Novel tacrine-ebselen hybrids with improved cholinesterase inhibitory, hydrogen peroxide and peroxynitrite scavenging activity

Article abstract of DOI:10.1016/j.bmcl.2013.10.034

A series of tacrine-ebselen hybrids were synthesised and evaluated as possible multifunctional anti-Alzheimer's disease (AD) agents. Compound 6i, which is tacrine linked with 5,6-dimethoxybenzo[d][1,2]selenazol-3(2H)-one by a six-carbon spacer, was the most potent acetylcholinesterase (AChE) and butylcholinesterase (BuChE) inhibitor, with IC₅₀ values of 2.55 and 2.80 nM, respectively. Furthermore, this compound demonstrated similar hydrogen peroxide and peroxynitrite scavenging activity as ebselen by horseradish peroxidase assay and peroxynitrite scavenging activity assay, indicating that this hybrid is a good multifunctional drug candidate for the treatment of AD.

Full text of DOI:10.1016/j.bmcl.2013.10.034

Bioorganic & Medicinal Chemistry Letters 23 (2013) 6737–6742
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel tacrine–ebselen hybrids with improved cholinesterase
inhibitory, hydrogen peroxide and peroxynitrite scavenging activity
⇑
⇑
Fei Mao, Jianwen Chen , Qi Zhou, Zonghua Luo, Ling Huang, Xingshu Li
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tacrine–ebselen hybrids were synthesised and evaluated as possible multifunctional anti-Alz-
Received 22 June 2013
Revised 5 October 2013
Accepted 18 October 2013
Available online 29 October 2013
heimer’s disease (AD) agents. Compound 6i, which is tacrine linked with 5,6-dimethoxybenzo[d][1,2]sel-
enazol-3(2H)-one by
a six-carbon spacer, was the most potent acetylcholinesterase (AChE) and
butylcholinesterase (BuChE) inhibitor, with IC50 values of 2.55 and 2.80 nM, respectively. Furthermore,
this compound demonstrated similar hydrogen peroxide and peroxynitrite scavenging activity as ebselen
by horseradish peroxidase assay and peroxynitrite scavenging activity assay, indicating that this hybrid is
a good multifunctional drug candidate for the treatment of AD.
Keywords:
Alzheimer’s disease
Cholinesterase inhibitor
Antioxidant
Ó 2013 Elsevier Ltd. All rights reserved.
Peroxynitrite scavenging activity
Alzheimer’s disease (AD) is one of the most common neuro-
degenerative diseases, causing progressive dementia amongst the
ageing population.1 It is characterised by irreversible neuronal
damage that causes memory loss, impaired cognitive functions,
loss of speech and behavioural abnormalities. The main features
of AD include low levels of acetylcholine, amyloid plaques
The brain of a man, which accounts for 2% of the body mass yet
consumes 20% of the total oxygen in a resting individual, is the
most aerobically active organ in the body because of its high
metabolic requirements.11–13 As a consequence of the high oxygen
demand, the brain inevitably induces the generation of large
amounts of ROS, which are considered to be important causative
