Organocatalyzed Solvent Free and Efficient Synthesis of 2,4,5-Trisubstituted Imidazoles as Potential Acetylcholinesterase Inhibitors for Alzheimer's Disease

Article abstract of DOI:10.1002/cbdv.201900493

The catalytic potential of pyridine-2-carboxlic acid has been evaluated for efficient, green and solvent free synthesis of 2,4,5-trisubstituted imidazole derivatives 3a–3m. The compounds 3a–3m were synthesized by one pot condensation reaction of substituted aromatic aldehydes, benzil, and ammonium acetate in good to excellent yields (74–96 %). To explore the potential of these compounds against Alzheimer's disease, their inhibitory activities against acetylcholinesterase (AChE) were evaluated. In this series of compounds, compound 3m, bearing one ethoxy and a hydroxy group on the phenyl ring on 2,4,5-trisubstituted imidazoles, proved to be a potent AChE inhibitor (102.56±0.14). Structure–activity relationship (SAR) of these compounds was developed. Molecular dockings were carried out for the compounds 3m, 3e, 3k, 3c, 3a, 3d, 3j, and 3f in order to further investigate the binding mechanism. The inhibitor molecule was molecularly docked with acetylcholinesterase to further study its binding mechanism. The amino group of the compound 3m forms an H-bond with the oxygen atom of the residue (i. e., THR121) which has a bond length of 3.051 ?.

Full text of DOI:10.1002/cbdv.201900493

Title: Organocatalyzed solvent free and efficient synthesis of 2,4,5-
trisubstituted imidazoles as potential acetylcholinesterase
inhibitors for Alzheimer’s disease
Authors: Sania Pervaiz, Sadaf Mutahir, Islam Ullah Khan, Muhammad
Ashraf, Xiao Liu, Sidrah Tariq, Bao-jing Zhou, and
Muhammad Asim Khan
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900493
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900493

10.1002/cbdv.201900493
Chemistry & Biodiversity
Organocatalyzed solvent free and efficient synthesis of 2,4,5-trisubstituted imidazoles as
potential acetylcholinesterase inhibitors for Alzheimer’s disease
Sania Pervaiz a, Sadaf Mutahir b*, Islam Ullah Khan a, c *, Muhammad Ashraf d Xiao Liu b, Sidrah
,
Tariq a, Bao-jing Zhou b, Muhammad Asim Khan b*.
a Department of Chemistry, Government College, University, Lahore 54000, Pakistan
bSchool of Chemical Engineering, Nanjing University of Science & Technology, Nanjing,
210094, China.
cDepartment of Chemistry, Faculty of Sciences, University of Sialkot- 51040, Sialkot, Pakistan
dDepartment of Chemistry, and Department of Biochemistry & Biotechnology, The Islamia
University of Bahawalpur, Bahawalpur 63100, Pakistan
Corresponding authors,
khanabdali@njust.edu.cn (M. A. Khan), sadafmutahir@njust.edu.cn (S. Mutahir) and
iukhan@gcu.edu.pk (I. U. Khan).
Abstract
The catalytic potential of pyridine-2-carboxlic acid has been evaluated for efficient, green and
solvent free synthesis of 2,4,5-trisubstituted imidazole derivatives. The compounds were
synthesized by condensation reaction of substituted aromatic aldehydes, benzil and ammonium
acetate (3a-3m) in one pot in a good to excellent yield (74-96 %). These compounds were
evaluated against acetylcholinesterase (AChE) enzyme activity to explore their potential against
Alzheimer’s disease. Among this series of compounds 3m bearing one ethoxy and a hydroxyl
group on the phenyl ring on 2,4,5-trisubstituted imidazoles proved potent AChE inhibitor
(102.56±0.14). Structure–activity relationship (SAR) of these compounds was developed.
Molecular dockings were carried out for the inhibitors 3m, 3e, 3k, 3c, 3a, 3d, 3j, and 3f in order
to further investigate the binding mechanism. The inhibitor molecule was molecularly docked
with acetylcholinesterase to further study its binding mechanism. The amino group of the
1
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10.1002/cbdv.201900493
Chemistry & Biodiversity
inhibitor 3m forms an H bond with the oxygen atom of the residue (i.e., THR121), and has a
bond length of 3.051 Å.
