In silico studies, synthesis and pharmacological evaluation to explore multi-targeted approach for imidazole analogues as potential cholinesterase inhibitors with neuroprotective role for Alzheimer's disease

Article abstract of DOI:10.1016/j.bmc.2018.01.029

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with multiple factors associated with its pathogenesis. Our strategy against AD involves design of multi-targeted 2-substituted-4,5-diphenyl-1H-imidazole analogues which can interact and inhibit AChE, thereby, increasing the synaptic availability of ACh, inhibit BuChE, relieve induced oxidative stress and confer a neuroprotective role. Molecular docking was employed to study interactions within the AChE active site. In silico ADME study was performed to estimate pharmacokinetic parameters. Based on computational studies, some analogues were synthesized and subjected to pharmacological evaluation involving antioxidant activity, toxicity and memory model studies in animals followed by detailed mechanistic in vitro cholinesterase inhibition study. Amongst the series, analogue 13 and 20 are the most promising multi-targeted candidates which can potentially increase memory, decrease free radical levels and protect neurons against cognitive deficit.

Full text of DOI:10.1016/j.bmc.2018.01.029

Accepted Manuscript
In silico studies, synthesis and pharmacological evaluation to explore multi-
targeted approach for imidazole analogues as potential cholinesterase inhibitors
with neuroprotective role for Alzheimer’s disease.
Archana S. Gurjar, Mrunali N. Darekar, Keng Yoon Yeong, Luyi Ooi
PII:
S0968-0896(17)31824-2
https://doi.org/10.1016/j.bmc.2018.01.029
BMC 14183
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 September 2017
27 January 2018
31 January 2018
Please cite this article as: Gurjar, A.S., Darekar, M.N., Yeong, K.Y., Ooi, L., In silico studies, synthesis and
pharmacological evaluation to explore multi-targeted approach for imidazole analogues as potential cholinesterase
inhibitors with neuroprotective role for Alzheimer’s disease., Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.01.029
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In silico studies, synthesis and pharmacological evaluation to explore multi-
targeted approach for imidazole analogues as potential cholinesterase
inhibitors with neuroprotective role for Alzheimer’s disease.
Archana S. Gurjara*, Mrunali N. Darekara, Keng Yoon Yeongb, Luyi Ooib
a Prin. K. M. Kundnani College of Pharmacy, 23 Jote Joy, R. S. Marg, Cuffe Parade, Mumbai
400005, India.
b School of Science, Monash University Malaysia Campus, Jalan Lagoon Selatan, Bandar
Sunway, 47500, Selangor, Malaysia.
*gurjar_a_s@yahoo.co.in

Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder with multiple
factors associated with its pathogenesis. Our strategy against AD involves design of multi-
targeted 2-substituted-4,5-diphenyl-1H-imidazole analogues which can interact and inhibit
AChE, thereby, increasing the synaptic availability of ACh, inhibit BuChE, relieve induced
oxidative stress and confer a neuroprotective role. Molecular docking was employed to study
interactions within the AChE active site. In silico ADME study was performed to estimate
pharmacokinetic parameters. Based on computational studies, some analogues were synthesized
and subjected to pharmacological evaluation involving antioxidant activity, toxicity and memory
model studies in animals followed by detailed mechanistic in vitro cholinesterase inhibition
study. Amongst the series, analogue 13 and 20 are the most promising multi-targeted candidates
which can potentially increase memory, decrease free radical levels and protect neurons against
cognitive deficit.
Keywords: Acetylcholinesterase; 2-substituted-4,5-diphenyl-1H-imidazole; Molecular docking;
in vitro antioxidant assay; in vitro Ellman assay.

1. Introduction
Alzheimer’s disease (AD) is one of the most common neurodegenerative disorder mainly
disrupting communication within neural circuits vital for memory and other cognitive
functions.1-2 Past two decades have witnessed extensive research dedicated to unravel the
molecular, biochemical, and cellular mechanisms of cognitive deficit.3 Various hypotheses have
been proposed in the pathogenesis of AD like Acetylcholine (ACh) imbalance, amyloid beta
(Aβ) production and its aggregation, tau hyperphosphorylation, oxidative stress and others
including dysfunctional calcium homeostasis, hormonal, inflammatory-immunological causes.
Hence, the development of an effectual therapeutics is of extreme importance.4
The deficiency of neurotransmitter ACh in the brain is a crucial factor associated with
AD pathogenesis. ‘Cholinergic hypothesis’ states severe failure of cholinergic function in the
central nervous system which contributes to cognitive symptoms. Acetylcholinesterase (AChE)
or true ChE predominantly hydrolyzes ACh and is found in high concentrations mainly in the
cholinergic brain synapses, at neuromuscular junctions and red blood cells. Butyrylcholinesterase
(BuChE) or pseudo/plasma ChE is a non-specific type of cholinesterase enzyme that hydrolyzes
different types of choline esters and exists ubiquitously throughout the body, most importantly,
in the human liver, blood serum, pancreas and the central nervous system.5 BuChE mostly
hydrolyzes ACh in later stages of AD due to increased BuChE activity and decreased AChE
activity, so search of BuChE inhibitors for progressed AD patients can be beneficial.6 However,
the most promising approach for symptomatic relief of AD is to inhibit the AChE, which
primarily catalyzes the hydrolysis of ACh, thereby increasing synaptic levels of ACh in the
brain. Research so far has not led to breakthrough drug candidate or a single pathway that can
cure AD. Till date four acetylcholinesterase inhibitors such as tacrine, rivastigmine, galantamine,

