Synthesis and biological evaluation of a new series of ebselen derivatives as glutathione peroxidase (GPx) mimics and cholinesterase inhibitors against Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2013.12.066

A series of ebselen derivatives were designed, synthesised and evaluated as inhibitors of cholinesterases (ChEs) and glutathione peroxidase (GPx) mimics. Most of the compounds were found to be potent against AChEs and BuChE, compounds 5e and 5i, proved to be the most potent against AChE with IC₅₀ values of 0.76 and 0.46 μM, respectively. Among these hybrids, most of the compounds were found to be good GPx mimics compare with ebselen. The selected compounds 5e and 5i were also used to determine the catalytic parameters and in vitro hydrogen peroxide scavenging activity. The results indicate that compounds 5e and 5i may be excellent multifunctional agents for the treatment of AD.

Full text of DOI:10.1016/j.bmc.2013.12.066

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a new series of ebselen
derivatives as glutathione peroxidase (GPx) mimics and
cholinesterase inhibitors against Alzheimer’s disease
Zonghua Luo a, Liang Liang b, Jianfei Sheng a, Yanqing Pang a, Jianheng Li a, Ling Huang a, Xingshu Li a,
⇑
a Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
b School of Chemical Engineering and Light Industry, Guangdong University of Technology, 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 ebselen derivatives were designed, synthesised and evaluated as inhibitors of cholinesterases
(ChEs) and glutathione peroxidase (GPx) mimics. Most of the compounds were found to be potent against
AChEs and BuChE, compounds 5e and 5i, proved to be the most potent against AChE with IC50 values of
Received 29 October 2013
Revised 19 December 2013
Accepted 30 December 2013
Available online xxxx
0.76 and 0.46 lM, respectively. Among these hybrids, most of the compounds were found to be good GPx
mimics compare with ebselen. The selected compounds 5e and 5i were also used to determine the
catalytic parameters and in vitro hydrogen peroxide scavenging activity. The results indicate that
compounds 5e and 5i may be excellent multifunctional agents for the treatment of AD.
Ó 2014 Published by Elsevier Ltd.
Keywords:
GPx mimics
Cholinesterase inhibitors
Alzheimer’s disease
1. Introduction
systems, selenium exists in the form of selenoproteins and is
primarily present in the form of selenocysteine (Sec), which is an
Alzheimer’s disease (AD) is a progressive brain neurological dis-
ease that leads to memory loss, steady deterioration of cognition,
and dementia.1 The disease become sufficiently severe to impede
social and occupational functioning and worsens as it develops.
At present, there is no satisfactory cure for AD; thus, its treatment
remains a challenge to pharmaceutical scientists. Although the
pathogenesis of AD is not completely known at present, several
hallmarks, for example, increased production and deposits of
b-amyloid peptide (Ab), reduced levels of acetylcholine (ACh),
and increased oxidative stress, are thought to play significant roles
in the process of the disease.2
Clinically, the primary current therapeutic options approved by
the US Food and Drug Administration for the treatment of AD are
cholinesterase inhibitors (AChEIs), namely, tacrine, donepezil, riv-
astigmine, and galantamine, based on the cholinergic hypothesis.3
However, many studies have shown that oxidative stress is another
major sign of AD. Oxidative stress due to an increase in free radical
generation or damage to the endogenous antioxidant mechanism
is an important factor that has been implicated in cognitive deficits
in the elderly.4 Therefore, the successful protection of neuronal
cells from oxidative damage may prevent AD.
analogue of cysteine. Selenium in the Sec residue acts as a strong
nucleophile in catalytic reactions. Glutathione peroxidase (GPx),
which protects cells by catalyzing the reduction of peroxides with
the stoichiometric reductant glutathione, is a typical selenoen-
zyme.6–9 This selenoprotein, which bears selenol (Enz-SeH) at the
active site, is responsible for the reduction of a peroxide molecule,
and the subsequent reduction of the corresponding selenenic acid
is catalyzed by glutathione. The resulting selenenyl sulfide then re-
acts with a second glutathione to regenerate the original selenol
and the disulfide of glutathione (Fig. 1).10 Ebselen, which has been
extensively studied as the GPx mimic, has undergone clinical trials
as an anti-inflammatory agent due to its antioxidant activity.11–14
Additionally, Ebselen could reduce brain damage in patients with
delayed neurological deficits and may be a promising neuroprotec-
tive agent.15–17
Because both ChE and oxidative damage may be crucial partic-
ipants in the pathological processes of AD, dual or multifunctional
drugs would be beneficial for the treatment of AD. Literatures de-
scribed that dimethoxy and benzylpiperidine group in donepezil or
its derivatives are important pharmacophores for inhibition activ-
ity of AChE.18,19 In addition, many researchers have reported that
the nitrogen heterocyclic ring is important pharmacophore for
ChE inhibition activity.20–22 In this paper, we disclose the design,
synthesis, and evaluation of a series of anti-AD molecules devel-
oped through the fusion of the pharmacophores of the AChEIs
and ebselen (Fig. 2).
Selenium has been recognized as an essential trace element,
and its deficiency has been linked to many diseases.5 In biological
⇑
Corresponding author. Tel.: +86 02039943050.
