Design, synthesis and evaluation of 4-dimethylamine flavonoid derivatives as potential multifunctional anti-Alzheimer agents

Article abstract of DOI:10.1016/j.ejmech.2016.06.022

A new series of 4-dimethylamine flavonoid derivatives were designed and synthesized as potential multifunctional anti-Alzheimer agents. The inhibition of cholinesterase activity, self-induced β-amyloid (Aβ) aggregation, and antioxidant activity by these derivatives was investigated. Most of the compounds exhibited potent acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activity. A Lineweaver-Burk plot and molecular modeling study showed that these compounds targeted both the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. The derivatives showed potent self-induced Aβ aggregation inhibition and peroxyl radical absorbance activity. Moreover, compound 6d significantly protected PC12 neurons against H₂O₂-induced cell death at low concentrations. Thus, these compounds could become multifunctional agents for further development for the treatment of AD.

Full text of DOI:10.1016/j.ejmech.2016.06.022

Accepted Manuscript
Design, synthesis and evaluation of 4-dimethylamine flavonoid derivatives as
potential multifunctional anti-Alzheimer agents
Wen Luo, Ting Wang, Chen Hong, Ya–Chen Yang, Ying Chen, Juan Cen, Song–
Qiang Xie, Chao–Jie Wang
PII:
S0223-5234(16)30500-1
10.1016/j.ejmech.2016.06.022
EJMECH 8683
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 April 2016
Revised Date: 12 June 2016
Accepted Date: 13 June 2016
Please cite this article as: W. Luo, T. Wang, C. Hong, Y. Yang, Y. Chen, J. Cen, S. Xie, C.
Wang, Design, synthesis and evaluation of 4-dimethylamine flavonoid derivatives as potential
multifunctional anti-Alzheimer agents, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.06.022.
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ACCEPTED MANUSCRIPT
Graphical Abstract
4
-Dimethylamino flavonoid derivatives were synthesized as potential multi-functional
anti-Alzheimer agents, they showed potent inhibition activity for cholinesterase,
anti-oxidation and anti-Aβ aggregation activities. Molecular modeling study and
kinetic analysis showed that these compounds interacted at both catalytic active site
and peripheral anionic site of AChE. Compound 6d significantly protected PC12
neurons against H O -induced cell death at low concentrations.
2
2

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Design, Synthesis and Evaluation of 4-Dimethylamine
Flavonoid Derivatives as Potential Multifunctional
Anti-Alzheimer agents
Wen Luo, §,a Ting Wang, §,a Chen Hong,a,b YaꢀChen Yang, Ying Chen, Juan
a
a
Cen, a,* SongꢀQiang Xie, ChaoꢀJie Wang a,*
c
a
Key Laboratory of Natural Medicine and Immuno-Engineering, Henan
University, Kaifeng 475004, Peopleꢁs Republic of China
b
Huaihe Hospital, Henan University, Kaifeng 475000, Peopleꢁs Republic of
China
c
Institute of Chemical biology, Henan University, Kaifeng 475004, Peopleꢁs
Republic of China
*
Juan Cen: Email 675711997@qq.com, Tel 086 0fl71-22864665, Fax 086
0
fl71ꢀ22864665
*
Chao-Jie Wang: Email wcjsxq@16fl.com, Tel 086 0fl71-22864665, Fax 086
fl71ꢀ22864665
0
§
The first two authors contributed equally to this work.

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ABSTRACT
A new series of 4-dimethylamine flavonoid derivatives were designed and
synthesized as potential multifunctional anti-Alzheimer agents. The inhibition
of cholinesterase activity, self-induced β
antioxidant activity by these derivatives was investigated. Most of the
compounds exhibited potent acetylcholinesterase (AChE’ and
-amyloid (Aβ’ aggregation, and
butyrylcholinesterase (BChE’ inhibitory activity. A LineweaverꢀBurk plot and
molecular modeling study showed that these compounds targeted both the
catalytic active site (CAS’ and peripheral anionic site (PAS’ of AChE. The
derivatives showed potent self-induced Aβ aggregation inhibition and peroxyl
radical absorbance activity. Moreover, compound 6d significantly protected
PC12 neurons against H
2
O -induced cell death at low concentrations. Thus,
2
these compounds could become multifunctional agents for further
development for the treatment of AD.
KEYWORDS
Flavonoid
Anti-Alzheimer agent

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Cholinesterase
Aβ aggregation
Antioxidation
1. Introduction
Alzheimerꢁs disease (AD’ is a deadly neurodegenerative brain disorder
characterized by dementia, cognitive impairment, and memory loss [1]. It has
been estimated that 36 million people were living with dementia in the world in 2010
and that the number will double every 20 years, leading to more than 115 million
people with AD in 2050 [2]. Although the etiopathogenesis of AD still remains
unknown, in the last decades, several factors that are involved in the onset of
AD have been identified. Among them, low levels of acetylcholine, oxidative
stress and β-amyloid (Aβ’ deposits play significant roles in AD [fl]. Because of
the multipathogenesis of AD, new therapeutic strategies are being developed
to arrest and reverse the progress of AD, one strategy is to develop novel
anti-AD agents with multiple potencies. This is the case of the
multi-target-directed ligand (MTDL’ approach [4, 5]. In this regard, a lot of
interest has recently regained the natural product such as flavonoids.
Flavonoids are a class of polyphenolic compounds present in plants, fruits
and vegetables, as well as in wine and tea [6]. They show diverse biological
activities such as antioxidant, anti-inflammatory, neuroprotection, and

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vasodilatory activity. Particularly, they are nontoxic and therefore could be
considered as leading compounds for AD treatment [7]. In the recent years,
some research groups selected flavonoids and optimized their chemical
structures to develop new anti-AD agents (Fig. 1, A and B’ [8, 9].
Among them, the N,N-dimethylated derivatives caught our attention,
because they showed potent antioxidant activity and excellent binding
affinities with Aβ in an in vitro binding assay (Fig. 1, CꢀE’ [10ꢀ12]. Previously,
we modified flavonoids with aminoalkyl group and evaluated their biological
activities, it was found that N,N-dimethylated derivatives showed multiple
potencies for cholinesterase (ChE’, Aβ aggregation and reactive oxygen
species (ROS’ inhibition (Fig. 2’. However, these derivatives exhibited a
moderate inhibitory potency for ChE and lacked good solubility [1fl, 14].
Tacrine was the first approved ChE inhibitor by FDA for AD treatment,
although its side effects, the search for tacrine conjugates is still of interest [15].
Tacrine can inhibit both ChE with potency in nanomolar range by binding to
their catalytic active site (CAS’. Therefore, in this study, a series of new
N,N-dimethylated flavonoid derivatives were designed as more potent
inhibitors for ChE, Aβ aggregation, and ROS by conjugating the
N,N-dimethylated flavonoid with tacrine using different long chain linker, in
order to study the structure-activity relationship (SAR’, some derivatives

