Design, synthesis, biological evaluation, and molecular modeling studies of quinoline-ferulic acid hybrids as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103310

A series of quinoline-ferulic acid hybrids has been designed, synthesized, and evaluated as cholinesterase inhibitors. Most of the compounds showed good inhibitory activities toward both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Among them, 10f was found to be the most potent inhibitor against AChE (IC₅₀ = 0.62 ± 0.17 μM), and 14 was the most potent inhibitor against BChE (IC₅₀ = 0.10 ± 0.01 μM). Representative compounds, such as 10f and 12g, act in a competitive manner when they inhibit AChE or BChE. Molecular docking and dynamic simulation revealed that the synthesized compounds bind to the target by simultaneously interacting with the catalytic active site (CAS) and the peripheral anionic site (PAS) of both AChE and BChE. The U-shaped confirmation was preferred when 12g bound to BChE, which was different from the linear conformation of 10f bound to AChE. Cell-based assays have confirmed the moderate neuroprotective effects of compounds 10f and 12g against H₂O₂-induced oxidative damage towards PC12 cells. Moreover, the hepatotoxicity of 12g was lower than that of tacrine, indicating its potential safety as an anti-Alzheimer's agent. In summary, we report a new chemotype of multifunctional hybrid, which may be further modified to develop new anti-Alzheimer's agents.

Full text of DOI:10.1016/j.bioorg.2019.103310

BioorganicChemistry93(2019)103310
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, biological evaluation, and molecular modeling studies of
quinoline-ferulic acid hybrids as cholinesterase inhibitors
Jun Mob,1, Hongyu Yangb,1, Tingkai Chenc, Qihang Lib, Hongzhi Linb, Feng Fengc,f,
⁎
⁎
Wenyuan Liud, Wei Quc, Qinglong Guoe, Heng Chif, Yao Chena, , Haopeng Sunb,f,
a School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China
b Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
c Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 211198, People’s Republic of China
d Department of Pharmaceutical Analysis, Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University,
Nanjing 210009, People’s Republic of China
e State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, School of Basic Medicine and Clinical Pharmacy, China
Pharmaceutical University, Nanjing 210009, People’s Republic of China
f Jiangsu Food and Pharmaceuticals Science College, Institute of Food and Pharmaceuticals Research, 223005, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of quinoline-ferulic acid hybrids has been designed, synthesized, and evaluated as cholinesterase in-
hibitors. Most of the compounds showed good inhibitory activities toward both acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE). Among them, 10f was found to be the most potent inhibitor against AChE
Alzheimer’s disease
Cholinesterase inhibitor
Quinoline-ferulic acid hybrid
Molecular docking
(IC50 = 0.62
0.17 μM), and 14 was the most potent inhibitor against BChE (IC50 = 0.10
0.01 μM).
Representative compounds, such as 10f and 12g, act in a competitive manner when they inhibit AChE or BChE.
Molecular docking and dynamic simulation revealed that the synthesized compounds bind to the target by
simultaneously interacting with the catalytic active site (CAS) and the peripheral anionic site (PAS) of both AChE
and BChE. The U-shaped confirmation was preferred when 12g bound to BChE, which was different from the
linear conformation of 10f bound to AChE. Cell-based assays have confirmed the moderate neuroprotective
effects of compounds 10f and 12g against H2O2-induced oxidative damage towards PC12 cells. Moreover, the
hepatotoxicity of 12g was lower than that of tacrine, indicating its potential safety as an anti-Alzheimer’s agent.
In summary, we report a new chemotype of multifunctional hybrid, which may be further modified to develop
new anti-Alzheimer’s agents.
1. Introduction
extensively studied for the design of anti-AD drugs [7,8]. Indeed, cho-
linergic dysfunction has been identified as one of the major causes of
Alzheimer’s disease (AD) is an age-related neurodegenerative disease
characterized by a progressive loss of cognitive abilities, such as memory,
language skills, and attention span, leading to disorientation and depres-
sion [1]. AD is estimated to incur a global annual expenditure of two
trillion dollars by 2030 [2]. Currently, only four drugs have been approved
by the U.S. Food and Drug Administration (FDA) for the treatment of AD,
namely donepezil, rivastigmine, galantamine, and memantine [3]. How-
ever, the therapeutic effects of these drugs are limited, so there is an ur-
gent demand to develop new therapeutic agents for AD [4,5].
AD. Acetylcholine (ACh) can be hydrolyzed by two types of cholines-
terases (ChEs): acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) [9]. During the early stage of AD, AChE plays a dominant role in
ACh hydrolysis, while BChE plays only a supportive role [10,11].
Therefore, inhibiting the activity of AChE is an effective way to ame-
liorate cognitive dysfunction in AD. However, studies have shown that
compounds that are able to inhibit BChE may offer an alternative role
for the treatment of AD, especially in its advanced stage [12]. During
the development of AD, the BChE/AChE ratio gradually increases,
partially as a consequence of a progressive loss of the cholinergic sy-
napses in which AChE resides [13].
The cholinergic system, which plays an important role in the reg-
ulation of learning, cognition, and memory processes [6], has been
⁎ Corresponding authors at: School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, People’s Republic of China (Y. Chen). Department of
Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 211198, People’s Republic of China (H. Sun).
E-mail addresses: clarissa0710@163.com (Y. Chen), sunhaopeng@163.com (H. Sun).
1 These two authors contributed equally to this paper.
https://doi.org/10.1016/j.bioorg.2019.103310
Received 6 December 2018; Received in revised form 4 September 2019; Accepted 22 September 2019
Availableonline23September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

J. Mo, et al.
BioorganicChemistry93(2019)103310
Fig. 1. Design of a series of quinoline-ferulic acid hybrids as cholinesterase inhibitors.
Scheme 1. Reagents and conditions: (a) PPA, 130 °C, 3 h; (b) POCl3, reflux, 2 h; (c) phenol, sodium iodide, 120 °C; (d) K2CO3, DMF, 85 °C, 5 h; (e) NaOH, MeOH, H2O,
85 °C, 3 h; (f) PyBOP, DIPEA, DMF, 4 h, rt.
Multi-target-directed ligands (MTDLs) can display multiple bioac-
tivities by simultaneously regulating different nodes or pathways.
Considering the multifactorial nature of AD, MTDLs are supposed to
exhibit promising therapeutic effects, therefore, it is considered to be an
important strategy for the development of anti-AD drugs [14]. Our
group has focused on the design and synthesis of new MTDLs in recent
years. Previously, we disclosed a series of tacrine-cinnamic acid hy-
brids/tacrine-ferulic acid hybrids (CYP36, Fig. 1) as MTDLs against AD
[15,16]. In these publications, we focused our attention on modifica-
tions of the cinnamic acid/ferulic acid moieties, as well as the choice of
linker in these hybrids. Although these hybrids showed good multi-
functional activity as desired, the chemotype of the hybrids remained
limited, and structure–activity relationships (SAR), especially of the
tacrine moiety, were not comprehensively studied. We surmised that
the complicated structure of these hybrids may lead to undesirable
physicochemical properties, which may hinder their further develop-
ment. Hence, further structural modification and SAR studies were
deemed necessary. Herein, we disclose the results of structural mod-
as new bioactive compounds for AD treatment. Additionally, the
slightly reduced molecular weight of the compounds, compared to
those reported previously, is also preferable for drug development.
In the present study, the inhibitory activities of the quinoline-cin-
namic acid hybrids on AChE and BChE have been investigated. To
further determine the binding mode of the target molecules on AChE
and BChE, kinetic, molecular docking, and molecular dynamics (MD)
experiments have been performed on selected compounds. The multi-
functional properties of the compounds, including their effects on self-
mediated Aβ aggregation, antioxidant activities, and neuroprotection,
have been determined in vitro or in cell-based assays. The hepatotoxi-
city of the hybrids has been evaluated to show their potential safety as
anti-AD agents.
This study identifies a new scaffold for the further design of MTDLs
against AD. The SAR results, especially the tolerance to modification of
the quinoline core, suggest that further optimization of the substituent
thereon, or even replacement of the ring with another heterocycle,
merits further study.
ification on
a 1,2,3,4-tetrahydroacridine moiety. To simplify the
structures of the hybrids, the cyclohexyl part of 1,2,3,4-tetra-
hydroacridine moiety was removed, leading to a series of compounds
with quinoline core. The resultant hybrids retained multifunctional
activity according to biological assays, indicating that removal of the
cyclohexyl part of the 1,2,3,4-tetrahydroacridine moiety is tolerable for
medicinal chemistry modification. The obtained anti-AD hybrids with a
new chemotype, such as a quinoline-cinnamic acid scaffold (Fig. 1),
may represent an alternative starting point for subsequent optimization
2. Results and discussion
2.1. Chemistry
The synthetic route to quinoline-ferulic acid hybrids 10a–10h is
shown in Scheme 1. 2-Methyl-4-hydroxylquinoline (3) was prepared by
the condensation of aniline with ethyl acetoacetate catalyzed by poly-
phosphoric acid (PPA) at 130 °C. Compound 3 was then treated with
2

J. Mo, et al.
BioorganicChemistry93(2019)103310
Scheme 2. Reagents and conditions: (b) POCl3, reflux, 2 h; (c) phenol, sodium iodide, 120 °C; (f) PyBOP, DIPEA, DMF, 4 h, rt.
Table 1
Structures and eeAChE and eqBChE inhibitory activities of the target compounds.
