Aurone Mannich base derivatives as promising multifunctional agents with acetylcholinesterase inhibition, anti-β-amyloid aggragation and neuroprotective properties for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.12.009

A series of aurone Mannich base derivatives were designed, synthesized and evaluated as multifunctional agents for the treatment of Alzheimer's disease. In vitro assays demonstrated that most of the derivatives were selective AChE inhibitors with good multifunctional properties. Among them, compound 7d exhibited outstanding inhibitory activity for RatAChE, EeAChE and HuAChE (IC₅₀= 0.00878 ± 0.0002 μM, 0.0212 ± 0.006 μM and 0.0371 ± 0.004 μM, respectively). Moreover, 7d displayed high antioxidant activity and could confer significant neuroprotective effect against H₂O₂-induced PC-12 cell injury. In addition, 7d also showed biometal chelating abilities, good self- and Cu²⁺-induced Aβ_1-42aggregation inhibitory potency and high BBB permeability. These multifunctional properties highlight 7d as promising candidate for further studies directed to the development of novel drugs against AD.

Full text of DOI:10.1016/j.ejmech.2016.12.009

Accepted Manuscript
Aurone Mannich base derivatives as promising multifunctional agents with
acetylcholinesterase inhibition, anti-β-amyloid aggragation and neuroprotective
properties for the treatment of Alzheimer's disease
Yan Li, Xiaoming Qiang, Li Luo, Xia Yang, Ganyuan Xiao, Qi Liu, Jiachen Ai,
Zhenghuai Tan, Yong Deng
PII:
S0223-5234(16)31015-7
10.1016/j.ejmech.2016.12.009
EJMECH 9100
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 October 2016
Revised Date: 28 November 2016
Accepted Date: 3 December 2016
Please cite this article as: Y. Li, X. Qiang, L. Luo, X. Yang, G. Xiao, Q. Liu, J. Ai, Z. Tan, Y. Deng,
Aurone Mannich base derivatives as promising multifunctional agents with acetylcholinesterase
inhibition, anti-β-amyloid aggragation and neuroprotective properties for the treatment of Alzheimer's
disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.009.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Aurone Mannich base derivatives as promising multifunctional agents with
acetylcholinesterase inhibition, anti-β-amyloid aggragation and neuroprotective
properties for the treatment of Alzheimer’s disease
a
a
a
a
a
b
b
Yan Li , Xiaoming Qiang , Li Luo , Xia Yang , Ganyuan Xiao , Qi Liu , Jiachen Ai , Zhenghuai
b,*
a,*
Tan , Yong Deng
a
Department of Medicinal Chemistry, Key Laboratory of Drug Targeting and Drug Delivery System
of the Education Ministry, West China School of Pharmacy, Sichuan University, Chengdu, 610041,
China
b
Institute of Traditional Chinese Medicine Pharmacology and Toxicology, Sichuan academy of
Chinese Medicine Sciences, Chengdu, 610041, China
Abbreviations: AD, Alzheimer’s disease; AChE, acetylcholinesterase; BuChE, butyrylcholinesterase;
EeAChE, Electrophorus electricus AChE; Aβ, β-amyloid; CAS, catalytic active site; PAS, peripheral
anionic site; ROS, reactive oxygen species; MAO, Monoamine oxidase; PBS, phosphate-buffered
saline; DTNB, 5,50-dithiobis-2-nitrobenzoic acid; TcAChE, Torpedo californica AChE; ORAC-FL,
Oxygen Radicals Absorbance Capacity by Fluorescence; AAPH, 2,2’-Azobis(amidinopropane)
dihydrochloride; ThT, thioflavin T; HEPES, 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid.
*Corresponding Author.
Phone, +86-28-85258982; E-mail: tanzhh616@163.com (Zhenghuai Tan)
Phone, +86-28-85503790; E-mail: dengyong@scu.edu.cn (Yong Deng)
1

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Abstract
A series of aurone Mannich base derivatives were designed, synthesized and evaluated as
multifunctional agents for the treatment of Alzheimer’s disease. In vitro assays demonstrated that
most of the derivatives were selective AChE inhibitors with good multifunctional properties. Among
them, compound 7d exhibited outstanding inhibitory activity for RatAChE, EeAChE and HuAChE
(
IC = 0.00878 ± 0.0002 µM, 0.0212 ± 0.006 µM and 0.0371 ± 0.004 µM, respectively). Moreover,
50
7d displayed high antioxidant activity and could confer significant neuroprotective effect against
H O -induced PC-12 cell injury. In addition, 7d also showed biometal chelating abilities, good self-
2
2
2
+
and Cu -induced Aβ
aggregation inhibitory potency and high BBB permeability. These
-42
1
multifunctional properties highlight 7d as promising candidate for further studies directed to the
development of novel drugs against AD.
Keywords:
Alzheimer’s disease; Aurone Mannich base derivatives; Acetylcholinesterase inhibitors; Aβ
aggregation inhibitors; Multifunctional agents
2