Keywords: 2,4,5-trisubstituted imidazoles, Organocatalyst, Acetylcholinesterase, Alzheimer’s
disease, Molecular docking
Introduction
Organocatalyst is an organic molecule with low molecular weight, capable of catalyzing a wide
variety of chemical reactions and has many advantages over traditional metal catalysts. It is
greener, has synthetic range, mild reaction conditions, economic and less toxic, hence highly
appreciated by pharmaceutical industry because it reduces the risk of metal contamination.
Organocatalyst having acidic hydrogen has ability to catalyze wide variety of reactions which
possibily interact with functional groups of substance, hence they play a valuable role in organic
synthesis. Organocatalysts transfer proton to the substrate in transition state and stabilizes the
intermediate products by hydrogen bonding.[1-4] Organocatalysis now-a-days has become the hot
topic in synthetic chemistry. Our research group is actively engaged in the synthesis of potent
organic motifs by organo-catalysis and so far we have explored the catalytic potential of N-acetyl
glycine in the synthesis of bis-biphenyl substituted thiazolidinones,[5] and pyrimidines,[6] a step
towards the green and one pot synthesis of organic compounds avoiding the risk of metal
contamination and obtaining good yield.
Alzheimer’s disease (AD) is a neurodegenerative brain turmoil, which is linked with the low
level of acetylcholine neurotransmitter. This disorder is associated by slow but sure loss of
memory and cognitive impairments. About 4-8 % of the elderly population of the world are
affected by this disorder.[7-10] In Alzheimer’s disease, cognitive impairment is due to the
reduction in levels of acetylcholine in synaptic cleft which occurs by the loss of basal forebrain
cholinergic cells. Acetylcholinesterase is a hydrolase enzyme that hydrolyzes acetylcholine,
which is the major constituent of central cholinergic pathways; hence impart an important role in
the nervous system.
Restraining the acetylcholinesterase (AChE) enzyme is one of the approaches for the treatment
of Alzheimer’s disease which increases the synaptic levels of acetylcholine. For clinical
treatment of AD galanthamine, donepezil, rivastigmine and tacrine are mainly used as AChE
inhibitors (Fig. 1). [11-13]
.
2
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10.1002/cbdv.201900493
Chemistry & Biodiversity
Figure 1: Structures of AChE inhibitors
The increasing mortality rate, complexities in disease and the limited option of drugs for the
curing of AD, demands new and more effective pharmacological products. Keeping these factors
in view researchers is aiming to develop new potential drugs. [14-16]
Substituted imidazole nucleus is well recognized for a wide range of therapeutic effects such as it
is used as farnesyltransferase inhibitors [17], potent BRAF kinase inhibitor,[18] orally active 5-
lipoxygenase (5-LO) inhibitors,[19] CSBP kinase inhibitors,[20], cytokine biosyhthesis inhibitor,[21]
transforming growth factor b1 (TGF-b1) type 1 activating receptor-like kinase (ALK5).[22]
Synthesis of 2,4,5-trisubstituted imidazoles is usually carried out by cyclocondensation of three
components i.e. an aldehyde and ammonium acetate with a 1,2-diketone, α-hydroxyketone or α-
ketomonoxime. To obtain good yield reaction is generally catalyzed by the selection of different
compounds such as silica gel, BF3. SiO2, molecular iodine, zirconium (IV) acetylacetonate
Zr(acac)4, silica sulfuric acid, scolecite, Ammonium Chloride, nanocrystalline magnesium
chloride, TiCl4.SiO2, tetrabutylammonium hexatungstate [TBA]2[W6O19], ZnO nanorods,
Eu(OTf)3, silica chloride, silica bonded propyl-N-sulfamic acid nanocatalyst (NHSO3H-KIT-5),
Ce(SO4)2. 4H2O, keggin-structured phosphotungstic acid (HPW).[23-38]
3
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10.1002/cbdv.201900493
Chemistry & Biodiversity
R
R
Scheme 1: Synthesis of 2,4,5-trisubstituted imidazoles.
