Mono- or di-substituted imidazole derivatives for inhibition of acetylcholine and butyrylcholine esterases

Article abstract of DOI:10.1016/j.bioorg.2019.01.044

Mono- or di-substituted imidazole derivatives were synthesized using a one-pot, two-step strategy. All imidazole derivatives were tested for AChE and BChE inhibition and showed nanomolar activity similar to that of the test compound donepezil and higher than that of tacrine. Structure activity relationship studies, docking studies to on X-ray crystal structure of AChE with PDB code 1B41, and adsorption, distribution, metabolism, and excretion (ADME) predictions were performed. The synthesized core skeleton was bound to important regions of the active site of AChE such as the peripheral anionic site (PAS), oxyanion hole (OH), and anionic subsite (AS). Selectivity of the reported test compounds was calculated and enzyme kinetic studies revealed that they behave as competitive inhibitors, while two of the test compounds showed noncompetitive inhibitory behavior. ADME predictions revealed that the synthesized molecules might pass through the blood brain barrier and intestinal epithelial barrier and circulate freely in the blood stream without binding to human serum albumin. While the toxicity of one compound on the WS1 (skin fibroblast) cell line was 1790 μM, its toxicity on the SH-SY5Y (neuroblastoma) cell line was 950 μM.

Full text of DOI:10.1016/j.bioorg.2019.01.044

Accepted Manuscript
Mono- or Di-Substituted Imidazole Derivatives for Inhibiton of Acetylcholine
and Butyrylcholine Esterases
Burak Kuzu, Meltem Tan, Parham Taslimi, İlhami Gülçin, Mehmet Taşpınar,
Nurettin Menges
PII:
S0045-2068(18)31309-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.044
YBIOO 2748
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 October 2018
18 January 2019
19 January 2019
Please cite this article as: B. Kuzu, M. Tan, P. Taslimi, I. Gülçin, M. Taşpınar, N. Menges, Mono- or Di-Substituted
Imidazole Derivatives for Inhibiton of Acetylcholine and Butyrylcholine Esterases, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.01.044
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Mono- or Di-Substituted Imidazole Derivatives for Inhibiton
of Acetylcholine and Butyrylcholine Esterases
Burak Kuzu,a,b Meltem Tan,a,b Parham Taslimi,c İlhami Gülçin,c Mehmet Taşpınar,d Nurettin
Mengesa,b,*
a.Pharmaceutical Chemistry Section, Van Yuzuncu Yil University, 65080, Van, Turkey,
b. SAFF Chemical Reagent R&D Lab. YYU-TEKNOKENT, 65080, Van, Turkey
c. Department of Chemistry, Atatürk University, 25240-Erzurum, Turkey
d. Department of Medicinal Biology, Van Yuzuncu Yil University, 65080, Van, Turkey
Keywords: SAR, Docking, Alzheimer’s disease, water solubility, ADME, enzyme kinetic study
ABSTRACT: Mono- or di-substituted imidazole derivatives were synthesized using a one-pot, two-
step strategy. All imidazole derivatives were tested for AChE and BChE inhibition and showed
nanomolar activity similar to that of the test compound donepezil and higher than that of tacrine.
Structure activity relationship studies, docking studies to on X-ray crystal structure of AChE with PDB
code 1B41, and adsorption, distribution, metabolism, and excretion (ADME) predictions were
performed. The synthesized core skeleton was bound to important regions of the active site of AChE
such as the peripheral anionic site (PAS), oxyanion hole (OH), and anionic subsite (AS). Selectivity of
the reported test compounds was calculated and enzyme kinetic studies revealed that they behave
as competitive inhibitors, while two of the test compounds showed noncompetitive inhibitory
behavior. ADME predictions revealed that the synthesized molecules might pass through the blood
brain barrier and intestinal epithelial barrier and circulate freely in the blood stream without binding
to human serum albumin. While the toxicity of one compound on the WS1 (skin fibroblast) cell line
was 1790 µM, its toxicity on the SH-SY5Y (neuroblastoma) cell line was 950 µM.
1. Introduction
Alzheimer’s disease (AD) is one of the most important disorders and is irreversible. It is a progressive
brain disorder that slowly destroys memory, thinking skills, and the ability to carry out simple tasks.1,2
Altered levels of acetylcholine (ACh) in hippocampal and cortical regions lead to cholinergic system
dysfunction, resulting in severe memory and learning deficits. One therapeutic approach to enhance
1

cholinergic neurotransmission is to increase ACh availability by inhibiting acetylcholinesterase
(AChE).3-7 Thus far, several anti-AD drugs targeting ChEs have become available, including tacrine,8
donepezil,9 rivastigmine,10 and the alkaloid galantamine.11
Figure 1. Clinically used drugs for inhibition of ChEs.
However, no drugs have significantly affected the symptoms or stopped the progression of the
disease in clinical studies. Many research groups are studying AChE inhibitors, as the level of ACh is
crucial for AD therapy. Indole-containing piperidine derivatives,12 tacrine-based and carbamate
derivatives,13 selenium-piperidine derivatives,14 p-aminobenzoic acid derivatives,15 and coumarin-
type carbamate derivatives16 were tested for AChE inhibition. In addition, Moon et al. researched
oxotremorine derivatives, which possess imidazole, pyrrole, and triazole rings, and achieved
nanomolar activity using N,N-di-imidazole propargyl amine derivatives.17a There are many studies in
progress on AD in which structural motifs such as piperidine, alkyl chain spacer, carbamate, and
methoxy groups serve as active pharmacophore groups. Gurjar et al. have found out that imidazole
derivatives showed good potencies on inhibition of ChEs.17b These studies prompted us to search C-2
and C-4 substituted or C-2 substituted imidazole derivatives for inhibition of AChE and BuChE.
2. RESULTS AND DISCUSSION
2.1. Synthesis
We synthesized imidazole derivatives 2a-o, 2s, and 2u using a one-pot, two-step procedure based on
our previous study (Scheme 1).18 To extend the study and the modulate structure-activity
relationship (SAR), the condensation reaction was performed via a reaction between compound 2b
and hydroxyl amine in propanol at reflux temperature, giving rise to product 3b. Compounds 2b and
2e were reduced by NaBH4 to obtain 4b and 4e, which are racemic.
2

Scheme 1. Synthesis of imidazole derivatives (synthetic strategy for 2r and 2t is explained in SI).
2.2.Cholinesterase activity and Structure-activity relationship (SAR) study
Imidazole molecules have similar cores with an aroyl group at the C-2 position and an aryl group at
the C-4 position. IC50 values of BChE were between 27.02 and 151.2 nM, whereas those of AChE were
between 17.3 and 120.9 nM. While the clinically used reference drug tacrine displayed inhibition at
698.6 nM,19 donepezil displayed inhibition at 32.9 nM.20 Thus, the reported imidazole molecules
showed approximately 40- and 20-fold higher activity than tacrine and similar or higher activity than
donepezil (Table 1).
Table 1. Human Acetylcholinesterase and Butyrylcholinesterase (AChE, BChE) inhibition and some
predicted values.
Entry IC50
(nM)
IC50
(nM)
BChE
Ki
Ki (nM) AChE/
Log BBa Log
KHSA
Log P
Caco-2
b
(nM) BChE
AChE
BChE
Permeability
c
AChE
2a
34.7±
1.56
61.27± 4.49± 30.08± 0.149
2.07 1.65 5.98
-0.366
0.299
3.396
1598
3

