Chromone derivatives bearing pyridinium moiety as multi-target-directed ligands against Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2021.104750

A new serise of 7-hydroxy-chromone derivatives bearing pyridine moiety were synthesized, and evaluated as multifunctional agents against Alzheimer's disease (AD). Most of the compounds were good AChE inhibitors (IC₅₀ = 9.8–0.71 μM) and showed remarkable BuChE inhibition activity (IC₅₀ = 1.9–0.006 μM) compared with donepezil as the standard drug (IC₅₀ = 0.023 and 3.4 μM). Compounds 14 and 10 showed the best inhibitory activity toward AChE (IC₅₀ = 0.71 μM) and BuChE (IC₅₀ = 0.006 μM), respectively. The ligand–protein docking simulations and kinetic studies revealed that compound 14 and 10 could bind effectively to the peripheral anionic binding site (PAS) of the AChE and BuChE through mixed-type inhibition. In addition, the most potent compounds showed acceptable neuroprotective activity on H₂O₂- and Aβ-induced.neurotoxicity in PC12 cells, more than standard drugs. The compounds could block effectively self- and AChE-induced Aβ aggregation. All the results suggest that compounds 14 and 10 could be considered as promising multi-target-directed ligands against AD.

Full text of DOI:10.1016/j.bioorg.2021.104750

Bioorganic Chemistry 110 (2021) 104750
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Chromone derivatives bearing pyridinium moiety as multi-target-directed
ligands against Alzheimer’s disease
Shahin Abdpoura, Leili Jalili-Balehb, Hamid Nadric, Hamid Forootanfard,
Syed Nasir Abbas Bukharie, Ali Ramazania, Seyed Esmaeil Sadat Ebrahimif,
,g,*
Alireza Foroumadif, Mehdi Khoobif
a Department of Chemistry, University of Zanjan, Zanjan, Iran
b The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 1417614411, Iran
c Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
d Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran
e Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf, Sakaka 2014, Saudi Arabia
f Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14176, Iran
g Biomaterials Group, Pharmaceutical Sciences Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran
1417614411, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new serise of 7-hydroxy-chromone derivatives bearing pyridine moiety were synthesized, and evaluated as
multifunctional agents against Alzheimer’s disease (AD). Most of the compounds were good AChE inhibitors
(IC50 = 9.8–0.71 µM) and showed remarkable BuChE inhibition activity (IC50 = 1.9–0.006 µM) compared with
donepezil as the standard drug (IC50 = 0.023 and 3.4 µM). Compounds 14 and 10 showed the best inhibitory
activity toward AChE (IC50 = 0.71 µM) and BuChE (IC50 = 0.006 µM), respectively. The ligand–protein docking
simulations and kinetic studies revealed that compound 14 and 10 could bind effectively to the peripheral
anionic binding site (PAS) of the AChE and BuChE through mixed-type inhibition. In addition, the most potent
compounds showed acceptable neuroprotective activity on H2O2- and Aβ-induced .neurotoxicity in PC12 cells,
more than standard drugs. The compounds could block effectively self- and AChE-induced Aβ aggregation. All
the results suggest that compounds 14 and 10 could be considered as promising multi-target-directed ligands
against AD.
Alzheimer’s disease
Cholinesterase inhibitors
Neuroprotective activity
Anti-amyloid aggregation
Chromone
Pyridinium salts
1. Introduction
plaques and tangles which is hallmarks in patients with AD. While the
activity of AChE declines in the hippocampus, BuChE activity tends to
Alzheimer’s disease (AD) is the most common cause of dementia and
one of the major causes of mortality in the world. It is one of the most
commonly debilitating disabilities in the elderly. The disease is patho-
physiology associated with the decline in the level of cholinergic neu-
rotransmitters, abnormal formation of amyloid-beta (Aβ) protein
plaques and neurofibrillary collapse, appearance of hyper-
increase. Recently, several evidences have been emerged indicating that
BuChE plays a pivotal role in regulating the ACh level of brain in the late
stage of AD. The AChE level in the brain decreases to 55–67% of normal
values, while BuChE increases to 165% of the normal levels [3–5].
Moreover, AChE knockout mice models showed that BuChE can possibly
substitute for AChE, to destroy ACh in synaptic cleft [6]. In addition,
BuChE inhibition does not lead to peripheral adverse effects [7].
Therefore, development of highly potent and selective BChE inhibitors
with the ability to reinstate ACh levels in the brain and reduced pe-
ripheral side effects, has gained much interest [8]. Furthermore, many
studies have shown that both AChE and BuChE facilitate amyloid fibril
formation resulting in the formation of more toxic ChE-Aβ complexes
phosphorylated
τ-protein aggregation, dyshomeostasis of biometals,
oxidative stress, and neuroinflammation [1,2].
There are two forms of cholinesterase in the brain of mammals
namely acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).
The two forms are different genetically, structurally and in their ki-
netics. BuChE is found in neurons and glial cells as well as in senile
* Corresponding author at: The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 1417614411, Iran.
E-mail address: m-khoobi@tums.ac.ir (M. Khoobi).
https://doi.org/10.1016/j.bioorg.2021.104750
Received 24 August 2020; Received in revised form 16 January 2021; Accepted 13 February 2021
Available online 19 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
[9,10]. Some studies have suggested that AChE forms a complex with Aβ
through the hydrophobic pocket near of peripheral anionic binding site
(PAS) promoting amyloid fibril formation [11]. Therefore, compounds
with the ability to inhibit PAS of the enzyme, not only block ACh
entrance into the catalytic core, but also inhibit the PAS-induced Aβ
oligomerization [12]. In vivo studies also revealed that Aβ plaques
accumulation in the brain tissue is associated with BuChE activity
[13–15].
chromone-based compounds have been evaluated against AD exhibiting
valuable biological activities [36]. Recent study has indicated that
chromone-2-carboxamido-alkylbenzylamine derivatives (Fig. 1) are
interesting inhibitors of AChE/BuChE and Aβ aggregation, with
acceptable antioxidant, and metal chelation activities [37]. The study
revealed that the chromone part has great role in antioxidant and neu-
roprotection activity as well as anti-Aβ aggregation potency of the target
compounds (Fig. 1) [37,38]. Moreover, pyridinium salt is a well-known
pharmacophore having strong binding affinity towards catalytic active
There are tangible shreds of evidence that toxic effect of the Aβ
plaques has great role in the disease initiation and progression [16]. It
has been revealed that during Aβ aggregation, accumulation of intra-
cellular hydrogen peroxide (H2O2) leads to the peroxidation of lipid
membrane of neurons and ultimately to the cell death [17–19]. There-
fore, neuroprotection against Aβ-induced oxidative stress as a key item
in the progression of the disease is recommended to combat against AD
[20]. During the past decades, valuable efforts have been devoted to
discovering disease-modifying drugs controlling the progression of AD,
mainly focused on Aβ peptide, as a widely accepted key problem of AD.
Notwithstanding all attempts, no drug candidates have been approved in
advanced clinical trials since the launching of Memantine, so far.
In modern medicinal chemistry, the multi-target-directed ligand
(MTDL) strategy is a well-established theory for the design and discov-
ery of the multifunctional compounds modulating various receptors or
targeting diverse enzymes to employ for the potential treatment of
complex disorder like AD [21]. Multi-factorial cause of AD requires a
multi-target approach for the treatment which could be achieved
through the combination of effective pharmacophoric groups in a
unique small molecule. This paradigm is more effective than the one-
target, one-drug concept [22,23]. Drug-combination therapies for the
treatment of AD lead to more beneficial therapeutic effects and result in
superior in vivo outcomes compared to the one-target compounds having
high affinity, even if the multi-target small molecules have mild activity
against one or several targets [24].
site (CAS) of AChE through
π-stacking and charge interactions and
various MTDLs bearing this moiety have been introduced so far (Fig. 1)
[39,40].
Considering these findings, and in continuation of our interest in
developing potent MTDLs against AD [41,42], we designed, synthesized,
and evaluated biological activity of a series of novel chromone-based
derivatives bearing pyridinium moiety as multi-targeted inhibitors of
ChEs. We aimed to improve the inhibitory activity of the chromone
scaffold against AChE and BuChE as well as AChE-induced Aβ peptide
aggregation by contribution of pyridinium part in the structure of the
target system. To reach this purpose, aliphatic carbon chain was
employed as cross-linker to endow the target structure with appropriate
flexibility and binding affinity towards ChEs. Docking simulation, in-
hibition mechanism, inhibitory kinetics, anti-Aβ aggregation, nour-
oprotectivity and toxicity of the selected compounds were investigated
to introduce promising multi-targeted-ligands against AD.
2. Results and discussion
2.1. Chemistry
As illustrated in Scheme 1, compound 1a was prepared via addition
of perchloric acid to a solution of 2,4-dihydroxyacetophenone in triethyl
orthoformate [43]. The ethyl ester analogue 1b was prepared from the
reaction of 2,4-dihydroxyacetophenone and diethyl oxalate in ethanolic
solution of sodium [44]. Also, 2,4-dihydroxyacetophenone underwent
cyclization with appropriate benzaldehyde derivatives in the presence of
KOH resulting in the formation of chalcone derivatives. The corre-
sponding flavonoid derivatives (1c-f) were synthesized by refluxing the
chalcone derivative in DMSO with catalytic amount of I2 for 45 min
[45,46]. The intermediates 2a-f were then synthesized via the reaction
of 1a-f with appropriate amount of dibromoalkanes in acetone solution
Chromones with γ-benzopyrone ring are an advantageous frame-
work for drug discovery in medicinal chemistry especially for the
treatment of AD [25]. The core fragment of various flavonoids have the
chromone skeleton representing a wide range of pharmacological ac-
tivities, including their ability to inhibit cholinesterase enzymes
[26,27], anti-Aβ aggregating property [28,29], neuroprotective [30,31],
free radicals scavenging, metal ions chelating [32–34], and anti-
inflammatory activities [35]. Based on MTDLs strategy, some
Fig. 1. Design strategy for the preparation of compounds 3–26.
