Discovery of methoxy-naphthyl linked N-(1-benzylpiperidine) benzamide as a blood-brain permeable dual inhibitor of acetylcholinesterase and butyrylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2020.112761

The cholinesterase enzymes play a vital role in maintaining balanced levels of the neurotransmitter acetylcholine in the central nervous system. However, the overexpression of these enzymes results in hampered neurotransmission. Both the major forms of cholinesterase enzymes viz. acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) play a crucial role in blocking neurotransmission; therefore, in recent years, a strategy of dual cholinesterase inhibition is being explored. Herein, we developed an energy-optimized e-pharmacophore hypothesis AHHPRR from AChE-donepezil complex and screened a set of 15 scaffolds that were designed imaginarily. The ligand with N-(1-benzylpyridinium) benzamide framework has shown the highest fitness and volume score, which was chosen for synthesis and validation. A series of pyridinium benzamides were synthesized and screened for cholinesterase inhibition that led to the identification of 7b, a naphthalene containing N-(1-benzylpiperidine) benzamide as a potent dual AChE and BChE inhibitor with IC₅₀ values of 0.176, and 0.47 μM, respectively. The kinetic study indicated that 7b inhibits AChE in a non-competitive manner with Ki value of 0.21 μM, and BChE in a mixed-fashion with Ki of 0.15 μM. The observed mode of inhibition was corroborated with molecular docking studies. The MD simulation studies pointed out that both AChE and BChE undergo low conformational changes in complex with 7b. The benzamide 7b displayed high BBB permeability in PAMPA assay, which indicates its potential for further exploration in preclinical studies for Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2020.112761

European Journal of Medicinal Chemistry 207 (2020) 112761
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of methoxy-naphthyl linked N-(1-benzylpiperidine)
benzamide as a blood-brain permeable dual inhibitor of
acetylcholinesterase and butyrylcholinesterase
,
,
, b, *
Mohd Abdullaha a b, Vijay K. Nuthakki a b, Sandip B. Bharate a
a Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, 180001, India
b Academy of Scientific & Innovative Research, Ghaziabad, 201002, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The cholinesterase enzymes play a vital role in maintaining balanced levels of the neurotransmitter
acetylcholine in the central nervous system. However, the overexpression of these enzymes results in
hampered neurotransmission. Both the major forms of cholinesterase enzymes viz. acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) play a crucial role in blocking neurotransmission; therefore, in
recent years, a strategy of dual cholinesterase inhibition is being explored. Herein, we developed an
energy-optimized e-pharmacophore hypothesis AHHPRR from AChE-donepezil complex and screened a
set of 15 scaffolds that were designed imaginarily. The ligand with N-(1-benzylpyridinium) benzamide
framework has shown the highest fitness and volume score, which was chosen for synthesis and vali-
dation. A series of pyridinium benzamides were synthesized and screened for cholinesterase inhibition
that led to the identification of 7b, a naphthalene containing N-(1-benzylpiperidine) benzamide as a
Received 2 August 2020
Received in revised form
15 August 2020
Accepted 15 August 2020
Available online 28 August 2020
Keywords:
Alzheimer’s disease
e-pharmacophore modeling
1-Benzylpiperidine benzamide
Acetylcholinesterase
Butyrylcholinesterase
potent dual AChE and BChE inhibitor with IC50 values of 0.176, and 0.47
study indicated that 7b inhibits AChE in a non-competitive manner with Ki value of 0.21
a mixed-fashion with Ki of 0.15 M. The observed mode of inhibition was corroborated with molecular
m
M, respectively. The kinetic
m
M, and BChE in
m
docking studies. The MD simulation studies pointed out that both AChE and BChE undergo low
conformational changes in complex with 7b. The benzamide 7b displayed high BBB permeability in
PAMPA assay, which indicates its potential for further exploration in preclinical studies for Alzheimer’s
disease.
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
cholinesterases. The human brain contains two types of cholines-
terase enzymes viz. acetylcholinesterase (AChE) and butyr-
The prevalence of Alzheimer’s disease (AD) is rapidly increasing
throughout the world, and has already affected more than 50
million people [1]. The cholinergic deficit is the dominant patho-
logical symptom in the brains of AD patients, resulting in problems
of memory, thinking, and behaviour [2,3]. The hydrolytic cleavage
of acetylcholine by cholinesterase enzymes results in a cognitive
impairment and memory deficits, which is associated with AD
[4,5]. Therefore, the current treatment of AD (donepezil, galant-
amine, and rivastigmine) predominantly involves the up-regulation
of the cholinergic system in the synaptic cleft by inhibiting
ylcholinesterase (BChE) that share 65% of the amino acid sequence,
and both possessing a deep hydrophobic active site gorge of 20 Å
[6]. The difference in their structural features contributes to the
substrate specificity. The substrate specificity difference is due to
the variations in the amino acid sequence in the active site gorge
[7e9]. The active site gorge of AChE is further divided into two sub-
sites called peripheral anionic site (PAS) and catalytic anionic site
(CAS) [10,11]. The PAS is located at the entrance of active-site gorge
that allosterically regulates the functions of AChE, and studies have
shown that PAS is involved in Ab aggregation [12,13]. Butyr-
ylcholinesterase (BChE), which is also called as pseudo-
cholinesterase, is mainly found in the blood plasma and in a
small quantity in the brain [14]. In the brain, BChE is predominantly
present in the glial cells [15], and is involved in the cholinergic
mediation [16]. With the advancement in the AD biology, the
* Corresponding author. Medicinal Chemistry Division, CSIR-Indian Institute of
Integrative Medicine, Canal Road, Jammu, 180001, India.
E-mail addresses: sbharate@iiim.res.in, sandipbharate@gmail.com (S.B. Bharate).
https://doi.org/10.1016/j.ejmech.2020.112761
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

2
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
Fig. 1. (A) E-pharmacophore screening of designed compounds; (B) Interaction of donepezil and scaffold c with the active site of AChE.
increased activity of BChE has been detected in the hippocampus
and temporal cortex parts of brain compared to its counterpart
AChE, whose concentration is decreased in the specified regions
[17,18]. Moreover, the activity of BChE is significantly increased
with the age in AD brain, but no such correlation is observed in
AChE enzyme [18]. Many studies have demonstrated that the in-
hibition of BChE is correlated with the improved memory in AD
patients [17,19,20]. Initially, the inhibition of AChE was mainly
taken into consideration to treat AD, however now several studies
have shown the benefits of inhibiting both AChE and BChE paral-
lelly in the treatment of AD [8]. Given the substantial involvement
of BChE in the cholinergic regulation, and ultimately in the pro-
gression of AD, the dual inhibition is likely to provide additional
advantage particularly in the long run [21,22]. Therefore, many
potential dual inhibitors have been developed over the past decade
[23e32].
Although numerous cholinesterase inhibitors are reported in
the literature; however, the search for newer drug-like BBB-
permeable scaffolds is a continuing process. The pharmacophore
modeling is one of the tools commonly employed in designing
potential drug-like inhibitors in drug discovery. Here, we applied
an e-pharmacophore approach for design and the scaffold with
highest fitness, volume, and dock scores was taken for synthesis,
validation, and further lead optimization. Thus, we report the dis-
covery of a naphthyl-linked piperidinyl benzamide as a dual
cholinesterase inhibitor that has high BBB permeability.
2. Results and discussion
e-Pharmacophore based design. The e-pharmacophore based
design approach has provided numerous lead structures in various
therapeutic areas [33,34]. As our objective was to identify a new
scaffold for dual inhibition of AChE and BChE inhibition, here we
employed an e-pharmacophore based design approach. Donepezil
was chosen for e-pharmacophore creation as it is an FDA approved
drug, and along with AChE inhibition, it also possesses a moderate
level of BChE inhibition (IC50 ¼ 5.5
mM). The e-pharmacophore
hypothesis AHHPRR was developed from the AChE-donepezil
complex. The hypothesis is composed of one acceptor group (A),
two hydrophobic groups (H), one positively charged group (P), and
two aromatic rings (R) features. The hypothesis was validated using
a database containing active and inactive set of ligands and was
able to successfully differentiate active and inactive ligands. Then,
the library of 15 scaffolds designed imaginarily was subjected to
screening using this hypothesis. The scaffold c showed good fitness
and volume score. The scaffold c was found to possess three fea-
tures one acceptor and two rings (ARR) with fitness score and
volume scores of 1.81, and 0.68. Scaffold c was found to superim-
pose with donepezil sharing various common interactions with the
AChE. The phenyl moiety was found oriented towards the PAS re-
gion showing p-p stacking with TRP 286. The carbonyl group dis-
played H-bonding with TYR 124 and ARG 224 residues while the
amine functionality displayed H-bonding interaction with TYR 337,
Furthermore, the poorefficacyof single-targeted inhibitors invivo
has developed a pressing need towards development of multi-target-
directed ligands (MTDLs) as potential therapeutics of AD.
and PHE 338 residues. Further, the nitrogen of pyridine formed a
cation interaction with TYR 337 and also stacked with TRP 86. The
benzyl ring was oriented towards CAS region showing stacking
p-
p-p

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
3
Fig. 2. Synthesis of pyridinium benzamides 4a-w. Reagents and conditions: (a) oxalyl chloride, Dry THF, N2, 0 ꢀC, 2 h, >90%; (b) 4-aminopyridine, dry THF, 0 ꢀC, 10 min, 75e90%; (c)
ACN, benzyl bromide, 80 ꢀC, 1 h, 87e95%.
with TRP 86. Based on the fitness, volume, and dock scores, the
scaffold c was selected for its synthesis and further optimization.
The e-pharmacophore based screening to identify a potential
scaffold for cholinesterase inhibition is depicted in Fig. 1.
However at 1 mM, seven compounds 4a, 4b, 4k, 4m, 4n, 4q 4w
showed >70% inhibition of AChE. In the BChE inhibition assay,
comparatively lesser inhibition was observed, with only two com-
pounds showing significant inhibition at 1
4w, which bears a naphthyl ring (ring A), substantially inhibited
BChE at 10 M and >35% at 1 M. Other compounds that showed
mM. Compounds 4g and
Synthesis and biological evaluation of benzamides 4a-w. The
synthesis of pyridinium benzamides was started from commer-
cially available aryl carboxylic acids 1a-w. The treatment of aryl
carboxylic acids 1a-w with oxalyl chloride yielded corresponding
acid chlorides 2a-w in quantitative yields. The reaction of acid
chlorides 2a-w with 4-aminopyridine produced pyridinyl benza-
mides 3a-w in 75-90% yield. Finally, refluxing the pyridine benza-
mides 3a-w in the presence of benzyl bromide in acetonitrile
resulted in the formation of pyridinium compounds 4a-w in
87e95% yield. In this step, the product was obtained as a precipitate
in the reaction mixture, in most of the cases. The obtained products
were characterized by using NMR, mass, IR, and melting point
analysis. The synthetic scheme for the preparation of products 4a-
w is depicted in Fig. 2.
m
m
>40% inhibition of BChE include 4e, 4j, 4p, 4q, and 4t. It was
interesting to note that the compounds bearing electron-
withdrawing groups on the ring A, generally do not inhibit BChE
(compounds 4h, 4i, 4k, 4n, 4o, 4s) or show only marginal inhibition
(compounds 4c, 4f, 4l). The significant difference in the BChE in-
hibition activity of compound 4g, 4w, and all other compounds
could be because of the presence of bicyclic ring in 4g and 4w. The
results of in-vitro inhibition of AChE and BChE by compounds 4a-w
are displayed in Table 1.
The IC50 values of best 7 compounds 4a, 4b, 4e, 4g, 4m, 4n, 4p,
and 4w was then determined for AChE inhibition, and results are
shown in Table 2. All 7 compounds displayed IC50 in the range of
The pyridinium benzamides 4a-w were tested for inhibition of
0.2e1.4
showing IC50 values of 0.2e0.22
inhibitor (compound 4p) and two most potent BChE inhibitors
m
M. The most active compounds were 4b, 4m, 4p, and 4w,
AChE and BChE at 1 and 10
mM using a modified Ellman assay [35].
mM. One of the most potent AChE
All compounds displayed >90% inhibition of AChE at 10
m
M.