2b
2c
2d
2e
2f
34.7±
2.83
70.16± 4.31± 43.01± 0.100
0.65 0.99 12.11
-0.979
-1.466
0.055
0.359
-0.137
0.462
0.155
0.184
0.952
0.928
0.047
0.330
1.925
0.378
0.168
3.159
1.624
4.266
3.328
3.485
5.157
5.040
3.013
4.165
7.597
3.849
2.020
673
34.6±
3.50
49.04± 14.32 33.83± 0.423
1.83 ±7.00 7.76
163
23.0±
0.02
36.64± 3.93± 15.13± 0.260
0.65 0.91 2.24
1804
1796
2141
2263
1800
1691
1684
2275
1605
25
17.3±
0.93
41.67± 3.89± 25.07± 0.155
1.85 0.44 2.95
-0.556
-0.474
-0.220
-0.362
-0.050
0.270
17.3±
1.75
47.04± 12.95 22.21± 0.583
0.98 ±3.74 4.38
2g
2h
2i
17.3±
1.06
27.02± 4.89± 9.31±0. 0.525
1.13 1.29 85
23.0±
0.44
49.82± 5.21± 33.61± 0.155
0.17 0.86 7.87
34.7±
2.47
72.25± 6.02± 24.21± 0.249
1.85 0.56 6.41
2j
17.3±
0.25
44.08± 10.17 31.57± 0.321
1.23 ±1.79 4.71
2k
2l
17.3±
1.40
64.28± 4.31± 37.65± 0.115
0.61 1.49 4.73
-0.284
-0.150
-2.387
61.82± 88.54± 39.06 36.16± 1.1
1.54 0.93 ±7.37 4.01
2m
89.36± 130.73 59.44 44.68± 1.33
0.41
±3.47
±12.4 13.23
6
2n
2o
2r
52.57± 118.72 50.52 68.22± 0.74
-2.462
-1.289
-0.217
-0.027
-0.263
-0.611
-0.744
0.174
-0.313
-0.506
0.525
-0.424
1.435
0.093
2.054
1.033
1.244
4.347
1.417
6.498
3.147
25
52.27
±1.70
±13.1 22.43
1
120.94 147.04 111.7 57.21± 1.95
136
±4.21
±3.30
1±19. 14.71
33
78.41± 95.93± 75.63 50.27± 1.50
1402
1600
1491
1600
1351
1.06
1.17
±14.0 11.28
8
2s
2t
72.43± 73.96± 38.31 29.67± 1.29
1.92 0.28 ±3.67 3.02
66.51± 80.37± 56.41 65.35± 0.86
1.68 1.10 ±6.33 13.96
2u
3b
106.88 151.24 53.51 51.35± 1.04
±0.94
±6.17
±14.7 3.36
8
23.1±
0.01
51.56± 7.02± 25.87± 0.271
1.30 3.46 6.48
4

4b
4e
34.7±
0.04
36.13± 9.98± 17.15± 0.582
1.35 3.72 7.92
-0.397
-0.542
0.025
0.082
0.164
0.156
0.072
0.617
3.282
3.392
2.580
4.426
2184
2217
2828
842
23.0±
1.84
49.78± 13.42 24.86± 0.540
1.17 ±6.19 3.34
Tacri 698.6± 124.73 282.5 55.22± 5.117
ne
0.12
±1.68
4±55. 12.41
36
Done 32.87± >1000b 22.81 >1000c 0.014
pezil
1.03
±1.25
a: The appropriate range for Log BB is between −3.0 and 1.2.
b: The appropriate range for Log KHSA is between -1.5 and 1.5
c: Higher than 500 nm/s means that the compound can be absorbed by human intestinal system.
For the SAR model, we chose compound 2a, which has benzoyl and phenyl groups, and showed
inhibition at 34.65 nM for AChE and at 61.27 nM for BChE. Compound 2b, which has one methoxy
group at the para-position of the phenyl ring, did not change the inhibition constant for AChE and
revealed less activity on BChE than that of 2a. Two methoxy groups at the meta- and para-positions
of the phenyl ring (compound 2e) increased the inhibition ability from 34.65 nM to 17.33 nM for
AChE and from 61.27 nM to 41.67 nM for BChE. The clinically used molecule donepezil possesses the
same sub-structure. SAR studies for donepezil show that two vicinal methoxy groups and a short
linker increased biological activity up to 25-fold.21,22 Replacement of methoxy groups with hydroxyl
groups at the same position did not change the AChE activity but increased BChE activity. We tested
the positional effect of the di-methoxy groups, and placement at both ortho-positions (compound 2f)
induced good inhibition, as seen in compound 2e. The conversion of the phenyl ring to α-naphthyl, β-
naphthyl, or pyrene was also evaluated. Compound 2g, which has α-naphthyl groups, showed good
inhibition for AChE and BChE. We hypothesized that more than one phenyl ring would increase the
inhibition due to a better fit in the active site of the enzyme through π-π interactions due to the
aromatic gorge of ChEs. We tested compound 2h, which has Ar = β-naphthyl, to assess the effect of
the position of the naphthyl group on inhibition activity. Surprisingly, we observed that the position
of the naphthyl ring is important for a correct match, which was confirmed by Linusson et al.23 To
understand the importance of the position of the phenyl ring with one more example, para-biphenyl
derivative (2u) sharply decreased inhibition activity in which BChE was inhibited with the worst
concentration (151.2 nM) within imidazole derivatives. The pyrene-substituted imidazole derivative
2k exhibited potent AChE and BChE inhibitory activities with IC50 values of 17.32 and 64.28 nM,
respectively, seen in compound 2g. A similar result was reported in the literature in which
caproctamine was modified by placing a pyrene-type bulky aromatic molecule, benzophenanthridine,
causing nanomolar inhibitor activity due to π-π interactions with aromatic residues in the gorge of
5