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1. Introduction
Alzheimer’s disease (AD), the most common cause of dementia, is a fatal and age-related
neurodegenerative disorder and a major public health concern. This disease is progressive and
irreversible, with the symptoms of memory loss, behavioral disturbances and many other cognitive
impairments [1]. Although the exact etiology of AD remains unknown, it is established that multiple
interconnected factors, including β-amyloid (Aβ) deposits, τ-protein aggregation, oxidative stress,
dyshomeostasis of biometals and degeneration of cholinergic neurons in the central nervous system
(CNS), are related to the initiation, development and evolution of the disease [2].
Among the various causative factors of AD, the impairment of the cholinergic
neurotransmission in brain, resulting from the decreased levels of acetylcholine (ACh), is considered
to be a critical determinant of AD pathogenesis and progression. Hence, one efficient approach to
improve the cholinergic neurotransmission in AD brain is to reduce the ACh metabolism [3].
Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are the two
types of cholinesterase enzymes responsible for the enzymatic hydrolysis of ACh. Although some
studies reported the potential role BuChE played in AD pathogenesis, the pharmacological
mechanism of BuChE was not yet completely understood. Moreover, BuChE primarily distributes in
plasma, liver and muscle tissues [4]. Accordingly, serious inhibition of BuChE may contribute to the
potential peripheral side effects of ChE inhibitors, for instance tacrine, the dual AChE and BuChE
inhibitor, has shown severe hepatotoxicity and many other adverse effects [5]. Therefore, the
development of AChE inhibitors, with expectation of lower side effects, may be a more rational
therapeutic strategy for AD.
The crystallographic structure of AChE from Torpedo californica (Tc) shows three crucial
binding sites: the catalytic triad (Ser200, His440 and Glu327) at the bottom of the active site gorge,
the catalytic anionic site (CAS) at the vicinity of the catalytic triad and the peripheral anionic site
(PAS) at the entrance of the gorge [4, 6]. Inhibitors binding to any one of the three sites can restrain
the AChE activity. Nevertheless, AChE is implicated in AD pathogenesis through not only
hydrolyzing ACh, but also influencing the Aβ deposition. It is demonstrated that AChE can
accelerate the formation and aggregation of Aβ fibrils through PAS, and further increase its
neurotoxicity [7, 8]. Therefore, inhibitors that could simultaneously bind two or more active sites of
AChE, including PAS, hold great promise for developing disease-modifying drugs against AD.
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Another important hypothesis suggests that the increased production and accumulation of Aβ
oligomeric aggregates in brain plays a pivotal role in AD pathogenesis. And these aggregates can
trigger the pathogenic cascade and finally cause the neuronal loss and dementia [9]. There are two
major isoforms of Aβ peptides: Aβ1-40 and Aβ1-42. Although Aβ1-40 presents in larger amounts in brain,
Aβ1-42 displays lower solubility and higher tendency to aggregate [10]. Aβ1-42 can aggregate into
oligomers and fibrils in the brain, and eventually cause severe neuronal toxicity [11]. Therefore,
preventing the Aβ1-42 aggregation in the brain may provide neuroprotective effects and serve as a
potent therapeutic strategy for AD treatment.
2
+
2+
2+
Recently, more evidence have indicated that excessive biometals, such as Cu , Fe , Zn and
3
+
Al , clearly exist in the brains of AD patients, especially in Aβ plaques [12, 13]. These biometals are
known to interact with Aβ peptides and promote their aggregation [11]. On the other hand, studies
showed that Aβ neurotoxicity depends on the catalytic generation of H O , which is enhanced by the
2
2
2
+
Aβ-Cu complexes. Excess of these redox-active metals could increase the production of reactive
oxygen species (ROS) and cause oxidative stress. Furthermore, oxidative stress is considered to be
one of the most important factors associated with AD pathogenesis. The “oxidative stress
hypothesis” states that the increasing production of ROS and free radicals leads to deleterious effects
on the cellular components and damages on biological molecules such as proteins, DNA and lipids [7,
8]. Unlike other organs, CNS is highly sensitive to oxidative stress due to its rich amount of
peroxidizable fatty acids, high demand for oxygen and relative paucity of classical antioxidant
systems [14]. Moreover, oxidative stress can facilitate Aβ aggregation by generating modified Aβ
species which are prone to aggregate and resistant to elimination [15]. So it is apparent that biometals
homeostasis and antioxidant protection are necessary in AD patients.
Another important contributor to the oxidative stress in AD is the catabolism of monoamine,
which is undertaken by the monoamine oxidases (MAOs). MAOs are flavin adenine dinucleotide
(FAD)-containing enzymes and exist as MAO-A and -B [16]. These enzymes are responsible for the
degradation of neurotransmitters and xenobiotic amines. However, this biochemical activity of
MAOs contributes to the generation of hydroxyl radicals, the harmful members of the oxygen free
radical group [17]. Recently, MAO inhibitors have been re-evaluated in many age-related
pathologies and studies indicated that they benefit the neurodegenerative diseases to various degrees.
For instance, selective MAO-A inhibitors such as clorgyline and moclobemide are effective
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antidepressants, whereas MAO-B inhibitors such as selegiline and rasagiline are beneficial for both
Alzheimer’s and Pakinson’s disease (PD) [17]. Therefore, drugs with MAO inhibitory activity might
be beneficial for AD treatment in multiple aspects [18].
Thus far, there are five drugs approved by FDA for clinical treatment of AD, including four
acetylcholinesterase inhibitors (donepezil, rivastigmine, galantamine and Huperzine A) and
memantine. These drugs only target the symptoms of AD and can temporarily improve the cognitive
and daily function. But, they cannot mitigate the progression of this disease. Since AD has been
acknowledged as a disorder with uncertain etiology and complex disease networks, it becomes clear
that the modulation of a single target seems insufficient. Therefore, medicinal chemists have devoted
great efforts into the development of molecules that could simultaneously interact with two or more
targets associated with AD pathogenesis. Such molecules are the so-called “multi-target-directed
ligands” (MTDLs).
Aurones, 2-benzylidenebenzofuran-3(2H)-ones, are naturally occurring structural isomerides of
flavones (Figure 1a) [19]. Numerous researches have focused on aurones because they exhibited
various bioactivities. Furthermore, many studies have indicated that both aurones and the synthesized
analogs showed high affinities toward Aβ aggregates [20-22] and good MAO inhibitory activities
[
23]. Recently, Sheng et al. have synthesized a series of aurone derivatives bearing various
dialkylbenzyl amine moieties (Figure 1b) [24]. These aurone derivatives exhibited good selective
AChE inhibitory activities. It revealed that aurone could serve as an excellent leading scaffold to
design new multifunctional agents against AD. In addition, phenolic Mannich bases have presented
numerous biological activities, many of which might benefit the AD treatment (Figure 1c). Studies
demonstrated that substances bearing phenolic Mannich base moieties exhibited good antioxidant [25,
26], anti-inflammatory [27] and AChE inhibitory [28, 29] activities. These results make Mannich
bases promising for the anti-AD drug design. Moreover, various benzylamine moieties have been
reported previously by our group as the defined AChE inhibitory pharmacophores [30, 31].
Collectively, we designed and synthesized a series of aurone Mannich base derivatives with the aim
to act as multifunctional AChE inhibitors, with anti-Aβ aggregation, MAO inhibition, antioxidant
and biometal chelating properties (Figure 2).
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Figure 1. Aurone scaffolds (a), aurone derivatives (b) and phenolic Mannich bases (c).
Figure 2. Structures of the designed aurone Mannich base derivatives 7-9.
2. Results and discussion
2.1. Chemistry
The synthetic pathways of target derivatives were summarized in Scheme 1. Compound 2
(3,4-dimethoxyphenol) was synthesized from 3,4-dimethoxybenzaldehyde (1) in good yield
according to an improved solvent-free Daking oxidation protocol [32]. Compound 4 was produced
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from 2 after Fries rearrangement and cyclization reaction. The key intermediates 5 and 6 were
afforded by the condensation of 4 with m- or p-hydroxybenzaldehyde, respectively, in ethanolic 50%
KOH solution. As reported in many literatures, only Z stereoisomers were isolated. The
stereochemistry of the aurone diastereomers has been elucidated by NMR spectroscopic
measurements and by X-ray diffraction analysis [33-36]. Due to the deshielding effect of the
carbonyl group, the vinyl protons should give a signal at a greater chemical shift than in the E isomer,
the value is higher than 7.0 [24]. Mannich reactions of compounds 5 and 6 with paraformaldehyde
and different secondary amines in EtOH gave the novel aurone Mannich base derivatives 7a-n, 8a-b
and 9a-b. The difference in reactivity allows the regioselectivity for Mannich reaction. The exclusive
formation of ortho-aminoalkylated products is attributed to the directing effect and the hydrogen
bonding between nitrogen and the o-hydroxyl group, which afford a stabilized six-membered ring
1
[
37]. Additionally, the H NMR signals of the aromatic hydrogens in B ring of 7a-n showed no triplet,
which indicated the aminoalkylation was not occurred at 2’-position. Moreover, according to the
1
relevant studies, the H NMR, HMBC data and X-ray crystallography results provided by the
literatures [29, 37, 38] demonstrated the specific structures of 7a-n with 4’-aminoalkylation. All of
1
the target compounds were characterized by H NMR and HR-ESI-MS, and most of them were
1
3
further characterized by C NMR.
Scheme 1. Synthesis of aurone Mannich base derivatives 7-9. Reagents and conditions: (a) mCPBA,
7

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then 10% NaOH, r.t., 4 h; (b) ClCH COCl, AlCl , EtOAc, r.t., overnight; then 50 °C, 6 h; (c) NaOAc,
2
3
MeOH, reflux for 4 h; (d) m- or p-hydroxybenzaldehyde, 50% KOH, EtOH, r.t., 4 h; (e)
paraformaldehyde, secondary amine, EtOH, reflux for 24-48 h.
2.2. Pharmacology
2.2.1. Evaluation of AChE and BuChE inhibitory activities
All the synthesized aurone Mannich base derivatives were evaluated for their inhibitory
activities toward AChE and BuChE in vitro according to modified Ellman’s method [39]. AChE
activity was measured from both rat cortex homogenate and Electrophorus electricus, and BuChE
activity was measured from rat serum. Moreover, some representative compounds with relatively
good potency were selected to re-evaluate using human erythrocyte AChE (HuAChE). Donepezil
was used as a reference compound. The ChE inhibitory results are summarized in Table 1.
Most of the tested compounds showed significant AChE inhibitory activities with IC values
5
0
ranging from nanomolar to micromolar. A structure-activity relationship analysis showed that the
AChE inhibitory potency was closely related to the amine moiety and its substituted position.
Generally, the aliphatic amine substituted compounds possessed greater AChE inhibitory activities
than the benzylamine substituted compounds. And the results of 7d, 7m, 8a and 8b demonstrated that
compounds with amines substituted at 4’-position were more appropriate for the AChE inhibitory
potency. The weakened potency of the bis-Mannich bases 9a and 9b might be the result of their steric
hindrance, which could interfere with the interaction between the compounds and the enzyme.
Among the tested derivatives, 7d exhibited the most potent inhibitory activity for RatAChE,
EeAChE and HuAChE (IC = 0.00878 ± 0.0002 µM, 0.0212 ± 0.006 µM and 0.0371 ± 0.004 µM,
5
0
respectively), which was more excellent than donepezil under our experimental condition.
All the aurone Mannich base derivatives were inactive or weak on BuChE inhibitory activities
at 50 µM, which made them AChE selective inhibitors. As mentioned in introduction, this selectivity
profile may be good for the reduction of the associated side effects and benefit the AD treatment.
Based on the above results, we selected 7d for kinetic analysis of AChE inhibition.
Table 1. In vitro inhibition of AChE and BuChE and Oxygen Radical Absorbance Capacity (ORAC,
Trolox Equivalents) by aurone Mannich base derivatives and donepezil.
a
e
f
Compd.
NR R
IC (µM) ± SD
RatBuChE (%)
ORAC
3
4
50
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b
c
d
RatAChE
EeAChE
HuAChE
g
h
h
h
h
5
6
—
—
A
B
131 ± 5.35
297 ± 10.8
36.3 ± 1.08
64.4 ± 2.10
8.93 ± 1.67
0.486 ± 0.011
2.70 ± 0.871
NT
n.a.
n.a.
n.a.
n.a.
1.51 ± 0.02
1.52 ± 0.07
2.02 ± 0.05
2.06 ± 0.03
1.79 ± 0.06
1.66 ± 0.02
1.58 ± 0.04
1.78 ± 0.02
1.61 ± 0.08
1.01 ± 0.02
1.62 ± 0.06
1.35 ± 0.03
1.17 ± 0.09
1.47 ± 0.05
1.56 ± 0.08
2.02 ± 0.11
1.69 ± 0.10
1.60 ± 0.07
1.56 ± 0.12
1.47 ± 0.06
—
g
NT
7
a
25.8 ± 0.514
0.108 ± 0.002
0.246 ± 0.007
24.3 ± 1.18
0.70 ± 0.032
1.32 ± 0.028
7
b
7
c
C
D
E
19.0 ± 0.6
12.9 ± 0.2
7
d
0.00878 ± 0.0002 0.0212 ± 0.006 0.0371 ± 0.004
h
7
e
8.66 ± 1.92
16.3 ± 0.812
24.0 ± 0.481
20.2 ± 0.442
11.9 ± 2.20
11.7 ± 1.90
6.0 ± 0.242
14.0 ± 0.280
32.1 ± 1.12
n.a.
h
7
f
F
n.a.
h
7
7
g
G
H
I
n.a.
h
h
g
h
h
n.a.
n.a.
NT
n.a.
h
7
i
20.0 ± 0.341
74.4 ± 1.22
94.1 ± 3.27
44.9 ± 2.21
7.32 ± 1.73
288 ± 12.1
9.10 ± 0.991
28.9 ± 1.28
35.8 ± 1.09
37.0 ± 2.02
6.40 ± 1.11
431 ± 20.1
26.3 ± 1.137
n.a.
g
h
7
j
J
NT
n.a.
g
h
7
k
K
L
NT
n.a.
g
7
l
NT
5.21 ± 0.3
19.6 ± 0.8
7
m
M
N
D
M
D
M
—
6.69 ± 0.722
g
h
7
n
a
b
a
b
NT
n.a.
h
8
0.127 ± 0.005
28.9 ± 0.562
23.3 ± 0.822
42.1 ± 1.15
0.019 ± 0.0003
0.411 ± 0.011
17.1 ± 0.233
57.2 ± 2.11
34.6 ± 1.09
0.51 ± 0.066
n.a.
g
h
8
9
9
NT
n.a.
g
h
NT
n.a.
g
NT
14.9 ± 0.9
Donepezil
0.0274 ± 0.001 0.0356 ± 0.0012 IC50 = 37.3 ± 1.12µM
a
IC values represent the concentration of inhibitor required to decrease enzyme activity by 50% and are the mean
5
0
of 3 independent experiments, each performed in triplicate (SD = standard deviation ).
b
From 5% rat cortex homogenate.
c
From Electrophorus electricus.
d
From human erythrocytes.
e
From rat serum. Tested compounds were used at 50 µM.
f
The mean ± SD of the three independent experiments. The data are expressed as µM of Trolox equiv/µM of tested
compound.
g
NT = not tested.
h
n.a. = no active. Compounds defined “no active” means that percent inhibition is less than 5.0% at a concentration
of 50 µM in the assay conditions.
2.2.2. Kinetic study for the inhibition of AChE
To gain further insight into the inhibitory mechanism of this family of compounds, 7d, the most
potent AChE inhibitor among these compounds, was chosen for a kinetic study using EeAChE. The
graphical presentation of the reciprocal Lineweaver-Burk plots (Figure 3) showed that with the
concentrations of 7d increasing, both the slopes (decreased Vmax) and intercepts (higher K ) were
m
increased. Replots of the slope versus concentration of compound 7d gave an estimate of the
9

