New 4-N-phenylaminoquinoline derivatives as antioxidant, metal chelating and cholinesterase inhibitors for Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2019.103328

A series of new 4-N-phenylaminoquinoline derivatives were designed, synthesized, and their anticholinesterase activities, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, metal-chelating ability were tested. Among them, compounds 11j, 11k and 11l had comparable inhibition activities to reference drug galantamine both in AChE and in BChE. Especially, compound 11j revealed the most potent inhibition to eeAChE and eqBChE with IC₅₀ values of 1.20 μM and 18.52 μM, respectively. Furthermore, both kinetic analysis of AChE inhibition and molecular docking study indicated that compound 11j was mixed-type inhibitor, binding simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE, and propidium iodide displacement assay showed significant displacement of propidium iodide with compound 11k (25.80%) from PAS of eeAChE. More importantly, compound 11l displayed excellent DPPH radical scavenging activity (84% at 1 mg/mL), and its EC₅₀ value was 0.328 μM. In addition, compounds 11a, 11j, 11k and 11l exhibited obvious biometal chelating abilities toward Al³⁺, Fe²⁺, Cu²⁺ and Zn²⁺ ions. Taken together, 4-N-phenylaminoquinoline derivatives targeting multiple pathogenetic factors deserve further investigation for treatment of AD.

Full text of DOI:10.1016/j.bioorg.2019.103328

Journal Pre-proofs
New 4-N-phenylaminoquinoline derivatives as antioxidant, metal chelating and
cholinesterase inhibitors for Alzheimer’s disease
Rong Cai, Li-Ning Wang, Jing-Jing Fan, Shang-Qi Geng, Yu-Ming Liu
PII:
S0045-2068(19)31119-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.103328
YBIOO 103328
Reference:
Bioorganic Chemistry
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Accepted Date:
14 July 2019
26 September 2019
27 September 2019
Please cite this article as: R. Cai, L-N. Wang, J-J. Fan, S-Q. Geng, Y-M. Liu, New 4-N-phenylaminoquinoline
derivatives as antioxidant, metal chelating and cholinesterase inhibitors for Alzheimer’s disease, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103328
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New 4-N-phenylaminoquinoline derivatives as antioxidant, metal
chelating and cholinesterase inhibitors for Alzheimer’s disease
Rong Caia, Li-Ning Wangb, Jing-Jing Fana, Shang-Qi Genga, Yu-Ming Liua,*
a Department of Pharmacy Engineering, Tianjin University of Technology, Tianjin 300384, PR
China
b
College of Traditional Chinese Medicine, Tianjin Univerisity of Traditional Chinese Medicine,
Tianjin 300193, PR China
ABSTRACT
A series of new 4-N-phenylaminoquinoline derivatives were designed, synthesized, and their
anticholinesterase activities, 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity,
metal-chelating ability were tested. Among them, compounds 11j, 11k and 11l had comparable
inhibition activities to reference drug galantamine both in AChE and in BChE. Especially,
compound 11j revealed the most potent inhibition to eeAChE and eqBChE with IC50 values of
1.20 μM and 18.52 μM, respectively. Furthermore, both kinetic analysis of AChE inhibition and
molecular docking study indicated that compound 11j was mixed-type inhibitor, binding
simultaneously to the catalytic anionic site (CAS) and the peripheral anionic site (PAS) of AChE,
and propidium iodide displacement assay showed significant displacement of propidium iodide
with compound 11k (25.80%) from PAS of eeAChE. More importantly, compound 11l displayed
excellent DPPH radical scavenging activity (84% at 1mg/mL), and its EC50 value was 0.328 μM.
In addition, compounds 11a, 11j, 11k and 11l exhibited obvious biometal chelating abilities
toward Al3+, Fe2+, Cu2+ and Zn2+ ions. Taken together, 4-N-phenylaminoquinoline derivatives
targeting multiple pathogenetic factors deserve further investigation for treatment of AD.
Keywords:
Alzheimer’s disease
Anti-cholinesterase activity
Antioxidant
Metal chelator
4-N-phenylaminoquinoline
1. Introduction
Alzheimer's disease (AD) is one of the most common types of dementia, and has seriously
threatened the health of old people. The illness progress is well-known by the advent of aphasia,
apraxia, executive disorders, mood disturbances, and psychiatric symptoms [1,2]. The exact origin
of AD is ambiguous [3] and combinatorial reasons such as genetic, lifestyle, and environmental
factors are involved in the onset and progression of the disease [4]. Now, such cellular processes
as neurotransmitter systems [5], excitotoxicity [6], beta-amyloid aggregation, abnormal tau
phosphorylation [7], oxidative stress, metal ion deregulation and inflammation [8] have been
considered as the main causes of AD. Though several novel therapeutic approaches were explored

in the last few decades like apolipoprotein E, CREB signaling pathways, insulin resistance, etc. to
identify the potential leads, but were unable to elicit significant clinical outcomes [6].
One therapeutic approach to enhance cholinergic neurotransmission is to increase
acetylcholine (ACh) availability by inhibiting acetylcholinesterase (AChE) [9–13]. Thus far,
several anti-AD drugs targeting AChEs have become available, including tacrine, donepezil, and
the alkaloid galantamine. Compared with AChE, butyrylcholinesterase (BChE), the sister enzyme
of AChE, plays a supportive role in the cholinergic neurotransmission. In the brain of healthy
adults, AChE is very more active than BChE, and cause almost 80% ACh hydrolysis in the brain
[14,15]. However, with the gradual severity of AD, AChE in the brain drops to 90% of the normal
level, while the level of BChE is about doubled and tends to increase continuously [16]. And then
the regulation of ACh is increasingly dependent on BChE in progressed AD [17–20]. Thus, the
synergistic inhibition of both AChE and BChE enzymes may be one more valuable approach in
the treatment of AD [21]. It is worth mentioning that AChE possesses two binding sites including
the catalytic anionic site (CAS) and the peripheral anionic site (PAS). The new agents that serve as
dual binding inhibitors for both CAS and PAS of AChE are more efficient in treating AD [22], as
they can alleviate cognitive deficits by restoring cholinergic activity and prevent the deposition of
beta-amyloid in the brain promoted by PAS of AChE [23]. As a result, novel AChE inhibitors
targeting both CAS and PAS attract the attentions of medicinal chemists throughout the world.
Among the various causative factors involved in the pathogenesis of AD, oxidative stress has
emerged as an important factor. Oxidative stress is caused by the imbalance of antioxidant defense
system and intracellular reactive oxygen species (ROS) accumulation [24]. Unregulated reaction
of molecular oxygen with the redox active metals can lead to the generation of ROS [25]. What’s
more, the excessive accumulation of metals promotes beta-amyloid fibril aggregates, which
induce inflammation and activate neurotoxic pathways, leading to the dysfunction and death of
brain cells [26,27]. Recent studies have shown that antioxidants can inhibit lipid peroxidation by
acting as metal ion-chelating and free-radical scavenging agents [28]. Clinical studies have shown
the particular therapeutic efficacy of several antioxidants. Therefore, antioxidation can be a key
factor in AD treatment.
Quinoline derivatives are important compounds that exhibited various biological activities,
such as anti-tumor, anti-malaria, and cholinesterase (ChE) inhibitory activity [29,30], and tacrine
was before used as the first AChE inhibitor in the treatment of AD [31]. Recently some
4-N-phenylaminoquinoline analogues were synthesized and evaluated as AChE inhibitors [32–34].
The previous investigations in our laboratory had reported two lead structures derived from
4-N-phenylaminoquinoline ‒ compounds I and II (Fig. 1). Compound I [16] was found to have
more potent effects on inhibiting AChE and BChE (IC50 values equal to 0.92 μM and 14.20 μM,
respectively) than galanthamine. Compound II [35] also showed significant inhibitory activities
on AChE and BChE with IC50 values of 0.86 and 2.65 μM, respectively, a lot better than that of
reference drug galanthamine. Furthermore, molecular modeling showed that compound II bind
concomitantly to both catalytic and peripheral active site of AChE. As part of our ongoing project,
we focused on their 4-N-phenylaminoquinoline core as a privileged skeleton for inhibiting AChE
and BChE. On the other hand, a literature survey revealed that most of ChE inhibitors usually
contain a tertiary amine unit in a ring or an open chain [36–39], which may play significant roles
in the ChE inhibition. To this end, we employ molecular hybridization and bioisosterism
replacement approaches to identify novel ChE inhibitors. Our design is based on bioisosteric

