Synthesis and evaluation of multi-target-directed ligands against Alzheimer's disease based on the fusion of donepezil and ebselen

Article abstract of DOI:10.1021/jm401047q

A novel series of compounds obtained by fusing the cholinesterase inhibitor donepezil and the antioxidant ebselen were designed as multi-target-directed ligands against Alzheimer's disease. An in vitro assay showed that some of these molecules did not exhibit highly potent cholinesterase inhibitory activity but did have various other ebselen-related pharmacological effects. Among the molecules, compound 7d, one of the most potent acetylcholinesterase inhibitors (IC₅₀ values of 0.042 μM for Electrophorus electricus acetylcholinesterase and 0.097 μM for human acetylcholinesterase), was found to be a strong butyrylcholinesterase inhibitor (IC₅₀ = 1.586 μM), to possess rapid H₂O₂ and peroxynitrite scavenging activity and glutathione peroxidase-like activity (ν₀ = 123.5 μM min^-1), and to be a substrate of mammalian TrxR. A toxicity test in mice showed no acute toxicity at doses of up to 2000 mg/kg. According to an in vitro blood-brain barrier model, 7d is able to penetrate the central nervous system.

Full text of DOI:10.1021/jm401047q

Article
pubs.acs.org/jmc
Synthesis and Evaluation of Multi-Target-Directed Ligands against
Alzheimer’s Disease Based on the Fusion of Donepezil and Ebselen
Zonghua Luo, Jianfei Sheng, Yang Sun, Chuanjun Lu, Jun Yan, Anqiu Liu, Hai-bin Luo, Ling Huang,*
and Xingshu Li*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
S
* Supporting Information
ABSTRACT: A novel series of compounds obtained by fusing
the cholinesterase inhibitor donepezil and the antioxidant
ebselen were designed as multi-target-directed ligands against
Alzheimer’s disease. An in vitro assay showed that some of
these molecules did not exhibit highly potent cholinesterase
inhibitory activity but did have various other ebselen-related
pharmacological effects. Among the molecules, compound 7d,
one of the most potent acetylcholinesterase inhibitors (IC50
values of 0.042 μM for Electrophorus electricus acetylcholines-
terase and 0.097 μM for human acetylcholinesterase), was
found to be a strong butyrylcholinesterase inhibitor (IC50
=
1.586 μM), to possess rapid H2O2 and peroxynitrite scavenging activity and glutathione peroxidase-like activity (ν0 = 123.5 μM
min−1), and to be a substrate of mammalian TrxR. A toxicity test in mice showed no acute toxicity at doses of up to 2000 mg/kg.
According to an in vitro blood−brain barrier model, 7d is able to penetrate the central nervous system.
become heightened.6−11 Given the complex nature of AD and
INTRODUCTION
■
the fact that a single drug acting on a specific target (AChE)
may have undesirable clinical effects, a variety of MTDLs acting
on very diverse targets have been developed by many research
groups.12−15
Alzheimer’s disease (AD), the most common form of dementia
associated with progressive loss of memory, speech, and
recognition, occurs most frequently in elderly people. It has
been estimated that 36 million people were living with
dementia in the world in 2010 and that the number will
double every 20 years, leading to more than 115 million people
with AD in 2050.1 The etiology of AD is not fully known, but
some hallmarks, such as low levels of acetylcholine, β-amyloid
(Aβ) deposits, τ-protein aggregation, oxidative stress, inflam-
mation, and dyshomeostasis of biometals, are considered to
play important roles in the pathogenesis of this disease.2 Many
different approaches to the treatment of AD have been
developed over the past several decades. At present, there is
no drug that can definitively cure Alzheimer’s disease, and the
primary therapeutic options currently approved by the U.S.
Food and Drug Administration for the treatment of AD are
acetylcholinesterase inhibitors (AChEIs), namely, tacrine,
donepezil, rivastigmine, and galantamine. Among them,
donepezil is the most effective pharmacological agent for AD
treatment.3,4 However, it is effective in reversing the symptoms
for only a short period of time.
Selenium (Se) is an essential trace mineral nutrient with
multiple roles in the growth and function of living animal cells.
Twenty-five selenoproteins in the human body exert specific
biological functions.16 Moreover, the mineral is known to
provide protection from free radical-induced cell damage.17 In
humans, selenium levels decrease with age, and there are
considerable data showing that selenium might play different
roles in the progression of AD.18,19 There has been a
heightened interest in the role of this trace element in health
and neurological disorders in recent years.20−23 Some animal
experiments indicate that organoselenium has a higher
biodisposability and biological activity than Se in inorganic
compounds.24 Recently, diphenyl diselenide [(PhSe)2] and
p,p′-methoxyldiphenyl diselenide [(MeOPhSe)2] were studied
for the treatment of a sporadic Alzheimer’s-type dementia in a
rat model, and the results demonstrated that (MeOPhSe)2
dietary supplementation reversed streptozotocin-induced mem-
ory impairment.25,26
In recent years, accumulated evidence has indicated that the
most successful treatment strategy will likely incorporate a
sequential, multifactorial approach to this multifaceted disease,
termed the multi-target-directed ligand (MTDL) strategy.5 The
“one-molecule, multiple-targets” paradigm is effective in
treating complex diseases because of the ability of the drug to
interact with multiple targets responsible for disease patho-
genesis. Thus, interest in the development of MTDLs has
Ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] is a
lipid-soluble cyclic selenenamide and a classic example of a
glutathione peroxidase (GPx) mimic,27 and it protects cells by
catalyzing the reduction of peroxides with glutathione. Ebselen
Received: July 11, 2013
Published: October 25, 2013
© 2013 American Chemical Society
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has also been demonstrated to be a substrate for human
thioredoxin reductase.28−30 It inhibits nonenzymatic and
enzymatic lipid peroxidation in cells and has anti-inflammatory
activity in various animal models. Because of its anti-
inflammatory activity, ebselen has been used in the treatment
of patients with acute ischemic stroke or delayed neurological
deficits after aneurismal subarachnoid hemorrhage. A recent
study indicates that ebselen could be used as a potential anti-
AD agent on the basis of its ability to inhibit iron-induced tau
phosphorylation.31
Because the pharmacological effects of ebselen, including its
antioxidant and anti-inflammatory activities, are closely related
to the etiology of AD, our design principle involved the fusion
of the important pharmacophores of donepezil and ebselen in a
new molecule, “selenpezil”, without the major structural
modifications (Figure 1) like those of other MTDLs.32 The
hybrid compounds possess strong AChE inhibitory activity and
many of the pharmacological effects of ebselen.
chloride and the amine intermediates efficiently to provide the
target compounds in good yields.
One of the possible metabolic products of selenide in vivo is
selenoxide. To evaluate the biological activity of the metabolic
products, we prepared selenoxide 10 by the oxidation of 7d
with 1.5 equiv of H2O2 at room temperature (Scheme 2).
In Vitro Inhibition Studies of AChE and BuChE. To
evaluate the potential application of target compounds 5a−d,
6a−d, 7a−d, 8, 9, and 10 for the treatment of AD, we
determined their levels of AChE inhibitory activity by the
method of Ellman et al.,33 using donepezil and ebselen as
reference standards (Table 1). Among these compounds, 7d, 8,
and 9, in which the piperidine groups were linked to the 5,6-
dimethoxybenzoselenazol-3(2H)-one moiety by two-, three-,
and four-carbon spacers, respectively, exhibited more potent
inhibition of AChE than other analogues (IC50 values of 0.042,
0.072, and 0.038 μM for 7d, 8, and 9, respectively). Structure−
activity relationship analysis showed that the methoxy group in
the benzoselenazol-3(2H)-one moiety is necessary for inhib-
itory activity. Compounds 7a, 7b, and 7c, whose methoxy
groups (R1 and R2) were replaced by H, F, and Cl, respectively,
exhibited weaker inhibitory activities than 7d (7a, R1 = R2 = H,
IC50 = 0.382 μM; 7b, R1 = H, R2 = F, IC50 = 0.224 μM; 7c, R1 =
H, R2 = Cl, IC50 = 0.206 μM; 7d, R1 = R2 = OCH3, IC50
=
0.042 μM). The length of the alkylene chain, especially with
zero to two carbons, is also important for inhibitory activity.
