Design, synthesis and evaluation of some N-methylenebenzenamine derivatives as selective acetylcholinesterase (AChE) inhibitor and antioxidant to enhance learning and memory

Article abstract of DOI:10.1016/j.bmc.2017.01.010

Series of some 3,5-dimethoxy-N-methylenebenzenamine and 4-(methyleneamino)benzoic acid derivatives comprising of N-methylenebenzenamine nucleus were designed, synthesized, characterized, and assessed for their acetylcholinesterase (AChE), butyrylcholinesterase (BChE) inhibitory, and antioxidant activity thereby improving learning and memory in rats. The IC₅₀values of all the compound along with standard were determined on AChE and BChE enzyme. The free radical scavenging activity was also assessed by in vitro DPPH (2,2-diphenyl-1-picryl-hydrazyl) and hydrogen peroxide radical scavenging assay. The selective inhibitions of all compounds were observed against AChE in comparison with standard donepezil. The enzyme kinetic study of the most active compound 4 indicated uncompetitive AChE inhibition. The docking studies of compound 4 exhibited the worthy interaction on active-site gorge residues Phe330 and Trp279 responsible for its high affinity towards AChE, whereas lacking of the BChE inhibition was observed due to a wider gorge binding site and absence of important aromatic amino acids interactions. The ex vivo study confirmed AChE inhibition abilities of compound 4 at brain site. Further, a considerable decrease in escape latency period of the compound was observed in comparison with standard donepezil through in vivo Spatial Reference Memory (SRM) and Spatial Working Memory (SWM) models which showed the cognition-enhancing potential of compound 4. The in vivo reduced glutathione (GSH) estimation on rat brain tissue homogenate was also performed to evaluate free radical scavenging activity substantiated the antioxidant activity in learning and memory.

Full text of DOI:10.1016/j.bmc.2017.01.010

Accepted Manuscript
Design, synthesis and evaluation of some N-methylenebenzenamine derivatives
as selective acetylcholinesterase (AChE) inhibitor and antioxidant to enhance
learning and memory
Sushant K. Shrivastava, Pavan Srivastava, T.V.R. Upendra, Prabhash Nath
Tripathi, Saurabh K. Sinha
PII:
S0968-0896(17)30043-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.010
BMC 13489
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 September 2016
9 January 2017
10 January 2017
Please cite this article as: Shrivastava, S.K., Srivastava, P., Upendra, T.V.R., Nath Tripathi, P., Sinha, S.K., Design,
synthesis and evaluation of some N-methylenebenzenamine derivatives as selective acetylcholinesterase (AChE)
inhibitor and antioxidant to enhance learning and memory, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.01.010
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Design, synthesis and evaluation of some N-
methylenebenzenamine derivatives as
selective acetylcholinesterase (AChE)
inhibitor and antioxidant to enhance
learning and memory
Leave this area blank for abstract info.
Sushant K Shrivastavaa, Pavan Srivastavaa, T V R Upendraa, Prabhash Nath Tripathia, and Saurabh K Sinhab
Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology
(Banaras Hindu University), Varanasi-221005, U.P., India.
b Department of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur-313001, Rajasthan, India.

Bioorganic & Medicinal Chemistry
Design, synthesis and evaluation of some N-methylenebenzenamine derivatives
as selective acetylcholinesterase (AChE) inhibitor and antioxidant to enhance
learning and memory
Sushant K Shrivastavaa, Pavan Srivastavaa, T V R Upendraa, Prabhash Nath Tripathia, and Saurabh K
Sinhab
a Pharmaceutical Chemistry Research Laboratory, Department of Pharmaceutics, Indian Institute of Technology (Banaras Hindu University), Varanasi-221005,
U.P., India.
bDepartment of Pharmaceutical Sciences, Mohanlal Sukhadia University, Udaipur-313001, Rajasthan, India.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Series of some 3,5-dimethoxy-N-methylenebenzenamine and 4-(methyleneamino)benzoic acid
derivatives comprising of N-methylenebenzenamine nucleus were designed, synthesized,
characterized, and assessed for their acetylcholinesterase (AChE), butyrylcholinesterase (BChE)
inhibitory, and antioxidant activity thereby improving learning and memory in rats. The IC50
values of all the compound along with standard were determined on AChE and BChE enzyme.
