Design, synthesis, and evaluation of some novel biphenyl imidazole derivatives for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.molstruc.2021.131152

The multi-targeted organized strategy provides effectual and significant results to cure progressive neurodegenerative diseases like AD. Treatment for this awful disease is in infancy till now. A series of novel 4, 5-diphenyl-1H-imidazole linked piperazin

Full text of DOI:10.1016/j.molstruc.2021.131152

Journal of Molecular Structure 1246 (2021) 131152
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Design, synthesis, and evaluation of some novel biphenyl imidazole
derivatives for the treatment of Alzheimer’s disease
Salunke Prashant Ramrao, Akash Verma, Digambar Kumar Waiker, Prabhash Nath Tripathi,
∗
Sushant Kumar Shrivastava
Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutical Engineering & Technology, Indian Institute of Technology (Banaras Hindu
University), Varanasi, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The multi-targeted organized strategy provides effectual and significant results to cure progressive neu-
rodegenerative diseases like AD. Treatment for this awful disease is in infancy till now. A series of novel 4,
Received 21 March 2021
Revised 17 July 2021
Accepted 19 July 2021
Available online 21 July 2021
5
-diphenyl-1H-imidazole linked piperazine hybrids (6a-g and 7a-g) were synthesized by utilizing the con-
cept of bio-isosteric replacement and hybrid pharmacophore approaches. The targeted compounds were
identified by spectroscopic techniques and assessed for in vitro cholinesterase, BACE-1 inhibition, propid-
ium iodide displacement, and Aβ disaggregation activities. Within the targeted inhibitors, 6f exhibited the
most significant and inhibitory capacity against eeAChE (IC50 = 0.416 ± 0.018 μM), eqBChE (IC50 = 0.474
± 0.054 μM), and BACE-1 (IC = 0.392 ± 0.021 μM) targets. Propidium iodide displacement showed that
Keywords:
Alzheimer’s disease
Aβ aggregation
50
β-Secretase-1 (BACE-1)
Acetylcholinesterase (AChE)
Multi-functional agents
Bio-isosteric
compound 6f could be effectively linked with AChE’s PAS (10 μM = 29.09 %, 50 μM = 41.43%). It showed
inhibitory potential against both self (21.28 to 30.85%) and AChE-induced (25.70 to 51.07%) Aβ aggrega-
tion in thioflavin T assay. Moreover, the most active compound 6f was evaluated by performing in vivo
behavioural studies with the help of the scopolamine-induced Y-maze model. The in vitro and in vivo in
collaboration with in silico molecular docking and molecular dynamics simulation leads to ascertain the
active site interaction of compound 6f with AChE and BACE-1.
©
2021 Elsevier B.V. All rights reserved.
Abbreviations
ACN
Acetonitrile
AD
Aβ
Alzheimer’s Disease
Amyloid-beta
K2CO3
DTNB
ATCI
Potassium carbonate
5,5-dithio-bis-(2-nitrobenzoic acid)
Acetylthiocholine iodide
NMDAR N-methyl D-aspartate receptor
CREB
ChE
cAMP response element-binding protein
Cholinesterase
BTCI
Butyrylthiocholine iodide.
eeAChE Electrical eel Acetylcholinesterase
1. Introduction
eqBChE Equine serum Butrylcholinesterase
BACE-1 Beta-site amyloid precursor protein cleaving enzyme-1
Alzheimer’s disease (AD) is considered a complex progressive
APP
Amyloid precursor protein
neurodegenerative disease, and it is identified by the gradual de-
cline of cholinergic neuronal cells and accumulation of amyloid-
beta (Aβ) proteins in the brain area like the cortex and hippocam-
pus. The progression starts with short-term memory impairment
that gradually progresses to complete loss of cognitive function,
weak performance, loss of logical thoughts, and ultimately death
[1]. Due to its complex nature, it may lead to the cause of both
emotional as well as financial load. One of its atrocious and crit-
ical aspects is the deprivation of cognitive capabilities that re-
cently calculate for more than 50 million cases globally and it is
expected that this number will cross more than triple by 2050
[2]. The preponderance of 10 – 30% and incidence of 1 – 3 % of
the AD belongs to the population having age more than 65 years
AChEIs Acetylcholinesterase inhibitors
MDLs
CAS
PAS
PI
Multitarget-directed ligands
Catalytic active site
Peripheral anionic site
Propidium iodide
Rf
Retention factor
MD
Molecular dynamics
RMSD
RMSF
Root mean square deviations
Root mean square fluctuations
∗
Corresponding author.
E-mail address: skshrivastava.phe@itbhu.ac.in (S.K. Shrivastava).
https://doi.org/10.1016/j.molstruc.2021.131152
0
022-2860/© 2021 Elsevier B.V. All rights reserved.

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
which represents the inexorable effect of aging [3]. AD is corre-
lated with various pathophysiological hallmarks like declination in
acetylcholine (ACh) levels [4], assembling of Aβ [5], N-methyl-D-
aspartate receptor (NMDAR) hyper-activation [6], tau protein hy-
perphosphorylation and assembling of neurofibrillary tangles [7],
inflammation of CNS and neuronal cells [8], gene transcription by
CREB (cAMP response element-binding protein) signaling and ox-
idative stress [9]. Cholinesterase (ChE) is considered as a serine hy-
drolytic enzyme including acetylcholinesterase (AChE) and butyryl-
cholinesterase (BChE) that help in the progression of AD by rapidly
hydrolyzing the ACh that results in the termination of signaling
at the cholinergic synaptic cleft. However, specific cholinesterase
inhibition by currently available drugs provides only symptomatic
relief [10]. AChE is also involved in the formation of Aβ aggre-
gates in Alzheimer’s disease. [11]. β-secretase-1 is also considered
as a beta-site amyloid precursor protein cleaving enzyme-1 (BACE-
Based on these prior studies and facts, a classic bio-isosteric bi-
valent approach for the design of new hybrid molecules was in-
troduced and it was planned to connect the 4, 5- diphenyl-1H-
imidazole ring and substituted piperazine with different lengths
of alkyl linkers to seek a novel 4, 5-diphenyl-1H-imidazole linked
piperazine hybrids. It was considered that the 4, 5 diphenyl-1H-
imidazole core of the constructed hybrids would bind with the pe-
ripheral anionic site (PAS) of AChE and the piperazine fragment
would interact with the CAS region of AChE. Thus, the designed 4,
5-diphenyl-1H-imidazole linked piperazine hybrids could demon-
strate multi-target-directed AD management potential (Fig. 1).
The proposed compounds were synthesized, identified, and bi-
ologically assessed against ChE and BACE-1 targets. The binding
ability of active compounds with PAS was achieved by propidium
iodide (PI) displacement assay. For their anti-Aβ activity, active
compounds were also evaluated in self and AChE-induced exper-
iments with the help of thioflavin T assay. Most promising com-
pounds were evaluated for their capability to cognitive impairment
in scopolamine-induced AD rat models. Molecular docking studies
were also performed in the most active compound.
1
) that disintegrates amyloid precursor protein (APP) and formed
amyloid peptides which are further accompanied by a combination
of oligomeric reactions and lead to the formation of oligomers, fib-
rils, and plaques [12]. All these reactions integrate and perform an
important role in the aggregation of Aβ and interrupt the various
cellular communications, inflammatory responses, neuronal abnor-
malities, behavior alterations, etc. [1314].
2. Results anddiscussion
Recently, no remedies are present for the cure of the
disease. FDA-approved medications such as acetylcholinesterase
inhibitors (AChEIs) (donepezil, rivastigmine, galantamine) and
NMDA-inhibitor (memantine) have been used in recent treatment
plans [15]. Moreover, treatment for the awful ailment in the de-
velopment phase till now. Recent findings are directed towards
a highly promising strategy that includes the evolution of multi-
targeted and multi-functional pharmacophore to cure this mul-
tifaceted disorder. Among several drug discovery strategies, the
multitarget-directed ligands (MDLs) strategy is the most promis-
ing recent approach as it involves targeting multiple enzymes re-
sponsible for AD at the same time by a single compound. Hence,
this MDLs approach provides not only symptomatic relief but also
proves appreciable in the mitigation of the disease progression
2.1. Chemistry and physicochemical characterization
The target compounds (6a-g and 7a-g) were prepared according
to reactions involved in the Scheme 1. Initially, compound 1 was
reacted with bromo chloro substituted alkanes along with the ad-
dition of dry potassium carbonate (K CO ) and acetonitrile (ACN)
2
3
gave 2 and 3 intermediates [32]. In 1H NMR spectra, compound 1
having singlet of (-OH) proton peak was disappeared and a triplet
signal of (-O-CH ) between was appeared in between 4.36 – 4.17
2
ppm. Also, 13C NMR spectra exhibited the characteristics signal of
(-O-CH ) and (-CH -Cl) at 67.96 – 64.62 ppm and 31.96 – 28.48
2
2
ppm, respectively, confirmed the formation of 2 and 3 intermedi-
ates. The obtained intermediates were further refluxed with ben-
zoin in presence of ammonium acetate, cerric ammonium nitrate,
and ethanol:water (1:1, v/v) to yield key intermediates 4 and 5
[33]. In 1H NMR spectra, the singlet peak of (-CH) proton of ben-
zaldehyde was disappeared and the appearance of singlet peak of
(-NH) proton of imidazole ring at 12.66 – 12.53 ppm and appear-
ance of multiplet of 10 biphenyl protons at 7.77 – 7.15 ppm con-
firmed the formation of 4 and 5 intermediates. Also in 13C NMR
spectra of 4 and 5 intermediates, the disappearance of carbonyl
(C=O) peak of benzaldehyde and appearance of resonance signal
[
16].
To design and develop the multi-functional molecular hybrid,
a literature survey of some recently reported compounds was uti-
lized. Literature study showed that advanced series with different
hetero-aromatic rings such as imidazole, benzimidazole, thiazole,
indanone, coumarin, piperidine derivatives showed promising in-
hibitory activity against AChE and BACE-1 inhibition [17–23]. Based
on the literature survey, diaryl 1, 2, 4-triazine scaffold was utilized
due to its interaction capabilities and better inhibition potential
against several enzymes especially cholinesterase enzymes [24–27].
Among diaryl 1, 2, 4-triazine scaffolds, 5, 6-biaryl of (1) proved to
be an appropriate moiety that gained the capability in anchoring
the peripheral anionic region of AChE by creating a hydrophobic
environment [28]. Replacement of 1, 2, 4-triazine group with thi-
azole moiety and linking with benzyl piperidine not only acquired
a significant improvement in the binding parameters but also
2
for (N=C-NH) sp hybrid carbon of imidazole ring in the range
of 158.54 – 153.23 ppm. By refluxing the 4 and 5 intermediates
with different substituted piperazine in the presence of potassium
iodide, anhydrous potassium carbonate, and acetonitrile, the fi-
nal compounds (6a-g and 7a-g) were obtained. The Dragendroff
reagent was used to recognize the compounds that were obtained
[34]. The absence of singlet peak of (-NH) of piperazine and pres-
ence of the triplet signal of (-N-CH ) in a range of 3.11 – 2.62
2
(
2) improve the inhibitory activity profile [29]. Further replace-
ppm, also the appearance of characteristic (-N-CH ) peak at 57.20
2
– 53.00 ppm in 13C NMR spectra confirmed the formation of final
compounds (6a-g and 7a-g). In 1H NMR spectra, the compounds
6b and 7b having 2-pyridyl moiety substituted on piperazine ex-
hibited a resonance signal of an aromatic proton between 8.18 –
6.58 ppm. Methoxy group of substituted piperazine of compound
ment of sulfur (S) of thiazole by nitrogen (N) (3) seeks the new
diaryl-imidazole scaffold with improved memory and cognitive
abilities, as well as a reduction in progressive neurodegeneration.
By employing the above-mentioned approaches, 4, 5-diphenyl-1H-
imidazole was developed as a core moiety and bounded with other
pharmacophoric characteristics [18]. FDA-approved drug containing
benzyl piperidine moiety named donepezil used to treat dementia
is conformed into the catalytic active site (CAS) of AChE [30]. Bio-
isosteric replacement of piperidine with piperazine proved a lower
affinity for AChE but improved the inotropic impact with signifi-
cant neuro-protective activity [31].
6c and 7c showed characteristic singlet peak of (CH -O-) at 3.66–
2
3.59 ppm. Methylene protons (N-CH -C H ) of benzyl piperazine
2
6
5
of compounds 6f and 7f displayed a singlet peak at 3.56 - 3.54
ppm. Compounds 6g and 7g having benzhydryl methyl (-CH) group
of substituted piperazine gives a diagnostic singlet peak at 5.18
– 5.15 ppm. In 13C NMR spectra, compounds 6c and 7c with 4-
methoxyphenyl substitution on piperazine exhibits NMR signal of
2

