Multifunctional cholinesterase inhibitors for Alzheimer's disease: Synthesis, biological evaluations, and docking studies of o/p-propoxyphenylsubstituted-1H-benzimidazole derivatives

Article abstract of DOI:10.1002/ardp.201800076

This study indicates the synthesis, cholinesterase (ChE) inhibitory activity, and molecular modeling studies of 48 compounds as o- and p-(3-substitutedethoxyphenyl)-1H-benzimidazole derivatives. According to the ChE inhibitor activity results, generally, para series are more active against acetylcholinesterase (AChE) whereas ortho series are more active against butyrylcholinesterase (BuChE). The most active compounds against AChE and BuChE are compounds A12 and B14 with IC₅₀ values of 0.14 and 0.22 μM, respectively. Additionally, the most active 16 compounds against AChE/BuChE were chosen to investigate the neuroprotective effects, and the results indicated that most of the compounds have free radical scavenging properties and show their effects by reducing free radical production; moreover, some of the compounds significantly increased the viability of SH-SY5Y cells exposed to H₂O₂. Overall, compounds A12 and B14 with potential AChE and BuChE inhibitory activities, high neuroprotection against H₂O₂-induced toxicity, free radical scavenging properties, and metal chelating abilities may be considered as lead molecules for the development of multi-target-directed ligands against Alzheimer's disease.

Full text of DOI:10.1002/ardp.201800076

Received: 12 March 2018
Revised: 14 June 2018
Accepted: 19 June 2018
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DOI: 10.1002/ardp.201800076
FULL PAPER
Multifunctional cholinesterase inhibitors for Alzheimer's
disease: Synthesis, biological evaluations, and docking studies
of o/p-propoxyphenylsubstituted-1H-benzimidazole
derivatives
Görkem Sarıkaya1
Güneş Çoban1
Mümin A. Erdoğan3
Sülünay Parlar1
Ayse H. Tarikogullari1
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Güliz Armagan2
Vildan Alptüzün1
Ayşe S. Alpan1
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1 Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, Ege University,
Bornova, İzmir, Turkey
Abstract
This study indicates the synthesis, cholinesterase (ChE) inhibitory activity, and
molecular modeling studiesof 48 compounds as o- andp-(3-substitutedethoxyphenyl)-
1H-benzimidazole derivatives. According to the ChE inhibitor activity results,
generally, para series are more active against acetylcholinesterase (AChE) whereas
ortho series are more active against butyrylcholinesterase (BuChE). The most active
compounds against AChE and BuChE are compounds A12 and B14 with IC50 values of
0.14 and 0.22 μM, respectively. Additionally, the most active 16 compounds against
AChE/BuChE were chosen to investigate the neuroprotective effects, and the results
indicated that most of the compounds have free radical scavenging properties and
show their effects by reducing free radical production; moreover, some of the
compounds significantly increased the viability of SH-SY5Y cells exposed to H2O2.
Overall, compounds A12 and B14 with potential AChE and BuChE inhibitory activities,
high neuroprotection against H2O2-induced toxicity, free radical scavenging proper-
ties, and metal chelating abilities may be considered as lead molecules for the
development of multi-target-directed ligands against Alzheimer's disease.
2 Faculty of Pharmacy, Department of
Biochemistry, Ege University, Bornova, İzmir,
Turkey
3 Faculty of Medicine, Department of
Physiology, İzmir Katip Çelebi University, Çiğli,
İzmir, Turkey
Correspondence
Prof. Dr. Vildan Alptüzün, Faculty of
Pharmacy, Department of Pharmaceutical
Chemistry, Ege University, 35040 Bornova,
İzmir, Turkey.
Email: vildan.alptuzun@ege.edu.tr
Funding information
TUBITAK, Grant number: 213S027
K E Y W O R D S
antioxidant activity, benzimidazole, cholinesterase inhibitors, molecular modeling
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1
INTRODUCTION
cognition, and memory through progressive degeneration of central
nervous system (CNS) neurons.[3,4] The etiology of AD is not yet fully
Alzheimer's disease (AD) is a dreadful neurodegenerative disorder
affecting over 35 million people worldwide and is expected to reach 66
million by 2030.[1,2] It is characterized by the loss of brain functions,
understood, but common hallmarks, such as intracellular tau-protein
aggregation, extracellular beta-amyloid plaques, the selective loss of
cholinergic neuron s, and increased oxidative stress are considered as
key pathological features.[5]
Based on the pathological features of AD, different hypotheses have
been developed. One of them is the cholinergic hypothesis defined by the
Görkem Sarıkaya and Güneş Çoban contributed equally to this article.
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Arch Pharm Chem Life Sci. 2018;e1800076.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800076

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SARIKAYA ET AL.
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decline of acetylcholine (ACh) in some parts of the brain such as
hippocampus and cortex.[4,6] In order to elevate ACh levels that are
lowered by acetylcholinesterase (AChE) and butyrylcholinesterase
(BuChE) in the CNS, inhibition of the cholinesterases is the main purpose.
Besides the acetylcholinesterase inhibitors (AChEI), butyrylcholinester-
ase inhibitors (BuChEI) also play an important therapeutic role in elevating
ACh because even if AChE decreases in CNS, BuChE increases to offset the
deficiency of AChE and to hydrolyze ACh during the progression of AD.[3,7]
Another hypothesis relies on the elevation of biometals in the
brain, which mediate the formation of amyloid plaques and oxidative
stress. Therefore, metal chelating agents also contribute to the
treatment of AD by reducing oxidative stress.[8,9]
On the other hand, docking studies and molecular dynamics (MD)
simulations were performed to analyze the ChEI activity in terms of
binding interactions.
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
Forty-seven 2-substitutedphenyl-1H-benzimidazole derivatives con-
sisting 30 compounds of 17 compound of 2-[p-(3-substitutedpropoxy)-
phenyl]-1H-benzimidazole (A series) and 2-[o-(3-substitutedpropoxy)-
phenyl]-1H-benzimidazole (B series) derivatives were synthesized.
Methyl, ethyl, pyrolydine, piperidine, and morpholine groups were
chosen as terminal substitution of o-propoxy derivatives but dimethyl-
amine andmorpholinegroups waseliminated inp-propoxy counterparts
due to weak biological activity in our previous study.[12]
There are many studies based on the treatment of AD with
different heterocyclic scaffolds.[10] Among them, 1H-benzimidazole
derivatives are considered as a new promising core for designing new
molecules.[11] In our previous studies, we synthesized 2-[p-(2-
substitutedethoxy)phenyl]-1H-benzimidazole and 2-[o-(2-substituted-
ethoxy)phenyl]-1H-benzimidazole derivatives in order to evaluate the
cholinesterase inhibitor activity. We observed that p-substituted
derivatives possessed more AChE inhibitory activity whereas o-
substituted analogues possessed more BuChE inhibitory activity.[12,13]
In this study, we extended the side chain and synthesized
o- and p-(2-substitutedpropoxyphenyl) derivatives with the purpose
of binding the key amino acids with the molecules at the entrance of the
gorge to improve the cholinesterase activity. Moreover, in our previous
study we pointed out that 5-substituted benzimidazole derivatives are
more active than their nonsubstituted counterparts so we added
different substituents on 1H-benzimidazole ring.
All of the final compounds were synthesized by the reported
methods and the synthetic pathways are given in Schemes 1 and 2.
In the IR spectra of the title compounds, the characteristic
frequencies for benzimidazole derivatives at 2360–3426 cm−1 for
N─H─H type H-bonds were observed. Additionally, N─H stretching
bands at 3270–3426 cm−1, C C and C N stretching bands at
1636–1507 cm−1 were observed.


The assessment of the chemical shifts in 1H NMR spectra
demonstrated that the aromatic and aliphatic protons were observed
in the expected regions with expected multiplicities confirming the
substitution patterns. The mass spectra of the title compounds were
recorded by positive ion mode electrospray ionization (ESI+)
technique. The [M+H]+ ions of title compounds are in complete
agreement with the calculated molecular weights.
Thus, we chose 16 active AChE and/or BuChE inhibitory
compounds and tested their potential neuroprotective effects,
antioxidative properties, and metal chelating potency based on the
“one molecule, multiple target” strategy.[8]
The purity levels of compounds were determined by elemental
analyses (C, H, N) and results were within 0.4% of the calculated values.
SCHEME 1 Synthesis pathway for the A series compounds

