Design, synthesis and pharmacological evaluation of novel tacrine-caffeic acid hybrids as multi-targeted compounds against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2012.08.036

A novel series of tacrine-caffeic acid hybrids (5a-f) were designed and synthesized by combining caffeic acid (CA) with tacrine. The antioxidant study revealed that all the hybrids have much more antioxidant capacities compared to CA. Among these compound

Full text of DOI:10.1016/j.bmcl.2012.08.036

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6498–6502
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and pharmacological evaluation of novel tacrine–caffeic
acid hybrids as multi-targeted compounds against Alzheimer’s disease
Xiaojuan Chao a, , Xixin He b, , Yilin Yang a, , Xie Zhou a, , Minghua Jin c, Shu Liu d, Zhiyi Cheng a,
Peiqing Liu a, Yuting Wang a, Jianchen Yu a, Yi Tan b, Yingjuan Huang c, Jian Qin c, Simona Rapposelli e,
Rongbiao Pi a,
⇑
a Department of Pharmacology & Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou 510006, China
b Department of Chinese Medicinal Chemistry, College of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
c Department of Traditional Chinese Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510006, China
d Zhongshan Medical School, Sun Yat-Sen University, Guangzhou 510080, China
e Dipartimento di Scienze Farmaceutiche, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of tacrine–caffeic acid hybrids (5a–f) were designed and synthesized by combining caffeic
acid (CA) with tacrine. The antioxidant study revealed that all the hybrids have much more antioxidant
capacities compared to CA. Among these compounds, 5e showed the highest selectivity in inhibiting ace-
tylcholinesterase (AChE) over butyrylcholinesterase (BuChE). Enzyme kinetic study had suggested that 5e
binds to both catalytic (CAS) and peripheral anionic sites (PAS) of AChE. Moreover, compound 5e also
inhibited self- or AChE-induced b-amyloid1–40 aggregation, as well as had potent neuroprotective effects
against H2O2- and glutamate- induced cell death with low toxicity in HT22 cells.
Received 12 June 2012
Revised 7 August 2012
Accepted 10 August 2012
Available online 16 August 2012
Keywords:
Caffeic acid
Dimer
Ó 2012 Elsevier Ltd. All rights reserved.
Alzheimer’s disease
Antioxidants
Acetylcholinesterase
Multi-targeted compounds
Alzheimer0s disease (AD) is the most common form of irrevers-
ible dementia in the elderly. However, no effective treatment for
AD is currently available. AD is thought to be a complex, multifac-
torial syndrome with many related molecular lesions contributing
to its pathogenesis.1,2 Besides its pathological hallmarks, the accu-
mulation of misfolded protein deposits as amyloid-b (Ab) plaques
and neurofibrillary tangles, AD brains exhibit constant evidence
of oxidative damage.3–5 In particular, oxidative stress-induced in-
jury is observed in the most cellular macromolecules of AD brains,
including nucleic acids, proteins, and lipids. These findings support
the ‘oxidative stress hypothesis’ of AD, of which reactive oxygen
species (ROS) plays a key role in AD onset and progression.6 This
hypothesis proposes antioxidants as beneficial therapeutic tools
in AD treatment.6
Anti-oxidants presenting additional pharmacological effects are
currently thought to offer a good chance of combating complex
diseases in which free radicals are significant but not exclusive
drivers.6,7 This concept has been recently rationalized in the field
of neurodegenerative diseases affording the multitarget-directed
ligands (MTDLs) design strategy.8–11 The strategy holds that single
molecules being properly combined two or more distinct pharma-
cological properties, including antioxidant properties and then able
to act at different targets in the neurodegenerative process, can
achieve greater effectiveness compared to single-targeted drugs
for investigating AD.8–11
Previously, we developed tacrine–ferulic acid hybrids (TAnFA),
one new series of the multifunctional antioxidants rationally de-
signed to combat AD.12–15 It was also argued that the cyclic moiety
of ferulic acid could interact with the peripheral anionic site (PAS)
of AChE, which was associated with the neurotoxic cascade under-
lying AD through AChE-induced Ab aggregation.12,13 Indeed,
kinetic analyses confirmed that tacrine–ferulic acid hybrids could
bind both catalytic and peripheral sites, thus acting as a dual-bind-
ing AChE inhibitor.12 In in vitro and in vivo models, tacrine–ferulic
acid hybrids effectively displayed the expected multiple biological
properties, namely inhibition of AChE activity, inhibition of
Abbreviations: AD, Alzheimer’s disease; Ab, amyloid b-protein; ROS, reactive
oxygen species; MTDLs, multitarget-directed ligands; CA, caffeic acid; TA, tacrine;
FA, ferulic acid; AChE, acetylcholine esterase; BuChE, butyrylcholinesterase; CAS,
catalytic anionic site; PAS, peripheral anionic site; CNS, central nervous system.
