Quinol fatty alcohols as promoters of axonal growth

Article abstract of DOI:10.1016/j.bmcl.2006.02.041

The synthesis of three series of quinol fatty alcohols (QFAs) and their biological activities on the promotion of axonal growth are described. Interestingly, the 15-(2,5-dimethoxyphenyl)pentadecan-1-ol, the QFA bearing 15 carbon atoms on the side chain (n = 15), shows the most potent promotion of axonal growth in the presence of both permissive and non-permissive naturally occurring substrates such as Sema3A and myelin proteins.

Full text of DOI:10.1016/j.bmcl.2006.02.041

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2637–2640
Quinol fatty alcohols as promoters of axonal growth
Mazen Hanbali,a Marta Vela-Ruiz,a Dominique Bagnardb and Bang Luua,*
aLaboratoire de Chimie Organique des Substances Naturelles, LC3-UMR7177 CNRS-ULP,
Universite´ Louis Pasteur, 67084 Strasbourg Cedex, France
`
´
Laboratoire de Physiologie du Systeme Nerveux, U575 INSERM, Universite Louis Pasteur, 67084 Strasbourg Cedex, France
b
Received 11 January 2006; revised 14 February 2006; accepted 14 February 2006
Available online 6 March 2006
Abstract—The synthesis of three series of quinol fatty alcohols (QFAs) and their biological activities on the promotion of axonal
growth are described. Interestingly, the 15-(2,5-dimethoxyphenyl)pentadecan-1-ol, the QFA bearing 15 carbon atoms on the side
chain (n = 15), shows the most potent promotion of axonal growth in the presence of both permissive and non-permissive naturally
occurring substrates such as Sema3A and myelin proteins.
Ó 2006 Elsevier Ltd. All rights reserved.
The adult CNS has limited capacity for regeneration.
After a lesion has occurred, the site concerned under-
goes many cellular and chemical changes. The site of
lesion is invaded by a multitude of nerve cells such as
astrocytes, microglial cells, oligodendrocytes and pro-
genitor cells. This constitutes a mechanical barrier,
called the ‘glial scar’,1 that axon extensions are unable
to cross. Moreover, these cells over-express many pro-
tein factors like myelin associated proteins (NOGO,
MAG and OMgp),2 Sema3A,3 chondroitin sulfate pro-
teoglycans all possessing strong inhibitory activity
towards axon extension.4,5 One therapeutic strategy
emerging from the above considerations is the use of
natural neurotrophic factors in order to improve neuron
survival, sprouting and axon extension. Nonetheless, the
major problems in applying neurotrophic factors are
their poor system penetration, relative instability and
wide range of local and systemic biological activities.6
To address these limitations, innovative therapeutics7
should consider the use of synthetic small molecules able
to mimic the biological effects of natural neurotrophic
factors while avoiding all the side effects.8
decyl)-2,4,4-trimethylcyclohexen-2-one). This com-
pound is able to promote neuron survival and axon-
specific growth on inhibitory substrates such as Sema3A
or myelin associated proteins.10,11
Despite the mechanical and chemical inhibitory proper-
ties of the glial scar, the local microglial activation re-
sults in an excessive cellular respiration that generates
an accumulation of reactive oxygen species1 (ROSs) as
well as nitric oxide.12 ROSs attack cellular membranes
through a process called ‘lipid peroxidation’ which re-
sults in apoptotic cell death. Compounds that are able
to scavenge these oxidizing agents would be of particu-
lar interest to therapy. Synergistic effects have been ob-
served in drug combination studies of trophic factors
with antioxidants.13 As a result, various synthetic com-
pounds comprising an x-alkanol chain have proven to
be active towards nerve cells while maintaining a strong
antioxidant activity.14,15
Ubiquinol, the antioxidant form of ubiquinone, is a
well-known antioxidant16,17 and its biological activity
within the CNS is significant.18,19 Since the ubiquinone
moiety is structurally closely related to the antioxidant
moiety of tCFA15, we designed a new series of neuro-
trophic compounds combining both the trophic effect
of the x-alkanol chain and the radical scavenging capac-
ity of the hydroquinone ring.
Initial screening of Chinese medicinal plants showed
that an x-alkanol, n-hexacosanol, possesses some neuro-
trophic effects.9 The combination of this x-alkanol with
a retinol moiety gave tCFA15 (3-(15-hydroxypenta-
Keywords: Ubiquinol; Quinol fatty alcohols; Brain lesion; Glial scar;
Axonal growth; Myelin proteins; Sema3A.
In this study, we analyze the effects of quinol fatty alco-
hols (QFAs) as free radical scavengers and as promoters
of axonal growth. To our great surprise, we found that
*
Corresponding author. Tel.: +33 0 3 88 41 16 72; fax: +33 0 3 88 60
74 64; e-mail: luu@chimie.u-strasbg.fr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.041