replacement of N-methylbenzylamine moiety of compound II with 4-methylpiperidine fragment,
with a lower structural weight and relatively small volume for improving brain exposure, to
produce a class of compounds III (Fig. 1), which was inspired by this pharmacophoric unit with
strong binding interactions to ChE [40–42].
Now in the present study, a series of new 4-N-phenylaminoquinoline derivatives (11a-11m)
were designed, synthesized, and their anticholinesterase activities, 1,1-Diphenyl-2-picrylhydrazyl
(DPPH) radical scavenging activity, metal-chelating ability were tested.
Fig. 1. Structures of previously reported compounds (I, II) and newly designed compounds III.
2. Results and Discussion
2.1 Chemistry
According to scheme 1, compounds 11a–11m were synthesized starting from the
commercially available material vanillic acid, which was esterifcated with methanol under acidic
conditions to obtain compound 2. Compound 2 was alkylated with 1-bromo-3-chloropropane in
acetone under basic condition to provide compound 3, which was converted to nitro compound 4
using fuming nitric acid as nitration reagent in dichloromethane for 5 h with 98.98% yield.
Compound 4 was reduced using iron powder and catalytic amounts of ammonium chloride in
ethanol to obtain compound 5, which was further hydrolyzed with NaOH to provide compound 6.
Next, intermediate 7 was formed by the reaction of compound 6 and 2-nitroacetaldoxime, which
was prepared beforehand from nitromethane in the presence of NaOH. Compound 7 was cyclized
with acetic anhydride to yield compound 8, which was subsequently treated with phosphorus
oxychloride to afford compound 9. Compound 9 was further reacted with the corresponding
aniline, affording compounds 10a–10m. Then, final compounds 11a–11m were achieved upon the
substitution of compound 10a–10m with excessive 4-methyl-piperidine, respectively. All target
1
compounds were purified by column chromatography and characterized by H NMR, 13C NMR,
and HR-ESI-MS.

Scheme 1. Synthesis of compounds 11a–11m. Reagents and conditions: (a) MeOH, HCl, 70 °C, 10 h (94.36%); (b)
Br(CH2)3Cl, K2CO3, acetone, 70 °C, 10 h (92.85%); (c) fuming HNO3, CH2Cl2, room temperature, 5 h (98.98%);
(d) Fe, NH4Cl, EtOH, reflux (66.67%); (e) 5% NaOH, EtOH, 50 °C, 10 h (80.68%); (f) HON=CHCH2NO2, HCl,
room temperature, 18 h (87.97%); (g) KOAc, Ac2O, 15 min, reflux (32.24%); (h) POCl3, 70 °C, 10 h (87.29%); (i)
corresponding aniline, isopropanol, 90 °C, 12 h (80.65%−95.63%); (j) 4-methyl-piperidine, NaI, K2CO3, CH3CN,
87 °C, 24 h, reflux (43.36%−56.79%).
2.2. Biological evaluation
2.2.1. AChE and BChE inhibition assay
All synthesized quinoline derivatives (11a–11m) were evaluated for their ChE inhibitory
activities on electric eel acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase
(eqBChE), by the Ellman’s method with galanthamine as the reference drug. The results showed
that most 4-N-phenylaminoquinoline derivatives have inhibitory effects on AChE. Compared with
compounds 11k and 11l, compounds 11e, 11f and 11g had relatively weaker inhibitory activities. It
seems that AChE inhibitory activities can be enhanced when there is electron-donating group
(such as hydroxyl) in 4-N-substituted aniline ring, and that relative activities are reduced when
electron-withdrawing group is present (such as chlorine atom).
As shown in Table 1, what exhibited more potent on AChE than galanthamine were
compound 11j with a para methoxy group (IC50 = 1.20 μM) and compound 11k with a meta
hydroxyl group (IC50 = 1.23 μM). According to the different substituents, the order of the
inhibitory potency of these derivatives bearing different o-substituted groups was hydrogen atom >
chloro group > methoxy group > methyl group; the order with different m-substituted groups was
as followed: hydroxyl group > hydrogen atom > chloro group > methyl group > methoxy group >
trifluoromethyl group; and the order with different p-substituted groups was: methoxy group >
hydrogen atom > hydroxyl group > methyl group > chloro group. On the other hand, based on the
substituted position in the 4-N-substituted aniline ring, the order of inhibitory potency against
AChE was: Meta > Ortho > Para, but it turned to be: Para > Meta > Ortho after methoxyl
substitution. Therefore, their anti-AChE activities directly depended on the electronic properties
and position of different substituents on the aniline ring.
In terms of inhibitory activity against eqBChE, although the activities of compounds 11a-11g,
11m were weak, compounds 11j, 11k, 11l showed considerable activities. And compared with
compound 11a, compounds with hydroxyl or methoxy group (i.e. compounds 11h-11l) were more