⇑
Corresponding author. Tel./fax: +86 20 3994 3122.
E-mail address: pirb@mail.sysu.edu.cn (R. Pi).
These authors equally contributed to this paper.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.036

X. Chao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6498–6502
6499
MTDLs against AD. To this end, we synthesized the hybrid com-
pounds 5a–f (Fig. 1), among which there was chlorine atom substi-
tution in position 6 of the tacrine core in compounds 5d–f. Thus, in
the present study, we linked CA with the anticholinesterase motifs
of the first marketed AD drug, tacrine. Synthesized compounds
were then investigated to assess their antioxidant, anticholinester-
ase, and anti-aggregating activities as well as neuroprotection.
Consequently, compound 5e showed a high AChE/BuChE selectiv-
ity. Interestingly, the compound 5e also inhibited self- or AChE-in-
duced b-amyloid1-40 aggregation, Cu2+ chelating property as well
as had neuroprotective effects against ROS-induced cell death in
HT22 cells.
Tacrine–caffeic acid derivatives 5a–f were prepared with a
sequence of reactions as shown in Scheme 1 and Scheme 2. The
intermediate 3 was prepared from anthranilic acid derivatives in
77% overall yield according to the reported procedure.24,25 The
intermediate 4 was afforded as a brown oil with the reaction of
compound 3 and alkylamine under reflux for 16 h in 1-pentanol
in 80–90% yield. The desired compound 5a–f was further synthe-
sized in a solution of caffeic acid, DCC,4-dimethylaminopyridine
(DMAP) and intermediate 4 in dichloromethane/N,N-dimethyl-
formamide at room temperature in 45–50% yield (Scheme 2).12,16
Synthesized compounds (5a–f) were firstly tested their radical
scavenging activities by using 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) assay. The IC50s (defined as the concentration resulting in
50% scavenging activity) were determined. The data are summa-
rized in Table 1. All new compounds (5a–f) have exhibited superior
radical scavenging activity comparing to TA3FA, a tacrine–ferulic
acid hybrids,13,16 the best candidate compound from hybrids of
tacrine and ferulic acid, which indicated that the antioxidative
activities of tacrine–caffeic acid hybrids should be stronger than
that of tacrine–ferulic acid ones as we hypothesized.
Moreover, synthesized compounds’ (5a–f) inhibitory of AChE
and BuChE were also assayed as experimental part described.
The ratios of BuChE to AChE were also calculated respectively.
The data are summarized in Table 1. The IC50s for BuChE and AChE
of 5a–f have exhibited a linkage to the length of the linker between
tacrine and caffeic acid, as reported before12,16,25,26 and compara-
ble or superior to that of TA3FA. Interestingly, 5e has the highest
ratio of BuChE/AChE IC50, indicating that chloro-substituted of tet-
rahydroacridine fragment may dramatically increase the selection
Figure 1. Design strategy for compounds 5a–f.