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M. Hanbali et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2637–2640
QFAs were able to counteract the inhibitory effects of
proteic factors present within the glial scar. Such effects
could lead to innovative therapeutic strategies for the
treatment of CNS injuries.
hydride. The C-16 diol was obtained by reduction of
the corresponding lactone, hexadecanolide.26
The synthesis was pursued by a Sonogashira-like palla-
dium catalyzed cross-coupling reaction between the ter-
minal alkynes 9a–e27,28 and the different arylbromides
51–3. Catalytic hydrogenation of 101–3a–e with palladi-
um on charcoal led to the quinol fatty alcohols 11–3a–e.
Brominated methoxybenzenes 51–3 were the first interme-
diates needed to prepare the quinol fatty alcohols by
means of a Sonogashira palladium catalyzed cross-cou-
pling reaction. Compounds 51 and 52 were obtained, as
described in the literature, in one step by the bromination
of 1,4-dimethoxybenzene 21 and 1,2,4-trimethoxyben-
zene 22 by either the use of a N-bromosuccinimide20 or
bromine21 in dichloromethane, respectively. Compound
53 was obtained in three steps from 2,3,4-trimethoxyben-
zene carboxaldehyde 3 which was oxidized to 2,3,4-tri-
methoxyphenol 4 through a Baeyer–Villiger reaction.22
It was subsequently methylated with dimethylsulfate23
in acetone and then brominated with NBS/TFA24,25
(Scheme 1).
In an attempt to study the biological activity of QFAs,
we analyzed the ability of each compound to promote
axonal growth on mouse cortical neurons (Table 1).
The experimental protocol has been described in a prior
publication.11 Briefly, mice embryos at the 15th day of
gestation were dissected and cortical extracts were disso-
ciated with trypsine. The neurons obtained were plated
in 6-well plates and cultured on poly-L-lysine coverslips
for 24 h at 37 °C, 5% CO2. On day 2, neurons were incu-
bated with the test compound and grown for 24 h at
37 °C, 5% CO2. On day 3, neurons were fixed and immu-
nostained with a primary anti-phosphoneurofilament
The terminal alkynes 9a–e were prepared from O-pro-
tected bromoalcohols 8a–e (Scheme 2). All bromoalco-
hols 7a–e were obtained by monobromination of their
respective diols 6a–e in a mixture of HBr–cyclohexane.26
antibody (SMi312, Sternberger) and
a secondary
Alexa488-coupled antibody. Coverslips were then
mounted on plates with aqua-polymount. In a first series
of experiments we determined which compounds exerted
the strongest biological effect. An initial screening of all
compounds at 10ꢀ9 M showed that compounds with a
15 carbon side chain were the most effective.
The C-10 and C-12 diols were commercially available.
The C-13 and C-14 diols were prepared by reduction
of the corresponding diacids with lithium aluminium
In a parallel experiment, we evaluated the ability of
QFAs to scavenge hydroxyl radicals present in an etha-
nolic medium. To do so, we used the 2,20-azinobis-(3-
ethylbenzothiazoline-6-sulfonic acid) (ABTS) competi-
tion assay.29 In the presence of hydroxyl radicals, ABTS
is oxidized to the stable ABTS cation radical (ABTSÅ+)
observed by its absorbance at 405 nm.
OMe
OMe
OMe
OMe
MeO
MeO
a
b
Br
Br
OMe
OMe
OMe
OMe
21
51
22
52
O
This method measures the relative ability of QFAs to
scavenge hydroxyl radicals and thus inhibits the forma-
tion of ABTSÅ+ as measured by the decrease of its absor-
bance at 405 nm. Results are shown in Table 1.
OMe
OH
OMe
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
c
d
e
Br
OMe
OMe
OMe
OMe
3
23
53
4
Examination of Table 1 suggested that compounds bear-
ing 15 carbon atoms on the side chain are the most
active in each family,30 with 11c, Q2FA15, being the
most active compound on the biological model tested
even though its antioxidant activity is low. No special
Scheme 1. Reagents and conditions: (a) NBS, CH2Cl2, reflux, 6 h,
80%; (b) Br2, CH2Cl2, 0 °C, Quant; (c) i—mCPBA, CH2Cl2, 0 °C to rt;
ii—NaOH 10%, MeOH, 95%; (d) Me2SO4, acetone, reflux, 4 h, 95%;
(e) NBS, TFA, ꢀ20 °C to rt, 55%.
HO
Br
BnO
HO
OH
BnO
Br
n-4
n-4
n-4
n-4
a
b
c
6a-e
7a-e
8a-e
9a-e
OMe
OMe
OMe
OMe
a : n=12
b : n=14
c : n=15
d : n=16
e : n=18
R
R
R
e
OH
d
Br
n
OBn
n-2
OMe
OMe
51-3
101-3a-e
11-3a-e
Scheme 2. Reagents and conditions: (a) HBr, cyclohexane, reflux, 6 h, 75–89%; (b) i—NaH, THF, rt, 30 min; ii—BnBr, THF, reflux, 24 h, 72–88%;
(c) lithium acetylide (ethylene diamine complex), DMSO, 0 °C to rt, overnight, 83–90%; (d) 8a–e, piperidine, Pd(PPh3)4, 80 °C, 24 h, 71–90%;
(e) Pd/C 5%, H2, EtOH, rt, overnight, 80–92%.