E-mail address: xsli219@yahoo.com (X. Li).
0968-0896/$ - see front matter Ó 2014 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmc.2013.12.066
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066

2
Z. Luo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
The obtained IC50 values are summarized in Table 1. The results
clearly show that most of the designed compounds exhibit moder-
ate to good inhibitory activities. The IC50 values ranged from >15 to
0.46 lM for AChE and from >15 to 3.62 lM for BuChE. Additionally,
all of the synthesized compounds, with the exception of com-
pounds 5b and 5d, exhibited more activity toward AChE than
toward BuChE. Among these compounds, compound 5i (the X is
oxygen and the linker is a two-alkylene chain) exhibited the most
potent inhibition for AChE (IC50 value = 0.46 lM). A simple struc-
ture–activity relationship analysis showed that the AChE inhibi-
tory potency is closely related to the substituted groups on the
benzo[d][1,2]selenazol-3(2H)-one moiety (A ring) and the side
chain linked to the B ring. The results also indicated that the dime-
thoxy on the A ring is important for the AChE inhibitory activity
Figure 1. Postulated catalytic cycle of the GPx-catalyzed reduction of peroxide by
glutathione.
(5a/5e, IC50 = 2.36/0.76
IC50 = 5.66/3.87 M). The piperidine group (linked to the B ring)
endows more potent AChE inhibitory activity than the pyrrolidine
group (5d/5e, IC50 = 1.06/0.76 M; 5g/5h, IC50 = 4.32/3.87 M).
lM; 5b/5g, IC50 = >15/4.32 lM; 5c/5i,
l
l
l
2. Results and discussion
2.1. Chemistry
Moreover, when the X was methylene, the AChE inhibitory activity
decreased as the length of the linker increased (5d/5g, IC50 = 1.06/
4.32 lM; 5e/5h, IC50 = 0.76/3.87 lM). However, when the X was
changed from methylene to oxygen, the activity was not very irreg-
ular. The inhibitory activity of these compounds against human
AChE were also determined using the same method with donepezil
as the reference (Table 1). Comparing the inhibition results with
AChE from the electric eel, slightly weaker activities were observed
except compounds 5i and 5k. The most two potent compounds 5e
and 5i still provided the best activity (IC50 = 1.44/0.45 lM). In con-
trast, most of the compounds exhibited similar BuChE inhibitory
activities in the micromolar range.
The general procedure for the synthesis of the target com-
pounds is shown in Scheme 1. Using o-aminobenzoic acid or its
analogues as the starting material, we initiated the reaction
through diazotization, and the subsequent reaction with Na2Se2
provided the diselenium intermediate 2. Compound 2 reacted with
thionyl chloride and then different phenylamine derivatives (4) in
CH2Cl2 to generate the target compounds (5a–5k) in good yields
(62–80%). The different phenylamine derivatives 4 were prepared
following previously reported methods (see the Supporting
information).
2.3. Glutathione peroxidase-like activity
2.2. In vitro evaluation of cholinesterase inhibition
The catalytic reduction of H2O2 using GSH as a cosubstrate in
the presence and absence of the catalysts 5a–5k was studied14,24
The in vitro activity of these new molecules against EeAChE and
eqBuChE were determined using the method developed by Ellman
et al.23 using tacrine and donepezil as the reference compounds.
(Table 2). The initial reduction rates (m0) for H2O2 by reduced glu-
tathione were calculated from a linear fit spanning the first 5–10%
of the reaction by following the oxidation of reduced nicotinamide
adenine dinucleotide (NADPH) in phosphate buffer at 340 nm.
Interestingly, it was found that 5a (166.1 l
M minꢀ1) and 5e
(149.0
(118.4
l
l
M minꢀ1) exhibited much higher activities than ebselen
M minꢀ1), and compounds 5b, 5c, 5d, 5j, 5h, 5i and 5j
exhibited GPx-like activities similar to that of ebselen, whereas
5f (59.87
l
M minꢀ1) and 5k (85.85 M minꢀ1) presented lower
l
activities. Based on the results shown in Table 2, we found that
compounds with piperidine exhibited much higher GPx-like activ-
ities than those with pyrrolidine and N,N-dimethyl-1-phenylme-
thanamine (5e > 5d > 5f, 5h > 5g), which is in accordance with
the AChE inhibitory activity results. In addition, a long linker
between the benzene ring and piperidine was unfavorable for
GPx-like activity (5i > 5j > 5k).