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Fig. 1. Designing strategy of targeted compounds.
Table 1
The results of eeAChE, eqBChE, and BACE-1 inhibition assay.
a
SI
Comp.
Code
IC50 (μM) ± SEM
ra-
tiob
n
Ar
eeAChE
eqBChE
BACE-1
6
6
6
6
6
6
6
7
7
7
7
7
7
7
a
b
c
d
e
f
2
2
2
2
2
2
2
3
3
3
3
3
3
3
-
-C6H5
3.056 ± 0.442
1.132 ± 0.067
0.751 ± 0.043
1.153 ± 0.179
0.917 ± 0.271
0.416 ± 0.018
>10
> 10
6.242 ± 0.530
4.785 ± 0.371
1.485 ± 0.095
1.685 ± 0.012
1.342 ± 0.078
0.392 ± 0.021
>10
-
-C5H4N
3.463 ± 0.013
1.217 ± 0.374
3.357 ± 0.054
2.498 ± 0.375
0.474 ± 0.054
>10
3.05
1.62
2.91
2.72
1.13
-
4-OCH3C6H5-
4-NO2C6H5-
4-FC6H5-
-CH2-C6H5
-CH(C6H5)2
-C6H5
g
a
b
c
d
e
f
1.549 ± 0.023
1.435 ± 0.164
0.467 ± 0.021
1.409 ± 0.054
1.313 ± 0.022
0.697 ± 0.047
>10
1.836 ± 0.054
1.922 ± 0.121
0.826 ± 0.038
4.481 ± 0.376
3.032 ± 0.081
2.354 ± 0.753
>10
3.811 ± 0.339
5.622 ± 0.346
0.922 ± 0.103
2.037 ± 0.249
1.934 ± 0.245
1.792 ± 0.287
>10
1.18
1.33
1.76
3.18
2.30
3.37
-
-C5H4N
4-OCH3C6H5-
4-NO2C6H5-
4-FC6H5-
-CH2-C6H5
-CH(C6H5)2
-
g
Donepezil
0.033 ± 0.01
1.4 ± 0.06
0.24 ± 0.03
42.4
a
-
Results are expressed as the mean IC50 ± SEM (n = 3);
SI (selectivity index) = IC50 of BChE/IC50 of AChE;
b
-
(
CH -O-) and carbon attached to it between 54.63 – 54.19 ppm and
Targeted compounds were analyzed for their ChE activity profile.
The ability of these compounds to prevent AChE was influenced by
the length of the bridge and the freedom of rotation around the
two-atom connecting structure [35,36]. Hence, it was found that
targeted compounds exhibited 2 carbon chain linkers provided bet-
ter output.
3
1
45.88 – 145.21 ppm, respectively. Compounds 6f and 7f having
methyl (-CH ) group of benzyl substituted piperazine showed char-
2
acteristics resonance signal at 63.72 – 62.82 ppm. The synthesized
compounds 6g and 7g bearing benzhydryl substitution on piper-
azine exhibited distinctive NMR signal for biphenyl methyl (-CH)
approximately between 77.67 – 77.04 ppm.
The compounds (6g, 7g) possessing benzhydryl piperazine moi-
ety at the endpoint did not provide any appropriate inhibition po-
tential due to enhanced hydrophobicity lowers the binding poten-
tial of targeted compounds to the AChE region [37].
2
2
.2. In vitro evaluation
Variation of terminal piperazine functionalities
.2.1. Cholinesterase inhibition assay (eeAChE and eqBChE)
The results exhibited moderate to excellent eeAChE inhibition
in the concentration range of micromolar to sub-micromolar ex-
cept for the compounds 6g, 7g (IC50 > 10 μM). The eeAChE in-
hibition was checked by both the electron-donating (methoxy, 6c)
and electron-withdrawing (nitro, 6d; and fluoro, 6e) groups at the
fourth position of phenyl piperazine ring (6c, IC50: 0.751 ± 0.043
μM; 6d, IC50: 1.153 ± 0.179 μM; 6e, IC50: 0.917 ± 0.271 μM)
and expressed that compound 6c with a 4-methoxyphenyl sub-
stituent on piperazine showed stronger inhibition of eeAChE com-
pared to 6d and 6e compounds. The increase in eeAChE inhibitory
The targeted compounds 6a-f and 7a-f were evaluated by the
Ellman technique using donepezil as beneficial reference norms for
their inhibitory capacity against eeAChE and eqBChE. The ChE en-
zymes inhibition activity of various targeted compounds were ex-
amined with regards to the variation of alkyl linkers (n = 2, 3) as
well as substituent linked with the endpoint of piperazine scaffold
(
Table 1).
Variation of alkyl chain linker
Alkyl linkers (n = 2, 3) constructed a bridge between 4, 5
diphenyl-1H-imidazole and substituted phenyl piperazine scaffold.
3