2

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
Scheme 1. Synthetic routes to compounds 3–26. Reagents and conditions: (a) HC(OEt)3, 70% HClO4 , then H2O, 100 ◦C; (b) Na, EtOH (abs), diethyl oxalate, reflux,
1 h; (c) KOH (30%), MeOH, appropriate benzaldehyde derive, stirr, 72 h, then DMSO, I2, refluxe, 45 min; (d) Br(CH)nBr (n = 3–5), anhydrous K2CO3, acetone, reflux,
4 h; (e) Pyridine, 110 ◦C, 24 h.
of anhydrous K2CO3 under refluxing condition for 4 h. Target com-
Table 1
pounds 3–26 were finally prepared through a reaction between bro-
Inhibitory activity of the synthesized compounds 3–26 against AChE and BuChE.
moalkoxy intermediates and pyridine derivatives at 110 ◦C for 24 h.
Comp.
n
R’
R
eelAChE
IC50 (µM)a
Equine serum BuChE
2.2. Biological assays
IC50 (µM)
%
inhib.b
2.2.1. Anti-cholinesterase activity
3
4
5
6
3
3
3
3
H
H
H
H
H
24.5 ± 0.6
3.1 ± 0.1
3.1 ± 0.1
6.1 ± 0.1
–
23.6
Ph
4.4 ± 0.7
1.4 ± 0.2
1.3 ± 0.2
–
–
–
The results of the anticholinesterase activity of the final compounds
3–26 are seen in Table 1. The activity was expressed as IC50 (µM). For
compounds having no appropriate activity, the percent of inhibition was
reported at 100 µM. Donepezil as a clinically approved ChEs inhibitor
used to treat AD was applied as the standard drug. To investigate the role
of linker’s length, chromone scaffold was attached to the pyridinium
part via 3-, 4- and 5-carbon length (n = 3–5). The role of substituents on
the chromone core and pyridinium part was also evaluated. The results
clearly confirmed that the presence of substituent at position 2 of the
chromone moiety (R) was necessary to improve the inhibitory effect of
the compounds. Most of the compounds bearing substituent at position 2
of the chromone ring showed good to moderare AChE inhibitory activity
(9.8 µM > IC50 > 0.71 µM). The results revealed that 4-carbon chain
length (n = 4) could be considered as the optimum length of the linker
for AChE inhibition, except for the compounds having no substitution at
position 2 of the chromone ring (compounds 3, 11 and 20). Shortening
or elongation of the linker led to decrease in the activity. Compounds 14,
16 and 22 were the best AChE inhibitors with IC50 value of about 0.7
µM. By comparison of the results, it could be concluded that the presence
of substituent (ethyl group) at para position of the pyridinium group
improved ChE inhibitory effect of the compounds having 3- or 4-carbon
linker length.
4-Cl-Ph
4-OMe-
Ph
7
8
3
3
H
COOEt
Ph
1.0 ± 0.1
2.7 ± 0.1
82.0 ± 1.7
0.090 ±
0.014
–
–
Et
9
3
3
Et
Et
4-Cl-Ph
4.1 ± 0.1
2.5 ± 0.1
0.097 ±
0.011
–
–
10
4-OMe-
Ph
0.006 ±
0.001
11
12
13
14
15
4
4
4
4
4
H
H
H
H
H
H
32.4 ± 2.5
2.5 ± 0.2
1.2 ± 0.1
0.71 ± 0.1
1.4 ± 0.1
–
46.3
Ph
1.9 ± 0.2
1.1 ± 0.1
1.5 ± 0.2
1.4 ± 0.2
–
–
–
–
2-F-Ph
4-Cl-Ph
4-OMe-
Ph
16
17
4
4
H
COOEt
Ph
0.75 ± 0.1
2.6 ± 0.2
8.4 ± 1.6
0.012 ±
0.001
–
–
Et
18
19
4
4
Et
Et
4-Cl-Ph
2.5 ± 0.2
9.8 ± 0.6
0.087 ±
0.005
–
–
4-OMe-
Ph
0.076 ±
0.009
20
21
22
23
5
5
5
5
H
H
H
H
H
50.2 ± 1.4
6.8 ± 0.5
0.79 ± 0.1
2.7 ± 0.2
27.8 ± 0.9
0.63 ± 0.12
0.46 ± 0.06
0.99 ± 0.12
–
–
–
–
Ph
4-Cl-Ph
4-OMe-
Ph
Also, most of the compounds had higher BuChE inhibition activity
than AChE inhibition activity. 17 out of 23 compounds were more active
than donepezil as standard drug and 10 derivatives showed BuChE in-
hibition activity in nanomolar range (compounds 8–10, 17–19 and
21–24). Compound 10 with IC50 = 0.006 µM was 566 times more active
than standard drug, donepezil (BuChE IC50 = 3.4 µM). Such compounds
with high selectivity for BuChE may represent an additional advantage
for long-term stabilization of cognitive and behavioral symptoms,
particularly in progressed or late stage of AD patients, through reducing
β-amyloid aggregation and increasing ACh levels in the brain without
triggering severe peripheral side effects [8]. Compound 14 as the best
24
5
Et
Ph
1.1 ± 0.1
0.055 ±
0.001
–
4.4 ± 0.8
–
25
26
5
5
Et
Et
4-Cl-Ph
4-OMe-
Ph
24.5 ± 0.8
3.1 ± 0.3
23.6
–
Donepezil
–
–
–
0.023 ±
3.4 ± 0.2
–
0.002
a
Inhibitor concentration (mean ± SEM of three experiments) for 50% inac-
tivation of AChE (from electrophorus electricus)/BuChE (from equine serum).
b
Percentage of inhibition of BuChE (mean ± SEM of three experiments).
AChE inhibitor having appropriate BuChE inhibition activity (IC50
=
2.2.2. Kinetic analysis of AChE and BuChE inhibition
0.71 and 1.5 µM, respectively) and compound 10 as the best BuChE
inhibitor (IC50 = 0.006 µM) were selected for further analyses.
The enzyme kinetic study was performed to understand the mode of
inhibition. For this purpose, compound 10 and 14 were selected for
kinetic analysis of AChE and BuChE inhibition, respectively. The
3

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
Fig. 2. Left) Lineweaver-Burk plot for the inhibition of AChE by compound 14 at different concentrations of substrate (ATCh). Right) Secondary plot for calculation of
steady-state inhibition constant of compound 14 (Ki = 1.01 µM).
Fig. 3. Left) Lineweaver-Burk plot for the inhibition of BuChE by compound 10 at different concentrations of substrate (BuTCh). Right) Secondary plot for calculation
of steady-state inhibition constant of compound 10 (Ki = 10.25 nM).
Lineweaver-Burke plot for AChE is shown in Fig. 2. Based on the plot,
compound 14 showed mixed type of inhibition. The Ki value was also
calculated using secondary plot (Ki = 1.01 µM). Accordingly, the inhi-
bition mode of compound 10 on BuChE was investigated. The results
revealed that compound 10 inhibited BuChE in the mixed type manner
(Fig. 3). The Ki for compound 10 was 10.25 nM. Based on the kinetic
results, it could be suggested that both the compounds showed non-
competitive inhibition which is kind of reversible inhibition and also
could occupy CAS and PAS, simultaneously.
2.2.3. Ligand-protein docking simulation
Docking study was performed to determine the key residues in the
ligand involved in the interaction with the enzymes. Compound 14 was
used as ligand for AChE and compound 10 for BuChE. The main ligand-
enzyme interactions between compound 14 and AChE was illustrated in
Fig. 4. As shown, compound 14 occupied both CAS and PAS. The posi-
tive charge of pyridine was fascinated to the central anionic site. Trp83
and Glu198 provide negative charge to interact with pyridinium ring.
Moreover, Gly116 in the oxyanion hole made an amide-
pyridine. The head of the ligand was directed to the rim of the gorge and
engaged in some -staking. Phenyl ring of chromone stacked against
Phe329 in the mid-gorge recognition site. The adjacent ring made two
π bond with
π
π
-staking with Tyr120 and Tyr333 in the PAS of the enzyme. The phenyl
at position 2 of the chromone ring was also involved in
Tyr333.
π-stacking with
Fig. 4. Interaction of compound 14 with AChE. For simplification the key
Compound 10 was also docked in the active site of BuChE (Fig. 5).
residues were shown.
4

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
The calculated RMSD was 0.42 Å (Fig. 6).
2.2.4. Inhibitory effects on AChE-induced and self-induced Aβ peptide
aggregation
The potential of compounds 10 and 14 to inhibit Aβ-aggregation was
evaluated using thioflavin T (ThT) assay. The obtained results indicated
that compounds 10 and 14 displayed significant inhibitory activity
against Aβ aggregation (31.7 and 27.5% inhibition, respectively) and
compound 10 was about 2-fold more effective than reference drug
donepezil (15.1% inhibition, Table 2). The potential of compounds 10
and 14 to inhibit Aβ aggregation induced by AChE were also evaluated
(Table 2). Compound 10 showed appropriate inhibition activity toward
AChE-induced Aβ aggregation (36.6% inhibition) more than donepezil
as standard drug (26.9% inhibition). Compound 14 also exhibited
acceptable inhibition activity (21.8% inhibition). The results confirmed
appropriate efficacy of the selected compounds to inhibit Aβ
aggregation.