2.4. Determination of the catalytic parameters
Compounds 5e and 5i, which exhibit good AChE inhibitory
activities and GPx-like activities, were selected to perform detailed
kinetic experiments in order to further understand their catalytic
behaviors. The Lineweaver–Burk (double reciprocal) plots for ebse-
len, 5e, and 5i were obtained by plotting the reciprocal of the initial
rate (1/m0) against the reciprocal of the substrate concentration (1/
[substrate]) and were used for the determination of the catalytic
parameters.25 The catalytic parameters, such as the maximum
velocity (Vmax), Michaelis constant (Km), catalytic constant (kcat),
and catalytic efficiency (g), for the reduction of H2O2 using ebselen,
5e, and 5i were obtained (Table 3). It is worth mentioning that 5e
Figure 2. The design strategy of the ebselen derivatives.
and 5i, which have a piperidine group linked to the benzene ring,
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066

Z. Luo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Scheme 1. Synthesis route for compounds 5a–5k. The reagents and conditions used are the following: (a) NaNO2, HCl; Na2Se2; (b) SOCl2, reflux; (c) (Et)3N, CH2Cl2.
presented a higher maximal velocity than ebselen, particularly
when the concentration of H2O2 is variable. The Vmax values of 5e
and 5i were determined to be 869.57, 308.64 and 316.46,
ion oxidation-xylenol orange (FOX) assay26 with ebselen as the
reference compound. The results shown in Figure 3 demonstrate
that ebselen and 5e have similar scavenging activities for hydrogen
peroxide at the different concentrations tested. However, 5i
showed a slightly lower scavenging activity than ebselen and 5e.
These results indicate that ebselen, 5e, and 5i possess antioxidant
activity against H2O2 in vitro.
276.24 l
M minꢀ1, respectively. The Km values of 5e (2.37 mM)
and 5i (2.47 mM) were similar to that of ebselen (2.34 mM) when
GSH is variable, and the inverse results were obtained when H2O2
is variable. The catalytic efficiencies of 5e and 5i were determined
to be 1.45, 1.63 and 0.74, 1.40 mMꢀ1 minꢀ1, respectively, whereas
the catalytic efficiency of ebselen was 2.85 and 1.64 mMꢀ1 minꢀ1
for varied H2O2 and GSH, respectively. The comparison of the
catalytic efficiencies of 5e and 5i, and ebselen suggests that the
GPX-like activity of 5e is similar to that of ebselen, whereas 5i
exhibits a slightly weaker activity.
3. Conclusion
In conclusion, we have developed a new series of multi-target-
directed ligands derived from ebselen for the treatment of AD.
These compounds were evaluated as potential multi-valent inhib-
itors of AChE and BuChE, as GPx mimics, and as superfast antioxi-
dant agents against H2O2. Among the synthesized compounds,
compounds 5e and 5i exhibited good inhibitory potency toward
2.5. In vitro hydrogen peroxide scavenging activity
Oxidative stress is an obvious feature of AD pathology, and dur-
ing the aging process, oxidative-induced apoptosis and increased
activity of b- and c-secretases potentially contribute to Ab genera-
tion. The removal of reactive oxygen species (ROS), such as hydro-
gen peroxide, is important for older individuals, particularly AD
patients, due to their declined endogenous antioxidant protection
system. To further evaluate the antioxidant activity of the ebselen
derivatives, compounds 5e and 5i were analyzed using the ferrous
AChE (IC50 values = 0.76 and 0.46 lM, respectively). These ebselen
derivatives exhibit good GPx-like activities, that is, these deriva-
tives can catalytically reduce harmful peroxides, and most of the
compounds, particularly 5a, 5e, 5g, and 5i, exhibited higher
activity than ebselen. The H2O2 scavenging activity of compounds
5e and 5i was then investigated in subsequent experiments, and
the activity of these compounds was similar to that of ebselen.
These results indicate that compounds 5e and 5i may be excellent
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066

4
Z. Luo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Table 1
Inhibition of AChE and equine serum butyrylcholinesterase (eqBuChE)
O
R
R
Y
n
N
Se
X
Compound
R
X
n
0
1
2
0
0
Y
IC50 (AChE inhibition)(
2.36 0.006
>15
l
M) SDa
IC50 (BuChE inhibition)(
5.56 0.191
6.38 0.672
1.97 0.177
13.40 1.711
>15
l
M) SDb
IC50 (hAChE inhibition)(
10.85 0.191
>15
l
M) SDc
5a
5b
5c
5d
5e
H
CH2
CH2
O
N
N
N
N
N
H
H
5.66 0.183
1.06 0.050
0.76 0.008
10.93 0.191
4.31 0.399
1.44 0.127
OCH3
OCH3
CH2
CH2
5f
OCH3
CH2
0
1.71 0.034
3.62 0.467
>15
N
5g
5h
5i
OCH3
OCH3
OCH3
OCH3
OCH3
CH2
CH2
O
1
1
2
3
4
N
N
N
N
N
4.32 0.130
3.87 0.095
0.46 0.004
4.50 0.080
4.32 0.030
>15
13.94 0.121
10.52 0.050
0.45 0.014
5.15 0.097
3.41 0.045
>15
9.69 0.276
5.80 0.566
5.72 0.438
5j
O
5k
O
Ebselen
Donepezil
Tacrine
>15
0.041 0.003
0.11 0.01
10.58 0.003
3.49 0.15
0.015 0.002
>15
0.011 0.001
0.534 0.022
a
b
c
50% Inhibitory concentration (means SD of three independent experiments) of AChE.
50% Inhibitory concentration (means SD of three independent experiments) of BuChE.
50% Inhibitory concentration (means SD of three independent experiments) of hAChE.
multifunctional agents for the treatment of AD. Further investiga-
tions of AD therapeutic candidates based on these results are in
progress.
