Design, synthesis, evaluation and molecular modeling study of 4-N-phenylaminoquinolines for Alzheimer disease treatment

Article abstract of DOI:10.1016/j.bmcl.2019.03.050

Dual binding site acetylcholinesterase (AChE) inhibitors and butyrylcholinesterase (BChE) inhibitors have recently emerged as two classes of new anti-Alzheimer agents to positively modify the disease's course. In this work, a new series of 4-N-phenylaminoquinolines was synthesized and evaluated for their abilities to inhibit AChE and BChE. Compound 11b 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, docking study showed that compound 11b interacted simultaneously not only with active and peripheral sites of AChE, but also with all five regions of BChE active site. These findings suggest that these derivatives could be regarded as promising starting points for further drug discovery developments.

Full text of DOI:10.1016/j.bmcl.2019.03.050

Accepted Manuscript
Design, synthesis, evaluation and molecular modeling study of 4-N-phenyla-
minoquinolines for Alzheimer disease treatment
Jun Zhu, Li-Ning Wang, Rong Cai, Shang-Qi Geng, Ya-Fei Dong, Yu-Ming
Liu
PII:
S0960-894X(19)30205-7
https://doi.org/10.1016/j.bmcl.2019.03.050
BMCL 26364
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 January 2019
22 March 2019
30 March 2019
Please cite this article as: Zhu, J., Wang, L-N., Cai, R., Geng, S-Q., Dong, Y-F., Liu, Y-M., Design, synthesis,
evaluation and molecular modeling study of 4-N-phenylaminoquinolines for Alzheimer disease treatment,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.03.050
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Design, synthesis, evaluation and molecular
modeling study of 4-N-phenylaminoquinolines
for Alzheimer disease treatment
a
b
a
a
a
a,
Jun Zhu , Li-Ning Wang , Rong Cai , Shang-Qi Geng , Ya-Fei Dong , Yu-Ming Liu *
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
*
Corresponding author at: Department of Pharmacy Engineering, Tianjin University of
Technology, Tianjin 300384, PR China.
E-mail address: coumarin1968@hotmail.com (Y.M. Liu).
1

Keywords:
Acetylcholinesterase inhibitors
Butyrylcholinesterase inhibitors
Alzheimer’s disease
4-N-phenylaminoquinoline
Molecular modeling
ABSTRACT
Dual binding site acetylcholinesterase (AChE) inhibitors and butyrylcholinesterase
(BChE) inhibitors have recently emerged as two classes of new anti-Alzheimer agents
to positively modify the disease’s course. In this work, a new series of 4-N-
phenylaminoquinolines was synthesized and evaluated for their abilitites to inhibit
AChE and BChE. Compound 11b 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, docking study showed that compound 11b
interacted simultaneously not only with active and peripheral sites of AChE, but also
with all five regions of BChE active site. These findings suggest that these derivatives
could be regarded as promising starting points for further drug discovery developments.
2

Alzheimer's disease (AD), as a neurodegenerative disease, accounts for about 50%
to 75% of the type of dementia and has seriously threatened the health of old people.
With the acceleration of the aging process of the world population, the incidence of AD
increases year by year, and it is estimated that the number of AD patients worldwide
will exceed 100 million by 2050.1,2 Now there is a huge market demand for AD
therapeutic drugs, but there are few available drugs on the market. Therefore, it is of
great realistic and social significance to accelerate the search for more effective AD
drugs.
The well-known cognitive impairments observed in AD patients, mainly in memory
and language, are due, among other causes, to β-amyloid (Aβ) aggregation, tau
hyperphosphorylation, acetylcholine (ACh) deficiency, inflammation, and oxidative
stress.2
Currently AChE inhibitors are the primary anti-AD drugs used in clinic, including
selective AChE inhibitors donepezil and galantamine. These AChE inhibitors mainly
act on the catalytic active site (CAS) at the bottom of AChE, which can relieve
symptoms of mild to moderate AD patients. Nevertheless, the recent studies found that
the peripheral active site (PAS), at the entrance to the AChE gorge active site, is proven
to have a close relationship with both the hydrolysis of acetylcholine and the neurotoxic
cascade of AD through Aβ aggregation induced by AChE,3,4 and that dual interaction
with two binding sites (CAS and PAS) leads to much better AChE inhibitory activity.5,6
Therefore, dual binding site AChE inhibitors have currently been recognized as a new
strategy to increase inhibitory activity through the enhancement of number of drug-
target interactions and to prevent from Aβ aggregation.5,6,7
The multifaceted factors of the AD led to the development of multitarget-directed
ligands (MTDLs) to act as agents for the treatment of this disease. In recent years the
pivotal role played by butyrylcholinesterase (BChE), the sister enzyme of AChE, has
gained increasing interest. It was found that BChE was distributed in the brain and could
also degrade the neurotransmitter ACh. There was strong evidence that BChE
compensated the loss of neuronal AChE in progressed AD and took over the function
3