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SCHEME 2 Synthesis pathway for the B series compounds
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2.2 AChE/BuChE inhibitory activity
Controversially, in ortho series compounds (B series), all of the
compounds exhibited more BuChEI activity than AChEI activity except
for the compounds bearing morpholine ring in accordance with our
previous study where we had pointed out the same observation.[13]
Also, the most active compounds were piperidine derivatives bearing
nonsubstituted, methyl and chloro substituents; and pyrrolidine
derivatives bearing nitro and nitrile substituents in terms of BuChE
inhibitory activity,
Inhibitor potency of the final compounds against AChE and BuChE
were evaluated by Ellman method[14] and ChEI activity results are
summarized in Table 1. According to AChEI activity results of the
para series compounds (compounds A1–18), all compounds ex-
hibited good to moderate AChEI activity with IC50 values of
0.14–8.43 μM. Among them, nitro, nitrile, and dimethoxy substitu-
ents (compounds A10–18) were clearly more active than the
nonsubstituted, methyl and chloro counterparts (compounds
A1–9) and also selective in terms of AChEI activity. The selectivities
of the compounds A1–9 are in accordance with our previous
study.[12] Additionally, chloro substituents had better activity than
the nonsubstituted and methyl analogues. On the other hand, diethyl
derivatives displayed higher activity in comparison to piperidine
derivatives. All of the para series compounds (A series) exhibited
moderate to weak BuChEI activity with IC50 values of
2.47–46.79 μM. Within these compounds, pyrrolidine and piperidine
derivatives had generally better BuChE inhibitory potency than the
diethyl derivatives where the nitro, nitrile, and dimethoxy sub-
stituents possessed better activity with piperidine ring analogues
and nonsubstituted, methyl and chloro substituents displayed better
activity with pyrolidine ring counterparts.
Among the para series compounds, compound A12 showed
highest inhibitory activity against both AChE (IC50 = 0.14 μM) and
BuChE (IC50 = 2.47 μM). Besides, in ortho series, compound B9
exhibited the best inhibitory activity against AChE (IC50 = 5.86 μM)
enzyme while compound B14 was the most active compounds against
BuChE (IC50 = 0.14 μM).
In ortho series, compounds B4, B12–14, B18, B19, and B29
were the most active compounds against BuChE enzyme with
0.22–0.71 μM inhibitory activity and were also selective.
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2.3 Neuroprotective activity
Neuroprotective activity studies were performed on 16 compounds
(compounds A10–18, B4, B12–14, B18, B19, B29) with the highest
activity as a result of enzyme activity studies.

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TABLE 1 In vitro inhibition of AChE and BuChE
IC50 SEM (μM)a
Compounds
A1c
A2c
A3
Position
Para
n
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
R1
R2
H
R3
AChE
BuChE
SIb
1
H
-N(C2H5)2
Pyrrolidine
Piperidine
-N(C2H5)2
Pyrrolidine
Piperidine
Pyrrolidine
Piperidine
-N(C2H5)2
Pyrrolidine
Piperidine
-N(C2H5)2
Pyrrolidine
Piperidine
-N(C2H5)2
Pyrrolidine
Piperidine
-N(CH3)2
7.56 0.243
7.45 0.073
8.43 0.357
2.76 0.087
6.40 0.317
4.41 0.112
2.69 0.140
2.64 0.400
0.28 0.009
0.30 0.035
0.14 0.021
0.51 0.029
0.95 0.104
0.69 0.013
0.88 0.028
0.44 0.017
1.79 0.076
7.29 0.108
6.54 0.538
72.79 3.523
8.04 0.250
11.19 0.199
6.92 0.103
7.64 0.146
72.32 5.026
5.86 0.075
82.80 4.978
7.37 0.084
6.49 0.266
63.99 1.661
6.20 0.071
>100
14.30 0.341
2.83 0.235
11.65 0.832
4.27 0.274
3.05 0.025
4.64 0.268
3.28 0.115
3.62 0.072
3.45 0.274
3.41 0.032
2.47 0.315
8.59 0.31
0.53
2.63
0.72
0.65
2.10
0.95
0.82
0.73
0.08
0.09
0.06
0.06
0.13
0.17
0.02
0.03
0.25
1.75
0.45
3.71
16.41
0.46
2.96
2.73
36.71
3.10
2
Para
H
H
3
Para
H
H
4
A4
Para
CH3
CH3
CH3
Cl
H
5
A5
Para
H
6
A6
Para
H
7
A8
Para
H
8
A9c
A10
A11
A12
A13
A14
A15
A16
A17
A18
B1c
Para
Cl
H
9
Para
NO2
NO2
NO2
CN
CN
CN
OCH3
OCH3
OCH3
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Para
H
Para
H
Para
H
Para
H
7.16 0.408
4.13 0.191
46.79 3.162
13.16 0.249
7.07 0.290
4.17 0.114
14.31 1.777
19.63 0.920
0.49 0.019
24.19 0.858
2.34 0.045
2.80 0.054
1.97 0.075
1.89 0.042
>100
Para
H
Para
OCH3
OCH3
OCH3
H
Para
Para
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
Ortho
B2
H
H
-N(C2H5)2
Pyrrolidine
Piperidine
Morpholine
-N(CH3)2
B3
H
H
B4c
H
H
B5
H
H
B6c
CH3
CH3
CH3
CH3
CH3
Cl
H
B7
H
-N(C2H5)2
Pyrrolidine
Piperidine
Morpholine
-N(CH3)2
B8
H
B9c
H
B10
B11c
B12
B13
B14c
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
H
H
1.48 0.033
0.28 0.014
0.30 0.017
0.22 0.005
>100
4.98
Cl
H
-N(C2H5)2
Pyrrolidine
Piperidine
Morpholine
-N(CH3)2
23.18
213.3
28.18
Cl
H
Cl
H
Cl
H
NO2
NO2
NO2
NO2
NO2
CN
CN
CN
CN
CN
OCH3
H
10.33 0.367
7.27 0.127
28.33 3.358
11.92 0.417
19.34 2.287
7.43 0.309
7.35 0.363
>100
2.93 0.124
3.04 0.128
0.35 0.015
0.62 0.023
34.13 0.621
7.86 0.189
5.39 0.217
2.78 0.046
6.27 0.754
88.64 6.524
54.81 3.159
3.53
2.39
80.94
19.23
0.57
0.95
1.36
H
-N(C2H5)2
Pyrrolidine
Piperidine
Morpholine
-N(CH3)2
H
H
H
H
H
-N(C2H5)2
Pyrrolidine
Piperidine
Morpholine
-N(CH3)2
H
H
55.90 5.235
7.47 0.323
65.70 4.693
8.92
0.08
H
OCH3
1.20
(Continues)

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TABLE 1 (Continued)
IC50 SEM (μM)a
Compounds
Position
Ortho
Ortho
Ortho
Ortho
n
3
3
3
3
R1
R2
R3
AChE
BuChE
SIb
44
45
46
47
B27
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
-N(C2H5)2
Pyrrolidine
Piperidine
Morpholine
7.62 0.171
73.68 2.969
8.72 0.293
8.07 0.199
0.075 0.02
0.43 0.03
37.52 5.041
>100
0.20
B28
B29
0.71 0.063
93.43 7.057
0.0098 0.0002
14.92 0.57
12.28
0.09
B30
Tacrine
Galanthamine
aData are means standard error of the mean of triplicate independent experiments.
bSI = selectivity index = IC50 (EeAChE)/IC50 (eqBuChE).
cRefs. [44–46].
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2.3.1 Cell viability
group was found 27.12% 9.57 and compounds A11, A12, A14–18,
B4, B12, B18, B19 at 0.1 μM; A13 at 1 µM concentrations provided
higher than 30% neuroprotection against H2O2-induced toxicity.
Moreover, compounds B13 and B14 significantly increased cell
viability at all selected concentrations.
The protective effects of newly synthesized compounds were
evaluated in cellular oxidative stress model. Human neuroblastoma
SH-SY5Y cell line is highly sensitive to oxidants, thus this cell line was
used in current study.[15] Cells were treated with four concentrations
(1–1000 μM) of H2O2 and assayed for cytotoxicity after 1 h
incubation. Figure 1 shows that H2O2 reduced the viability of SH-
SY5Y cells in a time- and concentration-dependent manner. IC50 value
of H2O2 was found 150 μM. Hence, this concentration was used for
further experiments to induce oxidative stress in SH-SY5Y cells.
To avoid possible toxicity, cells were exposed to three different
concentrations of synthesized compounds, then their effects on the
proliferation of cells were determined. An increase in activity of
dehydrogenase enzymes showed that treatments with indicated
concentrations alone for 24 h were not cytotoxic and did not alter
cell proliferation (data not shown). Following these experiments, SH-
SY5Y cells were pre-treated with compounds ranging from 0.1 to
10 µM for 24 h, followed by treatment with 150 µM H2O2 for 1 h.
Table 2 shows that selected compounds reduced H2O2-induced
neuronal death in a dose-dependent manner. The cell viability in H2O2
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2.3.2 Determination of ROS production
The changes in ROS production were determined by using cell-
permeable nonfluorescent reagent CellROX Deep Red Reagent
(Figure 2). As expected, the significant increase in dye-associated
fluorescence was determined following H2O2 treatment when
compared with control (control: 32.07 6.24% vs. H2O2:
100.06 16.98%) (p < 0.05, Figure 2). However, ROS production
was significantly decreased in cells after pre-treating with com-
pounds A10–18, B12–14, B19, B29 at indicated concentrations in
the presence of H2O2 when compared with H2O2-treated cells
(p < 0.05, Figure 2). ROS levels were determined in compounds A12,
A15, A16, B12, B13, B14-treated cells by 48.22, 41.12, 38.38,
40.92, 56.52, and 52.25%, respectively (p < 0.01 vs. H2O2-treated
cells). Our present data on cell viability and ROS production support
the findings that H2O2 triggers oxidative stress in brain cells and
leads to cell death.[16] Besides, the synthesized compounds, except
compounds B4 and B18, used in this study were found effective in
reducing free radical production and were shown to have free radical
scavenging properties.
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2.3.3 Antioxidant protein analysis
Induction of antioxidant system in brain is crucial for neuronal survival.
Heme oxygenase (HO-1) is a stress-responsive enzyme that has anti-
inflammatory, antioxidant, and cytoprotective functions. Targeting
and upregulation of HO-1 by drugs are assumed as a therapeutic
strategy for the treatment of neurodegenerative diseases, such as
Alzheimer's disease and Parkinson's disease.[17] Due to their critical
role in oxidative stress, in this study the potential effects of
synthesized compounds on HO-1 and catalase levels were determined
in H2O2-induced oxidative stress.
FIGURE 1 Evaluation of cell viability of SH-SY5Y cells exposed
to various concentrations (0–1000 μM) of H2O2 for 1 h. Data
represent the mean SD of at least six independent experiments in
triplicate and are expressed as percentage of control cells. *p < 0.05
significant difference from control cells