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conjugated with terminal amine group were also synthesized (Fig. 2’. The
synthetic route is shown in Scheme 1.
2. Results and discussion
2
.1. Chemistry
The synthetic route to the target compounds (6aꢀ8b’ starting from
commercially available 1,fl-benzenediol (1’ was shown in Scheme 1.
Condensation of 1,fl-benzenediol (1’ with chloroacetonitrile catalyzed by ZnCl
2
and HCl gas was followed by hydrolysis in water and produced ketone in 87%
yield. Treatment of ketone (2’ with 4-dimethylaminobenzaldehyde in the
presence of excess NaOH in H O/EtOH was followed by acidification with
2
aqueous HCl and resulted in the expected compound fl in 92% yield [16]. The
reaction of fl with ethyl chloroacetate in acetone provided 4 in 7fl% yield. Then
treatment of 4 with excess Na
2
CO
fl
in DMSO/H O was followed by acidification
2
with aqueous HCl and resulted in the intermediate (5’ in 70% yield. Finally, the
reaction of 5 with different amines (10a-10l’ produced the targets 6a-8b in
fl7-61% yields. The amines 10a-10j used in the final step were synthesized in
our
lab
according
to
Scheme
2
and
Scheme
and
fl,
10k
10l
1
1
(
N ,N -dimethylethane-1,2-diamine’
1
1
(
N ,N -dimethylpropane-1,fl-diamine’ were commercial available and used
without further purification. The structures and purities of the target
1
1fl
compounds were confirmed by H NMR, C NMR, MS and HPLC.

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2
.2. Inhibition studies on AChE and BChE
The inhibitory activity of the target compounds was evaluated against
acetylcholinesterase (AChE’ and butyrylcholinesterase (BChE’ with tacrine as
a positive control using the method of Ellman et al [17]. The IC50 values and
selectivity index for the inhibition of AChE and BChE were summarized in
Table 1. These compounds can be divided into three types: The first type of
compounds (6aꢀ6e’ contains a tacrine moiety, the second type of compounds
(7aꢀ7e’ contains a pyrrolidine group, and the third type of compounds (8a and
8
b’ contains an N,N-dimethylamino group. Generally, all of three types of
compounds showed significant ChE inhibitory activity, the IC50 of the first type
of compounds (6aꢀ6e’ was in the nanomolar range, and the IC50 of the second
and third type compounds (7aꢀ7e, 8a and 8b’ was in the micromolar range.
In the first type of compounds (6aꢀ6e’, compound 6d (n = 6’ showed the
most potent inhibition for AChE with an IC50 value of 59.61 nM, much better
than that of tacrine (192.6 nM’. Compound 6e (n = 8’ exhibited the strongest
inhibitory activity for BChE with an IC50 value of 24.67 nM, also better than
tacrine (fl7.08 nM’. In the second and third type (7aꢀ7e, 8a and 8b’, compound
7
2
e was the most potent inhibitor for both AChE and BChE with IC50 values of
295.10 nM and 2fl7.92 nM, respectively.
A comparison between the structure and activity of the first type of
compounds (6aꢀ6e’ shows that their inhibitory activity for AChE and BChE
was significantly affected by the chain length. When the chain length was

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increased, the AChE inhibitory activity of the compounds increased. Their
selectivity also changed from 1.9 to 0.26, indicating that a longer chain length
is beneficial for BChE inhibition. A similar trend was observed for the second
type of compounds. This is reasonable because these two enzymes have
different conformations: The active site of BChE is wider than that of AChE.
Therefore, BChE had less restriction to inhibitors with larger linker length.
A comparison of the first type of compounds (6aꢀ6e’ with the second and
third type of compounds (7aꢀ7e, 8a and 8b’ showed that compounds 6aꢀ6e
exhibited much more potent inhibitory activity on both AChE and BChE than
compounds 7aꢀ7e, 8a and 8b. This indicates that the tacrine moiety, not the
alkylamino group, was significant for ChE inhibition.
2
.fl. Kinetic studies for the inhibition of ChE
In order to investigate the mechanism of flavonoid derivatives against ChE,
kinetic study was performed using compounds (6d, 7e’ and the steady-state
inhibition data for AChE are shown in Fig. fl (A and B’. LineweaverꢀBurk plots
showed both increasing slopes and increasing intercepts at higher inhibitor
concentration. This pattern indicated a mixed-type inhibition, this result
showed that compound 6d could bind to both CAS and peripheral anionic site
(PAS’ of AChE, which was consistent with the results of molecular modeling
studies. The similar interaction pattern was observed between compound 7e
and AChE.

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The LineweaverꢀBurk plots for compounds 6d and 7e with BChE were also
performed (Fig. fl, C and D’, the pattern also indicated a mixed-type inhibition.
2
.4. Molecular modeling
Molecular docking simulations have become important tools in recent years
for understanding the interaction mode and the structure-activity relationships
of ligands with receptors. To obtain useful information about the binding
interactions between the flavonoid derivatives and AChE, molecular docking
simulations were performed using docking program AUTODOCK 4.0 package
with PyMOL program [18,19], the result is shown in Fig. 4.
Our docking studies indicated compound 6d exhibited multiple binding
modes with AChE. In the complex of TcAChEꢀ6d, compound 6d interacted
with the enzymatic CAS and the PAS along the mid-gorge. The tacrine moiety
was bound to CAS with a classic π-π stacking interaction between Trp84 and
Pheflfl0. Their ring-to-ring distance was fl.5 Å and fl.7 Å, respectively, the
protonated nitrogen atom of the quinoline ring had a hydrogen bond (fl.4 Å’
interaction with the carbonyl group of His440. In the PAS, there was a π-π
stacking with the distance of fl.8 Å between the flavonoid moiety and Trp279.
The docking studies showed that compound 6d had a mixed type of inhibition,
which was consistent with the kinetic analysis result.