4
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
Table 1
and pyridine ring (series B). Initially, the naphthyl portion was kept
constant, and modifications were imparted at other parts of the
structure. In synthesis, the first step in all pyridinium based
schemes involved activating the aromatic acids with EDC followed
by coupling with amines in dry DCM at room temperature. Finally,
refluxing pyridine amide intermediates with different benzyl ha-
lides/alkyl halides in ACN at 80 ꢀC yielded corresponding pyr-
idinium benzamides 4x-ap in 80e95% yield (Fig. 3AeB). To probe
the role of the positive charge on nitrogen, the pyridine ring was
replaced with a piperidine ring (7a-b, Fig. 3C). Further, the naph-
thalene ring was also replaced with different biphenyls such as
phenoxy phenyl, biphenyl, benzoyloxy phenyl, phenoxy benzyl
(4aq-at, Fig. 3DeF) to explore the role of naphthalene that in-
creases the potency against BChE. Furthermore, to check the role of
benzyl ring in the activity, it was replaced with ethyl moiety (4au,
Fig. 3G). A total of 26 new derivatives were further prepared as
depicted in Fig. 3.
In-vitro inhibition of AChE and BChE by benzamides 4a-w.
Entry
% Inhibition SEM
AChEa
BChEb
10
m
M
1
m
M
10
m
M
1 mM
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
91.45 0.31
96.93 0.39
91.36 0.94
93.21 0.71
95.70 0.34
92.06 0.13
99.29 0.10
96.55 0.51
94.90 0.21
96.15 0.36
96.27 0.11
96.05 0.46
94.95 0.16
94.42 0.4
90.13 0.51
92.39 0.36
97.08 0.39
93.96 0.05
92.01 0.21
92.14 0.20
90.41 0.32
96.93 0.39
96.36 0.03
99.42 1.58
72.92 0.24
86.82 0.42
60.8 0.14
nac
nac
nac
nac
nac
nac
nac
31.98 1.44
13.16 2.34
30.82 1.4
48.48 2.22
22.73 1.57
86.83 1.53
nac
61.01 0.93
67.87 0.92
63.31 1.00
49.91 1.15
62.45 1.28
36.85 0.46
68.17 1.34
70.12 0.99
51.45 1.84
74.92 0.24
78.71 1.72
55.8 0.70
62.36 0.92
86.09 0.41
66.67 1.66
57.67 0.83
58.94 0.84
58.29 1.75
86.82 0.42
92.36 0.94
98.36 1.52
35.22 1.92
nac
nac
nac
71.48 0.70
nac
nac
nac
7.64 0.23
6.65 0.67
nac
nac
nac
nac
All compounds from Fig. 3 were tested at 1 mM against AChE and
nac
nac
40.45 0.13
42.30 0.39
7.40 0.26
nac
47.94 0.28
8.57 0.28
31.98 1.44
88.83 1.59
67.85 2.66
nac
BChE. In the 6-methoxy-N-(pyridin-4-yl)-2-naphthamide series
(Table 3), it was observed that the position of substituent on the
pyridyl linked benzyl moiety has a significant effect on AChE as well
as BChE inhibition. As a general trend, the 2-substituted derivatives
(e.g., 4x, 4z, 4ac, 4af) were superior over other substitutions. The
comparison of this series of compounds with the parent lead 4w,
has indicated that only derivatives bearing substitution at ortho-
position in benzyl ring have displayed similar level of AChE inhi-
bition activity to that of 4w. However, in case of BChE inhibition, all
ortho-substituents do not display desirable level of BChE inhibition,
with only 2-halo substituted compounds (e.g. 4z, 4ac) possess same
level of BChE inhibition. The replacement of benzyl moiety with
alkyl (e.g., 4au) has resulted in drastic reduction in cholinesterase
inhibition activity. This indicated that the benzyl moiety is essential
for interaction at the catalytic anionic site of the AChE. Though few
compounds from this series (Table 3) displayed similar level of
activity profile to that of 4w, but none displayed superior activity.
Another series of compounds ‘6-methoxy-N-(pyridin-4-yl-
alkyl)-2-naphthamide 4 ag-ap’ (Table 4) was superior over 4x-4af
series (Table 3) with respect to dual inhibition. In this series, a
“small alkyl linker” is placed between pyridyl moiety and amide
linked naphthyl unit. This extra linker positively impacted the
cholinesterase inhibition activity. The ethylene linker (4ah) was
significantly superior to methylene linker (4 ag). Among the
ethylene linker containing compounds, the substitution on N-
benzyl was varied, where the 2-substitution was superior over
other positions. Though there was somewhat low AChE inhibition
in this series, however several compounds (4ah, 4aj, 4ao) have
displayed superior inhibition of BChE than benzamide 4w. This
series has provided critical information that placing a small
eCH2CH2- linker between naphthylamide and pyridine ring im-
parts significant positive impact on potency. This additional spacer
might be probably helping the compound to occupy the active site
gorge perfectly, and helping it to interact with all key residues of
both the active sites.
nac
nac
4s
4t
nac
nac
4u
4v
4w
Donepezil
nac
nac
36.88 1.29
24.71 2.29
a
Percentage inhibition (mean SEM of three experiments) of AChE from Elec-
trophorus electricus.
b
Percentage inhibition (mean SEM of three experiments) of BChE from equine
serum.
c
Not active.
Table 2
IC50 values for inhibition of AChE, and BChE by selected compounds.
Entry
IC50
(m
M
SEM)a
AChE
BChE
4a
4b
4e
4g
4m
4n
4p
4w
0.420 0.022
0.202 0.01
0.852 0.171
1.442 0.026
0.216 0.018
0.469 0.074
0.201 0.006
0.218 0.011
0.049 0.001
nd
nd
nd
nd
nd
nd
12.65 0.384
1.591 0.021
5.520 1.05
Donepezil
nd is Not determined.
a
The 50% inhibitory concentration of EeAChE, or eqBChE.
(compounds 4p, 4w) were then selected for IC50 determination
against BChE. These two compounds have shown inhibition of BChE
with IC50 values of 12.65, and 1.59
promising compound from this series was the naphthyl ring con-
taining compound 4w that was found to be a dual inhibitor of AChE
and BChE. The compound 4w was ~3 fold more potent BChE in-
hibitor than donepezil.
Lead optimization studies for benzamide 4w. The significant
difference in the BChE inhibition activity of compound 4g, 4w, and
all other compounds, could be attributed to the presence of bicyclic
ring in 4w and 4g. This initial screen provided us a naphthyl linked
benzamide having an ability to inhibit both cholinesterases; how-
ever, the BChE inhibition activity requires further improvement. As
depicted in Fig. 3, the compound 4w was further optimized by
substituting different electron-withdrawing and electron-donating
groups on N-benzyl moiety (series A) and also incorporating a
methyl linker (4 ag) and an ethyl linker (4ah-4ap) between amide
mM, respectively. The most
Next, when the pyridinium moiety of the first-stage lead com-
pound 4w was replaced with piperidine (compound 7a), the
cholinesterase inhibition activity was completely lost (7.7 and 1.8%
inhibition at 1 mM) (Table 5). However, interestingly with simply an
introduction of a ethylene linker between piperidine ring and
naphthyl amide moiety (compound 7b, Table 5) has resulted in the
gain of activity against both cholinesterases. In fact, compound 7b,
though it does not bear quaternary nitrogen, still displayed the
same level of AChE inhibition, and superior BChE inhibition
compared to 4w. Finally, we also explored the possibility of
replacing the naphthyl moiety from 4w with other biphenyl rings.
The replacement of naphthyl with phenoxy-phenyl, biphenyl,

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
5
Fig. 3. Lead optimization studies for naphthyl linked benzamide derivative 4w. Synthesis of naphthyl substituted pyridinium benzamides 4x-ap, 7a-b, 4aq-at, 4au. Reagents and
conditions: (a) EDC.HCl, Et3N, Dry DCM, N2, rt, 3 h, 77-91%; (b) ACN, substituted benzyl halides/alkyl halides, 80 ꢀC, 1 h, 80e95%.
benzoyloxy-phenyl, phenoxy-benzyl (4aq-at) has resulted in loss of
activity (Table 6), indicating that naphthalene ring is essential for
dual cholinetserase inhibition.
The compounds showing inhibition >50% against AChE and
BChE were subjected for IC50 determination (Table 7). Compounds
4ah, 4aj, and 7b were found to possess dual cholinesterase inhi-
The BBB permeability is the most vital parameter in CNS drug
discovery. Therefore, two best compounds from Table 7 were
selected for the BBB permeability analysis. A parallel artificial
membrane permeation assay for BBB (PAMPA-BBB) was used to
screen the permeability potential of 4ah, and 7b. The assay was
validated by comparing the experimental and reported perme-
ability values of three standards, including donepezil with higher
permeability, carbamazepine having moderate permeability, and
theophylline with low permeability. The Pe value of
>3.3 ꢁ 10ꢂ6 cm/s is considered as BBB permeable [36,37]. Thus, the
bition activity with IC50 values less than 1
Though compounds 4ac, 4ad, and 4am displayed potent inhibition
of AChE (IC50 < 0.3 M); however, their IC50 for BChE inhibition was
>1 M; therefore, these compounds were not investigated further.
mM for both enzymes.
m
m

6
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
Table 3
In-vitro inhibition of AChE and BChE by 6-methoxy-N-(pyridin-4-yl)-2-naphthamide series 4x-af, 4au.
Entry
% inhibition SD (@ 1 mM)
R
X
AChE
BChE
4w
4x
4y
4z
4aa
4ab
4ac
4ad
4ae
4af
-Ph
Br
Br
Cl
Cl
Cl
Cl
Br
Cl
Br
Br
I
92.36 0.94
88.59 0.79
48.63 0.79
91.67 0.36
18.05 4.63
67.30 0.69
90.68 0.66
91.46 0.49
78.04 4.22
49.27 2.7
36.88 1.29
20.97 0.93
7.94 0.49
35.21 0.49
13.39 8.56
4.27 1.39
33.24 1.19
35.56 8.86
10.85 6.35
8.470 0.43
22.39 1.64
24.71 2.29
-Ph (2-Me)
-Ph (4-OMe)
-Ph (2-Cl)
-Ph (3-Cl)
-Ph (4-Cl)
-Ph (2-F)
-Ph (4-F)
-Ph (2-NO2)
-Ph (4-NO2)
-CH3
4au
Donepezil
43.85 9.97
98.36 1.52
_
_
compound 7b with permeability (Pe) value of 6.9 ꢁ 10ꢂ6 cm/s falls
in the category of high BBB permeability. However, the other
pyridinium-based compound 4ah has low Pe value (1.3 ꢁ 10ꢂ6 cm/
s), indicating its low BBB permeability. Thus, compound 7b, pos-
sessing dual cholinesterase inhibition with IC50 values less than
of the X-axis intercept is the Ki. From the graph, Ki was found to be
0.21 M for hAChE. The LB plot constructed for eqBChE has shown
that with an increase in the inhibitor concentration has led to a
decrease in the Vmax and an increase in Km value, indicating that the
compound 7b is a mixed inhibitor of BChE. The replot of slopes of
the LB plot against inhibitor concentration has been constructed to
m
0.5
mM (for both cholinesterases) and ability to cross BBB, was
chosen for further studies. The compound 7b was also checked for
its inhibitory activity against human recombinant human AChE and
determine the Ki, which was found to be 0.15
To further investigate the multi-targeted nature of the identified
dual AChE/BChE inhibitor, its ability to inhibit A -aggregation was
studied. Following thioflavin-T based fluorometric assay, the
inhibitory activity of 7b was determined against self-induced A 42
aggregation. Compound 7b has shown A 42 anti-aggregating ac-
tivity of 25% at 10 M.
mM for eqBChE.
was found to have an IC50 value of 0.17
mM.
b
Enzyme kinetic analysis for dual cholinetserase inhibitor 7b.
In order to determine the type of hAChE and eqBChE inhibition by
compound 7b, we performed kinetic analyses using different con-
centrations of substrate (0.06 mMe1 mM) and inhibitor
b
b
m
(0.015
m
Me0.5
m
M) and evaluated the results using Lineweaver-
Molecular docking and MD simulation of 7b with AChE and
BChE. To further understand the observed non-competitive and
mixed-type of inhibition, the docking studies were performed for
7b with human AChE (PDB: 4EY7) [38] and human BChE (PDB:
6EP4) [39] using Glide module of the Schrodinger molecular
modeling software. The study demonstrated that the compound 7b
accommodates in the active site gorge interacting with the amino
acid residues of both PAS and CAS of both enzymes (Fig. 5AeD). The
naphthalene moiety of 7b orients towards the peripheral site and
its N-benzyl group towards the catalytic site of the AChE active site
gorge. The close packing of the molecule inside the active site gorge
Burk double reciprocal (LB) plot (Fig. 4). LB plot was generated by
plotting the reciprocal enzyme velocity (1/V) against reciprocal of
substrate concentration (1/[S]) and was used to determine the
mode of inhibition of the enzyme by the compound 7b. From the
graph, it was found that the increase in the inhibitor concentration
results in a decrease in the Vmax leaving the Km value constant,
indicating that the compound 7b is a non-competitive inhibitor of
AChE. Further, the slopes of the lines of the LB plot were plotted
against the inhibitor concentration to determine the inhibition rate
constant (Ki) for the enzyme-inhibitor complex, in which the value
Table 4
In-vitro inhibition of AChE and BChE by 6-methoxy-N-(pyridin-4-yl-alkyl)-2-naphthamides 4 ag-ap.
Entry
% inhibition SD (@ 1 mM)
n
R
X
AChE
BChE
4w
4ag
4ah
4ai
4aj
4ak
4al
4am
4an
4ao
0
1
2
2
2
2
2
2
2
2
2
_
H
H
H
Br
Br
Br
Br
Cl
Cl
Cl
Br
Cl
Br
Br
_
92.36 0.94
48.33 2.78
78.93 1.37
61 1.41
78.69 1.28
69.89 1.08
2.42 8.07
77.72 0.23
66.35 4.25
46.58 1.00
6.33 7.68
98.36 1.52
36.88 1.29
2.41 1.31
53.25 1.55
25.80 0.493
54.17 2.12
33.40 2.00
19.35 2.13
29.74 2.51
20.44 1.58
47.75 2.15
14.01 4.32
24.71 2.29
2-Me
2-Cl
3-Cl
4-Cl
2-F
4-F
2-NO2
4-NO2
_
4ap
Donepezil

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
7
Table 5
In-vitro inhibition of AChE and BChE by N-(1-benzylpiperidin-4-yl)-6-methoxy-2-naphthamides 7a-b.
Entry
% inhibition SD (@ 1 mM)
n
AChE
BChE
4w
7a
7b
P ¼ pyridinium, n ¼ 0
92.36 0.94
7.70 5.59
80.02 1.10
98.36 1.52
36.88 1.29
1.77 1.21
60.98 0.31
24.71 2.29
0
2
_
Donepezil
Table 6
H-bonding with Asp 74, which is present at the lining of the gorge.
The strong interactions of 7b with PAS could be attributed to its
non-competitive mode of inhibition as observed in kinetic studies.
Further, the docking investigation was also carried of 7b with BChE.
The NH of amide moiety was found engaged in H-bonding inter-
In-vitro inhibition of AChE and BChE by phenoxy phenyl, biphenyl, benzoyloxy
phenyl, phenoxy benzyl substituted benzamides 4aq-at.
Entry
% inhibition SD (@ 1 mM)
action with Asp 70, and nitrogen of piperidine display a cation-
p
interaction with Trp 82, and benzyl attains a position to stack with
the Trp 82. The surface view (Fig. 5C) of BChE with 7b depicts that
the molecule bends in a U-shaped format because of the available
space within the cavity. The interaction of 7b with PAS in both cases
corroborates with the kinetic analysis results.
The MD simulation studies were carried out for the most potent
compound 7b of the series with the targeted enzymes (AChE and
BChE) over a period of 10 ns (Fig. 6). In the course of this study,
R
n
AChE
BChE
4aq
4ar
4as
4at
-OBn
-OPh
-OBn
-Ph
0
0
1
0
_
87.56 0.51 12.74 1.83
34.21 0.99 23.19 5.06
6.16 3.10
60.07 1.14 33.29 1.35
98.36 1.52 24.71 2.29
7.94 0.49
Donepezil
_
RMSD of the protein backbone C-a atoms and individual inhibitor
was recorded that was found to be in a range between 2 and 2.5,
reflecting the stability of the protein-ligand complex. Further, the
RMSF in the individual amino acid side chain was below 3 Å
signifying conformational changes to a low degree in the protein
during entire MD simulations. The reliability of MD simulation was
also advocated by the constant temperature, pressure, volume, and
potential energy during the whole course of time. The final frame
analysis has indicated that 7b occupies both the CAS and PAS re-
gions of AChE during MD simulation time. In the PAS region, the
contact between Trp 286 and naphthalene moiety was retained for
>80% of the total simulation time. At the same time, the Phe 295
reproduced the H-bonding interaction with the carbonyl of the
amide that was conserved throughout the simulation time. Further,
the H-bonding interaction between Asp 74 and amine of piperidine
was retained during the entire course of simulation time. Trp 86
Table 7
IC50 values for inhibition of EeAChE and eqBChE by selected naphthyl series of
compounds.
Entry
IC50
(
m
M) SD
AChE
BChE
4x
4z
0.435 0.09
0.34 0.04
0.9 0.03
>1
>1
>1
>1
>1
>1
4 ab
4ac
4ad
4ae
4ah
4ai
0.21 0.00
0.21 0.00
0.48 0.01
0.77 0.07
1.08 0.06
0.50 0.04
0.61 0.02
0.28 0.04
0.74 0.06
0.28 0.04
0.74 0.06
0.41 0.09
0.36 0.03
0.77 0.03
0.035 0.023
0.68 0.04
>1
0.83 0.04
>1
>1
>1
>1
>1
4aj
was found engaged in a
p-cation, and p-p stacking interactions
4ak
4am
4an
4ao
4ap
7b
4aq
4 at
Donepezil
with an amine of piperidine and N-benzyl moiety, respectively
which were also conserved throughout the simulation time. At the
same time, contacts between Tyr 341 and amine of piperidine
persisted for>80% of the total simulation time. Further, in the case
of BChE, the RMSD value was found below 2.5 Å, indicating the
stability of the protein-ligand complex during a simulation. BChE
also experienced a low degree of conformation as the value of RMSF
was below 1.5. In addition, the interactions of 7b with Trp 82 and
Tyr 332 of BChE were also conserved throughout the simulation
time.
0.47 0.01
>1
>1
5.25 0.23
was observed by virtue of its various types of interactions such as
H-bonding, stacking, and -cation interactions with residues of
p-p
p
active site gorge. Further, it attains a position wherein carbonyl
moiety establishes H-bonding with the Phe 295 and Arg 296 and
eNH of amide forming a H-bonding with Asp 74 at the lining of the
gorge. In the CAS region of active site gorge, the piperidine nitrogen
3. Conclusion
In summary, the new scaffold predicted via e-pharmacophore
modeling was validated experimentally via synthesizing a total of
49 compounds. This effort has resulted in the identification of a
naphthyl linked benzylpiperidine-benzamide 7b as a BBB perme-
able dual inhibitor of AChE and BChE (IC50 values of 0.17 and
has reproduced the cation-
piperidine nitrogen also display cation-
CAS. The N-benzyl ring and naphthalene moiety show
p
interaction with Tyr 337 residue. The
interaction with Trp 86 of
inter-
p
p-p
action with Trp 86, and Trp 286 residues, respectively indicating
that the compound 7b has ability to interact with both sites of the
AChE active site gorge. Additionally, the NH of amide linker show
0.47
0.1
m
M). It inhibited AChE in a non-competitive fashion (Ki value of
mM) and BChE via a mixed-type of inhibition (Ki of 0.15 M).
m