2.2.5. Neuroprotection against H2O2-induced cell death in PC12 cells
The neuroprotective effect of compounds 10 and 14 against neuro-
toxicity caused by H2O2 at the concentrations of 0.1, 1, 5, 10, 20, 50 µM,
and prior to treatment with H2O2⋅(150 µM) were evaluated via MTT
assay. As seen in Table 3, all the tested concentrations significantly
increased PC12 cell viability in a concentration-dependent manner for
both compounds. Compound 10 had no significant effect on cell viability
just at concentration of 0.1 µM. Notably, the neuroprotective effect of
compound 14 at concentrations of 0.1, 1, 5, 10 and 20 µM was higher
than that of the reference drug Quercetin.
Fig. 5. Interaction of compound 10 with BuChE. For simplification the key
residues were shown.
2.2.6. Neuroprotective activity against Aβ1-42-induced cytotoxicity in PC12
cells
It has been reported that Aβ plaques induce oxidative stress on
neuronal cells through the formation of free radicals [47]. Therefore, the
potential neuroprotective effects of compounds 10 and 14 against Aβ1-
42-induced cytotoxicity in PC12 cells were evaluated at the concentra-
tions of 0.01, 0.1, 10 and 100 μM. As seen in Fig. 7, the tested com-
pounds increased cell viability in the presence of Aβ in a concentration-
dependent manner. According to the results, compounds 10 and 14
showed significant protective capability with the percentage of cell
viability of 79 and 76%, respectively, at the concentrations of 100 μM,
Fig. 6. Redocking of donepezil in the active site of AChE. The green is original
ligand (1EVE PDB) and the cyan is the redocked ligand. (For interpretation of
the references to color in this figure legend, the reader is referred to the web
version of this article.)
higher than reference drug Selegiline (52%) and showed slightly lower
protective effect at other concentrations. Compound 10 could protect
neuronal cells at concentration of 1–100 μM, greater than compound 14.
The results suggested that target compounds 10 and 14 could efficiently
protect neuronal cells against Aβ toxic effects.
Table 2
Inhibition of Aβ self and AChE-induced aggregation by the compound 10 and 14.
2.2.7. Cytotoxicity effect
To evaluate the cytotoxic effect of the selected compounds, the MTT
based colorimetric assay was employed. The IC50 values of compounds
10 and 14 compared with donepezil as the reference drug are seen in
Table 4. The results exhibited that both compounds 10 and 14 showed
lower cytotoxic effect on normal cell (HDF) compared with two cancer
cell lines (Table 4). In addition, compound 10 was somehow more toxic
than compound 14 against all applied three cell lines. Interestingly, the
cytotoxic effect of the selected compounds was lower than donepezil as a
clinically approved standard drug.
Compound
Inhibition of self-induced Aβ
Inhibition of AChE-induced Aβ
aggregationa (%)
aggregationb (%)
10
31.7 ± 1.5
27.5 ± 1.7
15.1 ± 1.8
36.6 ± 1.2
21.8 ± 2.4
26.9 ± 3.0
14
Donepezil
a
Inhibition of self-induced Aβ1-42 aggregation (25 µM) produced by the tested
compound at 10 µM concentration. Values are expressed as means ± SEM of
three experiments.
b
Co-aggregation inhibition of Aβ1-42 and AChE (2 µM, ratio 100:1) by the
tested compound at 100 µM concentration was detected by ThT assay. Values are
expressed as means ± SEM of three experiments.
2.2.8. ADMET prediction
The ADMET (absorption, distribution, metabolism, excretion and
toxicity) property of the compounds was predicted using the Swis-
sADME web server (http://www.swissadme.ch) [48]. Based on the
predicted values, all compounds could be able to penetrate brain and
therefore could be considered as CNS-active compounds, except for
compound 25. Also, the compounds showed well gastrointestinal (GI)
absorption property (Table 5). The computed physicochemical de-
scriptors, pharmacokinetic properties and druglike nature of compounds
Similar to compound 14, the pyridine ring of compound 10 was adjacent
to Trp82 and Glu197 by making attractive charges and the planar
chromone ring stacked against Tyr332 in the PAS.
To confirm the robustness of the docking simulations, the original
donepezil-AChE complex, retrieved from RCSB (1EVE PDB) was re-
docked and its superimposition with the original X-ray structure was
rechecked by calculation of the Root Mean Square Deviation (RMSD).
5

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
Table 3
The protective effect of compound 10 and 14 against H2O2-induced injury in PC12 cell line at different concentrations.
Compound
n
R
R’
PC12 cells viability (% of control)
H2O2
0.1 µM
1 µM
5 µM
10 µM
20 µM
50 µM
10
3
4
–
Et
H
–
4-OMe-Ph
4-Cl-Ph
–
33.0 ± 1.0
33.2 ± 1.3
31.9 ± 1.7
34.4 ± 0.8 ns
47.7 ± 1.8***
37.1 ± 2.0***
37.7 ± 1.5***
57.8 ± 1.3***
43.6 ± 1.6***
42.3 ± 0.9***
61.9 ± 1.1***
46.6 ± 0.4***
53.4 ± 1.3***
66.1 ± 1.3***
51.3 ± 1.6***
58.2 ± 0.4***
69.9 ± 6.5***
56.2 ± 2.0***
47.2 ± 1.8***
49.7 ± 1.3***
59.9 ± 2.3***
14
Quercetin
Cell viability was determined using MTT assay protocol. Data are expressed as the mean ± SEM of three independent replicates. The significant (***p < 0.001) and not
significant (ns) values with respect to the H2O2 group.
considered as promising multifunctional agents for further studies in the
field of AD. Study on the other potency of the target compounds in an-
imal models will be the objective of our future works.
4. Experimental
4.1. Chemistry
All starting materials and solvents for the synthesis were purchased
commercially from various suppliers and used without any purification.
The solvents were dehydrated according to the standard methods. The
melting points were recorded on a Kofler hot stage apparatus. The purity
of all products was confirmed by TLC, FTIR, 1H NMR and 13C NMR. TLC
Fig. 7. Protective effects of compounds 10 and 14 on cell injury induced by
was conducted on silica gel 250 µm, F254 plates (Merck). FTIR spectra
were recorded on a Bruker tensor 27 FT/IR spectrophotometer (KBr
disks), 1H NMR and 13C NMR spectra were recorded on a Brucker 500
MHz NMR and Bruker avence 250 MHz spectrometer using dime-
thylsulfoxide (DMSO‑d6) as solvent. The chemical shifts (δ) and coupling
constants (J) were presented in parts per million (ppm) and Hertz (Hz).
The elemental analysis of the compounds (C, H, and N) was determined
with CHN-Rapid Heraeus Elemental Analyzer agreed to within ±0.4% of
the calculated value. HPLC (Youngling 9100, South Korea) was served to
plot the chromatogram using a C18 column (150 × 4.6 mm) and the
mobile phase consisted of acetonitrile:water (60:40) was employed.
Methanol was applied as solvent. The flow rate was 1.2 mL/min. Ul-
traviolet absorption was read at 262 nm and the compound was iden-
tified using a diode array detector.
Aβ1-42 in PC12 cells. All groups were treated with 25 μM Aβ1-42 except for the
control group. The synthetic compounds and Selegiline were pre-incubated at
various concentrations (0.01–100 μM) in serum-free media for 24 h before the
addition of Aβ peptide. Cell viability is expressed as the mean percentage of
viable cells compared with the untreated cells. The data are the mean ± SE. (See
supporting information for details, Table S1).
Table 4
Calculated IC50 (µM) for compounds 10 and 14 and donepezil against PC12 (rat
pheochromocytoma), HepG2 (human hepatocarcinoma) and human dermal
fibroblast (HDF).
Compound
PC12
HepG2
HDF
10
336.2 ± 9.4
376.4 ± 10.1
314.2 ± 8.7
281.1 ± 7.2
312.9 ± 12.0
256.2 ± 4.9
543.2 ± 19.7
628.4 ± 19.6
363.4 ± 8.7
14
Donepezil
4.1.1. General procedure for the preparation of compound 1a
IC50s were determined using MTT assay protocol. Data are expressed as the
To a stirring solution of 2,4-dihydroxyacetophenone (1 mmol) in
triethyl orthoformate (1 mL), 70% perchloric acid (0.1 mL) was added
dropwise. The dark mixture was stirred until it was cooled to room
temperature. The anhydrous diethyl ether (3 mL) was added and then a
brown precipitate was filtered. The solid dissolved in hot water (2 mL),
and refluxed for 5 min. The mixture was allowed to cool overnight. A
dark product was filtered and recrystallized with water/ethanol [43].
mean ± SEM of three independent replicates.
10 and 14 as the most promising compound have been also provided in
supporting information (see supporting information, Figs. S1–S4).
3. Conclusion
In this work a series of chromone derivatives bearing pyridinium part
cross-linked to each other via flexible aliphatic carbon chain was pre-
pared and evaluated as multi-target directed ligands against AD. Most of
the compounds showed good to moderate AChE inhibitory activity and
excellent BuChE inhibition effect. A series of the compounds (17 de-
rivatives, compounds 5, 6, 8-10, 12-15, 17-19, 21-24) exhibited strong
BuChE inhibitory effect, more than standard drug, donepezil. Especially,
compound 10 was 566 times more active than donepezil. The most
active ChE inhibitors (compounds 10 and 14) revealed high neuro-
protective activity against H2O2-induced cytotoxicity in PC12 cells.
Especially, compound 14 as the best AChE inhibitor (IC50 = 0.71 µM)
showed higher neuroprotective activity than reference drug, Quercetin.
Both compounds could efficiently protect neuronal cells against Aβ toxic
effects and were more active than the standard drug, Selegiline, to
protect PC12 cells against Aβ1-42-induced cytotoxicity. Interestingly, the
selected compounds revealed valuable self- and AChE-induced anti-Aβ
aggregation activity, especially compound 10 was more active than
donepezil as standard drug. All the aforementioned findings suggest that
these new hybrids of chromone and pyridinium parts could be
4.1.2. General procedure for the preparation of compound 1b
A mixture of 2,4-dihydroxyacetophenone (2.28 g) and diethyl oxa-
late (7.3 g) in ethanol (10 mL) was added to a solution of in situe pre-
pared sodium ethoxide by adding 1.8 g sodium to EtOH (100 mL) and
the solution was then refluxed for 1 h. The solution was acidified and the
white precipitate was filtered off, then the solution was extracted with
ethylacetate and water. The product was recrystallized from ethyl-
acetate [44].