All these new methodologies have their own advantages but most of these are associated with
drawbacks like use of toxic solvents, prolong time, complicated work-up, purification
difficulties, poor yield, expensive reagents, hence there is a dire need to develop such synthetic
routes that are environmental friendly and economically. Various organocatalysts are being used
[39]
now-a-days to accelerate chemical reaction with easy handling.
We herein report the
organocatalyzed synthesis of 2,4,5-trisubstituted imidazole in solvent free system.
Result and discussion
2.1. Chemistry
For investigating the catalytic potential of various catalysts and for the choice of best catalyst
model reactions were carried out. We have selected the reaction of Benzil (1) with 4-
hydroxybenzaldehyde (2 d) and ammonium acetate for optimization of reactions. (Scheme 2).
Scheme 2: Model reaction for the optimization
Different catalysts were used to get comparative yields for model reaction and the obtained
results are summarized in table 1.
4
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Chemistry & Biodiversity
Table 1: Comparative study for the optimization of best catalyst (Scheme 2)
Entry
Catalyst
Yield
Time of reaction
(hours)
Nicotinic acid
Pyridine-2-carboxylic acid
Pyridine-2,3-dicarboxylic acid
Pyridine-2,4,6-tricarboxylic acid
Benzoic acid
1
2
3
4
5
6
72%
78%
54%
43%
67%
-
2-3
2-3
4-5
Overnight
3-4
-
No catalyst
Among all tested catalysts for the synthesis of 4-(4,5-diphenyl-1H-imidazol-2-yl)phenol,
pyridine-2-carboxylic acid has shown better catalytic ability resulting 78% yield in 2-3 hours
(Table 1, entry 2) so, it is selected for carrying out a whole series of compounds. Further
optimization of catalyst for minimum concentration and temperature was determined; results are
shown in Table 2.
Table 2: Optimization of the reaction conditions with the selected catalyst (pyridine-2-
carboxylic acid) (Scheme 2)
Entry
Catalyst (eq)
Time (hours)
Temperature (°C)
% yield
69
1
2
3
4
5
1.5
1.0
0.5
0.5
0.5
2-3
2-3
2-3
2-3
2-3
120
120
120
90
72
78
75
78
150
2,4,5-trisubstituted imidazoles 3a–3m were synthesized by condensation of the aromatic
aldehydes , benzil, and ammonium acetate in good to excellent yields (74 -96 %) as depicted in
scheme 1. The condensation reaction was catalyzed by pyridine -2- carboxylic acid. Physical
analysis and spectroscopic studies were applied for the conformation of synthesized compounds.
The plausible mechanism for the synthesis of 2,4,5-trisubstitued imidazoles is shown in Figure 2.
Organo- catalyst pyridine-2-carboxylic acid’s proton attacked on the carbonyl group of aldehyde
and benzyl. Nitrogen of ammonia attach as nucleophile on the protonated carbonyl group of
5
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10.1002/cbdv.201900493
Chemistry & Biodiversity
aldehydes, diamine intermediate formed. Benzil and diamine intermediate condensed to cyclic
intermediate state which rearranged by a [1, 5]-H shift to the trisubstituted imidazoles. [40-42]
Figure 2: Proposed mechanism for the synthesis of 2,4,5-trisubstituted imidazoles by catalytic
condensation using pyridine-2-carboxlic acid.