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 cooling, 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 cooling, the pH was adjusted
to <1 with HCl to obtain the product, which was separated by
filtration.
4. Experimental section
4.1. Chemistry
4.1.1. General remarks
All of the reagents used in the syntheses were commercial prod-
ucts and were used without further purification unless otherwise
specified. The 1H NMR and 13C NMR spectra were recorded using
TMS as the internal standard on a Bruker BioSpin GmbH spectrom-
eter at 400 and 101 MHz, respectively, and the coupling constants
are reported in hertz (Hz). 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 Shi-
madzu LCMS-IT-TOF mass spectrometer. The purity of the samples
was determined by HPLC, which was conducted using the Agilent
4.1.3. General procedure for the synthesis of compound 3
2,20-Diselenobisbenzoic acid (2) (0.01 mol) was added to thio-
nyl chloride (20 mL) using DMF (0.5 mL) as the catalyst. After the
reaction mixture was stirred at 85 °C for 3 h, the solvents were
evaporated under vacuum, and the crude products were purified
by recrystallisation from cyclohexane and used for the next step
without further purification.
4.1.4. Preparation of the aniline intermediates (4a–4h)
4-(Piperidin-1-ylmethyl)aniline (4a), 4-(pyrrolidin-1-ylmethyl)
aniline (4b), 4-((benzyl(methyl)amino)methyl)aniline (4c), 4-(2-
(piperidin-1-yl)ethyl)aniline (4d), 4-(2-(pyrrolidin-1-yl)ethyl)aniline
(4e), 4-(2-(piperidin-1-yl)ethoxy)aniline (4f), 4-(3-(piperidin-1-yl)
propoxy)aniline (4g), and 4-(4-(piperidin-1-yl)butoxy)aniline (4h)
were synthesized by methods previously described in the litera-
ture. For the detailed syntheses, refer to the Supplementary data.
1200 series system, a TC-C18 column (4.6 ꢁ 250 mm, 5
lm), and
acetonitrile/PBS (25 mM NaH2PO4, pH 3.0) at a flow rate of
1.0 mL/min for the elution.
4.1.2. General procedure for the synthesis of 2,20-
diselenobisbenzoic acid (2)
2-Amino benzoic acid (0.1 mol) was added to a mixture of 37%
HCl (20 mL) and H2O (20 mL). After the mixture was cooled to 0 °C,
a solution of sodium nitrite (0.11 mol, 30%) was slowly added, and
the reaction mixture was stirred for 30 min. The mixture was then
4.1.5. General procedure for the synthesis of compounds 5a–5k
A solution of compound 3 (2.5 mmol) in dry CH2Cl2 (5 mL) was
added dropwise over a period of 15 min to a stirred solution of the
corresponding amine (4a–4h; 2 mmol) in dry CH2Cl2 containing
dry triethylamine (5 mmol) at 0 °C. The mixture was stirred for
5 h at room temperature. Water was added to the mixture and
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066

Z. Luo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Table 2
Initial rates m0
and 5a–5k
(l
M minꢀ1) of the reduction of H2O2 by GSH in the presence of ebselen
O
R
R
Y
n
N
Se
X
a,b
Compound
R
X
n
Y
m0
(l )
M minꢀ1
5a
5b
5c
5d
5e
H
CH2
CH2
O
0
1
2
0
0
N
N
N
N
N
166.1 11.2
115.1 4.82
100.1 17.1
115.1 2.29
149.0 16.6
H
Figure 3. Scavenging effects of ebselen, 5e, and 5i on hydrogen peroxide, as
determined through the FOX assay (H2O2 = 100 lM). The values reported are the
means SD of three independent experiments. P < 0.01 compared with ebselen.
H
OCH3
OCH3
CH2
CH2
132.44, 130.00, 129.34, 127.61, 126.48, 125.10, 123.75, 63.19,
54.47, 25.91, 24.30; HRMS-ESI [M+H]+ calculated for C19H20N2OSe:
373.0814, found: 373.0827.
5f
OCH3
CH2
0
59.87 5.39
N
5g
5h
5i
OCH3
OCH3
OCH3
OCH3
OCH3
CH2
CH2
O
1
1
2
3
4
N
N
N
N
N
112.8 3.81
121.3 1.10
122.7 3.69
113.7 7.37
85.85 3.46
4.1.5.2. 2-(4-(2-(Pyrrolidin-1-yl)ethyl)phenyl)benzo[d][1,2]sele-
nazol-3(2H)-one (5b).
White solid; yield 72%; purity 99.3%;
8.19 (d,
mp: 228.2–230.6 °C; 1H NMR (400 MHz, DMSO)
d
J = 8.1 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H), 7.61–7.48 (m, 3H), 7.38 (t,
J = 7.4 Hz, 1H), 7.28 (d, J = 8.1 Hz, 2H), 3.04–2.86 (m, 4H), 2.41–
2.35 (m, 4H), 1.86 (s, 4H); 13C NMR (101 MHz, DMSO) d 164.91,
139.32, 138.87, 134.13, 131.69, 129.32, 129.16, 127.58, 126.53,
125.97, 124.57, 54.70, 53.12, 30.79, 22.64; HRMS-ESI [M+H]+ calcu-
lated for C19H20N2OSe: 373.0814, found: 373.0825.