favorable to the inhibitory activity. Interestingly, the position of methoxy substitution on phenyl
ring has an essential effect on inhibiting BChE. When the methoxy group was shifted from
2-position or 3-position to 4-positon of the phenyl ring, almost a 3-fold improvement in BChE
inhibition was observed, and the obtained compound 11j (IC50 = 18.52 μM) presented the most
potent BChE inhibitory activity in this series.
Taken as a whole, all SI values are greater than 10, so the activity characteristics of all
compounds is that their inhibitory activities on AChE are stronger than on BChE. It’s worth
mentioning that the SI value of compounds 11a-11c, 11e, 11f are all over 40, indicating that these
compounds possess rather high selectivity to AChE.
Table 1
Inhibition of cholinesterases activity and selectivity index (SI) of compounds 11a-11m.
Compd.
R
eeAChE IC50 (μM)a
1.35 ± 1.20
3.03 ± 1.34
1.65 ± 0.64
12.34 ± 3.00
1.81 ± 0.60
1.54 ± 0.50
14.05 ± 0.03
2.32 ± 0.03
1.70 ± 0.08
1.20 ± 0.18
1.23 ± 0.03
1.40 ± 0.23
2.95 ± 0.57
1.28 ± 0.01
eqBChE IC50 (μM)a
>150
SIb
>111.11
11a
H
11b
2-CH3
3-CH3
4-CH3
2-Cl
>150
>49.50
>90.90
11.51
67.09
82.25
>10.67
22.54
31.69
15.43
17.98
20.31
34.63
19.07
11c
>150
11d
142.40 ± 3.95
121.44 ± 1.52
126.67 ± 5.84
>150
11e
11f
3-Cl
11g
4-Cl
11h
2-OCH3
3-OCH3
4-OCH3
3-OH
4-OH
3-CF3
52.29 ± 2.81
53.88 ± 1.68
18.52 ± 1.21
22.11 ± 1.43
28.43 ± 4.70
102.17 ± 3.23
24.41 ± 2.01
11i
11j
11k
11l
11m
Galanthamine
a IC50 values are at least from three independent experiments and are expressed as the means ± SD.
b SI for AChE = IC50 eqBChE/IC50 eeAChE.
2.2.2. Enzyme kinetic analysis against AChE
The kinetics of more active compounds (11a, 11j, 11k, 11l) was further studied to understand
the nature of AChE inhibition. The results from Fig. 2 exhibited that all of the compounds (11a,
11j, 11k, 11l) are reversible inhibitors, as in the presence of different concentrations of compounds,
plots of the initial velocity versus enzyme concentration gave a series of straight lines, all of which
passed through the origin.

Fig. 2. Plots of initial velocity versus enzyme concentration for the inhibition of compounds 11a, 11j, 11k and 11l
(0.025U/mL, 0.04U/mL, 0.05 U/mL, 0.08 U/mL, 0.10 U/mL) on the hydrolysis activity of eeAChE.
The kinetic characterization of four active compounds (11a, 11j, 11k, 11l) against eeAChE
was also studied to elucidate the inhibition type and inhibition constant. The analysis was carried
out by measuring enzyme’s activity at different concentrations of substrate (0.5 μM – 2.0 μM).
The acquired inhibition data were further depicted with Lineweaver-Burk method followed by
inhibition constant (Ki) calculation using Lineweaver-Burk secondary plot. Results in Fig. 3
showed that compounds 11a, 11j and 11k had a family of lines with a common intercept on the
left of the vertical axis and above the horizontal axis, except compound 11l which lines were
below the horizontal axis. All of these lines had no intersection on the horizontal or vertical axis,
indicating that compounds 11a, 11j, 11k and 11l cause a mixed type of inhibition. This pattern of
inhibition is usually the result of a combination of partially competitive and non-competitive
inhibitions [43]. Then the inhibition constants Ki for compounds 11a, 11j, 11k and 11l were
calculated as 0.46 μM, 0.14 μM, 0.40 μM and 1.47 μM, respectively.

Fig. 3. Left: Lineweaver-Burk plots for the inhibition of eeAChE by compounds 11a, 11j, 11k and 11l at different
concentrations of substrate (ATCh), Right: Secondary plots for calculation of steady-state inhibition constants (Ki)
of compounds 11a, 11j, 11k and 11l.
2.2.3. Propidium iodide displacement assay
Propidium iodide assay is used to determine the displacement of propidium iodide (a
selective PAS-AChE inhibitor) from the propidium iodide-AChE enzyme complex. The results
obtained in Table 2 showed that compound 11k was more efficient to displace propidium (25.80%)
than donepezil (18.50%), although compounds 11j and 11l appeared to be somewhat less potent
(13.78−14.85%), with the exception of 11a, which only showed about half of displacement
capability of donepezil.
Table 2
Propidium iodide displacement (eeAChE) assay.
Compd.
Propidium Iodide
Displacement (%)a
9.73 ± 0.62
11a
11j
14.85 ± 0.59
11k
25.80 ± 1.37
11l
13.78 ± 0.96
Donepezil
18.50 ± 1.13
a All values are expressed as the mean ± SD (n = 3).
2.2.4. DPPH radical scavenging activity
As ROS support the development and progression of AD [44], the antioxidant activity of four
active compounds (11a, 11j, 11k, 11l) were evaluated by the DPPH test [45]. The result indicated
that the radical scavenging activity of compound 11l increased with increasing of its concentration.
However, compound 11j didn’t exhibit the similar capability, and even the percentage change of
compounds 11a and 11k in scavenging activity was decreased with negative values. As is shown
in Fig. 4, radical scavenging activity of ascorbic acid (Vc), as standard antioxidant, increased from
31% (at 0.0625 mg/mL) to 91% (at 0.25 mg/mL), and reached the maximum of 97% (at 1 mg/mL).
The sharp increase appeared relatively late for the activity of compound 11l, from 36% (at
0.25mg/mL) doubled to 78% (at 0.5mg/mL), and finally reached a maximum of 84% (at 1

mg/mL), only 13% lower than Vc. Nevertheless, when the concentration increased to 0.5mg/mL,
the radical scavenging activity of compound 11l was basically stable and quite close to Vc. And
the lower EC50 value (0.328 μM) of compound 11l manifests its remarkable antioxidant effect,
which was in submicromolar range similarly to Vc (EC50 = 0.095 μM).
11a
11j
11k
11l
Vc
100
80
60
40
20
0
-20
mg/mL
1
0.03125
0.0625
0.125
0.25
0.5
Fig. 4. Free radical scavenging activities of compounds 11a, 11j, 11k, 11l and Vc.
2.2.5. Metal-chelating properties
The ability of the synthesized compounds in metal chelation would be an added advantage in
the treatment of AD. Herein, compounds 11a, 11j, 11k and 11l were selected for their chelating
abilities toward Cu2+, Fe2+, Al3+ and Zn2+, using UV spectrophotometer with wavelength ranging
from 200 nm to 600 nm. As is seen in Fig. 5, the spectrum of compound 11l changed significantly
by adding CuCl2. The maximum absorption at 308 nm shifted to 282 nm, and the blue shift was 26
nm, which indicated the formation of 11l-Cu2+ complex. In addition, the absorption decreased
significantly when AlCl3, FeCl2, and ZnCl2 were added, indicating a possible interaction between
the compound 11l and these biometals. However, no remarkable shifts were observed upon the
addition of AlCl3, FeCl2 or ZnCl2. Similarly, the dramatic decreases in absorbance indicated a
possible interaction between these biometals and compounds 11a, 11j and 11k, which suggested
that all compounds 11a, 11j and 11k are also able to chelate biometals. The chelating ability of
compound 11l towards Cu2+ could be due to the donation of lone pair of electron on the nitrogen
atom present in 4-N-substituted aniline ring, which might be easily stabilised by resonance
through the phenol-quinone tautomerization, and strongly interfere in conjugated system between
4-N-substituted aniline ring and quinoline ring leading to a relative blue shift.

Fig. 5. UV-vis absorption spectra of test compound (20 μM in methanol) alone or in the presence of AlCl3, FeCl2,
CuCl2 or ZnCl2 (20 μM, in methanol); (A) compound 11a, (B) compound 11j, (C) compound 11k, (D) compound
11l.
To further determine the stoichiometry of 11l-Cu2+ complex, molar ratio method was used by
preparing the methanol solutions of compound 11l with increasing amounts of CuCl2. The UV
spectra were used to obtain the absorbance of the 11l complex and different concentrations of
CuCl2 at 282 nm (Fig. 6). Accordingly, the absorbance linearly decreased initially and then
plateaued. The two straight lines intersected at a mole fraction of 0.50, revealing a 2:1
stoichiometry for 11l-Cu2+ complex.