donepezil and NMDA receptor antagonist memantine are the only US FDA approved drugs for
its treatment.7-9
A unique structural aspect of AChE is a deep and narrow gorge, about 20Å long, which
penetrates more than halfway and widens out at its base, where the catalytic triad lies.10 The
peripheral anionic site (PAS) is located at the gorge entry lined by highly conserved 14 aromatic
amino acid residues.11 The X-ray crystallographic structure of AChE (PDB ID: 1EVE) has
revealed the main binding sites i.e. esteratic subsite (catalytic triad) at the bottom of gorge
(Ser200, His440, and Glu327); peripheral anionic site (PAS) with Tyr70, Asp72, Tyr121, Trp279
and Tyr334; an anionic substrate (AS) binding site having Trp84, Tyr130, Phe330 and Phe331,
oxyanion hole with Gly118, Gly119, Ala201 and an acyl pocket (Phe288, and Phe290).12
In the literature, a new non-cholinergic role of AChE has been suggested; the enzyme co-
localizes through its peripheral anionic site with the Aβ peptide deposits in the brain of AD
patients and promotes Aβ fibrillogenesis by forming stable AChE-Aβ complexes, so inhibition of
AChE will deter the fibrillogenesis.13-14 The accumulation of Aβ in various areas of the brain
exhibits its neurotoxic effects through inflammation, calcium dysregulation and activating
microglial cells which in turn cause oxidative stress and neuronal death.15 Due to the connection
of various factors in the disease progression, modulation of a single aspect might not be
satisfactory to achieve desired effectiveness. This has triggered our curiosity towards the design
of multi-targeted analogues which can interact with both the catalytic triad and peripheral
binding sites of AChE, thereby, increasing the synaptic availability of ACh, declining the
deposition of Aβ, relieving induced oxidative stress and conferring a neuroprotective role.
Literature survey indicates that series developed possessing various heteroaromatic rings
like coumarin, benzimidazole, thiazole, piperidine, indanone, spiro-indenoquinoxaline, spiro-

dihydropyridines derivatives showed promising acetylcholinesterase inhibitory activity.16-21
Rahim et al reported a series of thirty thiazole analogues (1) developed using structural-based
drug design tool and evaluated their AChE and BuChE inhibitory potential. All analogues
exhibited inhibition with an IC50 value ranging from 1.59±0.01 to 389.25±1.75μM with an
improvement in memory as well as cognitive functions including lowering of progressive
neurodegeneration.22 Prompted by these findings we replaced the sulphur moiety of thiazoles
with nitrogen and explored the imidazole scaffold and developed a series of 2-substituted-4,5-
diphenyl-1H-imidazole analogues (2) to target multiple factors simultaneously involved in the
etiopathogenesis of AD (Figure 1).
Figure 1: Substituted thiazole and 2-substituted-4,5-diphenyl-1H-imidazole scaffolds.
In this paper, we discuss computational studies which includes molecular docking within
AChE active site and in silico ADME studies, synthesis, evaluation of antioxidant activity,
toxicity and memory model studies in animals as well as in vitro cholinesterase inhibition
activity of 2-substituted-4,5-diphenyl-1H-imidazole analogues.

2. Results and discussion
2.1. Molecular Docking studies with AChE enzyme
In the current study, docking approach has been utilized to explore the AChE active site,
foresee orientation (pose) of the designed ligands in the binding pocket and assess the tightness
of target-ligand interactions (scoring) using GOLD (Genetic Optimization for Ligand Docking)
program with full range of ligand conformational flexibility and selective rotational flexibility of
the receptor.23-24 The docking protocol was validated by reproduction of binding pose of the co-
crystallized ligand donepezil in the enzyme active site (PDB: 1EVE; rmsd: 0.600). To further
substantiate the protocol, some reported AChE inhibitors (AChEIs): Physostigmine
(IC500.220μM), Galantamine (IC50 0.623μM), Alfuzosin (IC50 0.018μM), Dyclonine (IC50
0.181μM), Nefazodone (IC50 1.037μM), Indanone derivatives (IC50 0.0018μM) were also docked
within the active site. 25-28
The active marketed drug donepezil spans the entire gorge while the docked reported
AChEIs occupy bottom and entrance of the gorge. Donepezil exhibited hydrogen-bond
interactions through water molecules with the various active site residues such as Tyr70, Asp72,
Trp84, Gly117, Tyr121, Ser122, Tyr130 and Ser200. Hence, water molecules play a crucial role
in drug receptor interactions.29 Also; the cyclic ketone group of indanone moiety depicted
hydrogen-bond interaction with the Phe288 residue of the acyl pocket. The benzyl ring and
indanone ring of donepezil displayed a classical π–π stacking with the indole ring of Trp84 and
Trp279 respectively. N-benzyl piperidine fragment displayed π-sigma interaction with aromatic
ring of Phe330. Similarly, methoxy group exhibited π-sigma interaction with indole ring of
Trp279. π-alkyl interaction of the piperidine nitrogen was observed with Tyr334 residue of the
active site. These results indicate positively that the docking protocol was able to acceptably

reproduce the crystal pose for co-crystallized donepezil and retain nearly all the interactions that
were reported in the literature (Figure 2). Also, most of the reported AChEIs interacted with the
amino acid residues of catalytic triad especially Ser200, His440 and peripheral anionic subsite
(PAS) which play a decisive role in enzymatic catalysis and inhibitor binding. Thus, the docked
pose of donepezil and reported AChEIs reproduced analogous interactions as mentioned in the
literature within AChE active site. 11 From this it can be envisaged that the proposed docking
protocol will suitably predict the binding mode of the designed series.
Figure 2a&b: Docking pose of Donepezil within AChE active site
2a: H-bond formation
2b: Hydrophobic interactions
Subsequently, a series of 2-substituted-4,5-diphenyl-1H-imidazole analogues (Table 1)
was docked in the AChE active site. The test analogues have reached bottom of the gorge and
formed hydrogen bonds through water molecules with similar amino acid residues as observed
for donepezil. The docking analysis revealed that imidazole analogues interact with active site
primarily through hydrogen bonds and hydrophobic interactions such as pi-pi stacking, pi-pi