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Because the crystal structure of BChE from equine serum had not been
reported, and the sequence of BChE from equine showed high homology with
that of human BChE, so, the crystal structure of HuBChE was used in the
docking study. Similar interactions were found in 6d in complex with HuBChE
(PDB code: 1POI’.
2
.5. Studies of antioxidation activity
The antioxidant activities of the target compounds were determined by the
oxygen radical absorbance capacity (ORAC’ assay according to the method
originally described by Fang et al [20]. The vitamin E analogue Trolox was
used as the standard, and the antioxidant activity was expressed as the Trolox
equivalent. As shown in Table 1, all the compounds demonstrated excellent
antioxidant activity with ORAC values of 1.8ꢀfl.2 Trolox equivalents.
Compound 6a was the strongest antioxidant (ORAC value = fl.2’. Compounds
6
c, 6d and 8a showed a slightly lower activity (ORAC value = fl.0, 2.8, and fl.0’.
Tacrine exhibited almost no radical-capture capacity with ORAC value < 0.1.
These results indicate that the inclusion of 4-dimethylamino in the flavonoid is
responsible for the radical scavenging ability of the compounds.
2
.6. Inhibition of self-mediated Aꢂ(1-42’ aggregation
Aꢂ(1-40’ and Aꢂ(1-42’ are the main forms of Aꢂ peptides found in amyloid
plaques. They originated from the enzymatic cleavage of amyloid precursor
protein operated by ꢂ- and γ-secretases. Aꢂ(1-42’ is markedly more
fibrillogenic [21], therefore, Aꢂ(1-42’ was selected to study the inhibitory

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activity of the derivatives. The activity of the target compounds was assessed
by thioꢃavin T fluorescence assay using curcumin as the standard, due to poor
solubility, compounds 6e, 7e and 8b were not measured. As shown in Fig. 5,
most of the tested compounds exhibited significantly higher inhibitory activity
than curcumin. Compounds 6a, 6d, 7b and 7c showed the most potent
inhibitory activity with 7fl.8%, 79.fl%, 70.0% and 75.8% inhibition rates. Further,
the IC50 values of compound 6d and curcumin were measured, compound 6d
(
IC50= 9.47±0.44 ꢄM’ was approximately two-fold more potent than curcumin
IC50 = 18.25±0.15 ꢄM’. It was hypothesized that organic compounds with
(
antioxidant activity could specifically bind to Aꢂ or Aꢂ fibril; however, the
mechanisms are not known yet [22]. The current data are not sufficient to
establish a structureꢀactivity relationship for anti-Aꢂ aggregation activity.
Further studies are required to elucidate the mechanism.
2
.7. Protective effect against H
2
O
2
-induced cell death in PC12 neurons
The neuroprotective activity of compound 6d against oxidative
stress-induced cell death in PC12 cells was evaluated. The obtained data are
shown in Fig. 6. Compound 6d was evaluated at concentrations of 1, 2.5, 5 µM,
and quercetin was used as reference compound at a concentration of 5 µM.
Compound 6d exhibited no cytotoxicity towards PC12 cells at the tested
concentrations. H
2
O (200 µM’ reduced the cell viability to 52.8% compared to
2
the control. The pretreatment of PC12 cells with compound 6d significantly

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protected the neurons against cell death in all the concentrations (P value
<0.01’. Compound 6d showed slightly lower protective capability than
quercetin at a concentration of 5 µM. These observations indicated that
compound 6d has the potential to be an efficient multifunctional agent,
including antioxidant activity, for the treatment of AD.
2
.8. Measurement of intracellular ROS generation
The effect of compound 6d on the intracellular ROS generation of PC12
cells were investigated by dichloro-dihydro-fluorescein diacetate (DCFH-DA’
assay. The obtained results are shown in Fig. 7. The results show that the
DCFH-DA probe fluorescence emission significantly increased after treating
with H
2
O (B in Fig. 7’. After the preincubation with compound 6d or quercetin,
2
the fluorescence emission decreased (C and D in Fig. 7’. This indicates that
compound 6d has potential in vitro antioxidant activity, consistent with the
ORAC assay.
fl. Conclusion
In summary, a series of 4-dimethylamino flavonoid derivatives were
designed and synthesized as multifunctional anti-Alzheimer agents. Our
results showed that these compounds had a high inhibitory potency for both
AChE and BChE. Compounds 6d and 6e showed the highest inhibitory activity
for AChE (IC50=59.61 nM’ and BChE (IC50=24.67 nM’, respectively. The

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inhibition kinetic analysis and molecular modeling study indicated that
compound 6d had binding with both CAS and PAS of the AChE. Moreover,
these compounds had significant peroxyl radical absorbance capacity and
anti-Aβ aggregation activity. In addition, compound 6d showed neuroprotective
activity against the H
2
O -induced cell death in PC12 cells. Above all, the
2
results make the prototype compound 6d as a promising multiple potency
anti-AD agent for further developments.
4. Experimental section
4
.1. Chemistry
1
1fl
H and C NMR spectra were recorded using TMS as the internal standard
in CDCl with a Bruker BioSpin GmbH spectrometer at 400 MHz and 100 MHz,
fl
respectively. High resolution mass spectra (HRMS’ were recorded on
Shimadzu LCMS-IT-TOF. Flash column chromatography was performed with
silica gel (200ꢀfl00 mesh’ purchased from Qingdao Haiyang Chemical Co. Ltd.
The purities of synthesized compounds were confirmed to be higher than 95%
by using analytical HPLC with a dual pump Shimadzu LC-20AB system
equipped with a Ultimate XB-C18 column (4.6×250 mm, 5 µm’ and eluted
with methanol/water containing 0.1% TFA at a flow rate of 0.5 mL/min.
4
.2. Synthesis of intermediates 10aꢀ10j
Intermediates 10aꢀ10j were prepared in our lab (Supporting Information’.
.fl. Synthesis of intermediate fl
4