Compounds 5d, 6d, and 7d, with or without the one- and two-
carbon spacers between the 5,6-dimethoxybenzoselenazol-
3(2H)-one moiety and the piperidine group, exhibited IC50
values of 11.84, 4.231,M and 0.042 μM, respectively. The
inhibitory activities of compounds 7d, 8, 9, and 10 against
human AChE were determined using same method, with
donepezil as the reference. Via comparison of the results with
AChE from the electric eel, slightly weaker activities were
observed (Table ) (IC50 values of 0.097, 0.231, 0.103, and 0.163
μM for 7d, 8, 9, and 10, respectively).
Figure 1. Drug design strategy for multi-target-directed ligands.
RESULTS AND DISCUSSION
■
In recent years, numerous studies34−37 have indicated that
butyrylcholinesterase (BuChE) levels are unchanged or even
increase in advanced AD while AChE activity in certain brain
regions decreases. These studies implied that a balance of
inhibition of both AChE and BuChE may be beneficial in
treating the cognitive deficits observed in AD. Therefore, the
inhibitory activity of these compounds against BuChE was also
evaluated, and the results are listed in Table 1. The majority of
the compounds exhibited good BuChE inhibitory activities.
Chemistry. Amine intermediates 4a−e were obtained as
reported in the literature (see the Supporting Information), and
target compounds 5a−d, 6a−d, 7a−d, 8, and 9 were
synthesized according to the synthetic approaches in Scheme 1.
With various amine intermediates available, the target
molecules were easily synthesized by the route shown in
Scheme 1. The diazotization of o-aminobenzoic acid or its
analogues and their subsequent reaction with Na2Se2 provided
diselenium intermediate 2. Compound 2 reacted with thionyl
a
Scheme 1. Synthesis of Target Compounds 4a−d, 5a−d, 6a−d, 7, and 8
a
Reagents and conditions: (a) NaNO2, HCl; (b) Na2Se2; (c) SOCl2, reflux; (d) (Et)3N, CH2Cl2.
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a
Scheme 2. Synthesis of Selenoxide 10
a
Reagents and conditions: (a) 1.5 equiv of H2O2, CH2Cl2, room temperature.
Table 1. Inhibition of AChE from Electrophorus electricus (eeAChE) and Human Erythrocytes (hAChE), BuChE from Equine
Serum, and Human Recombinant AChE-Induced Aβ Aggregation by the Target Compounds
IC50 (μM)
IC50 (μM) SD
SD
selectivity for
% inhibition of AChE-induced Aβ1−40
a
b
c
d
f
compd
5a
R1
R2
n
eeAChE
eqBuChE
AChE
hAChE
e
aggregation
e
H
H
H
H
F
0
0
0
0
1
1
1
1
2
2
2
2
3
4
17.58 1.3
8.316 0.446
10.58 0.762
21.52 1.445
9.463 0.768
3.628 0.015
3.461 0.150
2.295 0.085
3.921 0.096
2.132 0.166
3.668 0.100
2.346 0.023
1.586 0.075
1.503 0.150
0.517 0.004
1.362 0.073
3.365 0.058
0.015 0.002
10.368 0.035
0.47
0.54
2.13
0.80
0.18
0.28
0.37
0.93
5.58
16.4
11.4
36.7
20.9
13.6
31.7
88.6
0.13
0.25
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
e
e
e
e
e
e
e
e
e
e
e
5b
19.72 1.4
nd
e
5c
Cl
10.10 0.3
nd
e
5d
OCH3
H
OCH3
H
11.84 0.8
nd
e
6a
19.80 0.7
nd
e
6b
H
F
12.27 0.4
nd
e
6c
H
Cl
6.160 0.5
nd
e
6d
OCH3
H
OCH3
H
4.231 0.10
0.382 0.012
0.224 0.016
0.206 0.026
0.042 0.003
0.072 0.002
0.038 0.002
0.043 0.001
0.038 0.003
0.113 0.012
41.01 0.672
nd
e
7a
nd
e
7b
H
F
nd
e
7c
H
Cl
nd
7d
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
0.097 0.007
0.231 0.03
0.103 0.01
0.163 0.01
0.011 0.001
0.534 0.022
21.4 2.0
26.3 4.0
46.3 9.3
8
9
e
10
nd
donepezil
tacrine
ebselen
21.1 3.3
10.3 2.8
e
e
nd
nd
a
b
The 50% inhibitory concentration (means SD of three experiments) of AChE from electric eel. The 50% inhibitory concentration (means SD
c
d
of three experiments) of BuChE from equine serum. Selectivity for AChE = IC50(eqBuChE)/IC50(eeAChE). The 50% inhibitory concentration
(means SD of three experiments) of AChE from human erythrocytes. Not determined. Inhibition of human recombinant AChE-induced Aβ1−40
aggregation produced by the tested compound at 100 μM.
e
f
Compound 9, which had the best AChE inhibitory activity, also
showed the best BuChE inhibitory activity (IC50 value of 0.517
μM). The structure−activity relationship between the length of
the alkylene chain and AChE inhibitory activity was also
observed for BuChE inhibitory activity. The effect of the
methoxy group observed with AChE inhibitory activity,
however, was not apparent with BuChE inhibitory activity. It
is notable that compound 10, one of the possible metabolic
products of 7d, exhibited inhibitions similar to those of AChE
and BuChE (IC50 values of 0.042 μM for AChE and 1.586 μM
for BuChE).
Kinetic Characterization of AChE Inhibition. Consider-
ing the requirement of the calculated logarithm of the octanol−
water partition coefficient (clogP) in Lipinski’s rules,
compound 7d (clogP = 4.611), one of the most potent
AChE inhibitors, was selected to further study the inhibitory
mechanism of AChE. The graphical presentation of the steady-
state inhibition data of compound 7d for AChE is shown in
Figure 2. The intersection point of the Lineweaver−Burk
reciprocal plot fell in the second quadrant, which is indicative of
mixed-type inhibition. The competitive inhibition constant (Ki)
of 7d is 0.017 0.003 μM.
Docking Study of Compounds 7d and 10 with AChE.
To evaluate the binding modes of the compounds with respect
to AChE, compound 7d was selected for docking simulations
using the CDOCKER program in Discovery Studio version 2.1
based on the X-ray crystal structure of the complex of the
Torpedo californica enzyme (TcAChE) and donepezil (Protein
Data Bank entry 1EVE).38 The position of compound 7d with
respect to the key residues in the binding site is shown in
Figure 3A. The results suggest that 7d binds in the central
catalytic pocket and the peripheral anionic site (PAS) along the
active site gorge of the enzyme with its three major functional
groups, the phenyl ring and the carbonyl group on the
dimethoxybenzo[d][1,2]selenazol-3(2H)-one moiety and the
phenyl ring of the N-benzyl group moiety. The phenyl ring with
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7d and 10 in AChE indicates that the selenoxide group does
not affect the binding mode, which agrees with the in vitro
activity assay (Figure 3B).
Inhibition of Human Recombinant AChE-Induced
Aβ1−40 Aggregation. Mounting evidence39,40 has indicated
that AChE colocalizes with Aβ in senile plaques, promoting the
assembly of Aβ into fibrils and accelerating Aβ peptide
deposition. It has been suggested that AChE achieves its
aggregation-promoting action through direct binding with Aβ
via its peripheral binding site. Inhibition of the peripheral site
might prevent the Aβ peptide aggregation induced by AChE.41
On the basis of the results of the kinetic and docking studies,
compound 7d could act as a dual-binding site AChE inhibitor.
Therefore, we selected 7d, 8, and 9 for evaluation of their
ability to inhibit Aβ1−40 aggregation induced by human
recombinant AChE using a thioflavin T-based fluorometric
assay42 (Table 1). The results showed that the selected
compounds were more effective inhibitors of AChE-induced
Aβ1−40 aggregation than tacrine (10.3%) and donepezil
(21.1%), with 21.4−46.3% inhibition at 100 μM. The results
also showed that a longer linker between the 5,6-dimethox-
ybenzoselenazol-3(2H)-one moiety and the piperidine group
was favorable for inhibition.
Figure 2. Kinetic study of the mechanism of eeAChE inhibition by
compound 7d. Overlaid Lineweaver−Burk reciprocal plots of the
AChE initial velocity at increasing substrate concentrations (0.1−1
mM) in the absence of inhibitor and in the presence of 7d are shown.
Lines were derived from a weighted least-squares analysis of the data
points. The experimental data are the means
SD of three
independent experiments.
