunoblotting after
SDS-PAGE separation. Major histocompatibility class
II molecules expression was examined by flow cytome-
try, using the rat anti-mouse MHC class II monoclonal
antibody IBL-3/5 from Serotec. The secondary fluores-
cent antibody (goat anti-rat) was also from Serotec.
Starting from either commercially available tetradecane-
,14-dioic acid 2c and oxacycloheptadecan-2-one 2d or
decane-1,10-diol 3a and dodecane-1,12-diol 3b, the cor-
responding TFAs 1a–d are synthesized through a four,
respectively, five step procedure.
1
According to Scheme 1, reduction of diacid 2c and lac-
tone 2d with lithium aluminium hydride affords the cor-
responding alcohols 3c (80%) and 3d (95%). Diols 3a–d
are then submitted to a monoiodation using a 57% aque-
ous solution of hydroiodic acid to give x-iodo-alkanols
Figure 1 shows the modulation of the induced TNF-a
production after 24h of exposure to LPS as well as the
modulating molecules. Final concentrations of TFAs
À5
À7
and a-tocopherol range from 10 to 10 M. a-Tocoph-
erol and TFA-10 1a show no effect. A slight concentra-
tion-dependent inhibition of TNF-a release is induced in
4
a–d (61–78%), which are readily protected with tert-
butyldimethylsilyl chloride in the presence of imidazole
to provide the corresponding silylated iodo-alkanols
À5
the presence of 10 M TFA-16 1d. TFA-12 1b and
1
1
5
a–d (96–99%).
TFA-14 1c show a major inhibition of the secretion at
0
À5 À6
M as well as at 10 M.
1
The key intermediates 6a–d are obtained in a single step
procedure starting from compounds 5a–d. Primary
alkyllithiums are prepared by lithium–iodine exchange
Figure 2 shows the modulation of the nitrite accumula-
tion after a 48h incubation in the presence of LPS. a-
Tocopherol and TFA-10 1a show no effect. TFA-16 1d
1
2
using tert-butyllithium. A subsequent in situ addition
of but-3-en-2-one provides the corresponding allylic
À6
shows a mild inhibition at 10 M. Strong inhibitions
are obtained with TFA-12 1b, TFA-14 1c and TFA-16
1d at 10 M.
1
3
alcohols 6a–d (45–58%). The chroman moieties are
formed racemically at carbon 2 by condensation of the
different allylic alcohols with trimethylhydroquinone in
À5
1
4
the presence of aluminium chloride and nitromethane.
The abundance of the enzyme responsible for the NO
production, NO-synthase-II (NOS-II) is strongly down
regulated by TFA-12 whereas no effect is observed after
TFA-10 treatment (Fig. 3).
Deprotection of the terminal alcohol occurs in situ to af-
ford the 2-(n-hydroxy-alkyl)-2,5,7,8-tetramethyl-chro-
man-6-ols 1a–d (36–53%).
HO C
CO H
2
2
(
CH2)
12
1
2
c
2
3
HO
OH
HO
I
TBDMSO
I
(
CH
2
)
n
(CH
2
)
n
(CH
2
)
n
O
3a-d
4a-d
5a-d
O
(CH2)14
2d
HO
OH
4i, ii
5
TBDMSO
(
CH2)n
6a-d
OH
O
(CH2)n
a: n = 10
b: n = 12
c: n = 14
d: n = 16
1a: TFA-10
1b: TFA-12
1c: TFA-14
1d: TFA-16
Scheme 1. General synthesis of TFAÕs 1a–d. Reagents and conditions: (1) LiAlH
1–78%;(3) TBDMSCl, imidazole, CH Cl
, rt, 3h, 96–99%;(4) (i) t-BuLi, ether, À78°C, 5min;(ii) but-3-en-2-one, ether, À78°C, 15min, 45–58%;(5)
trimethylhydroquinone, AlCl , nitromethane, CH Cl
, À20°C to rt, 3h, 36–53%.
4
, THF, 0°C to rt, 15h, 80–95%;(2) HI aq 57%, toluene, 90 °C, 6h,
6
2
2
3
2
2

T. Muller et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6023–6026
6025
1
1
1
20
10
00
LPS activation induces a minor rise in the expression
levels of the major histocompatibility class II molecules.
However, none of the molecules has an effect on the
expression levels of MHC class II molecules on the
microglial cells (results not shown).