AChE-induced Ab aggregation, and ability to protect cells against
ROS, emerging as a candidate for drug development.12,14–16
Caffeic acid (CA), which is abundant in nature, is an analogue of
ferulic acid and has more potent pharmacological activities, includ-
ing anti-oxidative stress, anti-inflammatory, anti-cancer, and anti-
viral activities, than that of ferulic acid.17 The caffeic acid scaffold is
found in a number of nature and synthetically active molecules,
such as caffeic acid phenethyl ester, a plant polyphenolic concen-
trated in honeybee propolis, has been found to be biologically ac-
tive in a variety of pathways including cytoprotection against
oxidative stress.18–20 Moreover, it was reported that chlorine atom
substitution in position 6 of the tacrine core is thought to enhance
the affinity toward AChE and selectivity in inhibiting AChE over
butyrylcholinesterase (BuChE).21–23
In addition, according to previous reports12,13,16, the linker
length n = 2, 3 and 6 should be the better ones to archeive the bet-
ter pharmacological activities. In light of this, we would like to ex-
pand our study by replacing the ferulic acid structure with CA
structure in the tacrine-based dimer in the search for better novel
Scheme 1. Reagents and conditions: (a) cyclohexanone, toluene, reflux,4 h, 85%; (b) POCl3, reflux, 2 h, 90%.
Scheme 2. Reagents and conditions: (a) 3 equiv NH2(CH2)nNH2, 1-pentanol, reflux, 16 h, 80–90%; (b) 1.2 equiv DCC, 1 equiv caffeic acid, cat. DMAP, CH2Cl2/DMF , room
temperature, 45–50%.

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X. Chao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6498–6502
Table 1
The inhibition of AChE and BuChE activities and DPPH redical scavenging activities (IC50, lM) of compounds
f
Compounds
IC50 SEMa
(l
M)
Ratio of BuChE/AChE IC50
DPPH radical scavenging activity (IC50 SEMa,
l
M)
AChEb
BuChEc
5a
5b
5c
5d
5e
5f
1.8 0.1
2.3 0.4
1.3
9.6
2.1
5.5
93.8
0.6
14.1 0.4
11.1 0.6
10.4 0.4
9.7 0.6
4.8 0.9
4.7 0.6
37.6 0.9
>1000
d
d
8.8
0.9
83.7
1.4
1.9 0.8
1.0 0.1
0.3 0.1
6.3 0.4
0.1 0.1
0.9 0.1
N/Ae
3.9 0.9
5.5 0.4
29.5 0.5
3.8 0.2
3.1 0.2
0.1 0.0
N/Ae
TA3FA
Tacrine
Caffeic acid
28.6
0.1
N/Ae
5.7 0.4
a
b
c
d
e
f
SEM: Standard Error of the Mean.
AChE from electric eel; IC50, inhibitor concentration (means SEM of three experiments) for 50% inactivation of AChE.
BuChE from horse serum; IC50, inhibitor concentration (means SEM of three experiments) for 50% inactivation of BuChE.
nM.
N/A: Not available.
IC50 was defined as the concentration resulting in 50% scavenging activity.
showed both increasing slopes and increasing intercepts at higher
inhibitor concentration. This pattern indicated
inhibition.
a mixed-type
Previous reports showed that polyphenolics, including CA, inhi-
bit the self-aggregation of amyloid protein and our previous study
demonstrated that tacrine–feulic acid hybrids not only inhibit
amyloid protein self-aggregation but also the AChE-induced aggre-
gation.12,14 Given the chloro-substituted compounds have better
radical scavenging activity, their inhibitory activities on amyloid
protein self-aggregation and also the AChE-induced aggregation
were further examined as described in experimental part. The data
are summarized in Table 2. All novel hybrids but not tacrine have
similarly strong inhibiting activities on AChE-induced amyloid pro-
tein aggregation and caffeic acid had mild activity to inhibit amy-
loid protein self-aggregation as well as AChE-induced aggregation.
The results indicated that tacrine–caffeic acid hybrids might bind
to the PAS and CAS of AChE.27,28 Dual binding AChE inhibitors have
been recognized as AD disease-modifying agents which may
simultaneously alleviate cognitive deficits and behave as by inhib-
iting the beta-amyloid (Ab) peptide aggregation29,30 suggesting 5e
might be a promising disease-modifying agents for AD.
Figure 2. Lineweaver-Burk plot for the inhibition of acetylcholinesterase by
compound 5e.
Table 2
It was shown that the hybrids were good radical scavengers by
using DPPH radical scavenging assay. So, the hybrids were tested
whether they protect against oxidative stress-induced cell death.