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TABLE 2 The protective effects of newly synthesized compounds against H2O2-induced toxicity
Compound
A10
Conc. (μM)
10
% Cell viability
34.61 2.87
50.13 14.19
35.54 15.62
27.22 2.99
61.09 8.55
59.98 4.31
27.17 5.23
73.73 13.92
67.27 3.01
17.98 2.41
60.72 13.90
51.57 9.56
25.22 2.87
33.22 3.91
59.42 7.11
23.74 7.32
48.69 4.88
73.26 13.51
28.38 10.01
66.94 14.01
67.08 8.48
36.93 3.52
38.98 5.13
58.77 9.27
100 10.38
Compound
A18
Conc. (μM)
10
% Cell viability
43.06 4.64
36.37 6.31
59.05 11.88
51.24 9.98
32.56 4.61
65.92 5.15
39.02 10.04
55.51 8.29
63.32 9.19
57.79 5.01
58.35 3.77
63.23 14.52
68.52 7.44
67.13 2.14
65.23 6.11
46.92 5.72
50.17 7.33
67.69 12.56
34.24 5.68
36.28 9.81
61.37 13.92
36.70 10.54
38.23 9.11
45.99 8.33
27.12 9.57
1
1
0.1
0.1**
10**
1
A11
A12
A13
A14
A15
A16
A17
Control
10
B4
1**
0.1**
10
0.1**
10
B12
B13
B14
B18
B19
B29
H2O2
1**
1**
0.1**
10
0.1**
10**
1**
1**
0.1**
10
0.1**
10**
1**
1
0.1**
10
0.1**
10
1
1
0.1**
10
0.1**
10
1**
1
0.1**
10
0.1**
10
1
1
0.1**
0.1
150*
*p < 0.05 significant difference from control cells. **p < 0.05 significant difference from H2O2-treated cells. All values are means SDs (n = 5).
According to the results obtained by cell viability analysis and ROS
measurements, compounds A12, A15, A16, B12–14 which exerted
maximum protection were used for protein analysis. Western blotting
and further semi-quantitative analysis of the blots revealed that the
relative amount of HO-1 expressions were significantly decreased in
H2O2-treated cells (p < 0.05, Figure 3). The decreased antioxidant
enzyme activities in brain appear to render neurons more susceptible
to oxidative stress. However, compounds A12, B13, and B14
significantly ameliorated H2O2-triggered downregulation of HO-1.
The increase in anti-oxidative capacity by these compounds may
contribute to its protection of SH-SY5Y cells against ROS.
Regarding to data obtained by human neuroblastoma cells studies,
it can be concluded that inhibition of cell death and ROS production
and induction of antioxidant proteins may have role in exhibiting the
neuroprotective effect of newly synthesized compounds, particularly
compounds A12 and B13, in oxidative stress conditions.
FIGURE 2 Effects of synthesized compounds against H2O2-
induced ROS production in SH-SY5Y cells. The concentrations used
for 1–4 were 1 μM, 5–16 were 0.1 μM (compounds A10–13 were
1 μM, the others were 0.1 μM). Percent changes in ROS generation
assessed by spectrofluorometric analysis, n = 5. *p < 0.05 significant
difference from control cells. **p < 0.05 significant difference from
H2O2-treated cells, #p < 0.01 significant difference from H2O2-
treated cells
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2.3.4 Metal chelating property
The chelating ability of 16 compounds with the highest ChEI activity
, , , was
for biologically related metal ions, Cu2+ Fe2+ and Zn2+
investigated by UV-vis spectrometry in methanol at 298 K with
wavelength ranging from 200 to 500 nm. The results demonstrated

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FIGURE 3 The changes in HO-1 protein levels following
treatments in H2O2-treated SH-SY5Y cells. Representative Western
blots showing protein expressions of HO-1 following treatments of
compounds A12 at 1 μM, A15 to 16, B12 or B13 at 0.1 μM, B14 at
10 μM against H2O2-induced toxicity. Graph indicates the relative
densitometric values of indicated protein. Quantification of protein
product was performed by densitometric scanning. Data are
normalized by using the β-actin signal and expressed as arbitrary
densitometric units. Values are means SD; n = 3 in each group.
*p < 0.05 significant difference from control cells. **p < 0.01
significant difference from H2O2-treated cells
FIGURE 4 UV spectra of compounds (a) A12 (40 µM) and (b)
B14 (40 µM) alone and in the presence of FeSO4 (20–100 µM)
molecular modeling technique. Therefore, Gold 5.2.1 program was
used for predicting the binding geometries of the titled compounds in
the gorge of the active site of AChE and BuChE. Donepezil as a
well-known AChE inhibitor and N-{[(3R)-1-(2,3-dihydro-1H-inden-2-
yl)piperidin-3-yl]methyl}-N-(2-methoxyethyl)naphthalene-2-carboxa-
mide (Ligand id: 3F9) obtained from the crystal structure of BuChE
(PDB id: 4TPK) were docked in eeAChE and homology model of BuChE
in order to validate docking methods, respectively. The first ranked
docking poses and the scoring values of the inhibitors inside the titled
proteins were figured and reported in Supporting Information
Figure S1 and S2 and Tables 3 and 4, respectively. It was observed
that the first ranked docking pose of and the crystal structure of
donepezil shared same binding orientation inside the active site of
AChE (Supporting Information Figure S1). A similar situation was
observed for the first ranked docking pose of and the crystal structure
of 3F9 which shared same binding orientation inside the homology
model of BuChE (Supporting Information Figure S2).
that all target compounds could interact with the Fe2+ effectively.
Similar results were also obtained when Cu2+ was added. However, no
significant shift was observed with the addition of Zn2+. The highest
metal chelating ability of the compounds was observed with Fe2+
(Figure 4A). The stoichiometry of compound A12-Cu2+ and compound
B14-Fe2+ complexes were determined by the molar ratio method.
Fixed concentrations of the compounds (40 µM) were mixed with
ascending doses of Fe2+ (20–100 µM) and the UV-vis absorption
spectra were recorded (Figure 4). The compounds showed a dose-
dependent increase in chelating ability.
|
2.4 Partition coefficent (log P) predictions
Log P is known as an important physicochemical parameter that
predicts the molecule's ability to cross blood–brain barrier (BBB).
According to literature survey, compounds that can penetrate central
nervous system generally have log P values around/more than two.[18]
For this purpose, the lipophilicities of the synthesized compounds
were calculated using MOE2016.08 (Molecular Operating Environ-
ment Chemical Computing Group) and the log P data show that all of
the compounds can pass the BBB (Tables 3 and 4).
As a result of the docking studies, it was determined that the
docking scores of the compounds of A series substituted from
4-position of the phenyl ring of 2-phenyl-1H-benzo[d]imidazole core
inside the active site of AChE were calculated in higher values in
comparison to the docking scores of them inside the active site of
BuChE. On the contrary, the docking score values of the compounds of
B series obtained with the changing of substitution from 4-position to
2-position of the phenyl ring inside BuChE were found better than the
calculated docking scores of the title compounds inside AChE. For all
series, the calculated docking scores were detected compatible for the
biological activity results except for the compounds bearing morpho-
line group (Table 1 and 4). For the morpholine-substituted compounds,
|
2.5 Docking studies
In our study, the non-covalent interactions of the synthesized 1H-
benzimidazole derivatives in the active sites of eeAChE (1EVE) and of
eqBuChE (homology model) enzymes have been evaluated using