the enzyme.24 This result highlights possible areas for further study in a more detailed approach to
the SAR. Halogenation of the para-position with bromine raised the inhibition activity for both
cholinesterases (compound 2d). In addition, fluorine- and chlorine-substituted derivatives (2l and 2s)
at para-position were also investigated and decreasing activity for ChEs was observed. The inhibitory
effect on BChE with halogenated derivatives revealed the array Br>Cl>F. On the other hand, hydroxy
and methoxy groups at the same position did not alter activity. Bromine is an atom that might
participate in both hydrogen bonding with the hydrogen of an amide group25 and halogen bonding
with OH groups.26 It was assumed the halogen and hydrogen bonds might occur on the same side,
causing increased inhibition, and these two abilities might be mediated by bromine. For instance, the
complex of the drug coded IDD594 with human aldose reductase was crystallized by Podjarny et al.
and showed a halogen bond between bromine at the para-position of IDD594 and the OH group of
threonine in the enzyme.27 We have assumed that similar bonding might occur between 2d and the
AChE and BChE, resulting in increased activity. Electron withdrawing group NO2 and its positions
(ortho, meta, and para) were examined. Within nitro-substituted derivatives (2m, 2n, and 2o), the
most active compound was found to be para-substituted molecules. Furthermore, C-2-substituted
imidazole derivatives (2r and 2t), which are without of aryl groups at the C-4 position, resulted in
decreasing biological activity about 2-fold. This means that aryl groups at the C-4 position are
essential for optimum biological activity.
In addition to the phenyl ring, we also tested a thienyl group (compound 2i) for inhibition. It showed
the same activity as compound 2a did. However, it was seen that the activity of compound 2i on
BChE decreased when compared with 2a. Bromine substituted at the C-5 position of the thienyl
groups (compound 2j) raised the inhibitory effect. The same explanation can be used for this
molecule as for compound 2d.
While reduction of the carbonyl group of 2e decreased the inhibition constant (compound 4e),
compound 4b did not yield any change in the inhibition of AChE but did in the inhibition of BChE.
Ketoxime derivative (compound 3b) increased inhibition from 34.65 to 23.10 nM for AChE and from
61.27 to 51.56 nM for BChE. Thus, we have seen that replacement of the carbonyl group with its
reduced alcohol form or its oxime form altered biological activity slightly.
The selectivity of the synthesized molecules was also analyzed and, according to Ki values, its
AChE/BChE ratio was between 0.10 and 1.95 (Table 1). The selectivity of donepezil was 0.014.
Imidazole derivatives have inhibitor potency on AChE and BChE with nanomolar concentrations
although donepezil, an important clinically used drug, inhibited AChE by 32.87 nM and BChE by 4030
nM (Table 1). Despite the fact that BChE activity is lower than that of AChE in the normal brain, the
BChE/AChE ratio is greatly increased in AD, which suggests that inhibition of BChE may become more
6

important as AD progresses. This raises the hypothesis that inhibitory action on both ChEs leads to
improved therapeutic benefits.5,6
Water solubility of drug candidates are important. Hence, we desired to determine the solubility data
of some synthesized compounds in water using HPLC (for technical details see SI). With this
experiment, we have obtained data shown in Table 2.
Table 2. Water solubility data of some compounds.
Compounds
Water
2c
2e
2f
4e
1.43 0.68 0.72 44.34
solubility(mg/mL)
2.3.Toxicity of the compound 2c on different cell lines
To test the toxicity of the newly synthesized molecules, compound 2c which was chosen due to its
being more soluble in cell medium, was incubated in WS1 (skin fibroblast) and SH-SY5Y
(Neuroblastoma ) cell lines. The toxicity value of 2c on the WS1 cell line was 1790 µM. This indicated
that our skeleton might be safe for non-cancerous cells. In addition, toxicity on the SH-SY5Y cell line
was 950 µM (Figures 2).
Toxicity of 2c on WS1 cell line
100
86
78
65
43 33 25
8
0
500 1000 1500 2000 3000 4000 8000
Dose (uM)
Toxicity of 2c on SH-SY5Ycell line
100
68
46 43 30 18 10
8
0
500 1000 1500 2000 3000 4000 8000
Dose (uM)
Figure2. Toxicity of 2c on WS1 and on SH-SY5Y cell lines.
2.4.Kinetic studies of synthesized molecules
7

To get an insight into the mechanism of ChEs inhibition, all imidazole derivatives were subjected to
an enzyme kinetic study (two examples of them was shown in Figure 3-4). Lineweaver−Burk
reciprocal plots showed that all tested compounds are competitive inhibitors of ChEs, except for
compounds 3b and 4e, which showed noncompetitive inhibition on AChE (for kinetic graphics, see
SI). Kinetic constants (Ki) calculated from the Lineweaver-Burk plots were between 3.89 and 111.71
nm for AChE, and between
9.31 and 68.22 nm for
120
100
y = 1.4068x + 7.094
y = 4.8704x + 6.424
BChE (Table 1).
y = 8.2323x + 6.4371
80
y = 9.8925x + 6.6231
60
40
20
0
-5
-2
1
4
7
10
1/[S] (nM)-1
Figure 3. Lineweaver-Burk plot of AChE for 2a.
y = 1.5867x + 10.314
100
y = 2.4535x + 15.549
y = 4.9583x + 31.683
y = 5.8214x + 37.543
80
60
40
20
0
-7
-4
-1
2
5
8
11
1/[S] (nM)-1
Figure 4. Lineweaver-Burk plot of AChE for 3b.
8

2.4.Computational studies
Potential inhibitory molecules should pass biological barriers such as the blood-brain barrier, should
not bind to serum albumin, and should traverse the gut membrane. To reveal theoretically these vital
parameters of the synthesized compounds, we utilized Schrödinger (qikprop) software.28 Log BB
(blood brain), Log KHSA (human serum albumin binding), cLog P (lipophilicity), and Caco-2 permeability
(predicted human intestinal permeability) were calculated. The predicted parameters of imidazole
molecules (except for 2k, which has two identical pyrene rings) are in good agreement with ranges,
which are reported by the software (see Table 1 footnotes). Thus, the imidazole analogues are
predicted to penetrate the blood brain barrier. Furthermore, Caco-2 permeability predicted that all
synthesized molecules could be given orally, except for 2m, 2n, and 2o (nitro derivatives). Log KHSA
was calculated to assess the binding of imidazoles to plasma proteins such as human serum albumin,
lipoprotein, and glycoprotein, because binding greatly reduces the quantity of the drug in general
blood circulation. Calculated values were between -0.137 and 1.435 for Log KHSA. In addition,
calculations showed that all synthesized molecules except for 2k are compliant with this parameter,
indicating that imidazole derivatives are likely to circulate freely within the blood stream and access
the target site (Table 1). cLog P is a popular descriptor to describe the lipophilicity of molecules. The
obtained cLog P values suggested that the molecule might pass the blood-brain barrier. cLog P values
for imidazoles are within the accepted range (range is reported by software and was given in Table
1), except for 2k.
Docking studies were done using the program Autodock with the AChE X-ray crystal structure with
PDB code 1B41. For the docking process, some of synthesized molecules (2a-2k, and 4b) were
optimized using density functional theory (DFT) through the DFT/B3LYP-6-311G (d, p) method (see
Table S2 in SI).31 The AChE active site consists of an aromatic gorge, catalytic triad (CT), peripheral
anionic site (PAS), omega loop (OL), oxyanion hole (OH), anionic subsite (AS), and acyl binding pocket
(ABP).29,30 CT side consists of Ser203, His447, and Glu334 while OH side includes Gly120, Ala204, and
Gly121. One of the important side of this protein is PAS and its residues are Trp286, Tyr124, Asp74,
Ser125, Tyr341, and Tyr337. Another important side of this protein is AS which includes Gly448,
Glu202, Ile451, Tyr133, and Trp86.
The docking study of compound 2e (inhibition constant 17.3 nM) was discussed in detail to observe
binding to the enzyme (Figure 5). The ligand interacted with Tyr341, Tyr124, and Tyr337 by π-sigma,
hydrogen bond, and π-π interactions, respectively which are PAS side of the enzyme. On the other
hand, two important interactions were observed between ligand-Glu202 (Van der Waals interaction)
and ligand-Trp86 (π-sigma interaction), which form the AS active site. In addition, Gly120, important
residue in the OH side, shown amide-π interaction and Van der Waals with benzene ring and its OMe
9