Glutathione Peroxidase-like Activity. Glutathione per-
oxidase (GPx) plays a crucial role in the detoxification of
hydroperoxides in aerobic living organisms. It catalytically
reduces harmful peroxides by reducing glutathione (GSH) or
other thiols and protects the lipid membranes and other cellular
components from oxidative damage.
The GPx-like catalytic activity of compounds 7d and 8−10
was studied using GSH as a thiol cofactor and hydrogen
peroxide (H2O2) as the substrate. The initial rates (ν0) were
determined for the reduction of peroxide at a concentration of
80 μM for all the tested compounds. The results listed in Table
2 showed that 7d (123.5 μM min−1) exhibited GPx-like activity
Table 2. Initial Rates for the Reduction of H2O2 by GSH in
the Presence of Ebselen, 7d, 8, 9, 10, and Donepezil
ab
,
compd
ν0 (μM min−1
49.5 2.6
)
b
control
7d
123.5 10.9
86.1 3.0
80.1 2.8
97.1 4.3
121.3 1.1
46.1 3.2
8
9
10
ebselen
donepezil
a
Reactions were conducted in phosphate buffer (100 mM) (pH 7.5)
with 1 mM ethylenediaminetetraacetate (EDTA), 2 mM GSH, 0.4
mM NADPH, 1.3 unit/mL glutathione reductase (GR), 80 μM
selenium compounds, and 1.6 mM H2O2. The control values were
obtained from the reduction of H2O2 by GSH in the absence of the
compound. All values were determined in triplicate for the initial 10 s,
and the average values with the standard deviation are reported.
Figure 3. Docking models of the compound−enzyme complex. (A)
Representation of compound 7d docked into the binding site of AChE
highlighting the protein residues that form the main interactions with
the inhibitor. The hydrogen bonding interaction between the ligand
and Arg289 is shown as a green line. (B) Overlap of 7d and 10 docked
with AChE.
b
the dimethoxy group is stacked above the peripheral anionic
site (PAS) residue Trp279 (4.327 Å). The piperidine ring is
located on the aromatic gorge connecting the PAS and CAS
(catalytic binding site). The carbonyl oxygen of 7d interacts
with Phe288 via a hydrogen bond (NH of Phe288 to O14 of
7d). The phenyl ring of the N-benzyl forms a π-stacking
interaction with the indole ring of Trp84 (4.297 Å). A docking
simulation of selenoxide 10 was also performed. The overlap of
similar to that of ebselen (121.3 μM min−1) but compounds 8,
9, and 10 had lower activities than ebselen. GPx-like activity
was associated with the length of the linker between the ebselen
moiety and benzylpiperidine (7d, n = 2, ν0 = 123.5 μM min−1;
8, n = 3, ν0 = 86.1 μM min−1; 9, n = 3, ν0 = 80.1 μM min−1).
Not surprisingly, selenoxide 10 still exhibited potent GPx-like
activity (97.1 μM min−1), but donepezil (46.1 μM min−1)
exhibited no activity compared to the control (49.5 μM min−1).
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Hydrogen Peroxide Scavenging Activity. Oxidative
stress is an early feature of AD pathology, and during the
aging process, oxidation-induced apoptosis and increased
activity of β- and γ-secretases may contribute to the generation
of Aβ. The antioxidant protection provided by removing
reactive oxygen species (ROS), such as hydrogen peroxide, is
important in older individuals, especially AD patients, because
the endogenous antioxidant protection system declines rapidly
with age. To evaluate the antioxidant activity of the donepezil
and ebselen derivatives, compounds 7d, 8, 9, and 10 were
selected for the ferrous ion oxidation−xylenol orange (FOX)
assay,43 and donepezil and ebselen were used as reference
compounds. The results in Figure 4 indicate that ebselen and
k
TC/kEB ratio could be calculated from the slope of the line
generated by plotting Δ[TC]0/Δ[EB] against [TC]/[EB]. For
the sake of simplicity, initial concentrations [TC]0 and [EB]0
were used in the calculations. In this work, instead of Δ[EB],
we measured the stoichiometry D = Δ[EB]/Δ[NaOONO3−]
to avoid the effect of slight deviations in peroxynitrite
concentration. The D0/DTC ratio is equivalent to Δ[TC]0/
Δ[EB]a, where D0 and DTC are the reaction stoichiometries in
the absence and presence of the test compound, respectively.
The D0/DTC values for different concentrations of antioxidant
were plotted against [TC]0/[EB]0 (Figure 5). Antioxidant
Figure 5. D0/DTC for different concentrations of test compounds
plotted vs [TC]/[EB]. The reaction conditions were as follows: room
temperature, phosphate buffer (50 mM), pH 7.4, and [EB]0 = 20 μM.
P < 0.01 comparing 7d and ebselen with donepezil; P < 0.05
comparing 10 with donepezil.
Figure 4. Scavenging effects on hydrogen peroxide in the FOX assay
(100 μM H2O2). Values reported are means
SD of three
independent experiments. P < 0.01 in comparison to donepezil.
7d show similar scavenging activities for hydrogen peroxide at
high concentrations, but 7d showed improved scavenging
activity at lower concentrations. Compounds 8 and 9 showed
scavenging activities lower than that of ebselen. Moreover,
selenoxide 10 demonstrated minimal antioxidant activity.
Donepezil showed very weak activity, even at high concen-
trations.
activity was measured as the slope of the straight line (kTC/kEB).
As indicated, compound 7d exhibited the most potent
antioxidant activity of all of the test compounds (kTC/kEB
values of 3.28 0.11, 2.42 0.08, and 0.21 0.01 for 7d,
ebselen, and 10, respectively). In contrast, donepezil showed
negligible activity under the same conditions (kTC/kEB = 0.04
0.004).
Compound 7d Is a Substrate of TrxR. Thioredoxin
reductase (TrxR) is a selenoenzyme that catalyzes the reduction
of thioredoxin (Trx) using NADPH as an electron source.
Maintaining full TrxR activity by adequate dietary selenium
intake has been proposed for the prevention of several
cardiovascular and neurological disorders.52 Organoselenium
compounds can be metabolized by TrxR to form selenol
intermediates28,29,53,54 that can imitate the function of the
antioxidant selenoenzymes.55,56
For this study, compound 7d was used according to the
method of Zhao and Holmgren28 to investigate the ability of
donepezil−ebselen hybrids to serve as substrates of TrxR.
Ebselen and donepezil were used as reference compounds. The
measurements were performed in buffer containing 50 mM
Tris-HCl, 1 mM EDTA, 100 μM NADPH, and 15 μM test
compound. The reactions were started via the addition of TrxR
to a final volume of 0.2 mL. The results shown in Figure 6
indicate that compound 7d and ebselen are substrates of
mammalian TrxR but that donepezil is not. Compound 7d and
ebselen show similar activities, as shown in Table 3.
Peroxynitrite Scavenging Activity. Peroxynitrite, which
is generated by the diffusion-limited reaction of nitric oxide and
the superoxide anion, has been detected in macrophages44 and
endothelial cells.45 Peroxynitrite is a strong oxidizing and
nitrating agent. Oxidation reactions include DNA damage
leading to base modification and mutations; single- and double-
stranded breaks;46,47 one- or two-electron oxidations of
sulfhydryl leading to thiyl radical formation and transmission,
which results in the depletion of thiol pools;48 lipid
peroxidation; and the hydroxylation of phenols.49,50 Antiox-
idants that may provide additional endogenous cellular defenses
against this species are of obvious interest.
The antioxidant activities of test compounds against
peroxynitrite were determined using the method reported by
Balavoineet et al.51 Peroxynitrite formation, resulting in the
bleaching of Evans blue (EB) dye, was measured at 608 nm (ε
= 70000 M−1 cm−1). Consumption of Evans blue dye, with or
without different concentrations of the test compound, was the
basis for the calculated amount of peroxynitrite. D0 and DTC are
the stoichiometries for the reaction of peroxynitrite with Evans
blue dye without and with the test compound, respectively. The
In Vitro Blood−Brain Barrier Permeation Assay. Brain
penetration is a major requirement for successful CNS drugs.