8
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
Fig. 4. Enzyme kinetics of 7b with AChE and BChE. (A) LB plot for the inhibition of hAChE by 7b (B) replot of slope vs 7b concentration to determine the Ki value for AChE inhibition.
(C) LB plot for the inhibition of eqBChE by 7b; (D) replot of slope vs 7b concentration to determine the Ki value for BChE inhibition.
Further, it is found that it is the naphthyl moiety that enhances
potency for BChE inhibition. The MD simulation studies revealed
that 7b does not induce conformational changes in both AChE and
BChE. Further, 7b also possesses high BBB permeability potential.
Because of the dual ability of 7b to inhibit both AChE and BChE
enzymes, along with ability to inhibit Ab-aggregation, and high BBB
permeability potential, the further exploration of this scaffold is
warranted in preclinical animal efficacy studies.
point data were same as described earlier [40]. The chemical shift
values in 1H and 13C spectra were reported downfield to tetrame-
thylsilane (TMS) in parts per million.
Generation of e-pharmacophore model and screening of a
scaffold library. The human AChE protein (PDB: 4EY7) was
retrieved and subjected for protein preparation under default set of
conditions using Maestro v10.2 and Impact v6.7 (Schrodinger Inc.,
New York, 2015-2). Further, the complex was minimized by OPLS
force field. Then, the grid file was constructed from the prepared
protein by selecting co-crystallized ligand (donepezil) as centroid
of cubic grid box. The donepezil was extracted from the prepared
protein-ligand complex, minimized and redocked using XP Glide
module under default setting. The PV file was used to develop the
energy optimized pharmacophore. Pharmacophore features were
4. Experimental section
General. All chemicals were procured from TCI and Sigma-
Aldrich Companies and were used as such without any further
purification. The instruments used to record 1H, 13C, IR, and melting

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
9
Fig. 5. Molecular docking of compound 7b with human AChE (PDB ID: 4EY7) and human BChE (PDB ID: 6EP4). (A) Surface view of AChE showing the active site gorge and
orientation of 7b inside the gorge; (B) Interaction of 7bwith the active site of AChE. (C) Surface view of BChE showing the active site gorge and orientation of 7b in the active site
gorge; (F) Interaction of 7b with the active site of BChE residues. The
p-p interactions are represented by dark blue dotted lines; whereas the red dotted lines indicate H-bonding
interactions. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
generated using Phase module, which uses the default set of six
chemical features containing hydrogen bond acceptor (A),
hydrogen bond donor (D), hydrophobic (H), negative ionisable (N),
positive ionisable (P) and aromatic ring (R). The advance pharma-
cophore screening panel of phase module was used to screen the
compounds against the developed hypothesis under the default set
of conditions. Out of the total six pharmacophore sites, ligands that
match at least three features were ranked according to the fitness
score.
General procedure for preparation of benzoyl chlorides 2a-w.
To the solution of benzoic acids (2.4 mmol) in anhydrous THF
(10 mL) under nitrogen atmosphere at 0 ꢀC was slowly added oxalyl
chloride (3.6 mmol). The reaction mixture was then stirred for
2e3 h under nitrogen. The reaction mixture was changed from
transparent to a slightly yellowish color. The solvent was evapo-
rated on vacuo rotavapor, and the obtained product was directly
used for the next reaction.
General procedure for the preparation of amides 3a-w from
benzoyl chlorides 2a-w. To the solution of benzoyl chlorides 2a-w
(1 equiv.) in anhydrous THF (10 mL) was added 4-aminopyridine (1
equiv.) under nitrogen atmosphere at 0 ꢀC. The mixture was stirred
for 5 min, and the reaction was monitored by TLC (Rf 0.5 in 10%
MeOH: CHCl3). After completion of the reaction, it was diluted with
methanol. The resulting solution was concentrated on vacuo rota-
vapor to get the crude product, which was purified by silica gel
(100e200) column chromatography using 5% MeOH: DCM as a
mobile phase to get amides 3a-w in 75e90% yield.
white solid; m.p. 90e92 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.63 (s,
1H), 8.38 (d, J ¼ 5.2 Hz, 2H), 7.55 (d, J ¼ 5.6 Hz, 2H), 7.39 (d, J ¼ 4.5
Hz,1H), 6.77 (d, J ¼ 8.3 Hz,1H), 3.82 (s, 3H), 3.80 (s, 3H). ESI-MS: m/z
259.0 [MþH]þ.
3,5-Dimethoxy-N-(pyridin-4-yl)benzamide (3b) [42]. Yield: 83%;
white solid; m.p.201e203 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 10.50
(s, 1H), 8.49 (d, J ¼ 6.1 Hz, 2H), 7.78 (d, J ¼ 6.2 Hz, 2H), 7.11 (d,
J ¼ 2.2 Hz, 2H), 6.76 (t, J ¼ 2.1 Hz, 1H), 3.84 (s, 6H). ESI-MS: m/z
259.0 [MþH]þ.
4-Chloro-N-(pyridin-4-yl)benzamide (3c) [43]. Yield: 85%; white
solid; m.p. 206e208 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 10.65 (s,
1H), 8.50 (d, 2H), 8.00 (d, J ¼ 8.5 Hz, 2H), 7.78 (d, J ¼ 4.3 Hz, 2H), 7.65
(d, J ¼ 8.5 Hz, 2H); ESI-MS: m/z 233.0 [MþH]þ.
N-(Pyridin-4-yl)benzamide (3d) [42,44]. Yield: 88%; white solid;
m.p. 198e200 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 10.59 (s, 1H), 8.49
(d, J ¼ 5.8 Hz, 2H), 7.97 (d, J ¼ 7.3 Hz, 2H), 7.80 (d, J ¼ 6.1 Hz, 2H),
7.64 (t, J ¼ 7.2 Hz, 1H), 7.57 (t, J ¼ 7.5 Hz, 2H). ESI-MS: m/z 199.0
[MþH]þ.
4-Isopropyl-N-(pyridin-4-yl)benzamide (3e). Yield: 83%; white
solid; m.p.109e111 ꢀC; 1H NMR (400 MHz, DMSO)
d 10.53 (s, 1H),
8.52 (d, J ¼ 6.2 Hz, 2H), 7.95 (d, J ¼ 8.3 Hz, 2H), 7.83 (dd, J ¼ 4.9,
1.4 Hz, 2H), 7.47 (d, J ¼ 8.3 Hz, 2H), 3.36 (s, 6H), 3.10e2.94 (m,
1H).ESI-MS: m/z 241.0 [MþH]þ.
3-Chloro-N-(pyridin-4-yl)benzamide (3f) [43]. Yield: 86%; white
solid; m.p. 172e174 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 10.72 (s,
1H), 8.55 (d, J ¼ 5.9 Hz, 2H), 8.07 (s, 1H), 7.97 (d, J ¼ 7.8 Hz, 1H), 7.82
(d, J ¼ 6.3 Hz, 2H),7.54 (d, J ¼ 8.0 Hz, 1H), 7.65 (t, J ¼ 7.9 Hz, 1H). ESI-
MS: m/z 233.0 [MþH]þ.
3,5-Dimethoxy-N-(pyridin-4-yl)benzamide (3a) [41]. Yield: 87%;

10
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
Fig. 6. MD simulation and interactions of compound 7b with AChE and BChE. (A) RMSD in AChE enzyme C-
MD simulation. (C) Histogram showing interactions of 7b with AChE; (D) 2D diagram for 7b interactions with AChE; (E) RMSD in BChE enzyme C-
RMSF in side chains of BChE during MD simulation; (G) Histogram showing interactions of 7b with BChE; (H) 2D diagram for 7b interactions with BChE.
a
carbon during MD simulation; (B) RMSF in side chains of AChE during
a
carbon during MD simulation; (F)
6-Bromo-N-(pyridin-4-yl)-2-naphthamide (3g). Yield: 84%; white
solid; m.p. 235e237 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
10.80 (s,
(d, J ¼ 8.3 Hz, 1H), 7.84 (d, J ¼ 4.5 Hz, 2H).ESI-MS: m/z 267.0
d
[MþH]þ.
1H), 8.62 (s, 1H), 8.52 (d, J ¼ 6.1 Hz, 2H), 8.34 (s, 1H), 8.09 (d,
J ¼ 10.8 Hz, 3H), 7.83 (d, J ¼ 6.2 Hz, 2H), 7.77 (dd, J ¼ 8.8, 1.8 Hz, 1H).
ESI-MS: m/z 327.0 [MþH]þ.
4-Methoxy-N-(pyridin-4-yl)benzamide(3m) [42].Yield: 81%;
white solid; m.p.139e141 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.43 (d,
J ¼ 4.7 Hz, 2H), 7.77 (d, J ¼ 8.8 Hz, 2H), 7.52 (d, J ¼ 5.3 Hz, 2H), 6.90
(d, J ¼ 3.4 Hz, 2H), 3.79 (s, 3H). ESI-MS: m/z 229.0 [MþH]þ.
4-Cyano-N-(pyridin-4-yl)benzamide (3n) [44]. Yield: 89%; white
3-Bromo-N-(pyridin-4-yl)benzamide (3i). Yield: 75%; white
solid; m.p.133e135 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.56 (brs, 2H),
8.19 (s, 1H), 8.02 (brs, 1H), 7.80 (d, J ¼ 7.7 Hz, 1H), 7.71 (d, J ¼ 8.0 Hz,
1H), 7.62 (d, J ¼ 5.3 Hz,1H), 7.38 (t, J ¼ 7.9 Hz,1H). ESI-MS: m/z 276.0
[MþH]þ.
solid; m.p. 203e205 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.59 (d,
J ¼ 5.5 Hz, 2H), 7.98 (d, J ¼ 8.2 Hz, 2H), 7.82 (d, J ¼ 8.1 Hz, 2H), 7.60
(d, J ¼ 5.9 Hz, 2H). ESI-MS: m/z 224.0 [MþH]þ.
2-Methoxy-N-(pyridin-4-yl)benzamide (3j) [42]. Yield: 78%;
3,4-Difluoro-N-(pyridin-4-yl)benzamide (3o).Yield: 88%; white
white solid; m.p.178e179 ꢀC; 1H NMR (400 MHz, CDCl3)
d
9.86 (s,
solid; m.p.174e176 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 10.47 (s,1H),
1H), 8.45 (d, J ¼ 6.0 Hz, 2H), 8.19 (d, J ¼ 9.3 Hz, 1H), 7.52 (d,
J ¼ 6.2 Hz, 2H), 7.46 (t, J ¼ 7.8 Hz, 1H), 7.08 (t, J ¼ 7.6 Hz, 1H), 6.98 (d,
J ¼ 8.3 Hz, 1H), 4.00 (s, 3H). ESI-MS: m/z 229.0 [MþH]þ.
8.33 (d, J ¼ 5.3 Hz, 2H), 7.91e7.86 (m, 1H), 7.71 (d, J ¼ 7.2 Hz, 1H),
7.59 (d, J ¼ 5.3 Hz, 2H), 7.52e7.45 (m, 1H). ESI-MS: m/z 235.0
[MþH]þ.
4-Nitro-N-(pyridin-4-yl)benzamide(3k) [43].Yield: 77%; white
4-Ethoxy-N-(pyridin-4-yl)benzamide (3p).Yield: 90%; white
solid; m.p. 248e250 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
10.87 (s,
solid; m.p.142e143 ꢀC; 1H NMR (400 MHz, DMSO)
d 10.44 (s, 1H),
1H), 8.50 (s, 2H), 8.37 (d, J ¼ 8.2 Hz, 2H), 8.17 (d, J ¼ 8.3 Hz, 2H), 7.77
8.50 (d, J ¼ 5.3 Hz, 2H), 8.01 (d, J ¼ 8.7 Hz, 2H), 7.83 (d, J ¼ 5.9 Hz,
2H), 7.12 (d, J ¼ 8.7 Hz, 2H), 4.21e4.12 (m, 2H),1.41 (t, J ¼ 7.0 Hz, 3H).
ESI-MS:m/z 243.0 [MþH]þ.
(s, 2H). ESI-MS: m/z 2244.0 [MþH]þ.
3,4-Dichloro-N-(pyridin-4-yl)benzamide (3l). Yield: 79%; white
solid; m.p. 208e210 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
10.78 (s,
3-Methoxy-N-(pyridin-4-yl)benzamide (3q). Yield: 87%; white
1H), 8.57 (d, J ¼ 4.0 Hz, 2H), 8.30 (s, 1H), 8.02 (d, J ¼ 8.2 Hz, 1H), 7.91
solid; 1H NMR (400 MHz, CDCl3)
d
8.56 (d, J ¼ 5.2 Hz, 2H), 7.98 (s,

