Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates

Article abstract of DOI:10.1016/j.bmcl.2008.03.073

Five tacrine-ferulic acid hybrids (6a-e) were designed and synthesized as multi-potent anti-Alzheimer drug candidates. All target compounds have better acetylcholinesterase inhibitory activity and comparable butyrylcholinesterase inhibitory activity in relation to tacrine. Interestingly, 6d showed a reversible and non-competitive inhibitory action for acetylcholinesterase indicating interaction with the peripheral anionic site, whereas a reversible but competitive inhibitory action for butyrylcholinesterase. The antioxidant study revealed that four target compounds have, compared to Trolox, high ability to absorb reactive oxygen species.

Full text of DOI:10.1016/j.bmcl.2008.03.073

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2905–2909
Design and synthesis of tacrine–ferulic acid hybrids
as multi-potent anti-Alzheimer drug candidates
Lei Fang,a,b Birgit Kraus,c Jochen Lehmann,a Joerg Heilmann,c
Yihua Zhangb,* and Michael Deckera,*
aLehrstuhl fu¨r Pharmazeutische/Medizinische Chemie, Institut fu¨r Pharmazie, Friedrich-Schiller-Universita¨t Jena,
Philosophenweg 14, D-07743 Jena, Germany
bCenter of Drug Discovery, China Pharmaceutical University, Tongjiaxiang 24, 210009 Nanjing, PR China
cLehrstuhl Pharmazeutische Biologie, Universita¨t Regensburg, Universita¨tsstraße 31, D-93053 Regensburg, Germany
Received 14 March 2008; revised 25 March 2008; accepted 27 March 2008
Available online 30 March 2008
Abstract—Five tacrine–ferulic acid hybrids (6a–e) were designed and synthesized as multi-potent anti-Alzheimer drug candidates.
All target compounds have better acetylcholinesterase inhibitory activity and comparable butyrylcholinesterase inhibitory activity
in relation to tacrine. Interestingly, 6d showed a reversible and non-competitive inhibitory action for acetylcholinesterase indicat-
ing interaction with the peripheral anionic site, whereas a reversible but competitive inhibitory action for butyrylcholinesterase.
The antioxidant study revealed that four target compounds have, compared to Trolox, high ability to absorb reactive oxygen
species.
Ó 2008 Elsevier Ltd. All rights reserved.
A case report of what was later named ‘Alzheimer’s
disease’ (AD) was first reported by Alois Alzheimer
in 1907 at a neurology conference, where he described
a 51-year-old woman with rapid and devastating mem-
ory degeneration. Although the disease was once con-
sidered rare, it is now established as the leading
cause of dementia and despite major research efforts
not curable. It has been estimated that the incidence
of AD varies from 12% to 19% for women and 6%
to 10% for men over the age of 65 years1 and rises
exponentially with age.2 Five pharmaceutical drugs
representing cholinesterase inhibitors (ChEIs), namely
galantamine, rivastigmine, donepenzil, tacrine (Fig. 1)
and an NMDA receptor antagonist (memantine) are
applied clinically. However, they can only offer little
more than short-term palliative effects and ChEIs suf-
fer from pronounced peripheral side effects.
NH2
OMe
OH
N
HOOC
Ferulic acid
Tacrine
Figure 1. Structures of ferulic acid and tacrine.
The difficulty for developing satisfactory therapy of AD
lies in the complex pathophysiology of the disease,
which involves numerous pathways. These include
deficiency in cholinergic neurotransmission, defective
beta-amyloid protein metabolism, abnormalities of
glutamatergic, adrenergic, serotonergic and dopaminergic
neurotransmission, and the involvement of inflamma-
tory, oxidative and hormonal pathways.3 Due to the
multi-pathogenesis of AD, one of the current strategies
to develop novel anti-Alzheimer agents focuses on com-
pounds with multiple potencies.4 Several, quite diverse
attempts have been made and some of them have shown
good performance.5 We here report a series of tacrine–
ferulic acid hybrids (6a–e) as multi-potent anti-Alzhei-
mer drug candidates. The target molecules consist of
three parts: the tacrine-like heterocycle, the ferulic acid
(Fig. 1) moiety and an alkylenediamine side chain to
link the former two parts.
Keywords: Tacrine; Ferulic acid; Cholinesterase inhibitor; Antioxidant;
Anti-Alzheimer drug; Hybrid molecule.
*
Corresponding authors. Present address: Medicinal Chemistry Pro-
gram, McLean Hospital, Harvard Medical School, 02478 Belmont,
USA. Tel.: +1 617 855 2057 (M.D.); e-mail addresses:
zyhtgd@hotmail.com; mdecker@mclean.harvard.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.073