Compound
n
R
IC50 (μM)
AChEb
SEMa
BChEc
10a
10b
10c
10d
10e
10f
10g
10h
12a
12b
12c
12d
12e
12f
12g
12h
12i
–
–
–
–
–
–
–
–
1
1
1
1
1
1
1
1
1
2
3
–
–
–
H
1.43
1.69
3.46
6.59
1.61
0.62
1.59
1.07
5.51
2.62
2.44
4.07
3.38
3.54
3.28
1.45
10.04
1.35
2.47
3.69
1.06
1.44
0.02
0.32
3.38
3.37
1.09
0.99
9.13
1.18
1.19
0.87
1.33
2.11
2.17
7.07
1.58
1.51
0.93
4.04
3.05
0.58
0.10
2.43
1.04
0.44
0.008
0.77
0.13
0.67
0.54
1.16
0.14
0.36
0.17
1.41
0.46
0.37
1.32
0.52
0.28
0.40
1.85
0.85
0.15
0.01
0.78
0.36
0.14
4-CH3
4-OCH3
4-F
0.62
0.67
0.95
0.16
0.17
0.58
0.47
2.90
0.88
1.27
1.77
0.95
1.12
1.32
0.62
4-Cl
4-Br
4-CF3
4-CN
H
4-CH3
4-OCH3
4-F
4-Cl
4-Br
4-CF3
4-NO2
3-CN
H
5.99
13
0.41
0.37
0.66
0.23
0.41
0.01
14
H
18
H
19
4-CH3
4-Br
20
Tacrine
0.004
a
b
c
Concentration required for 50% inhibition of ChEs; data are shown as the mean
AChE (EC 3.1.1.7) from electric eel.
SEM of triplicate independent experiments.
BChE (EC 3.1.1.8) from horse serum.
phosphoryl chloride at 120 °C for 2 h to give 4-chloro-2-methylquino-
line (4). Next, compound 4 was treated with ethylenediamine or 1,3-
propanediamine or 1,4-diaminobutane at 120 °C for 10 h to give in-
termediates 5a–5c. The intermediates 8a–8h were prepared by con-
densation reactions of ferulic acid (6) with various substituted benzyl
bromides (7a–7h) at 85 °C for 5 h. The benzyl groups of 8a–8h were
removed by treatment with 2 N aq. NaOH to obtain intermediates
9a–9h. Finally, the intermediates 9a–9h were reacted with compound
5a at room temperature to obtain the target molecules 10a–10h.
The synthetic route to quinoline-cinnamic acid hybrids 12a–12i, 13,
and 14 is shown in Scheme 2. The intermediates 5a–5c were reacted
with various cinnamic acid derivatives (11a–11i) at room temperature
to afford the target molecules 12a–12i, 13, and 14.
The synthetic route to quinoline-ferulic acid hybrids 18–20 is also
shown in Scheme 2. 4-Hydroxylquinoline (15) was treated with phos-
phoryl chloride at 120 °C for 2 h to give 4-chloroquinoline (16). Next,
compound 16 was treated with ethylenediamine at 120 °C for 10 h to
give N1-(quinolin-4-yl)ethane-1,2-diamine (17). The intermediate 17
was reacted with intermediates 9a, 9b, or 9f at room temperature to
afford the target molecules 18–20.
2.2. Pharmacology
2.2.1. AChE and BChE inhibitory activities of the target molecules
The inhibitory effects of all of the synthesized compounds on AChE
from eeAChE and BChE from eqBChE were assayed according to the
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J. Mo, et al.
BioorganicChemistry93(2019)103310
modified method of Ellman et al. [17]. Most of the compounds proved
to be potent inhibitors of AChE and BChE. The results are expressed in
terms of IC50 values (Table 1).
Table 2
Vmax and Km values for 10f and 12g in kinetic studies. Data are shown as
mean SEM of three independent experiments.
Firstly, we synthesized quinoline-ferulic acid hybrids (10a–10h).
Compared to 10a, we introduced 4-methyl (10b) and 4-methoxyl (10c)
groups on the benzyloxyl moiety. 10b showed comparable activity to
10a, whereas 10c showed slightly reduced activity towards AChE, but
improved inhibitory activity towards BChE. We then designed com-
pounds with different halogen atom substitutions. 10d showed se-
lectivity towards BChE, whereas 10e exhibited selectivity towards
AChE. Compared to 10a, 10f showed improved activity towards both
AChE and BChE. The activity towards AChE decreased in the order 4-Br
(10f) > 4-Cl (10e) > 4-F (10d). Subsequently, we explored the in-
ductive effects of substituents on ChE inhibitory activities. Compared to
the electron-donating groups 4-CH3 and 4-OCH3, electron-withdrawing
groups such as 4-CF3 (10g) and 4-CN (10h) enhanced AChE and BChE
inhibitory activities.
Vmax and Km at different concentrations of 10f
Concentration (μM)
Vmax (μM 7 min−1
)
Km (μM 7 min−1
)
R2
0
0.32
0.31
0.26
0.24
0.23
0.03
0.02
0.01
0.01
0.01
258.9
280.2
241.3
238.5
270.2
72.1
60.9
40.0
27.5
39.2
0.93
0.96
0.98
0.99
0.98
0.1
1
5
10
Vmax and Km at different concentrations of 12g
Concentration (μM)
Vmax (μM 7 min−1
)
Km (μM 7 min−1
)
R2
0
0.53
0.52
0.44
0.38
0.33
0.04
0.10
0.03
0.03
0.03
263.9
313.6
289.3
254.0
286.0
47.0
132.8
39.0
46.4
64.1
0.98
0.91
0.99
0.98
0.97
0.1
1
5
Next, we synthesized quinoline-cinnamic acid hybrids (12a–12i, 13,
and 14). Compared to quinoline-ferulic acid hybrids (10a–10h), they
showed similar activities and target selectivities. We introduced 4-me-
thyl and 4-methoxyl groups on the cinnamic acid moiety. 12b and 12c
showed reduced BChE inhibitory activities, but were favorable for
AChE inhibition. The impact of halogen atoms on the ChE inhibition
was also evaluated for this series of compounds. For the different halo-
substituted derivatives, the activities towards BChE decreased in the
order 4-Br (12f) > 4-Cl (12e) > 4-F (12d), as for the ferulic acid
counterparts mentioned above. It is noteworthy that compound 12d,
with 4-F as the substitution, showed reduced activity towards AChE
inhibition, similarly to compound 10d. The results indicate that F is not
favorable for enhancing the AChE inhibitory activity. Next, eCF3 (12g),
eNO2 (12h), and eCN (12i), as three electron-withdrawing groups,
were introduced. These three compounds showed activities comparable
to that of 12a. The results suggested that the electron-inductive effect
does not have an impact on the ChE inhibitory activities of the target
compounds, implying a tolerance for such structure modification. Next,
we explored the effect of linker length on ChE inhibitory activities.
Compound 13 incorporating a three-carbon chain showed increased
AChE and BChE inhibitory activities compared to compound 12a. When
a four-carbon chain was introduced, compound 14 also exhibited en-
hanced AChE and BChE inhibitory activities.
10
these compounds showed enhanced inhibitory activities compared to
10a, confirming the previous conclusion. Further optimization of the
substituent on the quinoline ring, or even replacement of the ring with
another heterocycle, merits further effort with the aim of obtaining
simplified hybrids as new MTDLs for AD therapy.
2.2.2. Kinetic studies of AChE and BChE inhibition
To determine the nature of the kinetics of AChE and BChE inhibi-
tion, the most active compounds toward AChE (10f) and BChE (12g)
were selected for kinetic studies using Lineweaver–Burk plots [17].
Graphical analyses of the steady-state inhibition data for 10f and 12g
are shown in Fig. 2 A, B. According to the results, increasing con-
centrations of the compounds (0.1, 1, 5, and 10 μM) resulted in gen-
erally stable Vmax values and increased Km, indicating a competitive
type of inhibition for both 10f and 12g. The values of Vmax and Km at
different concentrations of 10f and 12g are shown in Table 2.
2.2.3. Hepatotoxicity study in vitro
Compounds 10f and 12g showed good activities in ChE inhibition.
The hepatotoxicities of these two compounds were further evaluated
through MTT assays of the HepG2 cell line (Fig. 3) [18]. Cells were
incubated with 10f or 12g at 1, 2.5, 5, and 10 μM, respectively, for 24 h.
Concentration-dependent decreases in cell viability were observed for
tacrine, 10f, and 12g. However, the cell viability with 12g (93.0%) was
higher than that with tacrine (87.9%) at 10 μM. This suggested that 12g
Subsequently, we synthesized 18, 19, and 20, which lack the methyl
group on the quinoline ring of 10a. 18 showed comparable activity to
10a, indicating that the methyl group on the quinoline ring is not im-
portant for the activity. To confirm this result, we further synthesized 4-
methyl (19) and 4-Br (20) benzyloxy derivatives, respectively. Both of
Fig. 2. (A) Lineweaver–Burk plot for the inhibition of eeAChE by compound 10f at different substrate concentrations. (B) Lineweaver–Burk plot for the inhibition of
eqBChE by compound 12g at different substrate concentrations.
4

J. Mo, et al.
BioorganicChemistry93(2019)103310
damage. Treatment with 400 μM H2O2 for 24 h caused over 45% death
of PC12 cells compared with a control group (Fig. 4). Quercetine was
used as a positive control in the neuroprotective activity assay. When
pretreated with 10f and 12g for 24 h, the mortality rate of PC-12 cells
exposed to H2O2 was attenuated. This protective effect showed a dose
dependency for both compounds. 10f showed a better cytoprotective
effect (cell viability = 74% at a concentration of 20 μM) than 12g.