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TABLE 3 Docking scores for A series benzimidazole derivatives
inside AChE (PDB id: 1EVE) and BuChE (homology model) using
Goldscore
TABLE 4 Docking scores for B series benzimidazole derivatives
inside AChE (PDB id: 1EVE) and BuChE (homology model) using
Goldscore
Compounds
A1
eeAchE
eqBuChE
LogP (o/w)
4.0740
3.9240
4.3660
4.4090
4.2590
4.7010
4.5530
4.9950
4.0460
3.8960
4.3380
3.7710
3.6210
4.0630
5.0267
4.8767
5.3187
SLogP
Compounds
B1
eeAchE
eqBuChE
LogP (o/w)
3.3900
4.0720
3.9220
4.3640
2.9560
3.7250
4.4070
4.2570
4.6990
3.2910
4.0190
4.7010
4.5510
4.9930
3.5850
3.3620
4.0440
3.8940
4.3360
2.9280
3.0870
3.7690
3.6190
4.0610
2.6530
3.1257
3.8077
3.6577
4.0997
2.6917
SLogP
69.4044 (1)
75.4004 (1)
71.2654 (1)
69.8060 (1)
75.2212 (1)
72.2870 (1)
74.9731 (1)
72.4824 (1)
70.4250 (1)
75.3565 (1)
71.5385 (1)
74.1913 (1)
74.0482 (1)
71.6347 (1)
70.4153 (1)
70.2326 (1)
70.9553 (1)
79.7038 (1)
61.3371 (1)
62.2159 (1)
65.0349 (1)
62.9269 (1)
64.6994 (1)
69.3038 (1)
63.5093 (1)
67.8135 (1)
64.9897 (1)
63.4599 (1)
68.6276 (1)
61.1835 (1)
64.8108 (1)
68.2482 (1)
62.4826 (1)
65.3679 (1)
67.2536 (1)
2.9235
2.6775
3.0676
3.2319
2.9859
3.3760
3.3309
3.7210
2.8317
2.5857
2.9758
2.7952
2.5492
2.9393
3.4941
3.2481
3.6382
63.9776 (1)
60.6100 (1)
62.3583 (1)
59.0345 (1)
62.7902 (1)
57.5379 (1)
60.6396 (1)
57.0439 (1)
54.7265 (1)
56.0755(1)
56.9541 (1)
56.5172(1)
56.1283 (1)
64.1476 (1)
62.1223 (1)
61.4483(1)
56.6740 (1)
62.9744 (1)
70.8121 (1)
66.3160 (1)
58.7191 (1)
59.2004 (1)
51.8237 (1)
64.7433 (1)
63.9508 (1)
51.6589 (1)
54.9256 (1)
44.6938 (1)
45.0584 (1)
54.0803 (1)
67.3976 (1)
69.4207 (1)
68.1378 (1)
72.3041 (1)
68.0988 (1)
69.3457 (1)
68.3246 (1)
69.5202 (1)
74.6677 (1)
71.9423 (1)
70.5803 (1)
71.1736 (1)
73.3021 (1)
74.3642 (1)
73.5071 (1)
70.0103 (1)
67.3865 (1)
71.8285 (1)
71.1003 (1)
73.9741 (1)
71.4139 (1)
75.7295 (1)
73.3947 (1)
73.5357 (1)
73.2106 (1)
73.0449 (1)
69.9288 (1)
67.8756 (1)
72.4928 (1)
74.0361 (1)
71.1902 (1)
2.1433
2.9235
2.6775
3.0676
1.9139
2.4517
3.2319
2.9859
3.3760
2.2223
2.7967
3.5789
3.3309
3.7210
2.5673
2.0515
2.8317
2.5857
2.9758
1.8221
2.0150
2.7952
2.5492
2.9393
1.7856
2.1605
2.9407
2.6947
3.0848
1.9311
A2
B2
A3
B3
A4
B4
A5
B5
A6
B6
A8
B7
A9
B8
A10
A11
A12
A13
A14
A15
A16
A17
A18
Donepezil
B9
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28
B29
B30
3F9
Calculated LogP (octanol/water) and SLogP values. The absolute ranking
positions for the suggested binding poses are given inside brackets.
the biological inhibitory potencies of them on AChE were detected
more than on BuChE.
In the current study, it was determined that, in general, while A
series compounds displayed selective inhibitory potency against AChE,
B series showed this potency against BuChE. For instance, it was
observed that 2-{4-[3-(piperidin-1-y)propoxy]phenyl}-1H-benzimid-
azole and 2-{2-[3-(piperidin-1-y)propoxy]phenyl}-1H-benzimidazole
derivatives displayed the most active and selective inhibitory potency
against AChE and BuChE, respectively. The other derivatives excluded
the compounds bearing piperidine ring were found to display selective
inhibitory potency against AChE or BuChE in accordance with the
substitution position on phenyl ring apart from some compounds.
Among the A series compounds which displayed the most active
and selective inhibitory activity on AChE, 5-nitro-substituted benz-
imidazole derivatives as compounds A10–12 were detected as the
most active compounds with high selectivity and compound A12 was
found to have the best inhibitory potency. The first ranked docking
pose of compound A12 demonstrated the piperidine ring of compound
A12 lying in the quaternary ammonium binding domain of the catalytic
site of AChE formed by amino acid residues Trp84 and His440. In
addition, 5-nitro-substituted benzimidazole ring of compound A12
reached into the peripheral site of AChE and the phenyl ring bearing
ether group of compound A12 settled into hydrophobic pocket formed
by amino acid residues Tyr121, Phe290, Phe330, Phe331, and Tyr334
at the catalytic site of the enzyme (Figure 5). Also compounds A10 and
Calculated LogP (octanol/water) and SLogP values. The absolute ranking
positions for the suggested binding poses are given inside brackets.
A11 shared the similar binding orientation with A12 in the active site of
AChE. The 3-aminopropyl chains of these derivatives reached into the
pocket formed by Trp84, Gly117, Gly118, Gly119, Glu199,
Ser200, Phe330, and His440 that is present at the quaternary
ammonium binding domain of the catalytic site of AChE.
5-Nitro-substituted benzimidazole ring of the compounds lied toward
the peripheral site of AChE and the phenyl ring of the compounds
settled between amino acid residues as Asp72, Tyr121, Phe290,
Phe331, and Tyr334 at the catalytic site of enzyme (Supporting
Information Figures S9–S11). The binding orientation of compounds

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FIGURE 5 The first-ranked docking pose of compound A12 in AChE. Cyan and light gray sticks represent compound A12 and the active
site residues, respectively. The active site residues are named using three-letter code. For a clear image, all hydrogen atoms were ghosted.
H bonds are represented by black-dashed lines, respectively
A10–12 offered to form noncovalent interactions with the key
residues which contributed the binding process of the related
compounds. In addition, among the A series compounds, compounds
A6, A8, A9, A14, A15, A17, and A18 which were substituted with the
hydrogen bonding acceptor groups at 5 and/or 6 positions of
benzimidazole rings shared similar binding orientation and were the
most active inhibitors against AChE (Supporting Information Figures
S10 and S11).
obtained docking poses of both series compounds were analyzed, it
was assumed that the bulky B series compounds did not penetrate the
entrance of AChE gorge easily compared to A series compounds and
the calculated docking scores were also able to support the
suggestions. Replacement of several aromatic amino acid residues in
AChE with small and polar amino acid residues in BuChE lead to a larger
gorge, thus, the bulky B series compounds could pass into the active
site of BuChE (Supporting Information Figures S25 and S26). On the
other hand, it is suggested that the large gorge of BuChE reduces
the substrate selectivity.[19] For this reason, it was predicted that the
placements of the synthesized compounds inside the large gorge of
BuChE influenced the binding affinity and selectivity.
The other orientation has been determined for the 2-(p-
substituted)phenyl-1H-benzimidazole derivatives inside the active
site of AChE especially for the compounds bearing 3-diethylamino-
propyl chain except for compound A10 (compound A1, A4, A13, and
A16). The benzimidazole ring of the compounds reached into the
pocket formed by Trp84, Gly117, Gly118, Gly119, Glu199, Ser200,
Phe330, and His440, and the 3-diethylaminopropyl chains of the
compounds lied toward the peripheral site of AChE (Supporting
Information Figures S9–S11). Among these compounds, compounds
A13 and A16 which have hydrogen bonding acceptor groups
substituted on 5 and/or 6 positions of benzimidazole rings also
increased the inhibitory potency against AChE.
As stated by the enzyme inhibition assay, the B series compounds
were generally found to display the most active and selective inhibitory
activity on BuChE and compound B14 was the most active analogue
against BuChE. When the best ranked docking pose of compound B14
was visualized, the piperidine ring of compound B14 leaned toward the
hydrophobic pocket formed by Leu285, Ala328, Phe329, and Tyr332.
Besides, the phenyl ring of compound B14 reached into the pocket
formed by Trp82, Gly115, and Glu197 and the benzimidazole ring of
compound B14 settled close to Gly116 and Thr120 (Figure 6).
Among the synthesized compounds, it was detected that the B
series compounds bearing piperidine ring possessed selective inhibi-
tory activity against BuChE. While the docking poses of the
compounds B4, B9, B14, B19, B24, and B29 bearing piperidine ring
were compared, the 2-(o-substituted)phenyl-1H-benzimidazole skele-
ton of the compounds settled into the pocket formed by Gln67, Thr69,
Trp82, Asn83, Gly115, Gly116, Thr120, Gly121, Tyr128, Glu197, and
The main differences between AChE and BuChE are the absence
of several aromatic residues at the peripheral site, gorge entrance, and
in the acyl pocket of BuChE, which are located at the same positions of
AChE.[19] Regarding A series compounds, it was observed that A series
compounds occupied both catalytic and peripheral site of AChE as dual
binding. For B series compounds in AChE, it showed that they only
occupied the catalytic site of active site AChE in docking studies
(Supporting Information Figures S15–S19). In addition, when the