group (for overlay of the other ligands see Figures S1 and S4 in SI). In summary, it is noteworthy to
say that the selected ligands interacted with important amino acid residues in the PAS, AS, and OH
active sites of hAChE. These pivotal interactions might be responsible for the nanomolar activity.
Figure 5. Demonstration of docking study of 2e: Noncovalent interactions between compound 2e
and some residues in hAChE (PDB code: 1B41)
3. CONCLUSIONS
To sum up, the assayed compounds are simple, low cost structures compared with the molecules in
the literature. Enzyme kinetic studies presented the mechanism for inhibition of all compounds.
Most of the compounds showed competitive inhibition on ChEs and two of them showed
noncompetitive inhibition on AChE. The toxicity of one compound on the WS1 and SH-SY5Y cell lines
was 1790 and 950 µM, respectively. Selectivity (AChE/BChE) was between 0.10 and 1.95. ADME
predictions showed some biologically important barriers such as the BBB, serum albumin, and
intestinal membrane could be passed by the synthesized structures. The docking process showed
that the ligand could bind to the important regions of the hAChE active side, the PAS, AS, and OH
which might be a key area for further investigation. Docking scores revealed there is a common
skeleton responsible for the nanomolar activity. With this information, functionalization of the
skeleton might be progressed for further pre-clinical trials without losing the biological responses in
ChEs inhibition. In this study, we report a previously unknown imidazole core is a potent inhibitor for
ChEs with nanomolar activity. Further experiments using different functional groups and biological
tests are under way.
4. EXPERIMENTAL SECTION
10

4.1. General information for chemistry
1H- and 13C-NMR spectra were recorded on Varian NMR-400 MHz. 1H chemical shifts were referenced
to internal standard TMS (δ 0.00 ppm) or (solvent d6-DMSO). 13C NMR spectra was referenced to the
solvent (d6-DMSO) peak at 33 ppm. TLC was carried out on Kieselgel 60 mesh. FT-IR spectra was
recorded by Thermo Scientific, Nicoleti S10 FT-IR Spectrometer. HRMS data was recorded by Thermo
Scientific, TSQ-Quantum Access with electron spray ionization technique (ESI) at positive ion
detection.
4.2. General procedure for 2a-o, s,u
The commercially available acetyl substituted aromatic compounds (1 mmol) and 2.5 mmol SeO2
were added into 4 mL 1,4-Dioxane. The reaction flask was heated at reflux temperature of solvent
overnight and completion of the reaction was controlled with TLC method. Then after filtration of Se0
particules, solution of 5 mmol ammonium acetate in ethanol was poured into the former reaction
flask at room temperature. Final mixture was stirred for 1 hour at room temperature. The obtained
solid molecules were filtered, washed three times with water (30 ml) and dried in vacuo over P2O5.
4.3. General procedure for 2r and 2t
The imidazole (1 mmol) was dissolved in (10 mL) pyridine. Then 3 mmol triethylamine was added to
the solution. The mixure was cooled with ice-water medium and stirred for 30 minutes. Aroyl
chloride (1.5 mmol) solution which was prepared in 3.5 ml pyridine, was added to the imidazole
solution as dropwise. After the reaction was stirred 30 minutes, 5 M 20mL NaOH solution was added
and final mixture was refluxed for 1 hour. After reaction completed judged by TLC, acidic work-up
was progressed. Organic phase was collected with EtOAc. EtOAc phases were evaporated and crude
product was purified by column chromatography (1:5, EtOAc:n-hexane).
4.4. General procedure for 4b and 4e.
The obtained imidazole derivatives (1 mmol) were dissolved in methanole and NaBH4 (2 mmol) was
added in the solution at room temprature. The solution was stirred for 2 hours and the reaction
completion was controlled by TLC. The mixture was recrystallized with water. And the obtained solid
compound was dried with P2O5 at overnight.
4.5. Synthetic procedure for 3b
The synthesized compound 2b (1mmol) was dissolved in n-propanol (5 mL). 2 mmol NH2OH.HCl and
TEA (2 mmol) was added into the solution. Then reaction mixture was refluxed overnight and
controlled using TLC plate. After the starting compounds was disappeared on the TLC, reaction
11

solution was cooled to room temprature. The solution was extracted with ethyl acetate (3 X 20 mL)
and water. Organic layers were dried by MgSO4 and evaporated.
Phenyl(4-phenyl-1H-imidazol-2-yl)methanone1 (2a)
Light yellow solid, M.p: 190°C, yield: 68.5%. FTIR (ATR) cm-1:3647, 3269,
3092, 3061, 2980, 2888, 1669, 1615, 1597, 1570, 1020, 957, 935, 903,
868. 1H NMR (400 MHz, DMSO) δ 8.48 (d, J=7.11 Hz, 2H, H-19 and H-
15), 8.05 (s, 1H, H-8), 7.92 (d, J=7.11 Hz, 2H, H-3 and H-5), 7.68 (t,
J=7.50 Hz, 1H, H-17),7.60-7.58 (m, 2H, H-16 and H-18) 7.41 (t, J=7.49 Hz, 2H, H-2 and H-6), 7.29 (t,
J=7.49 Hz, 1H, H-1). 13C NMR (100 MHz, DMSO) δ 181.1, 144.7, 141.3, 136.3, 133.6, 132.5, 131.0,
129.2, 128.8, 128.1, 125.6, 120.9.
(4-methoxyphenyl)(4-(4-methoxyphenyl)-1H-imidazol-2-
yl)methanone2 (2b)
Yellow solid, M.p: 204-206°C, yield: 87.5% FTIR (ATR) cm-1: 3264,
3095, 2980, 2840, 1608, 1595, 1564, 1511, 1026, 972, 957, 903,
864. 1H NMR (400 MHz, DMSO) δ 8.50-8.48 (m, AA’BB’ system,
2H, Ar-H), 7.94 (s, 1H, H-8), 7.87-7.86 (m, AA’BB’ system, 2H, Ar-
H), 7.13-7.11 (m, AA’BB’ system, 2H, Ar-H), 7.00-6.68 (m, AA’BB’ system, 2H, H-2, H-6), 3.87 (s, 3H,
OMe), 3.78 (s, 3H, OMe). 13C NMR (100 MHz, DMSO ) δ 179.1, 163.9, 159.5, 144.1, 133.5, 128.8,
127.2, 124.2, 124.1, 119.8, 114.7, 114.3, 56.1, 55.6.
(4-hydroxyphenyl)(4-(4-hydroxyphenyl)-1H-imidazol-2-yl)methanone3 (2c)
Yellow solid, M.p: 298-305°C, yield: 56% FTIR (ATR) cm-1:3647,
3233, 2980, 2889, 1607, 1579, 1433, 1029, 963, 911, 819, 806. 1H
NMR (400 MHz, DMSO) δ 13.27 (bs, 2H, H-20, H-21), 8.59-8.50
(m, AA’BB’ system, 2H, Ar-H), 7.77 (s, 1H, H-8), 7.76-7.73 (m,
AA’BB’ system, 2H, Ar-H), 6.92-6.78 (m, AA’BB’ system, 4H, Ar-H).
13C NMR (100 MHz, DMSO ) δ 179.2, 162.6, 157.1, 145.1, 143.4, 133.8, 127.7, 126.6, 125.4, 116.7,
115.8, 115.5.
(4-bromophenyl)(4-(4-bromophenyl)-1H-imidazol-2-yl)methanone1 (2d)
Yellow solid, M.p: 298-305°C, yield: 56% FTIR (ATR) cm-1: 3266, 3094,
1
1612, 1580, 1557, 1483, 1010, 902, 869, 830, 766. H NMR (400
12