5j
O
5k
O
Ebselen
118.4 2.96
46.10 3.22
39.46 1.50
Donepezil
Controlc
4.1.5.3. 2-(4-(2-(Piperidin-1-yl)ethoxy)phenyl)benzo[d][1,2]sel-
a
Assay conditions: The reactions were performed in phosphate buffer (100 mM),
pH 7.5, with 1 mM ethylenediaminetetraacetate (EDTA), 2 mM GSH, 0.4 mM
enazol-3(2H)-one (5c).
White solid; yield 70%; purity 99.5%;
mp: 171.3–173.4 °C; 1H NMR (400 MHz, CDCl3) d 8.11 (d, J = 7.6 Hz,
1H), 7.71–7.60 (m, 2H), 7.53–7.41 (m, 3H), 6.97–6.95 (m, 2H), 4.13
(t, J = 6.0 Hz, 2H), 2.79 (t, J = 6.0 Hz, 2H), 2.52 (s, 4H), 1.76–1.35 (m,
6H); 13C NMR (101 MHz, CDCl3) d 165.90, 157.70, 137.83, 136.37,
132.35, 131.64, 129.35, 127.32, 126.46, 123.74, 115.21, 66.30,
57.83, 55.03, 25.92, 24.17; HRMS-ESI [M+H]+ calculated for C20H22-
N2O2Se: 403.0920, found: 403.0906.
NADPH, 1.3 U/mL glutathione reductase (GR), 80 lM selenium compounds, and
1.6 mM H2O2.
b
All of these values were measured in triplicate for the first 10 s, and the average
values and standard deviations were obtained.
c
The control values were obtained from the reduction of H2O2 by GSH in the
absence of selenium compounds.
extracted with CH2Cl2; the mixture was then dried and filtered to
obtain the crude products, which were purified by column chroma-
tography using silica gel and CH2Cl2/MeOH (62–80% yields).
4.1.5.4.
5,6-Dimethoxy-2-(4-(pyrrolidin-1-ylmethyl)phenyl)
White solid; yield
benzo[d][1,2]selenazol-3(2H)-one (5d).
76%; purity 98.6%; mp: 166.2–167.5 °C; 1H NMR (400 MHz, CDCl3)
d 7.58–7.53 (m, 3H), 7.40 (d, J = 6.8 Hz, 2H), 7.08 (s, 1H), 3.99–3.98
(m, 6H), 3.70 (s, 2H), 2.61 (s, 4H), 1.83 (s, 4H); 13C NMR (101 MHz,
CDCl3) d 164.78, 152.88, 148.14, 137.48, 135.42, 129.47, 128.92,
124.05, 119.06, 108.96, 104.22, 58.58, 55.37, 55.24, 52.79, 22.38;
HRMS-ESI [M+H]+ calculated for C20H22N2O3Se: 419.0869, found:
419.0861.
4.1.5.1.
2-(4-(Piperidin-1-ylmethyl)phenyl)benzo[d][1,2]sele-
nazol-3(2H)-one (5a).
White solid; yield 80%; purity 99.7%;
mp: 192.3–193.1 °C; 1H NMR (400 MHz, CDCl3) d 8.15–8.08 (m,
1H), 7.69–7.62 (m, 2H), 7.56 (d, J = 8.4 Hz, 2H), 7.51–7.43 (m,
1H), 7.39 (d, J = 8.4 Hz, 2H), 3.49 (s, 2H), 2.40 (s, 4H), 1.64 (m,
6H); 13C NMR (101 MHz, CDCl3) d 165.73, 137.80, 137.70, 137.36,
Table 3
Effect of H2O2 and GSH concentrations on Vmax, Km, kcat, and
g
for ebselen, 5e, and 5i
M minꢀ1
Compound
Vmax
(l
)
Km (mM)
kcat (minꢀ1
)
g )
(mMꢀ1 minꢀ1
Ebselen
H2O2 (variable)a
GSH (variable)b
188.68
307.69
0.83
2.34
2.36
3.85
2.85
1.64
5e
H2O2 (variable)a
GSH (variable)b
869.57
308.64
7.50
2.37
10.87
3.86
1.45
1.63
5i
H2O2 (variable)a
GSH (variable)b
316.46
276.24
5.36
2.47
3.96
3.46
0.74
1.40
a
Assay conditions: phosphate buffer (100 mM), pH 7.5, containing 2 mM GSH, 0.4 mM NADPH, 1 mM EDTA, 1.3 U/mL GR, variable H2O2, and 80
Assay conditions: phosphate buffer (100 mM), pH 7.5, containing variable GSH, 0.4 mM NADPH, 1 mM EDTA, 1.3 U/mL GR, 1.6 mM H2O2, and 80 lM test compound.
l
M test compound.
b
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066

6
Z. Luo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
4.1.5.5.
5,6-Dimethoxy-2-(4-(piperidin-1-ylmethyl)phenyl)
White solid; yield
d 7.52 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.07 (s, 1H), 6.92 (d, J = 8.8 Hz,
benzo[d][1,2]selenazol-3(2H)-one (5e).