The free radical scavenging activity was also assessed by in-vitro DPPH (2, 2-diphenyl -1-picryl
-hydrazyl) and hydrogen peroxide radical scavenging assay. The selective inhibitions of all
compounds were observed against AChE in comparison with standard donepezil. The enzyme
kinetic study of the most active compound 4 indicated uncompetitive AChE inhibition. The
docking studies of compound 4 exhibited the worthy interaction on active-site gorge residues
Phe330 and Trp279 responsible for its high affinity towards AChE, whereas lacking of the
BChE inhibition was observed due to a wider gorge binding site and absence of important
aromatic amino acids interactions. The ex vivo study confirmed AChE inhibition abilities of
compound 4 at brain site. Further, a considerable decrease in escape latency period of the
compound was observed in comparison with standard donepezil through in-vivo Spatial
Reference Memory (SRM) and Spatial Working Memory (SWM) models which showed the
cognition-enhancing potential of compound 4. The in-vivo reduced glutathione (GSH)
estimation on rat brain tissue homogenate was also performed to evaluate free radical
scavenging activity substantiated the antioxidant activity in learning and memory.
Available online
Keywords:
Learning and Memory
Acetylcholinesterase Inhibitor
Schiff base
Pterostilbene
Docking
2009 Elsevier Ltd. All rights reserved.
 Corresponding author. Tel.: +919452156527; fax: +91-542-2368428; e-mail: skshrivastava.phe@itbhu.ac.in

1.
Introduction
Pyrrolo-isoxazole derivatives are also mentioned in literature as
potent anticholinesterase and anti-amnesic agents. In our previous
studies, various Schiff bases of 4-aminopyridine16 and
semicarbazone17 were synthesized and evaluated for AChE
inhibitory activity. The Schiff bases of p-aminobenzoic acid have
been reported for various pharmacological activities, but it had
never been studied for selective AChE inhibition in cognitive
dysfunction. Schiff bases could be a promising pharmacophore
to treat the memory and learning.
The acetylcholine (ACh) plays
a significant role in
strengthening of synaptogenesis1 of active neurons for up-
regulating the memory and learning in humans.2 It is not only
parasympathetic neurotransmitter but also act as the memory
enhancer.3 Acetylcholinesterase (AChE) is a cholinesterase
enzyme that breaks the ACh, whereas inhibitors of AChE impede
the breakdown of acetylcholine (ACh) and improves the neuronal
acetylcholine amount. The crystal structure of AChE suggested
that its active site possessed a 20 Å deep gorge. Initially, the
binding of Ach at acetylcholinesterase enzyme occurs at the outer
gorge, known as the peripheral anionic site (PAS) lined with
Trp279, Tyr70, Tyr121, and Phe290. The bottom of the gorge
containing four main subsites, (i) the esteratic site, (ii) the
oxyanion hole, (iii) the anionic subsite and, (iv) the acyl pocket,
where the hydrolysis of Ach takes place.4 The
butyrylcholinesterase (BChE), a non-specific cholinesterase
enzyme found in both plasma and brain and capable of
hydrolyzing ACh and the other esters. The BChE also get
inhibited by the non-specific AChE inhibitors, leads to
hepatotoxicity and other side effects.5 Moreover, the primary
structural difference between AChE and BChE is the absence of
the PAS moiety in BChE at the top of the gorge; hence, it seems
clear that an inhibitor of cholinesterase, exhibiting strong
interaction with the PAS would have greater AChE specificity
over BChE.4 The kinetics of such inhibitors are reported as
reversible competitive, non-competitive, mixed and un-
competitive inhibition.6
Currently, some Schiff-bases have been investigated as
effective scavengers of Reactive Oxygen Species (ROS) that acts
as an antioxidant. The free radical scavenging effects of the
Schiff base may be due to its imino group which extends free
radical delocalization.18 Also, the nitrogen of the azomethine
group (>C=N) in Schiff bases acts as a good donor of the electron
due to the availability of loan pair of electrons on the nitrogen
atom. The electron donating character of the double bond, and
low electronegativity of nitrogen makes Schiff base an active
ligand for antioxidant activity.19
Recent studies proved that increased oxidative stress (an
imbalance between the generation of free radicals and the ability
of the body to counterattack or detoxify their damaging effects) is
one of the causes of impaired learning behavior that modestly
reduces the motor activity.20 Hence, the compounds like Schiff-
base having both AChE as well as free radical scavenging
activity could be more effective in the study of learning and
memory.