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inhibition constant (K = 0.012 µM). This pattern indicated a mixed-type inhibitory behavior for 7d
i
as the consequence of binding to both the CAS and PAS of AChE.
Figure 3. Kinetic study on the mechanism of EeAChE inhibition by compound 7d. Overlaid
Lineweaver-Burk reciprocal plots of AChE initial velocity at increasing substrate concentration
(0.1-0.4 mM) in the absence and presence of 7d are shown. Lines were derived from a weighted
least-squares analysis of data points.
2.2.3. Molecular modeling study
To explore the possible interacting mode of the aurone Mannich base derivatives with Torpedo
californica (Tc)AChE (PDB code: 1EVE), a molecular modeling study was performed using the
docking program, AutoDock 4.2 package with Discovery Studio 2.5 [40]. The results showed in
Figure 4 indicated that 7d occupied the entire enzymatic CAS, the mid-gorge sites and the PAS, and
binds simultaneously to both the catalytic site and peripheral site. In the TcAChE-7d complex, the
3’-OH group was involved in a hydrogen bond interaction with Asp72. Moreover, the
benzofuran-3(2H)-one moiety of 7d interacted with Trp279 and Tyr334 via parallel π-π interactions.
Similarly, the B ring was observed to adopt parallel π-π interactions with Phe330. And the piperidine
moiety potentially induced a hydrophobic interaction with Trp84. Other interactions could be
observed between the ligand 7d and Tyr70, Tyr121, Glu199, Phe331, His440 and Gly441. The
docking results, consistent with the kinetic analysis, indicated that compound 7d could
simultaneously bind to the CAS and PAS of AChE, and explained the high AChE inhibitory activities
1
0

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of these aurone Mannich base derivatives.
Figure 4. Representation of compound 7d (grey stick) interacting with residues in the binding site of
TcAChE (PDB code: 1EVE), highlighting the protein residues that participate in the main interactions
with the inhibitor.
2.2.4. Antioxidant activity assay
The reduction of the oxidative stress is another crucial aspect in designing agents for AD
treatment. The antioxidant activities of the aurone Mannich base derivatives were evaluated by the
well-established ORAC-FL method (oxygen radical absorbance capacity by fluorescein) [41] and the
results are shown in Table 1. The activities of tested compounds to scavenge radicals were expressed
as Trolox (a water-soluble vitamin E analog) equivalent. And their relative activity at concentration
of 5 µM was compared with the highly potent compound Trolox. All the tested compounds displayed
high peroxyl radical absorbance capacities, ranging from 1.01 to 2.06-fold of Trolox. Compound 7b
showed the most potent antioxidant activity of this family with ORAC-FL values of 2.06 ± 0.03
Trolox equivalents. In general, the aliphatic amine substituted compounds possessed higher
antioxidant activities than the benzylamine substituted compounds. However, the substituted
positions of the hydroxyl group and the amines showed no significant influence on the potency.
1
1

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2
.2.5. Neuroprotective effect on PC-12 cell injury induced by H O
2
2
The neuroprotective ability of our derivative against oxidative stress was evaluated using the
MTT assay [42], with compound 7d as representative. The active mitochondrias in living cells can
cleave MTT to produce formazan, the amount of which is a direct reflection of the living cell number.
We first evaluated the cytotoxicity of 7d toward PC-12 cells. As shown in Figure 5A, the nontoxic
concentration of 7d was below 50 µM. For the neuroprotective effect evaluation, cell viability
remarkably decreased after exposure of PC-12 cultures to H O , suggesting that PC-12 cells were
2
2
highly sensitive to H O induction (Figure 5B). However, when the cells were preincubated with
2
2
compound 7d (1-50 µM), H O -induced cell toxicity significantly attenuated. Compared with the
2
2
reference compound Trolox (100 µM), 7d was more efficient at 1 µM and showed similar potency at
0 and 50µM. The slightly decrease in the potency with higher dose might be the combined results of
1
the neuroprotective effect and cytotoxicity of 7d.
To further investigate the neuroprotective effect of 7d, lactate dehydrogenase (LDH) assay was
performed. The release of LDH to the supernatant suggests an increase of cell membrane damage.
Similar to the above PC-12 cell viability assay, after the exposure to H O , the LDH activities in the
2
2
culture supernatants were measured. As shown in Figure 5C, the LDH activity dramatically
increased after exposure of PC-12 cultures to H O . However, preincubation with 7d (0.1-50 µM)
2
2
could significantly reduce the LDH release and protect PC-12 cells from H O -induced injury. And
2
2
7
d was more potent than Trolox.
Collectively, according to the above results, compound 7d exhibited good neuroprotective effect
on PC-12 cell injury induced by H O .
2
2
1
2

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Figure 5. (A) Cytotoxicity of 7d toward PC-12 cells. (B and C) Attenuation of H O -induced PC-12
2
2
cell injury by compound 7d. Cell viability was assessed by measuring the MTT reduction. Three
independent experiments were carried out in triplicate. Data were expressed as mean ± SD and
percentage of control value. ## p < 0.01 vs. control; ** p < 0.01, * p < 0.05 vs. H O group.
2
2
2.2.6. Metal-chelating studies
2
+
2+
2+
The chelating abilities of compounds 7d and 7m toward biometals such as Cu , Zn , Fe and
3
+
Al were evaluated by UV-visual spectrometry, and the results are shown in Figure 6. It could be
seen that, the spectrum of 7d was significantly changed upon the addition of CuCl , FeSO and AlCl .
2
4
3
The maximum absorption at 348 nm shifted to 342 nm, 342 nm and 340 nm respectively and the
absorption increased, which indicated a possible interaction between these biometals and 7d. No
significant change was observed upon the addition of ZnCl . Similarly, the spectrum of 7m was
2
significantly changed upon the addition of these biometals. The absorption peak at 254 nm shifted to
2
66 nm, 264 nm, 264 nm and 272 nm respectively, with the addition of CuCl , ZnCl , FeSO and
2 2 4
AlCl . Similar to 7d, slight shifts of the maximum absorption at 346 nm were observed and the
3
absorption increased dramatically. These results revealed the ability of 7m to interact with biometals.
2
+
2+
To determine the stoichiometry of 7d-Cu and 7m-Cu complexes, molar ratio method was
performed by preparing the methanol solutions of 7d and 7m with ascending amounts of CuCl . The
2
UV spectra were used to obtain the absorbance of the 7d and 7m complexes and different
concentrations of CuCl at 346 nm and 344 nm respectively. As indicated in Figure 6C and 6D, the
2
absorbance linearly increased initially and then plateaued. The points for the straight lines to intersect
were determined to be at a mole proportion of 0.50 and 0.98, revealing 2:1 and 1:1 stoichiometry for
1
3

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2
+
2+
1
4e-Cu and 15b-Cu complexes, respectively.
Figure 6. UV spectra of compound 7d (A) and 7m (B) (37.5 µM in methanol) alone or in the
presence of CuCl , ZnCl , FeSO or AlCl (37.5 µM, in methanol). Determination of the
2
2
4
3
2
+
stoichiometry of complex-Cu by using the molar ratio method of titrating the methanol solution of
compound 7d (C) and 7m (D) with ascending amounts of CuCl . The final concentration of tested
2
2
+
compound was 37.5 µM, and the final concentration of Cu ranged from 3.75 to 150 µM.
2
+
2
.2.7. Inhibition of self- and Cu -induced Aβ aggregation
1-42
2
+
The inhibitory activities of the target compounds on self- and Cu -induced Aβ
aggregation
-42
1
were determined by a thioflavin T (ThT) fluorescence assay, with curcumin as a reference compound
43-46]. The results were summarized in Table 2. It indicated that these aurone Mannich base
[
2
+
derivatives exhibited moderate to good inhibitory activities of self- and Cu -induced Aβ
1
-42
1
4