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
capacity could be due to the O-CH3 group with non-polar features
that interacted with eeAChE hydrophobic pocket [3]. Comparing
compounds with distinct electron-withdrawing substituents, fluoro
substituted compound 6e showed a mildly good inhibitory ability
against eeAChE compared to nitro substituted compound 6d. An
improved inhibitory activity might be due to the elevated fluorine
atom electronegativity that influences the molecule’s lipophilic-
ity. The compound having a 2-pyridyl substituent on piperazine
(
6b) exhibited reduced inhibition of eeAChE (IC50: 1.132 ± 0.067
μM) compared to 6c, 6e, and 6f compounds. The compounds (6g
and 7g) comprising benzhydryl piperazine moiety at the endpoint
and having various linkers showed no compelling results. It was
already disclosed that elevated hydrophobicity blocks the bind-
ing of the compounds at the AChE’s region [37]. Among all the
evaluated compounds, compound 6f containing two carbon con-
nected to the phenyl piperazine end group showed significant in-
hibition of eeAChE (IC50: 0.416 ± 0.018 μM). Due to the benzyl
group which involved showed better interaction with AChE’s CAS
residues, Compound 6f eeAChE inhibitory potential was also com-
pared to that of donepezil (IC50: 0.033 ± 0.01 μM). All the tar-
geted compounds for their eqBChE inhibitory activity were further
evaluated. Compounds 6c and 6f demonstrated excellent eqBChE
Fig. 2. Result of Propidium iodide displacement assay.
2.2.4. Aβ inhibition- thioflavin T assay
The AChE has also been shown to be indulged in promoting Aβ
aggregation by interacting particularly with PAS [41]. AChEIs linked
to the PAS can not only inhibit AChE, but also help prevent Aβ de-
velopment and deposition. Therefore, a fluorometric assay based
on thioflavin T was conducted on compound 6f to determine their
binding to the PAS region which could be useful in the prevention
of Aβ aggregation. The experiment was performed at three distinct
Aβ: inhibitor ratios (10:5, 10:10, and 10:20 μM). The percentage
of Aβ aggregation inhibition was recorded (Fig. 3A and B). The in-
hibitor (6f) exhibited greater anti-aggregator property in self (21.28
to 30.85%) and AChE-induced (25.70 to 51.07%) studies compared
with donepezil (Self-induced: 14.59 to 21.17%; AChE-induced: 19.72
to 33.05%). The findings verified these inhibitors’ PAS-AChE binding
capacity.
inhibitory activity (6c, IC50: 1.217 ± 0.374 μM; 6f, IC : 0.474 ±
50
0
.054 μM). Compounds 6b, 6d and 6e showed moderate inhibitory
potential against eqBChE with an IC50 value ranging from 2.498 ±
.375 μM to 3.463 ± 0.013 μM and compounds 6a, 6g, and 7g did
not show important eqBChE inhibitory activity (IC > 10 μM). The
0
5
0
wider eqBChE gorge than eeAChE, which results from the replace-
ment of two aromatic amino acids by lower aliphatic amino acids,
may be the cause [38].
2
.2.2. BACE-1 inhibition assay
Using the FRET-based BACE-1 fluorescence assay kit, all targeted
compounds were analyzed for inhibitory activity against BACE-
(Table 1). The electron-donating (methoxy, 6c) and electron-
1
2.3. In vivo evaluation
withdrawing (nitro, 6d; and fluoro, 6e) groups at the para position
of phenyl piperazine fragment showed moderate inhibitory activity
against BACE-1 with an IC50 value ranging from 1.342 ± 0.078 to
2.3.1. Scopolamine-induced amnesia models for testing cognition
enhancement in rats
1.685 ± 0.012 μM. Compounds 6a, 6b, and 6g showed no impor-
Compound 6f was evaluated for cognition and memory by
performing behavioral studies with the help of the scopolamine-
induced Y-maze model. To persuade cognitive impairment, a gold
standard drug, scopolamine was administered [42]. Test compound
in a dose of 2.5, 5, and 10 mg/kg, p.o., was given daily to healthy
male Wistar rats. On the seventh day of the study, a Y-maze exper-
iment was performed to determine the effect of compound 6f, and
spontaneous alternations, a measure of spatial working memory,
were estimated. The proportion of spontaneous alternations was
significantly lower (Fig. 4A, p < 0.001) in the scopolamine group
of animals relative to the control group, indicating induction of
memory and learning disability. In comparison with scopolamine,
Donepezil (5 mg/kg) showed considerably enhanced spontaneous
alternations (Fig. 4A, p < 0.001). As compared to donepezil, com-
pound 6f (10 mg/kg) showed a statistically non-significant dif-
ference in the percentage of spontaneous alterations (Fig. 4A, p
< 0.001). The complete arm in all groups stayed unchanged in-
ferred that scopolamine did not interfere with locomotor capabil-
ity in rats. The general findings of scopolamine-induced models
supported compound 6f’s ability to improve spatial and immedi-
ate memory.
tant inhibitory activity of BACE-1.
Compound 6f with two carbon atoms connected to the terminal
group of benzyl piperazine showed a powerful inhibition of BACE-
1
(IC50: 0.392 ± 0.021 μM). Compound 6f was chosen for further
screening with promising inhibitory potential for both the targeted
enzymes (eeAChE and BACE-1).
2
.2.3. Propidium iodide displacement assay
Propidium iodide assay was used to check propidium iodide
displacement from the enzyme complex of propidium iodide-AChE
39]. Propidium iodide is a recognized AChE complex that binds
[
particularly at PAS and increases the intensity of fluorescence up
to several fold [40]. Binding compounds at AChE’s PAS results in
decreased fluorescence intensity with propidium iodide displace-
ment. Compound 6f showed a greater propidium iodide displace-
ment (10 μM = 29.09%, 50 μM = 41.43%) relative to donepezil (10
μM = 24.86%, 50 μM = 36.31%). Compounds 6c exhibited rela-
tively equal propidium iodide displacement (10 μM = 24.12%, 50
μM = 37.20 %) as compared to donepezil. Compounds 6b and 6e
seemed to be less able to displace the propidium iodide of PAS-
AChE (6b 10 μM = 15.03%, 50 μM = 21.77%; 6e 10 μM = 20.15%,
5
0 μM = 24.29%). The results showed that compound 6f could be
2.4. Computational studies
effectively linked to AChE’s PAS site (Fig. 2).
All the results are reported as the mean ± SEM (n = 3); Propid-
ium iodide displacement assay was performed on AChE to assess
the ability of compounds to displace propidium with reference to
donepezil at 10 and 50 μM.
2.4.1. In silico molecular docking studies
Compound 6f was monitored for its binding affinity and bind-
ing pattern on active site residues of AChE (PDB Code: 4EY7), BChE
(PDB Code: 4TPK), and BACE-1 (PDB Code: 2ZJM) by conducting
4

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Fig. 3. [A] Self-induced and; [B] AChE-induced Aβ aggregation inhibition experiment. Results are expressed as the mean ± SEM of three independent experiments.
Fig. 4. Effect of compound 6f on scopolamine-induced cognitive deficit in the Y-maze test. [A] Spontaneous alteration (%) and; [B] Total arm entries in the Y-maze ex-
periment. All result are showed as the mean ± SEM (n = 6). ###p < 0.0001 versus control; ∗∗∗p < versus scopolamine; πp < 0.05, ns = non-significant versus DONE5
(
donepezil 5 mg/kg).
molecular docking studies using Schrodinger Maestro’s Glide mod-
ule 2018–1. On AChE’s active site, compound 6f and donepezil
showed the docked scores -15.566 and -15 kcal/mole, respectively.
According to the docking study, the compound 6f was found to
bind likely with AChE’s CAS and PAS region. Benzyl piperazine por-
tion of the compound 6f exhibited polar interactions with AChE’s
(
CAS) residues His447 and Ser203. At the AChE’s (PAS), compound
f interacted with Tyr72, Tyr124, Tyr341 residues through hy-
6
drophobic interactions, with Trp286 residue through hydropho-
bic as well as π-π stacking interactions and with Asp74 residue
through charged interaction. Compound 6f’s benzyl piperazine por-
tion developed hydrophobic, π-π, and π-cation interactions with
the Trp86 residue of AChE’s anionic location.
At the same site, it also formed hydrophobic and charged
interactions with Phe338 and Glu202 residues, respectively. The
linker part of compound 6f showed hydrophobic interactions
with Phe295 and Phe297 residues in the acyl binding pocket of
AChE. Benzyl substituent of compound 6f binds with Gly120 and
Gly121 residues of the oxyanion site of AChE via glycine inter-
actions (Table 2). Other residues of AChE also showed interac-
tions with compound 6f, like Tyr133, Leu76, Val294, Tyr337, and
Ile451 residues interacted via hydrophobic interactions, Thr75, and
His287 residues via polar interactions, and Gly448 residue via
glycine interaction (Fig. 5).
Fig. 5. Molecular docking of donepezil and compound 6f in the gorge of AChE en-
zyme. 3D pictorial image representing the interaction of donepezil and compound
6f in ligand binding surface (magenta color) with AChE active site.
PAS of BChE interacted with compound 6f. At the anionic site of
hBChE, Trp82 (hydrophobic and π-cation interactions) and Glh197
(charged interaction) residues showed binding with benzyl piper-
azine moiety of compound 6f. Ala328 residue also showed hy-
drophobic interaction with compound 6f at this site. Compound 6f
interacted with Gly116 residue in the oxyanion hole of BChE via
glycine interaction. The overlaid dock poses of all the synthesized
compounds at both ChE enzymes were also depicted in Fig 6 to
highlight their relative position as well as their helpful interaction.
BACE-1 is a membrane-bound enzyme with catalytic aspartic
dyad residues at the N-terminus and C-terminus interfaces (Asp32
Compound 6f also showed their strong binding affection and
binding pattern with the BChE active site. The piperazine moiety of
compound 6f displayed polar interactions with Ser198 and His438
residues of BChE’s CAS. Also, the His438 residue displayed hydro-
gen bonding with compound 6f. Asp70 (charged interaction) and
Tyr332 (π-π stacking and hydrophobic interactions) residues of
5