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Table 4. Permeabilities in the PAMPA-BBB Assay for the
Selected Compounds and Their Predicted Penetration into
the CNS
a
b
compd
Pe (×10−6 cm s−1
)
prediction
7d
8
8.6 0.8
10.3 0.8
15.8 1.0
6.0 0.3
CNS+
CNS+
CNS+
CNS+
9
chlorpromazine
a
Compounds were dissolved in DMSO at a concentration of 5 mg/mL
b
and diluted with a PBS/EtOH mixture (70:30). Values are expressed
as the means SD of three independent experiments.
for any abnormal behavior and mortality changes, intermit-
tently for the next 24 h, and occasionally thereafter for 14 days
for the onset of any delayed effects. All mice were sacrificed on
the 14th day after drug administration and examined
macroscopically for possible damage to the heart, liver, and
kidneys. The results showed that the animals treated with
compound 7d did not show any acute toxicity and mortality
either immediately or during the post-treatment period.
Furthermore, no significant abnormal changes were observed
during the experimental period in terms of water or food
consumption or body weight. Therefore, compound 7d proved
to be nontoxic and well tolerated at doses of up to 2000 mg/kg.
Figure 6. Reduction of compounds by NADPH catalyzed by
mammalian thioredoxin reductase (TrxR). Test compounds at
concentrations of 15 μM in 0.2 mL of 50 mM Tris-HCl and 1 mM
EDTA (pH 7.5), containing 100 μM NADPH, were mixed with
thioredoxin reductase from rat liver, and the absorbance at 340 nm
(A340) was measured against an identical blank with enzyme but
without test compounds.
Table 3. Comparative Reduction of 7d, Donepezil, and
Ebselen by Thioredoxin Reductase (TrxR) from Rat Liver
CONCLUSION
■
a
b
compd
ΔAbs340 (NADPH oxidation/min)
activity
In conclusion, our study involved the synthesis of a new series
of multi-target-directed ligands for the treatment of AD that
were produced by the fusion of donepezil and ebselen. These
compounds were evaluated as potential multivalent inhibitors
of ChE, as superfast antioxidant agents against H2O2 and
peroxynitrite, and as GPx mimics. Among the synthesized
compounds, compound 7d exhibited very good inhibitory
potency toward hAChE (IC50 = 97 nM) and good GPx-like
activity (123.5 μM min−1). This compound is able to penetrate
the CNS, according to an in vitro blood−brain barrier model,
and toxicity tests in mice showed that 7d has no acute toxicity
at doses of up to 2000 mg/kg. These results showed that
compound 7d is a potential lead compound for the treatment
of AD. Further investigations of AD therapeutic candidates
based on these results are in progress.
control
donepezil
7d
0.0158 0.0014
0.019 0.0017
0.0514 0.0021
0.0517 0.0033
1.0 0.09
1.20 0.11
3.25 0.13
3.27 0.21
ebselen
a
Data are expressed as the means SD for three or more independent
experiments. Calculations were performed using a concentration of 15
b
μM for all tested compounds. Relative activities were calculated in
relation to the control (arbitrary value of 1.00) activity determined in
the absence of test compounds.
To evaluate the brain penetration of our new compounds, we
used a parallel artificial membrane permeation assay for the
blood−brain barrier (PAMPA-BBB), as recently described by
Di et al.57 We compared the permeability of 13 commercial
drugs with reported values to validate the assay (Table S8 of
the Supporting Information). A plot of experimental data
versus reported values produced a good linear correlation,
Pe(exp) = 1.4574Pe(bibil) − 1.0773 (R2 = 0.9427) (see Figure
S1 of the Supporting Information). From this equation and
considering the limit established by Di et al. for blood−brain
barrier permeation, we determined that compounds with
permeabilities above 4.7 × 10−6 cm s−1 could cross the
blood−brain barrier (see Table S9 of the Supporting
Information). The selected compounds were then tested in
the PAMPA-BBB assay, and the results are presented in Table
4. Thus, it is expected that they could penetrate into the CNS
and reach their biological targets located in the CNS.
Acute Toxicity of Compound 7d. The acute toxicity
profile is considered to be a very important criterion in the
development of new drugs. Compound 7d, which was perhaps
the most promising multifunctional anti-AD agent, was tested
in this assay. Twenty KM mice were randomly divided into four
groups, and the test compound was given in single oral doses of
0, 677, 1333, and 2000 mg/kg. After administration of the
compound, mice were observed continuously for the first 4 h
EXPERIMENTAL SECTION
■
Chemistry. All reagents used in the synthesis were obtained
commercially and used without further purification, unless otherwise
1
specified. The H NMR and 13C NMR spectra were recorded using
TMS as the internal standard on a Bruker BioSpin GmbH
spectrometer at 400 and 101 MHz, respectively, and the coupling
constants are reported in hertz. The reactions were followed by thin-
layer chromatography (TLC) on glass-packed precoated silica gel
plates and visualized in an iodine chamber or with a UV lamp. Flash
column chromatography was performed using silica gel (200−300
mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The high-
resolution mass spectra were obtained using a Shimadzu LCMS-IT-
TOF mass spectrometer. The term “dried” refers to the use of
anhydrous sodium sulfate. The purity (≥95%) of the samples was
determined by high-performance liquid chromatography (HPLC),
conducted on a Shimadzu LC-20AT series system, a TC-C18 column
(4.6 mm × 250 mm, 5 μm), eluted with a 30:70 acetonitrile/PBS
mixture [25 mM NaH2PO4 (pH 3.0)], at a flow rate of 0.5 mL/min.
General Procedure for the Synthesis of 2,2′-Diselenobisbenzoic
Acid (2). 2-Aminobenzoic acid (0.1 mol) was added to a mixture of
37% HCl (20 mL) and H2O (20 mL). After the mixture had cooled to
0 °C, a solution of sodium nitrite (0.11 mol, 30%) was added slowly,
9094
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Article
and the reaction mixture was stirred for 30 min. The mixture was then
slowly poured into an aqueous solution of disodium diselenide (0.05
mol), and the resulting solution was stirred at 50 °C for 2.5 h. After
the mixture had cooled, hydrogen chloride was added until the pH
reached <1. The solution was filtered and washed with H2O, and the
residue was dissolved in saturated NaHCO3. The solution was stirred
at 100 °C for 1 h. After the mixture had cooled, the pH was adjusted
with HCl to a value of <1 to produce the product, which was separated
by filtration.
General Procedure for the Synthesis of 3. 2,2′-Diselenobisbenzoic
acid (2, 0.01 mol) was added to thionyl chloride (20 mL), and DMF
(0.5 mL) was added as a catalyst. After the reaction mixture had been
stirred at 85 °C for 3 h, the solvents were evaporated under a vacuum,
and the crude products were purified by recrystallization from hexane.
General Procedure for the Synthesis of 5a−d, 6a−d, 7a−d, 8,
and 9. A solution of 3 (2.5 mmol) in dry CH2Cl2 (5 mL) was added
dropwise over 15 min at 0 °C to a stirred solution of the
corresponding amine (2 mmol) in dry CH2Cl2 containing dry
triethylamine (5 mmol). Stirring was continued for 5 h at room
temperature. Water was added, and the mixture was extracted with
CH2Cl2, dried, and filtered to produce the crude products, which were
purified by column chromatography using silica gel and a CH2Cl2/
MeOH mixture (58−82% yield).
2-(1-Benzylpiperidin-4-yl)benzo[d][1,2]selenazol-3(2H)-one (5a).
2-Aminobenzoic acid and 1-benzylpiperidin-4-amine (4a) were used
as reactants to give 5a (0.61 g, 82%) as a white solid: mp 182.4−183.5
°C; Rf = 0.42 (10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3)
δ 8.04 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.56 (t, J = 6.9 Hz,
1H), 7.41 (t, J = 7.1 Hz, 1H), 7.34−7.28 (m, 5H), 4.56−4.48 (m, 1H),
3.54 (s, 2H), 3.00 (d, J = 11.9 Hz, 2H), 2.20 (t, J = 11.0 Hz, 2H),
2.08−2.01 (m, 2H), 1.82−1.72 (m, 2H); 13C NMR (101 MHz,
CDCl3) δ 166.77, 138.05, 137.94, 131.71, 129.16 (2C), 128.62, 128.28
(3C), 127.14, 126.12, 123.87, 62.94, 52.72 (2C), 52.13, 33.21 (2C);
FT-IR 2922, 2803, 1591, 1446, 1340, 1251, 1019, 737, 700 cm−1;
HRMS (ESI) m/z [M + H]+ for C19H20N2OSe predicted 373.0814,
measured 373.0818; HPLC purity of 99.66%.