ACCEPTED MANUSCRIPT
aggregation (31.5-80.5% and 23.6-83.9% at 25 µM respectively), compared with that of curcumin
(41.3 ± 0.9% and 67.2 ± 1.3% respectively). And most of the derivatives possessed higher potency
than the key intermediates aurone 5 and 6. Contrary to the conclusion resulting from the AChE
inhibition assay, generally, the benzylamine substituted compounds possessed greater Aβ1-42
aggregation inhibitory activities than the aliphatic amine substituted compounds. The most potent
self-induced Aβ1-42 aggregation inhibitors were 7m and 7n (78.3 ± 3.0% and 80.5 ± 2.2%
2
+
respectively). As for the Cu -induced Aβ
aggregation, the most potent compounds were 7m and
-42
1
9b (72.6 ± 1.9% and 83.9 ± 2.3% respectively). Noticeably, 7d exhibited the most potent activity
among the aliphatic amine substituted compounds (58.1 ± 1.6% and 45.1 ± 2.1% for the self- and
2
+
Cu -induced Aβ
aggregation respectively). Consistent with the AChE inhibition assay,
-42
1
compounds with amines substituted at 4’-position were more appropriate for the potency.
2
+
Table 2. In vitro inhibition of self- and Cu -induced Aβ
aggregation and MAO inhibitory
-42
1
activities of aurone Mannich base derivatives and reference compounds.
a
Compd.
NR R % Inhibition of Aβ1-42 aggregation
% Inhibition of MAO
3
4
b,e
2+
c,e
d,e
d,e
Self-induced
33.6 ± 1.1
15.7 ± 0.8
46.9 ± 1.4
47.5 ± 1.8
57.2 ± 2.0
58.1 ± 1.6
31.5 ± 1.4
38.6 ± 1.1
49.1 ± 1.0
41.8 ± 0.9
51.5 ± 1.9
55.1 ± 1.2
72.5 ± 2.8
74.9 ± 2.2
78.3 ± 3.0
80.5 ± 2.2
40.2 ± 1.9
56.4 ± 1.6
38.7 ± 0.8
75.2 ± 2.2
41.3 ± 0.9
—
Cu -induced
MAO-A
17.2 ± 0.9
12.1 ± 0.2
20.2 ± 0.8
22.7 ± 1.1
62.9 ± 2.3
15.3 ± 1.1
12.1 ± 1.6
9.2 ± 0.9
MAO-B
5
6
—
—
A
B
28.9 ± 0.7
26.7 ± 1.1
32.2 ± 1.8
23.6 ± 2.0
38.8 ± 1.3
45.1 ± 2.1
28.5 ± 1.1
35.1 ± 0.7
39.7 ± 0.6
38.7 ± 0.9
70.1 ± 1.3
55.8 ± 1.9
60.9 ± 2.1
71.2 ± 2.3
72.6 ± 1.9
68.5 ± 1.2
44.6 ± 1.2
59.9 ± 2.0
42.7 ± 1.2
83.9 ± 2.3
67.2 ± 1.3
—
37.0 ± 1.3
36.1 ± 2.1
2.23 ± 0.3
7.2 ± 0.5
7a
7b
7
c
C
D
E
37.9 ± 1.5
32.0 ± 1.8
3.3 ± 0.2
7d
7e
7f
F
1.0 ± 0.1
7
g
G
H
I
15.1 ± 1.4
15.3 ± 2.0
19.4 ± 1.2
16.3 ± 1.0
17.0 ± 2.1
9.7 ± 0.7
2.6 ± 0.1
7h
6.9 ± 0.5
7
i
17.8 ± 0.9
8.4 ± 0.3
7j
J
7
k
K
L
23.9 ± 1.1
26.4 ± 1.0
38.1 ± 1.3
19.2 ± 1.0
16.6 ± 0.9
9.0 ± 0.2
7
l
7
m
M
N
D
M
D
M
15.3 ± 1.2
6.8 ± 0.8
7
n
a
b
a
b
8
8.3 ± 0.4
8
9
9
6.6 ± 0.3
7.7 ± 0.7
8.6 ± 0.2
37.3 ± 1.1
—
32.7 ± 0.9
—
Curcumin
Clorgyline
Rasagiline
f
f
0.0027 ± 0.0006 µM
4.19 ± 0.101 µM
f
f
—
—
1.42 ± 0.015 µM
0.0825 ± 0.002 µM
1
5

ACCEPTED MANUSCRIPT
f
f
Iproniazid
—
—
2.56 ± 0.023 µM
1.95 ± 0.074 µM
a
b
c
d
e
f
For inhibition of Aβ aggregation, the thioflavin-T fluorescence method was used.
Inhibition of self-induced Aβ aggregation (25 µM) by tested inhibitors at 25 µM.
1
-42
2+
2+
Inhibition of Cu -induced Aβ aggregation. The concentration of tested compounds and Cu were 25 µM.
1
-42
Percentages are the percent inhibition of MAO by tested compounds at 10 µM.
The mean ± SD of the three independent experiments.
IC values of the drugs.
50
2.2.8. Recombinant human MAO-A and –B inhibition studies
The synthesized derivatives were tested in vitro for their MAO-A and –B inhibitory activities
using clorgyline, rasagiline and iproniazid as reference drugs. Recombinant human MAO-A and -B
were employed as enzyme sources, and their activities were determined by a fluorimetric method
with kynuramine as a common substrate [47-49]. The derivatives were tested at the concentration of
10 µM and all determinations were carried out in triplicate. As it shown in Table 2, the results were
not so satisfactory. Most of the derivatives displayed weak to moderate potency at the concentration
of 10 µM and no obvious structure-activity relationship was observed. The most potent MAO-A
inhibitor was 7c (62.9 ± 2.3% at 10 µM) and the most potent MAO-B inhibitor was 7m (38.1 ± 1.3%
at 10 µM).
2.2.9. In vitro blood-brain barrier permeation assay
The ability to cross the blood-brain barrier (BBB) and reach the therapeutic targets is an
important prerequisite for drugs to act on neurodegenerative processes. To determine the BBB
penetration of the target compounds, the parallel artificial membrane permeation assay of the
blood-brain barrier (PAMPA-BBB) was performed [50, 51]. First, we compared the experimental
permeabilities of 11 commercial drugs with reported values to validate the assay (Table S1,
Supplementary Material). A plot of experimental data versus bibliographic values produced a good
2
linear correlation: P (exp.) = 0.9163 × P (bibl.) − 0.2247 (R = 0.9558) (Figure S1, Supplementary
e
e
Material). From this equation and considering the limit established by Di et al. [50] for BBB
-6
permeation, we determined that compounds with P values above 3.44 × 10 cm/s could cross the
e
BBB (Table S2, Supplementary Material). As it shown in Table 3, compounds 7d and 7m exhibited
good BBB permeabilities and would be able to reach the therapeutic targets in central nervous
system (CNS).
1
6

ACCEPTED MANUSCRIPT
-6
Table 3. Permeability results P (× 10 cm/s) from the PAMPA-BBB assay for selected aurone
e
derivatives with their predicted penetration into the CNS.
a
-6
b
Compd.
P (× 10 cm/s)
Prediction
CNS +
e
7
7
d
13.48 ± 0.42
12.25 ± 0.48
m
CNS +
a
Compound 7d and 7m were dissolved in DMSO at 5 mg/mL and diluted with PBS/EtOH (70:30). The final
concentration of each compound was 100 µg/mL.
b
Data are the mean ± SD of three independent experiments.
3. Conclusion
In summary, a series of aurone Mannich base derivatives were designed, synthesized and
evaluated as multifunctional agents for the treatment of AD. In vitro assays demonstrated that most
of these compounds were selective AChE inhibitors with good multifunctional properties. Among
them, compound 7d exhibited outstanding inhibitory activity for RatAChE, EeAChE and HuAChE
(
IC = 0.00878 ± 0.0002 µM, 0.0212 ± 0.006 µM and 0.0371 ± 0.004 µM, respectively), that was
50
more excellent than donepezil under our experimental condition. And both the kinetic analysis and
molecular modeling study suggested that 7d showed mixed-type inhibition, which figured out that
the compound could bind to both CAS and PAS of AChE.. In addition, 7d could confer significant
neuroprotective effect against H O -induced PC-12 cell injury and displayed high antioxidant activity,
2
2
2
+
biometal chelating ability and good self- and Cu -induced Aβ
aggregation inhibitory potency.
-42
1
Moreover, 7d could cross the BBB and penetrate into brain. These multifunctional properties
highlight 7d as a promising candidate for further studies directed to the development of novel drugs
against AD.
4. Experimental section
4.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used
without further purification. Melting points (uncorrected) were measured on YRT-3 melting-point
1
13
apparatus (China). The H NMR and C NMR spectra were recorded in CDCl on a Varian INOVA
3
spectrometer at 25 °C with TMS as the internal standard. Coupling constants are given in Hz. Mass
spectra were recorded on Agilent-6210 TOF LC-MS Spectrometer. All the reactions were monitored
17