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
11
1H), 7.61 (d, J ¼ 5.4 Hz, 2H), 7.43e7.40 (m, 2H), 7.12 (d, J ¼ 6.9 Hz,
1H), 3.88 (s, 3H). ESI-MS: m/z 229.0 [MþH]þ.
J ¼ 8.3 Hz, 2H), 7.00 (dd, J ¼ 8.5, 2.1 Hz, 2H), 5.08 (s, 2H), 3.69 (s, 2H).
ESI-MS: m/z 319.0 [MþH]þ.
3-Chloro-4-fluoro-N-(pyridin-4-yl)benzamide (3r). Yield: 88%;
N-(Pyridin-4-yl)-[1,10-biphenyl]-4-carboxamide (3ac). Yield: 77%;
white solid; m.p.168e170 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.56 (d,
white solid; m.p. 250e252 ꢀC; 1H NMR (400 MHz, CDCl3)
d 9.84 (s,
J ¼ 5.5 Hz, 2H), 8.07 (s, 1H), 7.97 (dd, J ¼ 6.8, 2.1 Hz, 1H), 7.80e7.77
(m, 1H), 7.59 (d, J ¼ 6.2 Hz, 2H). ESI-MS: m/z 251.0 [MþH]þ.
2,4-Dichloro-N-(pyridin-4-yl)benzamide (3s) [45]. Yield: 80%;
1H), 7.70 (d, J ¼ 4.3 Hz, 2H), 7.29 (d, J ¼ 8.1 Hz, 2H), 7.07 (d,
J ¼ 8.1 Hz, 2H), 7.03 (d, J ¼ 4.9 Hz, 2H), 6.97 (d, J ¼ 7.8 Hz, 2H), 6.72 (t,
J ¼ 7.5 Hz, 2H), 6.64 (t, J ¼ 7.2 Hz, 1H). ESI-MS: m/z 275.0 [MþH]þ.
white solid; m.p.172e174 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d
10.76
N-(1-Benzylpiperidin-4-yl)-6-methoxy-2-naphthamide
(7a).
(s, 1H), 8.33 (d, J ¼ 3.4 Hz, 2H), 7.63 (s, 1H), 7.52 (d, J ¼ 2.8 Hz, 1H),
7.50 (d, J ¼ 2.5 Hz, 2H), 7.42 (d, J ¼ 8.2 Hz, 1H). ESI-MS: m/z 267.0
[MþH]þ.
Yield 91%; white solid; m.p. 186e187 ꢀC; IR (nmax): 3303, 3061,
2924, 2852, 2799, 2758, 2355, 1626, 1602, 1536, 1502, 1482, 1453,
1265, 1215, 1114, 1028 cmꢂ1 1H NMR (400 MHz, CDCl3)
; d 8.18 (s,
4-Methyl-N-(pyridin-4-yl)benzamide (3t) [43]. Yield: 83%; white
1H), 7.81 (d, J ¼ 8.9 Hz, 1H), 7.77 (s, 2H), 7.33 (d, J ¼ 4.6 Hz, 4H), 7.20
(d, J ¼ 2.4 Hz,1H), 7.18 (d, J ¼ 2.4 Hz,1H), 7.15 (d, J ¼ 2.2 Hz,1H), 3.94
(s, 3H), 3.56 (s, 2H), 2.90 (d, J ¼ 11.8 Hz, 2H), 2.24 (t, J ¼ 10.9 Hz, 2H),
2.07 (d, J ¼ 10.5 Hz, 2H),1.68 (s,1H),1.63 (d, J ¼ 8.0 Hz, 2H); 13C NMR
solid; m.p.161e163 ꢀC; 1H NMR (400 MHz, DMSO‑d6)
d 9.65 (s, 1H),
7.63 (d, J ¼ 5.0 Hz, 2H), 7.05 (d, J ¼ 7.1 Hz, 2H), 6.95 (d, J ¼ 5.1 Hz,
2H), 6.53 (d, J ¼ 7.8 Hz, 2H), 1.56 (s, 3H). ESI-MS: m/z 213.0 [MþH]þ.
N-(Pyridin-4-yl)furan-2-carboxamide (3u) [43]. Yield: 81%;
(126 MHz, CDCl3)
d 166.95, 159.04, 136.23, 130.42, 129.74, 129.24,
white solid; m.p.158e160 ꢀC; 1H NMR (400 MHz, CDCl3)
d
10.28 (s,
128.31, 128.03, 127.24, 127.12, 124.15, 119.74, 105.66, 63.00, 61.03,
55.38, 55.01, 52.32, 51.53, 47.01, 32.25; ESI-MS: m/z 375.0 [MþH]þ.
N-(2-(1-benzylpiperidin-4-yl)ethyl)-6-methoxy-2-naphthamide
(7b).Yield: 89%; white solid; m.p. 140e142 ꢀC; IR (nmax): 3318, 3061,
3027, 2924, 2852, 2802, 2327, 1630, 1603, 1539, 1502, 1481, 1453,
1411, 1391, 1366, 1339, 1304, 1215, 1165, 1116 cmꢂ1;1H NMR
1H), 8.22 (d, J ¼ 5.8 Hz, 2H), 7.74 (s, 1H), 7.52 (d, J ¼ 6.2 Hz, 2H),7.17
(d, J ¼ 3.5 Hz, 1H), 6.50 (dd, J ¼ 3.4, 1.6 Hz, 1H). ESI-MS: m/z 189.0
[MþH]þ.
3,4,5-Trimethoxy-N-(pyridin-4-yl)benzamide (3v) [42,43]. Yield:
88%; white solid; m.p.165e167 ꢀC; 1H NMR (400 MHz, DMSO)
d
10.28 (s, 1H), 8.32 (d, J ¼ 5.2 Hz, 2H), 7.59 (d, J ¼ 5.3 Hz, 2H), 7.11 (s,
(400 MHz, CDCl3)
d
8.30 (s, 1H), 7.85 (d, J ¼ 8.5 Hz, 1H), 7.79 (d,
2H), 3.71 (s, 6H), 3.58 (s, 3H). ESI-MS: m/z 289.0 [MþH]þ.
General procedure for preparation of amides 3w-ac, 7a-b
from carboxylic acids. To the solution of carboxylic acid (1w-1ac, 1
equiv.) in anhydrous DCM (10 mL) was added EDC.HCl (1.5 equiv.)
and TEA (1equiv.) under nitrogen atmosphere at 0 ꢀC. The mixture
was stirred for 30 min, corresponding amines were added, and the
mixture was again stirred for 1 h. The reaction was monitored by
TLC (Rf: 0.5 in 10% MeOH: DCM). After completion of the reaction,
the mixture was diluted with methanol. The resulting solution was
concentrated on vacuo rotavapor to get the crude product, which
was purified by silica gel (100e200) column chromatography using
5% MeOH: DCM as a mobile phase to get amides 3w-3ac, 7a-b in
77e91% yield.
J ¼ 8.9 Hz, 1H), 7.72 (d, J ¼ 8.5 Hz, 1H), 7.46 (brs, 2H), 7.36 (brs, 3H),
7.16e7.12 (m, 2H), 6.98 (s, 1H), 3.95 (s, 2H), 3.91 (s, 3H), 3.49 (d,
J ¼ 6.2 Hz, 2H), 3.24 (d, J ¼ 11.0 Hz, 2H), 2.48 (t, J ¼ 10.7 Hz, 2H),
1.88e1.76 (m, 4H), 1.64e1.59 (m, 3H); 13C NMR (126 MHz, CDCl3)
d
167.64, 159.04, 136.22, 130.43, 129.72, 129.64, 128.42, 128.04,
127.65, 127.13, 124.16, 119.73, 105.65, 62.86, 55.39, 53.37, 37.76,
36.29, 33.22, 31.43. ESI-MS: m/z 403.0 [MþH]þ.
General procedure for preparation of N-benzylpyridinium
salts 4a-au. To the solution of benzamides (3a-3ac), 0.5 mmol) in
ACN (10 mL) was added halides (0.5 mmol) and resulting mixture
was stirred at 80 ꢀC for 1 h. The formation of white precipitate was
observed in the reaction mixture. The precipitate was filtered and
washed with acetone (10 mL ꢁ 3). The resulting residue was dried
to get the desired N-benzylpyridinium salts 4a-4au in 80e95%
yield.
6-Methoxy-N-(pyridin-4-yl)-2-naphthamide (3w).Yield: 89%;
white solid; m.p.187e189 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.57 (d,
J ¼ 6.0 Hz, 2H), 8.32 (s, 1H), 8.08 (s, 1H), 7.90e7.83 (m, 3H), 7.65 (d,
J ¼ 6.2 Hz, 2H), 7.19 (d, J ¼ 2.2 Hz, 1H), 3.96 (s, 3H). ESI-MS: m/z
279.0 [MþH]þ.
1-Benzyl-4-(3,4-dimethoxybenzamido)pyridin-1-ium
(4a).Yield: 90%; white solid; m.p.199e201 ꢀC; IR (nmax): 3584, 3396,
2913, 1684, 1600, 1604, 1513, 1461, 1324, 1265, 1173, 1134, 1019 cmꢂ1
1H NMR (400 MHz, DMSO‑d6)
bromide
;
6-Methoxy-N-(pyridin-4-ylmethyl)-2-naphthamide
(3x).Yield:
d
11.48 (s, 1H), 8.98 (d, J ¼ 6.8 Hz, 2H),
87%; white solid; m.p. 207e209 ꢀC; 1H NMR (400 MHz, CDCl3)
8.39 (d, J ¼ 6.8 Hz, 2H), 7.75 (d, J ¼ 8.4 Hz, 1H), 7.59 (s, 1H), 7.50 (d,
d
8.58 (d, J ¼ 5.9 Hz, 2H), 8.27 (s, 1H), 7.85e7.78 (m, 3H), 7.30 (d,
J ¼ 7.0 Hz, 2H), 7.46e7.41 (m, 3H), 7.15 (d, J ¼ 8.5 Hz, 1H), 5.72 (s, 2H),
J ¼ 5.8 Hz, 2H), 7.21 (dd, J ¼ 8.9, 2.4 Hz, 1H), 7.17 (d, J ¼ 2.2 Hz, 1H),
4.72 (d, J ¼ 6.0 Hz, 2H), 3.95 (s, 3H). ESI-MS: m/z 293.0 [MþH]þ.
6-Methoxy-N-(2-(pyridin-4-yl)ethyl)-2-naphthamide (3y). Yield:
3.86 (s, 6H); 13C NMR (126 MHz, CD3OD):
d 166.98, 161.26, 153.79,
153.59, 149.19, 144.49, 133.81, 129.36, 129.24, 128.43, 124.89, 122.23,
115.78,111.22,110.66,62.53,55.29,55.22.ESI-MS:m/z349.0[Mꢂ Br]þ.
89%; white solid; m.p.148e150 ꢀC; 1H NMR (400 MHz, CDCl3)
d
8.54
1-Benzyl-4-(3,5-dimethoxybenzamido)pyridin-1-ium
bromide
(d, J ¼ 5.5 Hz, 2H), 8.15 (s, 1H), 7.80e7.71 (m, 3H), 7.20e7.18 (m, 3H),
7.15 (d, J ¼ 2.3 Hz, 1H), 3.94 (s, 3H), 3.79 (dd, J ¼ 13.1, 6.8 Hz, 2H),
2.99 (t, J ¼ 7.0 Hz, 2H).ESI-MS: m/z 307.0 [MþH]þ.
(4b). Yield: 93%; white solid; m.p.232e235 ꢀC; IR (nmax): 3584,
3354, 2917, 2849, 2327, 1699, 1638, 1590, 1518, 1456, 1329, 1206,
1160, 1044 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d 11.35 (s, 1H), 8.81
4-(Benzyloxy)-N-(pyridin-4-yl)benzamide (3z). Yield: 87%; white
solid; m.p. 200e202 ꢀC; IR (nmax): 3358, 2906, 1692, 160500, 1605,
1511, 1422, 1345, 1276, 1167, 1122, 1020 cmꢂ1;1H NMR (400 MHz,
(d, J ¼ 6.7 Hz, 2H), 8.17 (d, J ¼ 6.6 Hz, 2H), 7.33e7.27 (m, 5H), 6.99 (d,
J ¼ 1.1 Hz, 2H), 6.67 (s, 1H), 5.55 (s, 2H), 3.68 (s, 6H); 13C NMR
(126 MHz, CD3OD)
d 167.42, 161.26, 153.40, 144.61, 134.73, 133.75,
CDCl3)
d
8.53 (d, J ¼ 5.3 Hz, 2H), 7.84 (d, J ¼ 8.5 Hz, 3H), 7.58 (d,
129.40, 129.26, 128.42, 115.96, 105.82, 104.63, 62.62, 54.80; ESI-
J ¼ 5.5 Hz, 2H), 7.44e7.34 (m, 4H), 7.06 (d, J ¼ 8.5 Hz, 2H), 5.15 (s,
MS:m/z 349.0 [M ꢂ Br]þ.
2H). ESI-MS: m/z 305.0 [MþH]þ.
1-Benzyl-4-(4-chlorobenzamido)pyridin-1-ium bromide (4c).
Yield: 93%; white solid; m.p.247e249 ꢀC; IR (nmax): 3073, 3029,
2923, 2850, 2337, 1695, 1639, 1596, 1455, 1327, 1256, 1205, 1162,
4-Phenoxy-N-(pyridin-4-yl)benzamide (3aa).Yield: 85%; white
solid; m.p. 157e159 ꢀC; 1H NMR (400 MHz, CDCl3)
d 8.49 (d,
J ¼ 6.1 Hz, 2H), 7.85 (d, J ¼ 8.7 Hz, 2H), 7.61 (d, J ¼ 6.1 Hz, 2H), 7.39 (t,
J ¼ 7.9 Hz, 2H), 7.20 (t, J ¼ 7.4 Hz, 1H), 7.07e7.01 (m, 4H). ESI-MS: m/
z 291.0 [MþH]þ.
1096 cmꢂ1 1H NMR (400 MHz, DMSO‑d6)
; d 11.46 (s, 1H), 8.78 (d,
J ¼ 6.7 Hz, 2H), 8.15 (d, J ¼ 5.7 Hz, 2H), 7.86 (d, J ¼ 8.3 Hz, 2H), 7.53
(d, J ¼ 8.5 Hz, 2H), 7.29e7.24 (m, 5H), 5.54 (s, 2H); 13C NMR
2-(4-(Benzyloxy)phenyl)-N-(pyridin-4-yl)acetamide (3 ab). Yield:
78%; white solid; m.p. 97e99 ꢀC; 1H NMR (400 MHz, CDCl3) 8.44 (d,
J ¼ 4.7 Hz, 2H), 7.43 (t, J ¼ 6.8 Hz, 3H), 7.39e7.33 (m, 4H), 7.22 (d,
(101 MHz, CD3OD) d 166.69, 153.38, 144.70, 139.31, 133.71, 131.56,
129.66, 129.42, 129.28, 128.78, 128.43, 115.99, 62.70. ESI-MS:m/z
323.0 [M ꢂ Br]þ.