agents in inducing oxidative damage in cellular structures, which
precedes the appearance of the other pathological hallmarks of
AD.14 Moreover, b-secretase expression is increased by oxidative
stress,15,16 which could explain why hypoxia up-regulates b-secre-
tase because hypoxia leads to local oxidative stress due to meta-
bolic inefficiency.
composed of b-amyloid (Ab), neuro-fibrillary tangles,
s-protein
aggregation,2 oxidative stress and reactive oxygen species (ROS)
formation.3
Because the pathogenesis of AD is complicated and is related to
the abnormality and dysfunction of multiple systems, no ideal drug
has been found for preventing and treating this disease. Currently,
the most efficacious treatment approach for AD is increasing cho-
linergic neurotransmission in the brain by inhibiting cholinesteras-
es (ChEs).4 Clinically, the acetylcholinesterase inhibitors (AChEIs),
such as tacrine, donepezil, rivastigmine and galantamine, have
been approved by the FDA. (In addition, memantine, an uncompet-
itive N-methyl-D-aspartate (NMDA) receptor antagonist, also has
been approved for treatment of moderately to severe AD in
20035,6). However, these drugs show modest improvements in
memory and cognitive function but do not appear to prevent or
slow the progressive neuro-degeneration.7 Moreover, tacrine was
withdrawn from the pharmaceutical market shortly after its ap-
proval due to hepatotoxicity issues.8 Therefore, a more appropriate
approach to addressing the multifaceted nature of AD may be the
development of multi-target directed ligands (MTDLs).9,10
ROS include superoxide (O2Åꢀ), dihydrogen peroxide (H2O2), and
hydroxyl radical (ÅOH).17,18 Another notable class of reactive oxi-
dants are the reactive nitrogen species (RNS), including nitric oxide
radical (ÅNO) and peroxynitrite (ONOOꢀ), which can impair protein
functioning and stability by causing the nitration of tyrosine
residues.19 The nitration of proteins results in the inactivation of
several important mammalian proteins, such as Mn superoxide
dismutase (SOD) and Cu/Zn SOD, which play an important role in
the clearance of ROS. Furthermore, it is believed that a deficiency
of SOD-2 has been shown to accelerate plaque deposition20 and in-
crease tau phosphorylation in AD mice models,21 and overexpres-
sion of Mn-SOD can protect against b-amyloid-induced neuronal
death and improve mitochondrial respiratory function.22 Thus,
drugs that specifically scavenge ROS and/or RNS could be useful
for either the prevention or the treatment of AD.23,24
Tacrine, the first inhibitor of both AChE and butylcholinesterase
(BuChE) approved by the FDA for the treatment of AD, suffers from
therapy-limiting side effects, such as liver toxicity.8 This side effect
⇑
Corresponding authors. Tel./fax: +86 20 39943050.
E-mail address: lixsh@mail.sysu.edu.cn (X. Li).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.10.034