MHz, DMSO) δ 13.71 (bs, 1H, H-9), 8.50-8.48 (m, AA’BB’ system, 2H, Ar-H), 8.13 (bs, 1H, H-8), 7.87-
7.85 (m, AA’BB’ system, 2H, Ar-H), 7.81-7.79 (m, AA’BB’ system, 2H, Ar-H), 7.60-7.59 (m, AA’BB’
system, 2H, Ar-H). 13C NMR (100 MHz, DMSO ) δ 180.1, 144.9, 142.3, 135.3, 133.2, 133.0, 132.0,
131.9, 127.9, 127.3, 120.6, 119.9.
(3,4-dimethoxyphenyl)(4-(3,4-dimethoxyphenyl)-1H-imidazol-2-yl)methanone1 (2e)
Brown-yellow mixture solid, M.p: 120-125°C, yield: 52% FTIR
(ATR) cm-1: 3217, 3086, 2980, 2836, 1609, 1588, 1567, 1511,
1017, 948, 894, 856, 846. 1H NMR (400 MHz, DMSO) δ 8.35 (d,
J=8.25 Hz, 1H, H-19), 7.86 (bs, 1H, H-9), 7.52 (s, 2H, H-5 and H-
15), 7.52 (s, 1H, H-8), 7.12 (d, J=8.25 Hz, 1H, H-3), 6.97 (d,
J=8.25 Hz, 1H, H-2), 3.86 (s, 3H, OMe), 3.85 (s, 3H, OMe), 3.81 (s, 3H, OMe), 3.75 (s, 3H, OMe).13C
NMR (100 MHz, DMSO ) δ 179.1, 153.5, 149.4, 148.7, 148.5, 145.4, 128.9, 125.9, 117.9, 113.7, 112.4,
111.2, 109.1, 56.1, 55.9, 55.8, 55.7.
(2,6-dimethoxyphenyl)(4-(2,6-dimethoxyphenyl)-1H-imidazol-2-yl)methanone (2f)
Light yellow solid, M.p, 239-241°C, yield %30, FTIR (ATR) cm-1: 3426,
3096, 3006, 2980, 2951, 1641, 1592, 1486, 1472, 1431, 950, 937, 899.
1H NMR (400 MHz, CDCI3) δ 11.64 (bs, 1 H, H-9), 7.93 (s, 1H, H-8), 7.34
(t, J17,18 =8.43 Hz, 1H, H-17), 7.26 (t, J1,6 =8.43 Hz, 1H, H-1), 6.69 (d,
J18,17 =8.43 Hz, 2H, H-18, H-16), 6.63 (d, J6,1 =8.43 Hz, 2H, H-6 and H-2),
3.97 (s, 6H, OMe) 3.75 (s, 6H, OMe).13C NMR (100 MHz, CDCI3 ) δ 184.6, 157.9, 157.5, 143.9, 134.3,
131.2, 129.4, 129.2, 117.0, 106.4, 104.4, 104.2, 56.02, 56.01. HRMS (ES) [M+H]; m/z
C20H20N2O5:368,1372; Found: 368,1389.
naphthalen-1-yl(4-(naphthalen-1-yl)-1H-imidazol-2-yl)methanone (2g)
1
Yellow solid, M.p: 105-107°C, yield: 61% H NMR (400 MHz, DMSO) δ
14.0 (s, 1H, N-H), 8.60 (d, J=8.35 Hz, 1H, Ar-H), 8.34-8.26 (m, 2H, Ar-H),
8.17 (d, J=8.21 Hz, 1H, Ar-H), 8.07-8.05 (m, 2H, Ar-H), 7.95-7.88 (m, 2H,
Ar-H), 7.75 (d, J=6.86 Hz, 1H, Ar-H), 7.69-7.62 (m, 4H, Ar-H), 7.53-7.45
(m, 2H, Ar-H).13C NMR (100 MHz, DMSO ) δ 184.8, 145.3, 142.8, 134.1, 133.5, 133.2, 131.8, 130.9,
130.4, 130.2, 128.5, 128.2, 127.8, 127.3, 126.6, 126.3, 126.2, 125.9, 125.8, 125.4, 125.2, 124.6, 121.7.
HRMS (ESI) [M+H] C24H17N2O: 349,1341; Found (M+H)+: 349.1344.
(Naphthalen-2-yl(4-(naphthalen-2-yl)-1H-imidazol-2-
13