2H), 4.00–3.97 (m, 8H), 2.46–2.29 (m, 6H), 1.82–1.76 (m, 2H),
71%; purity 98.9%; mp: 204.3–206.6 °C; 1H NMR (400 MHz, CDCl3)
d 7.49–7.38 (m, 3H), 7.28 (s, 1H), 7.16 (d, J = 1.8 Hz, 1H), 6.97 (s,
1H), 3.89–3.87 (m, 6H), 3.39 (s, 2H), 2.31 (s, 4H), 1.70 (s, 4H),
1.50–1.47 (m, 2H); 13C NMR (101 MHz, CDCl3) d 165.75, 153.83,
149.11, 138.07, 137.12, 130.41, 129.96, 124.95, 120.06, 109.97,
105.14, 63.18, 56.36, 56.25, 54.46, 25.92, 24.32; HRMS-ESI
[M+H]+ calculated for C21H24N2O3Se: 433.1026, found: 433.1027.
1.71–1.64 (m, 2H), 1.62–1.56 (m, 4H), 1.43 (s, 2H); 13C NMR
(101 MHz, CDCl3)
d 165.97, 157.83, 153.73, 149.09, 131.70,
130.55, 127.29, 119.78, 115.04, 109.98, 105.15, 68.10, 59.00,
56.35, 56.24, 54.57, 27.35, 25.92, 24.43, 23.39; HRMS-ESI [M+H]+
calculated for C24H30N2O4Se: 491.1445, found: 491.1455.
4.2. Determination of the inhibitory effect on AChE, and BuChE
activities
4.1.5.6. 2-(4-((Benzyl(methyl)amino)methyl)phenyl)-5,6-dim-
ethoxybenzo[d][1,2]selenazol-3(2H)-one (5f).
White solid;
We followed the method developed by Ellman et al. for the
determination. Acetylcholinesterase (from electric eel or human
erythrocytes), butyrylcholinesterase (from equine serum), 5,50-
dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine chloride
(ATC), and butyrylthiocholine chloride (BTC) were purchased from
Sigma Aldrich. Prototype tacrine and donepezil were used as the
reference compounds. Five different concentrations of each
compound ranging from 20% to 80% were used to determine the
inhibition of cholinesterase activity.
yield 79%; purity 97.5%; mp: 178.3–179.4 °C; 1H NMR (400 MHz,
CDCl3) d 7.60–7.51 (m, 3H), 7.42 (d, J = 6.9 Hz, 2H), 7.40–7.29 (m,
5H), 7.07 (s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.54 (d, J = 5.5 Hz, 4H),
2.21 (s, 3H); 13C NMR (101 MHz, CDCl3) d 165.74, 153.87, 149.15,
139.20, 138.12, 137.93, 130.31, 129.63, 128.88, 128.24, 126.97,
125.08, 120.06, 110.03, 105.06, 61.87, 61.23, 56.38, 56.27, 42.27;
HRMS-ESI [M+H]+ calculated for C24H24N2O3Se: 469.1026, found:
469.1004.
All of the assays were conducted in 0.1 M KH2PO4/K2HPO4 pH
8.0 using a Shimadzu UV-2450 spectrophotometer. The AChE solu-
tions were prepared to a concentration of 2.0 U/mL in 2 mL ali-
quots. The assay medium (1 mL) consisted of phosphate buffer
4.1.5.7.
5,6-Dimethoxy-2-(4-(2-(pyrrolidin-1-yl)ethyl)phenyl)
Gray solid; yield
benzo[d][1,2]selenazol-3(2H)-one (5g).
63%; purity 97.4%; mp: 176.2–179.4 °C; 1H NMR (400 MHz, CDCl3)
d 7.54–7.52 (m, 3H), 7.28 (s, 2H), 7.12 (s, 1H), 3.99 (s, 3H), 3.98 (s,
3H), 3.09–2.81 (m, 8H), 1.95 (s, 4H); 13C NMR (101 MHz, CDCl3) d
165.85, 153.70, 149.06, 137.90, 137.19, 130.90, 129.41, 125.40,
120.21, 109.77, 105.85, 57.50, 56.38, 56.22, 54.07, 33.97, 23.40;
HRMS-ESI [M+H]+ calculated for C21H24N2O3Se: 433.1026, found:
433.1042.
(pH 8.0), 50
eel). The test compounds were added to the assay solution and
pre-incubated at 37 °C for 15 min, and 50 L of 0.01 M ATC was
lL of 0.01 M DTNB, and 20 lL of AChE (from electric
l
then added immediately. The activity was determined by measur-
ing the increase in the absorbance at 412 nm at 37 °C; the
measurements were obtained at 1 min intervals. The data from
the concentration–inhibition experiments of the inhibitors were
calculated through nonlinear regression analysis using the Graph-
Pad Prism 5.0 program package (GraphPad Software Inc), and this
analysis provided estimates of the IC50 values (concentration of the
drug that inhibits 50% of the enzyme activity).
4.1.5.8.
5,6-Dimethoxy-2-(4-(2-(piperidin-1-yl)ethyl)phenyl)
White solid; yield
benzo[d][1,2]selenazol-3(2H)-one (5h).