TFA-10
TFA-12
TFA-14
TFA-16
Vit E
90
80
70
60
50
40
*
*
4. Discussion and conclusion
*
The microglial cells are readily activated after in vitro
stimulation with the Gram-negative bacterial wall com-
ponent lipopolysaccharide (LPS). Besides changing cell
morphology, the cells start to release pro-inflammatory
products like TNF-a and nitric oxide free radicals,
*
*
1
0.-5 M
10.-6M
Concentration
10.-7 M
5
which are known to induce neuronal death. Since epi-
demiological studies document the importance of these
inflammatory components during the course of Alzhei-
Figure 1. Effect of TFA-10–TFA-16 1a–d and vitamin E on TNF-a
production of MMGT12 cells in the presence of LPS (0.01lg/mL).
Culture supernatants were collected after a 24h incubation. TNF-a
production data are mean ± SEM (bars) values obtained from three
experiments, each conducted in independents cultures (n = 3). Results
1
7,18
merÕs disease,
the search for pharmacological tools
to modulate the pro-inflammatory activation of central
microglial cells appears as a necessary step towards a
more complete treatment of the disease. Two of the mole-
cules of the TFA family appear to be able to influence
significantly the activation of the microglial cells.
TFA-12 1b, TFA-14 1c and to a lesser extend TFA-16
are expressed as
00% = 3264pg/mL TNF-a). *p < 0.05, **p < 0.01, TNF-a expression
levels are significantly different from the corresponding control levels.
a percentage of control (0.01lg/mL LPS;
1
1d down-regulate two of the activation parameters stud-
ied in this work. The activity clearly depends on the
length of the side chain, TFA-10 1a having no effect
and TFA-16 1d showing only minor effects. The most
potent length seems to be obtained with TFA-12 1b.
1
1
1
20
10
00
TFA-10
TFA-12
TFA-14
TFA-16
Vit E
The chroman ring composed of a-tocopherol, does not
appear to be the sole responsible agent for this effect,
since under our experimental conditions, a-tocopherol
alone has no significant effect on the microglial response.
9
8
7
6
5
4
0
0
0
0
0
0
No effect was observed on the expression levels of the
MHC class II molecules. This aspect of our results seems
to hint at an effect of the TFA molecules on the signal
transduction pathway of the pro-inflammatory stimula-
tions. TNF-a secretion and NOS-II production depend
*
*
1
9,20
1
0.-5 M
10.-6M
Concentration
10.-7 M
on the NF-kB pathway.
Class II expression however
21
is regulated via different pathways.
2À
Figure 2. Effect of TFA-10–TFA-16 1a–d and vitamin E on NO
accumulation of MMGT12 cells in the presence of LPS (0.01lg/mL).
Culture supernatants were collected after a 48h incubation. NO
TFA molecules modulate, but have no inhibiting effect
on the pro-inflammatory activation of microglial cells.
Under pharmacological aspects, this property is particu-
larly important. Microgliocytes are important cells in
the maintenance of cerebral integrity. These cells play
a crucial role in the clearance of amyloid-b depositions
À
2
accumulation data are mean ± SEM (bars) values obtained from three
experiments, each conducted in independents cultures (n = 3). Results
are expressed as
a percentage of control (0.01lg/mL LPS;
À
À
expression levels are
100% = 8.34nmol/mL NO
significantly different from the corresponding control levels.
2
). *p < 0.05, NO
2
3
during the course of AlzheimerÕs disease. A complete
inhibition of their activation would therefore most prob-
ably present a deleterious effect on the course of the dis-
ease. Further studies on the biological properties, and
especially on the signalling cascades affected, of the mole-
cules of the TFA family are necessary to fully charac-
terize their pharmacological potentials.
In a first attempt, we decided to synthesize racemic
TFAs because most synthetic supplements of a-tocoph-
erol used in the food industry are a mixture of eight pos-
sible stereoisomers arising from three chiral centres. On
the other hand, the side chains bear an even number of
carbon atoms because most naturally occurring
Figure 3. Effect of TFA-10 1a and TFA-12 1b on NOS type II protein
expression of MMGT12 cells in the presence or absence of LPS
(0.01lg/mL). Cell lysates were prepared for immunoblotting after a
24h incubation.

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T. Muller et al. / Bioorg. Med. Chem. Lett. 14 (2004) 6023–6026
aliphatic chains derive from acetyl-CoA (two carbon
atoms) condensation.
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Nonetheless, it would be interesting to synthesize TFAs
with a fixed configuration at carbon atom 2 and/or an
odd number of carbon atoms at the side chain. This
would allow comparing TFA-11 and TFA-13 with the
most active compound TFA-12 1b and determining
whether there is a difference in the modulation of
microglial activation depending on the enantiomer at
carbon atom 2.
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