Fig. 6. Determination of the stoichiometry of complex 11I-Cu2+ by using molar ratio method through titrating
methanol solution of compound 11I with ascending amounts of CuCl2.
2.3 Docking studies
In order to explore the possible interaction mode in the active sites of AChE, the most active
compound 11j was selected for molecular modeling research using CDOCKER in Discovery
Studio 3.0 software. The crystal complex of AChE with galanthamine (PDB: 1DX6) was selected
for the docking research [35]. Fig. 7 showed that compound 11j interacted with the CAS site of
AChE by the following moieties: the propyl ether fragment attached to quinoline nucleus showed
one carbon hydrogen bond with His440; 4-N-phenylamine fragment created one π-π interaction
with Phe331; 4-methyl-piperidine fragment of compound 11j formed one π-alkyl interaction and
one carbon hydrogen bond concurrently with Trp84. It is worth mentioning that there are three
amino acids of PAS site of AChE binding to compound 11j: Asp72 interacted with nitro group
through one attractive charge, and with C-2 hydrogen through one carbon hydrogen bond; Tyr334
interacted with 4-N-phenylaminoquinoline skeleton through two π-π interactions; and Tyr121
interacted with the NH group via one conventional hydrogen bond. The binding mode suggests
that compound 11j behaves as dual binding site AChE inhibitors, which is consistent with our
kinetic analysis.
Noteworthily, nitrogen cation of the nitro group could form the attractive force with carboxyl
anion of Asp72, an ion of opposite charge. And the strong electron-withdrawing ability of the nitro
group could decrease the electron density of quinoline ring, and induces face-to-face π-π
electron-donor-acceptor interaction with the aromatic amino acid residue (Tyr334) of AChE.
Furthermore, other relative reports also suggested its important role in the AChE inhibition [46,47].
Unexpectedly, it was found from recent literatures that the nitro substituent of aromatic
compounds could cause surprisingly monoamine oxidase (MAO) inhibitory and
N-methyl-D-aspartate (NMDA) antagonistic effects [48,49]. So, the presence of nitro group might

represent a valuable pharmacophoric feature for the treatment of AD.
The interaction of compound 11j with BChE (PDB code: 4BDS) was also carried out. As
seen in Fig. 8, compound 11j was bound to the residues Gly117 and Gly116 from the oxyanion
hole (OAH), Ser198 from esteratic site (ES), Trp82 from anionic substrate binding site (AS), three
PAS amino acid residues such as Asp70, Pro285 and Tyr332, but also bound to Leu286, Ser287,
Trp231 and Thr120 residues, via two π-ion interactions, two conventional hydrogen bond, four
carbon hydrogen bonds, two amide-π interactions, one π-sigma interaction, one van der Waals,
and two hydrophobic interactions. Briefly, it is noteworthy to say that compound 11j interacted
with important amino acid residues in the OAH, ES, AS and PAS active sites of BChE, which
makes it worthy of further study.
Fig. 7. 3D binding mode of compound 11j with AChE (PDB code: 1DX6), highlighting the protein residues that
participate in the main interactions with the inhibitor.

Fig. 8. 3D binding mode of compound 11j with BChE (PDB code: 4BDS), highlighting the protein residues that
participate in the main interactions with the inhibitor.
3. Conclusion
In summary, a series of novel 4-N-phenylaminoquinoline derivatives were designed, synthesized
and evaluated as multifunctional agents for the treatment of AD. Among them, compounds 11j,
11k and 11l had comparable inhibition activities to galantamine in both AChE and BChE.
Especially, compound 11j demonstrated the most potent inhibition to eeAChE and eqBChE with
IC50 values of 1.20 μM and 18.52 μM, respectively. The kinetic analysis inferred that compound
11j was reversible inhibitor, and showed a mixed type of inhibition on AChE. What’s more,
through molecular docking studies, it was found that compound 11j interacted with CAS and PAS
of both AChE and BChE. Meanwhile, compound 11k exhibited significant displacement of
propidium iodide from the PAS of AChE (25.80%). More importantly, The DPPH radical
scavenging activity of compound 11l reached 84% (at 1mg/mL), and its EC50 value of compound
11l was 0.328 μM, which was almost as high level as that of Vc (EC50 = 0.095 μM). Additionally,
compounds 11a, 11j, 11k and 11l showed satisfactory metal-chelating properties toward Al3+, Fe2+,
Cu2+ and Zn2+ ions. From our in vitro and in silico results, 4-N-phenylaminoquinoline might be a
multifunctional potential scaffold for the treatment of AD, and compounds 11j, 11k and 11l could
provide a starting point for further developments of new therapeutic agents.
4. Experimental Section
4.1. Chemistry
The chemical reactions were monitored by TLC using commercially available alumina plates
coated with silica gel 60 F254 (Merck). Chromatographic separation was performed on
self-packed columns with silica gel from Qingdao Haiyang Chemical Group Co., Ltd. (PR China),
and MPLC was carried out on a BUCHI apparatus equipped with a C-605 pump. 1H and 13C NMR
spectra were recorded on a Bruker Avance III 400 spectrometer with TMS as an internal standard.