2.2. AChE inhibition activity
In vitro inhibition against AChE was performed for all synthesized 2,4,5-trisubstituted
imidazoles 3a–3m. Eserine was used as control in this assay. [43-45] The results were summarized
in table 3 which illustrates that 3m, 3k, and 3e are, the more potent AChE inhibitor as compared
to the rest of synthesized compounds that showed moderate activity. Compound 3m bearing one
ethoxy and one hydroxyl group on phenyl moiety at position 2 of imidazole nucleus proved most
active compound with IC50 value of 102.18±0.12 μM. When we replaced the group from ethoxy
to methoxy on the phenyl moiety in compound 3k there is a slight decrease in activity with IC50
value of 125.86±0.13 μM. There is slightly diminishing effect in activity when the phenyl moiety
have only methoxy group in 3c (128.22±0.17 μM). In compound 3e hydroxyl group is at C-2 of
phenyl moiety having IC50 value of 112.40±0.16 μM when hydroxyl group is at C-3 of
compound 3a it has the inhibitory value IC50 value of 158.14±0.19 while in compound 3d
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Chemistry & Biodiversity
hydroxyl group is at C-4 of phenyl moiety having IC50 value of 172.16±0.14 μM. Change in the
position of –OH group from carbon 2 to 4 of phenyl ring there is decreased in the AChE
inhibitory activity. It can be depicted from the activity pattern that phenyl group bearing
hydroxyl group along with ethoxy and methoxy groups are more effective AChE inhibitor as
compared to alone hydroxyl group (compound 3m vs. 3e). The unsubstituted phenyl ring 2i
proved inactive but when electron withdrawing group are attached either it act as moderate
AChE inhibitor (3j) or proved in active (3b, 3g, & 3l).
The overall order of reactivity is 3m>3e>3k>3c>3a>3d>3j>3f
Table 3
Yield (%) and in vitro AChE inhibitory activity of 2,4,5-trisubstituted imidazoles 3a–3m
[(inhibition percentage and IC50 values are means given with SEM, (ND, not determined)].
(3a–3m)
Yield
(%)
83
76
74
78
79
77
79
81
78
96
81
89
78
-
%
Entry compound
R
IC50 (μM)
Inhibition
87.46±0.21 158.14±0.19
23.75±0.16 ND
86.25±0.23 128.22±0.17
42.53±0.25 172.16±0.14
87.15±0.28 112.40±0.16
77.58±0.22 332.636±0.16
34.75±0.24
23.24±0.23
37.68±.025
81.27±0.19 243.55±0.16
87.51±0.18 125.86±0.13
14.52±0.12
76.54±0.22 102.56±0.14
91.27±1.17 0.04±0.0001
3-OH–C6H4
2-Cl–C6H4
4-OCH3–C6H4
4-OH–C6H4
2-OH–C6H4
C6H4N
1
2
3a
3b
3
3c
4
3d
5
3e
6
3f
4-Cl–C6H4
7
3g
ND
ND
ND
C6H5
4-(CH3)2N–C6H4
4-Br–C6H4
8
3h
9
3i
10
11
12
13
14
3j
3k
3l
3-OCH3-4-OH–C6H4
4-F–C6H4
3-OC2H5-4-OH–C6H4
ND
3m
Eserine
Molecular Docking Studies
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Chemistry & Biodiversity
Molecular dockings were carried out for the inhibitors 3m, 3e, 3k, 3c, 3a, 3d, 3j, and 3f in order
to further investigate the binding mechanism (Fig. 3). The inhibitor molecule was molecularly
docked with acetylcholinesterase to further study its binding mechanism. Obviously, the
different groups on the inhibitor molecule are the main reasons leading to different docking
results. Generally, the inhibitor molecule will be inserted into a residue pocket formed by
THR121, PHE290, PHE331, PHE330, TYP84, TYR334, ASP72, TYR70, TRP270. The amino
group of the inhibitor 3m forms an H bond with the oxygen atom of the residue (i.e., THR121),
and has a bond length of 3.051 Å. The amino group of the inhibitor 3e forms Hbond with the
oxygen atom of the residue (i.e., THR121), and has a bond length of 2.685 Å. The amino group
of the inhibitor 3k forms an H bond with the oxygen atom of the residue (i.e., THR121), and has
a bond length of 3.025 Å. The docking results of the inhibitor 3c and acetylcholinesterase have
two main conformations. One is that the inhibitor N atom forms a H bond with the oxygen atom
of the residue (i.e., THR121), and the bond length is 2.970 Å. Second, the inhibitor N atom
forms a H bond with the oxygen atom of the residue (ie, THR121), and the bond length is 3.197
Å. The amino group of the inhibitor 3a forms H bond with the oxygen atom of the residue (ie
SER122) with a bond length of 2.603Å; the hydroxyl group and the N atom of the residue
ASP72, respectively. It forms a bond with the O atom of the residue ASN85, and the bond
lengths are 2.479 Å and 1.718 Å, respectively. The amino group of the inhibitor 3d forms a H
bond with the oxygen atom of the residue (i.e., THR121), and has a bond length of 2.896 Å. The
amino group of the inhibitor 3j forms a H bond with the oxygen atom of the residue (i.e.,
THR121), and has a bond length of 3.783 Å. The amino group of the inhibitor 3f forms a H bond
with the oxygen atom of the residue (i.e., SER122) with a bond length of 2.450Å. The interaction
between the inhibitor and the enzyme is enhanced by the interaction of the H bond, resulting in
high binding affinity.