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Table 2
and Asp228). Compound 6f indicated charged interactions with the
In silico docking interaction analysis of compound 6f
Asp228 residue of BACE-1(Fig. 7).
and donepezil on AChE (4EY7).
Interacting residues#
Comp.
2
.4.2. Molecular dynamics (MD) simulation
The molecular dynamics simulation study used Desmond (DE
Other
in-
Shaw Research) to analyze the compound 6f-AChE complex bind-
ing stability. The simulations of molecular dynamics evaluated the
stability of the docked complex along with virtual water surround-
ing in a flexible protein. The values of the docked complexes’ root
mean square deviations (RMSD) were determined and compared
to the reference protein backbone structures within the range. Root
mean square fluctuations (RMSF) values for the compound 6f-AChE
complex showed stable ligand and protein residue trajectories dur-
ing the simulation analysis. The detailed analysis of interaction
was performed using the histogram, graphical, and time-line rep-
resentation of the protein-ligand. Using protein-ligand contacts, the
percentage of protein residue interactions with compound 6f was
identified. The findings of AChE molecular dynamics studies pro-
posed that compound 6f interacted considerably with PAS residues
of AChE (Fig. 7). At the PAS region, the Trp286 residue created π-
π stacking (62% time-scale) and hydrophobic interactions with im-
idazole moiety of compound 6f. Also, the Tyr341 residue formed
the same interactions as that of Trp286 residue with compound
6f in the PAS region for a 70% time-scale. Asp74, a PAS residue
that interacted with the nitrogen atom (N-1) of piperazine ring
of compound 6f through the creation of salt bridge (47% time-
scale) and charged interaction. The piperazine nitrogen atom (N-
1) and phenyl ring of benzyl piperazine moiety of compound 6f
displayed π-cation (80% time-scale) and π-π stacking interactions
Acyl
Anionic bind-
ter-
act-
sub-
site
ing
Oxyanio inn g
CAS
PAS
pocket hole
residues
6
f
a
c
b,c,e
c
f
c
Ser203 ,T yr72 , Trp86 Ph,e295 G, ly120 ,T yr133 ,
a
c
c
c
f
c
His447 Tyr124 P, he338 P, he297 Gly121 Leu76 ,
b,c
d
Val294c,
Tyr337c,
Gly448f,
Ile451c,
Thr75a,
Trp286 G l, u202
Tyr341c,
Asp74d
His287a
∗
a
c
b,c,e
c,g
c,f
c
Don
Ser203 ,T yr72 , Trp86 Ph,e295 Gl ,y 120 T y, r133 ,
a
c
b,c,e
c
c,f
c
His447 Tyr124 P, he338 Phe297 G, ly121 Leu289 ,
Trp286b,c
,
Val294c,
Tyr337c,
Gly448c,
Ile451c
Tyr341c,
#
All the interacting residues are within the 4 A dis-
tance with the ligand;
∗
a
Don – Donepezil;
–
polar interaction;
π-π stacking interaction;
hydrophobic interaction;
charged interaction;
π-cation interaction;
glycine interaction;
hydrogen bonding.
b
c
d
e
f
–
–
–
–
(
49% time-scale) with Trp86 residue of the anionic subsite, respec-
–
g
tively. At anionic subsite, Trp86 residue also exhibited hydropho-
bic interactions with the benzyl piperazine portion of compound
–
6f. Other interacting residues, like His287, formed π-π stacking
(
31% time-scale) as well as polar interactions with diaryl part of
the compound 6f.
The results of the BACE-1 molecular dynamics simulation
showed that Compound 6f created nominal interactions with as-
partate dyad residues (Asp32 and Asp228) with fluctuations. The
MD findings suggested that compound 6f showed more stable in-
teraction in AChE as compared to BACE-1 (Fig. 7 and 8)
2
.4.3. Drug-likeliness by QikProp module
The drug-likeliness properties of compound 6f were assessed
using Schrödinger’s QikProp module, and the results were deter-
mined to be comparable to ordinary donepezil (Table 3). Lipinski’s
rule of five (mol MW 500, QPlog Po/w 5, donorHB 0–6.0, accptHB
2
.0–20), as well as the other expected parameters (SASA 300–1000,
QPlogBB 3.0 to 1.2, QPlogPo/w 2.0 to 6.5), indicated that compound
6f elicited "drug-like" properties.
3. Conclusion
Continuing with the ideas of bioisosterism and hybrid pharma-
cophore and pursuance in the development of newer and safer
agents as ChE and BACE-1, the substituted piperazine nucleus teth-
ered with 4, 5-diaryl-1H-imidazole scaffold through different link-
ers were designed. The synthesized derivatives (6a-g and 7a-g)
were initially evaluated for ChE inhibition potential using Ellman’s
colorimetric protocol. Amongst the tested compounds, 6c, 6e, and
6f showed significant eeAChE inhibitory activity. Compound 6f
containing the benzyl piperazine moiety displayed most significant
eeAChE (IC50 = 0.416 ± 0.018 μM) and eqBChE (IC = 0.474 ±
50
0
.054 μM) inhibitory activity. Compound 6f showed a potent BACE-
Fig 6. The superimposed docking poses of all the synthesized compounds at a)
1 (IC50 = 0.392 ± 0.021 μM) inhibitory potential. Compounds 6b,
c, 6e, and 6f were evaluated for propidium iodide displacement
AChE (PDB: 4EY7) b) BChE (PDB: 2ZJM)
6
6

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Fig. 7. Molecular dynamics studies of 6f-AChE (4EY7) docked complex. [A] RMSD graph of the compound 6f-AChE complex for the period of 100 ns dynamics simulations; [B]
Interaction fractions of active amino acid residues are displayed in a histogram; [C] Graphical representation of compound 6f showing interacting residues for the simulations
of 100 ns; [D] At each time frame, a timeline representation of interaction with all amino acid residues was displayed.
Table 3
QikProp analysis of compound 6f.
Compound
Donepezil
Mol_ Wt
Donor HB
Acceptor HB
SASA
QplogBB
Qplogo/w
393.61
514.7
1
0
7.1
3.0
759.67
789.48
0.14
0.78
4.25
6
f
6.474
aMol_MW- Molecular weight of the molecule (130 to 725).
bDonor HB- number of hydrogen bonds (0.0 to 6.0).
cAcceptor HB- number of hydrogen bonds (2.0 to 20.0).
dSASA- total solvent accessible surface area (SASA) in square angstroms using a probe with 1.4
A radius (300 to1000).
eQPlogBB- predicted brain/blood partition coefficient (-3.0 to 1.2).
fQPlogPo/w- this gives the predicted octanol/water partition coefficient (-2.0 to 6.5).
capability from PAS-AChE. Compound 6f showed a greater propid-
terations. Compound 6f was monitored for its binding affinity and
binding pattern with an active site residue of AChE (PDB Code:
4EY7), BChE (PDB Code: 4TPK), and BACE-1 (2ZJM) by conduct-
ing molecular docking studies and found that compound 6f sig-
nificantly interact with all the respective enzymes. The stability
of the compound 6f was analyzed through a molecular dynamics
simulation study and compound 6f showed more stable interaction
in AChE as compared to BACE-1. Based on overall outcomes, com-
pound 6f could be regarded as a prospective lead candidate in the
management of AD.
ium iodide displacement (10 μM = 29.09%, 50 μM = 41.43%) rela-
tive to donepezil (10 μM = 24.86%, 50 μM = 36.31%). The inhibitor
(
(
6f) exhibited greater anti-aggregatory property in self-induced
21.28 to 30.85%) and AChE-induced (25.70 to 51.07%) studies com-
pared with donepezil (self-induced: 14.59 to 21.17%; AChE-induced:
9.72 to 33.05%). The in-vivo behavioral study revealed that the
1
scopolamine group induces memory impairment in rats, in com-
parison to donepezil (5 mg/kg), compound 6f (10 mg/kg) showed
a statistically non-significant difference in percent spontaneous al-
7

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Fig. 8. Molecular dynamics studies of 6f-BACE-1 (2ZJM) docked complex. [A] RMSD graph of the compound 6f-BACE-1 complex for the period of 100 ns dynamics simula-
tions; [B] Interaction fractions of active amino acid residues are displayed in a histogram; [C] Graphical representation of compound 6f showing interacting residues for the
simulations of 100 ns; [D] At each time frame, a timeline representation of interaction with all amino acid residues was displayed.
4
. Experimental
light, Dragendorff’s reagent, vapors of iodine were used for visual-
ization of TLC plates. FT-NMR spectrophotometer (Bruker Avance)
was used to record 1H NMR spectra (500 MHz) and 13C NMR spec-
tra (125 MHz) in DMSO-d6 or CDCl3 using tetramethylsilane as an
internal standard, and the spectra were interpreted with the help
of MestReNova version 6.0.2. The absorbance of inhibitors at differ-
ent concentrations was measured colorimetrically at 412 nm using
a Multimode Microplate Reader (BioTek Synergy H1M, USA). Using
an Infinity II 1260 High-Performance Liquid Chromatography (Ag-
ilent, USA) with an Eclipse Plus C8 column and a methanol/water
(90:10 v/v) mobile phase at a flow rate of 1 mL/min, the percent
purity of synthesised compounds was determined to be ≥ 95%.
4
.1. Chemistry
4.1.1. Instrumentation and chemicals
Avra Synthesis, TCI Chemicals, and Sigma Aldrich provided both
reagents and chemicals (India). Progress of chemical reactions was
monitored by using pre-coated aluminum sheets of silica gel 60
F254 of thickness 0.25 mm, (Merk, Germany), and the Rf values
are also determined using the same sheets. The IR spectra (wave
numbers in cm-1) were recorded on FT-IR spectrophotometer us-
ing potassium bromide discs. The melting points were calculated
using capillary tubes and Stuart Melting Point apparatus (SMP10)
using capillary tubes and listed as uncorrected. The Retention fac-
tor (Rf) values were determined using solvent system methanol:
dichloromethane (2:98) to monitor the progress of the reaction. UV
4
4
.1.2. Synthesis
.1.2.1. General procedure for the synthesis of intermediates 2, 3.
Compound 1 (8.18 mmol) was dissolved in 15 mL ACN in a round
8