yl)methanone3 (2h)
Light brown solid, M.p: 215-220°C, yield: 72% FTIR (ATR) cm-1: 3275, 3056, 1668, 1631, 1607, 1479,
1
1463, 1071, 1017, 961, 950, 924, 821. H NMR (400 MHz, DMSO) δ 13.77 (s, 1H, H-9), 9.45 (s, 1H, H-
8), 8.48 (bs, 1H, Ar-H), 8.26-8.24 (m, 1H, Ar-H), 8.15-8.09 (m, 2H, Ar-H), 8.03 (d, J=8.03 Hz, 1H, Ar-H),
7.98-7.97 (m, 2H, Ar-H), 7.90 (d, J=8.46, 1H, Ar-H), 7.70-7.65 (m, 3H, Ar-H), 7.51-7.46 (m, 3H, Ar-H).
13C NMR (100 MHz, DMSO ) δ 181.1, 145.5, 143.4, 135.4, 133.7, 133.5, 132.8, 132.4, 131.6, 130.5,
129.2, 128.7, 128.4, 128.3, 128.1, 127.3, 126.8, 126.3, 126.1, 124.3, 123.3, 119.7.
thiophen-2-yl(4-(thiophen-2-yl)-1H-imidazol-2-yl)methanone3 (2i)
Orange solid, M.p: 195-200°C, yield: 55% FTIR (ATR) cm-1: 3235, 3092,
1
1644, 1594, 1506, 1473, 1051, 997, 915, 889, 870. H NMR (400 MHz,
DMSO) δ 13.67 (bs, 1 H, H-8), 8.66 (dd, J17,16=3.84 Hz, J17,15= 1.09 Hz,
1H, H-17), 8.10 (dd, J2,3=4.47 Hz, J2,4= 1.07 Hz, 1H, H-2), 7.92 (s, 1H, H-7), 7.46 (dd, J4,3=3.54 Hz, J4,2=
1.07 Hz, 1H, H-4), 7.45 (dd, J15,16= 4.98 Hz, J15,17=1.09 Hz, 1H, H-15), 7.32 (dd, J16,15 =4.98 Hz, J16,17=3.84
Hz, 1H, H-16), 7.09 (dd, J3,2=4.47 Hz, J3,5=3.54 Hz, 1H, H-3). 13C NMR (100 MHz, DMSO) δ 173.0, 144.0,
141.1, 138.7, 137.6, 136.9, 136.6, 129.0, 128.3, 125.0, 123.4, 118.5.
(5-bromothiophen-2-yl)(4-(5-bromothiophen-2-yl)-1H-imidazol-2-yl)methanone (2j)
Brown solid, M.p: 170-171°C, yield: 92% 1H NMR (400 MHz, DMSO) δ
8.25 (bs, 1H, Ar-H), 7.93 (bs, 1H, Ar-H), 7.42 (d, J=4.05 Hz, 1H, Ar-H),
7.30 (bs, 1H, Ar-H), 7.19 (d, J=3.70 Hz, 1H, Ar-H). 13C NMR (100 MHz,
DMSO ) δ 170.8, 143.2, 141.1, 136.2, 131.7, 131.1, 123.9, 123.7, 109.9. HRMS (ESI) [M+H]
C12H7Br2N2OS2: 416.8367; Found (M+H)+: 416.8372.
Piren-1-il(4-(piren-4-il)-1H-imidazol-2-il)metanon (2k)
Dark brown solid, M.p: 145-149°C, yield; 72% FTIR (ATR)
cm-1: 3039, 1622, 1593, 1537, 1506, 1446, 1069, 991,
959, 921, 872, 840. 1H NMR (400 MHz, DMSO) δ 8.87 (d,
J=9.33 Hz, 1H, Ar-H), 8.80 (s, 1H, Ar-H), 8.63-8.61 (m,
1H, Ar-H), 8.52 (d, J=9.33 Hz, 1H, Ar-H), 8.44 (s, 1H, Ar-
H), 8.39 (d, J=9.68 Hz, 2H, Ar-H), 8.35-8.33 (m, 1H, Ar-H), 8.32-8.31 (m, 1H, Ar-H), 8.30-8.29 (m, 1H,
Ar-H), 8.28 (bs, 1H, Ar-H), 8.25-8.23 (m, 1H, Ar-H), 8.21-8.20 (m, 1H, Ar-H), 8.19-8.17 (m, 1H, Ar-H),
8.15-8.12 (m, 2H, Ar-H), 8.13-8.11 (m, 1H, Ar-H), 8.08 (bs, 1H, Ar-H), 8.03-8.01 (m, 1H, Ar-H). 13C NMR
(100 MHz, DMSO ) δ 185.5, 146.7, 133.3, 132.1, 131.4, 131.1, 130.8, 130.7, 130.5, 129.8, 129.7 129.5,
129.3, 128.0, 127.9, 127.8, 127.7, 127.6, 127.2, 126.8, 126.5 125.7, 125.4, 125.3, 125.0, 124.8, 124.4,
14

124.3, 124.2, 124.0. HRMS (ESI) [M+H] C36H21N2O: 497.1654; Found (M+H)+: 497.1649.
(4-fluorophenyl)(4-(4-fluorophenyl)-1H-imidazol-2-yl)methanone (2l)
1
Dark yellow solid, M.p: 224-226°C. Yield % 92. H NMR (400 MHz,d6-
DMSO) δ = 13.6 (bs, 1H, -NH), 8.71-8.67 (m, AA’BB’ system, 2H, Ar-H),
8.06 (d, J=2.51 Hz, 1H, Ar-H), 7.96-7.93 (m, AA’BB’ system, 2H, Ar-H),
7.44-7.39 (m, AA’BB’ system, 2H, Ar-H), 7.26-7.22 (m, AA’BB’ system,
2H, Ar-H). 13C NMR (100 MHz, d6-DMSO) δ= 179.5, 166.8, 164.3, 163.1, 160.7, 144.9, 142.5, 134.1 (d,
J=9.39 Hz), 133.5, 132.9, 130.6, 127.2 (d, J=8.05 Hz), 127.2, 119.0, 116.0 (d, J=6.26 Hz), 115.8 (d,
J=6.58 Hz). HRMS (ES) [M+H]; m/z C16H11F2 N2O:285.08394; Found [M+H]: 285.08385
(3-nitrophenyl)(4-(3-nitrophenyl)-1H-imidazol-2-yl)methanone (2m)
1
Light brown solid, M.p: 210-212°C. Yield % 89. H NMR (400
MHz,d6-DMSO) δ = 13.94 (bs, 1H, -NH), 9.49-9.48 (m, 1H, Ar-H),
8.87-8.85 (m, 1H, Sr-H), 8.70-8.66 (m, 1H, Ar-H), 8.51-8.47 (m,
1H, Ar-H), 8.36-8.30 (m, 2H, Ar-H), 8.11-8.08 (m, 1H, Ar-H),
7.89-7.85 (m, 1H, Ar-H), 7.71-7.66 (m, 1H, Ar-H). 13C NMR (100 MHz, d6-DMSO) δ= 178.6, 148.8,
148.0, 144.8, 141.3, 137.1, 136.9, 135.6, 131.4, 130.7, 130.6, 127.8, 126.0, 122.2, 121.3, 119.4. HRMS
(ES) [M+H]; m/z C16H11N4O5:339.07294; Found [M+H]: 339.07211
(4-nitrophenyl)(4-(4-nitrophenyl)-1H-imidazol-2-yl)methanone (2n)
1
Dark Brown solid, M.p: 280-282°C. Yield 88%. H NMR (400
MHz,d6-DMSO) δ = 8.71-8.68 (m, AA’BB’ system, 2H, Ar-H), 8.42
(bs, 1H, Ar-H),8.42-8.40 (m, AA’BB’ system, 2H, Ar-H), 8.28-8.26
(m, AA’BB’ system, 2H, Ar-H), 8.18-8.16 (m, AA’BB’ system, 2H, Ar-
H). 13C NMR (100 MHz, d6-DMSO) δ=180.1, 150.2, 146.6, 145.3, 141.5, 141.2, 132.4, 126.0, 124.6,
123.8. HRMS (ES) [M+H]; m/z C16H11N4O5:339.07294; Found [M+H]: 339.07288
(2-nitrophenyl)(4-(2-nitrophenyl)-1H-imidazol-2-yl)methanone (2o)
1
Light brown solid, M.p: 236-237°C. Yield % 75. H NMR (400 MHz,d6-
DMSO) δ = 13.12 (bs, 1H, -NH), 8.50 (dd, J=0.95 Hz, J=8.30 Hz, 1H, Ar-H),
15