4
,8
of AChE when AChE production was insufficient or its activity was inhibited. In fact,
the level of AChE drops down to 90 % in advanced AD compared to the healthy brain,
9
while the level of BChE is about doubled and tends to increase continuously. This is
the main reason why AD patients are resistant to AChE inhibitors. In addition,
butyrylcholinesterase-knockout reduced brain deposition of Aβ in Alzheimer mouse
10
model; while BChE inhibitors could significantly reduce the formation of Aβ and Aβ
11
9
precursor protein-α (APP) and ameliorated cognitive dysfunction. What has the
extreme advantage is that BChE inhibitors have no notable deleterious physiological
effects, as confirmed by more than 30 years of clinical use of the antiasthmatic drug
1
2
bambuterol, a prodrug of the β -adrenergic agonist terbutaline, which also effectively
2
and selectively inhibits BChE, and especially haven’t any significant peripheral side
effects associated with BChE inhibition.12‒15 Therefore BChE is an important drug
target for AD in the later stage.15-17 Now rivastigmine is the sole powerful inhibitor of
cholinesterase (ChE, id est BChE and AChE) used in the treatment of AD, which could
be attributed to the long-term neglect of the role of BChE. But rivastigmine is strongly
bound only with the CAS of AChE, and does not interact with the PAS of AChE. 18
Quinoline derivatives and their analogues exhibited a variety of pharmacological
activities, such as anti-tumor, anti-malaria, bacteriostatic and ChE inhibitory,19,20 and
2
1
tacrine was before used as an AChE inhibitor in the treatment of AD. Recently some
-N-phenylaminoquinoline analogues were synthesized and evaluated as AChE
4
inhibitors.22,23,24 The previous investigations in our laboratory suggested that a 4-N-
phenylaminoquinoline derivative with pyridinium salt side chain, lead compound 1
(Fig. 1), had more potent effects on inhibiting AChE and BChE (IC50 values equal to
0
.92 μM and 14.20 μM, respectively) than galanthamine.25
As part of our ongoing project, compound 1 was chosen for a more detailed
investigation and was subjected to structural modifications to further evaluate the
structure-activity relationship (SAR) of this series of compounds. Our design was based
on bioisosteric replacement of pyridinium salt of compound 1 with N-
methylbenzylamine fragment to produce new compounds, which was inspired by this
4

pharmacophoric unit with attractive ChE inhibition.26−31 Furthermore, different
substituents were now introduced at the 4-N-phenyl ring to investigate their effects on
ChE inhibition.
Fig. 1. Design strategy leading to compounds 11a–11n.
According to Scheme 1, compounds 11a–11n were synthesized starting from the
commercially available material vanillic acid, which was esterificated using methanol
to obtain compound 2. Compound 2 was alkylated with 1-bromo-3-chloropropane in
acetonitrile 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 86.25% 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.32 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–10n. Then, final compounds 11a–
1
1n were achieved upon the substitution of compound 10a–10n with excessive N-
methylbenzylamine, respectively. All target compounds were purified by column
chromatography and characterized by H NMR, 13C NMR, and HR-ESI-MS
1
(Supplementary material).
5