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Scheme 1. Reagents and conditions: (I) anhydrous potassium carbonate, Br(CH2)nCl (n = 2 and 3), ACN, reflux, 7-8 8 h; (II) benzoin, ammonium acetate, cerric ammonium
nitrate, and ethanol:water, reflux (III) substituted piperazines, potassium iodide, anhydrous potassium carbonate, and ACN, reflux, 16 h.
bottom flask at room temperature. An accurate quantity of dry
K2CO3 (10.63 mmol) was added, and after some time, compound 1
was precipitated in the form of potassium salt. Bromo chloro sub-
stituted alkane (8.18 mmol) (1, 2-dibromo ethane, 1, 3-bromochloro
propane) were added to the reaction mixture and refluxed for 7-8
h. The reaction was completed and it was affirmed by TLC using
ethyl acetate: hexane (20:80) as mobile phase. The ACN was evapo-
rated under reduced pressure, the solid residue was obtained. This
solid residue dissolved in ethyl acetate and washed with water fol-
lowed by passing over anhydrous sodium sulfate. The organic layer
was taken and evaporated under a vacuum. The crude product was
obtained, which was then purified by using mobile phase ethyl ac-
etate: hexane (1:99) and stationary phase silica gel through col-
umn chromatography to afford intermediates 2 and 3.
cooling of the reaction mixture at room temperature. The solid was
obtained by vacuum filtration, followed by water-washed and dried
to give crude solid. Crude solid was purified by using mobile phase
ethyl acetate:hexane (5:95) and stationary phase silica gel through
column chromatography to afford intermediates 4 and 5.
2-(4-(2-bromoethoxy)phenyl)-4, 5-diphenyl-1H-imidazole (4)
1H NMR (500 MHz, δH, DMSO-d ): 12.66 (s, 1H), 8.17 (d, 1H,
6
J = 2.1 Hz), 8.01 (dd, 1H, J = 8.6, 3.2 Hz), 7.56 – 7.41 (m, 7H), 7.37
(t, 1H, J = 7.3 Hz), 7.31 (dd, 3H, J = 7.9, 6.8 Hz), 7.23 (t, 1H, J = 7.3
Hz), 4.50 – 4.45 (m, 2H), 3.89 – 3.85 (m, 2H). 13C NMR (125 MHz,
δC, DMSO-d ): 153.23, 144.17, 130.98, 128.71, 128.33, 128.25, 127.12,
6
126.62, 121.79, 114.40, 68.85, 31.07.
Anal. C23H19BrN O: C,65.88; H,4.57; Br,19.06; N,6.68; O,3.82;
2
Found: C,65.86; H,4.54; Br,19.09; N,6.64; O,3.79.
4
-(2-bromoethoxy) benzaldehyde (2)
2-(4-(3-chloropropoxy)phenyl)-4,
5-diphenyl-1H-imidazole
1H NMR (500 MHz, δH, CDCl ): 9.88 (s, 1H), 7.84 (d, 2H, J = 8.8
(5)
3
Hz), 7.01 (d, 2H, J = 8.7 Hz), 4.36 (t, 2H, J = 6.2 Hz), 3.66 (t, 2H,
1H NMR (500 MHz, δH, DMSO-d ): 12.53 (s, 1H), 8.02 (d, 2H,
6
13
J = 6.2 Hz). C NMR (125 MHz, δ CDCl ): 190.71, 163.02, 132.04,
J = 8.6 Hz), 7.77 – 7.15 (m, 10H), 7.06 (d, 2H, J = 8.7 Hz), 4.15 (t,
2H, J = 6.0 Hz), 3.82 (t, 2H, J = 6.4 Hz), 2.20 (p, 2H, J = 6.1 Hz).
C,
3
1
30.50, 114.90, 67.97, 28.48.
13
Anal. C H BrO : C,47.19; H,3.96; Br,34.88; O,13.97; Found:
C NMR (125 MHz, δ DMSO-d ): 158.54, 145.59, 128.20, 127.56,
9
9
2
C,
6
C,47.17; H,3.95; Br,34.86; O,13.98.
-(3-chloropropoxy) benzaldehyde (3)
127.46, 126.92, 126.60, 126.09, 123.31, 114.63, 64.36, 42.00, 31.73.
4
Anal. C24H21ClN O: C,74.12; H,5.44; Cl,9.12; N,7.20; O,4.11;
2
1H NMR (500 MHz, δH, CDCl ): 9.85 (s, 1H), 7.80 (d, 2H, J = 8.7
Found: C,74.08; H,5.48; Cl,9.10; N,7.23; O,4.08.
3
Hz), 6.98 (d, 2H, J = 8.7 Hz), 4.17 (t, 2H, J = 5.9 Hz), 3.72 (t, 2H,
1
3
J = 6.3 Hz), 2.24 (p, 2H, J = 6.0 Hz). C NMR (125 MHz, δ CDCl ):
C,
3
1
90.82, 163.73, 132.01, 130.06, 114.77, 64.63, 41.24, 31.96.
Anal. C10H11 ClO2: C,60.46; H,5.58; Cl,17.85; O,16.11; Found:
C,60.47; H,5.55; Cl,17.84; O,16.13.
4.1.2.3. General procedure for the synthesis of compounds 6a-g and
7a-g. 10 ml ACN was taken in a round bottom flask and interme-
diate 4 or 5 (1.19 mmol), potassium iodide (1.78 mmol), various
substituted piperazines (1.19 mmol), and dry potassium carbonate
(1.78 mmol) were suspended into it and refluxed for 16 h. Comple-
tion of the reaction was monitored by TLC using MeOH:DCM (2:98)
as a mobile phase. The solvent was evaporated by the application
of a rota evaporator to get the solid residue. The obtained solids
were dissolved in ethyl acetate and washed with water. The resid-
ual water was removed with the help of dry sodium sulfate. The
organic layer was vaporized using a rota evaporator to yield crude
solid, which was purified by using mobile phase MeOH:DCM (2:98)
4
.1.2.2. General procedure for the synthesis of intermediates 4, 5.
Ammonium acetate (14.08 mmol), cerric ammonium nitrate (0.35
mmol), and benzoin (3.52 mmol) were dissolved in a 10 ml mix-
ture of ethanol:water (1:1, v/v). Intermediate 2 or 3 (3.52 mmol)
was added to the above mixture and refluxed till the completion
of the reaction, which was indicated by using TLC having ethyl ac-
etate:hexane (40:60) as mobile phase. The solid get precipitated
when the reaction mixture was added into ice-cold water after the
9