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L. Fang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2905–2909
Tacrine was the first approved ChEI by the FDA for the
treatment of AD. Its rationale mainly aims to stabilize
acetylcholine levels in the synaptic cleft by the inhibition
of the main degrading enzyme of the neurotransmitter
to maintain neurotransmission. However, because it
only targets at the cholinergic dysfunction, without
any effect on the other pathologic factors, it can retard
the progression of AD for only about a year.6
ferulic acid molecule), thus the target compounds could
hardly be isolated. Therefore, the phenol hydroxyl
functional group of ferulic acid was at first protected
by reacting with ethyl chloridocarbonate to give the es-
ter intermediate 4. Compound 4 was then coupled to
3a–e in the presence of DCC/DMAP. Finally, the pro-
tection group was removed by the treatment with 95%
ethanol/aminoethanol to yield the target compounds
(Scheme 1).
More and more attention is paid to oxidative stress for
its role in the progression of AD.7 A recent report sug-
gested that oxidative stress is involved in the early stage
of the pathologic cascade and represents a key factor to
initiate the aggregation of b-amyloid and s-protein
hyperphosphorylation.8 Accordingly, many antioxi-
dants were observed to be able to attenuate the syn-
drome of AD and prevent the progression of the
disease.9
To determine the therapeutic potential of 6a–e for the
treatment of AD, the acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE) inhibitory activities,
respectively, were measured for the target compounds
as well as for the free amine intermediates 3a–e using
the Ellman’s assay.16 The results are given in Table 1.
All the target compounds showed better AChE inhibi-
tory activity than tacrine (IC50 = 45 nM) with the IC50
values varying from 4.4 to 38.6 nM. Particularly, the po-
tency of 6c and 6d (IC50 = 7.6 nM and 4.4 nM) is 6- and
10-fold improved, respectively. As for BChE, all the
tested compounds also showed comparable activity to
tacrine (IC50 = 5.1 nM) with the IC50 values varying
from 5.9 to 34.1 nM. The results of the amine intermedi-
ates showed a trend consistent with the target com-
pounds, indicating that the ferulic acid moiety might
not obviously affect the extent of ChE inhibitory activ-
ity. Amongst all the target compounds, 6c and 6d
showed high activity toward both AChE and BChE,
suggesting the optimal distance between the tacrine-like
heterocycle and the ferulic acid moiety is 6–7 atoms
long. This result seems consistent with the previous re-
ports of lipoic acid/tacrine,17 huperzine A/tacrine18 and
lipoic acid/quinazolinimines hybrids, the latter ones rep-
resenting a novel class of BChE-selective compounds.19
Ferulic acid (Fig. 1) is one of the dominating natural
phenolic acids and occurs, often together with caffeic
acid, in the secondary metabolite spectrum of important
economic and medicinal plants such as wheat (Triticum
aestivum) or eucalyptus (Eucalyptus globulus). Further-
more, many secondary metabolites contain ferulic acid
substructures or ferulic acid esters and showed very po-
tent anti-oxidative activity depending on the molecular
structure of this part.10 It was shown in an in vivo study
in mice that long-term administration of ferulic acid in-
duced resistance to Ab1–42 toxicity in the brain, this che-
mopreventive effect could also be proved in an
behavioural assay.11 Therefore, connecting ferulic acid
to a tacrine template via an alkylenediamine side chain
may yield a novel class of target molecules, which might
exert a synergic action of the cholinesterase (ChE) inhib-
itory activity originating from the tacrine template and
the antioxidant activity originating from the ferulic acid
moiety. Besides, according to our previous investiga-
tion,12 an introduction of an alkylenediamine side chain
at 9-position of the tacrine-like heterocycle may also
contribute to a reduction of the hepatotoxicity of tacrine
and enhance the ChE inhibitory activity. In the context
of tacrine’s hepatotoxicity,13 the formation of a hybrid
with ferulic acid seems to be of special interest, since also
hepatoprotective effect has been described for ferulic
acid.14
Because 6d showed the highest activities towards both
AChE and BChE, it was selected for kinetic measure-
ments, in order to gain information about the mode of
inhibition and binding of the novel inhibitors. The
mechanism of inhibition was analyzed by recording sub-
strate–velocity curves in the absence and presence of dif-
ferent concentrations of 6d using 25, 50, 90, 150, 226 and
450 lM of substrate for both BChE and AChE curves.
The concentrations of 6d were varied between 1 and
10 nM comprising 1, 2, 4, 8 and 10 nM, respectively.
Figures 2 and 3 show the Lineweaver–Burk plots, which
are reciprocal rates versus reciprocal substrate concen-
trations for the different inhibitor concentrations result-
ing from the substrate–velocity curves for AChE and
BChE. For AChE, with increasing inhibitor concentra-
tions, the vmax value (i.e., the reciprocal of the Y inter-
cept) is obviously decreased. In contrast the Km values
(i.e., the negative reciprocal of the X intercept) are not
changed with different inhibitor concentrations. For
BChE, a different Lineweaver–Burk plot from AChE is
observed. In this Lineweaver–Burk plot the Km values
differ with the different inhibitor concentrations, while
the vmax value remains unchanged. The Lineweaver–
Burk plot for AChE clearly shows reversible and non-
competitive inhibition by tacrine–ferulic acid hybrids,
meaning that these compounds do not compete for the
same active site as the substrate acetylcholine. Based
Because the target molecules consist of three parts, the
synthetic strategy is firstly to prepare the heterocycle,
then to introduce the side chain, and finally to connect
it to the ferulic acid moiety. According to a previously
reported protocol,15 9-chloro-1,2,3,4-tetrahydroacridine
(2) was first prepared by the cyclization of anthranilic
acid with cyclohexanone. In order to introduce the side
chain, different alkylenediamines reacted with 9-chloro-
1,2,3,4-tetrahydroacridine to generate the 9-amin-
oalkylamino-1,2,3,4-tetrahydroacridines 3a–e. We had
tried direct coupling of 3a–e to ferulic acid in the pres-
ence of DCC/DMAP or treating CDI-activated ferulic
acid with 3a–e. However, many by-products were
formed during the reaction because of the possibility
of inter-molecular side reactions (such as the reaction
of the phenolic OH-group of ferulic acid with another