Coupling constant (J) values were presented in Hz, and spin multiplicities were given as s (singlet),
d (doublet), t (triplet), q (quartet), brs (broad) and m (multiplet). The purity of all compounds for
biological evaluation was confirmed to be higher than 95% by analytical HPLC performed on
Shimadzu SPD-M20A. (Column: WondaSil C18, 5 μm particle size, 4.6 mm×250 mm, S/N:
3k9701-01; mobile phase: methanol/H2O (30/70–100/0); flow rate = 1 mL/min; λ = 308 nm).
High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) was acquired on a
Bruker micrOTOF-Q II spectrometer.
Compounds 2–9, 10a–10m were synthesized as our early reported procedures [16,35].
Synthesis of 6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-phenylquinolin-4-amine
derivatives (11a–11m)
A mixture of 6-Methoxy-7-(3-chloropropoxy)-3-nitro-N-phenylquinolin-4-amine (10a–10m)
(0.59mmol), NaI (0.098 g, 0.65 mmol), K2CO3 (0.013 g, 1.77 mmol) was refluxed in CH3CN (45
mL) for 1 h, then added 4-methyl-piperidine (0.71 mmol) refluxed for 24 h. Excess CH3CN was
rotated off, and the residue was partitioned between CH2Cl2 and H2O and filtered through Celite.
The organic portion was worked up to give a solid which was chromatographed on silica gel.
CH2Cl2/MeOH (9:2) eluted products 11a–11m.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-phenylquinolin-4-amine (11a)
1
Yield: 46.78%, yellow solid, mp: 177.8–180.3 °C. Purity 95.65% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.93 (d, 3H, J = 6.3 Hz, CH3), 1.26 (m, 2H, H-3'''ax and H-5'''ax), 1.35 (m, 1H,
CH), 1.63 (d, 2H, J = 12.4 Hz, H-3'''eq and H-5'''eq), 1.95 (t, 2H, J = 11.4 Hz, H-2'''ax and 6'''ax),
2.10 (m, 2H, CH2CH2CH2), 2.52 (t, 2H, J = 7.1 Hz, NCH2), 2.91 (d, 2H, J = 11.4 Hz, H-2'''eq and
H-6'''eq), 3.34 (s, 3H, OCH3), 4.23 (t, 2H, J = 6.6 Hz, OCH2), 6.88 (s,1H, H-5), 7.19 (d, 2H, J= 7.7
Hz, H-2', H-6'), 7.25 (t, 1H, J = 7.7 Hz, H-4'), 7.35 (s, 1H, H-8), 7.41 (t, 2H, J = 7.7 Hz, H-3',
13
H-5',), 9.36 (s, 1H, H-2), 10.43 (s,1H, NH); C NMR (100 MHz, CDCl3) δ: 153.7, 148.3, 148.1,
145.2, 145.0, 141.2, 129.7 (2C), 128.5, 126.0, 124.0 (2C), 112.6, 109.9, 106.4, 67.8, 55.3, 55.2,
54.0 (2C), 34.3(2C), 30.8, 26.5, 21.9. HRMS (ESI) calculated for (C25H30N4O4+H+) 451.2345,
found 451.2351.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(o-tolyl)quinolin-4-amine (11b)
1
Yield: 43.36%, yellow solid, mp: 166.6–168.9 °C. Purity 99.85% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.92 (d, 3H, J = 6.3 Hz, CH3), 1.25 (m, 2H, H-3'''ax and H-5'''ax), 1.35 (m, 1H,
CH), 1.62 (d, 2H, J = 12.6 Hz, H-3'''eq and H-5'''eq), 1.93 (t, 2H, J = 11.4 Hz, H-2'''ax and 6'''ax),
2.08 (m, 2H, CH2CH2CH2), 2.35 (s, 3H, ArCH3), 2.50 (t, 2H, J = 7.0 Hz, NCH2), 2.89 (d, 2H, J =
11.4 Hz, H-2'''eq and H-6'''eq), 3.27 (s, 3H, OCH3), 4.21 (t, 2H, J = 6.6 Hz, OCH2), 6.77 (s,1H, H-5),
7.10 (m, 1H, ArH), 7.23 (m, 2H, ArH), 7.33 (s, 1H, H-8), 7.37 (m, 1H, ArH), 9.36 (s, 1H, H-2),
10.52 (s,1H, NH); 13C NMR (100 MHz, CDCl3) δ: 153.5, 148.3, 147.8, 146.1, 145.6, 139.6, 133.5,
131.4, 127.3, 127.2, 127.1, 125.7, 112.4, 110.0, 105.5, 67.8, 55.3, 55.1, 54.0 (2C), 34.3 (2C), 30.8,
26.5, 21.9, 18.3. HRMS (ESI) calculated for (C26H32N4O4+H+) 465.2502, found 465.2516.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(m-tolyl)quinolin-4-amine (11c)
1
Yield: 48.29%, yellow solid, mp: 120.0–122.3 °C. Purity 96.39% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.94 (d, 3H, J = 5.9 Hz, CH3), 1.37 (m, 2H, H-3'''ax and H-5'''ax), 1.42 (m, 1H,
CH), 1.67 (d, 2H, J = 11.3 Hz, H-3'''eq and H-5'''eq), 2.07 (t, 2H, J = 11.5 Hz, H-2'''ax and 6'''ax), 2.16
(m, 2H, CH2CH2CH2), 2.34 (s, 3H, ArCH3), 2.62 (t, 2H, J = 7.6 Hz, NCH2), 3.00 (d, 2H, J = 11.5
Hz, H-2'''eq and H-6'''eq), 3.34 (s, 3H, OCH3), 4.22 (t, J = 6.5 Hz, 2H, OCH2), 6.90 (s,1H, H-5),
6.98 (d, J = 8.0 Hz, 1H, H-4'), 7.01 (s, 1H, H-2'), 7.06 (d, 1H, J = 7.5 Hz, H-6'), 7.28 (t, 1H, J =

7.7 Hz, H-5'), 7.32 (s, 1H, H-8), 9.32 (s, 1H, H-2), 10.42 (s,1H, NH); 13C NMR (100 MHz, CDCl3)
δ: 153.3, 148.2, 147.9, 145.3, 145.1, 141.0, 139.9, 129.5, 128.4, 126.9, 124.7, 121.2, 112.9, 110.0,
106.6, 67.3, 55.2, 55.1, 53.6 (2C), 33.1 (2C), 31.8, 25.6, 21.4, 21.3. HRMS (ESI) calculated for
(C26H32N4O4+H+) 465.2502, found 465.2510.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(p-tolyl)quinolin-4-amine (11d)
1
Yield: 46.47%, yellow solid, mp: 187.3–190.1 °C. Purity 98.91% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.95 (d, 3H, J = 5.5 Hz, CH3), 1.40 (m, 2H, H-3'''ax and H-5'''ax), 1.41 (m, 1H,
CH), 1.67 (d, 2H, J =10.3 Hz, H-3'''eq and H-5'''eq), 2.10 (t, 2H, J = 11.1 Hz, H-2'''ax and 6'''ax), 2.18
(m, 2H, CH2CH2CH2), 2.36 (s, 3H, ArCH3), 2.65 (t, 2H, J = 7.6 Hz, NCH2), 3.03 (d, 2H, J = 11.1
Hz, H-2'''eq and H-6'''eq), 3.32 (s, 3H, OCH3), 4.22 (t, J = 6.4 Hz, 2H, OCH2), 6.87 (s,1H, H-5),
7.08 (d, 2H, J = 8.1 Hz, H-3', H-5'), 7.20 (d, 2H, J = 8.1 Hz, H-2', H-6'), 7.30 (s, 1H, H-8), 9.32 (s,
1H, H-2), 10.45 (s,1H, NH); 13C NMR (100 MHz, CDCl3) δ: 153.3, 148.0, 147.9, 145.5, 145.4,
138.5, 136.1, 130.2 (2C), 128.0, 124.3 (2C), 112.6, 109.9, 106.5, 67.4, 55.2, 55.1, 53.8 (2C), 33.5
(2C), 30.4, 25.9, 21.7, 21.0. HRMS (ESI) calculated for (C26H32N4O4+H+) 465.2502, found
465.2506.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(2-chlorophenyl)quinolin-4-amine
(11e)
1
Yield: 44.97%, yellow solid, mp: 135.4–137.7 °C. Purity 98.85% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.92 (d, 3H, J = 6.4 Hz, CH3), 1.25 (m, 2H, H-3'''ax and H-5'''ax), 1.35 (m, 1H,
CH), 1.62 (d, 2H, J =12.1 Hz, H-3'''eq and H-5'''eq), 1.93 (t, 2H, J = 11.4 Hz, H-2'''ax and 6'''ax), 2.10
(m, 2H, CH2CH2CH2), 2.51 (t, 2H, J = 7.1 Hz, NCH2), 2.90 (d, 2H, J = 11.4 Hz, H-2'''eq and
H-6'''eq), 3.41 (s, 3H, OCH3), 4.24 (t, 2H, J = 6.7 Hz, OCH2), 6.75 (s,1H, H-5), 6.97 (dd, 1H, J =
7.5 Hz, 1.8 Hz, H-6'), 7.18 (m, 2H, H-4', H-5'), 7.37 (s, 1H, H-8), 7.54 (dd, 1H, J = 7.6, 1.7 Hz,
13
H-3'), 9.35 (s, 1H, H-2), 10.14 (s,1H, NH); C NMR (100 MHz, CDCl3) δ: 154.0, 148.9, 148.0,
145.0, 143.9, 138.4, 130.5, 129.5, 128.0, 127.4, 126.3, 124.4, 113.0, 109.9, 105.2, 67.9, 55.4, 55.3,
54.1 (2C), 34.4 (2C), 30.8, 26.5, 21.9. HRMS (ESI) calculated for (C25H29ClN4O4+H+) 485.1956,
found 485.1956.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(3-chlorophenyl)quinolin-4-amine
(11f)
Yield: 43.49%, yellow solid, mp: 154.3–155.8 °C. Purity 96.83% by HPLC. 1H NMR (400
MHz, CDCl3) δ: 0.92 (d, 3H, J = 6.3 Hz, CH3), 1.26 (m, 2H, H-3'''ax and H-5'''ax), 1.35 (m, 1H,
CH), 1.63 (d, 2H, J =12.7 Hz, H-3'''eq and H-5'''eq), 1.94 (t, 2H, J = 11.4 Hz, H-2'''ax and 6'''ax), 2.11
(m, 2H, CH2CH2CH2), 2.52 (t, 2H, J = 7.1 Hz, NCH2), 2.90 (d, 2H, J = 11.4 Hz, H-2'''eq and
H-6'''eq), 3.44 (s, 3H, OCH3), 4.24 (t, 2H, J = 6.7 Hz, OCH2), 6.83 (s,1H, H-5), 7.01 (d, 1H, J = 8.0
Hz, H-6'), 7.15 (s, 1H, H-2'), 7.19 (d, 1H, J = 8.1, H-4'), 7.30 (t, J = 8.0 Hz, 1H, H-5'), 7.37 (s, 1H,
H-8), 9.34 (s, 1H, H-2), 10.19 (s,1H, NH); 13C NMR (100 MHz, CDCl3) δ: 154.0, 148.8, 148.2,
145.0, 143.8, 142.5, 135.3, 130.5, 129.3, 125.5, 123.2, 121.2, 112.7, 110.0, 105.8, 67.9, 55.4, 55.3,
54.1, 34.3, 30.8, 26.5, 21.9. HRMS (ESI) calculated for (C25H29ClN4O4+H+) 485.1956, found
485.1950.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(4-chlorophenyl)quinolin-4-amine
(11g)
1
Yield: 44.26%, yellow solid, mp: 219.8–220.9 °C. Purity 97.49% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.93 (d, 3H, J = 6.3 Hz, CH3), 1.26 (m, 2H, H-3'''ax and H-5'''ax), 1.36 (m, 1H,
CH), 1.63 (d, 2H, J = 12.8 Hz, H-3'''eq and H-5'''eq), 1.94 (t, 2H, J = 11.4 Hz, H-2'''ax and 6'''ax),