ACCEPTED MANUSCRIPT
by thin-layer chromatography (TLC) using silica gel GF254 plates from Qingdao Haiyang Chemical
Co. Ltd. (China) and the spots were detected under UV light (254 nm). Column chromatography was
performed using silica gel (230-400 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
(
China).
.1.1. 3,4-dimethoxyphenol (2)
,4-dimethoxyphenol (2) was synthesized from 3,4-dimethoxybenzaldehyde (1) in good yield
according to an improved solvent-free Daking oxidation protocol [32]. mp 73-75 °C (Lit. [52] 74-78
C).
4
3
°
4.1.2. 2-chloro-1-(2-hydroxy-4,5-dimethoxyphenyl)ethanone (3)
3
,4-dimethoxyphenol (2, 2.24 g, 14.5 mmol) was dissolved in EtOAc (40 mL). AlCl (7.80 g,
3
58.5 mmol) was added at room temperature to give a brown suspension. The reaction mixture was
stirred at room temperature for 30 min. Chloroacetyl chloride (1.33 mL, 17.7 mmol) was added, and
the reaction mixture was stirred at room temperature overnight and then heated at 50 °C for 6 h. The
reaction mixture was quenched by pouring into ice-water and the product was extracted with CH Cl2
2
(
4 × 20 mL), then washed with brine, dried over Na SO , filtered and concentrated under reduced
2 4
pressure to give compound 3 (3.60 g) as a yellow solid, which was used without further purification.
4.1.3. 5,6-dimethoxybenzofuran-3(2H)-one (4)
Compound 3 (3.60 g) was dissolved in methanol (50 mL) and anhydrous NaOAc (2.70 g, 32,9
mmol) was added. The reaction mixture was then refluxed under argon atmosphere for 4 h. The
methanol was evaporated and the residue was recrystallized from ethanol to give compound 4 (1.54 g,
54.4%) as a yellow needle crystal. mp 164-166 °C (Lit. [52] 163-165 °C)
4.1.4. General procedure for the synthesis of 5 and 6
To a suspension of 4 (1.00 g, 5.15 mmol) and m- or p-hydroxylbenzaldehyde (661 mg, 5.41
mmol) in ethanol (15 mL) was added an aqueous solution of potassium hydroxide (50%, 5.8 mL).
The solution was stirred under argon atmosphere for 4 h, until TLC showed complete disappearance
of the starting material. Ethanol was removed under reduced pressure. The residue was diluted into
distilled water and acidified with an aqueous solution of hydrochloric acid (10%). The precipitate
was filtered, washed with water and dried at room temperature to afford the corresponding crude
product 5 or 6, which was used without further purification.
4.1.4.1. (Z)-2-(3-hydroxybenzylidene)-5,6-dimethoxybenzofuran-3(2H)-one (5)
1
8

ACCEPTED MANUSCRIPT
Compound 5 was synthesized from 4 and m-hydroxylbenzaldehyde according to the general
1
procedure. The crude product 5 (1.60 g) was obtained as a bright yellow solid. H NMR (400 MHz,
DMSO-d ) δ 9.69 (s, 1H), 7.39 (s, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.20 (s, 1H),
6
7
.19 (s, 1H), 6.86 (d, J = 8.0 Hz, 1H), 6.73 (s, 1H), 3.95 (s, 3H), 3.81 (s, 3H).
.1.4.2. (Z)-2-(4-hydroxybenzylidene)-5,6-dimethoxybenzofuran-3(2H)-one (6)
Compound 6 was synthesized from 4 and p-hydroxylbenzaldehyde according to the general
4
procedure. The crude product 6 (1.55 g) was obtained as a yellow solid.
4.1.5. General procedure for the synthesis of 7a-n, 8a-b and 9a-b
The mixture of secondary amine (1.01 mmol) and paraformaldehyde (1.01 mmol) in ethanol (3
mL) was refluxed for 1 h, until paraformaldehyde was dissolved. Then compound 5 or 6 (100 mg,
.34 mmol) was added in one portion. The reaction mixture was refluxed for 24-48 h. Ethanol was
0
removed under reduced pressure. The residue was diluted with water and acidified with an aqueous
solution of hydrochloric acid (10%). The aqueous phase was washed with CH Cl , and then basified
2
2
with saturated NaHCO solution. The alkaline aqueous phase was again extracted with CH Cl . The
3
2
2
combined organic phase washed with brine, dried over Na SO , filtered and concentrated under
2
4
reduced pressure to afford the corresponding crude aurone Mannich base derivatives. Additionally,
compounds 8a-b and the corresponding 9a-b were obtained in one pot.
4
.1.5.1. (Z)-2-(4-((dimethylamino)methyl)-3-hydroxybenzylidene)-5,6-dimethoxybenzofuran-3(2H)-o
ne (7a)
Compound 7a was synthesized from 5 and dimethylamine hydrochloride according to the
general procedure. After purification by chromatography on silica gel using petroleum ether/acetone
1
(
2:1) as eluent, the pure product 7a was obtained as a yellow solid, yield 43.7%, mp 191-193 °C. H
NMR (400 MHz, CDCl ) δ 7.50 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.03 (d, J = 8.0 Hz,
3
13
1
H), 6.84 (s, 1H), 6.77 (s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 3.70 (s, 2H), 2.37 (s, 6H). C NMR (150
MHz, CDCl ) δ 183.3, 163.3, 158.1, 157.5, 147.7, 146.4, 132.8, 128.6, 123.8, 122.6, 118.1, 112.8,
3
+
1
3
4
12.0, 103.8, 95.5, 62.5, 56.5, 56.2, 44.4 (2C). HR-ESI-MS: Calcd. for C H NO [M + H] :
2
0
22
5
56.1498, found : 356.1502.
.1.5.2. (Z)-2-(4-((diethylamino)methyl)-3-hydroxybenzylidene)-5,6-dimethoxybenzofuran-3(2H)-on
e (7b)
Compound 7b was synthesized from 5 and diethylamine according to the general procedure.
1
9

ACCEPTED MANUSCRIPT
After purification by chromatography on silica gel using dichloromethane/ethyl acetate (2:1) as
1
eluent, the pure product 7b was obtained as a yellow solid, yield 15.6%, mp 73-75 °C. H NMR (600
MHz, CDCl ) δ 7.49 (s, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.17 (s, 1H), 7.04 (d, J = 7.8 Hz, 1H), 6.83 (s,
3
1
6
1
H), 6.76 (s, 1H), 4.01 (s, 3H), 3.90 (s, 3H), 3.83 (s, 2H), 2.68 (q, J = 7.2 Hz, 4H), 1.15 (t, J = 7.2 Hz,
1
3
H). C NMR (150 MHz, CDCl ) δ 183.4, 163.4, 158.4, 157.6, 147.8, 146.5, 132.8, 128.8, 124.1,
3
22.7, 118.3, 112.9, 112.2, 103.9, 95.6, 56.6 (2C), 56.3, 46.4 (2C), 11.0 (2C). HR-ESI-MS: Calcd.
+
for C H NO [M + H] : 384.1811, found : 384.1822.
2
2
26
5
4
.1.5.3. (Z)-2-(3-hydroxy-4-(pyrrolidin-1-ylmethyl)benzylidene)-5,6-dimethoxybenzofuran-3(2H)-on
e (7c)
Compound 7c was synthesized from 5 and pyrrolidine according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/acetone (1:1) as eluent, the pure
1
product 7c was obtained as a yellow solid, yield 11.8%, mp 204-206 °C. H NMR (400 MHz, CDCl )
3
δ 7.50 (s, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.17 (s, 1H), 7.07 (d, J = 8.0 Hz, 1H), 6.83 (s, 1H), 6.76 (s,
1
3
1
1
5
3
4
H), 4.01 (s, 3H), 3.90 (s, 5H), 2.72 (brs, 4H), 1.89 (brs, 4H). C NMR (150 MHz, CDCl ) δ 183.5,
3
63.5, 158.1, 157.6, 147.9, 146.5, 132.9, 128.5, 124.1, 122.7, 118.3, 112.9, 112.1, 103.9, 95.6, 58.2,
+
6.6, 56.3, 53.5 (2C), 23.6 (2C). HR-ESI-MS: Calcd. for C H NO [M + H] : 382.1654, found :
2
2
24
5
82.1666.
.1.5.4. (Z)-2-(3-hydroxy-4-(piperidin-1-ylmethyl)benzylidene)-5,6-dimethoxybenzofuran-3(2H)-one
7d)
(
Compound 7d was synthesized from 5 and piperidine according to the general procedure. After
purification by chromatography on silica gel using petroleum ether/ethyl acetate (1:1) as eluent, the
1
pure product 7d was obtained as a yellow solid, yield 22.9%, mp 174-176 °C. H NMR (400 MHz,
CDCl ) δ 7.48 (s, 1H), 7.20 (d, J = 7.6 Hz, 1H), 7.15 (s, 1H), 7.01 (d, J = 7.6 Hz, 1H), 6.81 (s, 1H),
3
6.74 (s, 1H), 4.00 (s, 3H), 3.89 (s, 3H), 3.72 (s, 2H), 2.61 (brs, 4H), 1.67-1.64 (m, 4H), 1.52 (brs, 2H).
1
3
C NMR (100 MHz, CDCl ) δ 183.5, 163.5, 158.2, 157.6, 147.9, 146.5, 132.8, 129.0, 123.5, 122.7,
3
118.3, 113.0, 112.2, 103.9, 95.6, 61.7, 56.6, 56.3, 53.9 (2C), 25.7 (2C), 23.8. HR-ESI-MS: Calcd. for
+
C H NO [M + H] : 396.1811, found : 396.1817.
23
26
5
4.1.5.5. (Z)-2-(3-hydroxy-4-(morpholinomethyl)benzylidene)-5,6-dimethoxybenzofuran-3(2H)-one
(7e)
Compound 7e was synthesized from 5 and morpholine according to the general procedure. After
2
0