stacking (T-shaped), pi-sigma, pi-alkyl. Mostly the interactions of ligands were observed with
residues present at bottom or middle of the gorge i.e with the catalytic triad (Ser200) and PAS
(Tyr70, Asp72, Tyr121 and Tyr334). Anne Imberty et al related the distance of hydrogen bonds
between a ligand and receptor with the type such as strong, average and weak interactions.30
Results of imidazole analogues depicted that hydrogen bond interactions were of strong or
average nature. In the designed test series, substituents have been varied at the 2-position in the
imidazolyl ring system from simple alkyl groups such as methyl, ethyl to phenyl and heteroaryl.
Further, the phenyl ring has been substituted with mono-, di-, and tri-substitutions of different
functional groups such as nitro, hydroxyl, methoxy, amino and halogens. In case of 2-hydrogen
and 2-methyl substituted analogues, interactions were mainly observed through the imidazole
ring nitrogen with active site residues Tyr121 and Tyr334. However, on increasing the carbon
chain length to ethyl and n-propyl showed no contacts. On substituting 2-position of imidazole
ring with heteroaryl furan group, depicted interaction with indole ring nitrogen of Trp279 while
phenyl ring substitution exhibited interaction with only one water molecule. Interestingly, on
substituting the 2-phenyl ring with various functional groups depicted increased interactions with
AChE active site residues. Now, instead of the imidazole ring nitrogen, the functional groups
were involved in the interactions. On substituting 2-phenyl ring with electron withdrawing group
such as nitro displayed less number of hydrogen bonds (Tyr70, Asp72 and Tyr121) whilst,
electron donating groups such as hydroxy and methoxy groups not only increased the number of
interactions but also exhibited hydrogen bonds with catalytic triad (especially Ser200) an
important active site residue for enzymatic machinery. The halogens (-Cl, -F) and amino group
substitution on 2-phenyl ring of imidazole displayed no contacts. A better insight of effect of
substitution pattern on number of interactions was observed in case of methoxy substituted

analogues. Trisubstituted methoxy derivative exhibited highest number of hydrogen bonds as
compared to disubstituted and monosubstituted methoxy analogues as well as compared with
other analogues in the series. The 2-phenyl ring trisubstituted methoxy analogue interacted with
Tyr70, Asp72, Trp84, Gly118, Gly119, Ser122, Tyr130 and Ser200 residues.
The analogues showing more interactions within AChE active site were remarkably
found to interact with one of the catalytic triad residue Ser200 like donepezil. Amongst them
notable observation was found for the methoxy substituted analogues. Specifically, the analogues
17, 18, 19 and 20 have shown increase in number of interactions with increase in number of
methoxy group substitutions. The phenyl ring of imidazole substituted with polarizable group
especially hydroxy has shown interaction with Ser200 which is important for enzymatic catalysis
(analogue 13). Thus, in the designed series, imidazole analogues possessing 2-phenyl ring
substituted with methoxy or hydroxy group can be considered as more promising AChE
inhibitors. The docking pose and hydrophobic interactions of representative analogue 20 have
been depicted in Figure 3a&b.

Figure 3 a&b: Docking pose of analogue 20 within AChE active site
3a: H-bond formation
3b: Hydrophobic interactions
2.2. In silico ADME studies
Drug kinetics and exposure of tissues to drug influences the pharmacological activity and
the performance of a drug candidate, which is eventually determined by its ADME properties.
The ultimate objective of the in silico ADME studies is to accurately predict in vivo
pharmacokinetics of a possible and potential drug molecule.31 Results of in silico ADME
descriptors calculated for the 2-substituted-4,5-diphenyl-1H-imidazole analogues using QikProp
software of Schrodinger have been depicted in Table 1.
ADME results for CNS parameter seem to be favourable and indicate that all analogues
can be targeted for CNS activity. All the designed analogues were small in size with molecular
weight less than 400 g/mol, donor HB (hydrogen bond) upto 3 and acceptor HB in the range of
2-4. Solvent Accessible Surface Area (SASA) can be used as a size related parameter which
affects the partition co-efficient and aqueous solubility which was observed to be in the range of
400-800.32 The QPlogPo/w is a significant aspect to determine bioavailability and seems to be

favourable. Further, the predicted values for blood–brain barrier (BBB) penetration (QPlogBB)
of all analogues were in the optimum range (–3 to 1.2). The QPPMDCK model is considered to
be a good mimic for the blood-brain barrier. The QPPMDCK descriptor values were found to be
great for most of the analogues whilst analogue 11 and 15 had moderate values. The percent
human oral absorption was found to be more than 90% for all analogues. None of these
imidazole analogues violated the Lipinski’s rule of five, thus interpreting these analogues as
suitable drug candidates. Overall, in silico ADME results indicate that all analogues can be
thought to be CNS active and the descriptor values were found to be in the QikProp specified
ranges so these analogues seem to possess favourable pharmacokinetic properties.33
Table 1: In Silico ADME results of 2-substituted-4,5-diphenyl-1H-imidazole analogues
% Human
Oral
Absorption
Rule
Of
Five
donor
HB
accpt
HB
QPlog
Po/w
QPlog
BB
MWa
No
R
CNS
SASA
QPPMDCK
H
1
1
1
1
1
220.27
234.30
248.33
262.35
296.37
1
1
1
1
1
1.5
1.5
1.5
1.5
1.5
465.52
502.99
535.29
560.43
593.10
3.66
4.12
4.56
4.93
5.59
0.21
0.30
0.30
0.34
0.41
2284.05
2982.82
3539.05
3952.59
4114.49
100
100
100
100
100
0
0
0
0
1
3
4
5
6
7
CH3
CH2CH3
CH2CH2CH3
(Continued on next page)

1
1
286.33
398.51
1
1
2
555.90
771.53
4.92
7.96
0.36
0.21
3557.92
4113.17
100
100
0
1
8
9
1.5
0
0
0
341.37
341.37
312.37
1
1
2
2.5
2.5
627.57
631.08
605.31
5.02
4.89
4.87
-0.46
-0.63
-0.06
618.42
415.02
1540.05
100
100
100
1
0
0
10
11
12
2.25
0
0
312.37
312.37
328.37
2
2
3
2.25
2.25
3
605.54
605.52
616.49
4.77
4.77
4.02
-0.18
-0.19
-0.72
1135.48
1131.78
377.29
100
100
100
0
0
0
13
14
15
-1
0
372.42
2
3.75
674.38
5.15
-0.12
1875.21
100
1
16
1
1
1
326.40
326.40
356.42
1
1
1
2.25
2.25
3
630.10
629.90
673.10
5.69
5.70
5.88
0.35
0.35
0.28
4117.04
4112.43
4114.65
100
100
100
1
1
1
17
18
19
1
386.46
1
3.75
698.17
5.93
0.22
4108.68
100
1
20
21
2
2
330.82
330.82
1
1
1.5
1.5
604.92
617.18
5.94
6.09
0.50
0.59
7236.91
10000
100
100
1
1
22
23
2
1
314.36
339.44
1
1
1.5
2.5
602.15
670.64
5.83
6.04
0.53
0.33
7445.97
3961.24
100
100
1
1
24
a gm/mol