2.10 (m, 2H, CH2CH2CH2), 2.52 (t, 2H, J = 7.1 Hz, NCH2), 2.90 (d, 2H, J = 11.4 Hz, H-2'''eq and
H-6'''eq), 3.43 (s, 3H, OCH3), 4.24 (t, J = 6.6 Hz, 2H, OCH2), 6.81 (s, 1H, H-5), 7.09 (d, 2H, J =
8.6 Hz, H-2', H-6'), 7.35 (d, 2H, J = 7.6 Hz, H-3', H-5'), 7.36 (s, 1H, H-8), 9.35 (s, 1H, H-2), 10.23
(s,1H, NH); 13C NMR (100 MHz, CDCl3) δ: 153.9, 148.6, 148.2, 145.1, 144.3, 139.9, 131.0, 129.7
(2C), 129.0, 124.7 (2C), 112.6, 110.0, 105.9, 67.9, 55.34, 55.28, 54.1 (2C), 34.4 (2C), 30.8, 26.5,
21.9. HRMS (ESI) calculated for (C25H29ClN4O4+H+) 485.1956, found 485.1956.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(2-methoxyphenyl)quinolin-4-amine
(11h)
1
Yield: 46.23%, yellow solid, mp: 103.2–105.7 °C. Purity 98.30% by HPLC. H NMR (400
MHz, CDCl3) δ: 1.03 (d, 3H, J = 6.3 Hz, CH3), 1.67-2.02 (m, 5H, H-3''', H-4''' and H-5''' of
piperidyl moiety), 2.56 (m, 2H, CH2CH2CH2), 2.91 (br.s, 2H, H-2'''ax and 6'''ax), 3.33 (t, 2H, J =
7.0 Hz, NCH2), 3.34 (s, 3H, OCH3), 3.63 (br.s, 2H, H-2'''eq and H-6'''eq), 3.81 (s, 3H, OCH3), 4.25
(t, 2H, J = 5.7 Hz, OCH2), 6.923 (td, 1H, J = 7.8, 1.2 Hz, H-4'), 6.924 (s, 1H, H-5), 7.00 (dd,1H, J
= 7.8, 1.2 Hz, H-6'), 7.03 (dd, 1H, J = 7.8, 1.2 Hz, H-3'), 7.21 (td, 1H, J = 7.8, 1.2 Hz, H-5'), 7.23
(s, 1H, H-8), 9.25 (s, 1H, H-2), 10.24 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ: 152.6, 152.4,
147.9, 147.2, 145.3, 145.2, 129.4, 128.4, 126.9, 124.0, 120.7, 113.6, 111.7, 110.0, 105.7, 66.1,
55.9, 55.3, 55.2, 53.3 (2C), 30.7 (2C), 29.0, 27.7, 23.6. HRMS (ESI) calculated for
(C26H32N4O5+H+) 481.2451, found 481.2463.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(3-methoxyphenyl)quinolin-4-amine
(11i)
1
Yield: 45.23%, yellow solid, mp: 104.9–105.7 °C. Purity 96.93% by HPLC. H NMR (400
MHz, CDCl3) δ: 1.03 (d, 3H, J = 6.3 Hz, CH3), 1.76 (m, 1H, CH), 1.88 (d, 2H, J = 13.6 Hz,
H-3'''eq and H-5'''eq), 2.02 (m, 2H, H-3'''ax and H-5'''ax), 2.58 (m, 2H, CH2CH2CH2), 2.88 (t, 2H, J =
10.7 Hz, H-2'''ax and 6'''ax), 3.33 (br.s, 2H, NCH2), 3.37 (s, 3H, OCH3), 3.69 (d, 2H, J = 10.7 Hz,
H-2'''eq and H-6'''eq), 3.77 (s, 3H, OCH3), 4.26 (br.s, 2H, OCH2), 6.72 (m, 1H, H-6'), 6.74 (s, 1H,
H-2'), 6.79 (dd, 1H, J = 8.2 Hz, 1.8 Hz, H-4'), 6.90 (s, 1H, H-5), 7.25 (s, 1H, H-8), 7.29 (t, 1H, J =
8.0 Hz, H-5'), 9.25 (s, 1H, H-2), 10.35 (s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ: 160.8, 152.8,
148.0, 147.4, 145.1, 144.9, 142.0, 130.5, 128.5, 116.2, 113.3, 111.8, 109.9, 109.7, 106.6, 66.2,
55.6, 55.3, 53.2, 53.4 (2C), 30.7 (2C), 29.2, 23.6, 21.0. HRMS (ESI) calculated for
(C26H32N4O5+H+) 481.2451, found 481.2444.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(4-methoxyphenyl)quinolin-4-amine
(11j)
1
Yield: 47.11%, yellow solid, mp: 102.6–104.7 °C. Purity 99.77% by HPLC. H NMR (400
MHz, CDCl3) δ: 1.04 (d, 3H, J = 6.3 Hz, CH3), 1.68-2.03 (m, 5H, H-3''', H-4''' and H-5''' of
piperidyl moiety), 2.56 (m, 2H, CH2CH2CH2), 2.90 (br.s, 2H, H-2'''ax and 6'''ax), 3.32 (t, 2H, J =
7.7 Hz, NCH2), 3.33 (s, 3H, OCH3), 3.64 (br.s, 2H, H-2'''eq and H-6'''eq), 3.83 (s, 3H, OCH3), 4.25
(t, J = 5.7 Hz, OCH2), 6.89 (s, 1H, H-5), 6.94 (d, 2H, J = 8.8 Hz, H-2', H-6'), 7.15 (d, J = 8.8 Hz,
2H, H-2', H-5'), 7.21 (s, 1H, H-8), 9.24 (s, 1H, H-2), 10.49 (s, 1H, NH); 13C NMR (100 MHz,
CDCl3) δ: 158.1, 152.5, 147.63, 147.56, 145.8, 145.4, 133.7, 127.6, 126.1 (2C), 115.0 (2C), 112.8,
110.1, 106.6, 66.2, 55.7, 55.2, 55.1, 53.3 (2C), 30.7 (2C), 29.0, 23.7, 20.9. HRMS (ESI) calculated
for (C26H32N4O5+H+) 481.2451, found 481.2475.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(3-hydroxylphenyl)quinolin-4-amine
(11k)
1
Yield: 46.20%, yellow solid, mp: 84.6–87.2 °C. Purity 97.90% by HPLC. H NMR (400