ACCEPTED MANUSCRIPT
purification by chromatography on silica gel using petroleum ether/ethyl acetate (1:1) as eluent, the
1
pure product 7e was obtained as a yellow solid, yield 26.3%, mp 205-207 °C. H NMR (400 MHz,
CDCl ) δ 7.50 (s, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.18 (s, 1H), 7.06 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H),
3
1
3
6
.77 (s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 3.77 (brs, 6H), 2.61 (brs, 4H). C NMR (150 MHz, CDCl ) δ
3
183.3, 163.4, 157.6, 157.5, 147.9, 146.5, 133.1, 129.1, 122.9, 122.6, 118.2, 112.8, 111.8, 103.9, 95.5,
+
6
6.6 (2C), 61.6, 56.6, 56.3, 52.8 (2C). HR-ESI-MS: Calcd. for C H NO [M + H] : 398.1604,
22
24
6
found : 398.1618.
4
.1.5.6. (Z)-2-(3-hydroxy-4-((4-methylpiperazin-1-yl)methyl)benzylidene)-5,6-dimethoxybenzofuran-
(2H)-one (7f)
Compound 7f was synthesized from 5 and 1-methylpiperazine according to the general
3
procedure. After purification by chromatography on silica gel using dichloromethane/methanol (40:1)
1
as eluent, the pure product 7f was obtained as a yellow solid, yield 23.4%, mp 165-167 °C. H NMR
(
400 MHz, CDCl ) δ 7.45 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.14 (s, 1H), 7.02 (d, J = 8.0 Hz, 1H),
3
1
3
6
.81 (s, 1H), 6.74 (s, 1H), 3.99 (s, 3H), 3.88 (s, 3H), 3.74 (s, 2H), 2.59 (brs, 8H), 2.32 (s, 3H). C
NMR (100 MHz, CDCl ) δ 183.4, 163.4, 157.8, 157.5, 147.8, 146.4, 132.9, 128.9, 123.1, 122.8,
3
118.2, 112.9, 111.9, 103.8, 95.5, 61.1, 56.6, 56.3, 54.7 (2C), 52.3 (2C), 45.7. HR-ESI-MS: Calcd. for
+
C H N O [M + H] : 411.1920, found : 411.1925.
23
27
2
5
4
.1.5.7. (Z)-2-(4-((4-ethylpiperazin-1-yl)methyl)-3-hydroxybenzylidene)-5,6-dimethoxybenzofuran-3(
H)-one (7g)
2
Compound 7g was synthesized from 5 and 1-ethylpiperazine according to the general procedure.
After purification by chromatography on silica gel using dichloromethane/methanol (40:1) as eluent,
1
the pure product 7g was obtained as a yellow solid, yield 28.2%, mp 170-172 °C. H NMR (600
MHz, CDCl ) δ 7.48 (s, 1H), 7.25 (d, J = 7.8 Hz, 1H), 7.18 (s, 1H), 7.05 (d, J = 7.8 Hz, 1H), 6.83 (s,
3
1
1
1
5
4
3
H), 6.77 (s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 3.77 (s, 2H), 2.70 (brs, 8H), 2.48 (q, J = 7.2 Hz, 2H),
1
3
.11 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl ) δ 183.3, 163.3, 157.8, 157.5, 147.8, 146.4,
3
32.9, 128.9, 123.1, 122.7, 118.1, 112.8, 111.9, 103.8, 95.5, 61.1, 56.5, 56.2, 52.4 (2C), 52.3 (2C),
+
2.0, 11.8. HR-ESI-MS: Calcd. for C H N O [M + H] : 425.2076, found : 425.2085.
2
4
29
2
5
.1.5.8. (Z)-2-(4-((4-benzylpiperazin-1-yl)methyl)-3-hydroxybenzylidene)-5,6-dimethoxybenzofuran-
(2H)-one (7h)
Compound 7h was synthesized from 5 and 1-benzylpiperazine according to the general
2
1

ACCEPTED MANUSCRIPT
procedure. After purification by chromatography on silica gel using dichloromethane/methanol (20:1)
1
as eluent, the pure product 7h was obtained as a yellow solid, yield 20.9%, mp 196-198 °C. H NMR
(
600 MHz, CDCl ) δ 7.47 (s, 1H), 7.33-7.32 (m, 4H), 7.28-7.26 (m, 1H), 7.23 (d, J = 7.2 Hz, 1H),
3
7
2
1
9
4
4
2
.17 (s, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.83 (s, 1H), 6.76 (s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 3.76 (s,
1
3
H), 3.56 (s, 2H), 2.62 (brs, 8H). C NMR (150 MHz, CDCl ) δ 183.4, 163.4, 157.9, 157.6, 147.8,
3
46.5, 137.4, 132.9, 129.1 (2C), 128.9, 128.2 (2C), 127.2, 123.2, 122.8, 118.2, 112.9, 112.0, 103.9,
+
5.6, 62.7, 61.2, 56.6, 56.3, 52.7 (2C), 52.4 (2C). HR-ESI-MS: Calcd. for C H N O [M + H] :
2
9
31
2
5
87.2233, found : 487.2248.
.1.5.9. (Z)-2-(4-((benzyl(methyl)amino)methyl)-3-hydroxybenzylidene)-5,6-dimethoxybenzofuran-3(
H)-one (7i)
Compound 7i was synthesized from 5 and N-methyl-1-phenylmethanamine according to the
general procedure. After purification by chromatography on silica gel using dichloromethane/ethyl
acetate (20:1) as eluent, the pure product 7i was obtained as a yellow solid, yield 18.1%, mp 80-82
1
°
C. H NMR (400 MHz, CDCl ) δ 7.55 (s, 1H), 7.39-7.29 (m, 5H), 7.24 (d, J = 8.0 Hz, 1H), 7.17 (s,
3
1H), 7.07 (d, J = 8.0 Hz, 1H), 6.84 (s, 1H), 6.77 (s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 3.81 (s, 2H), 3.65
1
3
(
s, 2H), 2.29 (s, 3H). C NMR (150 MHz, CDCl ) δ 183.3, 163.4, 157.9, 157.5, 147.8, 146.4, 136.4,
3
1
6
4
32.9, 129.3 (2C), 128.8, 128.5 (2C), 127.7, 123.9, 122.8, 118.2, 112.9, 111.9, 103.9, 95.5, 61.3,
+
0.6, 56.5, 56.2, 41.2. HR-ESI-MS: Calcd. for C H NO [M + H] : 432.1811, found : 432.1816.
2
6
26
5
.1.5.10.
(Z)-2-(3-hydroxy-4-(((2-methoxybenzyl)(methyl)amino)methyl)benzylidene)-5,6-dimetho
xybenzofuran-3(2H)-one (7j)
Compound 7j was synthesized from 5 and 1-(2-methoxyphenyl)-N-methylmethanamine
according to the general procedure. After purification by chromatography on silica gel using
petroleum ether/ethyl acetate (1.5:1) as eluent, the pure product 7j was obtained as a yellow solid,
1
yield 24.0%, mp 148-150 °C. H NMR (400 MHz, CDCl ) δ 7.48 (s, 1H), 7.30 (t, J = 7.6 Hz, 1H),
3
7
1
.23-7.19 (m, 2H), 7.17 (s, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.95-6.91 (m, 2H), 6.83 (s, 1H), 6.77 (s,
1
3
H), 4.02 (s, 3H), 3.90 (s, 6H), 3.78 (s, 2H), 3.67 (s, 2H), 2.22 (s, 3H). C NMR (150 MHz, CDCl )
3
δ 183.4, 163.4, 158.2, 158.1, 157.5, 147.8, 146.4, 132.7, 131.3, 129.3, 128.8, 124.7, 124.6, 122.6,
120.2, 118.2, 112.9, 112.2, 110.5, 103.9, 95.6, 60.4, 57.0, 56.6, 56.3, 55.1, 40.9. HR-ESI-MS: Calcd.
+
for C H NO [M + H] : 462.1917, found : 462.1921.
2
7
28
6
4.1.5.11.
(Z)-2-(4-((benzyl(ethyl)amino)methyl)-3-hydroxybenzylidene)-5,6-dimethoxybenzofuran
2
2