2.2.1 Correlation of in silico ADME studies with docking results
Molecular docking results have been found to be in consensus with the predicted in silico
ADME data. It shows that the analogues substituted with polarizable groups such as hydroxy,
methoxy on 2-phenyl ring have displayed maximum number of interactions and analogously
favourable CNS parameters and high human oral absorption. These analogues were subjected to
synthesis and further studies.
2.3 Synthesis
Based on the outcome of the computational studies from the series consisting of 24
34
analogues, 10 compounds were further synthesized using cerric ammonium nitrate as catalyst
(Scheme 1). The synthesized and purified analogues were characterized by spectral techniques:
1
13
IR, HNMR, CNMR and Mass spectroscopy. Purity of analogues was estimated using HPLC
technique. The IR spectra of the synthesized analogues depicted expected absorption bands for
the functional groups. The formation of imidazole ring was revealed by the presence of singlet
near 12 ppm and also exhibited predictable delta values for all other aliphatic and aromatic
1
protons in HNMR spectra. Thus, the spectroscopic techniques confirmed the correctness of
anticipated structures of the synthesized analogues.
Scheme1b: General reaction for synthesis of 2-substituted-4,5-diphenyl-1H-imidazole
b Reagents: (a) Cerric Ammonium nitrate (10 mol%); (b) Ammonium acetate (40 mmol); Ethanol-water reflux(24-48 hrs.)

2.4 Pharmacological evaluation
2.4.1 In vitro lipid peroxidation (LPO) assay
Oxidative stress and Aβ-accumulation leads to formation of lipid peroxidation product
malondialdehyde. Hence, anti-oxidant activity was determined by carrying out in vitro lipid
peroxidation (LPO) assay.35, 36 For the comparison of in vitro LPO assay results, ascorbic acid
was considered as the reference compound. The IC50 values for antioxidant activity of all the
synthesized imidazole analogues were estimated and found to be in the range of 59-264 μM
(Table 2). Amongst the series, analogue 15 exhibited maximum antioxidant activity followed by
analogue 20 and 13. These analogues possessed primarily hydroxy and methoxy groups as
substituents in the 2-phenyl ring. From the structure-activity relationship (SAR) point of view it
can be proposed that as analogue 15 possesses more hydroxyl groups so it can hinder the
oxidation process more effectively than the corresponding monohydroxy as well as methoxy
analogues. Graphs of the LPO assay results of reference compound and representative analogue
13 are depicted in Figure 4. Thus, overall in vitro LPO assay results showcase the
neuroprotective role of these analogues.
Table 2: In vitro LPO assay results
Analogues
IC50(µM) c
93.24
106.05
100.84
61.82
62.37
59.42
264.31
86.46
94.01
61.29
38.77
3
7
12
13
14
15
16
17
19
20
Ascorbic acid
c Values are the mean values of at least two experiments

Figure 4: LPO inhibitory assay for ascorbic acid (a) and analogue 13 (b)
a: Ascorbic acid b: Analogue 13
70
60
50
40
30
20
10
0
100
80
60
40
20
0
y = 0.510x + 18.47
R² = 0.953
y = 1.133x + 6.069
R² = 0.980
%
%
inhibition
inhibition
0
20
40
60
0
50
100
150
200
Concentration (µM)
Concentration (µM)
2.4.1.1 Correlation of in vitro LPO assay and computational studies
Comparison between docking and in vitro LPO assay results highlighted analogues 13,
14, 15, 19 and 20 in the series. These analogues exhibited good number of hydrogen bond and
hydrophobic interactions within AChE active site. Amongst these analogues; 13, 19 and
20interacted with one of the residues of catalytic triad (Ser200) of AChE which plays a
fundamental role in enzymatic catalysis and inhibitor binding. The IC50 value for analogue 19
was no doubt higher than the above highlighted ones but it depicted interaction with crucial
catalytic triad residue of AChE enzyme. The LPO assay result of analogue 19 was comparable to
analogues 3 and 7 but they did not exhibit good docking results as observed with analogue 19.
The in silico ADME results were favourable but QPPMDCK value of analogue 15 was not found
to be great. Collating all inputs analogue 13 and 20 were selected for further studies.
2.4.2 Acute toxicity studies
Oral administration of the representative test analogue 20 at the dose of 800 mg/kg did
not reveal any signs of toxicity, behavioral changes or mortality in a group of 6 animals (3 male
and 3 female Swiss albino mice) for 14 consecutive days, so the dose was considered to be safe
and non-lethal. Various parameters studied for toxicological observations of the test analogue 20

were awareness, mood, CNS excitation, CNS depression and autonomic reflexes. Further, on
increasing the dose of analogue 20 to 900mg/kg resulted in 50% mortality in the group of six
animals.
2.4.3 Memory model studies
The in silico ADME results indicated that all designed analogues of the 2-substituted-4,5-
diphenyl-1H-imidazole series can be thought to be CNS active. To authenticate this finding,
memory model studies were undertaken before venturing into the detailed mechanistic in vitro
cholinesterase inhibition study.
2.4.3.1 Elevated Plus Maze (EPM) model
This model was used to evaluate memory and learning in Swiss albino mice. Decrease in
transfer latency (TL) in seconds was observed for test analogues 20 (Test A) and 13 (Test B) as
compared to the toxicant group (scopolamine hydrobromide) which conveys us that both the
analogues were able to retain the memory of mice to travel in one of the closed arms of maze.
Results of EPM demonstrate that both the analogues helped successfully to retain the memory in
mice with values of analogue 20 close to the reference drug Piracetam (Figure 5).
Figure 5: Effect of test analogues on Transfer Latency