L. Fang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2905–2909
2907
Cl
NH2
HN
n
O
COOH
NH2
a
b
+
N
N
3a, n=2; 3b, n=3;
3c, n=4; 3d, n=5;
3e, n=8
1
2
+
OMe
OMe
OH
OCO2C2H5
c
C2H5O2CCl
+
HOOC
HOOC
4
d
O
O
HN
N
N
H
n
HN
N
H
n
e
OCO2C2H5
OH
OMe
OMe
N
6a, n=2; 6b, n=3;
6c, n=4; 6d, n=5;
6e, n=8
5a, n=2; 5b, n=3;
5c, n=4; 5d, n=5;
5e, n=8
Scheme 1. General method for the synthesis of 6a–e. Reagents and conditions: (a) POCl3, reflux, 3 h; (b) 3 equiv H2N(CH2)nNH2, pentanol, reflux,
18 h; (c) 1 equiv NaOH, water, 0 °C; (d) 1.2 equiv DCC, equimolar 3a–e, cat. DMAP, CH2Cl2, rt; (e) 95% ethanol, 1 equiv H2NCH2CH2OH, 3 h, rt.
Table 1. IC50 values of 3a–e and 6a–e against AChE and BChE
the PAS of AChE being the structural motif responsible
for Ab-peptide fibril formation catalyzed by AChE.23
Compound
IC50 (nM) SEMa
AChEb
BChEc
In contrast, the Lineweaver–Burk plot for BChE shows
reversible and competitive inhibition by tacrine–ferulic
acid hybrids, revealing that these compounds compete
for the same active site as the substrate acetylcholine.
The different mechanism of the inhibitory action may
be due to the difference of the structures of the two cho-
linesterases. Because AChE presents several aromatic
residues that are replaced by smaller aliphatic ones in
BChE, BChE has a larger electrostatic gradient than
AChE,24 which makes it possible to allow a large mole-
cule to approach the active site at the bottom of the
gorge.
3a
3b
54.8 10.4
69.5 14.7
23.6 5.0
3.2 0.9
3.5 1.4
38.6 9.7
33.0 8.3
7.6 1.9
4.4 1.7
9.6 2.1
45.1 6.9
12.3 4.1
5.5 1.4
7.1 1.1
5.0 0.7
1.4 0.4
34.1 8.1
27.0 9.9
5.9 0.7
6.7 1.6
12.7 2.6
5.1 1.0
3c
3d
3e
6a
6b
6c
6d
6e
Tacrine
a Data is the mean of at least three determinations.
b E.C. 3.1.1.7, type VI-S, from Electric Eel.
c E.C. 3.1.1.8, from equine serum.
In order to prove the antioxidant capacities of the hy-
brid molecules in comparison to tacrine, the ability of
compounds 6a–6e to reduce the amount of peroxyl rad-
icals was determined by the ORAC (oxygen radical
absorbance capacity) assay.25 Ferulic acid itself was se-
lected as positive control, whereas tacrine was also
tested to serve as a negative standard (Table 2). The
compounds’ ability to scavenge radicals is expressed as
Trolox equivalent, that is, their relative ability (at con-
centrations between 0.5 and 10 lM) compared to the
highly potent compound Trolox (6-hydroxy-2,5,7,8-tet-
ra-methyl-chroman-2-carboxylic acid). Ferulic acid itself
is in exact accordance with literature data, a potent rad-
ical scavenger of peroxyl radicals.26 After connecting the
ferulic acid to the tacrine moiety the antioxidant activity
decreased, but nevertheless all hybrids tested showed
moderate to good antioxidant activity. Interestingly,
on the analysis of the chemical structure of the target
compounds as well as the model of the enzyme, we pos-
tulate that, rather than the catalytic binding site, the
binding site of the test compound might be the periphe-
ral anionic site (PAS), which is reportedly a second bind-
ing site of AChE, at the lip of the gorge and around
18 A away from the active site. Molecular modelling
studies are necessary to validate these assumptions
though. Given the relatively narrow gap of the active
site and the considerable steric demand of the target
compounds, the hybrids may be inclined to bind at the
PAS rather than the active site. This would be a highly
desirable therapeutic effect, since the neurotoxic cascade
is mediated through AChE-induced Ab aggregation,21,22
20
˚