M. Hanbali et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2637–2640
Table 1. Compounds synthesized and tested
2639
Compound number
Compound name
Molecular structure
HRMS (M+H+)
Antioxidant activity
Axonal growth
Calculated
Found
ABTS assay IC50 (lM)
%/control (1 nM)
—
—
—
Ethanol
TroloxÒ
tCFA15a
—
—
—
—
—
—
—
—
—
600
100
—
C24H44O2
>10,000
150
11a
11b
11c
11d
11e
Q2FA12
Q2FA14
Q2FA15
Q2FA16
Q2FA18
C20H34O3
C22H38O3
C23H40O3
C24H42O3
C26H46O3
323.2581
351.2880
365.3050
379.3207
407.3520
323.2575
351.2884
365.3056
379.3205
407.3522
>10,000
>10,000
>10,000
>10,000
>10,000
109
110
181
120
109
12a
12b
12c
12d
12e
13a
13b
13c
13d
13e
Q3FA12
Q3FA14
Q3FA15
Q3FA16
Q3FA18
Q4FA12
Q4FA14
Q4FA15
Q4FA16
Q4FA18
C21H35O4
C23H39O4
C24H41O4
C25H43O4
C27H47O4
C22H38O5
C24H42O5
C25H44O5
C26H46O5
C28H50O5
352.2614
380.2927
394.3083
408.3240
436.3553
383.2972
411.3105
425.3262
439.3418
467.3731
352.2626
380.2928
394.3090
408.3243
436.3560
383.2788
411.3108
425.3265
439.3433
467.3735
720
720
720
720
108
109
152
135
106
100
108
158
127
103
720
***
***
***
***
***
Each compound is tested on the ABTS competition assay. The IC50 values were determined as the concentration of the compound required for a 50%
***
diminution of the ABTSÅ+
Tool 3.0 and is presented as the mean value of three different experiments. Variation was generally 5%.
.
stands for an antioxidant activity that did not reach the IC50. Axonal growth was measured with UTHSCSA Image
a Biological activity of tCFA15 is expressed at 10ꢀ9 M to be consistent with the remaining biological results presented.
correlation between the antioxidant and neurotrophic
activity was found.
the glial scar. Table 2 shows that the growth promoting
effect of Q2FA15 is maintained in the presence of both
myelin proteins (1 and 10 lg/mL) and Sema3 A
(100 ng/mL), suggesting that our compound is able to
counteract the inhibitory substrates of the glial scar.
After the initial screening of our library of QFAs, a
more triggered study was conducted. Figure 1 shows
the dose-dependent effect of Q2FA15 on the model of
axonal growth. Q2FA15 exerts its highest activity at
10–9 M. To be consistent with our model of a CNS
lesion, we tested the activity of Q2FA15 in the presence
of naturally occurring inhibitory factors present within
Lesioned axons within the CNS have little or no hope of
regeneration. Previous studies in our laboratories have
shown that tCFA15 was a potent promoter of axonal
growth, acting at 10ꢀ7 M.10,11 Herein, we have designed
a similar compound that exerts a promoting effect 100
times stronger than tCFA15s.
200
150
100
50
***
***
Though no synergistic activity is noted between the anti-
oxidant and the neurotrophic moieties, Q2FA15 still
bears a methylated quinol moiety which is highly antiox-
idant in its demethylated form31 (IC50 = 6 lM on the
ABTS assay). Multiple studies have shown that intracel-
lular demethylases, based on cytochrome P450 activity,
have the ability to demethylate such aromatic com-
pounds within cells.32
***
Control
10-12M
10-9M
10-7M
0
In our case, methylation not only confers higher stabil-
ity to Q2FA15 but also allows easier CNS penetration.
Demethylated compound within the cells should exert
Figure 1. Dose-dependent promoting effect of Q2FA15 on a model
assay of axonal growth. Data are shown as means SEM.
***p < 0.001.
Table 2. Promoting effect of Q2FA15 as compared to tCFA15 upon naturally occurring inhibitory factors present in the glial scar
No inhibitory substrate
Myelin proteins
Sema3A
1 lg/mL
10 lg/mL
100 ng/mL
No treatment
tCFA15 (10ꢀ7 M)
Q2FA15 (10ꢀ9 M)
0
ꢀ19.8
+28.8
+18.4
ꢀ37.6
+5.0
ꢀ30.2
+10.2
+12.4
+80.0
+81.0
ꢀ5.3
Data are shown in % of axonal growth as variations from control conditions.