2.2.5. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
Compounds 10f and 12g were further evaluated for their anti-
oxidant activities by using the 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging method. Trolox was used as the reference compound
[19]. 12g showed a better inhibitory rate than 10f. The inhibition rate
with 12g was 27% at 100 μM, at which 10f showed no discernible an-
tioxidant activity (Fig. 5).
Fig. 3. In vitro hepatotoxicities of 10f and 12g towards the HepG2 cell line.
Data are expressed as the mean
pared with a control group.
SD (n = 3). *p < 0.05, **p < 0.01, com-
2.2.6. Inhibition of self-induced Aβ1–42 aggregation
Compounds 10f and 12g were evaluated for their inhibitory capa-
cities on self-induced Aβ1–42 aggregation based on a thioflavin T-based
fluorimetric assay. Neither of the tested compounds showed any in-
hibition activity at concentrations below 33 μM.
2.2.7. Docking studies
Docking studies were performed to identify possible interactions
between the compounds and enzyme active sites. Again, 10f and 12 g
were selected as representative compounds. As shown in Fig. 6A, 10f
simultaneously occupies the CAS and PAS of AChE. The quinoline core
is located at the CAS through two π-π stacking interactions with Trp86
and Tyr124, respectively. The N atom of the quinoline ring interacts
with Ser125 through a hydrogen bond. The phenyl core of the ferulic
acid moiety is involved in π-π stacking interaction with Trp286 in the
PAS. The benzyloxyl moiety interacts with Leu289 through a π-alkyl
contact, and with Ser293 through van der Waals forces. The ethylene-
diamine linker shows van der Waals interactions with Tyr341 and
Asp72.
Fig. 4. Protective effects of 10f and 12g against H2O2-induced cell death in PC-
Molecular docking of 12g in the active site of BChE is shown in
Fig. 6B. The quinoline core interacts with Trp82 through a π-π stacking
contact. The N atom of the quinoline ring interacts with Asp70 through
a salt bridge. The phenyl ring of the cinnamic acid moiety is engaged in
π-alkyl interactions with Gly116. The carbonyl group of the cinnamic
acid moiety forms a hydrogen bond with the side chain of His438. The
fluorine atom forms a halogen bond with Ser287.
12 cells. Data are expressed as the mean
SD (n = 3). p# < 0.001 compared
to a control, p* < 0.05, p** < 0.01 compared to H2O2-treated cells.
had a lower hepatotoxicity profile with respect to tacrine. Conversely,
the cell viabilities with 10f (98.6%, 81.0%, 74.3%, and 53.7%) were
lower than those with tacrine (98.9%, 95.2%, 92.6%, and 87.9%) under
the same conditions, indicating a higher hepatotoxicity of 10f com-
pared to tacrine.
The active site cavity of BChE is broader and larger than that of
AChE. The active site cavity of AChE is narrow and long [14,20–21].
12g adopts a U-shaped conformation, which is obviously different from
the linear conformation of 10f. It is possible that the U-shaped con-
formation of 12g can better occupy the active site of BChE, leading to
its selective BChE inhibitory effect.
2.2.4. Neuroprotective effect against H2O2-induced cell death in PC12
neurons
To explain the effect of the linkers on the ChE inhibitory activities,
molecular docking was performed to predict the binding arrangement
and to calculate the binding scores of representative compounds 12a
and 14. As shown in Fig. 7B, 14 occupies the CAS and PAS of AChE. The
quinoline core is located at the CAS through three π-π stacking inter-
actions with Trp86 and Tyr124. The N atom of the quinoline ring in-
teracts with Ser125 through a hydrogen bond. The phenyl core of the
cinnamic acid moiety is engaged in a π-π stacking interaction with
Trp286 in the PAS. When the linker was extended from a two-carbon to
a four-carbon chain, the differences compared to 12a were that the
carbonyl group of the cinnamic acid moiety formed a hydrogen bond
with the side chain of Phe295, and the N atom of the quinoline ring
interacted with Ser125 through a hydrogen bond. The extra hydrogen
bonds increase the binding energy of compound 14 (CDOCKER EN-
ERGY = –44.0066 kcal/mol) compared to compound 12a (CDOCKER
ENERGY = –39.9426 kcal/mol). Increasing chain length may be bene-
ficial for increasing the interaction with key amino acids and better
occupying the active pocket, thereby enhancing the activity.
The PC12 cell line is a useful model for assessing neuronal differ-
entiation and other neurobiological studies. Hence, we evaluated the
neuroprotective activities of 10f and 12g against H2O2-induced cell
Fig. 5. Inhibitory activities of 10f and 12g in DPPH assay.
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BioorganicChemistry93(2019)103310
Fig. 6. (A) The AChE active site cavity (left) and an interaction map (right) displaying the binding and interactions of compound 10f. (B) The BChE active site cavity
(left) and interaction map (right) displaying the binding and interactions of compound 12g. Color coding: green: hydrogen bond; purple: π–π stacking; red: T-shaped
π–π interaction; orange: salt bridge; blue: halogen bond. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version
of this article.)
Fig. 7. Binding mode predictions of 12a (A) and 14 (B) with huAChE (PDB id: 4EY7), and 12a (C) and 14 (D) with huBChE (PDB id: 4TPK). Compounds are shown in
a stick representation and colored yellow. Key residues are shown in a thin stick representation and colored white. Intermolecular interactions are shown as dashed
lines of different colors according to their type: light-green, hydrophobic contact; pink, π–π stacking; purple, π–alkyl contact. (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
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BioorganicChemistry93(2019)103310
Fig. 8. MD results for 10f and AChE. (A) RMSD plot for the backbone atoms and 10f during a 50 ns MD simulation. (B) Binding energy differences of complex 10f and
AChE: delta total, total binding free energy; DELTA G solv, total solvation free energy; DELTA G gas, total gas-phase free energy; ESURF, nonpolar solvation energy;
EGB, polar solvation energy; EEL, electrostatic energy; VDWAALS, van der Waals energy. (C) Contribution analysis of potential hot residues. (D) Energy decom-
position analysis of potential hot residues. All energies are expressed in kcal/mol.
The binding mode of compound 12a with BChE is shown in Fig. 7C.
The quinoline core is located at the CAS forming two π-π stacking in-
teractions with Trp82. The N atom of the quinoline ring interacts with
Asp70 through a salt bridge. This may explain the better inhibitory
activity of compound 12a on BChE than that on AChE. The phenyl core
of the cinnamic acid moiety in the PAS is engaged in π-π stacking in-
teractions with Trp231 and Phe329. As shown in Fig. 7D, the quinoline
core of compound 14 forms π-π stacking and salt bridge interactions
with Tyr332, instead of Trp82 in the case of compound 12a. The extra
salt bridge is beneficial for increasing the binding energy of compound
14 (CDOCKER ENERGY = –45.2775 kcal/mol) compared to compound
12a (CDOCKER ENERGY = –38.6528 kcal/mol), which may explain
the drastically increased inhibitory activity of compound 14 towards
BChE.
and Fig. 9C, D, the hot residues for 10f–AChE binding are Asp74,
Trp86, Tyr124, Ser125, and Trp286, while those for 12g–BChE binding
are Trp82, Gly116, Tyr332, and His438. This information provides a
theoretical basis for further lead optimization.
3. Conclusions
In summary, in the present studies, a series of quinoline-ferulic acid
and quinoline-cinnamic acid hybrids has been designed and synthesized
as MTDLs for the treatment of AD. In vitro assays proved that most of
the compounds effectively inhibited ChEs in the micromolar range. The
detailed mechanisms of the AChE–10f and BChE–12g interactions at
the atomic level were investigated through kinetic experiments, mole-
cular docking, and molecular dynamics simulations. Kinetic studies
indicated
10f
(IC50 = 0.62
0.17 μM)
and
12 g
(IC50 = 0.93
0.40 μM) to be competitive inhibitors. Cell toxicity as-
2.2.8. Molecular dynamics (MD) simulations
says using HepG2 cells showed that compound 12g has a lower hepa-
totoxicity profile with respect to tacrine. 10f and 12g exhibited cyto-
protective effects against H2O2-induced cell damage. 12g also showed
antioxidant activity according to a DPPH assay. It is noteworthy that
when using compounds with longer linkers, such as 14 with a three-
carbon linker, AChE inhibitory activity was maintained, but with much
improved BChE inhibition, leading to enhanced targeted selectivity
towards BChE. We infer that the more flexible chain may lead to a
better induced-fit recognition between the compound and BChE.
Additionally, as BChE has a broader binding site compared to AChE, a
longer linker can provide more intermolecular contacts such as van der
Waals interactions. The results may a provide new strategy for de-
signing selective BChE inhibitors.
After 50 ns MD simulations, the MM/GBSA method was used to
calculate the binding free energies and to gain information on the dif-
ferent components of the interaction energies that contribute to com-
pounds 10f and 12g binding with AChE and BChE, respectively.
Detailed results are shown in Figs. 8 and 9. Fig. 8A and Fig. 9A show the
RMSDs of the systems during MD simulations. The RMSDs of ligands
after the MD and docking vary within 0.75 Å, implying stable binding of
the compounds. Fig. 8B and 9B indicate that electrostatic energy (EEL)
and van der Waals energy (VDWAALS) are both vital in the bindings of
10f and 12g to AChE and BChE, respectively. Nonpolar solvation en-
ergy (ESURF) also contributes to their binding (–7.30 kcal/mol for
10f–AChE and –5.63 kcal/mol for 12g–BChE). Meanwhile, polar sol-
vation energy (EGB) is not favorable for the binding of these com-
pounds. The binding free energy for compound 10f–AChE is higher
than that for compound 12 g–BChE, consistent with our bio-assay re-
sults (Table 1).