Scheme 1. Synthesis of compounds 11a–11n. Reagents and conditions: (a) MeOH, HCl, 70°C, 12
h; (b) Br(CH Cl, K CO , CH CN, 70°C, 10 h; (c) fuming HNO , CH Cl , room temperature, 5 h;
d) Fe, NH Cl, EtOH, reflux; (e) 5 % NaOH, EtOH, 50°C, 10 h; (f) HON=CHCH NO , HCl, room
temperature, 18 h; (g) KOAc, Ac O, 15 min, reflux; (h) POCl , 70°C, 10 h; (i) corresponding aniline,
isopropanol, 90°C, 8 h; (j) N-methylbenzylamine, CH CN, 10 h, reflux.
)
2 3
2
3
3
3
2
2
(
4
2
2
2
3
3
The inhibitory potencies of compounds 11a–11n toward AChE and BChE were
3
3
evaluated using Ellman’s assay. For comparison purposes, the IC50 values of lead
compound 1 and the reference drug galanthamine were also considered. As shown in
Table 1, all the tested compounds 11a–11n turned out to successfully inhibit AChE.
Among them, compounds 11a–11e, 11g, 11j, 11l and 11m with IC50 values within a
very close range (from 0.65 to 1.88 μM), showed comparable inhibitory activity as
those of compound 1 (IC50 = 0.92 μM) and galanthamine (IC50 = 1.28 μM). Generally,
the order of the inhibitory potency of these derivatives bearing different substituents
was as followed: hydroxyl group > chloro group > methoxy group > methyl group >
trifluoromethyl group, which implied that carrying electron-drawing substituents would
have an adverse impact on their activities. On the other hand, based on the substituted
position in the 4-N-phenyl ring, the order of inhibitory potency against AChE was:
Meta > Ortho > Para. Interestingly, compound 11a was the most potent AChE inhibitor,
with IC50 value of 0.65 μM, which seemed that small aromatic moiety in quinoline
scaffold would be optimal for AChE inhibitory activity.
In terms of inhibitory activity against BChE, all derivatives showed IC50 values higher
6

than those obtained for AChE inhibition. Therefore, as also seen with lead compound
, all the tested compounds 11a–11n acted as selective AChE inhibitors, with selectivity
1
index ranging from 1.10–31.75. Similarly, all Para-substituted derivatives in the 4-N-
phenyl ring were clearly less potent against BChE than Meta-substituted or Ortho-
substituted ones, however, polysubstitution (such as compound 11e) seemed to be very
detrimental to their activities. Notably, compound 11b, as the most active BChE
inhibitor, was 5-fold more potent than compound 1, and 9-fold more potent than the
reference drug galanthamine.
Table 1
Inhibition of cholinesterases activity and selectivity index (SI) of compounds.
a
BChE IC ( μM)a
SIb
Compound
1
R
AChE IC ( μM)
5
0
50
0.92 ± 0.05c
0.65 ± 0.01
0.86 ± 0.03
0.83 ± 0.02
1.88 ± 0.04
1.85 ± 0.09
10.13 ± 1.29
1.86 ± 0.01
11.84 ± 1.65
7.90 ± 1.64
1.72 ± 0.01
10.87 ± 1.44
0.71 ± 0.05
0.74 ± 0.01
7.78 ± 0.74
1.28 ± 0.01
14.20 ± 0.96c
10.11 ± 1.76
2.65 ± 0.52
15.17 ± 2.23
17.45 ± 2.77
58.74 ± 5.15
29.71 ± 1.13
2.78 ± 0.67
73.59 ± 4.57
8.67 ± 0.45
9.19 ± 0.36
50.37 ± 1.82
10.77 ± 1.25
17.38 ± 2.49
55.41 ± 3.46
24.41 ± 2.01
15.43
15.55
3.08
18.27
9.28
31.75
2.93
1.49
6.22
1.10
5.34
4.63
15.16
23.48
7.12
19.07
1
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
Galanthamine
H
1
2-Cl
3-Cl
4-Cl
2,4-di-Cl
1
1
1
1
2-CH
3-CH
4-CH
3
3
3
1
1
1
2-OCH
3-OCH
4-OCH
3-OH
3
3
3
1
1
1
1
4-OH
1
3-CF
3
a
b
c
IC50 values are at least from three independent experiments and are expressed as the means ± SD.
SI for AChE = IC50 BChE/IC50 AChE.
Data from Ref. 25.
Even though a somewhat lower AChE inhibitory activity compared with the most
potent AChE inhibitor 11a, compound 11b was endowed with its highest BChE
inhibitory potency. To clarify the interaction mode in the active sites of AChE,
molecular docking simulations (Supplementary material) were performed for
compounds 11b using the CDOCKER in Discovery Studio 3.0 software (Fig. 2 and Fig.
7

3
). The crystal complex of AChE with galanthamine (PDB: 1DX6) was selected for the
docking study. N-methylbenzylamine fragment of compound 11b interacted with the
CAS through π-π-stacked interaction with Trp84. Additionally, compound 11b
displayed a π-alkyl interaction between the 4-N-phenyl ring and the sec-butyl moiety
of Ile287, which was adjacent to acyl binding site Phe288. Furthermore, compound 11b
was involved in a series of interactions with the majority of amino acid residues in the
PAS: ortho-chloro group at the 4-N-phenyl ring established a π-alkyl interaction with
Trp279 and an alkyl interaction with Leu282; nitro group formed a π-cation and a π-
anion interactions both with Trp279; quinoline moiety made a carbon hydrogen bond
with Tyr70 and three π-π-stacked interactions with Trp279 and Tyr121; and the propyl
ether fragment attached to quinoline skeleton showed a carbon hydrogen bond with
Asp72. This is known to be an important feature for the inhibition of AChE-induced
Aβ aggregation.34
Fig. 2. 3D binding mode of compound 11b with AChE.
8