2640
M. Hanbali et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2637–2640
10. Gonzalez de Aguilar, J. L.; Girlanda-Junges, C.; Coowar,
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its antioxidant activity as required by the biological
system.
Finally, preliminary pharmacological studies have
shown that Q2FA15 requires cyclic nucleotides
(cAMP/cGMP) to exert its biological effect. Recent find-
ings have shown that the inhibitory effect of Sema3A
can be circumvented by intracellular increases of
cGMP,33 whereas myelin-triggered inhibition can be
circumvented by cAMP.34 Moreover, one of the many
targets of intracellular cGMP is cAMP-dependent phos-
phodiesterases resulting in an increase of intracellular
cAMP.35 In addition, cAMP-triggered axonal growth
is known to result in over-expression of specific
a1- and bIII-tubulin genes. CNS neurons express three
subtypes of a-tubulin (a1, a2 and a4) and four subtypes
of b-tubulin (bI, bII, bIII and bIV) genes. Microtubules
consisting of a1- and bIII-tubulin are found in post-
mitotic neurons and exert increased stability.36
21. Sutherland, H. S.; Higgs, K. C.; Taylor, N. J.; Rodrigo, R.
Tetrahedron 2001, 57, 309.
All these data suggest that Q2FA15 (11c) is a strong pro-
moter of axonal growth that can circumvent the inhibi-
tory properties of the glial scar. Its very strong
biological activity makes it a very promising candidate
for future therapies the aim of which is the promotion
of functional recovery from CNS lesions.
22. Tisdale, E. J.; Chowdhury, C.; Vong, B. G.; Li, H.;
Theodorakis, E. A. Org. Lett. 2002, 4, 909.
23. Raul, F.; Schneider, Y.; Brouillars, R.; Fougerousse, A.;
Chabert P. et al., WO Patent 03, 031, 381, 2003.
24. Syper, L.; Mlochowski, J.; Kloc, K. Tetrahedron 1983, 39,
781.
25. Rizzacasa, M. A.; Sargent, M. V. J. Chem. Soc., Perkin
Trans. 1 1987, 9, 2017.
26. Girlanda-Junges, C.; Keyling-Bilger, F.; Schmitt, G.; Luu,
B. Tetrahedron 1998, 54, 7735.
27. Hofman, S. Synthesis 1998, 479.
28. Negishi, E. Handbook of Organopalladium Chemistry for
Organic Synthesis; John Wiley & Sons, 2002, Vol 1, pp
493–529.
Acknowledgments
This research was partially supported by Meiji Dairies
Corporation (B.L. and M.H.) and ACI Jeunes Cherch-
eurs (D.B.).
29. Poeggeler, B.; Thuermann, S.; Dose, A.; Schoenke, M.;
Burkhardt, S., et al. J. Pineal Res. 2002, 33, 20.
30. Q2FA15: Calcd C, 75.77; H, 11.06, found C, 75.63; H,
11.05. Q3FA15: Calcd C, 73.05; H, 10.73, found C, 72.99;
H, 11.48. Q4FA15: Calcd C, 70.72; H, 10.44, found C,
70.95; H, 10.55.
31. Unpublished data. 2-(15-Hydroxypentadecyl)benzene-1,4-
diol is obtained by the use of BBr3 in ethylene chloride at
room temperature.
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