This study has identified a quinoline-based scaffold for the further
design of new MTDLs against AD. The SAR results, especially the tol-
erance to modification of the quinoline core, suggest that further op-
timization of the substituent thereon, or even replacement of the ring
with another heterocycle, merits further study.
To identify the key residues contributing to binding affinity, we also
conducted an energy decomposition analysis. As shown in Fig. 8C, D
7

J. Mo, et al.
BioorganicChemistry93(2019)103310
Fig. 9. MD results for 12g and BChE. (A) RMSD plot for the backbone atoms and 12g during a 50 ns MD simulation. (B) Binding energy differences of complex 12g
and BChE: delta total, total binding free energy; DELTA G solv, total solvation free energy; DELTA G gas, total gas-phase free energy; ESURF, nonpolar solvation
energy; EGB, polar solvation energy; EEL, electrostatic energy; VDWAALS, van der Waals energy. (C) Contribution analysis of potential hot residues. (D) Energy
decomposition analysis of potential hot residues. All energies are expressed in kcal/mol.
4. Experimental section
2.47 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 177.17, 150.11, 140.58,
131.88, 125.23, 124.95, 123.13, 118.21, 108.84, 19.93.
4.1. Chemistry
4.1.2. Synthesis of 4-chloro-2-methylquinoline (4)
All the chemicals were purchased from commercial companies and
used without purification. All reactions were monitored by analytical
thin layer chromatography (TLC) on silica gel 60 F254 precoated plates
(purchased from Qingdao Haiyang Inc. China). Visualization was
achieved using UV light (254 nm and 365 nm). Flash column chroma-
tography was performed with silica gel (200–300 mesh) purchased
from Qingdao Haiyang Chemical Co.Ltd. Melting points were de-
termined using a Mel - TEMP II melting point apparatus. 1H NMR and
13C NMR spectra were recorded on a Bruker Avance (300 MHz for 1H,
500 MHz for 13C, Billerica, MA) at 300 K dissolved in deuterated di-
methyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3) with
tetramethylsilane (TMS) as an internal standard. Tetramethylsilane
(TMS) was used as internal standard. All chemical shifts are reported in
parts per million (ppm), relative to the internal standard. The following
abbreviations are used to indicate the multiplicities of the respective
signals: s - singlet; s - broad singlet; d - doublet; dd - doublet of doublets;
t - triplet and m - multiplet. High-resolution mass spectrometry (HRMS)
was performed on a Mariner Mass Spectrum (ESI) or an LC/MSD TOF
HR-MS Spectrum.
A mixture of 3 (1.0 g, 6.28 mmol) and POCl3 (12.0 mL) was heated
at 120 °C for 4 h. The reaction was monitored by using TLC. After
completion of the reaction, excess of POCl3 was distilled off. The re-
sidue was stirred with ice water for 10 min, and then the pH value was
adjusted to 7 with aqueous NaOH. The compound was collected by
filtration and washed with water. The crude product was purified by
using flash column chromatography with CH2Cl2/methanol (50: 1)
elution to afford the white solid compound 4 in 54.0% yield. 1H NMR
(300 MHz, CDCl3): δ 8.14 (d, J = 8.3 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H),
7.71 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.6 Hz, 1H), 7.35 (s, 1H), 2.70 (s,
3H).
4.1.3. General procedure for the synthesis of the intermediates 5a-5c
A mixture of 4 (1.0 g, 5.63 mmol) and ethylenediamine or 1,3-pro-
panediamine or 1,4-diaminobutane (1.35, 22.5 mmol) and phenol
(1.0 g) and sodium iodide (catalytic amount) were heated at 120 °C for
10 h. The reaction was monitored by using TLC. After completion of the
reaction, the resulting mixture was quenched by the addition of water.
The aqueous phase was extracted with CH2Cl2 (3 × 15 mL). The CH2Cl2
layer was then washed with brine and dried over anhydrous Na2SO4.
After concentration, the crude product was purified by silica gel column
chromatograph (CH2Cl2/methanol = 50:1) to give intermediates 5a-5c
as a brown oil [16].
4.1.1. Synthesis of 2-methylquinolin-4-ol (3) [22]
Polyphosphoric acid (15.2 g) was added to a solution of aniline
(2.0 g, 21.4 mmol) and ethyl acetoacetate (2.8 g, 21.4 mmol). The re-
action mixture was stirred at 130 °C for 3 h. The reaction was monitored
by TLC. The reaction mixture was poured into ice water (100 mL)
slowly with vigorous stirring. Then the pH value was adjusted to 7 with
aqueous NaOH. The precipitated solid was filtered and washed thor-
oughly with petroleum ether to afford compound 3 as a yellow solid
with a yield of 56.0%. 1H NMR (300 MHz, CDCl3): δ 11.55 (s, 1H), 8.37
(d, J = 8.5 Hz, 1H), 7.61 (d, J = 7.0 Hz, 2H), 7.35 (s, 1H), 6.22 (s, 1H),
4.1.4. General procedure for the synthesis of benzyl (E)-3–(4-(benzyloxy)-
3-methoxyphenyl)acrylate (8a-8h)
Compound 6 (1.0 g, 5.15 mmol) and K2CO3 (2.85 g, 20.6 mmol)
were added to 10 mL of DMF and then stirred at room temperature for
20 min. Compounds 7a-7h were added dropwisely to the above mixture
solution. The reaction was stirred at 85 °C for 5 h and was monitored by
8

J. Mo, et al.
BioorganicChemistry93(2019)103310
TLC. After completion of the reaction, the reaction mixture was quen-
ched by water. The precipitate was filtrated and the filter cake was
washed using water to give the crude product which could be used in
the next step without further purification [16].
70.09, 55.97, 55.57, 43.24, 37.91, 20.56. HRMS (ESI) m/z calcd. for
C30H31N3O4 [M+H]+ 498.2393, found 498.2396.
4.1.6.4. (E)-3-(4-((4-fluorobenzyl)oxy)-3-methoxyphenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10d). White solid, yield:
48%, m.p. 215–217 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.10 (s, 1H),
8.47 (s, 1H), 8.15 (s, 1H), 7.90 (s, 1H), 7.64 (s, 1H), 7.50 (m, 3H), 7.21
(m, J = 12.1 Hz, 4H), 7.08 (s, 2H), 6.84 (s, 1H), 6.53 (d, J = 15.0 Hz,
1H), 5.10 (s, 2H), 3.81 (s, 3H), 3.61 (s, 2H), 3.53 (s, 2H), 2.63 (s, 3H).
13C NMR (126 MHz, DMSO-d6): δ 166.60, 165.89, 163.24, 161.30,
154.70, 149.66, 149.48, 139.49, 133.55, 130.57, 130.51, 128.43,
126.20, 123.42, 121.70, 120.59, 120.19, 116.35, 115.80, 115.63,
113.89, 110.87, 98.84, 69.58, 55.98, 43.18, 37.92, 20.88. HRMS
(ESI) m/z calcd. for C29H28FN3O3 [M+H]+ 486.2193, found 486.2193.
4.1.5. General procedure for the synthesis of (E)-3–(4-(benzyloxy)-3-
methoxyphenyl)acrylic acid (9a-9h)
To a mixture solution of 2 N × NaOH (30 mL) and MeOH (30 mL)
compounds 8a-8h were added. The reaction mixture was heated to
reflux for 3 h. Then, MeOH in the solution was removed and the pH was
adjusted to around 2 by adding concentrated HCl. The precipitate was
filtrated, washed with cold water, and dried over an infrared lamp to
get compounds (9a-9h) [16].
4.1.6. General procedure for the synthesis of (E)-3-(4-(benzyloxy)-3-
methoxyphenyl)-N-(2-((2-methylquinolin-4-yl)amino)ethyl)acrylamide
(10a-10h)
4.1.6.5. (E)-3-(4-((4-chlorobenzyl)oxy)-3-methoxyphenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10e). White solid, yield:
63%, m.p. 210–213 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.31 (s, 1H),
8.12 (d, J = 8.0 Hz, 1H), 7.71 (dd, J1 = 8.4, J2 = 1.1 Hz, 1H),
7.63–7.55 (m, 1H), 7.47 (m, 4H), 7.39 (m, 2H), 7.31 (s, 1H), 7.21 (d,
J = 1.8 Hz, 1H), 7.15–7.02 (m, 2H), 6.59–6.48 (m, 2H), 5.13 (s, 2H),
3.82 (s, 3H), 3.49 (m, 2H), 3.42 (d, 2H), 2.48 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.52, 158.90, 150.65, 149.68, 149.32,
147.96, 139.37, 136.40, 132.94, 130.03, 129.37, 128.91, 128.59,
128.33, 123.72, 121.81, 121.68, 120.40, 117.82, 113.94, 110.86,
98.55, 69.44, 55.98, 42.97, 38.12, 25.46. HRMS (ESI) m/z calcd. for
Compounds (9a-9 h) (1.49 mmol) and PyBOP (0.93 g, 1.79 mmol)
and DIPEA (0.38 g, 2.98 mmol) were added to 10 mL of DMF and stirred
at room temperature for 20 min. Then, compound 5a (0.3 g, 1.49 mmol)
was added and stirred at room temperature for 4 h. After completion of
the reaction, the reaction mixture was quenched with water. The aqu-
eous phase was extracted with CH2Cl2 (3 × 15 mL). The CH2Cl2 layer
was then washed with brine and dried over anhydrous Na2SO4. After
concentration, the crude product was purified by silica gel column
chromatograph (CH2Cl2/methanol = 80:1) to give compound (10a-
10h).