ACCEPTED MANUSCRIPT
-3(2H)-one (7k)
Compound 7k was synthesized from 5 and N-benzylethanamine according to the general
procedure. After purification by chromatography on silica gel using petroleum ether/ethyl acetate
1
(
3:1) as eluent, the pure product 7k was obtained as a yellow solid, yield 21.5%, mp 80-82 °C. H
NMR (400 MHz, CDCl ) δ 7.53 (s, 1H), 7.38-7.30 (m, 5H), 7.23 (d, J = 7.6 Hz, 1H), 7.17 (s, 1H),
3
7
2
4
4
.06 (d, J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.76 (s, 1H), 4.01 (s, 3H), 3.90 (s, 3H), 3.83 (s, 2H), 3.69 (s,
+
H), 2.64 (q, J = 7.2 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H). HR-ESI-MS: Calcd. for C H NO [M + H] :
2
7
28
5
46.1967, found : 446.1979.
.1.5.12. (Z)-2-(4-((ethyl(2-methoxybenzyl)amino)methyl)-3-hydroxybenzylidene)-5,6-dimethoxyb
enzofuran-3(2H)-one (7l)
Compound 7l was synthesized from 5 and N-(2-methoxybenzyl)ethanamine according to the
general procedure. After purification by chromatography on silica gel using petroleum ether/acetone
1
(
3:1) as eluent, the pure product 7l was obtained as a yellow solid, yield 22.0%, mp 104-106 °C. H
NMR (400 MHz, CDCl ) δ 7.46 (s, 1H), 7.31-7.19 (m, 3H), 7.16 (s, 1H), 7.02 (d, J = 7.6 Hz, 1H),
3
6
3
.95-6.90 (m, 2H), 6.82 (s, 1H), 6.75 (s, 1H), 4.00 (s, 3H), 3.90 (s, 3H), 3.89 (s, 3H), 3.80 (s, 2H),
.72 (s, 2H), 2.62 (q, J = 6.8 Hz, 2H), 1.15 (t, J = 6.8 Hz, 3H). HR-ESI-MS: Calcd. for C H NO
6
28
30
+
[
M + H] : 476.2073, found : 476.2070.
4.1.5.13.
(Z)-2-(4-(((2-(dimethylamino)benzyl)(ethyl)amino)methyl)-3-hydroxybenzylidene)-5,6-d
imethoxybenzofuran-3(2H)-one (7m)
Compound 7m was synthesized from 5 and 2-((ethylamino)methyl)-N,N-dimethylaniline
according to the general procedure. After purification by chromatography on silica gel using
petroleum ether/acetone (5:1) as eluent, the pure product 7m was obtained as a yellow solid, yield
1
3
7
6
=
1.1%, mp 95-97 °C. H NMR (400 MHz, CDCl ) δ 7.52 (brs, 1H), 7.38 (d, J = 7.2 Hz, 1H),
3
.28-7.24 (m, 1H), 7.21-7.15 (m, 3H), 7.10 (t, J = 7.2 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.84 (s, 1H),
.76 (s, 1H), 4.02 (s, 3H), 3.90 (s, 3H), 3.81 (s, 4H), 2.67 (s, 6H), 2.59 (q, J = 7.2 Hz, 2H), 1.15 (t, J
13
7.2 Hz, 3H). C NMR (150 MHz, CDCl ) δ 183.4, 163.3, 158.1, 157.5, 153.5, 147.7, 146.4, 132.6,
3
1
5
4
4
31.4, 130.6, 128.9, 128.3, 124.5, 123.6, 122.6, 119.7, 118.1, 112.9, 112.2, 103.8, 95.5, 56.9, 56.5,
+
6.2, 52.8, 46.7, 45.2 (2C), 10.7. HR-ESI-MS: Calcd. for C H N O [M + H] : 489.2389, found :
2
9
33
2
5
89.2378.
.1.5.14.
(Z)-2-(4-(((4-(dimethylamino)benzyl)(ethyl)amino)methyl)-3-hydroxybenzylidene)-5,6-d
2
3

ACCEPTED MANUSCRIPT
imethoxybenzofuran-3(2H)-one (7n)
Compound 7n was synthesized from 5 and 4-((ethylamino)methyl)-N,N-dimethylaniline
according to the general procedure. After purification by chromatography on silica gel using
petroleum ether/acetone (6:1) as eluent, the pure product 7n was obtained as a yellow solid, yield
1
2
5.6%, mp 88-90 °C. H NMR (400 MHz, CDCl ) δ 7.50 (s, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.17 (d, J
3
=
8.4 Hz, 2H), 7.16 (s, 1H), 7.04 (d, J = 8.0 Hz, 1H), 6.82 (s, 1H), 6.77 (s, 1H), 6.71 (d, J = 8.4 Hz,
2H), 4.01 (s, 3H), 3.90 (s, 3H), 3.80 (s, 2H), 3.60 (s, 2H), 2.94 (s, 6H), 2.62 (q, J = 7.2 Hz, 2H), 1.16
1
3
(
t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl ) δ 183.5, 163.4, 158.2, 157.6, 150.1, 147.8, 146.5,
3
132.7, 130.5 (2C), 128.9, 124.4, 123.6, 122.8, 118.3, 113.0, 112.4 (2C), 112.2, 104.0, 95.6, 56.7,
+
5
6.6, 56.3 (2C), 46.4, 40.5 (2C), 11.1. HR-ESI-MS: Calcd. for C H N O [M + H] : 489.2389,
2
9
33
2
5
found : 489.2383.
4
.1.5.15.
one (8a)
Compound 8a was synthesized from 6 and piperidine according to the general procedure. After
(Z)-2-(4-hydroxy-3-(piperidin-1-ylmethyl)benzylidene)-5,6-dimethoxybenzofuran-3(2H)
-
purification by chromatography on silica gel using petroleum ether/acetone (3:1) as eluent, the pure
1
product 8a was obtained as an orange-yellow solid, yield 43.6%, mp 178-180 °C. H NMR (600
MHz, CDCl ) δ 7.76 (d, J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.18 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.82 (s,
3
1
2
1
H), 6.78 (s, 1H), 4.02 (s, 3H), 3.91 (s, 3H), 3.76 (s, 2H), 2.58 (brs, 4H), 1.67 (brs, 4H), 1.54 (brs,
1
3
H). C NMR (150 MHz, CDCl ) δ 183.2, 162.9, 160.4, 157.2, 146.5, 146.4, 132.6, 132.0, 123.5,
3
21.9, 116.9, 113.4, 113.0, 103.9, 95.5, 61.9, 56.6, 56.3, 53.8 (2C), 25.7 (2C), 23.8. HR-ESI-MS:
+
Calcd. for C H NO [M + H] : 396.1811, found : 396.1813.
2
3
26
5
4.1.5.16.
(Z)-2-(3-(((2-(dimethylamino)benzyl)(ethyl)amino)methyl)-4-hydroxybenzylidene)-5,6-d
imethoxybenzofuran-3(2H)-one (8b)
Compound 8b was synthesized from 6 and 2-((ethylamino)methyl)-N,N-dimethylaniline
according to the general procedure. After purification by chromatography on silica gel using
petroleum ether/acetone (3:1) as eluent, the pure product 8b was obtained as an orange-yellow solid,
1
yield 36.2%, mp 81-83 °C. H NMR (400 MHz, CDCl ) δ 7.76 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.36
3
(d, J = 7.6 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 7.17-7.15 (m, 2H), 7.10 (t, J = 7.6 Hz, 1H), 6.89 (d, J =
8
6
.4 Hz, 1H), 6.83 (s, 1H), 6.77 (s, 1H), 4.02 (s, 3H), 3.90 (s, 3H), 3.84 (s, 2H), 3.83 (s, 2H), 2.67 (s,
1
3
H), 2.61 (q, J = 7.2 Hz, 2H), 1.16 (t, J = 7.2 Hz, 3H). C NMR (150 MHz, CDCl ) δ 183.2, 162.9,
3
2
4

ACCEPTED MANUSCRIPT
1
1
60.3, 157.2, 153.6, 146.4 (2C), 132.6, 132.2, 130.7, 128.5, 123.8, 123.4, 122.6, 119.9, 116.9, 113.4,
13.0, 112.6, 103.9, 95.5, 57.0, 46.6, 46.3, 52.9, 46.8, 45.3 (2C), 10.7. HR-ESI-MS: Calcd. for
+
C H N O [M + H] : 489.2389, found : 489.2378.
29
33
2
5
4
.1.5.17.
2H)-one (9a)
Compound 9a was synthesized from 6 and piperidine according to the general procedure. After
(Z)-2-(4-hydroxy-3,5-bis(piperidin-1-ylmethyl)benzylidene)-5,6-dimethoxybenzofuran-3
(
purification by chromatography on silica gel using petroleum ether/acetone (3:1) as eluent, the pure
1
product 9a was obtained as an orange-red semisolid, yield 12.6%. H NMR (600 MHz, CDCl ) δ
3
7.79 (s, 2H), 7.16 (s, 2H), 6.67 (s, 1H), 5.70 (brs, 1H), 4.05 (s, 3H), 3.90 (s, 4H), 3.89 (s, 3H), 2.73
1
3
(
brs, 8H), 1.77 (brs, 8H), 1.54 (brs, 4H). C NMR (150 MHz, CDCl ) δ 183.3, 163.4, 159.5, 157.5,
3
146.9, 146.4, 133.8 (2C), 123.5 (2C), 120.7, 113.1, 111.9, 103.7, 96.3, 58.2 (2C), 56.8, 56.3, 53.3
+
(
4C), 24.8 (4C), 23.3 (2C). HR-ESI-MS: Calcd. for C H N O [M + H] : 493.2702, found :
2
9
37
2
5
4
4
5
93.2705.
.1.5.18.
(Z)-2-(3,5-bis(((2-(dimethylamino)benzyl)(ethyl)amino)methyl)-4-hydroxybenzylidene)-
,6-dimethoxybenzofuran-3(2H)-one (9b)
Compound 9b was synthesized from 6 and 2-((ethylamino)methyl)-N,N-dimethylaniline
according to the general procedure. After purification by chromatography on silica gel using
petroleum ether/acetone (3:1) as eluent, the pure product 9b was obtained as an orange-red solid,
1
yield 20.2%, mp 69-71 °C. H NMR (400 MHz, CDCl ) δ 7.85 (s, 2H), 7.56 (d, J = 7.6 Hz, 2H), 7.28
3
(t, J = 7.6 Hz, 2H), 7.16-7.09 (m, 6H), 6.74 (s, 1H), 4.80 (brs, 1H), 4.06 (brs, 11H), 3.90 (s, 3H), 2.82
+
(
brs, 4H), 2.57 (brs, 12H), 1.23 (t, J = 7.2 Hz, 6H). HR-ESI-MS: Calcd. for C H N O [M + H] :
4
1
51
4
5
679.3859, found : 679.3865.
4.2. Pharmacology
4.2.1. Inhibition experiments of AChE and BuChE
To determine the in vitro cholinesterase activities of the compounds, the spectrophotometric
method of Ellman was performed [31], using AChE from 5% rat cortex homogenate, purified AChE
from Electrophorus electricus and human erythrocytes (Sigma-Aldrich Co.), and BuChE from rat
serum. The brain homogenate was preincubated with tetraisopropyl pyrophosphoramide (iso-OMPA,
selective inhibitor of BuChE, 4.0 mmol/L, Sigma-Aldrich Co.) for 5 min before use. For rat AChE or
2
5