Natural products polyhydroxy stilbenes like pterostilbene have
been extensively exploited for anticancer, anti-inflammatory,
neuroprotective, antioxidant and AChE inhibitory activities with
low neurotoxicity that inferred the potential tropism of
polyhydroxy stilbene in uplifting learning and memory.21(a,b)
Recent patent suggests that pterostilbene is effectively improvise
working memory and reversing memory deficit.22 The isosteric
replacement of ethenyl carbon of stilbene with nitrogen shows
cation-π stacking interaction with the AChE Trp-84 at anionic
subsite.23 This imine also incorporates the required features for
metal chelation and antioxidant activities as stated in the
literature. The Schiff bases of polymethoxy stilbenes are reported
as novel intermediate for heterocyclic’s (indazoles) and suggest
to be a promising lead scaffold for Rational Drug Design of
AChE inhibitors.24(a,b)
The deficits of working and reference memory due to the lack
of AChE are considered as a feature of many neuroinflammatory
disorders.7 These short-term (working) memory and long-term
(reference) memories are ushered to learn well under cognition
and behavioral studies.8(a,b)
In search of a novel drug, Schiff base (azomethine -C=N-
functional group) have been attracting scaffold because of its
various therapeutic applications in drug discovery such as
antioxidant,9 anti-Alzheimer,10 antituberculosis,11 anticancer,12
antiepileptic,13 etc.
The Schiff bases of p-aminobenzoic acid are reported to
possess a high tendency to form metal chelate complexes that
14(a-e)
elicit a plethora of pharmacological activities.
Several
amide- and imide derivatives of m- and p-aminobenzoic acid
have been synthesized and evaluated for their AChE activity.15
Figure 1. The Design of compounds as potential agents to enhance learning and memory (Series-1) 3,5-dimethoxy-N-Methylenebenzenamine derivatives
(Series-2) 4-(methyleneamino)benzoic acid derivatives.

R1
R1
R'2
R'1
NH2
R'2
R'1
N
MeOH, glacial acetic acid,
4-6 h, Reflux
O
R2
+
R'3
R'3
R2
Scheme 1
Table 1. Synthesized N-Methylenebenzenamine derivative
R’1
R’2
R’3
R1
R2
Yield (%)
Rf*
Compound
Code
Melting
Point (°C)
Series 1: 3,5-dimethoxy-N-Methylenebenzenamine derivative
1a
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
2-OH
H
79
72
76
92-94
0.84
0.85
0.89
2
4-NO2
4-OCH3
H
84-86
3
C6H5
115-117
3,4,5-tri
OCH3
4
H
H
OCH3
OCH3
OCH3
OCH3
H
H
55
59
79-81
0.82
0.83
5b
2- NO2
122-124
Series 2: 4-(methyleneamino)benzoic acid derivative
6c
7d
8e
9f
COOH
COOH
COOH
COOH
H
H
H
H
H
H
H
H
2- OH
H
H
H
H
H
84
79
77
75
276-278
121-123
220-222
180-182
0.81
0.78
0.80
0.86
2- NO2
4- NO2
3,4,5-tri
OCH3
10g
11
COOH
COOH
COOH
H
H
H
H
H
H
H
71
74
71
180-182
194-196
210-212
0.90
0.87
0.74
4-OCH3
C6H5
CH3
4-OH, 3-
OMe
12
Reference; 1a: 11a, 5 b: 11b, 6c: 12a, 7d: 12b, 8 e: 12c, 9 f: 12d, 10g: 12e, *: Solvent system: Chloroform / methanol (4.5:0.5)
In this paper, we have designed and synthesized some lipophilic
3,5-dimethoxy-N-methylenebenzenamine and 4-
showed a characteristic sharp singlet peak of imine (N=CH)
approximately at 8.34 δ value (except compound 3, 11 and 12).