69%; purity 98.4%; mp: 193.2–195.2 °C; 1H NMR (400 MHz, CDCl3)
d 7.53–7.50 (m, 3H), 7.27 (s, 2H), 7.08 (s, 1H), 3.99 (d, J = 4.0 Hz,
6H), 2.91–2.82 (m, 2H), 2.66–2.58 (m, 2H), 2.53 (s, 4H), 1.66 (d,
J = 4.6 Hz, 6H); 13C NMR (101 MHz, CDCl3) d 165.76, 153.80,
149.10, 138.99, 137.30, 130.36, 129.55, 125.41, 119.99, 109.96,
105.09, 61.02, 56.39, 56.27, 54.49, 32.99, 25.81, 24.28; HRMS-ESI
[M+H]+ calculated for C22H26N2O3Se: 447.1183, found: 447.1198.
The inhibition of BuChE was measured as described above, with
the exception that 0.02 M BTC was used as the substrate.
4.3. Coupled reductase assay
The GPx-like activity of the organoselenium compounds was
determined through a spectrophotometric method at 340 nm, as
described by Wilson et al.25 The test mixture contained GSH
(2 mM), EDTA (1 mM), glutathione reductase (1.3 U/mL), and
NADPH (0.4 mM) in 100 mM potassium phosphate buffer, pH 7.5.
4.1.5.9. 5,6-Dimethoxy-2-(4-(2-(piperidin-1-yl)ethoxy)phenyl)
benzo[d][1,2]selenazol-3(2H)-one (5i).
White solid; yield
65%; purity 99.4%; mp: 162.1–163.2 °C; 1H NMR (400 MHz, CDCl3)
d 7.53 (s, 1H), 7.48–7.46 (m, 2H), 7.11 (s, 1H), 6.96–6.94 (m, 2H),
4.20–4.12 (m, 2H), 3.98 (s, 6H), 2.87–2.80 (m, 2H), 2.57 (s, 4H),
1.65 (s, 4H), 1.47 (s, 2H); 13C NMR (101 MHz, CDCl3) d 165.96,
157.44, 153.73, 149.08, 131.98, 130.52, 127.30, 119.75, 115.16,
109.96, 105.13, 66.06, 57.73, 56.37, 56.26, 54.97, 25.71, 24.02;
HRMS-ESI [M+H]+ calculated for C22H26N2O4Se: 463.1132, found:
463.1114.
The GPx samples (80 lM) were added to the test mixture at
25 °C, and the reaction was started through the addition of H2O2
(1.6 mM). The initial reduction rates were calculated from the oxi-
dation 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 6.22 mMꢀ1 cmꢀ1 as the extinction
coefficient for NADPH.
4.1.5.10.
5,6-Dimethoxy-2-(4-(3-(piperidin-1-yl)propoxy)
White solid;
phenyl)benzo[d][1,2]selenazol-3(2H)-one(5j).
4.3.1. FOX assay
yield 62%; purity 99.5%; mp: 196.1–197.2 °C; 1H NMR (400 MHz,
CDCl3) d 7.52 (s, 1H), 7.45 (d, J = 8.3 Hz, 2H), 7.07 (s, 1H), 6.93 (d,
J = 8.4 Hz, 2H), 4.02 (t, J = 6.2 Hz, 2H), 3.97 (s, 6H), 2.50–2.44 (m,
2H), 2.40 (s, 3H), 2.02–1.93 (m, 2H), 1.75 (s, 1H), 1.64–1.55 (m, 4H),
1.44 (s, 2H); 13C NMR (101 MHz, CDCl3) d 165.97, 157.83, 153.72,
149.08, 131.75, 130.61, 127.25, 119.80, 115.07, 109.96, 105.22,
66.85, 56.32, 56.22, 55.87, 54.65, 26.81, 25.99, 24.44; HRMS-ESI
[M+H]+ calculated for C23H28N2O4Se: 477.1288, found: 477.1272.
The hydrogen peroxide scavenging activity was determined
through a ferrous ion oxidation-xylenol orange (FOX) assay with
minor modifications. The FOX reagent was prepared through the
addition of nine volumes of reagent 1 (4.4 mM butylated hydroxy-
toluene (BHT) in methanol) to one volume of reagent 2 (1 mM
xylenol orange and 2.56 mM ammonium ferrous sulfate in
250
trations were incubated with 1 mM H2O2 (100
in the dark, and the FOX agent (500 L) was then added. The reac-
l
M H2SO4). The test compounds (400
l
L) at different concen-
l
L) for 10 h at 37 °C
l
4.1.5.11. 5,6-Dimethoxy-2-(4-(4-(piperidin-1-yl)butoxy)phenyl)
tion mixture was then vortexed and incubated at room tempera-
benzo[d][1,2]selenazol-3(2H)-one (5k).
White solid; yield
ture for 30 min. The development of a violet color after the
64%; purity 99.5%; mp: 175.1–177.2 °C; 1H NMR (400 MHz, CDCl3)
addition of the test compounds indicates
a positive control
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066

Z. Luo et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
6. Claudio, S.; Caterina, T.; Claudia, S.; Marta, P.; Francesco, G. Curr. Chem. Biol.
2013, 7, 12.