4.1.3. General procedure for the preparation of compounds 1c-f
2,4-dihydroxyacetophenone (10 mmol) was mixed with appropriate
benzaldehyde derivatives (10 mmol) in the presence of KOH solution
(30%, in 30 mL methanol) and stirred for 72 h. After completion of the
reaction (TLC control), the mixture was acidified and the product
extracted with ethylacetate/water and purified by column chromatog-
raphy [45]. Then, the purified chalcone was refluxed in DMSO with
catalytic amount of I2 for 45 min. The mixture was washed with water
and the product was extracted with ethylacetate. The solvent was
6

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
Table 5
The ADMET properties of the compounds predicted using SwissADME web-based server.
Compound
n
R’
R
TPSA (Å2)
Consensus logP
BBB permeation
GI absorption
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
–
H
H
H
H
H
Et
Et
Et
H
H
H
H
H
H
Et
Et
Et
H
H
H
H
Et
Et
Et
–
H
43.32
43.32
43.32
52.55
69.62
43.32
43.32
52.55
43.32
43.32
43.32
43.32
52.55
69.62
43.32
43.32
52.55
43.32
43.32
43.32
52.55
43.32
43.32
52.55
33.77
1.79
3.26
3.81
3.27
2.10
3.93
4.43
3.94
2.14
3.59
3.75
4.15
3.58
2.41
4.25
4.76
4.21
2.56
3.92
4.37
3.93
4.57
5.91
4.56
4.00
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ꢀ
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
4
Ph
5
4-Cl-Ph
4-OMe-Ph
COOEt
Ph
6
7
8
9
4-Cl-Ph
4-OMe-Ph
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Donepezil
Ph
2-F-Ph
4-Cl-Ph
4-OMe-Ph
COOEt
Ph
4-Cl-Ph
4-OMe-Ph
H
Ph
4-Cl-Ph
4-OMe-Ph
Ph
4-Cl-Ph
4-OMe-Ph
–
removed under vacuum [46].
pyridine); 13C NMR (DMSO‑d6, 125 MHz) δ 29.75, 58.76, 65.9, 101.5,
106.8, 114,9, 117.3, 126.2, 128.0, 129.1, 131.1, 131.8, 145.1, 145.7,
157.4, 162.2, 162.5, 176.4. Anal. Calcd for C23H20NO3 (358.41): C,
77.08; H, 5.62; N, 3.91. Found: C, 77.92; H, 5.82; N, 3.84.
4.1.4. General procedure for the preparation of bromoalkoxy intermediates
2a-f
The hydroxylayed chromone derivative (1 mmol) with appropriate
dibromoalkane (10 mmol) and anhydrous K2CO3 (2 mmol) was refluxed
in acetone (5 mL) for 4 h. After cooling, the reaction mixture was filtered
and the organic phase was evaporated under reduced pressure. The
obtained residue was triturated with n-hexane to give the product as
white solid [49].
4.1.5.3. 1-(3-((2-(4-Chlorophenyl)-4-oxo-4H-chromen-7-yl) oxy) propyl)
pyridin-1-ium (5). Yellow solid; yield 90%; mp 150–151 ◦C; IR (KBr,
cmꢀ 1
)
υmax: 2852–2926 (C H), 1628 (C O), 1007 (C O); 1H NMR
– –
–
–
–
(DMSO‑d6, 250 MHz) δ 2.47 (bs, 2H, CH2), 4.25 (bs, 2H, O CH2), 4.85
–
(t, 2H, J = 7.5 Hz, N CH2), 6.79 (d, 1H, J = 8.7 Hz, H6), 6.94 (s, 1H,
′
′
H3), 7.23 (s, 1H, H8), 7.59 (d, 2H, J = 8.2 Hz, H1 , 5 ), 7.85 (d, 1H, J = 8.7
4.1.5. General procedure for the preparation of the final compounds
(3–26)
′
Hz, H5), 8.05 (d, 2H, J = 8.5 Hz, pyridine), 8.16 (t, 2H, J = 6.5 Hz, H2 ,
′
4 ), 8.62 (t, 1H, J = 7.5 Hz, pyridine), 9.20 (d, 2H, J = 5.0 Hz, pyridine);
13C NMR (DMSO‑d6, 62.9 MHz) δ 30.1, 59.0, 66.3, 101.9, 107.5, 115.1,
117.6, 126.64, 128.45, 129.2, 129.61, 130.4, 131.5, 136.9, 145.5,
146.1, 157.7, 161.4, 162.9, 176.7. Anal. Calcd for C23H19ClNO3
(392.85): C, 70.32; H, 4.87; N, 3.57. Found: C, 70.12; H, 5.13; N, 3.31.
The bromoalkoxy intermediate (2a-f, 1 mmol) was stirred in pyri-
dine (15 mL) at 110 ◦C for 24 h. After completion of the reaction (TLC
control), the reaction mixture was put in ice bath and cold ether was
added to the mixture. Then, the precipitated product was filtered off and
washed with ether [50].
4.1.5.4. 1-(3-((2-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl) oxy) pro-
pyl) pyridin-1-ium (6). Yellow solid; yield 95%; mp 130–132 ◦C; IR (KBr,
4.1.5.1. 1-(3-((4-Oxo-4H-chromen-7-yl) oxy) propyl) pyridin-1-ium (3).
Pink solid; yield 90%; mp 164–166 ◦C; IR (KBr, cmꢀ 1
)
υmax: 3039–3093
–
–
cmꢀ 1
)
υmax: 3057 (C H aromatic), 2927 (C H), 1621 (C O), 1023
–
–
(C H aromatic), 2882–2941 (C H), 1636 (C O), 1045 (C O); 1H
–
–
–
–
–
–
(C O); 1H NMR (DMSO‑d6, 250 MHz,) δ 2.47 (bs, 2H, CH2), 3.82 (s, 3H,
NMR (DMSO‑d6, 500 MHz) δ 2.45 (bs, 2H, CH2), 4.25 (t, 2H, J = 5.6 Hz,
–
OCH3), 4.25 (t, 2H, J = 5.0 Hz, O CH2), 4.84 (t, 2H, J = 6.5 Hz,
–
–
O
CH2), 4.83 (t, 2H, J = 7.0 Hz, N CH2), 6.28 (d, 1H, J = 6.0 Hz, H3),
–
′
′
N
CH2), 6.76–6.89 (m, 2H, H3,6), 7.07 (d, 2H, J = 8.7 Hz, H2 , 4 ), 7.20
6.84 (d, 1H, J = 7.5 Hz, H6), 7.07 (s, 1H, H8), 7.91 (d, 1H, J = 9.0 Hz,
H5), 8.17 (t, 2H, J = 7.0 Hz, pyridine), 8.24 (d, 1H, J = 5.5 Hz, H2), 8.63
(t, 1H, J = 8.0 Hz, pyridine), 9.16 (d, 2H, J = 6.0 Hz, pyridine); 13C NMR
(DMSO‑d6, 125 MHz) δ 26.4, 28.6, 31.9, 50.0, 109.4, 114.4, 122.3,
126.2, 127.8, 128.0, 128.7, 144.2, 162.9, 196.0; Anal. Calcd for
(d, 1H, J = 2.0 Hz, H8), 7.84 (d, 1H, J = 2.5 Hz, H5), 7.99 (d, 2H, J = 8.7
′
′
Hz, H1 , 5 ), 8.15 (t, 2H, J = 6.7 Hz, pyridine), 8.61 (t, 1H, J = 7.7 Hz,
pyridine), 9.18 (d, 2H, J = 7.2 Hz, pyridine); 13C NMR (DMSO‑d6, 62.9
MHz) δ 30.1, 55.9, 59.1, 66.2, 101.8, 105.6, 114.98, 117.6, 123.6,
126.5, 128.4, 145.5, 146.1, 157,6, 162.4, 162.7, 176.7. Anal. Calcd for
C
17H16NO3 (282.32): C, 72.33; H, 5.71; N, 4.96. Found: C, 72.55; H,
C
24H22NO4 (388.44): C, 74.21; H, 5.71; N, 3.61. Found: C, 74.51; H,
5.94; N, 5.18.
5.82; N, 3.82.
4.1.5.2. 1-(3-((4-Oxo-2-phenyl-4H-chromen-7-yl) oxy) propyl) pyridin-1-
4.1.5.5. 1-(3-((2-(Ethoxycarbonyl)-4-oxo-4H-chromen-7-yl) oxy) propyl)
ꢀ 1
◦
pyridin-1-ium (7). Yellow solid; yield 80%; mp 89–90 ◦C; IR (KBr, cmꢀ 1
)
ium (4). Yellow solid; yield 90%; mp 95–96 C; IR (KBr, cm
) υmax:
–
–
–
–
–
3051 (C H aromatic), 2925 (C H), 1604 (C O), 1172 (C O); 1H
–
–
–
υmax: 3053 (C H aromatic), 2936–2968 (C H), 1732 (C O), 1017
–
NMR (DMSO‑d6, 500 MHz) δ 2.5 (bs, 2H, CH2), 4.28 (t, 2H, J = 5.0 Hz,
–
(C O); 1H NMR (DMSO‑d6, 500 MHz) δ 1.33 (t, 3H, J = 6 Hz,
–
–
O
CH2), 4.86 (t, 2H, J = 6.0 Hz, N CH2), 6.84 (d, 1H, J = 9.0 Hz, H6),
– –
– –
–
O
O
CH2 CH3), 2.48 (bs, 2H, CH2), 4.28 (bs, 2H, O CH2), 4.37 (bs, 2H,
′
′
6.97 (s, 1H, H3), 7.27 (s, 1H, H8), 7.37 (t, 2H, J = 6.0 Hz, H1 , 5 ), 7.58 (m,
–
CH2 CH3), 4.84 (bs, 2H, N CH2), 6.88 (m, 2H, H6,8), 7.19 (m, 1H,
′
′
′
3H, H2 , 3 ,4 ), 8.08 (d, 1H, J = 7.0 Hz, H5), 8.18 (t, 2H, J = 6.8 Hz,
H3), 7.91 (d, 1H, J = 9.0 Hz, H5) , 8.17 (m, 2H, pyridine), 8.63 (t, 1H, J
= 7.5 Hz, pyridine), 9.18 (d, 2H, J = 5.0 Hz, pyridine); 13C NMR
pyridine), 8.64 (t, 1H, J = 7.5 Hz, pyridine), 9.20 (d, 2H, J = 6.0 Hz,
7

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
(DMSO‑d6, 125 MHz) δ 13.9, 29.6, 58.6, 62.7, 66.0, 101.5, 114.0, 115.9,
117.8, 126.4, 128.0, 145.1, 145.7, 152.0, 157.2, 160.0, 163.2, 176.3.