Lead compound 4 showed characteristic singlet peak of the 3,4,5-
trimethoxy group at 3.93 δ of nine protons and singlet peak of the
3,5 dimethoxy group at 3.82 δ of six protons. In second series
compounds showed a carboxylate peak near 12 δ value having
integration value of 1 as a singlet at down field.
(methyleneamino)benzoic acid derivatives having the N-
methylenebenzenamine nucleus (Figure 1) and evaluated for the
in-vitro AChE and BChE inhibitory activity. The enzyme kinetic
study was performed to assess its type of inhibition. Further, the
most active compound 4 was assessed for the in-silico docking
analysis followed by ex vivo studies of AChE inhibition and the
in-vivo Spatial Reference Memory (SRM), Spatial Working
Memory (SWM) evaluation. We have also performed an in-vitro
DPPH (2, 2-diphenyl -1-picryl -hydrazyl) and hydrogen peroxide
radical scavenging assay for free radical scavenging activity
further this study was supported by in-vivo reduced glutathione
estimation.
2.2.
Cholinesterase Inhibition
The strength to inhibit AChE and BChE was observed through
their IC50 values determination using Ellman method of all
synthesized compounds (series 1 & 2) and standard donepezil.
The designed compounds (1-12) were initially expected to
inhibit both AChE & BChE with the same potential. However,
the results of the Ellman test showed that compounds of series 1
& 2 were the selective inhibitor of electric eel AChE having IC50
value in the micromole (0.82 to 25.53 µM) over BChE IC50 value
in the millimole (0.21 to 0.83 mM). The compound 4 (Figure 2)
bearing 3,4,5 tri-methoxy group on aldehydic phenyl ring showed
a more potent AChE inhibition over synthesized compounds of
both series 1 & 2. The non-polar group (OCH3) has modulated
the AChE inhibitory potency due to increased lipophilic
characteristic.
2.
Results and discussion
2.1.
Chemistry
The compounds were synthesized by the nucleophilic addition
of the amine to the carbonyl group forming an unstable
aminomethanol intermediate followed by dehydration in an
acidic environment to generate an imine (Scheme 1 and Figure 1
of supporting information).25 Basically, in this synthetic
condition the 3,5-dimethoxyaniline and p-aminobenzoic acid
(PABA) acted as the nucleophile for various carbonyl
compounds as listed in Table 1. Preliminary identification of
imine was confirmed by positive Dragendroff test on TLC (Thin
layer chromatography). The purity of the compounds was
checked and verified by state of the art spectroscopic technique
and elemental analysis. The FT-IR of all the compounds showed
a diagnostic stretching vibration of (-C=N) imine group in the
All 4-(methyleneamino) benzoic acid derivatives of series 2
possessed a moderate inhibitory activity towards AChE owing to
the polar group (-COOH) of benzoic acid as the common
scaffold.