MHz, CDCl3) δ: 0.93 (d, 3H, J = 6.3 Hz, CH3), 1.30 (m, 2H, H-3'''ax and H-5'''ax), 1.38 (m, 1H,
CH), 1.63 (d, 2H, J = 12.4 Hz, H-3'''eq and H-5'''eq), 2.03 (m, 2H, H-2'''ax and 6'''ax), 2.06 (m, 2H,
CH2CH2CH2), 2.55 (t, 2H, J = 7.1 Hz, NCH2), 2.93 (d, 2H, J = 10.8 Hz, H-2'''eq and H-6'''eq), 3.39
(s, 3H, OCH3), 4.06 (t, 2H, J = 6.0 Hz, OCH2), 6.38 (s, 1H, H-2'), 6.75 (d, 2H, J = 7.6 Hz, H-4',
H-6'), 6.91 (s, 1H, H-5), 7.10 (s, 1H, H-8), 7.27 (t, 1H, J = 7.6 Hz, H-5'), 9.23 (s, 1H, H-2), 10.27
(s, 1H, NH); 13C NMR (100 MHz, CDCl3) δ: 158.5, 153.7, 148.2, 147.0, 145.2, 144.8, 142.1,
130.9, 128.3, 114.8, 114.0, 112.8, 110.9, 108.6, 106.5, 67.4, 55.3, 55.2, 53.9 (2C), 33.7 (2C), 30.6,
26.0, 21.7. HRMS (ESI) calculated for (C25H30N4O5+H+) 467.2294, found 467.2294.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(4-hydroxylphenyl)quinolin-4-amine
(11l)
1
Yield: 47.12%, brown solid, mp: 124.8–126.3 °C. Purity 96.70% by HPLC. H NMR (400
MHz, CDCl3) δ: 0.94 (d, 3H, J = 5.2 Hz, CH3), 1.35 (m, 2H, H-3'''ax and H-5'''ax), 1.43 (m, 1H,
CH), 1.68 (d, 2H, J = 11.4 Hz, H-3'''eq and H-5'''eq), 2.11 (m, 2H, CH2CH2CH2), 2.09 (m, 2H,
H-2'''ax and 6'''ax), 2.59 (t-like, 2H, NCH2), 3.04 (d, 2H, J = 10.0 Hz, H-2'''eq and H-6'''eq), 3.29 (s,
3H, OCH3), 4.11 (t-like, 2H, OCH2), 6.87 (d, 2H, J = 7.4 Hz, H-2', H-6'), 6.97 (s, 1H, H-5), 7.09
(d, J = 7.4 Hz, 2H, H-3', H-5'), 7.25 (s, 1H, H-8), 9.31 (s, 1H, H-2), 10.65 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) δ: 155.1, 152.5, 148.2, 146.5, 144.8, 143.3, 132.5, 127.8, 124.1 (2C), 115.8
(2C), 113.4, 109.6, 104.5, 66.6, 55.4, 54.1, 52.8 (2C), 32.7 (2C), 29.5, 25.1, 21.5. HRMS (ESI)
calculated for (C25H30N4O5+H+) 467.2294, found 467.2299.
6-Methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)-3-nitro-N-(3-(trifluoromethyl)phenyl)quinolin-4
-amine (11m)
1
Yield: 56.79%, brown solid, mp:136.0–139.3 °C. Purity 97.97% by HPLC. H NMR (400
MHz, CDCl3) δ: 1.05 (d, 3H, J = 6.2 Hz, CH3), 1.73-2.07 (m, 5H, H-3''', H-4''' and H-5''' of
piperidyl moiety), 2.60 (br.s, 2H, CH2CH2CH2), 2.90 (br.s, 2H, H-2'''ax and 6'''ax), 3.35 (t-like, 2H,
NCH2), 3.38 (s, 3H, OCH3), 3.68 (br.s, 2H, H-2'''eq and H-6'''eq), 4.29 (t-like, 2H, OCH2), 6.77 (s,
1H, H-5), 7.29 (s, 1H, H-8), 7.31 (d, 1H, J = 7.7 Hz, H-4'), 7.39 (s, 1H, H-2'), 7.46 (d, 1H, J = 7.7
Hz, H-6'), 7.52 (t, 1H, J = 7.7 Hz, H-5'), 9.30 (s, 1H, H-2), 10.20 (s, 1H, NH); 13C NMR (100
MHz, CDCl3) δ: 153.1, 148.7, 145.0, 143.5, 141.8, 132.1 (q, J = 32.9 Hz), 130.3 (2C), 129.6,
126.1, 123.5 (q, J = 270.9 Hz ), 121.9 (q, J = 4.2 Hz), 119.4 (q, J = 4.1 Hz ), 113.4, 110.5, 105.8,
66.3, 55.3, 55.2, 53.4 (2C), 30.8 (2C), 29.0, 23.7, 21.1. HRMS (ESI) calculated for
(C26H29F3N4O4+H+) 519.2219, found 519.2224.
4.2. Biological evaluation
4.2.1. AChE and BChE inhibition assay
AChE and BChE inhibitory activities of compounds were determined by using modified
Ellman’s method [50]. AChE (EC 3.1.1.7, from electric eel), BChE (EC 3.1.1.8, from equine
serum), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATCI), and
butyrylthiocholine iodide (BTCI) were purchased from Sigma-Aldrich. Compounds were
dissolved in DMSO (10 mM). Reaction mixture contained 140 μL of 100 mM sodium phosphate
buffer (pH 8.0), the enzyme solution (either 0.05 U/mL of AChE, 20 μL; or 0.05 U/mL of BChE,
20 μL), and test compound (20 μL). Assayed solutions of test compounds were pre-incubated with
corresponding ChE at 25 °C for 15 min. The reaction was initiated by addition of 10 μL of 10 mM
DTNB and 10 μL of 7.5 mM substrate (ATCI or BTCI). The activity was determined by
measuring the increase in absorbance at 412 nm at 37 °C in 10 min intervals using microplate
reader (BioTek Epoch). The percentage of inhibition was calculated from the measured data as