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F. Mao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742
Ebselen, a organoselenium found in 1996, is known as a classic
glutathione peroxidase mimic,32,33 reacts in a rapid reaction with
peroxynitrite,34 possesses antioxidant activity and anti-inflamma-
tory properties35 and exhibits neuroprotective effects in several
preclinical studies.36–38
In our previous work, we designed a series of hybrid molecules
by reacting N-(aminoalkyl)tacrine with salicylic aldehyde or deriv-
atives of 2-aminobenzaldehyde, which was exhibited multifunc-
tional anti-Alzheimer’s disease activities.39 In this work, we
disclose the synthesis and evaluation of a series of tacrine–ebselen
hybrids (6a–6i) by connecting tacrine and ebselen (it also takes
origin from the structures of donepezil–which is thought to be
the most effective drug for AD at present.40,41) as an inhibitor of
AChE and BuChE and a scavenger of hydrogen peroxide and perox-
ynitrite (Fig. 1).
The synthesis of tacrine–ebselen hybrids and their analogues
(6a–6i) is shown in Scheme 1. Intermediate 2 was synthesised
according to the reported procedure.42,43 Simultaneously, diso-
dium diselenide (Na2Se2) reacted with the benzenediazonium salt,
which was prepared from 2-aminobenzoic acid by a diazotisation
reaction, to give 2,20-diselenobisbenzoic acid, that is compound 4.
The subsequent reaction of compound 4 with thionyl chloride
under reflux yielded intermediate 5, which was then reacted with
amine intermediate 2 at room temperature to provide target
compound 6 in moderate yields (45–68%). The purities of all
synthesised compounds were determined by HPLC. It should be
noted that some of the hybrids do not show the chemical shift
value for the secondary amine NH as exchange with CDCl3.
The biological profiles of tacrine–ebselen hybrids (6a–6i) as
inhibitors of ChEs were evaluated in vitro using AChE from the
electric eel and BuChE from equine serum following the methods
of Ellman et al.44 with tacrine and bis(7)-tacrine as the references
compound. As shown in Table 1, all nine compounds proved to be
potent inhibitors of AChE and BuChE (with IC50 values ranging
from 2.55 to 657 nM for AChE and from 2.80 to 38.88 nM for
BuChE). The results also revealed that the length of the alkyl link-
age affects the inhibition of both AChE and BuChE. In addition to
Figure 1. Synthesis strategy for compounds 6a–6i (Table 1).
can be prevented by a free radical scavenger, such as anethole dith-
iolethione glutathione or vitamin E.25,26 Thus, tacrine derivatives
endowed with additional antioxidant properties might be benefi-
cial for reducing toxicity.27 Recently, this strategy has led to the
discovery of several anti-AD drug candidates, for example,
tacrine–ferulic acid hybrids,28 tacrineꢀmelatonin hybrids,29
tacrine-8-hydroxyquinoline hybrids,27 and tacrine-4-oxo-4H-chro-
mene hybrids.30
Selenium, an essential antioxidant nutrient for humans and
other higher animal species, is abundant in fish and is an ingredi-
ent of glutathione peroxidase, which is an important antioxidant
enzyme that converts H2O2 to water via GSH. Recently, a study
showed that feeding a selenium-deficient diet to Tg2576 trans-
genic mice resulted in more than a twofold increase in the total
area of the Ab plaques compared to that of mice with a sele-
nium-adequate diet.31 Thus, antioxidative selenoproteins and/or
selenium-containing species are one of the factors that may affect
the risk of cognitive decline.
Scheme 1. Synthesis of tacrine–ebselen hybrids. Reagents and conditions: (a) cyclohexanone, POCl3, 0 °C ? reflux; (b) diamine, KI, 1-pentanol, 160 °C; (c) HCl, H2O, NaNO2;
(d) Na2Se2; (e) SOCl2, DMFcat, reflux; (f) 2a–2h (Et)3N, CH2Cl2, rt.