The
3,5-dimethoxy-N-methylenebenzenamine
derivative of series 1 having bulky 3,5-dimethoxy group on
phenyl ring that played a significant role in the structural-activity
range of 1578-1610 cm-1. H NMR spectra of all compounds
1

Table 2. IC50 values of the synthesized derivatives
AChE
BChE
aSelectivity Index
Compound
code
IC50 (µM) ± SEM
IC50 (µM) ±
SEM
Donepezil *
Series 1
0.04 ± 0.01
15.24 ± 0.88
381
± 6.33
1
25.53 ± 1.12
7.01 ± 0.34
4.37 ± 0.23
0.82 ± 0.05
14.35 ± 0.71
746.7 ± 37.27
452.4 ± 3.04
284.2 ± 10.96
328.0 ± 1.53
456.7 ± 9.17
29.25 ± 1.12a,b
64.52 ± 2.23 a,b
65.06 ± 2.78 a,b
401.96 ± 9.45a
2
3
4**
5
31.83 ± 1.10 a,b
Series 2
6
18.03 ± 0.92
12.64 ± 0.59
6.00 ± 0.24
5.95 ± 0.27
11.55 ± 0.42
6.09 ± 0.29
3.93 ± 0.17
717.2 ± 5.88
502.1 ± 1.66
386.9 ± 1.94
217.1 ± 8.38
378.2 ± 19.10
291.5 ± 2.20
839.0 ± 1.53
39.77 ± 1.31 a,b
39.71 ± 1.22 a,b
64.46 ± 2.12 a,b
36.48 ± 1.88 a,b
32.73 ± 1.12 a,b
47.85 ± 1.92 a,b
213.70 ± 7.55 a,b
7
8
Figure 3. Lineweaver-Burk plot resulting from substrate [S]–velocity
[V] curves of AChE activity for compound 4
9
10
11
12
2.4. Molecular docking
To understand the cholinesterase inhibition profile, the docking
was done on AChE complexed with E2020 (donepezil) (PDB
Code: 1EVE) and BChE complexed with N- [(3R)-1-(2,3-
dihydro-1H-inden-2-yl)piperidin-3-yl]methyl-N-
*:Donepezil, ** compound 4. Values are expressed in the mean ± SEM
(n=6), ap< 0.05 and bp< 0.05 as compared to the donepezil and compound 4,
(one way ANOVA followed by Bonferronipost tests).
aSelectivity for AChE is defined as IC50(BChE)/IC50(AChE).26
(2methoxyethyl)naphthalene- 2-carboxamide (3F9) (PDB Code:
4TPK) (Maestro 10.5.014, Glide, Schrödinger, 2016-1)(Figure 2)
to provide a better interpretation of the biological profile of
compound 4 and donepezil toward enzymes. Validation of
docking protocol was done by measuring the root mean square
deviation (RMSD) between the actual pose and predicted pose of
ligand to the protein. The RMSD of the reported protocol was
found to be 0.68, and 1.2868 which fell within the acceptable
limit of 2.0 Å for AChE and BChE respectively.
relationship. The presence of methoxy group modulated the
AChE inhibitory potency due to the high lipophilic (log P)
characteristic of the molecule. The selectivity index of all
synthesized compounds was calculated and mentioned in Table
2. The compound 4 showed a significant selectivity index
(401.96 ± 9.45) in comparison to standard donepezil (381 ±
6.33).
O
.
Superposition of the best pose of compound 4 and donepezil at
the active site revealed that the binding mode of compound 4 at
the PAS region of AChE resembled very much to that of
donepezil. In donepezil, residue Trp84 was interacting with
ligand via π–π stacking interaction at the bottom, while Phe330
and Trp279 were interacting with the ligand through cation–π
and π–π stacking interaction at the midpoint and the entrance of
the gorge respectively (Figure 4A).The most active compound 4,
which has a Glide score of -7.7 kcal/mol exhibited hydrophobic
interaction at PAS resulting in strong binding affinity. The
binding affinity was observed through the aromatic ring of the
3,5-dimethoxy ring formed π−π parallel Edge to Face stacking
interactions with Phe330, and the tri-methoxybenzene aromatic
ring formed π−π Face to Face stacking interactions with Tyr279
(Figure 4B).