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Intermediate fl was prepared according to the previous literature procedure
[
1fl].
4
.4. Synthesis of intermediate 4
Intermediate fl (2.90 g, 10 mmol’, ethyl 2-chloroacetate (1.80 g, 15 mmol’,
anhydrous K
anhydrous acetone (50 mL’, the mixture was refluxed for 6 h. The solution was
evaporated, and the residue was poured into water and extracted with CHCl
The combined organic layers were dried over Na SO , and then concentrated
to give dark red solid 4 which was used without further purification. Yield 2.68 g
2
CO
fl
(2.76 g, 20 mmol’ and catalytic amount KI were added in
fl
.
2
4
+
+ 1
(
7fl%’. MS (ESI ’: m/z: fl68.1 [M+H] . H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.84 (d,
= 8.4 Hz, 1H, Ar-H’, 6.86, (s, 1H, Ar-H’, 6.80
6.75 (m, 4H, 4×Ar-H’, 4.74 (s, 2H, CH CO’, 4.fl2 (q, = 7.2 Hz, 2H, CH O’,
’, 1.fl4 (t, = 7.2 Hz, flH, CH CH ’.
.5. Synthesis of intermediate 5
Compound 4 (2.60 g, 7.1 mmol’, 5% Na
J
=
ꢀ
8.8 Hz, 2H, 2×Ar-H’, 7.74 (d, J
2
J
2
fl.09 (s, 6H, N(CH
’
fl 2
J
fl
2
4
2
CO (10 mL’ and DMSO (40 mL’
fl
were stirred at 85 °C for 10 h. Then the reaction mixture was poured into 10%
HCl (fl00 mL’, kept overnight and filtered. The filter cake was recrystallized
1
from EtOH to give red solid 2.fl7 g; Yield 70%. H NMR (400 MHz, DMSO-
d
6
’
ꢅ 7.79 (d,
Ar-H’, 6.80 ꢀ 6.7fl (m, 4H, 4×Ar-H’, 4.52 (s, 2H, CH
.6. General procedures for the preparation of target compounds 6aꢀ8b
J
= 8.8 Hz, 2H, 2×Ar-H’, 7.62 (d,
J
= 8.4 Hz, 1H, Ar-H’, 6.87 (s, 1H,
2
’, fl.00 (s, 6H, N(CH ’.
fl 2
’
4

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Compound
4
(0.fl4
g,
1
mmol’,
1
-[fl-(dimethylamino’propyl]-fl-ethylcarbodiimide (EDC, 0.29 g, 1.5 mmol’ and
N-hydroxysuccinimide (NHS, 0.17 g, 1.5 mmol’ were stirred at room
temperature for 1.5 h, and then different amines (1.2 mmol’ were added. The
mixture was stirred at room temperature overnight. Then the solvent was
poured into water and extracted with ethyl acetate (20 mL×fl’. The solution
was dried over anhydrous Na
2
SO and concentrated. The residues were
4
purified by flash chromatography with chloroform/methanol/ammonia (20:1:0.5,
v/v/v’ elution.
4
2
.6.1.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-((1,2,fl,4-tetrah
ydroacridin-9-ylamino’methyl’acetamide (6a’
1
Red oil (61% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.94 (d,
= 8.8 Hz, 2H, 2×Ar-H’, 7.54 (d,
= 8.0 Hz, 1H, Ar-H’, 7.fl6 (t, = 5.8 Hz, 1H,
= 7.6 Hz, 1H, Ar-H’, 6.79 (s, 1H, Ar-H’, 6.68 ꢀ 6.64 (m, flH,
fl×Ar-H’, 6.58 (d, = 8.4, 1H, Ar-H’, 4.70 (s, 1H, NH’, 4.56 (s, 2H, CH ’, fl.68
s, 4H, 2×CH ’, fl.0fl (s, 6H, N(CH ’, fl.00 (d, = 6.0 Hz, 2H, CH ’, 2.70 (s,
H, CH ’, 1.9fl ꢀ 1.80 (m, 4H, 2×CH ’. C NMR (100 MHz, CDCl ’ ꢅ: 182.2fl,
J = 8.4 Hz, 1H,
Ar-H’, 7.85 (d,
= 8.4 Hz, 1H, Ar-H’, 7.50 (t,
NH’, 7.fl0 (t,
J = 8.4 Hz, 1H, Ar-H’, 7.72 (d, J
J
J
J
J
J
2
(
2
’
fl 2
J
2
1fl
2
1
1
1
2
2
fl
68.fl8, 167.05, 16fl.52, 158.51, 151.fl8, 150.fl0, 147.04, 145.54, 1flfl.55,
28.54, 125.66, 124.12, 122.fl1, 120.17, 119.71, 116.92, 116.68, 114.96,
11.95, 111.67, 97.59, 67.60, 49.10, 40.fl9, 40.08, flfl.81, 25.20, 2fl.04, 22.71.

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+
Purity: 99.5% by HPLC, HRMS (ESI’: calcd for [M+H] (Cfl4
H
fl4
N
4
O ’ requires
4
m/z 56fl.2658, found 56fl.264fl.
4
.6.2
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-((1,2,fl,4-tetra
2
hydroacridin-9-ylamino’methyl’acetamide (6b’
1
Red oil (47% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.92 (d,
J
= 8.4 Hz, 1H,
Ar-H’, 7.85 (d,
J
= 8.4 Hz, 1H, Ar-H’, 7.71 (d,
8.4 Hz, 1H, Ar-H’, 7.47 (t, = 7.6 Hz, 1H, Ar-H’, 7.28 (t,
.99 (t, = 5.8 Hz, 1H, NH’, 6.77 (s, 1H, Ar-H’, 6.67 ꢀ 6.61 (m, 4H, 4×Ar-H’,
.75 (s, 1H, NH’, 4.55 (s, 2H, CH ’, fl.54 ꢀ fl.44 (m, 4H, 2×CH ’, 2.99 (s, 8H,
, CH ’, 2.68 (s, 2H, CH ’, 1.87 ꢀ 1.78 (m, 6H, fl×CH
MHz, CDCl ’ ꢅ: 182.21, 167.92, 167.11, 16fl.52, 151.fl9, 150.7fl, 145.57,
flfl.55, 128.67, 128.24, 125.77, 124.10, 122.50, 120.14, 119.77, 116.99,
J
= 8.8 Hz, 2H, 2×Ar-H’, 7.58 (d,
J
=
6
4
J
J
= 7.6 Hz, 1H, Ar-H’,
J
2
2
1fl
N(CH ’
fl 2
2
2
2
’. C NMR (100
fl
1
1
2
16.50, 114.91, 111.96, 111.61, 97.59, 67.56, 45.57, 40.10, fl6.69, flfl.6fl, fl1.24,
5.05, 2fl.0fl, 22.66. Purity: 99.0% by HPLC, HRMS (ESI’: calcd for [M+H]+
(C
fl5
H
fl6
N
4
O ’ requires m/z 577.2815, found 577.2806.
4
4
.6.fl.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-((1,2,fl,4-tetr
2
ahydroacridin-9-ylamino’methyl’acetamide (6c’
1
Red oil (52% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.91 (t,
= 8.8 Hz, 1H, Ar-H’, 7.5fl (t,
= 8.8 Hz, 1H, Ar-H’, 6.98 (t, = 8.8 Hz, 1H, NH’,
J = 8.8 Hz, 2H,
2
×Ar-H’, 7.75 (d,
J
= 8.8, 2H, 2×Ar-H’, 7.62 (t,
J
J
=
8.4 Hz, 1H, Ar-H’, 7.fl2 (t,
J
J