F. Mao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742
6739
Table 1
In vitro inhibition and selectivity of AChE and BuChE by tacrine, bis(7)-tacrine and tacrine–ebselen hybrids 6a–6i
R1
O
R2
N
Se
HN
N
n
Compound
R1
R2
n
IC50a(nM)
Selectivity for AChEd
AChEb
657 57.27
423 35.36
152 5.67
70.77 5.48
6.32 0.56
BuChEc
6a
6b
6c
6d
6e
H
H
H
H
H
H
H
H
H
H
1
2
3
4
5
38.88 0.82
28.53 2.48
4.94 0.11
4.52 0.22
4.37 0.28
0.06
0.07
0.03
0.06
0.69
(19.52 1.54)e
26.49 2.15
24.14 1.35
31.98 3.17
2.55 0.24
6f
H
H
H
OMe
H
H
H
OMe
6
7
8
5
13.97 1.02
16.55 0.72
25.77 2.32
2.80 0.21
0.53
0.69
0.81
1.10
6g
6h
6i
(15.13 1.36)e
109 10.31
(285 29.4)e
0.17 0.016
Tacrine
—
—
—
—
—
—
15.8 1.32
4.08 0.27
0.14
Bis(7)-tacrine
24.00
a
b
c
Mean SD of at least three independent measurements.
AChE from the electric eel.
BuChE from equine serum.
Selectivity for AChE = IC50 (BuChE)/IC50 (AChE).
Values in parentheses were IC50 against human AChE.
d
e
6a–6c, which possess two, three and four carbon spacers between
the tacrine and ebselen moiety and resulted in lower AChE inhibi-
tory activities (6a, IC50 = 657 nM, 6b, 423 nM, 6c, 152 nM, tacrine,
IC50 = 109 nM), all other tacrine–ebselen hybrids without substi-
tuted groups on ebselen moiety containing six to nine carbon
spacers provided stronger AChE inhibitory activities than tacrine
(6.32–70.77 nM). Compound 6e gave the best result (6.32 nM),
which implies that the six-alkyl linkage may be the optimal length.
In addition, the substituent groups in ebselen moiety also have
some effect on the AChE inhibitory activities; in particular, com-
pound 6i, whose R1 and R2 are methoxy groups, provided 2.55
nM of AChE inhibitory activity.
Generally, the BuChE inhibitory activity showed a similar trend
as AChE. As shown in Table 1, all the synthetic hybrids with varia-
tions in alkyl chain length exhibited good to excellent BuChE inhib-
itory activities, and compound 6e, which has a six alkyl chain
between tacrine and ebselen moiety, provided the best result with
an IC50 value of 4.37 nM. It was interesting that compound 6i,
which was the best inhibitor of AChE, also exhibited the best
BuChE inhibitory activity with an IC50 value of 2.80 nM. Some
evidence suggests that the inhibition of BuChE could raise ACh lev-
els and improve cognition in AD patients.45,46 Therefore, dual
inhibitors of AChE and BuChE may have additional benefits for
the treatment of AD.
Some evidence suggests that AChE interacts with Ab and pro-
motes amyloid fibril formation through a pool of amino acids lo-
cated in the proximity of the peripheral anionic binding site
(PAS) of the enzyme.47 Peripheral and mixed-type AChE inhibitors,
which bind to both the catalytic active site (CAS) and the periphe-
ral anionic site (PAS) of AChE, might represent a new therapeutic
option for Alzheimer’s disease.48 To study the inhibitory mecha-
nism of this class of tacrine–ebselen hybrids, we chose compound
6i, which provided the most potent and a good balance of AChE/
BuChE inhibitory activity, for further kinetic studies. As shown in
Figure 2, the Lineweaver–Burk plots of 6i against AChE revealed
an increasing slope and an increasing intercept with higher inhib-
itor concentrations, indicating mixed-type inhibitory behaviour for
compound 6i in the presence of AChE.
To explore the binding modes of tacrine–ebselen hybrids to the
active site of AChE, molecular docking simulations of compound 6i
were performed using the CDOCKER program in the Discovery Studio
2.1 software. In view of the similarities between the bis-tacrine
and tacrine–ebselen hybrids, the simulations were based on the
crystal structure of the TcAChE-bis-(7)-tacrine complex (PDB entry
2CMF), and the full images of the interaction between 6i and
TcAChE are shown in Figure 3a and b. The binding model suggested
that the tacrine fragment of compound 6i bound near the bottom
Figure 2. Lineweaver–Burk plot for the inhibition of acetylcholinesterase hydro-
lysis of ATCh by compound 6i. Reciprocal plots of initial velocity and substrate
concentrations (50–500
lM) are reported. Lines were derived from a weighted
of the gorge, interacting with CAS via a parallel
p–p interaction
least-squares analysis of the data points.