N
O
Donepezil
O
OCH3
H3CO
N
H3CO
OCH3
Compound 4
H3CO
N
O
N
O
3F9
Figure 2. Chemical structure of donepezil, Compound 4, and 3F9
Superposition of the best pose of compound 4 and 3F9 was
observed at the active site of BChE to gain insight on their high
selectivity for AChE over BChE. In 3F9, a strong cation−π
stacking interaction was observed between the nitrogen of the
piperidine and Tyr332, while the naphthalene moiety was fully
occupied the acyl-binding pocket where it stacks to the Trp231
via π−π stacking interaction. The docking of compound 4 on
4TPK (score -4.8 kcal/mol) was not shown any hydrophobic
interaction rather exhibited electrostatic interactions at the active
site resulted in its weak binding affinity. However, the tri-
methoxybenzene aromatic ring formed π−π Edge to Face
stacking interactions with Trp82 (Figure 4C). All these results
suggested that compound 4 was unable to bind efficiently owing
2.3.
Enzyme kinetics studies
Further, the compound 4 was subjected to enzyme kinetics
study. Lineweaver–Burk plot of compound 4 resulted in a line
parallel to the original enzyme-substrate plot, with a higher y-
intercept. This characteristic pattern indicated an uncompetitive
inhibition of AChE enzyme (Ki = 0.72 ± 0.06 µM) (Figure 3),
due to the possible interaction of methoxy group (OCH3) at the
peripheral anionic site of AChE.

Figure 4. (A) Docking model of donepezil at the active sites of AChE. (B) Docking model of compound 4 at the active sites of AChE. The interactions are
shown in green. (C) Superimposition docking model of 3F9 and compound 4 at the active sites of at the active sites of BChE.
Table 3. QikProp Analysis
Compound
CNSa
SASAb
563.19
714.49
QPlogBBc
-0.088
QPlogPo/wd
3.82
QPPCacoe
6411.31
884.31
QPPMDCKf
3686.07
QPlogKhsag
0.04
4
1
1
Donepezil
0.11
4.44
479.198
0.618
aCNS - This exhibits the predicted central nervous system activity.( –2 to +2)
bSASA- Total solvent accessible surface area (SASA) insquare angstroms using a probe with a 1.4 Å Radius. (300 to 1000)
c
QPlogBB - Predicted brain/blood partition coefficient. Note: QikProp predictions are for orally delivered drugs so, for example, dopamine and serotonin are
CNS negative because they are too polar to cross the blood-brain barrier (-3 to +1.2)
dQPlogPo/w - This gives the predicted octanol/water partition coefficient. (-2 to 6.5)
eQPPCaco - This gives the predicted apparent Caco-2 cell permeability in nm/sec. Caco-2 cells are a model for the gut-blood barrier. (<25 is considered poor and
>500 is considered excellent)
fQPPMDCK- Predicted apparent MDCK cell permeability in nm/sec using the Affymax scale. (<25 is considered poor and >500 is considered excellent)
gQPLog Khsa -Prediction of binding to human serum albumin. Predictions are for non-active transport. (–1.5 to 1.5)
to the (i) small molecular size of compound 4 relative to the
larger volume of the bottom gorge, good to accommodate bulkier
substrate. (ii) no major interaction at the peripheral site, choline
binding pocket, and catalytic site. (iii) lack of aromatic amino
acid at the bottom of the gorge for hydrophobic interaction.
a better log P value (3.27) than standard donepezil (4.24) and
considered optimal to cross the blood-brain barrier.
The combined result of in-vitro, and in-silico studies indicated
that compound 4 could be a good candidate for ex vivo and in-
vivo assessment of learning and memory.
This interaction study demonstrated that compound 4 strongly
bound to the binding PAS sites of AChE, supporting the potential
inhibitory activity of compound 4 which was supported by the
kinetic study exhibiting binding of compound 4 to the PAS sites.