Anal. Calcd for C20H20NO5 (354.38): C, 67.79; H, 5.69; N, 3.95. Found:
C, 67.41; H, 5.83; N, 3.76.
(DMSO‑d6, 125 MHz) δ 25.0, 27.6, 60.3, 67.8, 101.5, 106.8, 115.0,
117.1, 126.2, 128.2, 129.1, 131.7, 144.8, 145.6, 157.5, 162.2, 163.0,
176.4. Anal. Calcd for C24H22NO3 (372.44): C, 77.40; H, 5.95; N, 3.76.
Found: C, 77.65; H, 5.76; N, 3.91.
4.1.5.6. 4-Ethyl-1-(3-((4-oxo-2-phenyl-4H-chromen-7-yl) oxy) propyl)
4.1.5.11. 1-(4-((2-(2-Fluorophenyl)-4-oxo-4H-chromen-7-yl) oxy) butyl)
pyridin-1-ium (8). Yield 60%; Brown solid; mp 134–135 ◦C; IR (KBr,
pyridin-1-ium (13). Brown solid; yield 96%; mp 92–93 ◦C; IR (KBr,
cmꢀ 1
)
υmax: 3050 (C H aromatics), 2924 (C H), 1629 (C O), 1173
cmꢀ 1
) υmax: 3058 (C H aromatic), 2852–2924 (C H), 1743 (C O),
– –
–
–
–
–
–
–
1
1
–
–
1029 (C O); H NMR (DMSO‑d6, 500 MHz,) δ 1.80 (bs, 2H, CH2), 2.12
(C O). H NMR (DMSO‑d6, 250 MHz) δ 1.22 (t, 3H, J = 7.5 Hz, CH3),
–
–
–
–
2.60 (m, 2H, CH2), 2.89 (bs, 2H, CH3 CH2), 4.28 (bs, 2H, O CH2), 4.75
(bs, 2H, CH2), 4.18 (bs, 2H, O CH2), 4.71 (t, 2H, J = 5.5 Hz, N CH2),
–
(bs, 2H, N CH2), 6.98 (s, 1H, H3), 7.05 (bs, 1H, H6), 7.20 (s, 1H, H8),
6.71 (s,1H, H3), 7.07 (d, 1H, J = 8 Hz, H6), 7.27 (s, 1H, H8), 7.48–7.43
′
′
′
′
′
′
7.57 (m, 3H, H2 , 3 , 4 ), 7.77–8.18 (m, 4H, pyridine and H5), 9.01 (d, 2H,
J = 6.0 Hz, pyridine); 13C NMR (DMSO‑d6, 62.9 MHz) δ 13.9, 28.4, 29.9,
58.3, 66.3, 101.9, 107.2, 115.1, 126.6, 126.8, 127.5, 129.5, 132.2,
144.7, 164.3, 176.8. Anal. Calcd for: C25H24NO3 (386.46): C, 77.70; H,
6.26; N, 3.62. Found: C, 77.92; H, 6.03; N, 3.45.
(m, 2H, H2 , 4 ), 7.66–7.67 (m, 1H, H5), 7.95 (d, 1H, J = 8.5 Hz, H1 ) ,
′
8.00 (t, 1H, J = 7.5 Hz, H3 ), 8.18 (t, 2H, J = 6.5 Hz, pyridine), 8.62 (t,
1H, J = 7.5 Hz, pyridine), 9.15 (d, 2H, J = 5.0 Hz, pyridine); 13C NMR
(DMSO‑d6, 125 MHz) δ 25.0, 27.6, 60.4, 67.8, 101.4, 115.3, 116.9,
117.0, 125.2, 126.3, 128.2, 129.5, 144.8, 156.1 (C-F), 157.6, 163.2,
176.1. Anal. Calcd for C24H21FNO3 (390.43): C, 73.83; H, 5.42; N, 3.59.
Found: C, 73.43; H, 5.22; N, 3.44.
4.1.5.7. 1-(3-((2-(4-Chlorophenyl)-4-oxo-4H-chromen-7-yl)oxy)propyl)-
4-ethylpyridin-1-ium (9). Yield 60%; off Brown solid; mp 144–145 ◦C; IR
(KBr, cmꢀ 1
)
4.1.5.12. 1-(4-((2-(4-Chlorophenyl)-4-oxo-4H-chromen-7-yl) oxy) butyl)
–
– –
υmax: 3023 (C H aromatics), 2968 (C H), 1633 (C O),
–
1
–
1174 (C O). H NMR (DMSO‑d6, 250 MHz) δ 1.25 (t, 3H, CH3), 2.60
pyridin-1-ium (14). Yellow solid; yield 88%; HPLC tR 3.437 min (99%);
(2H, CH2), 2.91 (2H, CH3 CH2), 4.25 (bs, 2H, O CH2), 4.75 (bs, 2H,
mp 111–112 ◦C; IR (KBr, cmꢀ 1
)
υmax: 3038 (C H aromatic), 2957
–
–
–
1
–
–
–
–
–
N
CH2), 6.80 (d, 1H, J = 8.2 Hz, H6), 6.98 (s,1H, H3), 7.22 (bs, 1H, H8),
(C H), 1642 (C O), 1013 (C O); H NMR (DMSO‑d6, 500 MHz) δ 1.8
′
′
′
′
–
7.62 (d, 2H, J = 8.5 Hz, H1 , 5 ), 7.87 (d, 2H, J = 9.0 Hz, H2 , 4 ), 8.04 (m,
2H, pyridine), 8.42 (d, 1H, J = 5.5 Hz, H5), 9.05 (d, 2H, J = 6.2 Hz,
pyridine); 13C NMR (DMSO‑d6, 62.9 MHz) δ 13.9, 28.4, 30.0, 58.3, 66.3,
101.9, 107.5, 115.2, 117.6, 123.1, 126.6, 127.5, 128.4, 129.6, 130.4,
136.9, 144.7, 149.1, 157.7, 161.4, 162.9, 164.3, 176.7. Anal. Calcd for:
(m, 2H, CH2), 2.13 (bs, 2H, CH2), 4.19 (t, 2H, J = 6.0 Hz, O CH2), 4.74
–
(t, 2H, J = 7.5 Hz, N CH2), 6.99 (s,1H, H3), 7.04 (d,1H, J = 9.0 Hz, H6),
′
′
7.32 (s,1H, H8), 7.63 (d, 2H, J = 8.0 Hz, H1 , 5 ), 7.76 (d, 2H, J = 8 Hz,
′
′
H
2 , 4 ), 7.92 (d, 1H, J = 9.5 Hz, H5), 8.19 (t, 2H, J = 6.0 Hz, pyridine),
8.63 (t, 1H, J = 8.0 Hz, pyridine), 9.18 (d, 2H, J = 6 Hz, pyridine); 13C
NMR (DMSO‑d6, 125 MHz) δ 25.0, 27.6, 60.4, 67.8, 101.5, 107.1, 115.1,
117.1, 123.9, 126.2, 128.0, 128.2, 136.2, 144.8, 149.6, 157.4, 161.0,
163.1, 174.4. Anal. Calcd for C24H21ClNO3 (406.89): C, 70.85; H, 5.20;
N, 3.44. Found: C, 70.93; H, 5.21; N, 3.13.
C
25H23ClNO3 (420.91): C, 71.34; H, 5.51; N, 3.33. Found: C, 71.12; H,
5.88; N, 3.61.