2.4.3.2 Step down inhibitory avoidance test
Animals administered with test analogues 20 (Test A) and 13 (Test B) took more time to
step down from the block as compared with toxicant which indicates memory retention in them.
Results of step down inhibitory avoidance model ascertain that both the analogues helped
effectively to retain the memory and learning in mice very much close to the standard Piracetam
drug whilst very much distant from the toxicant scopolamine results (Figure 6).
Figure 6: Effect of test analogues on Step Down Latency
Data of EPM and step down inhibitory avoidance model were analyzed by one-way
ANOVA. A significant statistical difference was found between control and test groups (p value
> 0.05). So, Dunett multiple comparison tests as post-test was applied to the data. Thus, both the
analogues 13 and 20 exhibited effective memory retention in the animals and can be considered
as promising CNS target candidates.
2.4.4 In vitro cholinesterase inhibition
A total of ten synthesized 2-substituted-4,5-diphenyl-1H-imidazole analogues were
screened at 10µM concentration using Ellman’s method to test their effectiveness in
cholinesterase inhibition (AChE and BuChE). Thereafter, IC50 values of analogues which

exhibited more than 50% inhibition at 10µM concentration were estimated and compared with
reference drugs Donepezil and Galantamine for AChE and BuChE respectively (Table 3).
Previous results from computational and in vitro pharmacological evaluation studies furnished
five promising imidazole analogues (13, 14, 15, 19 and 20). These are mainly analogues which
possess 2-phenyl ring substituted with either methoxy or hydroxy group. This indicates that
electronic effect predominates in the SAR of the series. Analogue 13 and 19 were found to be the
most potent inhibitors for AChE in the series. In comparison, analogue 13 was deemed a better
candidate as analogue 19 had poor aqueous solubility. For BuChE inhibition assay, the results
demonstrated that multiple substitutions on the 2-phenyl ring gave better BuChE inhibitory
activity as compared to mono substitution, with analogue 20 to be the most potent one (Table 4).
Table 3: AChE and BuChE inhibition results
% AChE inhibition ± SD
at 10µM d
% BuChE inhibition ±
SD at 10µM d
Analogues
39.70 ± 3.44
13.01 ± 1.99
3
16.08 ± 2.52
15.08 ± 2.60
65.08 ± 4.34
6.53e
N.I.
13.70 ± 1.82
N.I.
7
12
13
N.I.
14
N.I
79.45 ± 6.60
N.I.
15
29.65 ± 2.01
23.62 ± 3.45
67.33e
16
19.18 ± 3.66
N.I.
17
19
N.I.
81.28 ± 6.85
N.D.
20
91.07 ± 8.23
N.D.
Donepezil
Galantamine
80.77 ±8.01
d Experiments were carried out in quadruplicate and repeated at least twice; e Precipitation was
observed at 10 µM (1% DMSO), N.I. No inhibition, N.D. Not determined

Table 4: IC50 values of the most potent AChE and BuChE inhibitor.
AChE inhibition
BuChE inhibition
Analogues
IC50 (µM) f
IC50(µM) f
13
20
5.33
N.I.
N.I.
0.1
4.99
N.D.
3.31
Donepezil
Galantamine
1.67
f Experiments were carried out in quadruplicate and repeated at least twice
N.I. No inhibition, N.D. Not determined
2.4.5 Structure activity relationship
Imidazole scaffold is the core moiety of the designed series. The improvements in the
activity can be achieved by modifications on the imidazole nucleus. Incorporation of phenyl
rings may provide additional hydrophobic sites for interaction between inhibitor and the enzyme.
Hence, phenyl rings were substituted at 4th and 5th position of imidazole ring. Structure activity
relationship analysis reveals that AChE inhibition potency is closely related to the substitution at
2-position of imidazole core. Analogues with various substituents such as electron-withdrawing,
electron-donating were considered. Altering substituents at the 2-position of imidazolyl nucleus
from simple alkyl groups such as methyl, ethyl to aryl and heteroaryl led to varying
consequences on binding and inhibitory activity. Substituting hydrogen and methyl group at 2-
position, showed lesser docking interactions and gave comparable LPO assay results. However,
on increasing the carbon chain length to ethyl and n-propyl showed no contacts within AChE
active site. Also substituting aryl ring with halogens (-Cl, -F) and amino group did not furnish
noteworthy docking results.

Interestingly, on substituting the 2-phenyl ring with various polarisable groups depicted
increased interactions with AChE active site residues. Substituting electron-donating hydroxy
group at 2nd/3rd/4th position of 2-phenyl ring (analogue 12, 13 and 14) displayed good
interactions as compared to electron-withdrawing nitro group (analogue 10, 11). Further,
incorporation of additional hydroxy group (analogue 15) exhibited maximum antioxidant
activity. Dihydroxy analogue can hinder the oxidation process more effectively as compared to
monohydroxy analogues and analogously exhibited better LPO assay results.
A better insight of effect of substitution pattern on number of interactions was observed
on inclusion of methoxy group at 2-phenyl ring. Trisubstituted methoxy analogues (analogue 20)
gave better docking results and antioxidant activity as compared to mono- and di-substituted
methoxy candidates. Monohydroxy analogues were found to have affinity towards AChE while
dihydroxy analogues selectively inhibited BuChE. It was interestingly noted that increasing the
number of substitutions led to selectivity and affinity towards BuChE. Accordingly, the
trisubstituted analogue 20 exhibited maximum BuChE inhibitory activity. Analogue 13 and 19
were found to be the most potent inhibitors for AChE in the series. Those analogues possessing
primarily hydroxy and methoxy groups as substituents in the 2-phenyl ring can be considered as
potential lead for further optimization and development of more promising imidazole analogues
with multiple roles of cholinesterase inhibition, antioxidant effect and neuroprotection.
3. Conclusion
In the designed 2-substituted-4,5-diphenyl-1H-imidazole series, the analogues chosen for
synthesis exhibited more number of interactions with AChE active site residues, interacted with
the catalytic triad and PAS; significant for enzymatic catalysis, showed favourable
pharmacokinetic properties, depicted neuroprotective effect against oxidative stress with