ACCEPTED MANUSCRIPT
6
4
.80 (d,
J
= 5.2 Hz, 1H, Ar-H’, 6.70 ꢀ 6.66 (m, 4H, 4×Ar-H’, 4.56 (s, 2H, CH ’,
2
.10 (s, 1H, NH’, fl.51 (s, 2H, CH
’, 2.65 (s, 2H, CH ’, 1.89 ꢀ 1.86 (m, 4H, 2×CH
’. 1flC NMR (100 MHz, CDCl
’ ꢅ: 182.1fl, 167.fl5, 167.02, 16fl.85,
2 2 2
’, fl.42 (s, 2H, CH ’, fl.04 (s, 2H, CH ’, fl.0fl
(s, 6H, N(CH
’
fl 2
2
2
’, 1.7fl ꢀ 1.70 (m,
4
1
1
4
H, 2×CH
2
fl
56.25, 151.9fl, 151.fl0, 145.48, 144.82, 1flfl.48, 129.44, 126.20, 125.44,
24.15, 12fl.26, 119.61, 118.96, 116.60, 114.74, 111.89, 111.81, 97.57, 67.68,
8.55, 40.06, fl8.7fl, fl2.fl6, 28.68, 26.92, 24.57, 22.68, 22.12. Purity: 98.5% by
+
HPLC, HRMS (ESI’: calcd for [M+H] (Cfl6
H
fl8
N
4
O ’ requires m/z 591.2971,
4
found 591.295fl.
4
.6.4.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-((1,2,fl,4-tetr
2
ahydroacridin-9-ylamino’methyl’acetamide (6d’
1
Red oil (58% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.92 (d,
= 9.2 Hz, 1H, Ar-H’, 7.52 (t,
= 8.0 Hz, 1H, Ar-H’, 6.79 (s, 1H, Ar-H’, 6.77 ꢀ
.6fl (m, 5H, 4×Ar-H, NH’, 4.55 (s, 2H, CH ’, 4.14 (s, 1H, NH’, fl.45 (t, = 7.2
’, fl.fl4 (q, = 6.8 Hz, 2H, CH ’, fl.08 ꢀ 2.99 (m, 8H, N(CH
’, 2.62 (s, 2H, CH ’, 1.87 (t, = 2.8 Hz, 4H, 2×CH ’, 1.66 ꢀ 1.51 (m, 4H,
’, 1.41 ꢀ 1.28 (m, 4H, 2×CH ’. C NMR (100 MHz, CDCl ’ ꢅ: 182.20,
J = 8.8 Hz, 2H,
2
J
6
×Ar-H’, 7.7fl (d,
J = 8.8 Hz, 2H, 2×Ar-H’, 7.64 (d, J
= 8.0 Hz, 1H, Ar-H’, 7.fl0 (t,
J
2
J
Hz, 2H, CH
2
J
2
fl 2
’ ,
fl×CH
2
2
J
2
1fl
2
1
1
1
×CH
2
2
fl
67.1fl, 16fl.77, 157.61, 151.fl6, 151.22, 146.45, 145.57, 1flfl.51, 128.7fl,
27.80, 125.68, 12fl.79, 12fl.0fl, 119.7fl, 116.85, 115.fl4, 114.82, 111.94,
11.85, 97.54, 67.75, 49.20, 40.06, fl9.0fl, flfl.fl9, fl1.60, 29.48, 26.57, 24.68,

ACCEPTED MANUSCRIPT
2
2.96, 22.57. Purity: 98.9% by HPLC, HRMS (ESI’: calcd for [M+H]+
(C
fl8
H
42
N
4
O ’ requires m/z 619.fl284, found 619.fl269.
4
4
.6.5.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-((1,2,fl,4-tetr
2
ahydroacridin-9-ylamino’methyl’acetamide (6e’
1
Red oil (fl7% yield’, H NMR (400 MHz, CDCl
fl
’ ꢅ: 8.fl2 (d,
= 8.8 Hz, 2H, 2×Ar-H’, 7.65 ꢀ
= 7.6 Hz, 1H, Ar-H’, 6.95 (t, = 5.6 Hz, 1H, NH’,
.82 ꢀ 6.72 (m, flH, fl×Ar-H’, 6.69 (d, = 8.8 Hz, 2H, 2×Ar-H’, 5.70 (s, 1H, NH’,
.62 (s, 2H, CH ’, fl.75 (d, = 5.6 Hz, 2H, CH ’, fl.fl5 (q, = 6.8 Hz, 2H, CH ’,
= 6.0 Hz, 2H, CH ’, fl.08 ꢀ 2.99 (m, 6H, N(CH ’, 2.61 (t, = 5.6 Hz,
’, 1.94 ꢀ 1.80 (m, 4H, 2×CH ’, 1.75 ꢀ 1.71 (m, 2H, CH ’, 1.58 ꢀ 1.50 (m,
’, 1.fl5 ꢀ 1.27 (m, 8H, 4×CH ’. C NMR (100 MHz, CDCl ’ ꢅ: 182.16,
J = 8.4 Hz, 1H,
Ar-H’, 8.1fl (d,
J
= 8.4 Hz, 1H, Ar-H’, 7.74 (d,
J
7
6
4
.61(m, 2H, 2×Ar-H’, 7.fl8 (t,
J
J
J
2
J
2
J
2
fl.19 (t,
J
2
fl
’
2
J
2
2
1
1
H, CH
2
2
2
1fl
H, CH
2
2
fl
67.1fl, 16fl.97, 151.fl5, 145.55, 1flfl.46, 1fl1.29, 125.55, 124.72, 124.06,
19.67, 116.69, 114.67, 111.92, 111.84, 97.64, 67.79, 48.64, 40.05, fl9.10,
fl1.12, 29.fl8, 29.06, 28.96, 26.64, 26.59, 2fl.97, 22.20, 21.16. Purity: 99.4% by
+
HPLC, HRMS (ESI’: calcd for [M+H] (C40
H
46
N
4
O ’ requires m/z 647.fl597,
4
found 647.fl580.
4
.6.6.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-(pyrrolidin-1-
ylmethyl’acetamide (7a’
2