4.1.5.8. 4-Ethyl-1-(3-((2-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)
oxy)propyl)pyridin-1-ium (10). Yield 70%; off yellow solid; mp
100–102 ◦C; IR (KBr, cmꢀ 1
)
υmax: 3046 (C H aromatic), 2972 (C H),
4.1.5.13. 1-(4-((2-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)
oxy)
–
–
1
1640 (C O), 1179 (C O). H NMR (DMSO‑d6, 250 MHz) δ 1.23 (t, 3H,
butyl) pyridin-1-ium (15). Yellow solid; yield 90%; mp 190–191 ◦C; IR
–
–
–
J = 5.5 Hz, CH3), 1.96 (bs, 2H, CH2), 2.91 (bs, 2H, CH3 CH2), 3.84 (s,
(KBr, cmꢀ 1
)
υmax: 3045 (C H aromatic), 2852–2926 (C H), 1630
–
–
–
1
–
–
–
–
3H, OCH3), 4.25 (bs, 2H, O CH2), 4.76 (bs, 2H, N CH2), 6.81–6.85 (m,
(C O), 1025 (C O); H NMR (DMSO‑d6, 500 MHz,) δ 1.81 (t, 2H, J =
–
′
′
′
′
2H, H3,6), 7.18–727 (m, 3H, H8 and H2 ,4 ), 7.86–8.03 (m, 5H, H5, H1 ,5
8.0 Hz, CH2), 2.12 (t, 2H, J = 6.0 Hz, CH2), 3.85 (s, 3H, OCH3), 4.19 (t,
and pyridine), 9.02 (d, 2H, J = 6.0 Hz, pyridine); 13C NMR (DMSO‑d6,
62.9 MHz) δ 13.9, 28.4, 29.9, 56.0, 58.3, 66.3, 101.9, 105.7, 115.0,
127.5, 128.4, 144.7, 149.0, 162.7, 164.3, 176.7. Anal. Calcd for:
2H, J = 5.0 Hz, O CH2), 4.74 (t, 2H, J = 7.5 Hz, N CH2), 6.86 (s, 1H,
–
–
′
′
H3), 7.02 (d, 1H, J = 13.6 Hz, H6), 7.11 (d, 2H, J = 8.5 Hz, H2 , 4 ), 7.30
′
′
(s, 1H, H8), 7.91 (d, 1H, J = 8.5 Hz, H8), 8.04 (d, 2H, J = 10.0 Hz, H1 , 5 ),
8.19 (t, 2H, J = 7.0 Hz, pyridine), 8.63 (t, 1H, J = 7.5 Hz, pyridine), 9.18
(d, 2H, J = 6.0 Hz, pyridine); 13C NMR (DMSO‑d6, 125 MHz) δ 25.0,
27.6, 55.5, 55.6, 60.3, 67.80, 101.4, 105.3, 114.8, 114.8, 117.1, 123.3,
126.1, 128.0, 128.2, 144.8, 145.6, 157.3, 162.0, 162.2, 162.9, 176.3.
Anal. Calcd for C25H24NO4 (402.47): C, 71.61; H, 6.01; N, 3.48. Found:
C, 71.82; H, 5.90; N, 3.78.
C
26H26NO4 (416.49): C, 74.98; H, 6.29; N, 3.36. Found: C, 75.06; H,
6.56; N, 3.62.
4.1.5.9. 1-(4-((4-Oxo-4H-chromen-7-yl) oxy) butyl) pyridin-1-ium (11).
Yellow solid; yield 92%; mp 140–143 ◦C; IR (KBr, cmꢀ 1
)
υmax: 3056
–
–
–
–
(C H aromatic), 2925 (C H), 1625 (C O), 1074 (C O); 1H NMR
–
(DMSO‑d6, 500 MHz,) δ 1.78 (t, 2H, J = 8.5 Hz, CH2), 2.10 (t, 2H, J =
–
6.5 Hz, CH2), 4.16 (t, 2H, J = 6.0 Hz, O CH2), 4.69 (t, 2H, J = 7.5 Hz,
4.1.5.14. 1-(4-((2-(Ethoxycarbonyl)-4-oxo-4H-chromen-7-yl) oxy) butyl)
N
CH2), 6.27 (d, 1H, J = 6.0 Hz, H3), 7.04 (d, 1H, J = 9.0 Hz, H6), 7.13
pyridin-1-ium (16). Yellow solid; yield 85%; mp 95–96 ◦C; IR (KBr,
–
cmꢀ 1
) υmax: 3053 (C H aromatic), 2936 (C H), 1732 (C O), 1083
– –
–
–
(s, 1H, H8), 7.93 (d, 1H, J = 9.0 Hz, H5), 8.16 (t, 2H, pyridine), 8.22 (d,
1H, J = 6.0 Hz, H2), 8.61 (t, 1H, J = 6.0 Hz, pyridine), 9.12 (d, 2H, J =
6.0 Hz, pyridine); 13C NMR (DMSO‑d6, 125 MHz) δ 24.9, 27.6, 60.3,
67.8, 101.3, 112.1, 115.0, 118.0, 126.4, 128.1, 144.8, 145.5, 156.6,
157.7, 162.9, 175.7. Anal. Calcd for C18H18NO3 (296.34): C, 72.95; H,
6.12; N, 4.73. Found: C, 72.86; H, 6.31; N, 4.62.
1
–
(C O); H NMR (DMSO‑d6, 250 MHz,) δ 1.30 (t, 3H, J = 7.5 Hz, CH3),
–
1.76 (bs, 2H, CH2), 1.96 (bs, 2H, CH2), 4.16 (bs, 2H, CH3 CH2), 4.36 (t,
–
–
2H, J = 5.0 Hz, O CH2), 4.72 (bs, 2H, N CH2), 6.83 (s, 1H, H3), 7.04
(d, 1H, J = 8.7 Hz, H6), 7.17 (s, 1H, H8), 7.88(d, 1H, J = 9.2 Hz, H5), 8.18
(bs, 2H, pyridine), 8.60 (t, 1H, J = 5.7 Hz, pyridine), 9.18 (d, 2H, J = 5.2
Hz, pyridine); 13C NMR (DMSO‑d6, 62.9 MHz) δ 14.3, 25.3, 28.0, 60.7,
63.1, 68.4, 101.8, 114.4, 116.5, 118.0, 126.8, 128.5, 145.2, 150.0,
150.3, 157.7, 164.2, 176.7. Anal. Calcd for C21H22NO5 (368.41): C,
68.47; H, 6.02; N, 3.80. Found: C, 68.31; H, 5.37; N, 4.10.
4.1.5.10. 1-(4-((4-Oxo-2-phenyl-4H-chromen-7-yl) oxy) butyl) pyridin-1-
ium (12). Yellow solid; yield 92%; mp 102–104 ◦C; IR (KBr, cmꢀ 1
)
υmax
:
3050 (C H aromatic), 2942 (C H), 1628 (C O), 1013 (C O); 1H
–
–
–
–
–
NMR (DMSO‑d6, 500 MHz) δ 1.81 (bs, 2H, CH2), 2.12 (bs, 2H, CH2), 4.19
–
–
(t, 2H, J = 9.4 Hz, O CH2), 4.73 (t, 2H, J = 8.5 Hz, N CH2), 6.97 (s,
4.1.5.15. 4-Ethyl-1-(4-((4-oxo-2-phenyl-4H-chromen-7-yl) oxy) butyl)
1H, H3), 7.05 (d, 1H, J = 8.5 Hz, H6), 7.33 (s, 1H, H8), 7.58 (m, 3H, H2 ,
pyridin-1-ium (17). Yield 83%; off Brown solid; mp 136–137 ◦C; IR (KBr,
′
3 , 4 ), 7.93 (d, 1H, J = 8.5 Hz, H5), 8.08 (d, 2H, J = 9.5 Hz, H1 , 5 ), 8.62
cmꢀ 1
)
υmax: 3037 (C H aromatic), 2936 (C H), 1712 (C O), 1176
–
–
–
′
′
′
′
–
(t, 2H, J = 7.5 Hz, pyridine), 9.14–9.17 (m, 3H, pyridine); 13C NMR
(C O). H NMR (DMSO‑d6, 250 MHz) δ 1.22 (t, 3H, J = 7.5 Hz, CH3),
1
–
8

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
–
1.70 (bs, 2H, CH2), 2.10 (bs, 2H, CH2), 2.86 (q, 2H, J = 7.5 Hz,
cmꢀ 1
) υmax: 3055 (C H aromatic), 2865–2937 (C H), 1637 (C O),
– –
–
1
–
–
–
–
CH3 CH2), 4.20 (bs, 2H, O CH2), 4.66 (d, 2H, J = 7.5 Hz, N CH2),
1008 (C O); H NMR (DMSO‑d6, 500 MHz) δ 1.45 (t, 2H, J = 6.5 Hz,
′
′
6.91 (s, 1H, H3), 7.01 (bs, 1H, H6), 7.22–7.56 (m, 4H, H8 and H2 -4 ),
CH2), 1.81 (t, 2H, J = 6.5 Hz, CH2), 2.01 (bs, 2H, CH2), 4.11 (t, 2H, J =
– –
6.0 Hz, O CH2), 4.69 (bs, 2H, N CH2), 6.95 (s, 1H, H3), 7.00 (d, 1H, J
′
′
8.07–8.02 (m, 5H, H1 ,5 , H5 and pyridine), 9.05 (t, 2H, J = 6.2 Hz,
pyridine); 13C NMR (DMSO‑d6, 62.9 MHz) δ 25.4, 27.5, 27.9, 28.4, 59.3,
59.8, 64.3, 68.2, 69.20, 101.9, 107.2, 111.3, 115.4, 121.7, 123.8, 126.8,
127.6, 128.6, 144.2, 144.51, 151.24, 157.2, 162.60, 163.46, 176.7.
Anal. Calcd for C26H26NO3 (400.50): C, 77.97; H, 6.54; N, 3.50. Found:
C, 78.12; H, 6.62; N, 3.88.
′
′
= 8.8 Hz, H6), 7.27 (s,1H, H8), 7.61 (d, 2H, J = 8.5 Hz, H1 ,5 ), 7.89 (d,
′
′
1H, J = 10.0 Hz, H5), 8.08 (d, 2H, J = 8.5 Hz, H2 ,4 ), 8.18 (t, 2H, J = 6.0
Hz, pyridine), 8.62 (t, 1H, J = 7.5 Hz, pyridine), 9.20 (t, 2H, J = 5.5 Hz,
pyridine); 13C NMR (DMSO‑d6, 125 MHz) δ 22.0, 27.7, 29.9, 60.1, 68.2,
101.4, 107.0, 115.1, 117.0, 126.2, 128.0, 128.1, 129.2, 130.0, 144.8,
145.5, 157.4, 160.9, 163.2, 176.4. Anal. Calcd for C25H23ClNO3
(420.91): C, 71.34; H, 5.51; N, 3.33. Found: C, 71.18; H, 5.15; N, 3.23.