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SARIKAYA ET AL.
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FIGURE 6 The first-ranked docking pose of compound B14 in BuChE. Cyan and light gray sticks represent compound B14 and the active
site residues, respectively. The active site residues are named using three-letter code. For a clear image, all hydrogen atoms were ghosted.
H bonds are represented by black-dashed lines, respectively
Ile442. In addition, 3-(piperidin-1-yl)propyl chains of the title
compounds settled into the hydrophobic pocket formed by Leu285,
Ala328, Phe329, Tyr332, Trp430, Met437, His438, and Gly439
(Supporting Information Figure S23). This binding orientation inside
the active site of BuChE was observed for the other compounds which
have the selective inhibitory activity to BuChE too. The settlement of
2-phenyl-1H-benzimidazole skeleton of the compounds inside the
pocket formed by Gln67, Thr69, Trp82, Asn83, Gly115, Gly116,
Thr120, Gly121, Tyr128, Glu197, and Ile442 allowed molecular
interactions with the key residues as Trp82, Glu197, and His438 of
the active site and restricted the movement of the compounds
(Supporting Information Figure S23). On the contrary, most of the
compounds bearing 5,6-dimethoxy substituted and nonsubstituted
benzimidazole rings and the compounds bearing morpholine groups
displayed different orientation inside the active site of BuChE, and
their 2-phenyl-1H-benzimidazole skeletons settled into the large
pocket formed by Gly117, Gln119, Trp231, Val277, Leu286, Ser287,
and Asn289 (Supporting Information Figures S20–S22, S24). There-
fore, some limitations have occurred about reaching to the key
residues for the binding process. Similar orientations were detected for
the A series compounds with 2-(p-substituted)phenyl-1H-benzimid-
azole core inside the active site of BuChE. The 2-(p-substituted)
phenyl-1H-benzimidazole skeleton of the compounds went toward
the large pocket formed by Gly117, Gln119, Trp231, Val277, Leu286,
Ser287, and Asn289 (Supporting Information Figures S12–S14). On
the other hand, 3-aminopropyl chains were observed to reach out the
key residues (Trp82, Glu197, and His438) so that this could be
considered to form the interactions between the compounds and the
title residues. Besides, these derivatives did not occupy the pocket
formed by Gln67, Thr69, Trp82, Asn83, Gly115, Gly116, Thr120,
Gly121, Tyr128, Glu197, and Ile442 (Supporting Information Figures
S12–S14). Therefore, it could have affected the interactions which
contributed to the binding process of the 2-(p-substituted)phenyl-1H-
benzimidazole derivatives inside the active site of BuChE. The docking
poses of the synthesized compounds inside AChE and BuChE are given
in supplementary materials.
|
2.6 MD simulations
In this study, MD simulations were executed to analyze the stability
and the hydrogen bonding network and also to calculate the average
free binding energies of compound A12 inside AChE and compound
B14 inside BuChE. For this purpose, the first ranked poses of
compound A12 inside AChE (PDB id: 1EVE resolved at 2.5 Å) and
compound B14 inside the homology model of BuChE obtained from
docking studies were selected for MD simulations. The compound
A12-AChE and compound B14-BuChE complexes and the apo forms
of title proteins were then exposed to 100 ns simulations after the
equilibrium step.
|
2.6.1 Structural stability analysis
As a result of structural stability analysis, the RMSD plot of compound
A12 in complex with AChE was seen stable with the average RMSD
value ∼1.5 Å during the whole MD simulations. Meanwhile it was
detected that the average RMSD value was ∼0.1 Å from beginning to

SARIKAYA ET AL.
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end of MD simulations in the RMSD plot of compound A12. For
compound B14 in complex with BuChE, the average RMSD value had
been slightly increasing ∼1.5 to ∼2.0 Å at the time slot of 0 ns and
around 82 ns. After 82 ns, the RMSD value increased from ∼2.0 to
∼2.5 Å and it has been staying at ∼2.5 Å until the end of MD
simulations. In addition, the average RMSD value of compound B14
was detected ∼0.1 Å during the entire MD simulations. The stability of
the apo forms of the titled enzymes identified the average RMSD
values for both proteins as fluctuating between ∼1.5 and ∼2.5 Å,
however, were mostly kept at ∼2.0 Å throughout the entire MD
simulations. To sum up, both compounds have preserved their
existence inside the active site of the related proteins during the
entire simulations (Figure 7).
played a key role for binding process of compound A12 was π–π
interaction monitored between the benzimidazole core and Tyr121,
Phe290, and Trp279 residues. The benzimidazole ring of the ligand
was sandwiched between Tyr121 and Phe290. In addition, it was
observed that compound A12 that has positively charged nitrogen
atom (N3) of piperidine ring occupied the pocket formed by Trp84,
Phe330, Phe331, Tyr334, and His440, and resulted in cation-π and
hydrophobic interactions between ligand and the pocket residues and
these interactions were also supported by energy calculations. The
cation-π interactions of compound A12 were mostly viewed between
the positively charged nitrogen atom (N3) of piperidine ring and Trp84,
however, they were also observed between the other pocket residues,
too (Supporting Information Figures S3–S5).
Regarding compound B14 in the active site of BuChE, the
noncovalent interactions such as hydrogen and ionic bond, π–π,
cation-π and hydrophobic interactions were the main contributors to
the binding process. A hydrogen bond was detected between the
positively charged amine group of piperidine ring and the nitrogen
(ND1) of the imidazole ring of His438 (Table 8). Also, nitrogen atoms of
the benzimidazole ring and the positively charged amine group of the
piperidine ring formed hydrogen bonds with water molecules and
compound B14 formed water-mediated bridges with Thr120, Glu325,
and His438 (Tables 9 and 10). Another important interaction formed
between compound B14 and the active site residues is π–π interaction
that was detected between the indole ring of Trp82 and the phenyl ring
of the studied compound (Supporting Information Figures S6–S8).
Besides the hydrogen bond between compound B14 and His 438, the
cation–π interaction was also observed between the positively
charged amine group of piperidine ring of compound B14 and
imidazole ring of His438. Ionic bond, one of the important noncovalent
|
2.6.2 Binding mode analysis
The binding mode of compound A12 in the active site of AChE
exhibited a hydrogen bonds, π–π, cation-π and hydrophobic interac-
tion that played an important role for molecular interactions. For
compound A12, nitrogen (N1) of benzimidazole ring formed hydrogen
bonds with active site residues Gly119 and Arg289; and the positively
charged nitrogen atom (N3) of piperidine ring formed hydrogen bonds
with active site residues as Gly118 and His440 (Table 5). As well as the
hydrogen bonds formed between compound A12 and the active site
residues, water-mediated hydrogen provided an important contribu-
tion for hydrogen bonding networks. The nitrogen atoms (N and N1) of
benzimidazole ring, the positively charged nitrogen atom (N3) of
piperidine ring and oxygen atoms (O and O1) of nitro group have
formed hydrogen bonds with Gly119, Arg289, and Tyr334 via water
molecules (Tables 6 and 7). The other noncovalent interaction that
FIGURE 7 (a) Plots of root-mean square deviation (RMSD) of AChE and compound A12-AChE complex. (b) Plots of RMSD of BuChE and
compound B14-BuChE complex. RMSDs were calculated using initial structures as templates. Green, red, and black RMSD plots are
represented for ligands, proteins, and complexes, respectively. The trajectories were captured every 1 ps until the simulations end

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TABLE 5 Hydrogen bonds are formed between compound A12 and AchE
Compound
A12
Acceptor
Gly119:O
Arg289:O
Gly118:O
His440:O
Donor-H
H3
Donor
N1
Frames
30948
22572
11436
7854
Frac
Average distance (Å)
2.8438
Average angle (°)
157.0734
0.3030
0.2211
0.1120
0.0769
H3
N1
2.8409
157.7624
H14
H14
N3
2.8280
163.6306
N3
2.8472
160.7191
Cpptraj module of Ambertools 16 was used to analyze hydrogen bonding using default criteria of cpptraj module for hydrogen bonds (a cutoff of 3.0 Å for
distance and 135° for angle).
TABLE 6 Hydrogen bonds are formed between compound A12 and water molecules
Compound
A12
Acceptor
Donor-H
SolventH
H14
Donor
Count
74613
49266
10681
8736
Frac
Average distance (Å)
2.8619
Average angle (°)
159.7557
N
SolventDnr
N3
0.7308
0.4823
0.1046
0.0856
0.0853
SolventAcc
SolventAcc
O
2.8587
155.6549
H3
N1
2.8746
156.4169
SolventH
SolventH
SolventDnr
SolventDnr
2.8661
152.1941
O1
8711
2.8672
151.9398
Cpptraj module of Ambertools 16 was used to analyze hydrogen bonding using default criteria of cpptraj module for hydrogen bonds (a cutoff of 3.0 Å for
distance and 135° for angle).
interaction of compound B14, was observed between carboxylate
group of Glu197 and the positively charged amine group of the
piperidine ring and played a key role for binding process (Supporting
Information Figures S6–S8). Hydrophobic interactions were moni-
tored between compound B14 and the catalytic site residues, too
(Supporting Information Figure S6).
As a result of energy decomposition analysis of MM-GBSA free binding
energy, the important energy contributions were provided by the
active site residues of AChE which are tabulated in Supporting
Information Table S1. Especially, Trp84, Phe330, and Phe331 were
provided more energy contributions for MM-GBSA free binding
energy compared to the other residues (Supporting Information Table
S1). Van der Waals and nonpolar solvation energies which supplied by
Trp84, Phe330, Phe331, Tyr334, and His440 were dominant
components for binding of the ligand to the active site of AChE, and
these energies had supported the contribution of titled residues to
form hydrophobic interactions with ligand in the active site of AChE.
Moreover, the electrostatic energy (in vacuum) contributions of the
title residues excluding Tyr334 effected the binding process of
compound A12 inside AChE (Supporting Information Table S1). In
addition, it was detected that van der Waals and nonpolar solvation
energies were the main factors for the contribution of free binding
energy provided by Gly118, Phe120, Tyr121, Trp279, and Phe290. It
was also calculated that the energy contribution of Gly119 and Arg289
had been nearly equal between the electrostatic energies (in vacuum
and solvent) and van der Waals and nonpolar solvation energies
(Supporting Information Table S1).
|
2.6.3 MM-GBSA binding free energy calculations
The MM-GBSA binding free energies were calculated for compound
A12 inside AChE and for compound B14 inside BuChE from the related
MD simulations, respectively. Both the average free binding energies
of the title compounds inside the related proteins are tabulated in
Table 11. According to free binding energy calculations, the
unfavorable contribution was provided from the electrostatic energies
(in vacuum and solvent) for both systems. Besides, the van der Waals
energies and nonpolar solvation energies were the main contributors
in contrast to the electrostatic energies (in vacuum and solvent) for the
binding of compound A12 to AChE and of compound B14 to BuChE.
TABLE 7 Hydrogen bonds are formed between compound A12 and
water-mediated bridging residues
In consequence of energy decompositions analysis of MM-GBSA
free binding energy, the significant energy contributions were
obtained from the interaction between amino acid residues as
Trp82, Gly115, Gly116, Thr120, Glu197, Ser198, Leu285, Ala328,
Phe329, Tyr332, Met437, His438, and Gly439, and ligand in the
compound B14-BuChE complex (Supporting Information Table S2).
The amino acid residues as Trp82, Glu197, and His438 provided the
largest contribution of free binding energy. The contents of free
binding energy displayed that van der Waals and nonpolar solvation
Compound
A12
Bridging residues
Gly119
Frames
14194
7725
Arg289
Tyr334
7338
Cpptraj module of Ambertools 16 was used to analyze hydrogen bonding
using default criteria of cpptraj module for hydrogen bonds (a cutoff of 3.0
Å for distance and 135° for angle).