8.26 (dd, J=0.95 Hz, J=8.30 Hz, 1H, Ar-H), 8.09 (dd, J=1.43 Hz, J=7.89 Hz, 1H, Ar-H), 7.98 (J=1.12 Hz,
J=7.51 Hz, 1H, Ar-H), 7.90-7.86 (m, 2H, Ar-H), 7.80 (dd, J=1.39 Hz, J=7.48 Hz, 1H, Ar-H), 7.62-7.58 (m,
1H, Ar-H), 7.26-7.22 (m, 1H, Ar-H), 13C NMR (100 MHz, d6-DMSO) δ= 188.9, 170.0, 158.0, 148.2,
135.7, 134.3, 133.2, 132.0, 131.9, 130.7, 124.5, 124.1, 119.9. HRMS (ES) [M+H]; m/z
C16H11N4O5:339.07294; Found [M+H]: 339.07278
(1H-imidazol-5-yl)(thiophen-2-yl)methanone (2r)
1
Light yellow solid, M.p: 162-164°C. Yield 75%. H NMR (400 MHz, CDCl3) δ =
8.72 (dd, J11,9=1.26 Hz, J11,10=3.86 Hz, 1H, H-11), 7.77 (dd, J9,11=1.26 Hz,
J9,10=4.95 Hz, 1H, H-9,), 7.35 (bs, 2H, H-4,5), 7.21 (dd, J10,11=3.86 Hz, J10,9=4.95
Hz, 1H, H-10). 13C NMR (100 MHz,CDCl3) δ= 174.1, 144.6, 140.3, 137.0, 135.8,
128.3. HRMS (ES) [M+H]; m/z C8H7N2OS:179.02791; Found [M+H]: 179.02701
(4-chlorophenyl)(4-(4-chlorophenyl)-1H-imidazol-2-yl)methanone (2s)
1
Light brown solid, M.p: 189-191°C. Yield 90%. H NMR (400 MHz,d6-
DMSO) δ = 13.72 (bs, 1H, -NH), 8.60-8.56 (m, AA’BB’ system, 2H, Ar-
H), 8.13 (d, J=2.53 Hz, 1H, Ar-H), 7.94-7.91 (m, , AA’BB’ system, 2H, Ar-
H), 7.67-7.64 (m, , AA’BB’ system, 2H, Ar-H), 7.49-7.44 (m, AA’BB’
system, 2H, Ar-H). 13C NMR (100 MHz, d6-DMSO) δ= 179.8, 144.9, 142.2, 138.6, 134.9, 132.9, 132.8,
132.1, 132.0, 129.1, 128.9, 127.0, 119.8. HRMS (ES) [M+H]; m/z C16H11Cl2N2O:317.02484; Found
[M+H]: 317.02446
(1H-imidazol-5-yl)(phenyl)methanone (2t)
Brown Viscous liquid, Yield 65%. 1H NMR (400 MHz, CDCl3) δ =11.33 (bs, 1H, -NH),
8.59-8.56 (m, 2H, Ar-H), 7.63-7.59 (m, 1H, Ar-H), 7.53-7.49 (m, 2H, Ar-H), 7.41 (bs,
1H, Ar-H), 7.27 (bs, 1H, Ar-H). 13C NMR (100 MHz,CDCl3) δ=182.2, 145.2, 135.6,
133.4, 131.8, 131.0, 128.3, 120.3. HRMS (ES) [M+H]; m/z C10H9N2O:173.07149; Found [M+H]:
173.07149
[1,1'-biphenyl]-4-yl(4-([1,1'-biphenyl]-4-yl)-1H-imidazol-2-yl)methanone (2u)
16

1
Brown solid, M.p: 284-286°C. Yield 86%. H NMR (400 MHz,d6-
DMSO) δ =8.69-8.67 (m, AA’BB’ system, 2H, Ar-H), 8.10 (bs, 1H,
Ar-H), 8.04-8.02 (m, AA’BB’ system, 2H, Ar-H), 7.92-7.90 (m,
AA’BB’ system, 2H, Ar-H), 7.80-7.79 (m, AA’BB’ system, 2H, Ar-
H), 7.76-7.70 (m, 4H, Ar-H), 7.53-7.43 (m, 5H, Ar-H), 7.37-7.34
(m, 1H, Ar-H). 13C NMR (100 MHz, d6-DMSO) δ=180.5, 144.9,
135.2, 131.9, 129.6, 139.4, 128.9, 127.9, 127.5, 127.4, 127.0, 126.9, 126.0. HRMS (ES) [M+H]; m/z
C28H21N2O:401.16539; Found [M+H]: 401.16531
(Z-E)-(4-methoxyphenyl)(4-(4-methoxyphenyl)-1H-imidazol-2-
yl)methanone oxime (3b)
1
Light brown viscous, yield: 89.2% H NMR (400 MHz, DMSO) δ
12.29 (s, 1H, N-H), 11.30 (s, 1H, O-H), 7.73-7.65 (m, 1H, Ar-H),
7.63-7.60 (m, 2H, Ar-H), 7.55-7.53 (m, 1H, Ar-H), 7.51-7.47 (m, 1H,
Ar-H), 6.99-6.94 (m, 2H, Ar-H), 6.92-6.90 (m, 1H, Ar-H), 6.87-6.85
(m, 1H, Ar-H), 3.80-3.78 (m, 3H, OMe), 3.76-3.72 (m, 3H, OMe). 13C NMR (100 MHz, DMSO ) δ 160.1,
159.8, 158.3, 147.9, 143.8, 141.2, 132.0, 127.7, 126.0, 123.9, 114.6, 114.4, 114.2, 113.6, 113.5, 113.2,
55.6, 55.5. HRMS (ESI) [M+H] C18H18N3O3: 324.1348; Found (M+H)+: 324.1350.
(4-methoxyphenyl)(4-(4-methoxyphenyl)-1H-imidazol-2-
yl)methanol (4b)
Light brown solid, M.p: 180-181°C, yield: 87% 1H NMR (400 MHz,
DMSO) δ 11.91 (s, 1H, H-9), 7.63 (d, J=8.76 Hz, 2H, Ar-H), 7.35 (d,
J=8.67 Hz, 3H, Ar-H), 6.89-6.85 (m, 4H, Ar-H), 6.10 (d, J13,12=3.96,
1H, H-13), 5.70 (d, J12,13=3.96, 1H, H-12), 3.72 (s, 3H, OMe), 3.70 (s, 3H, OMe). 13C NMR (100 MHz,
DMSO ) δ 158.8, 158.1, 135.7, 128.1, 125.9, 114.2, 113.8, 69.7, 55.5, 55.4. HRMS (ESI) [M+H]
C18H19N2O3: 311.1396; Found (M+H)+: 311.1397.
17