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10.1002/cbdv.201900493
Chemistry & Biodiversity
Figure 3: Illustrations of close contacts between the residues and inhibitors: (a) 3m, (b) 3e, (c)
3k, (d) 3c, surface representations are employed for the enzyme.
Conclusions
In summary, We report the new methodology for the synthesis of 2,4,5-trisubstituted imidazoles
by utilizing the pyridine-2-carboxlic acid as an organocatalyst. The remarkable catalytic activity
of pyridine-2-carboxlic acid exhibited one pot synthesis, short time of reaction, amount of
catalyst, easy work up and pure products were obtained by simple crystallization. Products (3a-
3m) were evaluated for in vitro AChE inhibitory activity. The substituents on triphenyl
imidazole had a profound effect on the acetylcholinesterase inhibitory activity. Compound 3m
proves the most active compound with IC50 value of 102.18±0.12 μM and this could be a good
drug candidate for the treatment of AD. Molecular dockings were carried out for the inhibitors
3m, 3e, 3k, 3c, 3a, 3d, 3j, and 3f in order to further investigate the binding mechanism. The
inhibitor molecule was molecularly docked with acetylcholinesterase to further study its binding
mechanism. The amino group of the inhibitor 3m forms an H bond with the oxygen atom of the
residue (i.e., THR121), and has a bond length of 3.051 Å.
Experimental
General methods
All chemicals and solvents used were of analytical grade and were purchased from Sigma,
Aldrich and Merck Chemical Company and used without further purification. IR spectra in KBr
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10.1002/cbdv.201900493
Chemistry & Biodiversity
pellets were recorded on FT-IR Perkin Elmer spectrum BX spectrophotometer at the frequency
range of 4000–400 cm-1. Melting points were taken in open capillary tubes and are uncorrected.
TLC was performed on silica coated aluminum sheets (silica gel, 0.2 mm, E. Merk, Germany)
and visualized in low UV light. Elemental analysis was carried by CHNS analyzer (vail micro
1
cube, Elementary Germony). H NMR spectra were recorded in solvents such as DMSO-d6,
CDCl3 and MeOD. Splitting patterns were as follows s (singlet), d (doublet), dd (double
doublets), t (triplet), and m (multiplet). Chemical shifts values are reported in δ-units and
coupling constants are given in Hz.
General procedure for synthesis of 2,4,5-trisubstituted imidazoles (3a-3m)
For the synthesis of 2,4,5-trisubstituted imidazoles (3a-3m) benzil (1) (1eq), ammonium acetate
(2 eq), corresponding aldehydes (2a-2m) (1 eq) and pyridine 2 carboxylic acid ( 0.5eq) were
grounded in a mortar and pestle. The finely ground mixture was transferred into a round bottom
flask and stirred for two to three hours at 120 °C. The reaction was observed by TLC. After the
completion of reaction 5 % NaHCO3 was added. Through solvent extraction, product was
extracted with ethyl acetate which was dried over MgSO4 and evaporated. Products were purified
by recrystallization from methanol
3-(4,5-diphenyl-1H-imidazol-2-yl)phenol [46] (3a)
Shiny brown solid, yield: 83%; m.p: 249-251 °C; FT-IR (υmax, cm-1): 3048 (N-H), 3088 (Ar
CH), 2900 (Alkyl C-H), 1684 (C=N), 1652 (C=C); 1H-NMR (300 MHz, DMSO-d6): δ 12.65
(s, NH ,1H), 9.56 (s, O-H, 1H), 9.05 (s, Ar-H, H-5, 1H), 8.75 (s, Ar-H, 1H), 8.2 (s, Ar-H, 1H),
7.7-7.2 (m, Ar-H, 10H), 6.7 (d, J=5.96, Ar-H, 1H). Anal. Calcd for C21H16N2O: C, 80.75; H,
5.17; N, 8.97; O, 2.12. Found: C, 80.68; H, 5.62; N, 8.83.