ACCEPTED MANUSCRIPT
BuChE inhibition assays, the reaction mixture (100 µL) consisted of acetylthiocholine iodide (1
mmol/L, 30 µL) (J&K Scientific) or butyrylthiocholine iodide (1 mmol/L, 30 µL) (TCI Shanghai
Development), phosphate-buffered solution (0.1 mmol/L, pH = 7.40, 40 µL), 5% homogenate or
2
5% serum (10 µL) and different concentrations of test compounds (20 µL). The mixture was
incubated at 37 °C for 15 min, followed by the addition of 5,5’-dithiobis-2-nitrobenzoic acid (DTNB,
.2%, 30 µL) (J&K Scientific) to produce the yellow anion of 5-thio-2-nitrobenzoic acid. Changes in
0
absorbance were detected at 405 nm in a Varioskan Flash Multimode Reader (Thermo Scientific).
For EeAChE and HuAChE inhibition assay, EeAChE or HuAChE (0.05 U/mL, final concentration)
was used and the assay was carried out in a phosphate buffer (0.01 mmol/L, pH = 8.00). Changes in
absorbance were detected at 412 nm. The other procedure was the same as above. The inhibitory
activities of compounds toward AChE or BuChE would reduce the color generation and IC values
5
0
were calculated as the concentration of compound that produces 50% AChE or BuChE activity
inhibition. Donepezil was applied as the positive drug. All samples were assayed in triplicate.
4.2.2. Kinetic study for the inhibition of AChE
Kinetic characterization of AChE inhibition was carried out based on a reported method using
purified AChE from E. Electricus (EeAChE) [53]. The assay solution (100 µL) consists of 0.1 M
phosphate buffer (pH 8.00), with the addition of 30 µL of 0.2% DTNB, 10 µL of 0.5 U/mL EeAChE,
and 20 µL of substrate (ATCh). Three different concentrations of inhibitors were added to the assay
solution and pre-incubated with the EeAChE for 15 min at 37°C, followed by the addition of
substrate in different concentrations. Kinetic characterization of the hydrolysis of ATCh catalyzed by
EeAChE was done spectra-metrically at 412 nm. The parallel control experiments were performed
without inhibitor. The plots were assessed by a weighted least square analysis that assumed the
variance of v to be a constant percentage of v for the entire data set. Slopes of reciprocal plots were
then plotted against the concentration of 7d in a weighted analysis, and K was determined as the
i
intercept on the negative x-axis.
4.2.3. Molecular modeling study
The crystal structure of AChE complexed with donepezil (PDB code: 1EVE) was obtained from
the Protein Data Bank after removing the original inhibitors and water molecules. The 3D Structure
of 7d was built and performed geometry optimization by molecular mechanics. After addition of
Gasteiger charges, removal of hydrogen atoms, addition of their atomic charges to skeleton atoms,
2
6

ACCEPTED MANUSCRIPT
and the assignment of proper atomic types, the further preparation of the inhibitor was accomplished.
Autotors was then used to define the rotatable bonds in the ligands. Docking studies were performed
using the AUTODOCK 4.2 program. By using Autodock Tools (ADT; version 1.5.6), polar hydrogen
atoms were added to amino acid residues, and Gasteiger charges were assigned to all atoms of the
enzyme. The resulting enzyme structure was used as an input for the AUTOGRID program.
AUTOGRID performed a pre-calculated atomic affinity grid maps for each atom type in the ligand,
plus an electrostatics map and a separate desolvation map presented in the substrate molecule. All
maps were calculated with 0.375 Å spacing between grid points. The center of the grid box was
placed at the center of donepezil with coordinates x = 2.023, y = 63.295, z = 67.062. The dimensions
of the active site box were set at 60 × 60 × 60 Å. Flexible ligand docking was performed for the
compounds. Each docked system was performed by 100 runs of the AUTODOCK search by the
Lamarckian genetic algorithm (LGA). Other than the referred parameters above, the other parameters
were accepted as default. A cluster analysis was performed on the docking results using a root mean
square (RMS) tolerance of 1.0 and the lowest energy conformation of the highest populated cluster
was selected for analysis. Graphic manipulations and visualizations were done by Autodock Tools or
Discovery Studio 2.5 software.
4.2.4. Antioxidant activity assay
The antioxidant potency was evaluated by the oxygen radical absorbance capacity-fluorescein
(ORAC-FL) assay [54]. Fluorescein (FL) and 6-hydroxy-2,5,7,8-tetramethylchromane-2- carboxylic
acid (Trolox) were purchased from TCI (Shanghai) Development. 2,2’-Azobis(amidinopropane)
dihydrochloride (AAPH) was purchased from Accela ChemBio Co., Ltd. All the assays were
performed with 75 mM phosphate buffer (pH = 7.40), and the final reaction mixture was 200 µL. FL
(120 µL, 150 nM final concentration) and antioxidant (20 µL) were added in the wells of a black
96-well plate using Trolox as a standard (1-8 µM, final concentration). The plate was incubated for
15 min at 37 °C and then placed in a Varioskan Flash Multimode Reader (Thermo Scientific). AAPH
solution (60 µL, 12 mM final concentration) was added rapidly using an autosampler and the
fluorescence recorded every minute for 90 min with excitation at 485 nm and emission at 535 nm.
The plate was automatically shaken prior to each reading. A blank (FL + AAPH) using phosphate
buffer instead of antioxidant and Trolox calibration were carried out in each assay. The samples were
measured at different concentrations (1-10 µM). All the reaction mixture was prepared in duplicate,
27

ACCEPTED MANUSCRIPT
and at least three independent assays were performed for each sample. Antioxidant curves
(fluorescence versus time) were normalized to the curve of the blank in the same assay, and then the
area under the fluorescence decay curve (AUC) was calculated. The net AUC of a sample was
obtained by subtracting the AUC of the blank. ORAC-FL values were expressed as Trolox
equivalents by using the standard curve calculated for each sample, where the ORAC-FL value of
Trolox was taken as 1, indicating the antioxidant potency of the tested compounds.
4
.2.5. Neuroprotective effect on PC-12 cell injury induced by H O [42]
2 2
For the cell viability assay, briefly, PC-12 cells were seeded into 96-well plates at a density of 1
5
×
10 cells per mL in DMEM medium (GIBCO.) supplemented with 10% heat-inactivated bovine
calf serum (Hyclone). After incubation overnight, the medium was replaced with fresh DMEM
medium without calf serum and phenol red. And tested compounds at various concentrations were
added and incubated with the cells for further 2 h. Then, H O (100 µM) was added and the cells
2
2
were further incubated for 24 h. At the end of the assay, the cells were incubated with 0.5 mg/mL
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4 h at 37 °C. The
3
supernatants were carefully removed, and 150 µL DMSO was added into each well. The absorbance
at 570 nm of the formazan was determined using a Varioskan Flash Multimode Reader (Thermo
Scientific). Data were expressed as mean ± SD. To determine the cytotoxicity of the compound,
tested compound was incubated with PC-12 cells for 48 h without the addition of H O , and other
2
2
procedures were similar to the above mentioned.
As for the LDH assay, the procedures were same with the cell viability assay. After incubation
with H O for 24 h, the LDH activities were measured with a LDH kit (Nanjing Jiancheng
2
2
Bioengineering Institute, A020-1). Data were expressed as mean ± SD.
4.2.6. Metal-chelating studies [54]
The chelating studies were performed using a Varioskan Flash Multimode Reader (Thermo
Scientific). The UV absorption spectra of tested compound (7d or 7m) alone or in the presence of
CuCl , ZnCl , AlCl , and FeSO were recorded with wavelength ranging from 200 to 600 nm after
2
2
3
4
incubating for 30 min in methanol at room temperature. The final volume of reaction mixture was
00 µL, and the final concentrations of tested compound and metals were 37.5 µM. The difference
2
UV-vis spectra due to complex formation were obtained by numerical subtraction of the spectra of
the metal alone and the compound alone from the spectra of the mixture.
2
8