(3,4-dimethoxyphenyl)(4-(3,4-dimethoxyphenyl)-1H-
imidazol-2-yl)methanol (4e)
Light pink solid, M.p: 210-211°C, yield: 75% FTIR (ATR) cm-1:
3288, 3113, 3008, 2954, 2904, 1591, 1532, 1511, 1451, 1320,
1
944, 871, 854. H NMR (400 MHz, d6-DMSO) δ 11.96 (bs, 1H,
N-H), 7.35 (bs, 1H, Ar-H), 7.31 (d, J=1.96 Hz, 1H, Ar-H), 7.24 (dd, J=1.96, J=8.29 Hz, 1H, Ar-H), 7.14 (d,
J=1.58 Hz, 1H, Ar-H), 6.92-6.88 (m, 3H, Ar-H), 6.13 (d, J=3.39 Hz, 1H, OH), 5.70 (d, J=3.39 Hz, 1H, CH)
3.78 (s, 3H, OMe), 3.75(s, 3H, OMe), 3.73 (s, 3H, OMe), 3.71 (s, 3H, OMe). 13C NMR (100 MHz, d6-
DMSO ) δ 151.2, 149.2, 148.9, 148.4, 147.7, 136.1, 119.0, 116.8, 112.4, 111.8, 110.7, 108.6, 69.9,
56.0, 55.9, 55.8, 55.7. HRMS (ESI) [M+H] C20H23N3O5: 371. 1607; found (M+H)+ : 371.1600.
4.6. hAChE and hBChE activity determination and inhibition studies
In bioactivity studies, colorimetric Ellman method was slightly modified and enzyme inhibition was
examined.4 Acetylthiocholine iodide (AChI) and butyrylthiocholine iodide (BChI) were used as
substrates for the reaction. 5,5’-Dithio-bis(2-nitro-benzoic)acid (DTNB) was used for the
measurement of the AChE and BChE activities. In vitro enzyme activity studies was performed using
recombinant human AChE and human serum BChE enzymes. Activity process was utilized as
followed; To deionized water, 1.0 mL of Tris/HCl buffer (1.0 M, pH 8.0), and 10 mL of sample solution
at different concentrations were added. And then 50 mL AChE or BChE solution were mixed and the
o
final solution was incubated for 10 min at 25 C. After incubation, 50 mL of DTNB (0.5 mM) was
added. The reaction was allowed to be iniciated upon addition of 50 mL of AChI or BChI. The
hydrolysis of these substrates was monitored spectrophotometrically by the formation of the yellow
5-thio-2-nitrobenzoate anion, as a result of the reaction of DTNB with thiocholine, which released by
enzymatic hydrolysis of AChI or BChI, with absorption maximum at 412 nm. IC50 values were obtained
from activity (%) versus compounds plots. On the other hand, Ki values were obtained from
Lineweaver-Burk graph. All the experiments were performed at least three times (N=3).
18

4.7. Docking studies
For some of synthesized compounds, the structure-based docking method was applied to determine
the binding and interaction points of the enzymes in the active regions and protein-ligand interaction
analysis was carried out on the crystal structures, coded 1B41. Autodock program was used and the
Protein Preparation Wizard protocol was followed for docking studies. The compounds to be
analyzed were optimized by DFT calculation (DFT/B3LYP-6-311G (d,p)) with Gaussian 09. When
docking process was applied, grid box was determined. Grid box was used to construct a grid with
single precision (SP) and enzyme interaction energies (docking scores) were recorded.
8. Cytotoxicity assay with MTT
Preparation of the MTT solution: For the method to be used in the study, the 5 mg MTT dye
contained in the package will be vortexed after dissolving in 1 mL of sterile phosphate buffer (PBS)
and the insoluble parts will be centrifuged and the supernatant will be taken to a sterile ependorum.
o
The MTT solution that is sensitive to light will be stored in the dark at + 4 C until use. Prior to the
application of the MTT protocol, the cells must be prepared for this assay.
Preparation of Cell Cultures in 96-well plates: The cell lines (WS1 and SH-SY5Y) were firstly counted
and planted in cell plates (96-well plates at 8 x 103 cells/well density). Sterile water or PBS was added
to the outermost of the wells to prevent evaporation. Other fields was used for the experiment. Cells
were regularly and evenly distributed between the wells and whether they are healthy or not was
examined under microscope. The cells shown in the wells was incubated for 24 hours.
Addition of synthesized compound (2c) to cells: After 24 hours of incubation, the cells were re-
incubated with the final concentration of 2c (1,500,1000,1500,2000,3000, 4000, 8000 µM) for 96
hours.
Making the MTT experiment: After 96 hours, the medium was pipetted in the wells removed from
the incubator. 200 mL PBS was added to the wells to thoroughly clean the wells. Then addition of 100
uL of fresh medium to each well and 10μL of MTT solution were progressed and the final solution
was incubated in the incubator for 4 hours. At the end of 4 hours, 100 ul of sodium dodecyl sulfate
(SDS) was added to the wells and incubated for 12 hours at 37 ° C in a CO2 incubator to dissolve
formazone crystals formed with MTT. The culture plate was placed on the microplate reader and
absorbance values was read at 570 nm. This value was accepted as 100% by taking the average of the
absorbance values obtained from the control cultures with no added substances. The absorbance
values obtained from the cultures containing the synthesis compounds was proportioned to the
control absorbance value and the viability rates of the cells was expressed as%. These experiments
were carried out with at least 4 replicates.
19

ASSOCIATED CONTENT
Supporting Information
Synthesis and characterization of compounds; description of enzymatic assay and theoretical
methods
AUTHOR INFORMATION
Corresponding Author
* phone: + 90 432 2251025 (extension:21155); e-mail: nurettinmenges@yyu.edu.tr
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to
the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENT
This study was partly funded by Scientific and Technologic Research Agency of Turkey (TÜBİTAK)
(grant numbers:115Z112, 115Z894)
ABBREVIATIONS
AChE; Acetylcholinesterase, BChE; Butyrylcholinesterase, ACh; Acetylcholine, AD; Alzheimer’s
disease, BBB; Blood-brain barrier; CAS; Catalytic site ES; Acetyl ester, PAS; Peripheral anionic site, Log
P; Lipophilicity, LogKHSA; Human serum albumin binding, WS1; skin fibroblast cell line, SH-SY5Y;
Neuroblastoma cell line, ADME; adsorbtion, distribution, metabolism, excretion
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HIGHLIGHTS
1. C-2 and C-4 substituted imidazole derivatives were synthesized via a one pot and two steps
strategy
2. Activity of imidazole derivatives against AChE and BChE were found in a range of nanomolar
3. Selectivities, kinetic and docking studies were performed
4. Cytotoxycities of selected compound were tested on WS1 and SH-SY5Y whose values are 1790 and
950 µM, respectively
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