retention of memory in memory model studies as well as inhibited the AChE enzyme at 10 µM
concentration. Thus, it can be foreseen that the synthesized 2-substituted-4,5-diphenyl-1H-
imidazole analogues can potentially increase memory, decrease free radical levels and protect
neurons against cognitive deficit. Therefore, amongst the series, analogue 13 can be considered
as potential lead for further optimization and development of more promising imidazole
analogues with multiple roles of AChE inhibition, antioxidant and neuroprotection. Further,
analogue 20 can be explored for search of newer BuChE inhibitors. This biological profile
highlights the importance of these analogues as promising multi-targeted prototypes in the search
of new protective and regenerative drugs for the potential treatment of Alzheimer’s disease.
4. Materials and Method
4.1. Molecular Docking studies with AChE enzyme
4.1.1. Materials
Sybyl version 8.1.1 (Tripos International, Portugal) running on Red Hat Enterprise Linux
(RHEL) workstation was utilized for ligand preparation and its Biopolymer module for protein
preparation.37 GOLD version 5.2.2 (CCDC Ltd., UK) running on RHEL workstation was
employed to carry out docking studies.38
4.1.2. Structure based designing of ligands
All the structures of reported AChE inhibitors and test ligands of imidazole series were
sketched and subjected to energy minimization using steepest descent (SD) and Powell methods
with TRIPOS force field to attain gradient convergence of 0.0001 kcal/mol/Å. Gasteiger-Hückel
partial atomic charges were assigned to all compounds.

4.1.3. Preparation of protein for docking
The x-ray crystal structure of enzyme AChE (PDB ID: 1EVE,resolved at 2.5Å)
complexed with the ligand Donepezil was retrieved from protein data bank for structure aided
drug design.12 The ligand donepezil was extracted along with undesired water molecules,
hydrogen atoms were added, atom types and partial charges were assigned. Based on site points
of the complexed ligand, active site of protein was defined and confirmed from literature. The
active site was defined as a sphere with a radius of 7Å from the bound ligand which was found to
be equivalent to 10Å in GOLD. 20 key structural water molecules were retained in the active
site. The side chains were subjected to minimization using AMBER7FF99 force field toa
gradient of 0.001 kcal/mol/Å.12, 38
4.1.4. Docking protocol
The refined protein, minimized reported ligands and imidazole analogues were imported
into GOLD. The default parameters in GOLD v5.2.2 were utilized for docking studies. Docking
was carried out for 20 genetic algorithm (GA) runs with 1,00,000 operations and GoldScore as
the fitness function. The docking protocol was validated by reproduction of the binding pose of
donepezil in 1EVE. Further, the protocol was substantiated by docking reported AChEIs within
the active site. The best ranked pose of ligands was studied to determine their binding as well as
interactions within the AChE active site.
4.2. In silico ADME studies
4.2.1. Materials
In silico ADME studies were carried out with QikProp version 3.3 (Schrodinger LLC,
New York, USA) software running on Red Hat Enterprise Linux (RHEL) workstation.33

4.2.2. Method
Each designed structure was optimized by LigPrep using OPLS-2005force field. Out file
of LigPrep was used as input file for QikProp to determine various pharmacokinetic descriptors.
QikProp jobs were monitored and results were obtained in the form of OUT file.
4.3 Synthesis
4.3.1 Materials
All chemicals and solvents used were obtained from commercial sources. The starting
materials were assessed for purity by physical constant determination and thin layer
chromatography (TLC) on Merck silica gel F254 plates. TLC was employed to monitor all
reactions. Physical constants were determined in Analab melting point apparatus μThermoCal10.
1
Infrared (IR) spectra were recorded on Shimadzu IR 8400S. HNMR, 13CNMR spectra’s in
DMSO (Dimethyl sulfoxide) solvent were recorded on Mercury Plus 300 MHz (Varian, USA)
NMR Spectrometer. Chemical shifts have been reported in parts per million (ppm) using
tetramethylsilane (TMS) as an internal standard. Mass spectra were obtained using 410 Prostar
Binary LC with 500 MS IT PDA detectors (Varian Inc, USA) mass spectrometer. Percent purity
of samples was obtained using HPLC technique by Agilent 1200 Series HPLC System.
4.3.2 General procedure
A mixture of benzoin (25, 10 mmol), cerric ammonium nitrate (10 mol %) and ammonium
acetate (40 mmol) was dissolved in ethanol-water (20 ml, 1:1 v/v) and substituted aldehyde (26,
10 mmol) was added to reaction mixture. This was subjected to reflux (75-80 oC) till completion
of the reaction as indicated by TLC. The reaction mixture was allowed to cool to room
temperature and then poured on ice-water to precipitate the solid compound. The crude product

was filtered and washed with water to free from impurities. It was purified by dissolving in
dimethyl sulfoxide and the solution was poured on ice with stirring to precipitate the desired
product. Further, purification was done by recrystallisation using ethanol or column
chromatography (n-hexane: Ethyl acetate, 7: 3). All synthesized analogues were characterized
1
using IR, HNMR, 13C NMR and Mass spectroscopy. Purity of compounds was assessed by
HPLC technique. HPLC was performed using Inertsil-C18 reverse phase column (0⋅46 × 25 cm)
on instrument equipped with an automatic injector with UV-PDA detector. The mobile phase
consists of 0⋅05% TFA and acetonitrile (1:1, v/v). Products were eluted at flow rate of 1 ml/min
using isocratic method. Detection was carried out at 254 nm. The melting points were also
recorded. The plausible mechanism has been depicted in Scheme 2.
Scheme 2: Plausible reaction mechanism for synthesis of 2-substituted-4,5-diphenyl-1H-
imidazole analogues