Two oxidative stress inducers, H2O2 and glutamate, were used in
this study. The latter induced oxidative toxicity through depleting
intracellular glutathione in HT22, a mouse hippocampal cell line,
which makes it an endogenous ROS-induced neuronal death mod-
el.31,32 Compounds 5d–f were found to protect against cell death
induced by glutamate as well as that by H2O2 in HT22 cells
Inhibition of self-induced and AChE-induced Ab(1-40) aggregation
Compounds
Inhibition of Ab aggregation(%) SEMa
self-inducedb
AChE-inducedc
5d
5e
5f
31.0 5.8
36.2 3.3
55.7 5.1
40.5 3.2
22.8 1.8
32.3 3.7
60.3 4.8
54.3 1.4
67.7 1.9
74.1 1.8
75.0 2.2
22.1 2.2
31.2 2.2
93.1 1.1
TA3FA
Tacrine
Caffeic acid
Congo Red
(Fig. 3). Surprisingly, when at high concentration (30 lM), both
5e and 5f alone seemly have cellular toxicity while both of them
still can prevent the cell death induced by glutamate and H2O2.
In addition, 5f shows no difference between 10 lM and 30 lM.
a
SEM: Standard Error of the Mean.
Inhibition of self-mediated Ab (1–40) aggregation. The thioflavin-T fluorescence
b
method was used, and the measurements were carried out in the presence of 20 lM
It was reported that caffeic acid and caffeic acid derivatives
were capable of inhibiting cell proliferation.33–35 Treatment with
the novel hybrids alone caused a slight decrease in cell viability
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay comparing to that of control (Fig. 3) in HT22 cells.
Hence lactate dehydrogenase (LDH) release assay was adopted to
test whether the novel hybrids induced cytotoxicity or anti-prolif-
eration. With the most potent protective effects among 5d–f, 5e
was used for the LDH release assay. As shown in Figure 4.
Compound 5e did not increase the LDH release at the concentra-
inhibitor.
c
Inhibition of AChE-induced Ab (1–40) aggregation. The thioflavin-T fluores-
cence method was used, and the concentration of the tested inhibitor and Ab(1–40)
was 100
lM and 230 lM, respectively, whereas the Ab(1–40):AChE ratio was equal
to 100:1.
of cholinesterase inhibition and also compound 5e could be a dual
binding inhibitor binding both catalytic anionic site (CAS) and PAS
of AChE basing on its ability to simultaneously inhibit AChE and
AChE-induced b-amyloid aggregation.
tion as high as 100 lM, suggesting that 5e inhibits cell prolifera-
tion but not induces toxicity in HT22 cells. This might be the
explaination of the paradoxical activity of 5e and 5f.
The nature of AChE inhibition by the best inhibitor compound
5e was studied through the graphical analysis of steady state inhi-
bition data, as shown in Figure 2. The Lineweaver-Burk plots

X. Chao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6498–6502
6501
Figure 3. Protective effects of compounds on H2O2- or glutamate- induced cell death. HT22 cells were pretreated with each compounds for 30 min and then inclubated with
H2O2 (100
M) for 12 h or glutamate (5 mM) for 24 h. Cell viability was analyzed using the MTT assay. The data are shown as means SD, n = 6. ⁄p <0.05, ⁄⁄p <0.01 versus
glutamate (5 mM) treated alone cells, #p <0.05, ##p <0.01 versus H2O2 (100 M) treated alone cells.
l
l
porotein self- or AChE-induced aggregation and the ability to pro-
tect cells against oxidative stress. Taken together, compound 5e
that is endowed with a MTDL profile, a well-balanced AChE/BuChE
inhibitory activity and low toxicity, may represent a valuable
anti-AD candidates for further development.
Acknowledgments
This study was supported in part by Fundamental Research
Funds for the Central Universities (No 10ykpy23), Guangdong
Provincial International Cooperation Project of Science
&
Technology (No 2012B050300015) and National Natural Science
Foundation of China/RGC Hong Kong Joint Research Scheme (No
30731160617) to Pi R.
Figure 4. Effects of 5e on LDH release in HT22 cells. Cells were treated with 5e for
24 h. Cell death was analyzed using the LDH release assay. The data are shown as
means SD, n = 6.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.08.
036.
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