follows: (Ac−Ai)/Ac×100%, where Ai and Ac represent the change in the absorbance in the
presence of inhibitor and without inhibitor, respectively.
4.2.2. In vitro propidium iodide displacement assay
150 μL of a 20 μM solution of the test compound or standard donepezil (from Sigma) was
incubated with 5 units of eeAChE at 25 °C for 6 h in the 96-well plate. After incubation, 50 μL of
1 μM propidium iodide solution was added to make the final assay volume of 200 μL. After 10
min, the fluorescence intensity was observed at an excitation wavelength (λex) of 535 nm and an
emission wavelength (λem) of 595 nm using a fluorescence plate reader. The percentage inhibition
was calculated by following expression: 100 ‒ (IFi/IF0 × 100), where IFi and IF0 are the
fluorescence intensities with and without the test compounds, respectively. Each assay was
repeated at three different times [51,52].
4.2.3. Enzyme kinetic analysis against AChE
In order to investigate the reversibility of the compounds (11a, 11j, 11k, 11l) against AChE,
o
the absorbance was measured at 412 nm after two minutes of incubation at 37 C for different
concentrations of the compounds (0.0-2.0μM) and five different concentrations of AChE
(0.025-0.10 u/mL). The resulting values were measured in triplicate for the velocity and enzyme
concentration curves. The velocity (V) was calculated from the measured data as follows: V =
(Ac−Ai)/2, where Ai and Ac represent the change in the absorbance in the presence of AChE and
without AChE, respectively. All data were analyzed by OriginPro 8.
For estimates of the inhibition model and inhibition constant Ki, the rate of enzymatic
reaction was obtained with different concentrations of compounds (0.0-2.0 μM) and at least five
different concentrations of ATCI (3.75-15.00 mM). For each experiment, reaction was started by
adding substrate and progress curves were recorded at 412 nm after two minutes of incubation at
o
37 C. Next, double reciprocal plots (1/v vs 1/[s]) were made using the slopes of progress curves
to obtain the type of inhibition. Slopes of these reciprocal plots were then drawn against the
concentration of a compound in a related analysis, and Ki was determined as the intercept on the
negative x axis [53]. All rate measurements were performed in triplicate and data analysis was
performed with OriginPro 8.0.
4.2.4. DPPH radical scavenging activity
The antioxidant activity was determined by the DPPH radical scavenging assay. The mixture
was incubated for 30 min in the dark. The absorbance of the resulting solution was measured at
517 nm. Different concentrations (0.03125–1 mg/mL) of the extract were tested. The control
sample was a mixture of methanol and DPPH, and Vc was used as reference. Percentage of DPPH
radical scavenging activity was obtained as (1 – As/Ac) × 100. As and Ac represent the absorbance
of the sample and control, respectively [45], and then EC50 (effective concentration of a compound
that decreases the initial concentration of DPPH by 50%) can be calculated.
4.2.5. Metal-chelating properties
All solutions used in metal-chelating study were prepared in methanol, and Fe2+, Cu2+,
Al3+and Zn2+ solutions were prepared from FeCl2, CuCl2, AlCl3 and ZnCl2 respectively. To study
the metal binding ability, a mixture of test compound (1 mL) and metal solution (1 mL) with the
same concentration (20 μM) in a 1 cm quartz cuvette was incubated at room temperature for 30
min. Then, the absorption spectra were recorded with wavelength ranging from 200–600 nm [44].
The stoichiometry of complex 11l-Cu2+ was also studied using the molar ratio method [4,54].
The concentration of tested compound 11l was 20 μM and the final concentration of Cu2+ ranged

from 0 to 60 μM at 282 nm. The plot was obtained by the corresponding absorption versus mole
fraction of Cu2+.
4.3. Docking study
4.3.1. Molecular docking studies on AChE
The binding modes were generated by using the Discovery Studio CDOCKER software
(Accelrys, San Diego, USA). The crystal structure of AChE from Torpedo californica (TcAChE;
Code ID: 1DX6) was obtained from the Protein Data Bank. The binding pattern of galanthamine
in TcAChE is similar to that observed in human recombinant AChE (rhAChE) [55], hence
TcAChE was selected for docking studies [43,56]. TcAChE was prepared for receptor protein by a
sequence of operations including Clean Protein, Hydrogen Add and Apply Forcefield. All ligands
were performed using the default settings, and docked in all possible stereoisomeric forms in an
active site located sphere with 10Å radius for TcAChE. Then receptor-ligand interactions were
operated by the CDOCKER protocol with the default parameters. By using the DS CDOCKER
program, the crystallographic binding mode of galanthamine was reproduced into the TcAChE
binding site: the best ranked solution presented a root-mean-square deviations (RMSD) value of
0.50 Å with respect to the experimental pose, indicating that the docking methods and parameters
used in this study were approximate for the AChE system.
4.3.2. Molecular docking studies on BChE
Flexible docking was conducted using Discovery Studio 2017 R2 (Accelrys, San Diego,
USA). The crystal structure of BChE from Homo sapiens (Code ID: 4BDS) was extracted from
the Protein Data Bank. And all water molecules and co-crystallized ligand were removed, which
was followed by protein preparation protocol with CHARMm force field. The prepared ligands
were subjected to minimization also with CHARMm force field before being used for docking
analyses. The docking results were analyzed using Discovery Studio Visualizer 17.2.0.16349. The
docking parameters were first validated by re-docking of cocrystallized ligand into the active site
of the enzyme. The re-docked tacrine was found to bind in a similar manner as its respective
crystallographic conformation, and the RMSD value for tacrine conformation is 0.41 Å, indicating
that the selected docking parameters are acceptable.
Conflict of Interest
The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this
paper.
Acknowledgement
This research was supported by the Training Project of Innovation Team of Colleges and
Universities in Tianjin (TD13-5020).

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Highlights
• A series of new 4-N-phenylaminoquinoline derivatives were synthesized.
• Compounds 11j, 11k and 11l had comparable inhibition activities to reference drug galantamine
in both AChE and BChE.
• Compound 11k showed excellent displacement of propidium iodide from the PAS of AChE.
• Compound 11l exhibited significant radical scavenging activity.
• Compounds 11a, 11j, 11k and 11l displayed satisfactory metal-chelating properties, and the
possible mechanism for metal chelation of compound 11l towards Cu2+ was deduced.

Graphical abstract