10.0 Hz, 2H), 2.21 (t, J = 10.8 Hz, 2H), 2.03 (s, 2H), 1.76 (d, J = 12.1
Hz, 2H); 13C NMR (101 MHz, CDCl3) δ 166.85, 153.27, 148.92,
138.19, 130.50, 129.14 (2C), 128.25 (2C), 127.11, 120.82, 109.39,
105.23, 62.94, 56.31, 56.21, 52.73 (2C), 52.20, 33.26 (2C); FT-IR
2931, 2803, 1596, 1491, 1457, 1410, 1359, 1272, 1214, 1035, 745, 700
cm−1; HRMS (ESI) m/z [M + H]+ for C21H24N2O3Se predicted
433.1026, measured 433.1022; HPLC purity of 98.61%.
2-[(1-Benzylpiperidin-4-yl)methyl]benzo[d][1,2]selenazol-3(2H)-
one (6a). 2-Aminobenzoic acid and (1-benzylpiperidin-4-yl)-
methanamine (4b) were used as reactants to give 6a (0.564 g, 73%)
as a white solid: mp 132.7−134.8 °C; Rf = 0.31 (10:1 CH2Cl2/
1
CH3OH); H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 7.9 Hz, 1H),
7.62−7.55 (m, 2H), 7.41 (t, J = 7.2 Hz, 1H), 7.29 (d, J = 3.1 Hz, 4H),
7.23 (d, J = 3.4 Hz, 1H), 3.74 (d, J = 5.6 Hz, 2H), 3.49 (s, 2H), 2.87
(d, J = 11.2 Hz, 2H), 1.96 (t, J = 11.5 Hz, 2H), 1.81−1.66 (m, 3H),
1.45−1.37 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 167.37, 138.52,
137.81, 131.91 129.07, 128.97 (2C), 128.13 (2C), 127.39, 126.89,
126.17, 123.90, 63.22, 53.12 (2C), 50.37, 37.11, 29.91 (2C); FT-IR
2918, 2801, 1605, 1445, 1343, 1309, 1146, 738, 672 cm−1; HRMS
(ESI) m/z [M + H]+ for C20H22N2OSe predicted 387.0971, measured
387.0970; HPLC purity of 98.90%.
2-[(1-Benzylpiperidin-4-yl)methyl]-6-fluorobenzo[d][1,2]-
selenazol-3(2H)-one (6b). 2-Amino-4-fluorobenzoic acid and (1-
benzylpiperidin-4-yl)methanamine (4b) were used as reactants to
give 6b (0.606 g, 75%) as a white solid: mp 165.0−166.4 °C; Rf = 0.34
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J =
8.5, 5.3 Hz, 1H), 7.34−7.28 (m, 5H), 7.25−7.20 (m, 1H), 7.13 (t, J =
8.6 Hz, 1H), 3.72 (d, J = 6.8 Hz, 2H), 3.49 (s, 2H), 2.88 (d, J = 11.2
Hz, 2H), 1.96 (t, J = 11.5 Hz, 2H), 1.71 (d, J = 11.7 Hz, 3H), 1.44−
1.36 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 166.44, 165.10 (d, J =
256.5 Hz), 139.32 (d, J = 11.1 Hz), 138.45, 130.67 (d, J = 10.1 Hz),
129.08 (2C), 128.14 (2C), 126.92, 123.71, 114.76 (d, J = 23.2 Hz),
110.85 (d, J = 26.3 Hz), 63.21, 53.09 (2C), 50.48, 37.07, 29.87 (2C);
FT-IR 2921, 2803, 1602, 1463, 1417, 1340, 1245, 1174, 874, 828
cm−1; HRMS (ESI) m/z [M + H]+ for C20H21N2OFSe predicted
405.0877, measured 405.0863; HPLC purity of 99.96%.
2-[(1-Benzylpiperidin-4-yl)methyl]-6-chlorobenzo[d][1,2]-
selenazol-3(2H)-one (6c). 2-Amino-4-chlorobenzoic acid and (1-
benzylpiperidin-4-yl)methanamine (4b) were used as reactants to give
6c (0.630 g, 75%) as a white solid: mp 198.6−200.2 °C; Rf = 0.28
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.95 (d, J =
8.2 Hz, 1H), 7.62 (s, 1H), 7.43−7.36 (m, 1H), 7.34−7.23 (m, 5H),
3.73 (s, 2H), 3.49 (s, 2H), 2.88 (d, J = 9.5 Hz, 2H), 1.96 (t, J = 11.3
Hz, 2H), 1.73−1.66 (m, 3H), 1.45−1.36 (m, 2H); 13C NMR (101
MHz, CDCl3) δ 166.48, 138.92, 138.57, 138.44, 129.75, 129.07 (2C),
128.14 (2C), 127.05, 126.93, 125.86, 123.75, 63.21, 53.08 (2C), 50.48,
37.07, 29.87 (2C); FT-IR 2918, 2796, 1588, 1398, 1302, 1086, 753,
700 cm−1; HRMS (ESI) m/z [M + H]+ for C20H21N2OClSe predicted
421.0579, measured 421.0581; HPLC purity of 99.76%.
2-[(1-Benzylpiperidin-4-yl)methyl]-5,6-dimethoxybenzo[d][1,2]-
selenazol-3(2H)-one (6d). 2-Amino-4,5-dimethoxybenzoic acid and
(1-benzylpiperidin-4-yl)methanamine (4b) were used as reactants to
give 6d (0.624 g, 70%) as a white solid: mp 165.1−166.9 °C; Rf = 0.27
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H),
7.30−7.24 (m, 5H), 7.03 (s, 1H), 3.96 (s, 3H), 3.95 (s, 3H), 3.72 (d, J
= 6.5 Hz, 2H), 3.49 (s, 2H), 2.88 (d, J = 11.6 Hz, 2H), 1.97 (t, J = 11.4
Hz, 2H), 1.72 (d, J = 13.4 Hz, 3H), 1.45−1.36 (m, 2H); 13C NMR
(101 MHz, CDCl3) δ 167.44, 153.41, 148.94, 138.45, 130.20, 129.08
(2C), 128.13 (2C), 126.90, 119.86, 109.66, 105.21, 63.22, 56.26, 56.20,
53.13 (2C), 50.42, 37.17, 29.85 (2C); FT-IR 2923, 2801, 1598, 1490,
1456, 1270, 1213, 1144, 1031, 755, 700 cm−1; HRMS (ESI) m/z [M +
H]+ for C22H26N2O3Se predicted 447.1183, measured 447.1172;
HPLC purity of 98.76%.
2-(1-Benzylpiperidin-4-yl)-6-fluorobenzo[d][1,2]selenazol-3(2H)-
one (5b). 2-Amino-4-fluorobenzoic acid and 1-benzylpiperidin-4-
amine (4a) were used as reactants to give 5b (0.624 g, 80%) as a white
solid: mp 199.8−201.4 °C; Rf = 0.33 (10:1 CH2Cl2/CH3OH); H
1
NMR (400 MHz, CDCl3) δ 8.00 (dd, J = 8.6, 5.2 Hz, 1H), 7.33−7.31
(m, 5H), 7.28 (d, J = 3.9 Hz, 1H), 7.12 (td, J = 8.6, 2.1 Hz, 1H), 4.53−
4.46 (m, 1H), 3.54 (s, 2H), 2.99 (d, J = 11.9 Hz, 2H), 2.19 (t, J = 11.0
Hz, 2H), 2.04 (d, J = 9.9 Hz, 2H), 1.80−1.70 (m, 2H); 13C NMR (101
MHz, CDCl3) δ 165.84, 165.57 (d, J = 255.5 Hz), 139.51 (d, J = 10.1
Hz), 138.17, 130.45 (d, J = 10.1 Hz), 129.11 (2C), 128.27 (2C),
127.14, 124.66, 114.69 (d, J = 24.2 Hz), 110.81 (d, J = 26.3 Hz), 62.90,
52.67 (2C), 52.35, 33.19 (2C); FT-IR 2916, 2804, 1589, 1463, 1410,
1341, 1213, 1074, 872, 740 cm−1; HRMS (ESI) m/z [M + H]+ for
C19H19N2OFSe predicted 391.0720, measured 391.0731; HPLC purity
of 99.58%.