4.1.5.16. 1-(4-((2-(4-Chlorophenyl)-4-oxo-4H-chromen-7-yl)oxy)butyl)-
4-ethylpyridin-1-ium (18). Yield 82%; off Brown solid; mp 122–123 ◦C;
IR (KBr, cmꢀ 1
)
υmax: 3032 (C H aromatic), 2955 (C H), 1627 (C O),
4.1.5.21. 1-(5-((2-(4-Methoxyphenyl)-4-oxo-4H-chromen-7-yl)
oxy)
–
–
–
–
1
–
1117 (C O). H NMR (DMSO‑d6, 250 MHz) δ 1.22 (t, 3H, J = 7 Hz,
pentyl) pyridin-1-ium (23). Yellow solid; yield 91%; mp 170–172 ◦C; IR
– –
υmax: 3049 (C H aromatic), 2872–2945 (C H), 1625
CH3), 1.76 (bs, 2H, CH2), 2.07 (bs, 2H, CH2), 2.86 (d, 2H, J = 7 Hz,
(KBr, cmꢀ 1
)
–
–
–
–
CH3 CH2), 4.15 (bs, 2H, O CH2), 4.62 (bs, 2H, N-CH2), 6.97 (bs, 2H,
(C O), 1026 (C O); 1H NMR (DMSO‑d6, 500 MHz) δ 1.45 (bs, 2H,
–
′
′
′
′
H
3,6), 7.28 (d, 2H, J = 2.0 Hz, H1 ,5 ), 7.62 (bs, 2H, H2 , 4 ), 7.88–8.09 (m,
CH2), 1.8 (bs, 2H, CH2), 2.02 (bs, 2H, CH2), 3.83 (s, 3H, OCH3), 4.12 (t,
– –
2H, J = 7.0 Hz, O CH2), 4.69 (t, 2H, J = 8.5 Hz, N CH2), 6.83 (s, 1H,
3H, H8 and pyridine), 8.41 (bs, 1H, H5), 9.00 (d, 2H, J = 6.0 Hz, pyri-
dine); 13C NMR (DMSO‑d6, 62.9 MHz) δ 13.8, 25.4, 28.4, 59.9, 68.2,
101.9, 107.5, 115.5, 123.9, 126.6, 127.7, 128.4, 129.6, 130.4, 136.9,
144.5, 149.5, 157.8, 161.4, 16.51, 176.7. Anal. Calcd for: C26H25ClNO3
(434.94): C, 71.80; H, 5.79; N, 3.22. Found: C, 71.42; H, 6.09; N, 3.18.
′
′
H3), 6.99 (d,1H, J = 10.0 Hz, H6), 7.09 (d, 2H, J = 8.5 Hz, H2 ,4 ), 7.26 (s,
′
′
1H, H8), 7.89 (d, 1H, J = 10.0 Hz, H5), 8.01 (d, 2H, J = 13.0 Hz, H1 ,5 ),
8.18 (t, 2H, J = 6.5 Hz, pyridine), 8.62 (t,1H, J = 9.0 Hz, pyridine), 9.19
(t, 2H, J = 6.0 Hz, pyridine); 13C NMR (DMSO‑d6, 125 MHz) δ 22.0, 27.7,
30.4, 55.6, 60.1, 60.5, 101.3, 105.2, 114.8, 117.0, 123.2, 123.9, 126.1,
128.0, 144.8, 145.5, 157.3, 162.2, 163.0, 176.3. Anal. Calcd for
4.1.5.17. 4-Ethyl-1-(4-((2-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)
oxy)butyl)pyridin-1-ium (19). Yield 85%; Brown liquid; IR (KBr, cmꢀ 1
)
C
26H26NO4 (416.50): C, 74.98; H, 6.29; N, 3.36. Found: C, 74.86; H,
–
–
–
6.41; N, 3.28.
υmax: 3045 (C H aromatics), 2934 (C H), 1627(C O), 1179.11
–
1
–
(C O). H NMR (DMSO‑d6, 250 MHz) δ 1.22 (t, 3H, J = 7.5 Hz, CH3),
–
1.77–2.04 (m, 4H, CH2), 2.56 (m, 2H, CH3 CH2), 3.81 (s, 3H, OCH3),
4.1.5.22. 4-Ethyl-1-(5-((4-oxo-2-phenyl-4H-chromen-7-yl) oxy) pentyl)
–
4.14 (bs, 2H, O CH2), 4.64 (bs, 2H, N-CH2), 6.82 (s, 1H, H3), 6.97–7.25
pyridin-1-ium (24). Yield 78%; off yellow solid; mp 137–139 ◦C; IR
′
′
′
′
–
–
(m, 3H, H6,2 ,4 ), 7.87–8.02 (m, 2H, H8,1 ,5 ), 7.90 (m, 3H, H5 and pyri-
dine), 9.01 (d, 2H, J = 5 Hz, pyridine); 13C NMR (DMSO‑d6, 62.9 MHz) δ
14.6, 25.4, 27.7, 55.9, 59.9, 68.1, 101.8, 105.6, 114.9, 115.2, 117.5,
123.8, 126.5, 127.6, 128.4, 144.4, 149.8, 153.0, 157.7, 162.4, 163.3,
164.2, 176.7. Anal. Calcd for: C27H28NO4 (430.52): C, 75.33; H, 6.56; N,
3.25. Found: C, 75.61; H, 6.45; N, 3.08.
(KBr, cmꢀ 1
)
υmax: 3047 (C H aromatic), 2870–2944 (C H), 1629
1
–
–
(C O), 1171 (C O). H NMR (DMSO‑d6, 250 MHz) δ 1.10 (t, 3H, CH3),
–
1.40 (bs, 2H, CH2), 1.80–2.00 (m, 4H, CH2), 2.87 (q, 2H, J = 7.5 Hz,
–
–
CH3 CH2), 4.12 (t, 2H, J = 6.0 Hz, O CH2), 4.58 (t, 2H, J = 7.5 Hz, N-
CH2), 6.95 (s, 1H, H3), 7.02 (d, 1H, J = 9.2 Hz, H6), 7.28 (s, 1H, H8),
′
′
′
′
′
7.55–7.57 (m, 3H, H2 ,3 ,4 ), 7.90–8.06 (m, 5H, H1 ,5 ,5 and pyridine), δ:
8.99 (d, 2H, J = 6.0 Hz, pyridine); 13C NMR (DMSO‑d6, 62.9 MHz) δ
13.8, 22.4, 28.1, 28.42, 30.6, 60.1, 68.5, 101.8, 107.2, 115.4, 117.5,
126.6, 127.6, 129.5, 131.60 132.1, 144.5, 157.9, 162.5, 164.1, 176.8.
Anal. Calcd for C27H28NO3 (414.52): C, 78.23; H, 6.81; N, 3.38. Found:
C, 78.42; H, 7.03; N, 3.11.
4.1.5.18. 1-(5-((4-Oxo-4H-chromen-7-yl) oxy) pentyl) pyridin-1-ium
(20). White solid; yield 95%; mp 95–96 ◦C; IR (KBr, cmꢀ 1
)
υmax: 3051
(C H aromatic), 2864–2934 (C H), 1644 (C O), 1077 (C O); 1H
–
–
–
–
–
NMR (DMSO‑d6, 500 MHz,) δ 1.42–1.48 (m, 2H, CH2), 1.78–1.82 (m,
–
2H, CH2), 1.83–1.20 (m, 2H, CH2), 4.11 (t, 2H, J = 6.5 Hz, O CH2),
–
4.66 (t, 2H, J = 8.5 Hz, N CH2), 6.26 (d, 1H, J = 8.5 Hz, H3), 7.01 (d ,
4.1.5.23. 1-(5-((2-(4-Chlorophenyl)-4-oxo-4H-chromen-7-yl)oxy)pentyl)-
1H, J = 9.0 Hz, H6), 7.10 (s, 1H, H8), 7.92 (d, 1H, J = 8.5 Hz, H5), 8.18 (t,
2H, J = 6.5 Hz, pyridine), 8.22 (d, 1H, J = 7.5 Hz, H2), 8.62 (t, 1H, J =
8.0 Hz, pyridine), 9.14 (d, 2H, J = 6.0 Hz, pyridine); 13C NMR
(DMSO‑d6, 125 MHz) δ 21.9, 27.7, 30.3, 60.5, 68.0, 101.2, 112.1, 114.9,
118.0, 126.4, 128.1, 144.7, 145.5, 156.5, 157.7, 163.0, 175.7. Anal.
Calcd for C19H20NO3 (310.37): C, 73.53; H, 6.50; N, 4.51. Found: C,
73.68; H, 6.60; N, 4.43.
4-ethylpyridin-1-ium (25). Yield 90%; Brown solid; mp 123–124 ◦C; IR
(KBr, cmꢀ 1
)
υmax: 3045 (C H aromatic), 2865–2930 (C H), 1642
–
–
1
–
–
(C O), 1176 (C O). H NMR (DMSO‑d6, 250 MHz) δ 1.20 (t, 3H, J =
–
7.5 Hz, CH3), 1.42 (bs, 2H, CH2), 1.77–1.96 (m, 4H, CH2), 2.87 (q, 2H, J
–
–
–
= 7.5 Hz, CH3 CH2), 4.09 (bs, 2H, O CH2), 4.58 (bs, 2H, N CH2),
′
′
6.92–6.98 (m, 2H, H3,6), 7.23 (s, 1H, H8), 7.57 (d, 2H, J = 7.5 Hz, H1 ,5 ),
7.83–8.02 (m, 6H, H5,8,2 ,4 and pyridine), 9.02 (bs, 2H, pyridine); 13C
NMR (DMSO‑d6, 62.9 MHz) δ 13.8, 22.3, 28.1, 28.4, 30.6, 60.1, 68.5,
101.7, 107.4, 115.5, 117.4, 126.5, 127.6, 128.4, 129.5, 130.4, 136.8,
144.4, 157.8, 161.3, 163.6, 164.1, 176.7. Anal. Calcd for C27H27ClNO3
(448.97): C, 72.23; H, 6.06; N, 3.12. Found: C, 72.48; H, 5.86; N, 3.48.