C
29H28ClN3O3 [M+H]+ 502.1897, found 502.1899.
4.1.6.1. (E)-3-(4-(benzyloxy)-3-methoxyphenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10a). White solid, yield:
53%, m.p. 121–123 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.08 (s, 1H),
8.59 (s, 1H), 8.53 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 7.83 (t,
J = 7.3 Hz, 1H), 7.58 (m, 1H), 7.46–7.41 (m, 3H), 7.39 (m, J = 1.7 Hz,
2H), 7.35 (m, J = 5.5 Hz, 1H), 7.18 (d, J = 1.5 Hz, 1H), 7.12–7.01 (m,
2H), 6.79 (s, 1H), 6.57 (d, J = 15.8 Hz, 1H), 5.11 (s, 2H), 3.80 (s, 3H),
3.60 (t, 2H), 3.54 (t, J = 5.5 Hz, 2H), 2.62 (s, 3H). 13C NMR (126 MHz,
DMSO-d6): δ 166.59, 155.43, 154.38, 149.63, 149.59, 139.48, 138.71,
137.28, 133.42, 128.89, 128.38, 128.30, 126.33, 123.60, 121.71,
120.27, 120.14, 116.24, 113.78, 110.85, 98.85, 70.27, 55.98, 43.14,
4.1.6.6. (E)-3-(4-((4-bromobenzyl)oxy)-3-methoxyphenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10f). White solid, yield:
35%, m.p. 198–200 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.30 (s, 1H),
8.10 (d, J = 8.3 Hz, 1H), 7.73–7.66 (m, 1H), 7.60 (m, 3H), 7.46–7.33
(m, 4H), 7.21 (d, J = 1.6 Hz, 2H), 7.10 (d, J = 1.6 Hz, 1H), 7.04 (d,
J = 8.4 Hz, 1H), 6.57–6.45 (m, 2H), 5.11 (s, 2H), 3.82 (s, 3H), 3.50 (d,
J = 5.8 Hz, 2H), 3.41 (d, J = 5.5 Hz, 2H), 2.47 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.51, 159.04, 150.48, 149.69, 149.31,
148.27, 139.36, 136.84, 131.83, 130.32, 129.23, 128.62, 123.63,
121.74, 121.67, 121.48, 120.42, 117.87, 113.99, 110.91, 98.54,
69.50, 56.01, 42.97, 38.13, 25.62. HRMS (ESI) m/z calcd. for
37.92, 20.50. HRMS (ESI) m/z calcd. for
C
29H29N3O3 [M+H]+
C
29H28BrN3O3 [M+H]+ 548.1372, found 548.1376.
468.2287, found 468.2283.
4.1.6.2. (E)-3-(3-methoxy-4-((4-methylbenzyl)oxy)phenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10b). White solid, yield:
30%, m.p. 167–169 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.30 (s, 1H),
8.11 (d, J = 7.8 Hz, 1H), 7.75–7.67 (m, 1H), 7.62–7.52 (m, 1H), 7.45
(s, 1H), 7.40 (s, 1H), 7.37 (s, 1H), 7.34 (s, 1H), 7.32 (s, 1H), 7.27 (s,
1H), 7.22–7.20 (m, 1H), 7.19 (s, 1H), 7.07 (m, J = 12.8, 5.0 Hz, 2H),
6.49 (s, 1H), 5.07 (s, 2H), 3.80 (s, 3H), 3.49 (m, 2H), 3.41 (m,
J = 5.4 Hz, 2H), 2.48 (s, 3H), 2.31 (s, 3H). 13C NMR (126 MHz,
4.1.6.7. (E)-3-(3-methoxy-4-((4-(trifluoromethyl)benzyl)oxy)phenyl)-N-
(2-((2-methylquinolin-4-yl)amino)ethyl)acrylamide (10g). White solid,
yield: 54%, m.p. 162–165 °C. 1H NMR (500 MHz, DMSO-d6): δ 13.82
(s, 1H), 9.27 (d, J = 5.3 Hz, 1H), 8.48 (d, J = 8.5 Hz, 1H), 8.43 (s, 1H),
7.91 (s, 2H), 7.78 (d, J = 8.0 Hz, 2H), 7.67 (d, J = 7.7 Hz, 2H), 7.41 (d,
J = 15.7 Hz, 1H), 7.21 (s, 1H), 7.11 (d, J = 8.2 Hz, 1H), 7.06 (d,
J = 8.4 Hz, 1H), 6.89 (s, 1H), 6.52 (d, J = 15.7 Hz, 1H), 5.26 (s, 2H),
3.83 (s, 3H), 3.64 (d, J = 5.8 Hz, 2H), 3.53 (s, 2H), 2.65 (s, 3H). 13C
NMR (126 MHz, DMSO-d6): δ 165.76, 155.66, 154.30, 139.31, 138.40,
137.78, 133.67, 128.66, 126.50, 126.30, 125.11, 123.58, 120.06,
116.21, 98.89, 42.98, 37.96, 20.43. MS (ESI) m/z = 536.7.
DMSO-d6):
δ 166.54, 158.87, 150.68, 149.68, 149.62, 139.42,
137.62, 134.27, 129.43, 128.38, 123.73, 121.80, 121.74, 120.25,
117.81, 113.86, 110.82, 98.55, 70.20, 55.97, 42.99, 38.12, 25.43,
21.25. HRMS (ESI) m/z calcd. for C30H31N3O3 [M+H]+ 482.2443,
found 482.2444.
4.1.6.8. (E)-3-(4-((4-cyanobenzyl)oxy)-3-methoxyphenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10h). White solid, yield:
60%, m.p. 193–195 °C. 1H NMR (500 MHz, DMSO-d6): δ 13.75 (s,
1H), 9.07 (s, 1H), 8.50–8.36 (m, 2H), 7.89 (s, 2H), 7.88 (s, 2H), 7.64 (s,
2H), 7.63 (s, 1H), 7.21 (s, 1H), 7.10 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H),
6.85 (s, 1H), 6.52 (d, J = 15.8 Hz, 1H), 5.25 (s, 2H), 3.83 (s, 3H), 3.62
(d, J = 5.5 Hz, 2H), 3.52 (d, J = 5.9 Hz, 2H), 2.63 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.58, 154.95, 149.71, 149.14, 143.20,
139.42, 132.89, 129.96, 128.76, 128.53, 127.89, 126.10, 123.27,
122.30, 121.64, 120.38, 119.21, 116.43, 114.06, 111.04, 111.01,
98.85, 69.36, 56.08, 43.21, 37.93, 21.17. MS (ESI) m/z = 493.7.
4.1.6.3. (E)-3-(3-methoxy-4-((4-methoxybenzyl)oxy)phenyl)-N-(2-((2-
methylquinolin-4-yl)amino)ethyl)acrylamide (10c). White solid, yield:
72%, m.p. 211–213 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.24 (s, 1H),
8.56–8.35 (m, 2H), 7.91 (m, J = 5.6 Hz, 2H), 7.66 (m, J = 2.5 Hz, 1H),
7.43–7.28 (m, 2H), 7.17 (s, 1H), 7.08 (m, 3H), 6.95 (m, J = 8.7 Hz,
2H), 6.87 (s, 1H), 6.52 (d, J = 15.8 Hz, 2H), 5.03 (s, 2H), 3.78 (s,3H),
3.76 (s, 3H), 3.64 (t, J = 5.6 Hz, 2H), 3.53 (t, J = 5.8 Hz, 2H), 2.64 (s,
3H). 13C NMR (126 MHz, DMSO-d6): δ 166.64, 159.53, 155.50, 154.40,
149.72, 149.68, 139.56, 133.51, 130.12, 129.14, 128.20, 126.40,
123.50, 121.75, 120.04, 116.24, 114.27, 113.85, 110.84, 98.88,
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BioorganicChemistry93(2019)103310
4.1.7. General procedure for the synthesis of N-(2-((2-methylquinolin-4-yl)
amino)ethyl)cinnamamide (12a-12i, 13, 14)
4.1.7.6. (E)-3-(4-bromophenyl)-N-(2-((2-methylquinolin-4-yl)amino)
ethyl)acrylamide (12f). White solid, yield: 73%, m.p. 175–177 °C. 1H
NMR (500 MHz, DMSO-d6): δ 8.71 (d, J = 5.2 Hz, 1H), 8.18 (d,
J = 8.3 Hz, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H),
7.53 (d, J = 7.5 Hz, 3H), 7.46 (d, J = 15.8 Hz, 1H), 7.33 (d, J = 7.7 Hz,
2H), 6.77–6.70 (m, 1H), 6.47 (s, 1H), 3.49 (d, J = 5.8 Hz, 2H), 3.42 (d,
J = 5.4 Hz, 2H), 2.46 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 165.90,
159.12, 150.39, 148.50, 137.82, 134.68, 132.33, 129.94, 129.10,
128.68, 123.55, 123.49, 123.03, 122.10, 117.98, 98.49, 42.57, 38.15,
25.71. MS (ESI) m/z = 410.6.
Compounds 11a-11i (1.49 mmol) and PyBOP (0.93 g, 1.79 mmol)
and DIPEA (0.38 g, 2.98 mmol) were added to 10 mL of DMF and stirred
at room temperature for 20 min. Then, compounds 5a-5c (0.3 g,
1.49 mmol) were added and stirred at room temperature for 4 h. After
completion of the reaction, the reaction mixture was quenched with
water. The aqueous phase was extracted with CH2Cl2 (3 × 15 mL). The
CH2Cl2 layer was then washed with brine and dried over anhydrous
Na2SO4. After concentration, the crude product was purified by silica
gel column chromatograph (CH2Cl2/methanol = 80:1) to give the
target compounds (12a-12i, 13, 14).