2-(1-Benzylpiperidin-4-yl)-6-chlorobenzo[d][1,2]selenazol-3(2H)-
one (5c). 2-Amino-4-chlorobenzoic acid and 1-benzylpiperidin-4-
amine (4a) were used as reactants to give 5c (0.658 g, 81%) as a white
1
solid: mp 217.2−219.4 °C; Rf = 0.33 (10:1 CH2Cl2/CH3OH); H
NMR (400 MHz, CDCl3) δ 7.94 (d, J = 8.4 Hz, 1H), 7.63 (s, 1H),
7.38 (d, J = 8.4 Hz, 1H), 7.34−7.28 (m, 5H), 4.55−4.45 (m, 1H), 3.54
(s, 2H), 2.99 (d, J = 12.0 Hz, 2H), 2.19 (t, J = 11.3 Hz, 2H), 2.04 (d, J
= 10.2 Hz, 2H), 1.79−1.71 (m, 2H); 13C NMR (101 MHz, CDCl3) δ
166.87, 139.07, 138.35, 138.15, 129.53, 129.11 (2C), 128.28 (2C),
127.15, 126.99, 126.80, 123.71, 62.90, 52.65 (2C), 52.35, 33.19 (2C);
FT-IR 2916, 2801, 1573, 1446, 1368, 1298, 1079, 736, 696 cm−1;
HRMS (ESI) m/z [M + H]+ for C19H19N2OClSe predicted 407.0422,
measured 407.0408; HPLC purity of 98.17%.
2-[2-(1-Benzylpiperidin-4-yl)ethyl]benzo[d][1,2]selenazol-3(2H)-
one (7a). 2-Aminobenzoic acid and 2-(1-benzylpiperidin-4-yl)-
ethanamine (4c) were used as reactants to give 7a (0.536 g, 67%)
as a white solid: mp 110.0−112.3 °C; Rf = 0.27 (10:1 CH2Cl2/
2-(1-Benzylpiperidin-4-yl)-5,6-dimethoxybenzo[d][1,2]selenazol-
3(2H)-one (5d). 2-Amino-4,5-dimethoxybenzoic acid and 1-benzylpi-
peridin-4-amine (4a) were used as reactants to give 5d (0.657 g, 76%)
as a white solid: mp 177.7−180.3 °C; Rf = 0.33 (10:1 CH2Cl2/
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.47 (s, 1H), 7.34−7.27 (m,
5H), 7.05 (s, 1H), 4.49 (s, 1H), 3.96 (s, 6H), 3.55 (s, 2H), 3.00 (d, J =
1
CH3OH); H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 7.8 Hz, 1H),
7.66−7.54 (m, 2H), 7.42 (t, J = 7.4 Hz, 1H), 7.30−7.29 (m, 4H),
7.26−7.20 (m, 1H), 3.89 (t, J = 7.3 Hz, 2H), 3.48 (s, 2H), 2.87 (d, J =
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11.7 Hz, 2H), 1.94 (t, J = 11.1 Hz, 2H), 1.74 (d, J = 10.3 Hz, 2H), 1.66
(dd, J = 13.8, 6.7 Hz, 2H), 1.37−1.27 (m, 3H); 13C NMR (101 MHz,
CDCl3) δ 167.12, 138.39, 137.66, 131.86, 129.23 (2C), 128.80, 128.13
(2C), 127.62, 126.91, 126.20, 123.99, 63.42, 53.63 (2C), 42.56, 37.20,
33.14, 32.16 (2C); FT-IR 2918, 2800, 1602, 1444, 1346, 1267, 743,
702 cm−1; HRMS (ESI) m/z [M + H]+ for C21H24N2OSe predicted
401.1128, measured 401.1121; HPLC purity of 98.61%.
2-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-fluorobenzo[d][1,2]-
selenazol-3(2H)-one (7b). 2-Amino-4-fluorobenzoic acid and 2-(1-
benzylpiperidin-4-yl)ethanamine (4c) were used as reactants to give
7b (0.552 g, 66%) as a white solid: mp 171.2−173.5 °C; Rf = 0.31
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J =
8.5, 5.4 Hz, 1H), 7.34−7.24 (m, 5H), 7.14 (t, J = 8.7 Hz, 2H), 3.87 (t,
J = 7.2 Hz, 2H), 3.48 (s, 2H), 2.87 (d, J = 10.6 Hz, 2H), 1.94 (t, J =
10.9 Hz, 2H), 1.73 (d, J = 10.4 Hz, 2H), 1.66 (s, 2H), 1.39−1.29 (m,
3H); 13C NMR (101 MHz, CDCl3) δ 165.16, 164.04 (d, J = 255.53
Hz), 138.06 (d, J = 10.1 Hz), 137.42, 129.59 (d, J = 9.1 Hz), 128.19
(2C), 127.10 (2C), 125.88, 122.92, 113.75 (d, J = 23.2 Hz), 109.89 (d,
J = 25.3 Hz), 62.41, 52.60 (2C), 41.68, 36.14, 32.16, 31.16 (2C); FT-
IR 2920, 2855, 1602, 1458, 1413, 1343, 1255, 1026, 749 cm−1; HRMS
(ESI) m/z [M + H]+ for C21H23N2OFSe predicted 419.1033,
measured 419.1023; HPLC purity of 96.14%.
2-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-chlorobenzo[d][1,2]-
selenazol-3(2H)-one (7c). 2-Amino-4-chlorobenzoic acid and 2-(1-
benzylpiperidin-4-yl)ethanamine (4c) were used as reactants to give
7c (0.547 g, 63%) as a white solid: mp 194.2−196.8 °C; Rf = 0.31
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.94 (d, J =
8.1 Hz, 1H), 7.68 (s, 1H), 7.39 (s, 1H), 7.29 (s, 4H), 7.25 (s, 1H),
3.87 (d, J = 13.0 Hz, 2H), 3.47 (s, 2H), 2.86 (s, 2H), 1.93 (s, 2H),
1.71−1.62 (m, 4H), 1.33 (s, 3H); 13C NMR (101 MHz, CDCl3) δ
166.21, 138.77, 138.49, 138.43, 129.66, 129.21 (2C), 128.13 (2C),
127.06, 126.91, 126.11, 123.83, 63.42, 53.61 (2C), 42.69, 37.16, 33.18,
32.17 (2C); FT-IR 2913, 2846, 1584, 1448, 1398, 1300, 1085, 736,
697 cm−1; HRMS (ESI) m/z [M + H]+ for C21H23N2OClSe predicted
435.0735, measured 435.0731; HPLC purity of 97.2%.
2-[2-(1-Benzylpiperidin-4-yl)ethyl]-5,6-dimethoxybenzo[d][1,2]-
selenazol-3(2H)-one (7d). 2-Amino-4,5-dimethoxybenzoic acid and 2-
(1-benzylpiperidin-4-yl)ethanamine (4c) were used as reactants to
give 7d (0.561 g, 61%) as a white solid: mp 175.1−177.2 °C; Rf = 0.31
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.46 (s, 1H),
7.30 (s, 2H), 7.29 (s, 2H), 7.25−7.22 (m, 1H), 7.04 (s, 1H), 3.96 (s,
3H), 3.95 (s, 3H), 3.87 (t, J = 7.2 Hz, 2H), 3.48 (s, 2H), 2.87 (d, J =
11.7 Hz, 2H), 1.94 (t, J = 11.2 Hz, 2H), 1.74 (d, J = 10.4 Hz, 2H),
1.68−1.65 (m, 2H), 1.37−1.29 (m, 3H); 13C NMR (101 MHz,
CDCl3) δ 167.19, 153.35, 148.95, 138.44, 130.10, 129.21 (2C), 128.10
(2C), 126.88, 120.14, 109.60, 105.35, 63.43, 56.32, 56.20, 53.64 (2C),
42.65, 37.28, 33.14, 32.19 (2C); FT-IR 2920, 2801, 1597, 1490, 1456,
1413, 1352, 1270, 1214, 1146, 1031, 866, 738, 699 cm−1; HRMS (ESI)
m/z [M + H]+ for C23H28N2O3Se predicted 461.1339, measured
461.1340; HPLC purity of 97.07%.
6H), 3.82 (t, J = 7.0 Hz, 2H), 3.47 (s, 2H), 2.85 (d, J = 10.3 Hz, 2H),
1.93−1.88 (m, 3H), 1.70−1.61 (m, 4H), 1.39 (s, 2H), 1.22−1.26 (m,
4H); 13C NMR (101 MHz, CDCl3) δ 167.22, 153.33, 148.93, 138.53,
130.10, 129.23 (2C), 128.09 (2C), 126.85, 120.13, 109.60, 105.31,
63.51, 56.31, 56.20, 53.90 (2C), 44.90, 36.20, 35.59, 32.32 (2C), 30.86,
23.82; FT-IR 2923, 2851, 1598, 1490, 1450, 1270, 1214, 1145, 1030,
754 cm−1; HRMS (ESI) m/z [M + H]+ for C25H32N2O3Se predicted
489.1652, measured 489.1623; HPLC purity of 98.50%.