′
′
4.1.5.19. 1-(5-((4-Oxo-2-phenyl-4H-chromen-7-yl) oxy) pentyl) pyridin-
ꢀ 1
◦
–
–
1-ium (21). Brown solid; yield 87%; mp 145–146 C; IR (KBr, cm
)
–
–
–
υmax: 3052 (C H aromatic), 2947 (C H), 1633 (C O), 1013 (C O);
1H NMR (DMSO‑d6, 500 MHz) δ 1.45 (bs, 2H, CH2), 1.82 (bs, 2H, CH2),
–
–
2.01 (bs, 2H, CH2), 4.13 (bs, 2H, O CH2), 4.67 (bs, 2H, N CH2), 6.97
4.1.5.24. 4-Ethyl-1-(5-((2-(4-methoxyphenyl)-4-oxo-4H-chromen-7-yl)
′
′
′
(s, 1H, H3), 7.02 (bs, 1H, H6), 7.31 (s, 1H, H8), 7.59 (bs, 3H, H2 ,3 ,4 ),
oxy)pentyl)pyridin-1-ium (26). Yield 92%; Brown liquid; IR (KBr, cmꢀ 1
)
–
–
′
′
–
–
–
7.77 (bs, 2H, H1 ,5 ), 7.92 (bs, 1H, H5), 8.08 (bs, 2H, pyridine), 8.62 (bs,
1H, pyridine), 9.17 (bs, 2H, pyridine); 13C NMR (DMSO‑d6, 125 MHz) δ
21.9, 27.7, 30.3, 60.5, 68.1, 101.3, 106.7, 115.0, 117.0, 123.9, 126.2,
128.1, 129.1, 131.1, 144.7, 145.5, 157.5, 162.1, 163.1, 176.4. Anal.
Calcd for C25H24NO3 (386.47): C, 77.70; H, 6.26; N, 3.62. Found: C,
77.45; H, 6.38; N, 3.78.
υmax: 3035 (C H aromatic), 2939 (C H), 1628 (C O), 1177 (C O).
1H NMR (DMSO‑d6, 250 MHz) δ 1.22 (t, 3H, J = 7.5 Hz, CH3), 1.42 (bs,
–
2H, CH2), 1.78–1.97 (m, 4H, CH2), 2.87 (q, 2H, J = 7.5 Hz, CH3 CH2),
–
–
3.40 (s, 3H, OCH3), 4.08 (bs, 2H, O CH2), 4.60 (bs, 2H, N CH2), 6.79
′
′
(s, 1H, H3), 6.93–7.07 (m, 3H, H6,2 ,4 ), 7.21 (s, 1H, H8), 7.83–8.08 (m,
5H, H5,1 ,5 and pyridine), 9.04 (d, 2H, J = 6.0 Hz, pyridine); 13C NMR
(DMSO‑d6, 62.9 MHz) δ 13.8, 22.4, 28.1, 28.4, 30.7, 56.0, 60.1, 68.5,
101.7, 105.6, 114.9, 115.1, 126.5, 127.6, 128.4, 144.5, 149.8, 162.6,
163.4, 164.1, 178.9. Anal. Calcd for: C28H30NO4 (444.55): C, 75.65; H,
′
′
4.1.5.20. 1-(5-((2-(4-Chlorophenyl)-4-oxo-4H-chromen-7-yl) oxy) pen-
◦
tyl) pyridin-1-ium (22). Pink solid; yield 96%; mp 94–95 C; IR (KBr,
9

S. Abdpour et al.
Bioorganic Chemistry 110 (2021) 104750
6.80; N, 3.15. Found: C, 75.14; H, 5.92; N, 3.35.
1640 media containing heat-inactivated FBS (10%) and pen-
icillin–streptomycin (1%) and incubated at 37 ◦C under 5% CO2 atmo-
sphere and treated with the tested compound (0.01–100 µM) and
4.2. Biological assays
incubated for 3 h. After that, the cells were exposed to Aβ1–42 (25 μM).
After 24 h of incubation, MTT solution (5 mg/mL) was added and the
formazan crystals were dissolved with DMSO. The assay was performed
by a micro plate reader (Model 680; Bio-Rad Laboratories, Hercules, CA,
USA) at 570 nm.
4.2.1. In vitro AChE/BuChE inhibition assay
The method of Ellman et al. was used to determine the inhibitory
activity of the compounds against ChEs [51]. All compounds were dis-
solved in DMSO and the stock solution was prepared at 4.3 mM con-
centration. The serial dilution was used to obtain 4 test solutions. At
first, the compounds were tested at 100 µM in well concentration. If the
percent of inhibition was greater than 50%, the assay was performed at
10, 1, 0.1, 0.01 and 0.001 µM for each compound. Briefly, in each well
phosphate buffer (2 mL, pH = 8.0) was added followed by adding 20 µL
4.2.5. Determination of cytotoxicity effect
Two cancer cell lines of PC12 (rat pheochromocytoma) and HepG2
(human hepatocarcinoma) as well as normal cell of human dermal
fibroblast (HDF) were provided by the IBRC cell bank (Tehran, Iran) and
cultured in appropriate medium (RPMI-1640 fortified by 10% FBS)
using a CO2 incubator at 37 ◦C. At the exponential phase of growth, an
amount of 10,000 cells were harvested and separately inserted into each
well of a 96-well microplate and incubated for 24 h to adhere the cells.
Compounds 10 or 14 in the desired concentration range (0–750 µM) and
donepezil in corresponding concentrations (0–500 µM) was added to the
related wells and the wells were incubated for further 24 h. The media
was subsequently removed from each well and MTT solution (5 mg/mL
dissolved in RPMI-1640 medium) was added to each well and the well
was incubated for 4 h followed by replacing the media with DMSO (100
µL) to dissolve the formed formazan crystals and reading the absorbance
at 570 nm and calculating the viability percent and IC50 of each
compound.
́
of the AChE or BuChE (2.5 IU/mL), 50 µL of inhibitor and 60 µL of 5,5-
dithio-bis(2-nitrobenzoic acid) (DTNB). The as-prepared cocktail was
incubated for 15 min, 20 µL of substrate (acetylthiocholine iodide or
butyrylthiocholine iodide) was then added and the absorbance was
measured at 412 nM using Biotek Synergy HT microplate reader.
Microsoft Excel 2016 was used for calculation of IC50s. Each experiment
was repeated at least three independent times and each assay was run in
triplicate.
4.2.2. Determination of the inhibitory potency of the compounds against
self-induced and AChE-induced Aβ1-42 aggregation
The self- and AChE-induced Aβ1-42 aggregation was performed based
on ThT fluorometric assay [52,53]. Aβ sample (Bachem company,
Switzerland) was treated with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)
and diluted in assay sodium phosphate buffer (0.215 M, pH 8) to make a
stock solution (50 µM). The peptide was incubated in phosphate buffer
(pH 7.4) at 37 ◦C for 48 h with or without inhibitor (final Aβ concen-
tration was 25 µM). The inhibitor was dissolved in DMSO and diluted in
the assay buffer at a final concentration of 10 µM. For co-incubation
experiments, the mixtures with a volume of 20 µL, including Aβ1-42
peptide and AChE (Sigma, Electrophorus electricus) in presence or
absence of the test inhibitor were incubated for 24 h at room tempera-
ture. The final concentrations of Aβ1-42 peptide, AChE and the samples
were 200 µM, 2 µM and 100 µM in assay buffer, respectively. After in-
cubation, samples were diluted with 180 µL of ThT (5 mM in 50 mM
glycine-NaOH buffer, pH = 8.5) to a final volume of 200 µL. Blank
containing Aβ/AChE plus inhibitor and ThT was used in the experiment.
The fluorescence intensity was measured (at λex = 446 nm; λem = 490
nm) with multi-mode plate reader (EnSpire, PerkinElmer Waltham,
Massachusetts, United States), each reaction was repeated at least three
independent times and each assay was run in triplicate. The percentage
of inhibition was calculated by the following equation: 100 - [(IFi - IFb)/
(IFo - IFb) ꢀ 100], which IFi, IFo and IFb are the fluorescence intensities
for Aβ ± AChE aggregation in the presence of inhibitors, in the absence
of inhibitors and the blanks, respectively.
4.2.6. Docking simulations
For docking study, the structure of the compounds was sketched by
Chemdraw 19. The smiles were copied to MOE 2019.0102 and prepared
by Quickprep module. 1EVE PDB code of AChE and 4BDS code for
BuChE were used as enzyme templates for docking due to the high
resolution of these codes.
The active site was constructed using Site-Finder module. Rigid body
docking was run using default parameters of Autodock Vina program
[56]. The best pose of the docking was chosen for ligand-enzyme
interaction study by Discovery Studio Modeling [57].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by a grant from National Institute for
Medical Research Development (NIMAD; grant number: 983662).
4.2.3. Neuroprotection assay against H2O2-induced cell death in PC12 cells
The cell viability was determined with the MTT [3-(4,5-dime-
thylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay [54]. PC12
cells (from the Iranian Biological Resource Center, IBRC) were seeded at
1 × 104 cells/well. Cells were incubated at 37 ◦C under 5% CO2 atmo-
sphere for 24 h, then treated with the tested compound (0.1–50 µM) and
incubated for 3 h. After that, the cells were exposed to H2O2⋅(150 µM)
for 2 h again. MTT solution (20 µL, 5 mg/mL) was next replaced with the
medium and incubated for 4 h. The formazan crystals were dissolved
with DMSO (100 µL) and the assay was performed by a multi-mode plate
reader (Biotek, Winooski, VT) at 570 nm.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104750.
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