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
and stationary phase silica gel with the help of column chromatog-
130.71, 129.57, 128.47, 127.97, 127.19, 126.82, 126.23, 118.11, 117.33,
114.97, 68.13, 55.48, 51.24, 49.37.
raphy to yield pure compounds 6a-g and 7a-g.
1-(2-(4-(4,
5-diphenyl-1H-imidazol-2-yl)phenoxy)ethyl)-4-
Anal. C34H33FN O: C,76.42; H,6.03; F,3.66; N,10.80; O,3.08;
4
phenylpiperazine (6a)
Found: C,76.39; H,6.05; F,3.3.67; N,10.82; O,3.07. HPLC purity:
97.54%, retention time: 3.613 min.
Yield: 71%; mp: 156-158 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3346, 3114, 1654, 1276, 1136; 1H NMR (500 MHz,
−1
1-benzyl-4-(2-(4-(4,
5-diphenyl-1H-imidazol-2-
δH, CDCl ): 12.50 (s, 1H), 7.85 (d, 2H, J = 8.7 Hz), 7.56 (d, 4H,
yl)phenoxy)ethyl)piperazine (6f)
3
J = 7.0 Hz), 7.35 (t, 4H, J = 7.4 Hz), 7.32 – 7.28 (m, 4H), 6.99 (d,
Yield: 69%; mp: 158-160 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3344, 3119, 1648, 1281, 1129; 1H NMR (500 MHz,
−1
2
4
2
H, J = 8.8 Hz), 6.95 (d, 2H, J = 8.0 Hz), 6.89 (t, 1H, J = 7.3 Hz),
.21 (t, 2H, J = 5.7 Hz), 3.29 – 3.22 (m, 4H), 2.92 (t, 2H, J = 5.7 Hz),
.81 – 2.76 (m, 4H). 13C NMR (125 MHz, δ CDCl ): 159.31, 151.24,
δH, CDCl ): 12.49 (s, 1H), 7.85 (d, 2H, J = 8.8 Hz), 7.57 (d, 4H,
3
J = 7.1 Hz), 7.35 – 7.27 (m, 11H), 6.95 (d, 2H, J = 8.8 Hz), 4.16 (t,
2H, J = 5.5 Hz), 3.56 (s, 2H), 2.87 (t, 2H, J = 5.5 Hz), 2.68 – 2.56 (m,
8H). 13C NMR (125 MHz, δ CDCl ): 159.21, 146.07, 129.37, 128.56,
C,
3
146.06, 129.13, 128.58, 127.82, 127.59, 126.82, 122.97, 119.82, 116.13,
114.95, 66.01, 57.20, 53.69, 49.07.
C,
3
Anal. C33H32N O: C,79.17; H,6.44; N,11.19; O,3.20; Found:
128.31, 127.83, 127.30, 126.82, 123.00, 114.89, 65.76, 62.82, 57.03,
53.37, 52.62, 29.70.
4
C,79.21; H,6.47; N,11.15; O,3.17. HPLC purity: 99.19%, retention time:
3
.147 min.
-(2-(4-(4,
Anal. C34H34N O: C,79.35; H,6.66; N,10.89; O,3.11; Found:
4
1
5-diphenyl-1H-imidazol-2-yl)phenoxy)ethyl)-4-
C,79.32; H,6.65; N,10.87; O,3.09. HPLC purity: 96.52%, retention
time: 4.653 min.
(pyridin-2-yl)piperazine (6b)
Yield: 68%; mp: 157-159 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
1-benzhydryl-4-(2-(4-(4,
5-diphenyl-1H-imidazol-2-
f
IR (KBr, cm ): 3352, 3090, 1662, 1682, 1273, 1128; 1H NMR (500
−1
yl)phenoxy)ethyl)piperazine (6g)
MHz, δH, DMSO-d ): 12.51 (s, 1H), 8.13 – 8.02 (m, 3H), 7.60 – 7.47
Yield: 72%; mp: 167-169 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
6
f
IR (KBr, cm ): 3349, 3123, 1655, 1269, 1147; 1H NMR (500 MHz,
−1
(
m, 7H), 7.34 – 7.28 (m, 4H), 7.10 (d, 2H, J = 8.7 Hz), 6.76 – 6.58 (m,
2
2
H), 4.09 (t, 2H, J = 6.4 Hz), 3.64 -3.51 (m, 4H), 3.13 – 2.94 (m, 4H),
δH, DMSO-d ): 12.50 (s, 1H), 8.07 – 7.92 (m, 2H), 7.55 –7.45 (m,
6
13
.75 (t, 2H, J = 6.5 Hz). C NMR (125 MHz, δ DMSO-d ): 160.97,
10H), 7.31 –7.12 (m, 10H), 7.05 – 6.97 (m, 2H), 5.18 (s, 1H), 4.21
(t, 2H, J = 5.7 Hz), 3.11 (t, 2H, J = 5.7 Hz), 2.71 – 2.52 (m,
8H). 13C NMR (125 MHz, δ DMSO-d ): 159.93, 146.17, 142.91,
C,
6
1
57.83, 148.14, 145.89, 142.51, 139.65, 130.21, 129.27, 128.77, 127.87,
127.34, 126.73, 115.30, 114.01, 108.19, 67.76, 54.57, 52.66, 47.31.
C,
6
Anal. C32H31N O: C,76.62; H,6.23; N,13.96; O,3.19; Found:
137.33, 130.35, 129.75, 129.42, 128.57, 127.99, 127.54, 127.13, 126.87,
126.33, 114.91, 77.04, 67.19, 55.57, 53.67, 51.01.
5
C,76.64; H,6.27; N,13.93; O,3.16. HPLC purity: 95.47%, retention
time: 3.173 min.
Anal. C40H38N O: C,81.32; H,6.48; N,9.48; O,2.71; Found:
4
1
-(2-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)ethyl)-4-(4-
methoxyphenyl)piperazine
6c)
C,81.29; H,6.46; N,9.45; O,2.74. HPLC purity: 99.90%, retention
time: 4.053 min.
(
1-(3-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)propyl)-4-
phenylpiperazine (7a)
Yield: 74%; mp: 162-164 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3428, 3092, 1664, 1283, 1269, 1147; 1H NMR (500
−
1
Yield: 66%; mp: 158-160 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3339, 3110, 1648, 1269, 1129; 1H NMR (500 MHz,
−1
MHz, δH, DMSO-d ): 12.51 (s, 1H), 7.99 (d, 2H, J = 8.7 Hz), 7.56
6
–
–
7.47 (m, 6H), 7.39 – 7.28 (m, 4H), 7.01 (d, 2H, J = 8.7 Hz), 6.71
6.60 (m, 4H), 3.99 (t, 2H, J = 5.3 Hz), 3.59 (s, 3H), 3.13 (t, 4H,
δH, CDCl ): 12.49 (s, 1H), 7.95 (d, 2H, J = 8.8 Hz), 7.58 – 7.54 (m,
3
4H), 7.32 (t, 4H, J = 7.4 Hz), 7.27 (d, 4H, J = 5.9 Hz), 6.97 (d, 2H,
J = 5.5 Hz), 2.90 (t, 4H, J = 5.3 Hz), 2.32 (t, 2H, J = 5.3 Hz).
J = 8.7 Hz), 6.93 (d, 3H, J = 7.9 Hz), 4.12 – 4.09 (m, 2H), 3.31 – 3.28
13
13
C NMR (125 MHz, δ DMSO-d ): 159.16, 152.88, 145.21, 143.56,
(m, 4H), 2.82 (dd, 6H, J = 15.8, 11.5 Hz), 2.15 – 2.11 (m, 2H).
C
C,
6
1
30.13, 129.39, 128.67, 127.78, 127.47, 127.28, 126.66, 126.18, 116.07,
14.98, 66.37, 55.93, 54.63, 52.56, 49.02.
Anal. C34H34N O : C,76.95; H,6.46; N,10.56; O,6.03; Found:
NMR (125 MHz, δ CDCl ): 162.29, 140.57, 129.38, 128.77, 128.08,
C, 3
1
127.57, 120.46, 116.56, 115.01, 66.39, 55.25, 53.25, 48.81, 26.17.
Anal. C34H34N O: C,79.35; H,6.66; N,10.89; O,3.11; Found:
4
2
4
C,76.94; H,6.49; N,10.53; O,6.07. HPLC purity: 99.83%, retention
time: 2.613 min.
C,79.38; H,6.69; N,10.87; O,3.13. HPLC purity: 96.44%, retention
time: 5.733 min.
1
-(2-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)ethyl)-4-(4-
1-(3-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)propyl)-4-
(pyridin-2-yl)piperazine (7b)
nitrophenyl)piperazine (6d)
Yield: 76%; mp: 164-166 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
Yield: 70%; mp: 158-160 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
f
f
IR (KBr, cm ): 3323, 3124, 1523, 1649, 1354, 1271, 1128; 1H NMR
−1
IR (KBr, cm ): 3347, 3115, 1653, 1659, 1265, 1139; 1H NMR (500
−1
(
500 MHz, δH, DMSO-d ): 12.48 (s, 1H), 8.09 – 7.92 (m, 4H),
MHz, δH, CDCl ): 12.50 (s, 1H), 8.18 (d, 1H, J = 3.3 Hz), 7.83 (d,
6
3
7
.63 –7.36 (m, 10H), 7.10 – 6.89 (m, 4H), 4.17 (t, 2H, J = 5.6
Hz), 3.40 (t, 4H, J = 5.7 Hz), 3.32 – 2.94 (m, 4H), 2.73 (t, 2H,
2H, J = 8.5 Hz), 7.59 – 7.45 (m, 6H), 7.32 (t, 4H, J = 7.4 Hz),
7.28 (s, 1H), 6.94 (d, 2H, J = 8.6 Hz), 6.64 (dd, 2H, J = 10.5,
6.1 Hz), 4.06 (t, 2H, J = 6.2 Hz), 3.59 – 3.54 (m, 4H), 2.62
13
J = 5.6 Hz). C NMR (125 MHz, δ DMSO-d ): 160.87, 157.12,
C,
6
13
1
45.47, 143.51, 137.24, 130.45, 129.33, 128.79, 127.87, 127.25, 126.83,
26.30, 114.89, 112.65, 67.76, 54.26, 52.58, 49.12.
Anal. C33H31N O : C,72.64; H,5.73; N,12.84; O,8.80; Found:
(t, 6H, J = 7.1 Hz), 2.08 – 2.00 (m, 2H). C NMR (125 MHz,
1
δ
CDCl ): 159.69, 148.15, 146.40, 137.80, 128.80, 128.06, 127.56,
C, 3
127.08, 122.89, 115.03, 113.72, 107.43, 66.39, 55.40, 53.16, 45.22,
26.66.
5
3
C,72.67; H,5.70; N,12.85; O,8.78. HPLC purity: 96.80%, retention
time: 3.360 min.
Anal. C33H33N O: C,76.87; H,6.45; N,13.58; O,3.10; Found:
5
1
-(2-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)ethyl)-4-(4-
C,76.89; H,6.42; N,13.57; O,3.12. HPLC purity: 97.13%, retention
time: 3.387 min.
fluorophenyl)piperazine (6e)
Yield: 73%; mp: 162-164 °C; R = 0.52 (MeOH:DCM, 2:98 v/v);
1-(3-(4-(4,5-diphenyl-1H-imidazol-2-yl)phenoxy)propyl)-4-(4-
methoxyphenyl)piperazine (7c)
f
−1
1
IR (KBr, cm ): 3344, 3132, 1645, 1323, 1267, 1163; H NMR (500
MHz, δH, DMSO-d ): 12.50 (s, 1H), 8.03 (d, 2H, J = 8.6 Hz), 7.61 –
Yield: 62%; mp: 161-163 °C; R = 0.45 (MeOH:DCM, 2:98 v/v);
6
f
IR (KBr, cm ): 3338, 3119, 1656, 1284, 1279, 1144; 1H NMR (500
−1
7
.43 (m, 6H), 7.31 – 7.24 (m, 4H), 7.11 – 6.96 (m, 4H), 6.67 (d, 2H,
J = 8.6 Hz), 4.10 (t, 2H, J = 5.8 Hz), 3.49 (t, 4H, J = 5.6 Hz), 2.83
MHz, δH, DMSO-d ): 12.51 (s, 1H), 8.01 (d, 2H, J = 8.5 Hz), 7.50
6
13
(
t, 4H, J = 5.7 Hz), 2.68 (t, 2H, J = 5.8 Hz). C NMR (125 MHz,
(d, 4H, J = 7.4 Hz), 7.31 (d, 6H, J = 35.7 Hz), 7.04 (d, 2H, J = 8.6
Hz), 6.89 (d, 2H, J = 8.9 Hz), 6.81 (d, 2H, J = 8.9 Hz), 4.08 (t, 2H,
δ
DMSO-d ): 160.01(d, J C-F = 224.2 Hz), 158.52, 149.21, 145.09,
C,
6
10