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L. Fang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2905–2909
AChE inhibitory activity compared to tacrine. The
Lineweaver-Burk plot
AChE inhibition
inhibitory mechanism of 6d was analyzed by determin-
ing the Lineweaver–Burk plots. Interestingly, the results
indicated that the hybrid exerts a reversible and non-
competitive inhibitory action for AChE, whereas a
reversible but competitive inhibitory action for BChE.
The previous result strongly suggests the inhibition of
the PAS of AChE and therefore the possibility to inhibit
Ab-peptide fibril formation also, but this has to be
unambiguously proven in additional investigations. All
the target compounds were screened for their antioxi-
dant activity using the ORAC-Fluorescein assay. Com-
pared to Trolox, most of the target compounds, except
6c, showed a high ability to absorb reactive oxygen spe-
cies. The results suggest that these tacrine–ferulic acid
hybrids may be considered to be a kind of novel anti-
Alzheimer’s drug candidates, which are multi-target-di-
rected ligands with not only ChE inhibitory (both acting
at the active centre and the PAS of AChE) yet also anti-
oxidant activity.
by compound 6d
20
[6d] = 10 nM
15
10
5
[6d] = 4 nM
[6d] = 2 nM
[6d] = 1 nM
[6d] = 0 nM
-0.005
0.005 0.010 0.015 0.020
1/[S] in μM-1
-5
Figure 2. Lineweaver–Burk plots resulting from substrate–velocity
curves of AChE activity with different substrate concentrations (50–
450 lM) in the absence and presence of 1, 2, 4 and 10 nM 6d.
Lineweaver-Burk Plot
BChE Inhibition
by Compound 6d
Acknowledgment
75
50
25
[6d] = 10 nM
[6d] = 8 nM
[6d] = 4 nM
[6d] = 2 nM
[6d] = 1 nM
[6d] = 0 nM
Financial support for L. Fang by the ‘DAAD Sandwich
Program’ is very gratefully acknowledged.
Supplementary data
Supplementary data is available online. Supplementary
data associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2008.03.073.
0.01
0.02
0.03
0.04
1/[S] in μM-1
References and notes
Figure 3. Lineweaver–Burk plots resulting from substrate–velocity
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