12
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
4-Benzamido-1-benzylpyridin-1-ium bromide (4d).Yield: 91%;
Yield: 95%; yellow solid; m.p. 205e208 ꢀC; IR (nmax): 3584, 3384,
white solid; m.p.272e275 ꢀC; IR (nmax): 3378, 3127, 3067, 2951,
3018, 2954, 2917, 2894, 2327, 1693, 1604, 1517, 1456, 1251,
2852, 1694, 1518, 1258, 1206, 1164 cmꢂ1; 1H NMR (400 MHz, MeOD)
1172 cmꢂ1 1H NMR (400 MHz, DMSO‑d6)
; d 11.85 (s, 1H), 9.04 (d,
d
8.86 (d, J ¼ 7.3 Hz, 2H), 8.42 (d, J ¼ 7.3 Hz, 2H), 8.07e8.04 (m, 2H),
7.71 (t, J ¼ 7.4 Hz,1H), 7.60 (t, J ¼ 7.6 Hz, 2H), 7.52e7.49 (m, 3H), 5.72
(s, 1H); 13C NMR (126 MHz, CD3OD)
167.76, 153.46, 144.65, 133.77,
J ¼ 6.8 Hz, 2H), 8.37 (d, J ¼ 6.7 Hz, 2H), 8.19 (d, J ¼ 8.1 Hz, 2H), 8.11
(d, J ¼ 8.2 Hz, 2H), 7.52e7.43 (m, 5H); 13C NMR (126 MHz, CD3OD)
d
d 166.12, 153.08, 150.51, 144.86, 138.44, 133.66, 129.44, 129.39,
133.05, 132.92, 129.39, 129.25, 128.55, 128.45, 127.94, 115.91, 62.62.
129.27, 128.47, 123.47, 116.20, 62.80. ESI-MS:m/z 334.0 [M ꢂ Br]þ.
ESI-MS:m/z 289.0 [M ꢂ Br]þ.
1-Benzyl-4-(3,4-dichlorobenzamido)pyridin-1-ium
bromide
1-Benzyl-4-(4-isopropylbenzamido)pyridin-1-ium bromide (4e).
Yield: 90%; white solid; m.p. 247e250 ꢀC; IR (nmax): 3390, 3188,
3029, 2966, 2782, 2327, 1691, 1641, 1608, 1591, 1519, 1461, 1327,
(4l).Yield: 91%; white solid; m.p. 299e301 ꢀC; IR (nmax): 3583, 3396,
2917, 2849, 2351, 1692, 1518, 1455, 1326, 1164 cmꢂ1 1H NMR
(400 MHz, DMSO‑d6)
;
d
11.56 (s, 1H), 8.85 (d, J ¼ 6.7 Hz, 2H), 8.17 (d,
1259, 1161 cmꢂ1
;
1H NMR (400 MHz, CD3OD)
d
8.82 (d, J ¼ 7.5 Hz,
J ¼ 6.8 Hz, 2H), 8.12 (s,1H), 7.83 (d, J ¼ 8.4 Hz,1H), 7.72 (d, J ¼ 8.4 Hz,
2H), 8.40 (d, J ¼ 7.4 Hz, 2H), 7.99 (d, J ¼ 8.4 Hz, 2H), 7.52e7.46 (m,
1H), 7.33e7.26 (m, 5H), 5.58 (s, 2H); 13C NMR (126 MHz, CD3OD)
4H), 5.70 (s, 2H), 3.05 (m, 1H), 1.33 (d, J ¼ 6.9 Hz, 6H). 13C NMR
d
163.99, 152.28, 143.27, 135.91, 132.19, 131.61, 131.25, 129.32,
(126 MHz, CD3OD)
d
167.59, 154.97, 153.52, 144.59, 133.80, 130.39,
129.20, 128.53, 127.95, 127.78, 126.94, 126.19, 114.61. ESI-MS:m/z
129.37, 129.25, 128.45, 128.23, 126.62, 115.84, 62.57, 34.11, 22.65.
357.0 [M ꢂ Br]þ.
ESI-MS:m/z 331.0 [M ꢂ Br]þ.
1-Benzyl-4-(4-methoxybenzamido)pyridin-1-ium bromide (4m)
[46]. Yield: 92%; white solid; m.p. 261e265 ꢀC; IR (nmax): 3584,
1-Benzyl-4-(3-chlorobenzamido)pyridin-1-ium
bromide
(4f).Yield: 92%; white solid; m.p.274e277 ꢀC; IR (nmax): 3584, 3330,
3013, 2917, 1692, 1639, 1604, 1517, 1456, 1321, 1251, 1173 cmꢂ1 1H
;
2922, 2851, 2342, 1740, 1695, 1638, 1517, 1453, 1323, 1248, 1207,
NMR (400 MHz, DMSO‑d6)
d
11.48 (s, 1H), 8.98 (d, J ¼ 6.8 Hz, 2H),
1160 cmꢂ1
;
1H NMR (400 MHz, DMSO‑d6)
d
11.66 (s, 1H), 8.98 (d,
8.37 (d, J ¼ 6.6 Hz, 2H), 8.06 (d, J ¼ 8.4 Hz, 2H), 7.51e7.42 (m, 5H),
J ¼ 6.6 Hz, 2H), 8.32 (d, J ¼ 6.8 Hz, 2H), 8.08 (s, 1H), 7.97 (d,
J ¼ 8.2 Hz, 1H), 7.80 (d, J ¼ 8.0 Hz, 1H), 7.66 (t, J ¼ 7.9 Hz, 1H),
7.48e7.45 (m, 5H), 5.75 (s, 2H); 13C NMR (126 MHz, CD3OD)
7.41 (d, J ¼ 8.8 Hz, 2H), 5.73 (s, 2H), 3.87 (s, 3H); 13C NMR (126 MHz,
CD3OD)
d 165.51, 162.56, 152.15, 143.00, 132.32, 128.72, 127.87,
127.75, 126.92, 123.25, 114.23, 112.30, 61.03, 53.30. ESI-MS:m/z
d
164.82, 151.75, 143.20, 133.33, 133.05, 132.20, 131.32, 128.67,
319.0 [M ꢂ Br]þ.
127.89, 127.74, 126.90, 126.40, 124.82, 114.51, 61.16. ESI-MS:m/z
1-Benzyl-4-(4-cyanobenzamido)pyridin-1-ium bromide (4n).
Yield: 95%; white solid; m.p. 235e237 ꢀC; IR (nmax): 3396, 3072,
2918, 2849, 2231, 1698, 1639, 1594, 1518, 1326, 1257, 1206,
323.0 [M ꢂ Br]þ.
1-Benzyl-4-(6-bromo-2-naphthamido)pyridin-1-ium
bromide
(4g). Yield: 91%; white solid; m.p. 336e338 ꢀC; IR (nmax): 3584,
1163 cmꢂ1 1H NMR (400 MHz, CD3OD)
; d 11.85 (s, 1H), 8.89 (d,
3238, 2923, 2852, 2342, 1741, 1687, 1627, 1588, 1517, 1454, 1322,
J ¼ 7.4 Hz, 2H), 8.42 (d, J ¼ 7.4 Hz, 2H), 8.11 (d, J ¼ 8.2 Hz, 2H), 7.98
1287, 1230 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d 11.82 (s, 1H), 8.99
(d, 2H), 7.52e7.43 (m, 5H), 5.74 (s, 2H);13C NMR (126 MHz, CD3OD)
(d, J ¼ 6.8 Hz, 2H), 8.72 (s, 1H), 8.39e8.37 (m, 3H), 8.14e8.08 (m,
d 166.32, 153.09, 144.84, 136.93, 133.74, 132.39, 129.43, 129.27,
3H), 7.81 (d, J ¼ 8.8 Hz, 1H), 7.51e7.46 (m, 5H), 5.74 (s, 2H); 13C NMR
128.78, 128.50, 117.38, 116.14, 62.72. ESI-MS:m/z 314.2[M ꢂ Br]þ.
1-Benzyl-4-(3,4-difluorobenzamido)pyridin-1-ium bromide (4o).
Yield: 89%; white solid; m.p.215e217 ꢀC; IR (nmax): 1584, 3074,
3029, 2952, 2852, 2062, 1699, 1640, 1594, 1515, 1456, 1326,
(126 MHz, DMSO‑d6):
d 167.58, 153.15, 146.98, 145.72, 140.65,
136.42, 135.13, 131.85, 130.88, 130.33, 129.97, 129.70, 129.00, 128.84,
128.25, 125.99, 122.71, 116.59, 62.26. ESI-MS:m/z 418.0 [M ꢂ Br]þ.
1-Benzyl-4-(2-bromo-4-flourobenzamido)pyridin-1-ium bromide
(4h).Yield: 89%; white solid; m.p.225e228 ꢀC; IR (nmax): 3069,
2922, 2852, 2343, 2046, 1704, 2638, 1597, 1518, 1462, 1326,
1277 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
8.21 (d, J ¼ 6.9 Hz, 2H), 8.02e7.97 (m, 1H), 7.79 (d, J ¼ 6.6 Hz, 1H),
7.52 (dd, J ¼ 18.5, 8.6 Hz, 1H), 7.34 (d, J ¼ 7.2 Hz, 2H), 7.28e7.23 (m,
d
8.88 (d, J ¼ 6.9 Hz, 2H),
1204 cmꢂ1
;
1H NMR (400 MHz, DMSO‑d6)
d
11.95 (s, 1H), 9.00 (d,
3H), 5.60 (s, 2H); 13C NMR (126 MHz, CD3OD)
d 165.40, 154.45,
J ¼ 6.7 Hz, 2H), 8.23 (d, J ¼ 6.7 Hz, 2H), 7.99 (d, J ¼ 5.9 Hz, 1H),
154.36, 153.22, 152.33, 151.15, 144.74, 133.71, 130.21, 129.41, 129.26,
128.46, 125.51, 117.70, 117.55, 116.05, 62.70. ESI-MS:m/z 325.1
[M ꢂ Br]þ.
7.90e7.88 (m, 1H), 7.50e7.42 (m, 6H), 5.75 (s, 2H); 13C NMR
(126 MHz, CD3OD)
d 163.36, 160.02, 158.01, 152.58, 145.00, 137.00,
136.93, 133.65, 132.78, 129.43, 129.27, 128.51, 124.28, 124.16, 118.50,
1-Benzyl-4-(4-ethoxybenzamido)pyridin-1-ium
bromide
118.31, 116.74, 116.71, 115.97, 62.79. ESI-MS:m/z 385.0 [M ꢂ Br]þ.
(4p).Yield: 90%; white solid; m.p. 261e263 ꢀC; IR (nmax): 2926,
1-Benzyl-4-(3-bromobenzamido)pyridin-1-ium
bromide
2848, 2328, 1690, 1639, 1605, 1518, 1455, 1319, 1248, 1173 cmꢂ1; 1H
(4i).Yield: 93%; white solid; m.p. 247e250 ꢀC; IR (nmax): 3069, 3014,
NMR (400 MHz, DMSO‑d6)
d
11.43 (s, 1H), 8.94 (d, J ¼ 4.8 Hz, 2H),
2925, 2852, 1694, 1638, 1597, 1518, 1455, 1280, 1248, 1205,
8.33 (d, J ¼ 5.4 Hz, 2H), 8.03 (d, J ¼ 8.2 Hz, 2H), 7.48e7.41 (m, 5H),
7.13 (d, J ¼ 8.7 Hz, 2H), 5.70 (s, 2H), 4.16 (q, J ¼ 6.9 Hz, 2H), 1.37 (t,
1162 cmꢂ1 1H NMR (400 MHz, DMSO‑d6)
; d 11.66 (s, 1H), 8.98 (d,
J ¼ 5.7 Hz, 2H), 8.33 (d, J ¼ 6.9 Hz, 2H), 8.21 (s, 1H), 8.01 (d,
J ¼ 7.8 Hz, 1H), 7.92 (d, J ¼ 8.0 Hz, 1H), 7.59 (t, J ¼ 7.9 Hz, 1H),
7.48e7.45 (m, 5H), 5.73 (s, 2H); 13C NMR (126 MHz, CD3OD)
J ¼ 6.9 Hz, 3H); 13C NMR (126 MHz, CD3OD)
d 167.02, 163.38, 153.67,
144.48, 133.81, 130.19, 129.37, 129.25, 128.38, 124.55, 115.69, 114.21,
63.68, 62.51, 13.56. ESI-MS:m/z 333.0 [M ꢂ Br]þ.
d
166.24, 153.25, 144.72, 135.83, 135.02, 133.72, 130.86, 130.38,
1-Benzyl-4-(3-methoxybenzamido)pyridin-1-ium bromide (4q).
Yield: 93%; white solid; m.p. 170e172 ꢀC; IR (nmax): 2926, 2848,
2328, 1690, 1639, 1605, 1518, 1455, 1319, 1248, 1173 cmꢂ1; 1H NMR
129.41, 129.26, 128.44, 126.78, 122.34, 116.05, 62.69. ESI-MS:m/z
368.0 [M ꢂ Br]þ.
1-Benzyl-4-(2-methoxybenzamido)pyridin-1-ium
bromide
(400 MHz, DMSO)
d
11.56 (s, 1H), 8.96 (d, J ¼ 6.0 Hz, 2H), 8.34 (d,
(4j).Yield: 94%; white solid; m.p. 192e194 ꢀC; IR (nmax): 3295, 3163,
J ¼ 6.2 Hz, 2H), 7.60 (d, J ¼ 7.6 Hz, 1H), 7.54 (t, J ¼ 7.7 Hz, 2H),
7.48e7.43 (m, 5H), 7.29 (d, J ¼ 8.1 Hz, 1H), 5.72 (s, 2H), 3.86 (s, 3H);
3125, 3027, 2917, 2849, 1694, 1639, 1591, 1519, 1464, 1328,
1162 cmꢂ1
;
1H NMR (400 MHz, DMSO‑d6)
d
11.51 (s, 1H), 8.94 (d,
13C NMR (126 MHz, CD3OD)
d 167.53, 160.07, 153.40, 144.63, 134.17,
J ¼ 7.0 Hz, 2H), 8.25 (d, J ¼ 6.8 Hz, 2H), 7.61 (t, J ¼ 8.1 Hz, 2H),
133.76, 129.68, 129.38, 129.25, 128.47, 120.03, 118.84, 115.96, 113.15,
7.49e7.39 (m, 5H), 7.25 (d, J ¼ 8.1 Hz,1H), 7.12 (t, J ¼ 7.4 Hz,1H), 5.71
62.62, 54.72. ESI-MS:m/z319.0 [M ꢂ Br]þ.
(s, 2H), 3.89 (s, 3H); 13C NMR (126 MHz, CD3OD)
d
166.39, 157.74,
1-Benzyl-4-(3-chloro-4-fluorobenzamido)pyridin-1-ium bromide
(4r). Yield: 88%; white solid; m.p. 245e247 ꢀC; IR (nmax): 3387,
3026, 2923, 2851, 2347, 1695, 1640, 1600, 1517, 1456, 1325,
152.61, 144.69, 134.44, 133.75, 130.99, 129.38, 129.35, 128.42, 121.37,
120.89, 115.87, 111.85, 62.61, 55.48. ESI-MS:m/z 319.0 [M ꢂ Br]þ.
1-Benzyl-4-(4-nitrobenzamido)pyridin-1-ium bromide (4k) [46].
1207 cmꢂ1 1H NMR (400 MHz, DMSO‑d6)
; d 11.47 (s, 1H), 8.79 (d,