4.1.7.7. (E)-N-(2-((2-methylquinolin-4-yl)amino)ethyl)-3-(4-
(trifluoromethyl)phenyl)acrylamide (12g). White solid, yield: 62%, m.p.
158–160 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.34 (s, 1H), 8.80 (s, 1H),
8.56 (d, J = 8.3 Hz, 1H), 8.26 (d, J = 8.8 Hz, 2H), 7.94 (m, 2H), 7.84
(d, J = 8.8 Hz, 2H), 7.67 (d, J = 8.3, 1.7 Hz, 1H), 7.58 (d, J = 15.8 Hz,
1H), 6.93–6.79 (m, 2H), 3.68 (d, J = 5.9 Hz, 2H), 3.55 (d, J = 5.6 Hz,
2H), 2.65 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 166.58, 155.70,
154.25, 149.67, 139.48, 138.29, 133.73, 128.52, 126.54, 125.84,
123.73, 121.69, 120.25, 120.03, 116.17, 114.07, 110.95, 98.90,
69.36, 56.04, 43.22, 37.87, 20.41. HRMS (ESI) m/z calcd. for
4.1.7.1. N-(2-((2-methylquinolin-4-yl)amino)ethyl)cinnamamide
(12a). White solid, yield: 57%, m.p. 110–114 °C. 1H NMR (300 MHz,
DMSO-d6): δ 9.34 (s, 1H), 8.65 (s, 1H), 8.55 (d, J = 8.3 Hz, 1H),
8.03–7.84 (m, 2H), 7.65 (s, 1H), 7.56 (d, J = 6.3 Hz, 2H), 7.50 (s, 1H),
7.46–7.34 (m, 3H), 6.88 (s, 1H), 6.65 (d, J = 15.7 Hz, 1H), 3.66 (d,
J = 5.1 Hz, 2H), 3.53 (d, J = 5.5 Hz, 2H), 2.65 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.25, 155.64, 154.26, 139.45, 138.39,
135.21, 133.63, 130.03, 129.41, 128.02, 126.48, 123.61, 122.30,
120.03, 116.20, 98.88, 43.05, 37.93, 20.39. HRMS (ESI) m/z calcd.
for C21H21N3O [M+H]+ 332.1763, found 332.1765.
C
22H20F3N3O [M+H]+ 400.1636, found 400.1655.
4.1.7.8. (E)-N-(2-((2-methylquinolin-4-yl)amino)ethyl)-3-(4-nitrophenyl)
acrylamide (12h). White solid, yield: 73%, m.p. 182–185 °C. 1H NMR
(300 MHz, DMSO-d6): δ 9.33 (s, 1H), 8.79 (s, 1H), 8.54 (d, J = 8.5 Hz,
1H), 8.27 (s, 1H), 8.24 (s, 1H), 7.92 (t, J = 3.2 Hz, 2H), 7.85 (s, 1H),
7.82 (s, 1H), 7.66 (m, 1H), 7.57 (d, J = 16.0 Hz, 1H), 6.92–6.80 (m,
4.1.7.2. (E)-N-(2-((2-methylquinolin-4-yl)amino)ethyl)-3-(p-tolyl)
acrylamide (12b). White solid, yield: 53%, m.p. 100–103 °C. 1H NMR
(500 MHz, DMSO-d6): δ 13.83 (s, 1H), 9.29 (d, J = 5.4 Hz, 1H), 8.51 (d,
J = 15.8, 7.2 Hz, 2H), 7.91 (s, 2H), 7.68 (s, 1H), 7.55–7.43 (m, 2H),
7.23 (d, J = 7.9 Hz, 2H), 6.89 (s, 1H), 6.57 (d, J = 15.8 Hz, 1H), 3.65
(d, J = 5.8 Hz, 2H), 3.52 (d, J = 5.9 Hz, 2H), 2.64 (s, 3H), 2.32 (s, 3H).
13C NMR (126 MHz, DMSO-d6): δ 166.42, 155.67, 154.21, 139.79,
139.39, 138.33, 133.63, 132.46, 129.99, 127.99, 126.48, 123.64,
121.26, 119.96, 116.19, 98.87, 43.09, 37.94, 21.41, 20.33. MS (ESI)
m/z = 346.3.
2H), 3.67 (d, J = 5.7 Hz, 2H), 3.55 (d, J = 5.5 Hz, 2H), 2.65 (s, 3H). 13
C
NMR (126 MHz, DMSO-d6): δ 165.54, 155.71, 154.27, 148.03, 141.82,
138.31, 137.13, 133.71, 129.76, 129.09, 126.57, 124.58, 123.60,
119.98, 116.20, 98.90, 42.95, 38.00, 20.39. HRMS (ESI) m/z calcd.
for C21H20N4O3 [M+H]+ 377.1613, found 377.1616.
4.1.7.9. (E)-3-(3-cyanophenyl)-N-(2-((2-methylquinolin-4-yl)amino)
ethyl)acrylamide (12i). White solid, yield: 68%, m.p. 124–126 °C. 1H
NMR (300 MHz, DMSO-d6): δ 9.35 (s, 1H), 8.69 (s, 1H), 8.55 (d,
J = 8.2 Hz, 1H), 8.06 (s, 1H), 7.92 (m, 3H), 7.84 (d, J = 8.0 Hz, 1H),
7.64 (m, 2H), 7.50 (d, J = 15.9 Hz, 1H), 6.91–6.70 (m, 2H), 3.62 (d,
J = 20.2 Hz, 2H), 3.54 (d, J = 5.2 Hz, 2H), 2.65 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 165.77, 155.70, 154.27, 138.32, 137.34,
136.59, 133.70, 133.23, 132.23, 131.75, 130.62, 126.52, 124.74,
123.60, 119.99, 118.92, 116.19, 112.57, 98.89, 42.99, 37.96, 20.40.
HRMS (ESI) m/z calcd. for C22H20N4O [M+H]+ 357.1715, found
357.1717.
4.1.7.3. (E)-3-(4-methoxyphenyl)-N-(2-((2-methylquinolin-4-yl)amino)
ethyl)acrylamide (12c). White solid, yield: 62%, m.p. 96–99 °C. 1H
NMR (500 MHz, DMSO-d6): δ 8.47 (s, 1H), 8.26 (d, J = 8.3 Hz, 1H),
7.95 (s, 1H), 7.78 (d, J = 8.3 Hz, 1H), 7.68 (s, 1H), 7.52 (d, J = 8.6 Hz,
2H), 7.45 (s, 2H), 6.98 (d, J = 8.6 Hz, 2H), 6.61 (s, 1H), 6.51 (d,
J = 15.8 Hz, 1H), 3.79 (s, 3H), 3.49 (s, 4H), 2.52 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.55, 160.81, 157.30, 152.34, 139.05,
130.77, 129.59, 127.84, 124.62, 122.53, 119.95, 117.29, 114.84,
98.64, 55.72, 42.96, 38.05, 23.70. MS (ESI) m/z = 362.5.
4.1.7.4. (E)-3-(4-fluorophenyl)-N-(2-((2-methylquinolin-4-yl)amino)
4.1.7.10. N-(3-((2-methylquinolin-4-yl)amino)propyl)cinnamamide
(13). Yellow solid, yield: 69%, m.p. 107–109 °C. 1H NMR (500 MHz,
DMSO-d6): δ 8.29 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.70 (d, J = 7.9 Hz,
1H), 7.57 (d, J = 7.3 Hz, 3H), 7.46 (d, J = 15.8 Hz, 1H), 7.41 (d,
J = 7.5 Hz, 2H), 7.39–7.34 (m, 2H), 7.21 (s, 1H), 6.67 (d, J = 15.8 Hz,
1H), 6.40 (s, 1H), 3.37–3.29 (m, 4H), 2.47 (s, 3H), 1.91–1.85 (m, 2H).
13C NMR (126 MHz, DMSO-d6): δ 165.61, 158.77, 150.72, 147.80,
139.06, 135.38, 129.88, 129.39, 128.14, 127.95, 123.68, 122.66,
121.95, 117.88, 98.53, 55.38, 37.13, 28.41, 25.34. HRMS (ESI) m/z
calcd. for C22H23N3O [M+H]+ 346.1919, found 346.1917.
ethyl)acrylamide (12d). White solid, yield: 78%, m.p. 108–110 °C. 1H
NMR (300 MHz, DMSO-d6):
δ 9.33 (s, 1H), 8.65–8.44 (m, 2H),
8.06–7.87 (m, 2H), 7.63 (m, 3H), 7.47 (d, J = 15.8 Hz, 1H), 7.25 (t,
J = 8.8 Hz, 2H), 6.88 (s, 1H), 6.60 (d, J = 15.9 Hz, 1H), 3.66 (d,
J = 5.8 Hz, 2H), 3.54 (d, J = 5.6 Hz, 2H), 2.65 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.19, 162.21, 155.68, 154.24, 138.32,
138.27, 133.68, 131.86, 130.23, 130.16, 126.51, 123.60, 122.19,
119.97, 116.47, 116.30, 116.19, 98.88, 43.06, 37.92, 20.37. HRMS
(ESI) m/z calcd. for C21H20FN3O [M+H]+ 350.1668, found 350.1684.