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TABLE 8 Hydrogen bonds are formed between compound B14 and BuChE
Compound
B14
Acceptor
Donor-H
Donor
Frames
Frac
Average distance (Å)
Average angle (°)
HIS438:ND1
H14
N2
25406
0.2488
2.9052
157.7189
Cpptraj module of Ambertools 16 was used to analyze hydrogen bonding using default criteria of cpptraj module for hydrogen bonds (a cutoff of 3.0 Å for
distance and 135° for angle).
TABLE 9 Hydrogen bonds are formed between compound B14 and water molecules
Compound
B14
Acceptor
N
Donor-H
SolventH
H3
Donor
SolventDnr
N1
Count
129774
65771
42099
Frac
Average distance (Å)
2.8281
Average angle (°)
160.8705
1.2710
0.6442
0.4123
SolventAcc
SolventAcc
2.8644
156.1187
H14
N2
2.8469
162.1174
Cpptraj module of Ambertools 16 was used to analyze hydrogen bonding using default criteria of cpptraj module for hydrogen bonds (a cutoff of 3.0 Å for
distance and 135° for angle).
energies were favorable contributions supported by Trp82 for the
binding process (Supporting Information Table S2). These energy
contributions supported the π–π interactions between Trp82 and
compound B14 (Supporting Information Figure S6). It was observed
that total binding energy yielded from His438 had been the highest
than the energy obtained from other residues. The energy decompo-
sition showed that the electrostatic energy (in vacuum) had been major
favorable energy contribution yielded from His438. On the other hand,
it was calculated that the contributions of van der Waals and nonpolar
solvation energies had been much as the electrostatic energies (in
vacuum and solvent) (Supporting Information Table S2). As a result of
energy calculations yielded from Glu197, the electrostatic energies (in
vacuum and solvent) had been favorable for the contribution of
binding process. They contributed the ionic bonding between the
carboxylate group of Glu197 and the positively charged amine group
of the piperidine ring of compound B14. The energy contribution of
His438 showed that the electrostatic energies (in vacuum and solvent)
and van der Waals and nonpolar solvation energies were equally
efficient for binding process. The calculated van der Waals and
nonpolar solvation energies supported hydrophobic interactions
between compound B14 and His438, however, the electrostatic
energies (in vacuum and solvent) supplied the cation–π interactions
between the positively charged amine group of piperidine ring and the
imidazole ring of His438 (Supporting Information Figures S6–S8 and
Table S2). The residues mentioned above excluded Trp82, Glu197, and
His438 provided van der Waals and nonpolar solvation energies for
binding process of compound B14.
|
3
CONCLUSION
New 2-[o/p-(3-substitutedpropoxy)phenyl]-1H-benzimidazole ana-
logues were designed, synthesized, and evaluated for their potential
to inhibit ChE and neuroprotective effects. According to ChEI activity
results, para (A) series generally displayed better inhibitor activity
against AChE while ortho (B) series showed higher BuChEI activity. In
our previous studies, we have mentioned the same observation for the
derivatives with ethoxy side chain so we can speculate that extending
the side chain from ethoxy to propoxy did not make a change in terms
of the selectivity of ChE activity.[12,13] In the para series, nitro, nitrile,
dimethoxy derivatives that are newly added in this study exhibited
excellent AChE inhibitory activity compared with nonsubstituted,
methyl and chloro derivatives. It was observed that these three
substituents improved the AChEI activity.[12]
On the other hand, when the evaluation of cell viability of SH-
SY5Y cells exposed to H2O2, compounds B13 and B14 remarkably
increased cell viability. In addition, most of the synthesized compounds
reduced free radical production and compounds A12, B13, and B14
improved H2O2-triggered downregulation of HO-1.
Taken together, compounds A12 and B14 exhibited potential
AChE and BuChE inhibitory activities, high neuroprotection against
H2O2-induced toxicity, free radical scavenging properties, and metal
chelating abilities and also the ability to cross the BBB. As a result,
this study indicated that these compounds could be considered as
multi-targeted lead compounds for the further study in the
treatment of AD.
TABLE 10 Hydrogen bonds are formed between compound B14
and water-mediated bridging residues (BuChE)
Compound
B14
Bridging residues
Glu325 His438
His438
Frames
26666
10104
8466
In the current study, docking guided MD simulations of compound
A12 inside AChE displayed that hydrogen bonding network, cation–π,
π–π, and hydrophobic interactions have played key roles for the
stability of compound A12 in the active site of AChE. In addition to the
noncovalent interactions mentioned for compound A12-AChE
Thr120
Cpptraj module of Ambertools 16 was used to analyze hydrogen bonding
using default criteria of cpptraj module for hydrogen bonds (a cutoff of 3.0
Å for distance and 135° for angle).

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SARIKAYA ET AL.
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TABLE 11 The calculated MM-GBSA free binding energies (Delta total energy) of compound A12 inside AChE and of compound B14 inside
BuChE
A12
B14
Van der Waals energy (kcal/mol)
−50.6990 0.1525
−275.6734 0.4594
283.8478 0.4282
−5.9132 0.0107
−326.3724 0.5230
277.9347 0.4243
−48.4377 0.1682
−9.407a
−47.4014 0.0735
−98.0244 0.2716
108.0376 0.2775
−5.2987 0.0064
−145.4258 0.2872
102.7388 0.2756
−42.6870 0.1306
−9.138a
Electrostatic energy (in vacuum) (kcal/mol)
Polar solvation energy (in solvent) (kcal/mol)
Non-polar solvation energy (kcal/mol)
Delta gas phase free energy (kcal/mol)
Delta solvation free energy (kcal/mol)
Delta total energy (kcal/mol)
Experimental binding energy (kcal/mol)
aExperimental binding free energy was calculated from IC50 values according to ΔG ≈ RT ln IC50
.
complex, the ionic bonding was also observed on evaluating of MD
simulations of the compound B14-BuChE complex. The analysis of
the components of MM/GBSA free binding energies proposed that
van der Waals and nonpolar solvation energies have been favorable
for binding process of the title compounds inside their targeted
proteins. It was indicated that the contributions of the electrostatic
energies (in vacuum and solvent) obtained by the energy calculations
were unfavorable for the calculated binding energies of the title
compounds inside their targeted proteins. For this situation, it could
be excluded the contributions of some active site residues as
especially Trp84, Gly119, Phe330, and His440 for the compound
A12-AChE complex, and Glu197 and His438 for the compound B14-
BuChE complex.
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
|
4.1.2 Synthesis of 2-[p-(3-substitutedpropoxy)phenyl]-
1H-benzimidazole derivatives
A mixture of 1-bromo-3-chloropropane (20 mmol) and secondary
amines (30 mmol) (diethylamine, pyrolidine, piperidine) in anhydrous
THF (10 mL) was stirred at room temperature for 24 h. After
concentration in vacuo, the resultant residue was taken up in 2 N
HCl and washed with ethyl acetate. The aqueous layer was basicified
with 2 N NaOH to pH 14. The compound was extracted with ethyl
acetate and the organic phase dried over anhydrous magnesium
sulfate. The solution was then concentrated in vacuo to give the
desired compound as oil.[20]
|
4
EXPERIMENTAL
To a solution of 4-hydroxybenzaldehyde (1 mmol) in DMF, K2CO3
(5 mmol), NaI (2.5 mmol), and 3-chloropropylamine derivatives
(2.5 mmol) were added. The suspension was stirred at 110°C for 4 h.
The reaction mixture was filtered and the organic phase was
concentrated in vacuo. The resultant residue was then purified by
column chromatography.[21]
|
4.1 Chemistry
|
4.1.1 General
Melting points were determined with capillary melting point apparatus
(Stuart SMP30, Staffordshire, UK). The IR spectra of the compounds
were monitored by attenuated total reflectance (ATR) (PerkinElmer
Spectrum 100 FT-IR, Shelton, USA). The nuclear magnetic resonance
(NMR) spectra (400 MHz for 1H NMR and 100 MHz for 13C NMR)
were recorded in the deuterated solvent on AS400 Mercury Plus NMR
Varian (Varian Inc., Palo Alto, CA, USA). Chemical shifts were measured
in parts per million (δ). Coupling constants (J) are reported in hertz (Hz).
LC/MS was recorded on a Thermo MSQ Plus (San Jose, CA, USA) mass
spectrometer using ESI (+) method. Elemental analyses were
performed by Leco TruSpec Micro CHNS (Leco, St. Joseph, MI,
USA) and were within 0.4% of the theoretical values. Analytical thin-
layer chromatography (TLC) was run on Merck silica gel plates
(Kieselgel 60 F254) with detection by UV light (254 nm); column
chromatography was performed using Merck silica gel 60 (63–200 mm
diameter). All starting materials and reagents were high-grade
commercial products.
Substituted-o-phenylenediamine (1 mmol) and an appropriate
aldehyde (1 mmol) were placed in a 50 mL Schlenk tube and dissolved
in DMF (10 mL). TMSCl (2.5 mmol) was added dropwise to solution and
tube was sealed and stirred at 90°C for 4 h. After cooling, the reaction
mixture was poured into water (20 mL) and kept to stand at room
temperature in an ultrasonic bath for 1 h. The DMF–water mixture was
concentrated in vacuo. The resultant residue was then purified by
column chromatography and crystallized with an appropriate
solvent.[22]
|
4.1.3 Synthesis of 2-[o-(3-substitutedpropoxy)phenyl]-
1H-benzimidazole derivatives
A mixture of 1-bromo-3-chloropropane (20 mmol) and secondary
amines (30 mmol) (diethylamine, pyrolidine, piperidine, morpholine) in
anhydrous THF (10 mL) was stirred at room temperature for 24 h. After