2-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole [47] (3b)
White solid, yield: 69%; m.p: 242-244 °C; FT-IR (υmax, cm-1): 3057 (N-H), 3092 (Ar CH),
1
2951 (Alkyl C-H), 1640 (C=N), 1600 (C=C); H-NMR (300 MHz, DMSO-d6): δ 9.05 (s, O-H,
1H), 8.75 (d, J=6.98, Ar-H, 1H), 8.25 (t, J=8.93, Ar-H, 1H), 7.7 (t, J=5.69, Ar-H, 1H) 7.6-7.0 (m,
Ar-H, 10H). Anal. Calcd for C21H15ClN2: C, 76.24; H, 4.57; N, 8.47; Cl, 10.72. Found: C, 76.53;
H, 4.82; N, 8.75.
2-(4-methoxyphenyl)-4,5-diphenyl-1H-imidazole [41] (3c)
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Chemistry & Biodiversity
White solid, yield: 74%; m.p: 230-232 °C; FT-IR (υmax, cm-1): 3034 (N-H), 3066 (Ar CH),
2837 (Alkyl C-H), 1657(C=N), 1635 (C=C); 1H-NMR (DMSO-d6, 300MHz): 8.0 (d, J= 4.9, Ar-
H, 2H) 7.5-6.9 (m, Ar-H, 10H), 7.0 (d, J= 5.31, Ar-H, 2H) 3.75 (s, CH3, 3H). Anal. Calcd for
C22H18N2O: C, 80.96; H, 5.56; N, 8.58; O, 4.90. Found: C, 80.82; H, 5.76; N, 8.69.
4-(4,5-diphenyl-1H-imidazol-2-yl)phenol [48] (3d)
White solid, yield: 78%; m.p: 239-241 °C; FT-IR (υmax, cm-1): 3046 (N-H), 3071 (Ar CH),
2870 (Alkyl C-H), 1683 (C=N), 1625 (C=C); 1H-NMR (300 MHz, DMSO-d6): δ 12.41 (s, N-H,
1H), 9.15 (s, O-H, 1H), 8.2 (s, H-1, 1H), 7.9 (s, Ar-H, 1H), 7.6-7.1 (m, Ar-H, 10H), 6.8 (s, Ar-
H, 2H). Anal. Calcd for C21H16N2O: C, 80.75; H, 5.17; N, 8.97; O, 2.12. Found: C, 80.93; H,
5.47; N, 8.76.
2-(4,5-diphenyl-1H-imidazol-2-yl)phenol [48] (3e)
White solid, yield: 71%; m.p: 205-207 °C; FT-IR (υmax, cm-1): 3044 (N-H), 3120 (Ar CH),
1
2836 (Alkyl C-H), 1683(C=N), 1614 (C=C); H-NMR (300 MHz, DMSO-d6): δ 12.5 (s, N-H,
1H), 8.0 (d, J = 6.21, Ar-H, 2H), 7.50-7.1 (m, Ar-H, 12H). Anal. Calcd for C21H16N2O: C, 80.75;
H, 5.17; N, 8.97; O, 2.12. Found: C, 80.58; H, 5.45; N, 8.78.
3-(4,5-diphenyl-1H-imidazol-2-yl)pyridine [49] (3f)
White solid, yield: 77%; m.p: 234-236 °C; FT-IR (υmax, cm-1): 3245 (N-H), 3080 (Ar CH),
2910 (Alkyl C-H), 1698 (C=N), 1599 (C=C); 1H-NMR (300 MHz, DMSO-d6): δ 12.92 (s, N-H,
1H), 9.3 (s, Ar-H, H-1, 1H), 8.5(d, J = 8.74, Ar-H, 1H), 8.4 (d, J = 3.29, Ar-H, 1H), 7.6-7.2 (m,
Ar-H, 11H). Anal. Calcd for C20H15N3: C, 80.78; H, 5.08; N, 14.13. Found: C, 80.83; H, 5.25; N,
8.45.