Fig. 3. 2D diagram of the ligand-protein interaction for compound 11b with AChE.
The interaction of compound 11b with BChE (PDB code: 4BDS) was also carried
out (Supplementary material). The modeling results (Fig. 4 and Fig. 5) suggested that
compound 11b was bound to the residues Trp82 from the choline binding site, Gly117
from the oxyanion hole, Phe329 from the acyl pocket, and His438 from catalytic triad,
but also bound to Ser287 residue and two PAS amino acid residues such as Asp70 and
Pro285, via one hydrogen bond, three charge interactions, and eight hydrophobic
interactions, which interacted with all five regions of BChE active site.35,36 Significantly,
the presence of ortho-chloro group at the 4-N-phenyl ring was important for BChE
inhibition, as the chlorine atom was stabilized by the formation of two π–alkyl
interactions both with the electron density of the imidazole ring of His438 and the
benzene ring of Trp82. Instead, the lower inhibitory potency of compounds 11c and 11d
compared with compound 11b may be ascribed to less favorable specific interactions
in the binding site, which separately contain Meta-chloro group or Para-chloro group
at the 4-N-phenyl ring.
9

Fig. 4. 3D binding mode of compound 11b with BChE.
Fig. 5. 2D diagram of the ligand-protein interaction for compound 11b with BChE.
As a potential compound for treatment of AD, lipophilicity (logP) was thought as
an important physical chemistry parameter to valuate or predict the ability to cross
blood brain barrier (BBB). It was reported that the logP with appropriate central
3
7
nervous system (CNS) penetration was within the range of 2–5. After calculated using
ChemDraw Ultra 11.0, the logP value of active compound 11b was 4.58, which
1
0

indicated that the active compounds had sufficient lipophilicity to pass the BBB in vivo.
Polar surface area (PSA) is another important predictor for BBB. Generally, drugs
2
37
aimed at the CNS tends to have lower PSAthan 95 Å . After assessed using Discovery
Studio 2017 R2, the PSA value for compound 11b was 88.107, which also supported
its ability to penetrate the BBB. As p-glycoprotein is also closely related to BBB
penetration, it is now underway in our laboratory to evaluate whether compound 11b is
a substrate or an inhibitor of P-glycoprotein.
In summary, a new series of 4-N-phenylaminoquinoline derivatives was designed,
synthesized, and evaluated in vitro for their ability to inhibit AChE and BChE.
Biological assays demonstrated that compound 11b is a dual AChE and BChE inhibitor
with IC50 values (0.86 and 2.65 μM), which are a lot better than that of the commercially
available drug galanthamine. Molecular modelling studies suggested not only the
ability of the N-methylbenzylamine fragment of 11b to establish interactions with the
CAS residue Trp84 but also the ability to establish interactions with the majority of
amino acid residues in the PAS, leading to the speculation of a possible reduction of
AChE-induced Aβ aggregation. On the other hand, as treatment with a selective AChE
inhibitor reduced AChE activity, BChE could compensate for the lack of AChE in the
late stages of AD. So more importantly, compound 11b, being also highly active on
BChE, could shift to inhibiting this enzyme during the progression of the disease.
Finally, the comprehensive analysis of SARs indicated that compound 11g also showed
rather better activities simultaneously on both AChE and BChE than the rest. Further
studies on structural optimization and Aβ aggregation inhibition about these derivatives
are still in progress.
Acknowledgment
This work was supported by the Training Project of Innovation Team of Colleges
and Universities in Tianjin (TD13-5020).
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1
3

eel), BChE (EC 3.1.1.8, from equine serum), Acetylthiocholine iodide, butyrylthiocholine
iodide, and 5,5'-dithiobis-(2-nitrobenzoic)acid (DNTB) were purchased from Sigma-Aldrich.
To determine the percent of inhibition, 20 μl of the test compound, 20 μl of enzyme solution
(0.05 U/ml) and 10 μl of DTNB 10 mM were mixed in 140 μl sodium phosphate buffer 0.1
M (pH 8). After 15 min of incubation at 25°C, 20 μl substrate (7.5 mM) was added. Afterward,
absorption was measured using BioTek Epoch microplate reader at 412 nm. Each
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