7. Tan, S. M.; Sharma, A.; Yuen, D. Y. C.; Stefanovic, N.; Krippner, G.; Mugesh, G.;
Chai, Z.-L.; deHaan, J. B. PLoS ONE 2013, 8, 14.
8. Alberto, E. E.; Soares, L. C.; Sudati, J. H.; Borges, A. C. A.; Rocha, J. B. T.; Braga, A.
L. Eur. J. Org. Chem. 2009, 25, 4211.
9. Bhabak, K. P.; Mugesh, G. Acc. Chem. Res. 2010, 43, 1408.
10. Back, T. G.; Moussa, Z.; Parvez, M. Angew. Chem. 2004, 43, 1268.
11. Glass, R. S.; Farooqui, F.; Sabahi, M.; Ehler, K. W. J. Org. Chem. 1989, 54, 1092.
12. Saito, I.; Asano, T.; Sano, K.; Takakura, K.; Abe, H.; Yoshimoto, T.; Kikuchi, H.;
Ohta, T.; Ishibashi, S. Neurosurgery 1998, 42, 9.
reaction, whereas discoloration is associated with scavenging
activity. The FOX reagent without compound/H2O2 served as a
blank, and the FOX reagent with H2O2 served as a control. The
absorbance of the ferric–xylenol orange complex was measured
at 560 nm.
Acknowledgments
We thank the Natural Science Foundation of China (Nos.
21302235, 20972198), the Ph.D. Programs Foundation of the Min-
istry of Education of China (20120171120045) and Distinguished
Young Talents in Higher Education of Guangdong (2012LYM_003)
for financial support of this study.
13. Wilson, S. R.; Zucker, P. A.; Huang, R. R. C.; Spector, A. J. Am. Chem. Soc. 1989,
111, 5936.
14. Singh, V. P.; Singh, H. B.; Butcher, R. J. Eur. J. Org. Chem. 2011, 28, 5485.
15. Nakamura, Y.; Feng, Q.; Kumagai, T.; Torikai, K.; Ohigashi, H.; Osawa, T.;
Noguchi, N.; Niki, E.; Uchida, K. J. Biol. Chem. 2002, 277, 2687.
16. de Freitas, A. S.; Rocha, J. B. T. Neurosci. Lett. 2011, 503, 1.
17. Nascimento, V.; Alberto, E. E.; Tondo, D. W.; Dambrowski, D.; Detty, M. R.;
Nome, F.; Braga, A. L. J. Am. Chem. Soc. 2012, 134, 138.
18. Tong, W.; Collantes, E. R.; Chen, Y.; Welsh, W. J. J. Med. Chem. 1996, 39, 380.
19. Cardozo, M. G.; Iimura, Y.; Sugimoto, H.; Yamanishi, Y.; Hopfinger, A. J. J. Med.
Chem. 1992, 35, 584.
20. Sheng, R.; Lin, X.; Zhang, J.; Chol, K. S.; Huang, W.; Yang, B.; He, Q.; Hu, Y. Bioorg.
Med. Chem. 2009, 17, 6692.
21. Luo, W.; Su, Y.-B.; Hong, C.; Tian, R.-G.; Su, L.-P.; Wang, Y.-Q.; Li, Y.; Yue, J.-J.;
Wang, C.-J. Bioorg. Med. Chem. 2013, 21, 7275.
22. Rizzo, S.; Bartolini, M.; Ceccarini, L.; Piazzi, L.; Gobbi, S.; Cavalli, A.; Recanatini,
M.; Andrisano, V.; Rampa, A. Bioorg. Med. Chem. 2010, 18, 1749.
23. Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Featherstone, R. M. Biochem.
Pharmacol. 1961, 7, 88.
24. Singh, V. P.; Singh, H. B.; Butcher, R. J. Eur. J. Inorg. Chem. 2010, 4, 637.
25. Selvakumar, K.; Shah, P.; Singh, H. B.; Butcher, R. J. Chem. Eur. J. 2011, 17,
12741.
Supplementary data
Supplementary data (the experimental procedures for the
synthesis of the amine intermediates (4a–4h) and the full data
regarding the GPx-like activity of the test compounds) associated
with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.bmc.2013.12.066.
References and notes
1. Goedert, M.; Spillantini, M. G. Science 2006, 314, 777.
2. Scarpini, E.; Schelterns, P.; Feldman, H. Lancet. Neurol. 2003, 2, 539.
3. Terry, A. V.; Buccafusco, J. J. J. Pharmacol. Exp. Ther. 2003, 306, 821.
4. Zhao, Y.; Zhao, B. L. Front. Biosci. 2012, E4, 15.
26. Gul, M. Z.; Bhakshu, L. M.; Ahmad, F.; Kondapi, A. K.; Qureshi, I. A.; Ghazi, I. A.
BMC Complement. Altern. Med. 2011, 11, 64.
5. Papp, L. V.; Lu, J.; Holmgren, A.; Khanna, K. K. Antioxid. Redox Signaling 2007, 9,
775.
Please cite this article in press as: Luo, Z.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2013.12.066