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
13
J ¼ 5.4 Hz, 2H), 8.13 (d, J ¼ 6.3 Hz, 2H), 7.87 (s, 1H), 7.51 (t, J ¼ 8.9 Hz,
1H), 7.29e7.26 (m, 5H), 5.54 (s, 2H); 13C NMR (126 MHz, CD3OD)
1653, 1635, 1624, 1570, 1559, 1540, 1483, 1331, 1278, 1200, 1190,
1158, 1081, 1028 cmꢂ1 1H NMR (400 MHz, MeOD)
12.71 (s, 1H),
;
d
d
165.38, 162.14, 159.89, 153.24, 144.74, 144.74, 133.72, 133.72,
9.75 (d, J ¼ 6.9 Hz, 2H), 9.54 (s, 1H), 9.25 (d, J ¼ 6.9 Hz, 2H),
8.87e8.77 (m, 3H), 8.0e8.26 (m, 3H), 8.11 (d, J ¼ 8.9 Hz, 1H), 7.81 (d,
J ¼ 8.4 Hz, 2H), 6.43 (s, 2H), 4.73 (s, 3H), 4.57 (s, 3H); 13C NMR
130.91, 130.91, 129.42, 129.26, 129.14, 129.14, 129.07, 129.07, 128.43,
116.97, 116.79, 116.04, 62.70. ESI-MS: m/z 341.0 [M ꢂ Br]þ.
1-Benzyl-4-(2,4-dichlorobenzamido)pyridin-1-ium bromide (4s).
Yield: 90%; white solid; m.p. 247e249 ꢀC; IR (nmax): 3383, 2921,
(101 MHz, DMSO)
d 169.11, 161.81, 161.38, 154.72, 146.62, 138.72,
132.83, 132.28, 131.47, 129.42, 129.07, 128.34, 126.84, 121.73, 117.87,
116.48, 108.05, 63.27, 57.38, 57.17. ESI-MS:m/z 399.0[M ꢂ Cl]þ.
1-(2-Chlorobenzyl)-4-(6-Methoxy-2-naphthamido)pyridin-1-ium
chloride (4z).Yield: 90%; white solid; m.p. 255e257 ꢀC; IR (nmax):
3420, 2355, 2348, 1734, 1684, 1653, 1593, 1517,1481, 1457, 1324,
2848, 1706, 1639, 1593, 1518, 1455, 1326, 1273 cmꢂ1 1H NMR
;
(400 MHz, DMSO‑d6)
d
12.06 (s, 1H), 9.02 (d, J ¼ 6.9 Hz, 2H), 8.23 (d,
J ¼ 6.9 Hz, 2H), 7.86 (s, 1H), 7.77 (d, J ¼ 8.3 Hz, 1H), 7.66 (dd, J ¼ 8.3,
1.3 Hz, 1H), 7.51e7.43 (m, 5H), 5.77 (s, 2H); 13C NMR (126 MHz,
CD3OD)
d
166.29, 152.55, 145.08, 137.46, 133.65, 133.22, 131.99,
1269, 1201, 1164, 1073 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d 12.11
130.21, 129.79, 129.43, 129.27, 128.49, 127.45, 115.79, 62.80. ESI-
(s, 1H), 8.78 (d, J ¼ 6.6 Hz, 2H), 8.75 (s, 1H), 8.46 (d, J ¼ 6.5 Hz, 2H),
7.98 (d, J ¼ 8.6 Hz, 1H), 7.90e7.83 (m, 2H), 7.47 (d, J ¼ 7.3 Hz, 1H),
7.38e7.32 (m, 4H), 7.16 (d, J ¼ 8.9 Hz, 1H), 5.72 (s, 2H), 3.80 (s, 3H);
MS:m/z 357.0 [M ꢂ Br]þ.
1-Benzyl-4-(4-methylbenzamido)pyridin-1-ium bromide (4t) [47].
Yield: 91%; white solid; m.p. 276e278 ꢀC; IR (nmax): 3123, 3068,
3030, 2952, 2918, 2849, 2355, 1693, 1639, 1591, 1518, 1455, 1327,
13C NMR (126 MHz, DMSO‑d6)
d 167.76, 159.93, 153.77, 145.84,
137.32, 133.58, 132.45, 131.70, 131.46, 130.54, 130.41, 128.61, 127.85,
127.63, 127.57, 125.58, 120.30, 116.36, 106.54, 60.01, 55.95.ESI-MS:
m/z403.0 [M ꢂ Cl]þ.
1259, 1205, 1163, 1084 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d 11.52
(s, 1H), 8.95 (d, J ¼ 6.4 Hz, 2H), 8.34 (d, J ¼ 7.0 Hz, 2H), 7.95 (d,
J ¼ 7.9 Hz, 2H), 7.48e7.42 (m, 7H), 5.71 (s, 3H), 2.43 (s, 2H); 13C NMR
1-(3-Chlorobenzyl)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
chloride (4aa). Yield: 91%, white solid; m.p. 260e262 ꢀC; IR (nmax):
2920, 2352, 2344, 1734, 1700, 1683, 1653, 1640, 1623, 1554, 1558,
(126 MHz, DMSO‑d6) d 167.47, 153.22, 145.59, 144.25, 135.19, 130.42,
129.77, 129.68, 129.63, 128.99, 128.95, 116.46, 62.13, 21.63. ESI-MS:
m/z 303.0 [M ꢂ Br]þ.
1521, 1476, 1456, 1395, 1317, 1278, 1263, 1195, 1163, 1068, 1032 cmꢂ1
;
1-Benzyl-4-(furan-2-carboxamido)pyridin-1-ium bromide (4u).
Yield: 93%; white solid; m.p. 239e241 ꢀC; IR (nmax): 3583, 3387,
2918, 2849, 2343, 1691, 1641, 1596, 1518, 1467, 1327, 1273, 1218,
1H NMR (400 MHz, DMSO)
d
12.27 (s, 1H), 9.11 (d, J ¼ 6.9 Hz, 2H),
8.94 (s,1H), 8.64 (d, J ¼ 6.9 Hz, 2H), 8.18 (d, J ¼ 8.5 Hz,1H), 8.09e8.02
(m, 2H), 7.75 (s, 1H), 7.56 (s, 3H), 7.52 (s, 1H), 7.36 (dd, J ¼ 8.8, 1.7 Hz,
1173 cmꢂ1
;
1H NMR (400 MHz, DMSO‑d6)
d
11.61 (s, 1H), 8.97 (d,
1H), 5.82 (s, 2H), 4.00 (s, 3H); 13C NMR (126 MHz, DMSO‑d6)
d 167.72,
J ¼ 6.4 Hz, 2H), 8.35 (d, J ¼ 6.6 Hz, 2H), 8.11 (s, 1H), 7.66 (s, 1H),
159.93, 153.64, 145.55, 137.48, 137.31, 134.13, 131.55, 131.44, 130.35,
129.59, 129.08, 127.87, 127.81, 127.63, 127.58, 125.55, 120.29, 116.52,
106.56, 61.16, 55.95.ESI-MS: m/z403.0 [M ꢂ Cl]þ.
7.49e7.44 (m, 5H), 6.82e6.81 (m, 1H), 5.72 (s, 2H); 13C NMR
(126 MHz, CD3OD)
d 157.37, 153.06, 147.12, 146.17, 144.63, 133.73,
129.39, 129.25, 128.43, 118.05, 115.83, 112.63, 62.62; ESI-MS:m/
1-(4-Chlorobenzyl)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
chloride (4 ab).Yield: 91%; white solid; m.p. 256e258 ꢀC; IR (nmax):
2918, 2355, 2340, 1729, 1691, 1641, 1623, 1554, 1517, 1482, 1453,
z279.2[M ꢂ Br]þ.
1-Benzyl-4-(3,4,5-trimethoxybenzamido)pyridin-1-ium bromide
(4v). Yield 91%; white solid, m.p. 180e181 ꢀC; IR 3418, 2954, 2924,
1333, 1279, 1215, 1190, 1160, 1026 cmꢂ1 1H NMR (400 MHz,
;
2350,1691,1639,1586,1518,1503,1461,1416,1202,1171,1125 cmꢂ1
;
DMSO‑d6)
d
12.04 (s, 1H), 8.98 (d, J ¼ 7.0 Hz, 2H), 8.79 (s, 1H), 8.51
1H NMR (400 MHz, DMSO‑d6)
d
11.48 (s, 1H), 8.97 (d, J ¼ 7.2 Hz, 2H),
(d, J ¼ 7.0 Hz, 2H), 8.08e7.97 (m, 3H), 7.58e7.50 (m, 4H), 7.46 (s,1H),
8.33 (d, J ¼ 6.1 Hz, 2H), 7.48e7.43 (m, 5H), 7.33 (d, J ¼ 2.9 Hz, 2H),
7.30 (d, J ¼ 8.6 Hz, 1H), 5.73 (s, 2H), 3.93 (s, 3H); 13C NMR (101 MHz,
5.71 (s, 2H), 3.89 (s, 6H), 3.77 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
DMSO‑d6)
d 167.74, 159.98, 153.66, 145.51, 137.34, 134.96, 134.45,
d
167.10, 153.20, 153.11, 145.51, 142.08, 135.09, 129.70, 129.67,
134.19, 131.47, 131.11, 130.42, 129.66, 127.88, 127.68, 127.59, 125.59,
120.29, 116.54, 106.62, 61.18, 55.99.ESI-MS:m/z 403.0 [M ꢂ Cl]þ.
1-(2-Fluorobenzyl)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
bromide (4ac). Yield: 93%; white solid; m.p. 276e277 ꢀC; IR (nmax):
3386, 2377, 2343, 1824, 1767, 1729, 1712, 1690, 1680, 1659, 1642,
1538, 1516, 1502, 1486, 1469, 1415, 1322, 1262, 1194, 1023 cmꢂ1; 1H
128.99, 128.22, 116.53, 106.62, 62.17, 60.72, 56.74; ESI-MS:m/z 379.0
[M ꢂ Br]þ.
1-Benzyl-4-(6-methoxy-2-naphthamido)pyridin-1-ium bromide
(4w). Yield: 87%; white solid; m.p. 296e298 ꢀC; IR (nmax): 3286,
2954, 2922, 2885, 2870, 2367, 2351, 2344, 2312, 1624, 1583, 1517,
1454, 1377, 1317, 1286, 1265, 1197, 1158, 1024 cmꢂ1
;
1H NMR
NMR (400 MHz, MeOD)
d
8.81 (d, J ¼ 7.2 Hz, 2H), 8.57 (s, 1H), 8.43
(400 MHz, MeOD)
d
8.82 (d, J ¼ 7.1 Hz, 2H), 8.58 (s, 1H), 8.43 (d,
(d, J ¼ 7.2 Hz, 2H), 8.02 (d, J ¼ 8.6 Hz,1H), 7.96 (t, J ¼ 8.3 Hz, 2H), 7.61
(t, J ¼ 7.4 Hz, 1H), 7.58e7.53 (m, 1H), 7.38 (d, J ¼ 2.2 Hz, 1H), 7.34 (d,
J ¼ 7.3 Hz, 1H), 7.29 (dd, J ¼ 9.1, 2.0 Hz, 2H), 5.77 (s, 2H), 3.99 (s, 3H);
J ¼ 7.0 Hz, 2H), 8.02 (d, J ¼ 8.6 Hz,1H), 7.96 (t, J ¼ 8.8 Hz, 2H), 7.50 (s,
5H), 7.38 (d, J ¼ 1.7 Hz, 1H), 7.29 (dd, J ¼ 9.0, 2.1 Hz, 1H), 5.69 (s, 2H),
3.98 (s, 3H); 13C NMR (101 MHz, DMSO‑d6)
d
167.72, 159.93, 153.25,
13C NMR (126 MHz, DMSO‑d6)
d 167.68, 161.96, 159.94, 153.46,
145.51, 137.30, 135.05, 131.40, 129.88, 129.72, 129.69, 128.99, 128.05,
127.76, 127.60, 125.33, 120.43, 116.45, 106.56, 62.22, 55.96. ESI-MS:
m/z 369.0 [M ꢂ Br]þ.
145.75, 137.32, 132.42, 131.78, 131.40, 130.04, 128.00, 127.69, 125.77,
125.40, 122.18, 122.07, 120.40, 116.56, 116.40, 106.60, 56.76,
55.97.ESI-MS: m/z387.0[M ꢂ Br]þ.
4-(6-Methoxy-2-naphthamido)-1-(2-methylbenzyl)pyridin-1-ium
bromide (4x).Yield: 90%; white solid; m.p. 189e191 ꢀC; IR (nmax):
3500, 2920, 2343, 2148, 1684,1623, 1590, 1515, 1479, 1454, 1434,
1-(4-Fluorobenzyl)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
chloride (4ad). Yield: 91%; white solid; m.p. 275e277 ꢀC; IR(nmax):
3625, 3584, 3418, 2356, 2092, 1658, 1650, 1643, 1633, 1518, 1481,
1395, 1323, 1288, 1265, 1066, 1041 cmꢂ1
;
1H NMR (400 MHz,
1459, 1396, 1326, 1157, 1071, 1046, 1027 cmꢂ1 1H NMR (400 MHz,
;
DMSO‑d6)
2H), 8.01e7.95 (m, 3H), 7.45e7.24 (m, 5H), 7.18 (d, J ¼ 7.4 Hz, 1H),
5.65 (s, 2H), 2.25 (s, 3H); 13C NMR (126 MHz, DMSO‑d6)
167.71,
d
8.65 (d, J ¼ 6.6 Hz, 2H), 8.53 (s, 1H), 8.29 (d, J ¼ 5.0 Hz,
DMSO‑d6)
d
12.01 (s,1H), 8.88 (d, J ¼ 7.1 Hz, 2H), 8.71 (s, 1H), 8.41 (d,
J ¼ 7.0 Hz, 2H), 7.96 (d, J ¼ 8.6, 1.4 Hz, 1H), 7.89e7.82 (m, 2H),
d
7.50e7.47 (m, 2H), 7.31 (d, J ¼ 2.0 Hz, 1H), 7.19e7.14 (m, 3H), 5.59 (s,
159.94, 153.32, 145.72, 137.33, 133.13, 131.39, 129.99, 129.70, 129.48,
127.71, 127.63, 127.16, 125.40, 120.42, 116.41, 106.61, 60.40, 55.98,
19.26.ESI-MS:m/z 383.0 [M ꢂ Br]þ.
2H), 3.79 (s, 3H); 13C NMR (126 MHz, DMSO‑d6)
d 167.70, 163.91,
161.96, 159.93, 153.53, 145.39, 137.30, 131.66, 131.59, 131.48, 131.44,
130.34, 127.87, 127.63, 127.57, 125.55, 120.29, 116.63, 116.49, 116.46,
106.55, 61.17, 55.95.ESI-MS: m/z387.0 [M ꢂ Cl]þ.
1-(4-Methoxy)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
chloride (4y).Yield: 91%, white solid; m.p. 259e261 ꢀC; IR (nmax):
2955, 2923, 2852, 2350, 2327, 1830, 1792, 1729, 1700, 1691, 1663,
1-(2-Nitrobenzyl)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
bromide (4ae). Yield: 89%; white solid; m.p. 280e282 ꢀC; IR (nmax):