4.3.3 Spectral and analytical data
1. 4,5-diphenyl-1H-imidazole (3): White solid, Yield: 97.08%; 226.4OC, HPLC purity: 98.69%.
1
FTIR (cm−1): 3447 (NH), 3134 (C-H), 1493(C=C), 1400 (C-N), 1630 (C=N); H NMR (300
MHz, DMSO-d6): δ 12.770 (s, 1H), 8.252 (s, 1H), 7.744-7.762 (d, 4H, J=6.4 Hz), 7.353-7.330
(d, 4H, J=6.690 Hz), 7.465-7.404 (d, 2H, J=8.32 Hz); ESI-MS (acetone) [M+1]: 221.3
2. 2,4,5-triphenyl-1H-imidazole (7): Off-white solid, Yield: 79.13%; 272.3OC, HPLC purity:
1
98.45%. FTIR (cm−1): 3445 (NH), 3082 (C-H), 1462(C=C), 1386 (C-N), 1641 (C=N); H NMR
(300 MHz, DMSO-d6): δ 12.758 (s, 1H), 7.929-7.903 (d, 2H, J=7.7 Hz), 7.822-7.774 (t, 2H,
J=7.3 Hz), 7.376-7.354 (d, 1H, J=6.6 Hz), 7.583-7.536 (d, 4H, J=7.0 Hz), 7.656-7.605 (t, 4H,
J=7.7 Hz), 7.519-7.511 (d, 2H, J=2.6 Hz); 13C NMR (300 MHz, DMSO-d6): δ 194.898, 135.611,
132.289, 129.646, 129.570, 129.273, 129.081, 128.813, 128.355, 127.741, 126.525, 126.180;
ESI-MS (acetone) [M+1]: 297.4
3. 2-(2-hydroxyphenyl)-4,5-diphenyl-1H-imidazole (12): Yellowish solid, Yield: 69.38%;
206.8OC, HPLC purity: 99.34%. FTIR (cm−1): 3416 (NH), 3175 (OH), 2926 (C-H), 1449 (C=C),
1
1398 (C-N), 1620 (C=N); H NMR (300 MHz, DMSO-d6): δ 12.651 (s, 1H), 10.268 (s, 1H),
7.802-7.776 (dd, 1H, J=2.3 Hz), 7.397-7.356 (t, 1H, J=6.2 Hz), 7.345-7.305 (t, 1H, J=5.8 Hz),
7.280-7.195 (dd, 1H, J=8.9 Hz), 7.979-7.935 (d, 2H, J=13.1 Hz), 7.910-7.905 (d, 2H, J=1.4 Hz),
7.632-7.607 (t, 2H, J=3.6 Hz), 7.521-7.484 (t, 2H, J=5.4 Hz), 7.600-7.545 (t, 1H, J=3.6 Hz),
13
7.472-7.424 (t, 1H, J=6.7 Hz); C NMR (300 MHz, DMSO-d6): δ 194.851, 143.424, 138.024,
136.904, 135.564, 135.114, 132.251, 131.542, 130.230, 130.202, 129.666, 129.608, 128.766,
128.220, 127.205; ESI-MS (acetone) [M+1]: 313.3

4. 2-(3-hydroxyphenyl)-4,5-diphenyl-1H-imidazole (13): Brownish solid, Yield: 91.15%;
215.2OC, HPLC purity: 97.81%. FTIR (cm−1): 3441 (NH), 3132 (OH), 3233 (C-H), 1493 (C=C),
1
1402 (C-N), 1609 (C=N); H NMR (300 MHz, DMSO-d6): δ 12.758 (s, 1H), 8.357 (s, 1H),
7.455 (s, 1H), 7.430-7.404 (d, 1H, J=8.5 Hz), 7.374-7.348 (d, 1H, J=7.7 Hz), 7.159-7.137 (d, 1H,
13
J=6.6 Hz), 7.298-7.260 (d, 6H, J=8.7 Hz), 7.244-7.195 (t, 4H, J=7.5 Hz); C NMR (300 MHz,
DMSO-d6): δ 177.24, 129.484, 128.354, 128.181; ESI-MS (acetone) [M+1]: 313.4
5. 2-(4-hydroxyphenyl)-4,5-diphenyl-1H-imidazole (14): Brown solid, Yield: 72.10%;
261.4OC, HPLC purity: 97.44%. FTIR (cm−1): 3449 (NH), 3179 (OH), 2857 (C-H), 1491 (C=C),
1
1398 (C-N), 1609 (C=N); H NMR (300 MHz, DMSO-d6): δ 12.429 (s, 1H), 9.755 (s, 1H),
7.782-7.736 (dt, 2H, 8.2 Hz), 6.938-6.909 (d, 2H, J=8.4 Hz), 7.934-7.905 (dd, 4H, J=1.0 Hz),
7.822-7.802 (d, 2H, J=6.2 Hz ), 7.659-7.607 (t, 4H, J=7.7 Hz); 13C NMR (300 MHz, DMSO-d6):
δ 194.822, 135.535, 132.241, 129.589, 129.503; ESI-MS (acetone) [M+1]: 313.4
6. 2-(3,4-dihydroxyphenyl)-4,5-diphenyl-1H-imidazole (15): Dark brown solid, Yield:
68.83%; 226.8OC, HPLC purity: 99.25%. FTIR (cm−1): 3448 (NH), 3132 (OH), 3032 (C-H),
1628 (C=C), 1496 (C-N), 1628 (C=N); 1H NMR (300 MHz, DMSO-d6): δ 10.166 (s, 1H), 9.689
(s, 1H), 9.607 (s, 1H), 7.287-7.254 (dd, 1H, 4.5 Hz), 7.231-7.224 (d, 1H, 2.2 Hz), 6.916-6.889 (d,
1H, 8.0 Hz), 7.934-7.925(d, 2H, 7.0 Hz), 7.910-7.905 (d, 2H, 7.0 Hz), 7.826-7.781 (t, 2H, 6.8
13
Hz), 7.657-7.607 (t, 2H, 7.5 Hz), 7.422-7.375 (dd, 2H, 6.9 Hz); C NMR (300 MHz, DMSO-
d6): δ 194.860, 146.910, 145.895, 145.521, 135.564, 132.270, 129.618, 129.531, 128.584,
128.095, 117.592, 115.744, 113.542; ESI-MS (acetone) [M+1]: 329.4
7. 2-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-diphenyl-1H-imidazole (16): White solid, Yield:
72%; 225.8OC, HPLC purity: 98.69%. FTIR (cm−1): 3418 (NH), 3130 (OH), 1203, 1070 (C-O-