2-(4-chlorophenyl)-4,5-diphenyl-1H-imidazole [41] (3g)
White solid, yield: 78%; m.p: 259-261 °C; FT-IR (υmax, cm-1): 3038 (N-H), 3065 (Ar CH),
2825 (Alkyl C-H), 1640 (C=N), 1600 (C=C); 1H-NMR (300 MHz, CDCl3): δ 13.45 (s, N-H, 1H),
9.3 (s, Ar-H, 2H), 8.6 (s, Ar-H, 2H), 8.2-7.7 (m, Ar-H, 10H). Anal. Calcd for C21H15ClN2: C,
76.24; H, 4.57; N, 8.47; Cl, 10.72. Found: C, 76.68; H, 4.78; N, 8.64.
2,4,5-triphenyl-1H-imidazole [41] (3h)
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Chemistry & Biodiversity
White solid, yield: 81%; m.p: 271-273 °C; FT-IR (υmax, cm-1): 3036 (N-H), 3055 (Ar CH),
1
2850 (Alkyl C-H), 1680 (C=N), 1602 (C=C); H-NMR (300 MHz, DMSO-d6): δ 12.6 (s, N-H,
1H), 8.1 (d, J = 4.75, Ar-H, 2H), 7.6-7.2 (m, Ar-H, 13H). Anal. Calcd for C21H16N2: C, 85.11; H,
5.44; N, 9.45. Found: C, 85.38; H, 5.85; N, 9.78.
4-(4,5-diphenyl-1H-imidazol-2-yl)-N,N-dimethylaniline [41] (3i)
White solid, yield: 78%; m.p: 256-258 °C; FT-IR (υmax, cm-1): 3048 (N-H), 3090 (Ar CH),
1
2938 (Alkyl C-H), 1653 (C=N), 1633 (C=C); H-NMR (CDCl3): δ 12.4 (s, N-H, 1H), 8.0 (d J =
5.31, Ar-H, 2H), 7.5-7.2 (m, Ar-H, 10H), 7.0 (d, J = 4.94, Ar-H, 2H), 3.8 (s, H-(CH3)2, 6H).
Anal. Calcd for C23H21N3: C, 81.38; H, 6.24; N, 12.38. Found: C, 81.45; H, 6.12; N, 12.43.
2-(4-bromophenyl)-4,5-diphenyl-1H-imidazole [41] (3j)
White solid, yield: 93%; m.p: 247-248 °C FT-IR (υmax, cm-1): 3030 (N-H), 3078 (Ar CH), 2956
(Alkyl C-H), 1665 (C=N), 1620 (C=C); 1H-NMR (300 MHz, DMSO-d6): δ 8.0 (d, J = 5.31, Ar-
H, 2H), 7.6 (d, J = 5.13, Ar-H, 2H), 7.5-7.2 (m, Ar-H, 10H). Anal. Calcd for C21H15BrN2: C,
67.28; H, 4.03; N, 7.47; Br, 21.29. Found: C, 67.31; H, 4.15; N, 7.38.
4-(4,5-diphenyl-1H-imidazol-2-yl)-2-methoxyphenol [33] (3k)
FT-IR (υmax, cm-1): 3031 (N-H), 3073 (Ar CH), 2895 (Alkyl C-H), 1698 (C=N), 1600 (C=C);
1H-NMR (500 MHz, MeOD): δ 7.6 (d, J = 2.0, Ar-H, 1H), 7.4-7.2 (m, Ar-H, 11H), 6.8 (d, J =
8, Ar-H, 1H) 3.9 (s, CH3, 3H). Anal. Calcd for C22H18N2O2: C, 77.17; H, 5.30; N, 8.81; O, 9.35.
Found: C, 77.24; H, 5.38; N, 8.78.