ACCEPTED MANUSCRIPT
1
Red oil (41% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.78 ꢀ 7.67 (m, flH,
’, fl.49 (d, = 5.6 Hz,
= 6.0 Hz, 2H, CH ’, 2.57 (s, 4H,
’. C NMR (100 MHz, CDCl ’ ꢅ: 182.24,
fl×Ar-H’, 6.87 ꢀ 6.67 (m, 5H, 5×Ar-H’, 4.59 (s, 2H, CH
2
J
2
2
1
1
H, CH
2
’, fl.04 (s, 6H, N(CH
fl
2
’ ’, 2.70 (t,
J
2
1fl
×CH ’, 1.79 ꢀ 1.76 (m, 4H, 2×CH
2
2
fl
67.19, 16fl.90, 151.28, 145.59, 1flfl.47, 125.61, 119.81, 116.72, 114.64,
11.91, 111.86, 97.64, 67.69, 54.42, 5fl.89, 40.0fl, fl7.56, 2fl.54. Purity: 98.5%
+
by HPLC, HRMS (ESI’: calcd for [M+H] (C25
H
29
N
fl
O ’ requires m/z 4fl6.22fl6,
4
found 4fl6.22fl1.
4
.6.7.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-(pyrrolidin-1-yl
methyl’acetamide (7b’
2
1
Red oil (fl9% yield’, H NMR (400 MHz, CDCl
.80 (d, = 9.2 Hz, 2H, 2×Ar-H’, 7.71 (d, = 8.4 Hz, 1H, Ar-H’, 6.82 (d,
Hz, flH, fl×Ar-H’, 6.7fl (d, = 9.2 Hz, 2H, 2×Ar-H’, 4.59 (s, 2H, CH ’, fl.57 ꢀ fl.45
m, 2H, CH ’, fl.06 (s, 6H, N(CH ’, 2.89 ꢀ 2.68 (m, 6H, fl×CH ’, 1.90 (t, = 6.4
Hz, 6H, fl×CH ’. C NMR (100 MHz, CDCl ’ ꢅ: 182.16, 167.18, 167.1fl, 164.14,
51.24, 145.5fl, 1flfl.44, 125.51, 119.70, 116.6fl, 114.61, 111.9fl, 111.87, 97.58,
8.00, 54.96, 54.06, 40.00, fl8.85, 26.99, 2fl.49. Purity: 99.2% by HPLC, HRMS
’ requires m/z 450.2fl9fl, found 450.2fl7fl.
fl
’ ꢅ: 8.04 (t,
J
= 4.8, 1H, NH’,
7
J
J
J
= 8.4
J
2
(
2
’
fl 2
2
J
1fl
2
fl
1
6
fl1 fl
(ESI’: calcd for [M+H]+ (C26H N O
4
4
.6.8.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-(pyrrolidin-1-
ylmethyl’acetamide (7c’
2

ACCEPTED MANUSCRIPT
1
Red oil (5fl% yield’; H NMR (400 MHz, CDCl
×Ar-H’, 7.72 (d, = 9.2 Hz, 1H, Ar-H’, 6.97 (t, = 5.2 Hz, 1H, NH’, 6.85 (s, 1H,
Ar-H’, 6.79 ꢀ 6.71 (m, 4H, 4×Ar-H’, 4.58 (s, 2H, CH ’, fl.fl9 (q, = 6.4 Hz, 2H,
CH ’, fl.07 (s, 6H, N(CH ’, 2.5fl ꢀ 2.46 (m, 6H, fl×CH ’, 1.80 ꢀ 1.76 (m, 4H,
×CH ’, 1.65 ꢀ 1.56 (m, 4H, 2×CH ’. C NMR (100 MHz, CDCl ’ ꢅ: 182.fl1,
fl
’ ꢅ: 7.80 (d, J = 9.2 Hz, 2H,
2
J
J
2
J
2
’
fl 2
2
1fl
2
1
1
2
2
2
fl
67.22, 167.10, 16fl.85, 151.fl5, 145.62, 1flfl.52, 125.77, 119.85, 116.88,
14.79, 111.96, 111.85, 97.62, 67.85, 55.8fl, 54.09, 40.07, fl9.0fl, 27.5fl, 26.12,
+
fl.41. Purity: 97.8% by HPLC, HRMS (ESI’: calcd for [M+H] (C27
H N O ’
flfl fl 4
requires m/z 464.2549, found 464.2542.
.6.9
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-(pyrrolidin-1-
4
2
ylmethyl’acetamide (7d’
1
Red oil (41% yield’, H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.78 (d,
J
= 8.8 Hz, 2H,
2
2
2
2
×Ar-H’, 7.69 (d,
J
= 8.8 Hz, 1H, Ar-H’, 6.82 (s, 1H, Ar-H’, 6.78 ꢀ 6.75 (m, 2H,
= 9.2 Hz, 2H, 2×Ar-H’, 6.6fl (t, = 6.0 Hz, 1H, NH’, 4.57 (s,
= 6.8 Hz, 2H, CH ’, fl.05 (s, 6H, N(CH ’, 2.47 (s, 4H,
= 7.6 Hz, 2H, CH ’, 1.78 ꢀ 1.74 (m, 4H, 2×CH ’, 1.59 ꢀ 1.49
’, 1.fl6 ꢀ 1.25 (m, 6H, fl×CH ’. C NMR (100 MHz, CDCl ’ ꢅ:
82.20, 177.25, 167.16, 166.96, 16fl.76, 151.fl2, 145.58, 1flfl.48, 125.69,
19.79, 116.8fl, 114.69, 111.9fl, 111.78, 97.65, 67.70, 56.28, 54.10, 40.04,
×Ar-H’, 6.72 (d,
J
J
H, CH
×CH ’, 2.fl9 (t,
m, 4H, 2×CH
2
’, fl.fl6 (q,
J
2
’
fl 2
2
J
2
2
1fl
(
2
2
fl
1
1
fl9.11, 29.fl9, 28.16, 27.14, 26.70, 2fl.fl8. Purity: 99.1% by HPLC, HRMS (ESI’:
+
calcd for [M+H] (C29
H
fl7
N
fl
O ’ requires m/z 492.2862, found 492.2864.
4