14
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
3385, 2391, 2351, 2304, 2318, 1703, 1643, 1621, 1588, 1520, 1485,
1329, 1282, 1269, 1245, 1214, 1192, 1167, 1078, 1043 cmꢂ1; 1H NMR
(
nmax): 3374, 2955, 2924, 2853, 2356, 2327, 1744, 1641, 1536, 1462,
1377, 1302, 1274, 1214, 1191, 1160, 1026 cmꢂ1 1H NMR (400 MHz,
MeOD)
;
(400 MHz, DMSO‑d6)
d
11.76 (s, 1H), 8.82 (d, J ¼ 4.8 Hz, 2H), 8.61 (s,
d
8.99 (d, J ¼ 6.5 Hz, 2H), 8.78 (s, 1H), 8.28 (s, 1H), 8.14 (d,
1H), 8.39 (d, J ¼ 6.2 Hz, 2H), 8.26 (d, J ¼ 8.1 Hz, 1H), 8.04 (d,
J ¼ 9.0 Hz, 1H), 8.01 (s, 2H), 7.84 (t, J ¼ 7.6 Hz, 1H), 7.75 (t, J ¼ 7.8 Hz,
1H), 7.46 (s, 1H), 7.32e7.27 (m, 2H), 6.05 (s, 2H), 3.92 (s, 3H); 13C
J ¼ 6.4 Hz, 2H), 7.92e7.88 (m, 2H), 7.81 (d, J ¼ 8.5 Hz, 1H), 7.62 (s,
1H), 7.53e7.50 (m, 1H), 7.46 (t, J ¼ 8.2 Hz, 1H), 7.36 (s, 1H), 7.28 (dd,
J ¼ 9.0, 2.3 Hz, 1H), 5.85 (s, 2H), 4.02 (s, 3H), 3.92e3.93 (m, 2H); 13
C
NMR (101 MHz, DMSO‑d6)
d
167.77, 160.01, 153.69, 148.11, 146.19,
NMR (126 MHz, MeOD) d 168.99, 161.69, 159.46, 143.77, 136.64,
137.41, 135.42, 131.44, 130.99, 130.09, 128.07, 127.69, 126.12, 125.45,
122.96, 120.43, 116.41, 106.70, 59.52, 56.02. ESI-MS: m/z414.0
[M ꢂ Br]þ.
135.49, 135.00, 130.83, 130.11, 129.62, 128.82, 128.59, 128.51, 127.92,
127.19, 126.89, 126.85, 123.78, 119.46, 105.31, 62.75, 54.55, 39.02,
35.55. ESI-MS: m/z 431.0 [M ꢂ Cl]þ.
1-(4-Nitrobenzyl)-4-(6-methoxy-2-naphthamido)pyridin-1-ium
bromide (4af). Yield: 90%; white solid; m.p. 264e266 ꢀC; IR(nmax):
3020, 2919, 3030, 2851, 1669, 1624, 1439, 1328, 1215, 1116 cmꢂ1; 1H
1-(4-Chlorobenzyl)-4-(2-(6-methoxy-2-naphthamido)ethyl)pyr-
idin-1-ium chloride (4al). Yield: 93%; white solid; m.p. 115e116 ꢀC;
IR (nmax): 3374, 2921, 2372, 2350, 2320, 1700, 1695, 1658, 1635,
NMR (400 MHz, DMSO‑d6)
d
11.68 (s, 1H), 8.93 (d, J ¼ 7.2 Hz, 2H),
1539,1533, 1516, 1456, 1216, 1162, 1029 cmꢂ1 1H NMR (400 MHz,
;
8.60 (s, 1H), 8.36 (d, J ¼ 7.2 Hz, 2H), 8.23 (d, J ¼ 8.7 Hz, 2H),
7.98e7.93 (m, 4H), 7.67 (d, J ¼ 8.7 Hz, 2H), 7.39 (s, 1H), 7.23 (dd,
J ¼ 9.0, 2.3 Hz, 1H), 5.84 (s, 2H), 3.85 (s, 3H); 13C NMR (126 MHz,
MeOD)
d
8.78 (d, J ¼ 6.4 Hz, 2H), 8.08 (s, 1H), 7.95 (d, J ¼ 6.4 Hz, 2H),
7.74e7.70 (m, 2H), 7.61 (d, J ¼ 8.6 Hz, 1H), 7.32 (s, 4H), 7.19 (d,
J ¼ 2.1 Hz, 1H), 7.11 (dd, J ¼ 9.0, 2.4 Hz, 1H), 5.65 (s, 2H), 3.84 (s, 3H),
DMSO‑d6)
d
167.71, 159.97, 153.55, 148.29, 145.94, 142.27, 137.35,
3.75 (t, J ¼ 6.4 Hz, 2H); 13C NMR (101 MHz, MeOD)
d 170.43, 163.05,
131.40, 130.24, 130.00, 128.03, 127.73, 127.63, 125.37, 124.64, 120.44,
116.53, 115.48, 106.62, 61.04, 55.98.ESI-MS: m/z414.0[M ꢂ Br]þ.
1-Benzyl-4-((7-methoxy-2-naphthamido)methyl)pyridin-1-ium
bromide (4ag). Yield: 88%; brownish sticky-solid; IR (nmax): 3850,
3743, 3301, 2920, 2850, 1628, 1602, 1540, 1503, 1483, 1390, 1415,
160.90, 145.11, 138.05, 137.00, 133.51, 131.57, 131.52, 130.77, 130.23,
129.95, 129.36, 128.59, 128.27, 125.17, 120.86, 106.84, 64.20, 56.00,
40.39, 36.96. ESI-MS:m/z 431.0 [M ꢂ Cl]þ.
1-(2-Fluorobenzyl)-4-(2-(6-methoxy-2-naphthamido)ethyl)pyr-
idin-1-ium bromide (4am). Yield: 87%; white solid; m.p.
125e127 ꢀC; IR (nmax): 3414, 3048, 2923, 2849, 2356, 2327, 1824,
1767,1756,1746,1729,1712,1700,1679,1658,1652,1641,1635,1565,
1301,1266,1216,1165,1020 cmꢂ1; 1H NMR (400 MHz, MeOD)
d 8.98
(d, J ¼ 6.7 Hz, 2H), 8.42 (s, 1H), 8.09 (d, J ¼ 6.5 Hz, 2H), 7.92e7.90 (m,
4H), 7.49 (d, J ¼ 2.1 Hz, 3H), 7.34 (d, J ¼ 2.4 Hz, 1H), 7.25 (dd, J ¼ 9.0,
2.5 Hz, 1H), 5.82 (s, 2H), 3.97 (s, 3H); 13C NMR (126 MHz, MeOD)
1533, 1495, 1456, 1393, 1302, 1276, 1161, 1026 cmꢂ1 1H NMR
;
(400 MHz, MeOD)
d
8.78 (d, J ¼ 6.3 Hz, 2H), 8.09 (s, 1H), 7.93 (d,
d
169.24, 160.76, 159.61, 144.12, 136.88, 133.32, 130.25, 129.59,
J ¼ 6.4 Hz, 2H), 7.69 (t, J ¼ 9.0 Hz, 2H), 7.61 (d, J ¼ 8.6 Hz, 1H),
7.47e7.38 (m, 2H), 7.18e7.14 (m, 2H), 7.08 (t, J ¼ 8.8 Hz, 2H), 5.74 (s,
2H), 3.81 (s, 3H), 3.72 (t, J ¼ 6.6 Hz, 2H); 13C NMR (126 MHz, MeOD)
129.31, 128.68, 127.96, 127.83, 127.75, 126.98, 126.17, 123.93, 119.55,
105.38, 63.74, 54.59, 42.63. ESI-MS: m/z383.0 [M ꢂ Br]þ.
1-Benzyl-4-(2-(6-methoxy-2-naphthamido)ethyl)pyridin-1-ium
bromide (4ah). Yield 91%; brownish sticky solid; IR (nmax): 3123,
3068, 3030, 2952, 2918, 2849, 2355, 1693, 1639, 1591, 1518, 1455,
d 169.00, 161.66, 159.46, 143.83, 136.64, 132.35, 132.28, 131.23,
130.10, 128.70, 128.54, 127.92, 126.83, 125.18, 123.79, 120.52, 119.44,
115.92, 115.75, 105.30, 58.07, 54.53, 38.99, 35.48. ESI-MS: m/z 415.0
[M ꢂ Br]þ.
1327, 1259, 1205, 1163, 1084 cmꢂ1 1H NMR (400 MHz, MeOD)
;
d
8.91 (d, J ¼ 6.6 Hz, 2H), 8.22 (s, 1H), 8.06 (d, J ¼ 6.6 Hz, 2H),
1-(4-Fluorobenzyl)-4-(2-(6-methoxy-2-naphthamido)ethyl)pyr-
idin-1-ium chloride (4an). Yield: 95%; white solid; m.p. 120e122 ꢀC;
IR (nmax): 3374, 2955, 2924, 2869, 2853, 2351, 2313, 1735, 1638,
7.86e7.81 (m, 2H), 7.76e7.74 (m, 1H), 7.45 (s, 4H), 7.30 (d, J ¼ 2.4 Hz,
1H), 7.22 (dd, J ¼ 9.0, 2.5 Hz, 1H), 5.79 (s, 2H), 3.95 (s, 3H), 3.86 (t,
J ¼ 6.6 Hz, 2H); 13C NMR (126 MHz, MeOD)
d
168.97, 161.35, 159.44,
1537, 1491, 1461, 1377, 1246, 1211, 1190, 1160, 1082 cmꢂ1 1H NMR
;
143.69, 136.63, 133.41, 130.13, 129.50, 129.27, 128.71, 128.53, 127.92,
127.22, 126.84, 123.82, 119.42, 105.33, 63.63, 54.56, 39.02, 35.44.
ESI-MS: m/z 397.0 [M ꢂ Br]þ.
(400 MHz, MeOD)
d
8.77 (d, J ¼ 6.1 Hz, 2H), 8.08 (s, 1H), 7.92 (d,
J ¼ 6.0 Hz, 2H), 7.71e7.66 (m, 2H), 7.61 (d, J ¼ 8.6 Hz, 1H), 7.41e7.37
(m, 2H), 7.15 (s, 1H), 7.04 (dd, J ¼ 17.3, 8.8 Hz, 1H), 5.63 (s, 1H), 3.80
(s, 3H), 3.71 (t, J ¼ 5.9 Hz, 2H); 13C NMR (126 MHz, DMSO‑d6)
4-(2-(6-Methoxy-2-naphthamido)ethyl)-1-(2-methylbenzyl)pyr-
idin-1-ium bromide (4ai). Yield: 90%; white solid; m.p. 130e132 ꢀC;
IR (nmax): 3583, 3388, 2955, 2925, 2351, 2322, 1729, 1640, 1536,
1502, 1390, 1301, 1216, 1164, 1025 cmꢂ1; 1H NMR (400 MHz, MeOD)
d
168.95, 164.47, 162.49, 161.43, 159.45, 143.63, 136.62, 131.06,
130.99, 130.12, 129.52, 128.74, 128.53, 127.92, 127.21, 126.83, 123.81,
119.44, 116.16, 115.98, 105.33, 62.76, 54.55, 39.01, 35.45.ESI-MS: m/z
415.0 [M ꢂ Cl]þ.
d
8.84 (d, J ¼ 6.6 Hz, 2H), 8.30 (s, 1H), 8.12 (d, J ¼ 6.4 Hz, 2H),
7.92e7.84 (m, 3H), 7.83 (d, J ¼ 8.6 Hz,1H), 7.46e7.42 (m, 1H), 7.36 (s,
4-(2-(6-Methoxy-2-naphthamido)ethyl)-1-(2-nitrobenzyl)pyr-
idin-1-ium bromide (4ao). Yield: 90%; white solid; m.p.
200e202 ꢀC; IR (nmax): 3584, 3564, 3361, 2920, 2371, 2355, 2319,
1844, 1767, 1734, 1729, 1700, 1695, 1690, 1684, 1667, 1652, 1635,
1554, 1539, 1331, 1213, 1029, 869, 814, 767, 732, 667, 631, 604, 595,
2H), 7.33e7.27 (m, 2H), 5.91 (s, 2H), 4.01 (s, 3H), 3.93 (t, J ¼ 6.6 Hz,
2H), 2.34 (s, 3H); 13C NMR (101 MHz, MeOD)
d 168.95, 161.43,
159.49, 143.69, 137.43, 136.67, 131.10, 130.15, 129.88, 129.76, 128.70,
128.55, 127.96, 127.25, 126.87, 126.80, 123.84, 119.46, 105.37, 61.77,
54.58, 39.03, 35.56, 17.82.ESI-MS: m/z411.0 [M ꢂ Br]þ.
586, 577, 568, 559, 550, 532, 523, 515, 506 cmꢂ1
;
1H NMR
1-(2-Chlorobenzyl)-4-(2-(6-methoxy-2-naphthamido)ethyl)pyr-
idin-1-ium chloride (4aj). Yield: 91%; white solid; m.p. 173e175 ꢀC;
IR(nmax): 3123, 3068, 3030, 2952, 2918, 2849, 2355, 1693, 1639,
(400 MHz, MeOD)
d
8.92 (d, J ¼ 6.5 Hz, 2H), 8.38 (d, J ¼ 8.2 Hz, 1H),
8.30 (s,1H), 8.16 (d, J ¼ 6.5 Hz, 2H), 7.93e7.88 (m, 3H), 7.85e7.81 (m,
2H), 7.51 (d, J ¼ 7.5 Hz, 1H), 7.37 (d, J ¼ 2.0 Hz, 1H), 7.28 (dd, J ¼ 8.9,
2.4 Hz, 1H), 6.22 (s, 2H), 4.02 (s, 3H), 3.95 (dd, J ¼ 12.0, 5.9 Hz, 2H);
1591, 1518, 1455, 1327, 1259, 1205, 1163, 1084 cmꢂ1 1H NMR
;
(400 MHz, MeOD)
d
8.85 (d, J ¼ 6.4 Hz, 2H), 8.21 (s, 1H), 8.05 (d,
13C NMR (126 MHz, MeOD)
d 168.99, 161.87, 159.44, 147.95, 144.10,
J ¼ 6.3 Hz, 2H), 7.84e7.80 (m, 3H), 7.75 (d, J ¼ 8.6 Hz, 2H), 7.55e7.44
136.63, 134.72, 131.93, 131.06, 130.14, 128.60, 127.93, 127.71, 127.26,
126.84, 125.82, 123.84, 119.43, 105.30, 60.79, 54.55, 39.04,
35.60.ESI-MS: m/z442.0 [M ꢂ Br]þ.
(m, 4H), 5.91 (s, 2H), 3.93 (s, 3H), 3.85 (t, J ¼ 6.5 Hz, 2H); 13C NMR
(101 MHz, MeOD)
d 161.72, 159.44, 143.92, 136.63, 134.31, 131.85,
131.62, 130.60, 130.21, 130.12, 128.60, 128.54, 127.97, 127.93, 127.22,
126.83, 123.81, 119.42, 105.34, 61.35, 54.55, 38.96, 35.48. ESI-MS: m/
z431.0 [M ꢂ Cl]þ.
4-(2-(7-Methoxy-2-naphthamido)ethyl)-1-(4-nitrobenzyl)pyr-
idin-1-ium bromide (4ap).Yield: 95%; white solid; m.p. 184e186 ꢀC;
IR (nmax): 3417, 3338, 3273, 3046, 2921, 2849, 2351, 2327, 1767, 1729,
1712, 1700, 1695, 1684, 1680, 1634, 1620, 1565, 1554, 1518, 1481,
1468, 1453, 1426, 1382, 1346, 1320, 1289, 1260, 1243, 1216, 1156,
1-(3-Chlorobenzyl)-4-(2-(6-methoxy-2-naphthamido)ethyl)pyr-
idin-1-ium chloride (4ak). Yield: 80%; white solid; m.p. 80e82 ꢀC; IR