4.1.7.5. (E)-3-(4-chlorophenyl)-N-(2-((2-methylquinolin-4-yl)amino)
ethyl)acrylamide (12e). White solid, yield: 62%, m.p. 158–160 °C. 1H
NMR (300 MHz, DMSO-d6): δ 14.03 (s, 1H), 9.34 (s, 1H), 8.65 (s, 1H),
8.55 (d, J = 8.3 Hz, 1H), 8.06–7.87 (m, 2H), 7.65 (s, 1H), 7.56 (d,
J = 6.3 Hz, 2H), 7.46–7.35 (m, 2H), 6.88 (s, 1H), 6.65 (d, J = 15.7 Hz,
4.1.7.11. N-(4-((2-methylquinolin-4-yl)amino)butyl)cinnamamide
(14). Yellow solid, yield: 74%, m.p. 97–99 °C. 1H NMR (500 MHz,
DMSO-d6): δ 8.19 (d, J = 8.5 Hz, 2H), 7.69 (d, J = 8.3 Hz, 1H), 7.55 (d,
J = 7.2 Hz, 3H), 7.44 (d, J = 12.3 Hz, 1H), 7.42–7.39 (m, 2H), 7.36 (m,
2H), 7.15 (s, 1H), 6.65 (d, J = 15.8 Hz, 1H), 6.38 (s, 1H), 3.34–3.24 (m,
4H), 2.46 (s, 3H), 1.77–1.68 (m, 2H), 1.64–1.56 (m, 2H). 13C NMR
(126 MHz, DMSO-d6): δ 165.35, 158.84, 150.72, 138.91, 135.41,
129.83, 129.38, 129.30, 128.24, 127.91, 123.53, 122.78, 122.01,
117.87, 98.48, 42.55, 38.84, 27.36, 25.80, 25.42. HRMS (ESI) m/z
1H), 3.66 (d, J = 5.1 Hz, 2H), 3.53 (d, J = 5.5 Hz, 2H), 2.65 (s, 3H). 13
C
NMR (126 MHz, DMSO-d6): δ 166.02, 156.10, 153.67, 142.77, 138.04,
134.41, 134.22, 130.36, 129.72, 129.43, 125.38, 123.20, 122.92,
120.79, 116.85, 98.74, 42.92, 38.02, 22.40. MS (ESI) m/z = 366.4.
10

J. Mo, et al.
BioorganicChemistry93(2019)103310
147.73, 139.44, 137.62, 134.26, 129.65, 129.43, 128.64, 128.38,
128.35, 124.61, 122.18, 121.76, 120.26, 119.07, 113.84, 110.82,
98.60, 70.21, 55.97, 43.03, 38.01, 21.24. HRMS (ESI) m/z calcd. for
C29H29N3O3 [M+H]+ 468.2287, found 468.2277.
calcd. for C23H25N3O [M+H]+ 346.1919, found 346.1917.
4.1.8. Synthesis of the target compounds 18-20
4.1.8.1. Synthesis of 4-chloroquinoline (16). A mixture of 15 (1.0 g,
6.89 mmol) and POCl3 (12 mL) was heated at 120 °C for 4 h. The
reaction was monitored by using TLC. After completion of the reaction,
excess of POCl3 was distilled off. The residue was stirred with ice water
for 10 min, and then the pH value was adjusted to 7 with aqueous
NaOH. The compound was collected by filtration and washed with
water. The crude product was purified by using flash column
chromatography with CH2Cl2/methanol (100: 1) elution to afford a
white solid compound 16. 1H NMR (300 MHz, DMSO-d6): δ 8.85 (d,
J = 4.6 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.88
(t, J = 7.6 Hz, 1H), 7.81–7.72 (m, 2H).
4.1.8.6. (E)-3-(4-((4-bromobenzyl)oxy)-3-methoxyphenyl)-N-(2-
(quinolin-4-ylamino)ethyl)acrylamide (20). White solid, yield: 67%,
m.p. 256–258 °C. 1H NMR (500 MHz, DMSO-d6):
δ 8.43 (d,
J = 5.4 Hz, 1H), 8.34 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.80 (d,
J = 8.3 Hz, 1H), 7.67–7.62 (m, 1H), 7.60 (d, J = 8.2 Hz, 2H), 7.50 (s,
1H), 7.45 (d, J = 10.1 Hz, 1H), 7.41 (d, J = 8.1 Hz, 2H), 7.21 (s, 1H),
7.10 (s, 1H), 7.05 (s, 1H), 6.60 (d, J = 5.5 Hz, 1H), 6.54 (d,
J = 15.7 Hz, 1H), 5.11 (s, 2H), 3.82 (s, 3H), 3.50 (d, J = 6.0 Hz, 2H),
3.44 (d, J = 5.6 Hz, 2H). 13C NMR (126 MHz, DMSO-d6): δ 166.52,
150.89, 150.35, 149.69, 149.32, 147.71, 144.18, 139.38, 136.82,
131.82, 130.32, 129.66, 128.62, 124.61, 122.16, 121.70, 121.48,
120.42, 119.06, 113.97, 110.91, 98.60, 69.50, 56.01, 46.00, 43.03,
38.01. HRMS (ESI) m/z calcd. for C28H26BrN3O3 [M+H]+ 534.1236,
found 534.1212.
4.1.8.2. Synthesis of N1-(quinolin-4-yl)ethane-1,2-diamine (17). A
mixture of 16 (1.0 g, 6.11 mmol) and ethylenediamine (1.47,
24.45 mmol) and phenol (1.0 g) and sodium iodide (catalytic amount)
were heated at 120 °C for 10 h. The reaction was monitored by using
TLC. After completion of the reaction, the resulting mixture was
quenched by the addition of water, the aqueous phase was extracted
with CH2Cl2 (3 × 15 mL). The CH2Cl2 layer was then washed with brine
and dried over anhydrous Na2SO4. After concentration, the crude
product was purified by silica gel column chromatograph (CH2Cl2/
methanol = 40:1) to give compound 17 as a brown oil. 1H NMR
(300 MHz, DMSO-d6): δ 8.40 (d, J = 5.2 Hz, 1H), 8.25 (d, J = 8.4 Hz,
1H), 7.79 (d, J = 8.3 Hz, 1H), 7.60 (t, J = 7.6 Hz, 1H), 7.41 (t,
J = 7.5 Hz, 1H), 7.16 (s, 1H), 6.48 (d, J = 5.2 Hz, 1H), 3.19 (d,
J = 0.8 Hz, 2H), 2.87 (t, J = 6.3 Hz, 2H).
4.2. AChE and BChE inhibition assay
The inhibitory effects of the target compounds were evaluated fol-
lowed the method of Ellman et al [17]. AChE (EC 3.1.1.7, Type VI-S,
from electric eel, C3389) and BChE (EC 3.1.1.8, from equine serum,
C0663), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB, D218200), sub-
strates acetylthiocholine iodide (ATC, A5751) and butyrylthiocholine
iodide (BTC, B3253) were obtained from Sigma-Aldrich (St. Louis, MO,
USA). Briefly, the assay phosphate buffer solution was prepared as
follow: 1) 13.6 g potassium dihydrogen phosphate dissolved in 1 L
4.1.8.3. General synthesis of the target compounds 18–20. Compound 9a,
9b or 9f (1.60 mmol) and PyBOP (1.0 g, 1.92 mmol) and DIPEA (0.41 g,
3.20 mmol) were added to 10 mL of DMF and stirred at room
temperature for 20 min. Then, compound 17 (0.3 g, 1.60 mmol) was
added and stirred at room temperature for 4 h. After completion of the
reaction, the reaction mixture was quenched with water. The aqueous
phase was extracted with CH2Cl2 (3 × 15 mL). The CH2Cl2 layer was
then washed with brine and dried over anhydrous Na2SO4. After
concentration, the crude product was purified by silica gel column
chromatograph (CH2Cl2/methanol = 80:1) to give the target
compounds.
distilled water; 2) the pH of buffer solution was adjusted to 8.0
0.1
using 10% KOH solution (w/v). For AChE / BChE stock solution, the
protein from Sigma was diluted using 0.2 M phosphate buffer (pH 8.0)
to prepare 2.5 units per mL. For the preparation of the ATC/BTC ioide
solution (0.075 M), they were diluted with distilled water to give the
substrate solution. DTNB was diluted with 0.15% (w/v) sodium bi-
carbonate solution to give 10 μM per mL. The test smaples were firstly
dissolved in DMSO to give an initial concentration of 100 μM, which
was then diluted using methanol to the target concentration ranging
from 10 μM to 1 nM. These samples with different concentrations were
used in the subsequent IC50 determination.
For measurement, 3 mL of phosphate buffer, 100 μL of AChE/BChE,
100 μL of DTNB, and 100 μL test compound solution was added into a
cuvette in order. The enzymatic reaction was started when adding 20 μL
of ATC or BTC, and the mixture was immediately stirred. After two
minutes, the absorption was measured at 25 °C at 412 nm using a
Varioskan Flash 3001 spectrophotometer (from Thermo, USA) as de-
tector. For the control group, 100 μL of methanol was used to replace
test compound solution, while a mixture of 80 μL distilled water and
20 μL DTNB solution was used to replace enzyme solution as the blank
group. The determination of all the test samples was performed in tri-
plicate. GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA)
was used for data processing. The inhibition curve was fitted by plotting
percentage enzyme activity (100% for the reference) versus the loga-
rithm of the concentration of tested compound. The half maximal in-
hibitory concentration (IC50) value was calculated on the basis of the
inhibition curve. The data was calculated and shown in the form of
4.1.8.4. (E)-3-(4-(benzyloxy)-3-methoxyphenyl)-N-(2-(quinolin-4-
ylamino)ethyl)acrylamide (18). White solid, yield: 65%, m.p.