1
C), 2926 (C-H), 1485 (C=C), 1402 (C-N), 1605 (C=N); H NMR (300 MHz, DMSO-d6): δ
10.021 (s, 1H), 8.529 (s, 1H), 3.814 (s, 6H), 7.403-7.401 (d, 2H, 0.7 Hz), 7.609-7.551 (t, 2H, 8.6
Hz), 7.524-7.512 (d, 2H, 3.7 Hz), 7.503 (s, 2H), 7.287-7.277 (d, 2H, 2.9 Hz), 7.269-7.247 (t, 2H,
3.2 Hz); 13C NMR (300 MHz, DMSO-d6): δ 198.247, 198.055, 183.578, 176.972, 167.349,
166.660, 153.102, 152.489, 140.492, 138.864, 137.983, 137.926, 129.883, 129.672, 124.262,
124.071, 123.496, 122.616, 118.786, 118.039, 26.756, 13.878, 13.686; ESI-MS (acetone) [M+]:
372.4.
8. 2-(3-methoxyphenyl)-4,5-diphenyl-1H-imidazole (17): Yellow solid, Yield: 73.20%;
235.6OC, HPLC purity: 99.98%. FTIR (cm−1): 3439 (NH), 1247,1030 (C-O-C), 2985 (C-H),
1491 (C=C), 1402 (C-N), 1612 (C=N); 1H NMR (300 MHz, DMSO-d6): δ 12.677 (s, 1H), 2.386
(s, 3H), 7.375 (s, 1H), 7.821 (bs, 1H), 7.797-7.772 (d, 2H, 7.3 Hz), 7.927-7.902 (d, 6H, 7.7 Hz),
7.655-7.604 (t, 4H, 7.7 Hz); 13C NMR (300 MHz, DMSO-d6): δ 176.113, 152.386, 148.116,
148.068, 140.619, 139.064, 130.240, 130.087, 125.156, 124.688, 117.659, 116.720, 113.044,
112.527, 18.503; ESI-MS (acetone) [M+]: 326.7
9. 2-(3,4-dimethoxyphenyl)-4,5-diphenyl-1H-imidazole (19): Off-white, Yield: 79.04%;
217.2OC, HPLC purity: 98.88%. FTIR (cm−1): 3418 (NH), 3132 (C-H), 1254, 1024 (C-O-C),
1497 (C=C), 1445 (C-N), 1607 (C=N); 1H NMR (300 MHz, DMSO-d6): δ 12.603 (s, 1H), 3.860
(s, 3H), 3.820 (s, 3H), 7.572-7.566 (d, 1H, 1.8 Hz), 7.545-7.539 (d, 1H, 1.8 Hz), 7.384-7.378 (d,
6H, 1.8 Hz), 7.180 (s, 4H), 7.153(s, 1H); 13C NMR (300 MHz, DMSO-d6): δ 175.577, 129.618,
128.967, 128.316; ESI-MS (acetone) [M+1]: 357.4
10. 2-(3,4,5-trimethoxyphenyl)-4,5-diphenyl-1H-imidazole (20): White amorphous powder,
Yield: 75.82%; 285.2OC, HPLC purity: 98.89%. FTIR (cm−1): 2960 (NH), 1128, 1006 (C-O-C),

1
2934 (C-H), 1493 (C=C), 1464 (C-N), 1587 (C=N); H NMR (300 MHz, DMSO-d6): δ 11.884
(s, 1H), 3.863 (s, 3H), 3.853 (s, 6H), 7.250 (s, 2H), 7.546-7.522 (d, 2H, 7.3 Hz), 7.497-7.475 (d,
4H, 6.6 Hz), 7.403 (s, 4H); 13C NMR (300 MHz, DMSO-d6): δ 153.162, 145.464, 137.708,
136.933, 135.104, 131.198, 129.599, 128.727, 128.526, 128.181, 127.071, 126.506, 125.864;
ESI-MS (acetone) [M+1]: 387.4
4.4 Pharmacological evaluation
4.4.1 In vitro lipid peroxidation (LPO) assay
4.4.1.1 Materials
All chemicals and solvents used were obtained from commercial sources. The test
solutions were centrifuged using Remi centrifuge (R-4C DX). Absorbances were recorded on
Thermoscientific UV visible spectrophotometer (Thermosci. Evolution 300).
4.4.1.2 Procedure
The malondialdehyde content; a measure of lipid peroxidation, was assayed in the form
of thiobarbituric acid-reactive substances by the method of Wills. Briefly, 0.2 ml brain tissue
homogenate, 0.1 ml test sample at five different concentrations, 0.2 ml of 8.1% sodium dodecyl
sulfate, 1.5 ml of 20% acetic acid and 1.5 ml of 8% 2-Thiobarbituric acid (TBA) were taken
together and final volume of the mixture was adjusted to 4 ml with distilled water and then
heated at 95°C for 60 minutes. Then the tubes were cooled to room temperature and final volume
was made to 5 ml in each tube using distilled water. 5 ml of butanol: pyridine (15:1) mixture was
added to extract the contents and was centrifuged at 4000rpm for 10 minutes. After
centrifugation, the upper organic layer was collected and its absorbance was recorded at 532 nm

against an appropriate blank. The above mentioned procedure was followed in duplicate for
ascorbic acid (reference standard) and all the test analogues.35
Percentage inhibition of TBA reacting substances (TBARS) formation was calculated
with respect to control. The inhibition of lipid peroxidation was determined by calculating the
percent (%) decrease in the formation of TBARS.
Percentage inhibition = (Abscontrol – Abssample) X 100/Abscontrol
4.4.2 Acute toxicity studies
4.4.2.1. Animals
Swiss albino mice weighing 20-25 g of both male and female sex were procured
(Bombay Veterinary College, Mumbai) for this study. The animals were housed in group of six
in clean plastic cages andmaintained under standard laboratory conditions with temperature
(22±2) C, relative humidity (60±10)%, natural light and dark (12:12 hour) cycle with free access
to food and water. Animals were acclimatized to laboratory conditions for 15 days before the
test. The experimental protocols were approved by the Institutional Animal Ethics Committee
(Protocol number: KMKCP/IAEC/161710).
4.4.2.2 Method
Acute toxicity studies were performed on 6 albino mice (3 males+3 females per group) at
initial dose of 900 mg/kg p.o. and subsequently thereafter with 800 mg/kg p.o. to study
morbidity and mortality, if any, as per OECD-423 guidelines. The test substance was formulated
as suspension in an aqueous vehicle using 1% w/v of sodium carboxymethylcellulose as
suspending agent. The mice were critically observed for clinical signs, gross behavioral changes
and mortality; if any, following administration of the test formulation at different time intervals