M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
15
1116, 1016 cmꢂ1;1H NMR (400 MHz, DMSO‑d6)
d
9.06 (d, J ¼ 5.7 Hz,
used for cholinesterase inhibition studies. Enzymes and substrates
were diluted using 0.1 M phosphate buffer pH: 7.2 (PBS) to get the
desired stocks. 10 mM stock solutions of compounds were prepared
using biological grade DMSO and diluted with PBS to get the
desired final concentration. All experiments were carried out in 96
2H), 8.72 (t, J ¼ 5.6 Hz, 1H), 8.26 (d, J ¼ 6.0 Hz, 2H), 8.23 (s, 1H), 8.12
(d, J ¼ 6.0 Hz, 2H), 7.88 (d, J ¼ 9.0 Hz, 1H), 7.84 (d, J ¼ 8.6 Hz, 1H),
7.78 (d, J ¼ 8.6 Hz, 1H), 7.67 (d, J ¼ 8.2 Hz, 2H), 7.36 (s, 1H), 7.22 (dd,
J ¼ 9.0 Hz, 1H), 5.95 (s, 2H), 3.89 (s, 3H), 3.72 (dd, J ¼ 12.3, 6.2 Hz,
2H), 3.23 (t, J ¼ 6.4 Hz, 2H); 13C NMR (126 MHz, DMSO‑d6)
d
166.96,
well plates. The assay solution consists of 20
mL enzyme solution
161.63, 159.03, 148.28, 144.70, 141.96, 136.26, 130.82, 130.23, 129.58,
129.25, 127.85, 127.71, 127.13, 124.98, 124.59, 106.30, 61.92, 55.81,
39.19, 35.56.ESI-MS: m/z442.0 [M ꢂ Br]þ.
(AChE or BChE), 20 L test compound solution and 140 m
m
L 0.3 mM
5,5-dithiobis-(2-nitrobenzoic acid) solution (DTNB). This assay
mixture was incubated for 20 min at 37 ꢀC followed by addition of
1-Benzyl-4-(4-(benzyloxy)benzamido)pyridin-1-ium
bromide
20 mL 10 mM substrate (acetyl thiocholine iodide or S-butyryl thi-
(4aq). Yield 91%, brown sticky solid; IR (nmax): 3353, 2922, 2350,
ocholine iodide). Immediately after addition of the substrate, the
reaction was monitored by recording the absorbance at 412 nm
every minute for 10 min using a 96-well microplate plate reader.
Donepezil was used as reference standard and each concentration
was assayed in triplicate. Finally, percent inhibition and IC50 values
were determined. The detailed protocol has been described earlier
[30].
Enzyme kinetics. The kinetic studies of compound 7b with
rHuAChE and eqBChE was carried out using the same protocol as
described above. For each concentration of 7b, five different con-
centrations of substrate were used. The procedure of kinetic
experiment for Ki determination is described earlier in our previous
paper [30].
2337, 2306, 1653, 1591, 1506, 1453, 1415, 1382, 1331, 1258, 1213,
1093, 1108 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d 11.33 (s, 1H), 8.84
(d, J ¼ 6.8 Hz, 2H), 8.22 (d, J ¼ 6.8 Hz, 2H), 7.92 (d, J ¼ 8.5 Hz, 2H),
7.35e7.19 (m, 10H), 7.08 (d, J ¼ 8.5 Hz, 2H), 5.59 (s, 2H), 5.11 (s, 2H);
13C NMR (126 MHz, DMSO‑d6)
d 166.84, 162.77, 153.34, 145.51,
136.88, 135.20, 131.17, 129.67, 129.61, 128.98, 128.52, 128.29, 125.36,
116.34, 115.35, 70.08, 62.06.ESI-MS: m/z395.0 [M ꢂ Br]þ.
1-Benzyl-4-(4-phenoxybenzamido)pyridin-1-ium bromide (4ar).
Yield: 93%: white solid; m.p. 174e176 ꢀC; IR (nmax): 3393, 3066,
3027, 2925, 2318, 2351, 1962, 1693, 1639, 1606, 1588, 1518, 1504,
1488, 1455, 1375, 1326, 1286, 1242, 1206, 1161, 1118, 1081, 1043,
1022, 1010 cmꢂ1;1H NMR (400 MHz, MeOD)
d
8.81 (d, J ¼ 7.4 Hz,
2H), 8.38 (d, J ¼ 7.4 Hz, 2H), 8.07e8.05 (m, 2H), 7.51e7.44 (m, 6H),
In vitro PAMPA-BBB assay. The test compound was dissolved in
DMSO (10 mM stock), and was further diluted with PBS to get
100 mM stock. The 0.2 ml of each test compounds and PBS were
7.26 (t, J ¼ 7.4 Hz, 1H), 7.13e7.08 (m, 4H), 5.69 (s, 2H); 13C NMR
(101 MHz, DMSO-d6)
d
166.84,161.76, 155.42, 153.19,145.52, 135.08,
131.43, 130.89, 129.70, 129.67, 128.97, 127.53, 125.35, 120.39, 117.82,
added to the donor and acceptor plates, respectively. The absor-
bance of both plates was recorded using microplate reader, and the
effective permeability was calculated as described earlier [36].
116.44, 62.19. ESI-MS: m/z 381.0 [M ꢂ Br]þ.
1-Benzyl-4-(2-(4-(benzyloxy)phenyl)acetamido)pyridin-1-ium
bromide (4as). Yield: 89%; white solid; m.p. 187e189 ꢀC; IR (nmax):
3251, 3166, 3064, 3032, 3005, 2955, 2870, 2351, 2314, 1870, 1697,
1682, 1593, 1511, 1454, 1416, 1380, 1332, 1297, 1243, 1106, 1063,
Ab42 self-aggregation inhibition assay. Amyloid beta 1e42 rat
peptide (Sigma-Aldrich, Saint Louis) was pretreated with 10% (w/v)
NH4OH (0.5 mg/ml) in order to produce an aggregate free solution.
After brief sonication and incubation for 10 min at room temper-
ature, NH4OH was removed using vacuum concentrator resulting in
a fluffy white peptide [50]. The obtained peptide was reconstituted
1024, 1001 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d 11.62 (s, 2H), 8.88
(d, J ¼ 7.1 Hz, 2H), 8.10 (d, J ¼ 7.1 Hz, 2H), 7.44 (t, J ¼ 3.3 Hz, 7H), 7.39
(t, J ¼ 7.4 Hz, 2H), 7.33 (t, J ¼ 7.1 Hz, 1H), 7.25 (d, J ¼ 8.5 Hz, 2H), 6.98
(d, J ¼ 8.6 Hz, 2H), 5.68 (s, 2H), 5.10 (s, 2H), 3.76 (s, 2H); 13C NMR
in
(48.4:48.4:3.2) to obtain a 200
in 96-well black microtiter plates at a final assay volume of 200
with final A and inhibitor concentrations of 20 and 10 mM,
a
mixture of CH3CN/0.3 mM Na2CO3/250 mM NaOH
(101 MHz, DMSO‑d6)
d
172.57, 157.85, 152.61, 145.77, 137.57, 135.15,
mM solution. Assays were carried out
130.94, 129.65, 129.58, 128.91, 128.90, 128.26, 128.07, 126.81, 115.65,
mL
115.24, 69.61, 62.07, 42.94.ESI-MS:m/z409.0 [M ꢂ Br]þ.
b
4-([1,10-Biphenyl]-4-ylcarboxamido)-1-benzylpyridin-1-ium bro-
mide (4at). Yield: 94%; white solid; m.p. 337e339 ꢀC; IR(nmax):
3418, 2954, 2924, 2350, 1691, 1639, 1586, 1518, 1503, 1461, 1416,
respectively. Incubation of Ab with and without inhibitors were
carried out for 24 h at 30 ꢀC in 10 mM phosphate buffer (pH ¼ 8.0)
containing 10 mM NaCl. All the assays were carried out in duplicate.
1202, 1171, 1125 cmꢂ1; 1H NMR (400 MHz, DMSO‑d6)
d
11.63 (s, 1H),
Quantification of aggregates of A
(ThT) fluorescence method. ThT (final concentration of 40
added to the assay solutions after incubation and fluorescence in-
tensity was recorded for 300 with an interval of 30
exc ¼ 446 nm; em ¼ 490 nm) [51]. Background signal was
b
was done using Thioflavin T
8.97 (d, J ¼ 6.9 Hz, 2H), 8.37 (d, J ¼ 6.9 Hz, 2H), 8.14 (d, J ¼ 8.1 Hz,
2H), 7.94 (d, J ¼ 8.3 Hz, 2H), 7.80 (d, J ¼ 7.8 Hz, 2H), 7.54 (t, J ¼ 7.5 Hz,
2H), 7.50e7.44 (m, 6H), 5.73 (s, 2H); 13C NMR (101 MHz, DMSO)
mM) was
s
s
d
167.34, 153.16, 145.64, 145.17, 139.13, 135.15, 131.97, 129.69, 129.62,
(l
l
128.99, 127.52, 127.37, 116.54, 62.20. ESI-MS:m/z 365.0 [M ꢂ Br]þ.
1-Ethyl-4-(6-methoxy-2-naphthamido)pyridin-1-ium iodide (4au).
Yield: 85%; white solid; m.p. 241e243 ꢀC; IR (nmax): 3301, 2955,
2924, 2869, 2852, 2357, 2337, 1735, 1695, 1625, 1590, 1518, 1461,
recorded in blank wells containing only tested inhibitors and ThT.
The percentage inhibition due to the presence of test compound
was calculated by the following expression: 100 - (IFi/IFo ꢁ 100),
where IFi and IFo are the respective fluorescence intensities ob-
tained in the presence and in the absence of inhibitor, respectively.
Molecular modeling. The docking studies were carried in the
same way as described earlier [30]. Briefly, the crystal structures of
human AChE (PDB ID: 4EY7) and human BChE (PDB ID: 6EP4) were
retrieved from RCSB-PDB and the proteins were prepared, mini-
mized, optimized and grids were generated in Schrodinger pro-
gram. The ligand 7b was sketched in ChemDraw and prepared
using Ligprep module of Schrodinger program. Finally docking was
carried out using XP glide docking module of Schrodinger program.
The PV file retrieved from XP docking of proteinꢂligand docked
complex was used for the MD simulation studies. The protein-
ligand complex was solvated and neutralized under default
setting of Desmond program. The ligandꢂprotein complex was
minimized by steepest descent method followed by the BFGS
1377, 1325, 1272, 1205, 1190, 1159, 1124, 1026 cmꢂ1 1H NMR
;
(400 MHz, DMSO‑d6)
d
11.62 (s, 1H), 8.89 (d, J ¼ 7.1 Hz, 2H), 8.63 (s,
1H), 8.34 (d, J ¼ 7.1 Hz, 2H), 8.06 (d, J ¼ 9.0 Hz, 1H), 8.02 (s, 2H), 7.48
(d, J ¼ 2.2 Hz, 1H), 7.33 (dd, J ¼ 9.0, 2.4 Hz, 1H), 4.50 (q, J ¼ 7.3 Hz,
2H), 3.95 (s, 3H), 1.53 (t, J ¼ 7.3 Hz, 3H); 13C NMR (101 MHz,
DMSO‑d6)
d 167.70, 159.92, 152.82, 145.19, 137.29, 131.38, 129.77,
128.16, 127.76, 127.63, 125.31, 120.43, 116.18, 106.60, 55.98, 55.17,
16.48. ESI-MS: m/z 307.0 [M ꢂ I]þ.
In vitro AChE and BChE inhibition assay. In vitro cholines-
terase inhibitory assay was performed following Ellman assay
protocol [35,48,49]. Electrophorus electricus acetylcholinesterase
(EeAChE, EC 3.1.1.7, from electric eel, Type VeS, 827 U/mg solid;
1256 U/mg of protein), and butyrylcholinesterase (EqBChE, E.C.
3.1.1.8, from equine serum, 246 U mg/solid, 855 U/mg protein) were

16
M. Abdullaha et al. / European Journal of Medicinal Chemistry 207 (2020) 112761
[4] S. Haider, S. Saleem, T. Perveen, S. Tabassum, Z. Batool, S. Sadir, et al., Age-
related learning and memory deficits in rats: role of altered brain neuro-
transmitters, acetylcholinesterase activity and changes in antioxidant defense
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Author contributions
The manuscript was written through the contributions of all
authors. All authors have approved the final version of the paper. M.
Abdullaha performed the synthesis of compounds and docking
studies; VKN performed biological evaluations; SBB designed, and
coordinated all this work. SBB wrote the manuscript. IIIM Publi-
cation number. CSIR-IIIM/IPR/00214
[8] R.M. Lane, S.G. Potkin, A. Enz, Targeting acetylcholinesterase and butyr-
ylcholinesterase in dementia, Int. J. Neuropsychopharmacol. 9 (1) (2006)
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linesterase and of its complexes with anticholinesterase drugs, Chem. Biol.
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Interest statement
ꢀ
[12] N.C. Inestrosa, A. Alvarez, C.A. Perez, R.D. Moreno, M. Vicente, C. Linker, et al.,
Authors do not have any conflict of interest.
Declaration of competing interest
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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.
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Acknowledgement
M. Abdullaha thanks UGC for the research fellowship. The
financial support from the CSIR YSA grant (P90807) is gratefully
acknowledged.
Abbreviations
AD
Alzheimer’s disease
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butyryl-cholinesterase in the cerebrospinal fluid of patients with Alzheimer’s
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Rate of progression of cognitive decline in Alzheimer’s disease: effect of
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(2005) 640e643.
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for Alzheimer’s disease, Mini Rev. Med. Chem. 20 (8) (2020) 703e715.
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disease, Arch Pharm. Res. (Seoul) 36 (4) (2013) 375e399.
AChE
ACh
BBB
ACN
CAS
CIs
DMF
DMSO
EeAChE
acetylcholinesterase
acetyl choline
Blood Brain Barrier
acetonitrile
catalytic anionic site
cholinesterase inhibitors
dimethyl formamide
dimethyl sulphoxide
acetylcholinesterase from Electrophorus electricus
(electric eel)
Equine serum butyrylcholinesterase
Michaelis-Menten constant
inhibition constant
eqBChE
km
Ki
PAS
PDB
peripheral anionic site
protein data bank
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antioxidant activities of phenol derivatives from 1,6-bis(dimethoxyphenyl)
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rHuAChE recombinant human acetylcholinesterase
SEM standard error of mean
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Molecular-docking-guided design and synthesis of new IAA-tacrine hybrids as
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112761.
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