253–255 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.41 (d, J = 5.3 Hz,
1H), 8.33 (m, 1H), 8.17 (d, J = 7.8 Hz, 1H), 7.81–7.74 (m, 1H),
7.66–7.57 (m, 1H), 7.44 (m, J = 5.2, 2.6 Hz, 4H), 7.40 (s, 2H), 7.35
(m, J = 5.9, 2.1 Hz, 1H), 7.34–7.30 (m, 1H), 7.20 (m, J = 1.6 Hz, 1H),
7.08 (m, 2H), 6.64–6.47 (m, 2H), 5.13 (s, 2H), 3.82 (s, 3H), 3.51 (t,
J = 6.0 Hz, 2H), 3.42 (t, J = 5.6 Hz, 2H). 13C NMR (126 MHz, DMSO-
d6): δ 166.52, 151.19, 150.29, 149.67, 149.57, 148.76, 139.41, 137.31,
129.53, 129.19, 128.90, 128.42, 128.38, 128.29, 124.35, 121.97,
121.76, 120.32, 119.26, 113.81, 110.80, 98.61, 70.29, 55.97, 42.98,
38.01. HRMS (ESI) m/z calcd. for C28H27N3O3 [M+H]+ 454.2130,
found 454.2128.
4.1.8.5. (E)-3-(3-methoxy-4-((4-methylbenzyl)oxy)phenyl)-N-(2-
(quinolin-4-ylamino)ethyl)acrylamide (19). White solid, yield: 65%,
mean
SEM.
m.p. 245–247 °C. 1H NMR (500 MHz, DMSO-d6):
δ 8.43 (d,
J = 5.4 Hz, 1H), 8.34 (s, 1H), 8.21 (d, J = 8.3 Hz, 1H), 7.80 (d,
J = 8.3 Hz, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.51 (s, 1H), 7.48–7.40 (m,
2H), 7.33 (d, J = 7.9 Hz, 2H), 7.20 (d, J = 8.2 Hz, 2H), 7.09 (d,
J = 1.6 Hz, 1H), 7.05 (d, J = 8.4 Hz, 1H), 6.60 (d, J = 5.5 Hz, 1H),
6.54 (d, J = 15.7 Hz, 1H), 5.07 (s, 2H), 3.80 (s, 3H), 3.50 (d,
J = 6.0 Hz, 2H), 3.43 (d, J = 5.7 Hz, 2H), 2.31 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 166.54, 150.88, 150.36, 149.68, 149.62,
4.3. Kinetic studies of AChE and BChE inhibition
Kinetic studies were performed in the same manner as the de-
termination of ChEs inhibition, while the substrate (ATC/BTC) was
used in concentrations of 90, 150, 226, 452 and 904 μM. The con-
centrations of test compounds were set to 0, 0.1, 1, 5, 10 μM for both
10f and 12g. The enzymatic reaction was extended to 7 min for eeAChE
11

J. Mo, et al.
BioorganicChemistry93(2019)103310
and eqBChE before the determination of the absorption. Vmax and Km
values of the Michaelis–Menten kinetics were calculated by nonlinear
regression from substrate-velocity curves using GraphPad Prism 6.0.
Linear regression was used for fitting the Lineweaver–Burk plots.
4.8. Molecular docking study
The crystal structures of the hAChE (PDB ID: 4EY7) [25] and hBChE
(PDB ID: 4TPK) [26] were derived from the RCSB Protein Data Bank
(PDB). Docking studies were carried out using the discovery studio (DS,
version 3.0, BIOVIA, San Diego, CA) for compound 10f and compound
12g. Two protein structures were preprocessed by “prepare protein”
module in DS to give the structures suitable for docking. “Prepare li-
gands” module in DS was applied for the structural preparation of the
test compounds. The native ligand in the crystal structure was used to
define the binding site. The binding site was defined as a site sphere (in
10 Å radius) around the original ligands in the co-crystal structures. The
docking program CDOCKER encoded in DS 3.0 was applied to identify
the potential binding pattern of compound 10f to the hAChE and
compound 12g to the hBChE. Other CDOCKER parameters were set as
default.
4.4. Hepatotoxicity study in vitro
HepG2 cells (human hepatocellular liver carcinoma cell line pur-
chased from Cell Bank of the Chinese Academy of Sciences (Shanghai,
China), were cultivated in DMEM supplemented with 10% FBS at 37 °C
in a humidified atmosphere containing 5% CO2. For the determination,
cells (6 × 103 cells/well) were seeded in 96-well plate in complete
medium. After 24 h, the medium was removed, and cells were exposed
to the increasing concentrations of compounds 10f and 12g or tacrine
(1, 2.5, 5 and 10 μM) in DMEM for further 24 h. Cell survival was
measured through MTT assay.
4.9. Molecular dynamics (MD) simulations
4.5. Neuroprotection assay
MD simulations were performed using the PMEMD module in
AMBER 16 accelerated by GPU system consist of the NVIDIA CUDA
processor [27]. The initial structures of 10f and 12g binding in AChE or
BChE from molecular docking were used for MD. The proteins were
assigned with the AMBER ff99SB force field [28]. The ANTECHAMBER
module and the general AMBER force field [29] was applied to the li-
gands. The systems were solvated in a TIP3P water box in a 9 Å hex-
ahedron. Sodium ions were added with the purpose of neutralization of
the systems. To remove possible steric stresses, the systems were
minimized for 1000 steps with the steepest descent method, followed
by application of conjugate gradients for another 1000 steps. All two
systems were linearly heated from 0 to 300 K using a Langevin ther-
mostat and weak restraints of 10 kcal/mol on the protein backbone
atoms over 1 ns. Finally, dynamics simulations of 20 ns NPT ensemble
at 1 atm and 300 K. After MD simulation, 2000 frames were extracted
from the 20 ns of trajectory for CPPTRAJ analysis [30]. The MMGBSA
method in the AMBER 16 was used to calculate the binding free en-
ergies and energy decomposition [31].
PC-12 cells were plated in 96-well plates, raised to a population of
1 × 104 cells per well, and incubated overnight. The assay was initially
performed by treating the cells with the test compounds or DMSO for
24 h at 37 °C, and then the cells were further incubated with 400 μM
H2O2 for another 24 h. After that, 20.0 μL of MTT solution was added
into each well of the plate and incubated for 4 h. Then the solution was
removed and 150.0 mL of DMSO was added into each well to dissolve
the MTT formazan crystals. DMSO was used as a negative control. The
absorbance values (OD value) were read at 570 nm by Elx800
Absorbance Microplate Reader (BioTek, Vermont, USA). Results were
adjusted considering OD measured in the blank.
4.6. Antioxidant activity in vitro
The antioxidant activities of compounds 10f and 12g were eval-
uated by DPPH free-radical scavenging assay according to the method
of Lee et al. with slightly modifications [23]. Briefly, 95 mL of DPPH
radical solution (100 μM) was added in a 96-well plate containing 5 mL
of different concentrations of test compounds dissolved in DMSO, and
incubated for 30 min at 37 °C in the dark. The absorbance of each well
was measured at 517 nm using a Varioskan Flash 3001 microplate
reader (Thermo, USA).
Acknowledgements
We gratefully thank the support from the grants (Nos. 81872728
and 81573281) of National Natural Science Foundation of China, (Nos.
BK20191411) of Natural Science Foundation of Jiangsu Province. We
thank the support from Double First-Class“ initiative Innovation team
project of China Pharmaceutical University (Nos. CPU2018GF11
and CPU2018GY34), and Jiangsu Qinglan Project (Yao Chen).
Finally, we thank International Science Editing (http://www.
internationalscienceediting.com) for editing this manuscript.
4.7. Inhibition of self-induced Aβ1–42 aggregation
Inhibitory effects of the compounds on self-induced Aβ1–42 ag-
gregation were tested through a Thioflavin T (ThT, T3516, Sigma-
Aldrich) binding assay [24]. Firstly, aliquots of 2 μL of Aβ1–42 (AS-
64129–05 Anaspec Inc.) containing 2 mg/mL HFIP (1,1,1,3,3,3-hexa-
fluoro-2-propanol, 52517, Sigma-Aldrich) were stocked in DMSO. Then,
they were diluted with 0.215 M sodium phosphate buffer (pH 8.0) to
the final concentration of 500 μM. Test compounds were dissolved in
DMSO and then prepared at a concentration of 33 μM by the buffer.
Resveratrol was used as a positive control. The Aβ1-42 and the test
sample solutions were incubated in a 96-well plate for 24 h at room
temperature. After the incubation, the tested compounds were diluted
to a final volume of 150 μL with 50 mM glycine-NaOH buffer (pH 8.5)
containing 5 mM ThT. Fluorescence intensity was read (excitation wa-
velength 450 nm, emission wavelength 485 nm) on a SpectraMax
Paradigm Multimode Reader (Molecular Device, Sunnyvale, CA).
The calculation of the inhibitory rate of Aβ1–42 self-induced ag-
gregation was performed as the following equation: (1-IFi/IFc) × 100%.
IFi and IFc were the fluorescence intensities measured in the presence
and absence of inhibitors, respectively, after subtracting the back-
ground fluorescence of the 5 mM ThT solution. Each compound was
measured in triplicate.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103310.
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