ACCEPTED MANUSCRIPT
4
.6.10.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-(pyrrolidin-1-
2
ylmethyl’acetamide (7e’
1
Red oil (fl8% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.76 (d,
J
= 8.8 Hz, 2H,
2
×Ar-H’, 7.67 (d,
Ar-H, NH’, 6.70 (d,
Hz, 2H, CH ’, fl.04 (s, 6H, N(CH
’, 1.79 (s, 4H, 2×CH ’, 1.55 ꢀ 1.51 (m, 4H, 2×CH
’. 1flC NMR (100 MHz, CDCl
’ ꢅ: 182.17, 167.1fl, 166.9fl, 16fl.75,
J
= 8.8 Hz, 1H, Ar-H’, 6.80 (s, 1H, Ar-H’, 6.76 ꢀ 6.74 (m, 2H,
= 9.2 Hz, flH, fl×Ar-H’, 4.56 (s, 2H, CH ’, fl.fl5 (q, = 6.8
’, 2.5fl (s, 4H, 2×CH ’, 2.4fl (t, = 7.6 Hz,
’, 1.29 (s, 8H,
J
2
J
2
’
fl 2
2
J
2
4
1
9
2
H, CH
2
2
2
×CH
2
fl
51.28, 145.55, 1flfl.46, 125.6fl, 119.76, 116.79, 114.66, 111.90, 111.77,
7.61, 67.69, 56.5fl, 54.15, 40.02, fl9.24, 29.49, 29.fl9, 29.14, 28.70, 27.52,
6.81, 2fl.fl8. Purity: 98.5% by HPLC, HRMS (ESI’: calcd for [M+H]+
(C
fl1
H
41
N
fl
O ’ requires m/z 520.fl175, found 520.fl167.
4
4
.6.11.
N-(2-(Dimethylamino’ethyl’-2-(2-(4-(dimethylamino’phenyl’-4-oxo-4H-chro
men-7-yloxy’acetamide (8a’
1
Red oil (41% yield’, H NMR (400 MHz, CDCl
fl
’ ꢅ: 7.80 (d,
J
= 8.0 Hz, 2H,
2
×Ar-H’, 7.72 (d,
fl×Ar-H’, 6.72 (d,
H, CH ’, fl.06 (s, 6H, Ar-N(CH
N(CH
’. 1flC NMR (100 MHz, CDCl
51.26, 145.61, 1flfl.51, 125.72, 119.80, 116.76, 114.71, 114.67, 111.9fl,
J
= 8.0 Hz, 1H, Ar-H’, 7.19 (s, 1H, NH’, 6.86 ꢀ 6.76 (m, flH,
= 8.0 Hz, 2H, 2×Ar-H’, 4.60 (s, 2H, CH ’, fl.46 (d, = 5.6 Hz,
’, 2.51 (t, = 5.2 Hz, 2H, CH ’, 2.27 (s, 6H,
’ ꢅ: 182.fl5, 167.19, 167.15, 16fl.80,
J
2
J
2
2
fl
’
2
J
2
fl
’
2
fl
1

ACCEPTED MANUSCRIPT
9
7.58, 67.6fl, 57.69, 45.10, 40.06, fl6.fl4. Purity: 98.8% by HPLC, HRMS (ESI’:
+
calcd for [M+H] (C2fl
H
27
N
fl
O ’ requires m/z 410.2080, found 410.2062.
4
4
.6.12.
-(2-(4-(Dimethylamino’phenyl’-4-oxo-4H-chromen-7-yloxy’-N-(fl-(dimethyl
2
amino’propyl’acetamide (8b’
1
Red oil (57% yield’; H NMR (400 MHz, CDCl
fl
’ ꢅ: 8.41 (s, 1H, NH’, 7.81 (d,
J
= 8.8 Hz, 2H, 2×Ar-H’, 7.7fl (d,
6.77 (m, 2H, 2×Ar-H’, 6.74 (d,
= 5.6 Hz, 2H, CH ’, fl.07 (s, 6H Ar-N(CH
’, 2.28 (s, 6H, N(CH ’, 1.81 ꢀ 1.7fl (m, 2H, CH
CDCl ’ ꢅ: 182.fl7, 167.fl2, 167.2fl, 164.12, 151.flfl, 145.67, 1flfl.52, 125.77,
19.86, 116.71, 114.71, 111.97, 111.91, 97.45, 67.7fl, 58.29, 44.92, 40.1fl,
fl9.51, fl8.94, 25.09. Purity: 99.fl% by HPLC, HRMS (ESI’: calcd for [M+H]+
’ requires m/z 424.22fl6, found 424.2216.
J
= 9.2 Hz, 1H, Ar-H’, 6.85 (s, 1H, Ar-H’, 6.80
= 9.2 Hz, 2H, 2×Ar-H’, 4.58 (s, 2H, CH ’,
’, 2.52 (t, = 6.0 Hz, 2H,
’. 1flC NMR (100 MHz,
ꢀ
J
2
fl.48 (q,
CH
J
2
’
fl 2
J
2
fl
’
2
2
fl
1
(C
24
H
29
N
fl
O
4
5. Biological activity
5
.1. In vitro inhibition studies on AChE and BChE
Acetylcholinesterase (AChE, E.C. fl.1.1.7, from electric eel’,
butyrylcholinesterase (BChE, E.C. fl.1.1.8, from equine serum’,
,5ꢁ-dithiobis-(2-nitrobenzoic acid’ (DTNB’, acetylthiocholine chloride (ATC’,
5
butylthiocholine chloride (BTC’ were purchased from Sigma-Aldrich. Test
compounds were dissolved in a minimum volume of DMSO (1%’ and then

ACCEPTED MANUSCRIPT
diluted in 0.1 M KH
2
PO
4
/K
2
HPO
4
buffer (pH 8.0’ to provide a final
concentration range.
All the assays were under 0.1 M KH
2
PO
4
/K
2
HPO buffer, pH 8.0, using a
4
Shimadzu UV-2450 Spectrophotometer. Enzyme solutions were prepared to
give 2.0 units/mL in 2 mL aliquots. The assay medium contained phosphate
buffer, pH 8.0 (1 mL’, 50 µL of 0.01 M DTNB, 10 µL of enzyme, and 50 µL of
0
.01 M substrate (ATC’. The substrate was added to the assay medium
containing enzyme, buffer, and DTNB with inhibitor after 15 min of incubation
time. The activity was determined by measuring the increase in absorbance at
4
12 nm at 1 min intervals at fl7 °C. In vitro BChE assay use the similar method
described above. Each concentration was assayed in triplicate.
5
.2. Kinetic characterization of AChE inhibition
Six different concentrations of substrate were mixed in the 1 mL 0.1 M
2
KH PO
4
/K
2
HPO buffer (pH 8.0’, containing 50 µL of DTNB, 10 µL AChE, and
4
5
0 µL substrate. Test compound was added into the assay solution and
pre-incubated with the enzyme at fl7°C for 15 min, followed by the addition of
substrate. Kinetic characterization of the hydrolysis of ATC catalyzed by AChE
was done spectrometrically at 412 nm. A parallel control with no inhibitor in the
mixture, allowed adjusting activities to be measured at various times. Kinetic
characterization of BChE assay use the similar method described above.
5
.fl. Molecular modeling