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
J = 5.6 Hz), 3.66 (s, 3H), 3.09 (s, 4H), 2.80 (d, 6H, J = 22.7 Hz), 2.01
4.2.1.4. Cholinesterase inhibition assay (eeAChE and eqBChE). Tar-
geted compounds were evaluated for their cholinesterase in-
hibitory potency against eeAChE and eqBChE as per the Ellman
method [43]. Thiolated substrates are specifically hydrolyzed into
thiocholine by the ChE enzyme. 5, 5-dithio-bis-(2-nitrobenzoic
acid) (DTNB) is reduced by thiocholine results in the formation
of the yellow color compound which can be identified colori-
metrically at 412 nm. All the inhibitors were dissolved in DMSO,
and eight rising concentrations of inhibitors were prepared. The
eeAChE from electrophorus electricus (electric eel) and eqBChE
from equine serum were purchased from Sigma Aldrich India. 2.5
U/ml solution of cholinesterase was prepared in Tris buffer pH 8.
The solution of cholinesterase enzyme (eeAChE & eqBChE) (25 μL)
and inhibitors of different concentrations (10 μL) were taken into
a 96-well microplate and incubated for 10 min. Further, DTNB (40
μL) (1 mM), acetylthiocholine iodide (ATCI) or butyrylthiocholine
iodide (BTCI) substrate (10 μL) (7.5 mM), and Tris buffer pH 8
(240 μL) were added into above microplate. The absorbance was
measured at 412 nm wavelength. The rate of non-enzymatic reac-
tion was assessed by a blank assay contains all components except
AChE and BChE. The change in reaction rate and percent inhibitory
potency of inhibitors were assessed. The assay was performed in
triplicate, and Graph Pad Prism 5.01 was used to calculate the IC50
values of inhibitors [44].
(
s, 2H). 13C NMR (125 MHz, δ DMSO-d ): 158.88, 153.34, 145.88,
H, 6
144.98, 128.64, 127.89, 127.02, 123.27, 117.77, 114.84, 114.84, 65.77,
5
5.37, 54.19, 52.47, 48.84, 25.41.
Anal. C35H36N O : C,77.18; H,6.66; N,10.29; O,5.87; Found:
4
2
C,77.15; H,6.69; N,10.25; O,5.84. HPLC purity: 97.89%, retention
time: 3.440 min.
1-(3-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)propyl)-4-
(4-nitrophenyl)piperazine (7d)
Yield: 71%; mp: 162-164 °C; R = 0.39 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3336, 3109, 1648, 1549, 1338, 1267, 1129; 1H NMR
−1
(
500 MHz, δH, DMSO-d ): 12.49 (s, 1H), 8.11 (d, 2H, J = 8.6 Hz),
6
7
.97 (d, 2H, J = 7.4 Hz), 7.54 –7.51 (m, 6H), 7.31 – 7.26 (m, 4H),
.02 (d, 2H, J = 8.7 Hz), 6.87 (d, 2H, J = 7.5 Hz), 4.09 (t, 2H,
J = 5.7 Hz), 3.35 – 3.27 (m, 4H), 3.27 – 2.84 (m, 6H), 2.03 (t,
7
1
3
2
H, J = 5.7 Hz). C NMR (125 MHz, δ DMSO-d ): 160.59, 157.65,
C,
6
1
45.71, 143.40, 137.52, 130.79, 129.19, 128.46, 127.88, 127.37, 126.53,
25.35, 115.58, 112.47, 67.17, 53.46, 52.28, 50.30, 28.17.
Anal. C34H33N O : C,72.97; H,5.94; N,12.51; O,8.58; Found:
1
5
3
C,73.00; H,5.96; N,12.49; O,8.56. HPLC purity: 99.82%, retention
time: 2.933 min.
1-(3-(4-(4, 5-diphenyl-1H-imidazol-2-yl)phenoxy)propyl)-4-
(4-fluorophenyl)piperazine (7e)
Yield: 69%; mp: 161-163 °C; R = 0.53 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3344, 3124, 1651, 1332, 1271, 1139; 1H NMR (500
−1
MHz, δH, DMSO-d ): 12.48 (s, 1H), 7.98 (d, 2H, J = 8.7 Hz), 7.50 –
4.2.1.5. BACE-1 inhibition assay. A FRET-based BACE-1 activity de-
tection kit was used to evaluate all of the compounds for their
BACE-1 inhibitory potency. The assay involved the breaking of the
substrate by BACE-1, resulted in an increase in the fluorescent
signal, and was done following the protocol given by the manu-
facturer. The detection kit includes human BACE-1 enzyme, stop
solution, BACE-1 substrate (7-Methoxycoumarin-4-acetyl-[Asn670,
Leu671]-Amyloid β/A4 precursor protein 770 fragments 667-676-
(2, 4-dinitrophenyl) Lys-Arg-Arg amide trifluoroacetate salt), fluo-
rescent assay buffer, and assay standard. Inhibitors with various
concentrations were used, likely to suppress enzymes in a par-
ticular range of 20-80%. The intensity of fluorescence was imme-
diately monitored after the BACE-1 enzyme was introduced, with
the excitation and emission wavelengths set at 320 and 405 nm,
respectively. Plates were incubated for about 2 h at 37°C and Mul-
timode Microplate Reader was used to record the intensity of flu-
orescence. Following expression were used to determine the% in-
hibition: [(IFo-IFi)/IFo] × 100, where IFi and IFo are the intensi-
ties of fluorescence obtained with or without the presence of in-
hibitor, respectively, and linear regression graph (Graph Pad Prism
5.1) were used to calculate the IC50 values.
6
7
.27 (m, 10H), 6.77 – 6.89 (m, 4H), 6.51 (d, 2H, J = 8.6 Hz), 4.02
(
(
t, 2H, J = 5.7 Hz), 3.28 – 3.14 (m, 4H), 2.78 – 2.53 (m, 6H), 1.99
13
t, 2H, J = 5.7 Hz). C NMR (125 MHz, δ DMSO-d ): 160.30 (d,
C,
6
J C-F = 228.7 Hz), 157.98, 148.87, 145.29, 130.52, 129.77, 128.52,
27.84, 127.15, 126.84, 125.99, 118.23, 116.18, 115.07, 67.74, 53.00,
1
5
2.78, 51.21, 27.48.
Anal. C34H33FN O: C,76.67; H,6.24; F,3.57; N,10.52; O,3.00;
4
Found: C,76.69; H,6.21; F,3.55; N,10.50; O,3.03. HPLC purity:
8.85%, retention time: 3.680 min.
-benzyl-4-(3-(4-(4,
yl)phenoxy)propyl)piperazine (7f)
9
1
5-diphenyl-1H-imidazol-2-
Yield: 72%; mp: 163-165 °C; R = 0.42 (MeOH:DCM, 2:98 v/v);
f
IR (KBr, cm ): 3345, 3122, 1661, 1281, 1132; 1H NMR (500 MHz,
−1
δH, DMSO-d ): 12.50 (s, 1H), 8.02 (d, 2H, J = 8.7 Hz), 7.49 – 7.47
6
(
m, 6H), 7.29 –7.28 (m, 5H), 7.24 – 7.21 (m, 4H), 7.01 (d, 2H, J = 8.7
Hz), 4.15 (t, 2H, J = 5.7 Hz), 3.54 (s, 2H), 2.47 (t, 2H, J = 5.8 Hz),
.51 – 2.49 (m, 8H), 2.05 – 1.99 (m, 2H). 13C NMR (125 MHz, δC,
DMSO-d ): 160.16, 145.82, 143.39, 138.72, 130.15, 129.79, 128.78,
2
6
1
28.43, 127.83, 127.45, 127.33, 126.24, 126.07, 115.59, 67.20, 63.72,
3.51, 52.46, 28.14.
Anal. C35H36N O: C,79.50; H,6.86; N,10.60; O,3.03; Found:
5
4
C,79.53; H,6.82; N,10.57; O,3.06. HPLC purity: 98.98%, retention
time: 3.880 min.
4.2.1.6. Propidium iodide displacement assay. Propidium iodide is
tending to bind to the peripheral active site of eeAChE, where
the eeAChE inhibitor competitively displaces the propidium io-
dide. This displacement was measured by the propidium iodide
displacement assay to determine the capability of binding of in-
hibitors to the PAS of eeAChE. The solution of eeAChE enzyme (5
U) in tris buffer pH 8.0 was prepared. Various test compounds with
different concentrations were incorporated into the solution and
incubated for 6 h at 25 °C. Propidium iodide was added after incu-
bation, and fluorescence was measured at wavelengths of 535 nm
(excitation) and 595 nm (emission) after 10 minutes.
1
-benzhydryl-4-(3-(4-(4,
yl)phenoxy)propyl)piperazine (7g)
Yield: 67%; mp: 167-169 °C; R = 0.61 (MeOH:DCM, 2:98
5-diphenyl-1H-imidazol-2-
f
v/v); IR (KBr, cm 1): 3334, 3121, 1663, 1268, 1128; H NMR
−
1
(
500 MHz, δH, DMSO-d ): 12.51 (s, 1H), 8.07 (d, 2H, J = 8.7
6
Hz), 7.54 – 7.48 (m, 10H), 7.28 –7.27 (m, 4H), 7.24 – 7.23 (m,
H), 7.05 (d, 2H, J = 8.7 Hz), 5.15 (s, 1H), 4.18 (t, 2H, J = 5.6
Hz), 2.71 (t, 2H, J = 5.7 Hz), 2.67 – 2.59 (m, 4H), 2.53 – 2.47
6
13
(
m, 4H), 2.12 – 2.03 (m, 2H). C NMR (125 MHz, δ DMSO-
C,
d6): 159.96, 145.06, 143.44, 140.68, 130.95, 129.29, 129.18, 128.35,
27.83, 127.48, 127.40, 126.73, 126.57, 114.49, 77.67, 67.25, 53.65,
1
4.2.1.7. Aβ inhibition- thioflavin T assay. The compound 6f was
tested for the AChE-induced Aβ1-42 aggregation inhibitory activ-
ity by the thioflavin T assay. 2000 mM stock solution was obtained
by dissolving Aβ1-42 in 1% v/v solution of ammonium hydroxide
and stored at 80°C. Initially, DMSO was used to dissolve the test
compounds, and further dilutions were prepared using PBS pH 7.4.
For self-induced Aβ1-42 aggregation inhibition experiment, Aβ1-42
5
2.52, 52.28, 28.11.
Anal. C41H40N O: C,81.42; H,6.67; N,9.26; O,2.65; Found:
4
C,81.45; H,6.70; N,9.23; O,2.68. HPLC purity: 98.97%, retention
time: 5.667 min.
4
4
.2. Pharmacology
.2.1. In vitro studies
11