SARIKAYA ET AL.
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concentration in vacuo, the resultant residue was taken up in 2 N HCl
and washed with ethyl acetate. The aqueous layer was basicified with
2 N NaOH to pH 14. The compound was extracted with ethyl acetate
and the organic phase dried over anhydrous magnesium sulfate. The
solution was then concentrated in vacuo to give the desired compound
as oil.[20]
incubation, required aliquots of the DTNB solution (0.01 M, 100 mL)
and of the ATC/BTC (0.075 M, 20 mL) were added. After a rapid and
immediate mixing, the absorption was measured at 412 nm.
|
4.2.2 Cell culture and treatments
To a solution of 2-hydroxybenzaldehyde (1 mmol) in DMF, K2CO3
(5 mmol), NaI (2,5 mmol), and 3-chloropropylamine derivatives
(2.5 mmol) were added. The suspension was stirred at 110°C for 4 h.
The reaction mixture was filtered and the organic phase was
concentrated in vacuo. The resultant residue was then purified by
column chromatography.[21]
The human neuroblastoma cell line (SH-SY5Y) used in this study was
purchased from the American Type Culture Collection (ATCC, Catalog
#CRL-2266). Cells were suspended in complete Dulbecco's modified
Eagle Medium (DMEM) (Life Technologies, Gibco BRL, Grand Island,
NY) supplemented with 10% fetal bovine serum (FBS, Hyclone), 1%
penicillin and streptomycin (100 U/mL, Invitrogen) and plated in cell
culture dishes. The cultures were maintained at 37°C in 5% CO2 95%
humidified atmosphere. After reaching 85% confluence, cells were
transferred to 96-well plates or culture dish. The stock solutions of the
test compounds (5 mM) were prepared in sterile DMSO and these
stocks were then appropriately diluted with the complete culture
medium and DMSO levels were maintained below 1% in the test
concentrations.
Equimolar amount of 2-substitutedbenzaldehyde (3 mmol) in 2 mL
ethanol was added to the water solution of NaHSO3 and stirred at
room temperature for 15 min. After the ethanol–water mixture was
evaporated in vacuo, sodium hydroxy(2-hydroxysubstitutedphenyl)-
methane sulfonate salt was obtained. Sodium hydroxy(2-hydroxysub-
stitutedphenyl)methane sulfonate salt (3 mmol), substituted-o-phenyl-
enediamine derivatives and DMF (9 mL) were placed in a round bottom
flask. The mixture was refluxed for 2–3 h with a CaCl2 drying tube on it
in an oil bath. Then the mixture was concentrated in vacuo. The
resultant residue was purified by column chromatography and
crystallized with an appropriate solvent.[23]
|
4.2.3 H2O2-induced toxicity
H2O2-induced toxicity was determined as previously described.[24]
Briefly, SH-SY5Y cells were seeded at a density of 2 × 103 cells per well
into 96 well plates and incubated for 24 h. After 24 h, the medium was
aspirated and the cells were washed twice with sterile PBS and then
treated with a range of concentrations (1, 10, 100, and 1000 μM) of
H2O2 in sterile PBS (with Ca2+ and Mg2+) for 1 h. After 1 h, H2O2
solution in the well was replaced with the warm medium and incubated
for 17 h. After the 18 h exposure, the cell viability was determined
using the MTT assay.
The spectral data of all final compounds are given in the
Supporting Information.
|
4.2 Biological activity assays
|
4.2.1 In vitro inhibition of AChE and BuChE
AChE (E.C.3.1.1.7., type VI-S, from electric eel) and BuChE (E.C.
3.1.1.8, from equine serum) were purchased from Sigma–Aldrich (St.
Louis, MO, USA). 5,5′-Dithiobis(2-nitrobenzoic acid) (Ellman's reagent,
DTNB), buffer compounds (potassium dihydrogen phosphate, potas-
sium hydroxide), sodium hydrogen carbonate and acetylthiocholine
iodide (ATC), butyrylthiocholine iodide (BTC) used as substrates were
obtained from Sigma–Aldrich. Spectrophotometric measurements
were performed on a Shimadzu UV/160-A spectrophotometer.
AChE/BuChE enzyme activities were investigated using a slightly
modified colorimetric method of Ellman et al.[14] As the product of the
enzymatic hydrolysis, the thiocholine does not possess a significant
chromophore for UV detection; the evaluation of enzyme activity was
|
4.2.4 Cell viability assay against H2O2-induced
toxicity
SH-SY5Y cells were seeded at a density of 2 × 103 cells/well into 96
well plates and incubated for 24 h. After 24 h, the cells were treated
with synthesized compounds at a range of concentrations (0, 0.1, 1,
and 10 μM) for 24 h. After 24 h, the medium was aspirated and the cells
were washed twice with sterile PBS and then treated with 150 μM of
H2O2 in sterile PBS (with Ca2+ and Mg2+) for 1 h. After 1 h, the H2O2
solution in the well was replaced with the warm medium and incubated
for 17 h. After the 18 h exposure, the cell viability was determined
using the MTT assay.
performed using
a specific chromogenic reagent, DTNB. Stock
solutions of the inhibitor compounds were prepared in 2% dimethyl-
sulfoxide (DMSO) which were diluted with aqueous assay medium to a
final content of organic solvent always lower than 0.2%. The enzyme
activity was determined in the presence of at least five different
concentrations of an inhibitor, generally between 10−3 and 10−8, in
order to obtain the inhibition of AChE or BuChE activity between 0 and
100%. Each concentration was assayed in triplicate. Prior to use, all
solutions were adjusted to 20°C. Enzyme solution (2.5 units/mL,
100 mL) and inhibitor solution (100 mL) were added into a cuvette
containing the phosphate buffer (3.0 mL, 0.1 M; pH 8.0). After 5 min of
|
4.2.5 Cell proliferation assay
SH-SY5Y cells were seeded at a density of 2 × 103 cells/well into 96
well plates and incubated for 24 h. After 24 h, the cells well treated
with synthesized compounds at 1 and 10 μM concentrations for 24 h.
Following the 24 h exposure, the cell viability was determined using
the MTT assay.