2-[2-(1-Benzylpiperidin-4-yl)ethyl]-5,6-dimethoxybenzo[d][1,2]-
selenazol-3(2H)-one 1-Oxide (10). A solution of 30% H2O2 (65 μL,
0.65 mmol) was added to a solution of 7d (0.2 g, 0.43 mmol) in 10
mL of CH2Cl2, which was stirred for 30 min at room temperature.
After the reaction was complete, the solvents were evaporated under
vacuum, and the crude product was purified by column chromatog-
raphy to provide a colorless oil (0.133 g, 65%): Rf = 0.21 (10:1
1
CH2Cl2/CH3OH); H NMR (400 MHz, DMSO) δ 7.78 (d, J = 5.2
Hz, 1H), 7.32 (s, 6H), 3.90 (t, J = 5.5 Hz, 6H), 3.80−3.71 (m, 1H),
3.68−3.52 (m, 3H), 2.83 (s, 2H), 1.99 (s, 2H), 1.71 (s, 2H), 1.59 (s,
2H), 1.24 (s, 3H); 13C NMR (101 MHz, DMSO) δ 168.06, 152.87,
151.87, 139.27, 130.03, 129.07 (2C), 128.14 (2C), 127.13, 123.58,
109.57, 108.37, 62.04, 56.16, 56.01 (2C), 52.81, 48.09, 36.08, 32.39,
31.21 (2C); FT-IR 2923, 2809, 1589, 1490, 1448, 1422, 1271, 1214,
1045, 843 cm−1; HRMS (ESI) m/z [M + H]+ for C23H28N2O4Se
predicted 477.1288, measured 477.1276; HPLC purity of 96.0%.
Biological Assays. Determination of the Inhibitory Effects on
eeAChE, BuChE, and hAChE Activities. We followed the method of
Ellman et al. Acetylcholinesterase (from electric eel or human
erythrocytes), butyrylcholinesterase (from equine serum), 5,5′-
dithiobis(2-nitrobenzoic acid) (DTNB), acetylthiocholine chloride
(ATC), and butyrylthiocholine chloride (BTC) were purchased from
Sigma-Aldrich. The prototypes tacrine and donepezil were used as
reference compounds. Five different concentrations of each compound
were used to obtain between 20 and 80% inhibition of cholinesterase
activity.
All assays were conducted in 0.1 M KH2PO4/K2HPO4 buffer at pH
8.0 using a Shimadzu UV-2450 spectrophotometer. AChE solutions
were prepared to produce concentrations of 2.0 units/mL in 2 mL
aliquots. The assay medium (1 mL) consisted of phosphate buffer (pH
8.0), 50 μL of 0.01 M DTNB, and 20 μL of AChE (from electric eel or
human serum). The test compounds were added to the assay solution
and preincubated at 37 °C for 15 min, and 50 μL of 0.01 M ATC was
added immediately. The activity was determined by measuring the
increase in absorbance at 412 nm at 1 min intervals at 37 °C. Data
from concentration−inhibition experiments with the inhibitors were
subjected to nonlinear regression analysis using GraphPad Prism
version 5.0 (GraphPad Software Inc.), which gave estimates of the IC50
(concentration of the drug resulting in 50% inhibition of enzyme
activity).
Inhibition of BuChE was measured as described above, substituting
0.02 M BTC for the substrate.
Kinetic Characterization of AChE Inhibition. Kinetic character-
ization of AChE was performed using the reported method. The test
compound was added to the assay solution and preincubated with the
enzyme at 37 °C for 15 min, followed by the addition of substrate
(0.1−1.0 mM). Kinetic characterization of the hydrolysis of ATC
catalyzed by AChE was performed spectrometrically at 412 nm. A
parallel control with no inhibitor in the mixture allowed the activities
measured at various times to be adjusted. The plots were assessed by a
weighted least-squares analysis that assumed the variance of V to be a
constant percentage of V for the entire data set. The slopes of these
reciprocal plots were then plotted against the concentrations of the
inhibitors in a weighted analysis, and Ki was determined as the ratio of
the replot intercept to the replot slope.
Inhibition of Human Recombinant AChE-Induced Aβ1−40 Peptide
Aggregation Assay. The thioflavin T (ThT) fluorescence method was
used as previously described.42 Aβ1−40 (Sigma-Aldrich), lyophilized
from a 2 mg/mL 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solution,
and a test compound were dissolved in DMSO to produce 2.3 mM
and 1 mM solutions. Two microliters of Aβ1−40 was incubated with 16
μL of human recombinant AChE (Sigma-Aldrich) in the presence of 2
2-[3-(1-Benzylpiperidin-4-yl)propyl]-5,6-dimethoxybenzo[d][1,2]-
selenazol-3(2H)-one (8). 2-Amino-4,5-dimethoxybenzoic acid and 3-
(1-benzylpiperidin-4-yl)propan-1-amine (4d) were used as reactants to
give 8 (0.521 g, 55%) as a white solid: mp 151.1−152.4 °C; Rf = 0.33
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J =
3.1 Hz, 1H), 7.30−7.27 (m, 5H), 7.04 (d, J = 3.1 Hz, 1H), 3.96 (s,
6H), 3.81 (s, 2H), 3.48 (d, J = 2.9 Hz, 2H), 2.86 (d, J = 10.3 Hz, 2H),
1.93 (t, J = 9.9 Hz, 2H), 1.72 (s, 2H), 1.65 (s, 4H), 1.32−1.29 (m,
3H); 13C NMR (101 MHz, CDCl3) δ 167.24, 153.32, 148.91, 138.55,
130.14, 129.20 (2C), 128.09 (2C), 126.85, 120.11, 109.56, 105.34,
63.49, 56.31, 56.19, 53.84 (2C), 45.06, 35.46, 33.30, 32.26 (2C),
27.94; FT-IR 2923, 2850, 1598, 1490, 1456, 1419, 1270, 1144, 1030,
867, 740 cm−1; HRMS (ESI) m/z [M + H]+ for C24H30N2O3Se
predicted 475.1496, measured 475.1468; HPLC purity of 98.86%.
2-[4-(1-Benzylpiperidin-4-yl)butyl]-5,6-dimethoxybenzo[d][1,2]-
selenazol-3(2H)-one (9). 2-Amino-4,5-dimethoxybenzoic acid and 4-
(1-benzylpiperidin-4-yl)butan-1-amine (4e) were used as reactants to
give 9 (0.566 g, 58%) as a white solid: mp 141.4−144.2 °C; Rf = 0.31
(10:1 CH2Cl2/CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.46 (s, 1H),
7.30 (d, J = 3.8 Hz, 4H), 7.25−7.20 (m, 1H), 7.04 (s, 1H), 3.95 (s,
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μL of a test compound to give final concentrations of 230 μM Aβ1−40
,
Substrates for Hepatic Mammalian Thioredoxin Reductase
(TrxR). TrxR activity was determined according to the method of
Zhao and Holmgren.21 Measurements of TrxR activity were performed
in a buffer containing 140 μL of 50 mM Tris-HCl, 1 mM EDTA (pH
7.5), 20 μL of NADPH (1 mM), and 20 μL of the test compound
(150 μM). Reactions were initiated by the addition of 20 μL of TrxR
(3.5 units/mL), and the absorbance at 340 nm was monitored for 10
min.
2.3 μM hAChE, and 100 μM test compound. Following co-incubation
at room temperature for 48 h, 180 μL of 1.5 μM ThT in 50 mM
glycine-NaOH buffer (pH 8.5) was added. The fluorescence was
monitored at 446 nm, and the emission was monitored at 490 nm
using a Molecular Devices SpectraMax spectrofluorometer. The
percent inhibition of the AChE-induced aggregation was calculated
by the following expression: 100 − (IFi/IF0 × 100), where IFi and IF0
are the fluorescence intensities in the presence and absence of the test
compound, respectively, minus the fluorescence intensities due to the
respective blanks. Each assay was conducted in triplicate, and each
experiment was repeated at least three independent times.