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
(
10 μM, 10 ml) were incubated for 48 h at 37°C in the presence
grid generation, the prepared co-crystallized protein structure was
or absence of inhibitor (5 μM, 10 μM, and 20 μM; 10 ml). Blank
assays were carried out without Aβ1-42, only with PBS pH 7.4 in
the presence or absence of inhibitor. Thioflavin T (5 μM) in the
glycine-NaOH buffer (50 μM) pH 8.0 was added to the assay mix-
ture and intensities of fluorescence were measured at excitation
and emission wavelengths of 485 nm and 528 nm, respectively.
Following expression were used to determine the % inhibition of
self-induced Aβ1-42 aggregation: (IFc-IFi/IFc) × 100, where IFi and
IFc are the intensities of fluorescence found with or without the
presence of inhibitor, respectively. Every experiment was executed
in triplicates. For the Aβ1-42 AChE-induced aggregation inhibition
experiment, Aβ1-42 (10 μM, 2 ml) and AChE (230 μM, 16 ml) were
incubated for 48 h at 37°C with or without the presence of in-
hibitor (5 μM, 10 μM, and 20 μM; 2 ml).
used to determine the active sites present in the 10 × 10 × 10
˚
A distance from the centric co-crystallized ligand (donepezil). By
utilizing the LigPrep module, the generation of stable conformers
(compound 6f and donepezil) was incorporated, further which was
docked using Schrodinger Maestro’s Glide module 2018–1. A thor-
ough assessment of interactions was carried out using the Glide XP
visualizer tool.
4
.3.2. Molecular dynamics
To confirm the binding strength and pattern of the compound
f-AChE and 6f-BACE-1 complex using Desmond, a molecular dy-
6
namics simulation run of 100 ns was conducted. The clear-cut wa-
ter environment was created by soaking the ligand-protein com-
plex in the TIP3P molecules of water encompassed by the or-
thorhombic water box. The prepared complex system was neutral-
ized by adding the necessary counter ions, and the isosmotic salt
environment was maintained by adding 0.15 M NaCl. The system’s
energy minimization was achieved by using a combined gradient
algorithm with maximal 2000 interactions with convergence crite-
ria of 1 kcal/mol/A. Simulation run of 100 ns with periodic bound-
ary conditions under isothermal-isobaric ensemble (NPT) was per-
formed after energy minimization, and the system temperature
and pressure were set respectively at 300 K and 1,013 atmospheric
bar.
4
4
.2.2. In vivo studies
.2.2.8. Animals. The rats of either sex having weight 220-270 g
of both the sexes were obtained from the Institute of Medical Sci-
ences (IMS), Banaras Hindu University (BHU), Varanasi. They were
kept in a polyacrylic cage in a group of six per cage, a balanced
diet and water were provided. Temperature condition (25 ± 2°C)
and relative humidity (55 ± 10%) were maintained for rats with
light/dark cycles for 12 h. The Committee on Institutional Animal
Ethics (Dean/2017/CAEC/93) approved the study protocol.
4
.2.2.9. Cognition enhancement testing in rats by using scopolamine-
4
.3.3. In silico determination of drug-like properties
induced amnesia model. The solution of scopolamine hydrobro-
mide was prepared in sterilized normal saline. Test compound 6f
and donepezil (standard) were dissolved in 0.3% w/v solution of
sodium carboxymethyl cellulose. The rats were grouped as control,
scopolamine, donepezil, and 6f. Each group contains six animals.
Donepezil and 6f were administered orally queue die to the ani-
mal groups for seven days. Only 0.3% w/v solution of sodium car-
boxymethyl cellulose was given as a vehicle for scopolamine group
rats.
Using Schrodinger Maestro’s QikProp module 2018–1, the fea-
tures of drug-likeliness were discovered. The properties of drug-
likeliness in the molecule were determined by predicting many de-
scriptors, such as QPlogBB, PSA (polar surface area), and others, us-
ing Lipinski’s rule of five (accept HB ≤10, donor HB < 5, QPlogPo/w
<
5, mol MW < 500).
4
.4. Statistical analysis
4
.2.2.10. Y-maze test. The Y-maze apparatus comprises 3 arms
The values are displayed as Mean ± SEM for each experimental
maze. Instant and short working memory was estimated with the
help of this apparatus. After 30 min of the treatment on the sev-
enth day, all animals were injected with scopolamine hydrobro-
mide intra-peritoneally except the control group. The rats were in-
dependently placed at the center of the maze. In the beginning,
the rats were often going into the arm of facing, so the first entry
was deleted from the calculations. The spontaneous alternations
and total arm entries were recorded for about 8 min. The mem-
ory betterment score was estimated by using following expression,
percent spontaneous alteration = [number of spontaneous alterna-
tions / (total arm entries-2)] × 100 [45].
group. One-way ANOVA or two-way ANOVA was used by statisti-
cal analysis software (Graph Pad Software, Inc., San Diego, CA) to
evaluate differences between two groups (study and test).
Credit author statement
Salunke Prashant Ramrao: His research contribution includes
planning, carried out the experiments, design, synthesis, and char-
acterization, in-vitro evaluation of novel molecules.
Akash Verma: His research contribution includes rationale de-
signing, writing, analyze the data, in-vitro evaluations of novel
molecules.
4
4
.3. Computational studies
Prabhash Nath Tripathi: His research contribution includes ra-
tionale designing, and in-vivo evaluation.
.3.1. In-silico docking simulations
Digambar Kumar Waiker: His research contribution includes
rationale designing, writing, in-silico estimation of various novel
molecules.
The study of molecular docking was conducted to evaluate the
binding nature and interactions of the active site for compound
6
f on AChE (PDB Code: 4EY7), BChE (PDB Code:4TPK), and BACE-
Sushant Kumar Shrivastava (Co-Author): His research con-
tribution includes planning, supervised the work, rationale de-
signing, characterization, writing, biological evaluation of various
molecules.
1
(PDB Code: 2ZJM) [46]. The protein’s structure was processed
by utilizing the Protein Preparation Wizard module. Hydrogen was
initially incorporated and partial charges were allocated by utiliz-
ing the force field OPLS-2005. Using Prime the missing side chains
and loops were incorporated. The water molecules from the het-
˚
eroatoms with a range greater than 5 A were extracted, and het-
Declaration of Competing Interest
ero atom states were generated using the Epik at pH 7.0. Besides,
the PROPKA method was used to optimize the protein structure
at pH 7.0 and minimized by maintaining the convergence heavy
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
˚
RMSD atoms at 0.30 A at a restricted minimization. For receptor
12

S.P. Ramrao, A. Verma, D.K. Waiker et al.
Journal of Molecular Structure 1246 (2021) 131152
Acknowledgement
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, 6-diaryl-1, 2, 4-triazinyloxyacetyl-4-substituted thiosemicarbazides, triazoles
For conducting NMR experiments, the authors gratefully ac-
knowledge the Central Instrument Facility (CIF), Indian Institute of
Technology (Banaras Hindu University), Varanasi.
[
[
[
H
2 O 2
5
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