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SARIKAYA ET AL.
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|
4.2.6 MTT assay
Cell Signaling Technology) at room temperature for 1 h. After
washing, the membrane was incubated with infrared labeled
secondary antibodies (Odyssey Western Blotting kit, LI-COR
Biosciences) at room temperature for 1 h. Proteins were visualized
and quantified by scanning the membrane on an Odyssey Infrared
Imaging System (LI-COR Biosciences, USA) with both 700- and 800-
nm channels.
Percent cell viability was assessed using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, following
exposures, MTT (in final concentration 0.5 mg/mL) was added to each
well and plates were incubated for 3 h at 37°C in 5% CO2 humidified
incubator. The reaction mixture was carefully removed and 200 μL
DMSO was added to each well. The plate was shaken at room
temperature for 1 h and then the absorbance was measured at 570 and
630 nm using a microplate reader (VersaMax, Molecular Devices,
USA). Percent survival was plotted relative to vehicle control cells,
which were normalized to 100% survival.
|
4.2.10 Statistical analysis
Data were expressed as means standard deviation (SD) for five or
three independent experiments in triplicate. Comparisons of means
between groups were performed by one-way analysis of variance
(ANOVA) followed by Tukey's post-hoc test. p < 0.05 was considered
statistically significant.
|
4.2.7 Intracellular ROS measurement
ROS production was measured by monitoring the oxidation of the
cell-permeable nonfluorescent reagent CellROX Deep Red Reagent
(Molecular Probes, Carlsbad, CA), which, upon oxidation, exhibits a
strong fluorogenic signal. Briefly, SH-SY5Y cells were seeded at a
density of 2 × 103 cells/well into 96 well plates and incubated for
24 h. After 24 h, the cells were treated with synthesized
compounds at indicated concentrations for 24 h. After 24 h, the
medium was aspirated and the cells were washed twice with sterile
PBS and then treated with 150 μM of H2O2 in sterile PBS (with
Ca2+ and Mg2+) for 1 h. Subsequently, cells were incubated with
5 μM CellROX Deep Red Reagent for 30 min at 37°C and, after
three washes with PBS, the fluorescence was measured at ex 640/
em 665 nm using Varioskan Flash Multimode Reader (Thermo
Scientific, USA). ROS production was expressed as a percentage of
control cells.
|
4.2.11 Metal chelating property
The chelating ability of compounds for biometals such as Cu2+, Fe2+
,
and Zn2+ was studied by UV-visible spectrophotometer (Shimadzu
160A) in ethanol at 298 K with wavelength ranging from 200 to
500 nm. ZnSO4, CuSO4, or FeSO4 solutions were prepared in 200 μM
using ethanol. The tested compounds were prepared in 400 μM using
ethanol. To a mixture of 300 μL tested compound solution and
2100 μL ethanol, 600 μL ZnSO4 solution (CuSO4 or FeSO4) was added.
The solution was incubated at 298 K for 30 min and then the
absorption spectra were recorded at 298 K in a 3 cm quartz cell.
The control was prepared by mixing 300 μL tested compound solution
and 2700 μL ethanol. All the test solutions were measured against the
blank.[25]
|
4.2.8 Protein analysis
|
4.3 Molecular modeling study
SH-SY5Y cells (0.5 × 106 cell/well) were seeded into 6 well plates and
incubated for 24 h. After 24 h, the cells were treated with synthesized
compounds at indicated concentrations (A12 at 1 μM; A15 to 16, B12
to 13 at 0.1 μM; B14 at 10 μM) for 24 h. After 24 h, the medium was
aspirated and the cells were washed twice with sterile PBS and then
exposed to 150 μM of H2O2 in sterile PBS (with Ca2+ and Mg2+) for 1 h.
Following H2O2 induction, the cells were washed with ice-cold 1 × PBS
and lysed in cell lysis buffer containing phosphatase and protease
inhibitors. These lysates were quantified using the BCA protein assay,
then, Western blot assay was done.
|
4.3.1 Homology modeling
Sequence of Equus caballus (horse) BuChE (Q9N1N9) was taken from
The Universal Protein Resource Knowledgebase (UniProtKB).
Homology model of eqBuChE was built using SWISS-MODEL
server.[26] For this aim, the crystal structure of huBuChE (PDB id:
4TPK resolved at 2.7 Å) obtained from the RCSB Protein Data Bank
has been selected as the template structure to construct the
homology model. The selected PDB file displays a primary amino
acid sequence identity of 90.80% with eqBuChE. Following the
construction of the model, the crude model was aligned with chain A
of the crystal structure of huBuChE (PDB id: 4TPK) for indicating the
binding site residues of the model. The crude model was
parametrized with AMBER99SB force field, solvated in an octahedral
box with TIP3P water molecules with 10 Å distance between the
protein surface and the box boundary and neutralized with
appropriate number of chlorine counter ions.[27,28] The preparation
of the model for energy minimization was practiced using xleap
module of AmberTools 16. The model was exposed to an energy
minimization with Sander.MPI module of AmberTools 16.[29]
|
4.2.9 Western blotting
Western blotting was performed by loading 30 µg protein on 10%
(w/v) Tris-glycine denaturing gels and separating proteins by
electrophoresis, then transferring to PVDF membrane. After block-
ing, the membrane was incubated with primary antibody, anti-HO-1
rabbit monoclonal antibody (1:1000, Cell Signaling Technology) at
+4°C overnight. Following the incubation, the membrane was
incubated with anti-β-actin mouse monoclonal antibody (1:1000,

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|
4.3.2 Ligand docking
equilibration (2 ns), and production (100 ns). Firstly, the systems
were heated from 0 to 300 K with 10 kcal/mol/Å restraint force
permitting water molecules and ions to move freely. Secondly, the
temperature was equilibrated at 300 K using Langevin dynamics with
the collision frequency of 1.0 ps−1 in constant volume periodic
boundary for the entire systems. The pressure was equilibrated at 1
bar with keeping positional restrains for the solute using constant
pressure periodic boundary conditions with isotropic position scaling
method at 300 K. Lastly, the positional constrains were gradually
removed keeping the temperature at 300 K and pressure at 1 bar. In
the equilibration and production steps, the SHAKE algorithm was
performed to constrain band vibrations involving hydrogen atoms.[37]
The Particle Mesh Ewald (PME) method was practiced for long-range
electrostatic interactions.[38] The time step for all MD simulations has
been 2 fs and the non-bonded interactions were truncated using cutoff
of 10 Å. All systems were subjected to the production step for 100 ns.
Xmgrace program was used for visualization of the trajectories.[39] The
hydrogen bonding was detected with Cpptraj module of AmberTools
16 using default parameters.[40] The calculations of free binding
energies of compounds and the energy decomposition analysis were
executed with MMPBSA.py.MPI module of AmberTools16 using the
Generalized-Born (GB) model from 100 spaced snapshots of the
unrestrained MD simulations.[41] MD snapshots were extracted from
free MD simulations using UCSF Chimera package.[42] Figures
associated with MD snapshots in supplementary materials section
were set up with MOE2016.08 program.
The chemical structures of benzimidazole derivatives were generated
with the builder panel of MOE2016.08 and protonated using the
protonate 3D protocol. The protonated structures were exposed to an
energy minimization with MOE.2016.08 using MMFF94x force
field.[30,31] The crystal structure of AChE (PDB id: 1EVE resolved at
2.5 Å) was yielded from the RCSB Protein Data Bank (http://www.rcsb.
org/pdb). Chain A was kept for docking study and chain B, heteroatoms,
water molecules (excluded the water molecules existed in the binding
site) were deleted from the pdb file. Chain A was exposed to an energy
minimization with MOE.2016.08 using AMBER99 force field.[32]
The homology model of BuChE was prepared for the docking study
removing water molecules (excluded the water molecules existed in the
binding site) and chlorine counter ions. The docking study of the ligands
was performed using GOLD 5.2.1 program with default generic algorithm
parameters.[33,34] The studied compounds were docked within a radius of
22 Å around the nitrogen atom (N1) of the imidazole group of His440 of
AchE and of His438 of BuChE. Hundred conformations were generated
per structure. GoldScore fitness function was used as scoring
functions.[33,34] Figures 5 and 6 were created with MOE2016.08 program.
|
4.3.3 MD simulations
MD simulations were executed for the apo form of proteins (AChE and
BuChE) and protein-ligand complexes using AMBER12.[29] The starting
protein-ligand complexes were prepared using first ranked docking
poses of compound A12 in AChE and of compound B14 in BuChE
yielded from docking studies. The partial atomic charges of compounds
A12 and B14 were calculated with AM1-BCC charge model using
antechamber module of AmberTools 16.[29,35] The apo form of
proteins and the protein-ligand complexes were prepared for the
energy minimizations and MD simulations using xleap module of
AmberTools 16.[29] General AMBER force field (gaff) for ligand and
AMBER ff99SB force field for proteins were used to become the
parameterization of the complex systems.[27,36] The apo form of AChE
and the ligand-AChE complex were neutralized with the appropriate
number of sodium counter ions. The apo form of BuChE and the ligand-
BuChE complex were neutralized with the appropriate number of
chlorine counter ions. All systems were solvated in an octahedral box
with TIP3P water molecules with 10 Å distance between the protein
surface and the box boundary.[28] Sander.MPI module of AmberTools
16 and pmemd.cuda module of AMBER12 were used to practice the
energy minimizations and MD simulations of the systems, respec-
tively.[29] To avoid bad steric contacts, the starting systems were
subjected to energy minimization in two steps. In the first step, the
energy minimizations were performed to the restrained initial
structures using the steepest descent algorithm at 1000 iterations
and the conjugate gradient methods at 1000 iterations. Following the
first step, the energy minimization was carried out in all systems using
steepest descent algorithm at 2500 iterations and using conjugate
gradient methods at 2500 iterations. The MD simulations for the
systems were practiced at the three steps as heating (0.1 ns),
|
4.4 LogP and SLogP predictions
LogP and SLogP values of the compounds were calculated using
MOE2016.08.[43]
ACKNOWLEDGMENTS
This study was supported by TUBITAK (Turkish Scientific and
Technical Research Council) (Project number: 213S027). We appre-
ciate Pharmaceutical Sciences Research Centre (FABAL) of Ege
University Faculty of Pharmacy for spectral analyses of the compounds
and molecular modeling software support.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
Vildan Alptüzün
http://orcid.org/0000-0002-1477-4440
http://orcid.org/0000-0003-4412-3048
Ayşe S. Alpan
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