Coupled Reductase Assay. The GPx-like activity of the organo-
selenium compounds was determined using a spectrophotometric
method at 340 nm as described by Wilson et al.27 The test mixture
contained GSH (2 mM), EDTA (1 mM), glutathione reductase (1.3
units/mL), and NADPH (0.4 mM) in 100 mM potassium phosphate
buffer (pH 7.5). GPx samples (80 μM) were added to the test mixture
at 25 °C, and the reaction was initiated by the addition of H2O2 (1.6
mM). The initial reduction rates were calculated from the oxidation
rate of NADPH at 340 nm. The initial reduction rate was determined
at least three times and calculated from the first 5−10% of the reaction
using a value of 6.22 mM−1 cm−1 as the extinction coefficient for
NADPH.
FOX Assay. The hydrogen peroxide scavenging activity was
determined by the ferrous ion oxidation−xylenol orange (FOX)
assay with minor changes. FOX reagent was prepared by adding 9
volumes of reagent 1 to 1 volume of reagent 2, where reagent 1 was
4.4 mM butylated hydroxytoluene (BHT) in methanol and reagent 2
was 1 mM xylenol orange and 2.56 mM ammonium ferrous sulfate in
250 μM H2SO4. Test compounds (400 μL) at different concentrations
were incubated with 1 mM H2O2 (100 μL) for 10 h at 37 °C in the
dark, and the FOX agent (500 μL) was added. The reaction mixture
was then vortexed and incubated at room temperature for 30 min. The
development of violet color indicated a positive control reaction, and
discoloration was considered scavenging activity after addition of the
test compound. The FOX reagent without compound or H2O2 served
as a blank or control. The absorbance of the ferric−xylenol orange
complex was measured at 560 nm.
Peroxynitrite Scavenging Activity. Peroxynitrite, whose formation
results in the bleaching of Evans blue (EB) dye, was measured at 608
nm (ε = 70000 M−1 cm−1). Consumption of Evans blue dye, without
or with different concentrations of test compound, was the basis for
the calculated amount of peroxynitrite. D0 and DTC are the
stoichiometries for the reaction of peroxynitrite with Evans blue dye
without and with the test compound, respectively. The kTC/kEB ratio
could be calculated from the slope of the straight line plotting
Δ[TC]0/Δ[EB] versus [TC]/[EB]. For the sake of simplicity, initial
concentrations [TC]0 and [EB]0 were used in the calculations. In this
work, instead of Δ[EB], we measured the stoichiometry D = Δ[EB]/
Δ[NaOONO3−] to avoid the effect of slight deviations in peroxynitrite
concentration. The ratio D0/DTC is equivalent to Δ[TC]0/Δ[EB]a,
where D0 and DTC are the reaction stoichiometries in the absence and
presence of the test compound, respectively. The antioxidant activity
was measured as the slope of the straight line (kTC/kEB).
In Vitro Blood−Brain Barrier Permeation Assay. Brain penetration
of compounds was evaluated using a parallel artificial membrane
permeation assay (PAMPA), in a manner similar to that described by
Di et al.44 as the basis for the calculated amount of peroxynitrite.
Commercial drugs were purchased from Sigma and Alfa Aesar. Porcine
brain lipid (PBL) was obtained from Avanti Polar Lipids. The donor
microplate (PVDF membrane, pore size of 0.45 mm) and the acceptor
microplate were both from Millipore. The 96-well UV plate
(COSTAR) was obtained from Corning Inc. The acceptor 96-well
microplate was filled with 300 μL of a PBS/EtOH mixture (7:3), and
the filter membrane was impregnated with 4 μL of PBL in dodecane
(20 mg/mL). The compound was dissolved in DMSO at a
concentration of 5 mg/mL and diluted 50-fold in a PBS/EtOH
mixture (7:3) to yield a concentration of 100 mg/mL, and 200 μL was
added to the donor wells. The acceptor filter plate was carefully placed
on the donor plate to form a sandwich, which was left undisturbed for
10 h at 25 °C. After incubation, the donor plate was carefully removed,
and the concentrations of the compound in the acceptor wells were
determined using a UV plate reader (Flexstation 3). Every sample was
analyzed at five wavelengths in four wells and in at least three
independent runs, and the results are given as the means standard
deviation (Table S8 of the Supporting Information). In each
experiment, 13 quality control standards of known BBB permeability
were included to validate the analysis set. A plot of the experimental
data versus literature values gave a good linear correlation, Pe(exp) =
1.4574Pe(bibl) − 1.0773 (R2 = 0.9427) (Figure S1 of the Supporting
Information). From this equation and considering the limit established
by Di et al. for blood−brain barrier permeation, we found that
compounds with permeabilities above 4.7 × 10−6 cm s−1 are able to
cross the blood−brain barrier (Table S9 of the Supporting
Information).
Acute Toxicity of Compound 7d. Twenty KM mice (22 days, 18−
20 g), purchased from the laboratory animal center of Sun Yat-sen
University (Guangzhou, China), were used to evaluate the acute
toxicity of compound 7d. Mice were maintained on a 12 h light/dark
cycle (light from 7:00 a.m. to 7:00 p.m.) at 20−22 °C and 60−70%
relative humidity. Sterile food and water were provided according to
institutional guidelines. Prior to each experiment, mice were fasted
overnight and allowed free access to water. Compound 7d was
dissolved in a 0.5% carboxymethyl cellulose sodium (CMC-Na) salt
solution and given via oral administration to different experimental
groups. After administration of the compound, mice were observed
continuously for the first 4 h for any abnormal behavior and mortality
changes, intermittently for the next 24 h, and occasionally thereafter
for 14 days for the onset of any delayed effects. All animals were
sacrificed on the 14th day after drug administration and macroscopi-
cally evaluated for possible damage to the heart, liver, and kidneys.
Peroxynitrite synthesis was conducted by modifying the method
described by Beckman et al.44 Acidified H2O2 (8.2 M in 1.85 M
HNO3, 6.6 mL) and sodium nitrite (2 M, 6 mL) solutions were drawn
into two separate syringes. The contents of both syringes were
simultaneously injected into an ice-cooled beaker containing 4.2 M
sodium hydroxide (6 mL) through a Y-shaped junction. Excess
hydrogen peroxide was removed by MnO2 treatment. The
concentration of the resultant stock solution was measured
spectrophotometrically at 302 nm (ε = 1670 M−1 cm−1). The typical
yield ranged from 70 to 80 mM. Peroxynitrite was used to induce the
bleaching of Evans blue dye, which was measured at 608 nm (ε =
70000 M−1 cm−1). Consumption of Evans blue (20 μM) in the
absence and presence of the different concentrations of the test
compound was measured over the peroxynitrite (20 μM). Antioxidant
activities were determined against the control.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures for the synthesis of the amine
intermediates (4a−e), the HPLC data and FT-IR spectra of
the target compounds, the full data regarding the GPx-like
activity of the test compounds, and the method for PAMPA.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Journal of Medicinal Chemistry
Article
(10) Samadi, A.; Estrada, M.; Per
́
ez, C.; Rodríguez-Franco, M. I.;
AUTHOR INFORMATION
Corresponding Authors
*Phone: +086-20-3994-3051. Fax: +086-20-3994-3051. E-mail:
Huangl72@mail.sysu.edu.cn.
*Phone: +086-20-3994-3050. Fax: +086-20-3994-3050. E-mail:
lixsh@mail.sysu.edu.cn.
■
Iriepa, I.; Moraleda, I.; Chioua, M.; Marco-Contelles, J. Pyridonepezils,
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation of China
(21302235 and 20972198) and the Guangdong Engineering
Research Center of Chiral Drugs for financial support of this
study.
ABBREVIATIONS USED
■
AD, Alzheimer’s disease; AChE, acetylcholinesterase; hAChE,
human acetylcholinesterase; BuChE, butyrylcholinesterase;
GPx, glutathione peroxidase; CNS, central nervous system;
Aβ, β-amyloid; AChEIs, acetyl cholinesterase inhibitors;
MTDLs, multi-target-directed ligands; PAS, peripheral anionic
site; CAS, catalytic binding site; GSH, glutathione; EDTA,
ethylenediaminetetraacetate; GR, glutathione reductase; ROS,
reactive oxygen species; FOX, ferrous ion oxidation−xylenol
orange; EB, Evans blue; PAMPA-BBB, parallel artificial
membrane permeation assay for the blood−brain barrier;
DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); ATC, acetylthio-
choline chloride; BTC, butyrylthiocholine chloride; ThT,
thioflavin T; TLC, thin-layer chromatography; PBL, porcine
brain lipid; SD, standard deviation
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