2-(4-fluorophenyl)-4,5-diphenyl-1H-imidazole [50] (3l)
Pale white solid, yield: 89%; m.p: 191-193 °C FT-IR (υmax, cm-1): 3029 (N-H), 3069 (Ar CH),
1
2908 (Alkyl C-H), 1683 (C=N), 1607 (C=C); H-NMR (300 MHz, DMSO-d6): δ 12.6 (s, N-H,
1H), 8.1 (t, J = 8.6, Ar-H, 2H), 7.5-7.2 (m, Ar-H, 12H). Anal. Calcd for C21H15FN2: C, 80.24; H,
4.81; N, 8.91; F, 6.04. Found: C, 80.35; H, 4.84; N, 8.84.
4-(4,5-diphenyl-1H-imidazol-2-yl)-2-ethoxyphenol [49] (3m)
White solid, yield: 78%; m.p: 255-257°C. FT-IR (υmax, cm-1): 3031 (N-H), 3061 (Ar CH),
2890 (Alkyl C-H), 1698 (C=N), 1600 (C=C); 1H-NMR(600 MHz DMSO ): δ 12.5 (s, N-H,1H),
9.1 (s, O-H, 1H) , 7.1-7.6 (m, Ar-H, 12H), 6.9 (d, J = 7.9, Ar-H, 1H) 4.1 (q, J = 6.7, H-CH2,
12
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Chemistry & Biodiversity
2H), 1.3 (t, J = 6.4, H-CH3 3H,). Anal. Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86; O,
8.98. Found: C, 77.42; H, 5.58; N, 8.93.
Acetylcholinesterase assay
Ellman’s method with minor modifications was used for the determination of AChE inhibition
activity.[51] Total volume of the reaction mixture was 100 µl, 60µl was buffer solution
(Na2HPO4, 50 mM) for maintaining pH at 7.7. Test compound (10 µl, 0.5 mM well) was added,
followed by the addition of 10 µl (0.005 unit well) enzyme. The reactants were mixed and pre-
read at 405 nm. At 37 °C contents were pre-incubated for 10 min. The reaction was initiated by
the addition of 10 µl of substrate (acetylthiocholine iodide, 0.5 mM), then 10 µl DTNB (0.5
mM). Absorbance was measured at 405 nm after 30 minutes of incubation at 37 °C. In all
experiments synergy HT (BioTek, USA) 96-well plate reader was used. As a positive control
eserine (0.5 mM) was used. Following equation was used for the calculation of percent AChE
inhibition activity.
Inhibition (%) = (absorbance of control- absorbance of Test)
absorbance of Control * 100
EZ–Fit Enzyme kinetics software (Perella Scientific Inc. Amherst, USA) was used for estimation
of IC50 values of synthesized compounds.
Molecula Docking
The structure of acetylcholinesterase was obtained from the protein data bank (PDB code:
1EVE). The structures of inhibitors were optimized at B3LYP/6-31G level using Gaussian 09.
Docking studies were performed for four inhibitors using Autodock package. We used Chimera
software to determine the hydrogen bonds. The angstroms and degrees were set to 1.0 Å and 90
Å, respectively, except for the seventh (the angstroms and degrees were set to 2.0 Å and 90 Å),
in order to identify the H bonds.
Acknowledgement
We are thankful to the Office of Research Innovation and Commercialization Government
College University Lahore, Pakistan for funding this project.
Supplementary Data
Supplementary data related to this article can be found in supporting file.
13
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10.1002/cbdv.201900493
Chemistry & Biodiversity
Author Contribution Statement
Sania Pervaiz, Islam Ullah Khan and Sadaf Mutahir designed and planned the experiments.
Sania Pervaiz and Sidrah Tariq synthesized the compounds. Muhammad Ashraf evaluated all
synthesized compounds for AChE inhibition. Sania Pervaiz wrote the manuscript. Bao-jing Zhou
and Xiao Liu performed molecular docking. Sadaf Mutahir and Muhammad Asim Khan wrote
molecular docking report, and provided guidance in writing manuscript. Islam Ullah Khan
supervised the whole project.
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Table of Contents:
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