ACCEPTED MANUSCRIPT
The crystal structure of the torpedo AChE (code ID: 1ACJ’ and the human
BChE (code ID: 1POI’ were obtained in the Protein Data Bank after eliminating
the inhibitor and water molecules. Docking studies were carried out using the
AUTODOCK 4.0 program using ADT, polar hydrogen atoms were added and
Gasteiger charges were assigned to the enzyme. The resulting enzyme
structure was used as an input for the AUTOGRID program. AUTOGRID
performed a precalculated atomic affinity grid maps for each atom type in the
ligand plus an electrostatics map and a separate desolvation map present in
the substrate molecule. All maps were calculated with 0.fl75 Å spacing
between grid points. The center of the grid box was placed at the bottom of the
active site gorge (AChE [2.781 64.fl8fl 67.971]; BChE [1fl5.628 111.fl75
fl9.905]’. The dimensions of the active site box were set at 50 × 46 × 46 Å.
5
.4. Inhibition of self-mediated Aꢂ(1-42’ aggregation
Aꢂ(1-42’ peptide (Anaspec Inc’ was dissolved in phosphate buffer (pH 7.40,
.01 M’ to obtain a 20 ꢄM solution. Compounds were firstly prepared in DMSO
0
to obtain a 10 mM solution. The final concentration of Aꢂ(1-42’ and inhibitors
were 20 ꢄM. After incubated in fl7 °C for 48 h, thioflavin-T (5 ꢄM in 50 mM
glycine-NaOH buffer, pH 8.00’ was added. Fluorescence was measured at
4
50 nm (ꢆex’ and 485 nm (ꢆem’. Each inhibitor was run in triplicate. The
fluorescence intensities were recorded, and the percentage of inhibition on
aggregation was calculated by the following expression: (1-IFi/IFc’*100% in
which IFi and IFc were the fluorescence intensities obtained for absorbance in

ACCEPTED MANUSCRIPT
the presence and absence of inhibitors, respectively, after subtracting the
fluorescence of respective blanks.
5
.5. Measurement of the anti-oxidation activity
The antioxidation activity was determined by using the oxygen radical
absorbance capacityꢀfluorescein (ORAC-FL’ assay. The ORACꢀassay
measures antioxidant scavenging activity against peroxyl radical induced by
2
,2ꢁ-azobis(2-amidinopropane’ dihydrochloride (AAPH’ at fl7 °C.
The reaction was carried out in 75 mM phosphate buffer (pH 7.4’, and the
final reaction mixture was 200 µL. Antioxidant (20 µL’ and fluorescein (120 µL,
fl00 nM final concentration’ were placed in the wells of a black 96 well plate,
and the mixture was incubated for 15 min at fl7 °C. Then AAPH solution (60
µL; 12 mM final concentration’ was added rapidly. The plate was immediately
placed into a Spectrafluor Plus plate reader (BMG, German’, and the
fluorescence was measured every 60 seconds for 120 minutes with exitation at
4
6
85
nm
and
emission
at
5fl5
nm.
-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox’ was used as
a standard (1-5 µM, final concentration’. 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 (1-5 µM’, and
at least three independent runs were performed for each sample.
Fluorescence measurements were normalized based on the curve of the blank

ACCEPTED MANUSCRIPT
(without antioxidant’. From the normalized curves, the area under the
fluorescence decay curve (AUC’ was calculated as:
i=90
(1) AUC = 1+
fi/f
0
∑
Where f
0
is the initial fluorescence at 0 min, and f is
i
i= 1
the fluorescence at time i. The net AUC for a sample was calculated as
following:
(2’ Net AUC = AUCantioxidant ꢀ AUCblank; The ORAC-FL values were
calculated as following:
(fl’ [(AUCSample ꢀ AUCblank’/(AUCTroloxꢀAUCblank’] × [(concentration of
Trolox/concentration of sample’], and expressed as Trolox equivalents by
using the standard curve calculated for each assay. Final results were in µM of
Trolox equivalent / µM of pure compound.
5
.6. Protective effect against H
PC12 cells were cultured in DMEM media with 10% FCS containing 100
units/mL penicillin and 100 mg/mL streptomycin under 5% CO at fl7 °C. To
produce oxidative stress, H was freshly prepared from a fl0% stock solution
2
O
2
-induced cell death in PC12 neurons
2
2
O
2
prior to each experiment. PC12 cells were plated in cell culture plates at a
5
density of fl×10 cells per well (Corning Incorporated Life Sciences, Lowell,
MA’. In our study, 0.2 mM H
2
O were treated with PC12 cells for 24 h in the
2
injury module, while the protective-group cells were pretreated with different
concentrations (1, 2.5 and 5 µM’ of the compound 6d for 2 h. The
control-group cells were treated with the same medium without H
2
O or
2

ACCEPTED MANUSCRIPT
compound 6d. The cell viability of PC12 cells was evaluated using MTT assay.
After drug treatments PC12 cells were cultured with 5 mg/mL MTT and
incubated for 4 h at fl7 °C. Then the cells were lysed in dimethyl sulfoxide
(DMSO’ (150 uL’. The absorbance was detected at 570 nm by a microplate
reader. PC12 cells were cultured without 6d or H
2
O as control group and the
2
results were expressed by percentage of control.
5
.7. Measurement of ROS generation
Intracellular esterase can deacetylate DCFH-DA to DCFH, which can be
oxidized by reactive oxygen species to fluorescent compound 2ꢁ,
ꢁ-dichloroflorescein (DCF’. Thus, the fluorescence intensity of DCF is in direct
proportion to the amount of reactive oxygen species generation. Cells of 1×106
were exposed to H for 24 h, while the protective group cells were
7
2
O
2
pretreated with compound 6d for 2 h. After exposure, collected cells were
incubated with 100 ꢄmol/L DCFH-DA then 10 ꢄg/L DAPI at fl7 °C for 15 min
respectively in the darkness. Then cells were washed again and maintained in
1
mL PBS. The reactive oxygen species generation was analyzed by
fluorescence microscope with excitation and emission wavelengths of 488 and
fl0 nm, respectively.
5
Acknowledgements

ACCEPTED MANUSCRIPT
We thank the Natural Science Foundation of China (No. 21fl02041,
117205fl’, the Postdoctoral Science Foundation of China (No.
012M521fl91’, the Educational Commission of Henan Province (No.
4Afl50008’, and the Postdoctoral Science Foundation of Henan Province (No.
011015’ for financial support of this study.
2
2
1
2
Conflict of Interest
We declare that we have no conflict of interest.
References and notes
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