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F. Mao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742
Figure 3. Docking models of the compound–enzyme complex. (a) The binding pattern of compound 6i (pink stick) to TcAChE. (b) Representation of compound 6i interacting
with residues at the binding site of TcAChE, highlighting the protein residues that participate in the main interactions with the inhibitor.
between Trp84 (4.0 Å) and Phe330 (4.0 Å) in a ‘sandwich’ form. The
benzene ring of 1,2-benzisoselenazol-3(2H)-one interacted with
middle of the gorge, interacting with Tyr334, Tyr70 and Asp72
through hydrophobic interactions.
Trp279 of the PAS through
p
–
p
stacking with a distance of 4.8 Å.
Cells have protective mechanisms against oxidative damage
through antioxidant-scavenging enzymes, for example, superoxide
dismutases, catalases, and glutathione peroxidases. However, oxi-
dative stress occurs when the balance between the amount of
The carbonyl oxygen of 1,2-benzisoselenazol-3(2H)-one linked
with the hydroxy group of Tyr70 by a hydrogen bond (3.5 Å). In
addition, the methylene moiety of the long chain bound to the

F. Mao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742
6741
Figure 4. Scavenging effects on hydrogen peroxide through the horseradish
peroxidase assay, [H2O2] = 100 M. Values are expressed as the mean SD from
l
at least three independent measurements.
Figure 6. Cell viability determined by MTT after treatment of HSC with different
concentrations of tacrine and compound 6i. Data were subjected to one-way
ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism 5.0
Software (levels of significance ⁄p <0.05;⁄⁄p <0.01;⁄⁄⁄p <0.001).
ROS and/or RNS and the antioxidant defence system is affected.49
Therefore, antioxidants that can scavenge ROS should benefit AD
patients. To evaluate the antioxidant activity of the tacrine–ebse-
len hybrids, we chose 6e and 6i, which exhibited good ChE inhib-
itory activities, for the hydrogen peroxide scavenging activity
assay by the horseradish peroxidase assay method50 with ebselen
as the reference compound. The data summarised in Figure 4
indicated that ebselen, 6e and 6i had similar hydrogen peroxide
scavenging activities at all the tested concentrations in the pres-
at 542 nM (e = 24,000/M/cm). First, peroxynitrite was prepared
by the method described by Radi et al.53 Consumption of PR in
the presence and absence of the test compounds was measured
over a range of peroxynitrite concentrations (0–62.5
lM). The ratio
D0/DA is equivalent to [PR]0/D[PR]A, where D0 and DA are the
D
reaction stoichiometries of peroxynitrite with PR in the absence
and presence of the test compound, respectively. The ratio kA/kPR
(Table 2), which could be calculated from the slope of the straight
line plot of D0/DA against [compound]/[PR], represented the activ-
ity of the compounds scavenging peroxynitrite, where a greater ra-
tio of kA/kPR is related to more potent peroxynitrite scavenging
activity of the tested compounds. As shown in Table 2, the ta-
crine–ebselen hybrids 6e and 6i exhibited higher kA/kPR values of
1.17 and 1.26 times that of ebselen, respectively.
To evaluate the hepatotoxicity of the tacrine–ebselen hybrids
compared to tacrine, the effect of compound 6i on human hepatic
stellate cells (HSC) was investigated54 by the colorimetric MTT
[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]
assay.55 The results in Figure 6 indicated that at a concentration
ence of 100
no activity at 100
We also conducted the test in the presence of 25
peroxide with 25 M (black bars) and 12.5 M (red bars) of the
lM hydrogen peroxide. As expected, tacrine had nearly
lM.
l
M hydrogen
l
l
compounds (Fig. 5). All of the three tested compounds, ebselen,
6e and 6i, could scavenge all most of the hydrogen peroxide at
the 25
ide scavenging activities were 89.7%, 70.0% and 83.7%, respectively.
When the concentration of antioxidants was 12.5 M, approxi-
mately half of the hydrogen peroxide was scavenged.
lM concentration, and the percentages of hydrogen perox-
l
The peroxynitrite scavenging activities of the tacrine–ebselen
hybrids were determined according to the method reported by
Balavoine51 and Hsu and co-workers.52 Briefly, peroxynitrite in-
duced the bleaching of pyrogallol red dye, which was measured
of 20
lM, tacrine shows slightly higher cell viability (lower toxic-
ity). However, at the concentration of 10 and 5
l
M, compound 6i
exhibited obviously higher cell viability (lower toxicity) than ta-
crine. Considering its AChE inhibitory activity is much higher than
that of tacrine, compound 6i may overcome the weakness of
hepatotoxicity of tacrine in a certain extent.
In conclusion, we have designed, synthesised and evaluated a
series of tacrine–ebselen hybrids as multifunctional anti-AD agents
with cholinesterase and antioxidant activities. Most of these com-
pounds were potent inhibitors of AChE and BuChE. Among them,
compound 6i exhibited superior activity and a better balance of
AChE and BuChE activities than tacrine. Horseradish peroxidase
Table 2
Ratio of the rate constants for the reaction of test compounds and pyrogallol red with
peroxynitrite
a
Compound
kA/kPR
Activity relative to ebselen
Ebselen
6e
6i
0.436 0.073
0.511 0.089
0.55 0.0012
1.00
1.17
1.26
Figure 5. Scavenging effects on hydrogen peroxide through the horseradish
a
Results are means SD of three experiments and calculations were made using
the concentration of 50 M for the peroxynitrite.
peroxidase assay, [H2O2] = 25
lM. Values are expressed as the mean SD from at
l
least three independent measurements.

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F. Mao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742
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