2.6. Free radical scavenging activity
Recent studies proved that increased oxidative stress causes
impaired learning behavior and reduces motor activity. It is also
reported that the antioxidants and vitamin-rich diet could
improve the cerebellar physiology and motor learning of aged
rats and also improve the performance of learning in rat on water
maze model at the lower dose.29
2.5. Log P determination and in silico pharmacokinetic
parameters
It is a well-known fact that lipophilicity (log P) is an important
physicochemical parameter and it has the strong relationship with
in-vivo brain penetration of drugs. Compounds possess moderate
lipophilicity (lower than 4) often exhibit the highest uptake in the
brain.27 Other physicochemical parameters e.g. Caco-2 cell
permeability (PPCaco), brain/blood partition coefficient (log
BB), and MDCK cell permeability (PPMDCK) are also the
indicators of the lipophilicity of the molecule.28 We have
determined the partition coefficient of all the synthesized
The free radical scavenging activity of the synthesized
compound 4 was assessed by established in-vitro DPPH and
hydrogen peroxide methods. The in-vitro activity was performed
to determined the scavenging capability through DPPH (2,2-
diphenyl-1-picryl-hydrazyl)30 and Hydrogen peroxide radical
scavenging activity.31 The in-vitro results showed that compound
4 have better antioxidant activity compared to donepezil in both
assays. The result is summarized in Figure 5.
compounds
and standard
by octanol/water
system
2.7.
Biological Evaluation
(Supplementary Table 1) and QlogBB, QlogPo/w, QPPCaco,
QlogKhsa, and QPPMDCK were calculated through QikProp
software tool (Table 3). QikProp analysis resulted in an accurate,
quick, and comprehensive, prediction of adsorption, distribution,
metabolism, and excretion. The in-silico ADME/Tox Predictions
were analyzed through QikProp (Maestro 10.5.014, Schrödinger,
LLC, and New York-1). The result showed that compound 4 had
2.7.1. Ex vivo AChE inhibition
Ex vivo cholinesterase inhibition was determined by Ellman
method. The result showed that the compound 4 significantly
inhibited AChE activity in the brain compared to that of control
(Table 4). These results confirmed the ability of compound 4 to
cross the blood brain barrier.

differences [F (4, 24) = 17. 14, p < 0.05] among the groups in the
probe trial (Figure 6B). Post-hoc analysis indicated that the mean
time spent in the target quadrant of the maze by rats treated with
compound 4 and donepezil was significantly longer than the
control group. A significant time spent in the target quadrant
during the probe trial indicated the retention of spatial memory.
For the test of spatial working memory, the rat was expected to
find a platform placed in a new position during the first trial
(acquisition), whereas in the retrieval trial, the platform was kept
in its previous position. Statistical analysis using repeated
measure two-way ANOVA revealed that there were significant
differences among treatment [F (4, 100) = 10.94, p < 0.05] and
time [F (3, 100) = 51.76, p < 0.05]. Further there was a
significant interaction [F (12, 100) = 2.458, p < 0.05] between
treatment and time (Table 5). Post-hoc analysis showed that there
was no significant interaction among groups on day one but on
succeeding days the most active compound 4 (10, 20 and
30mg/Kg; p.o.) and donepezil (10 mg/Kg; p.o.) showed
significant differences in the escape latency compared to the
control. However, there was no decrease in escape latency
between the doses tested. Similarly, analysis of the retrieval trial
data revealed significant differences among treatment [F (4, 100)
= 45.15, p < 0.05] and time [F (3, 100) = 77.84, p < 0.05].
Further, there was a significant interaction [F (12, 100) = 3.220, p
< 0.05] between treatment and time. Post-hoc analysis revealed
that on day one, only the donepezil-treated group showed
reduced escape latency compared to the control. On days two,
three and four, the compound 4 (10, 20 and 30 mg/Kg; p.o.)
treated groups, and the donepezil-treated group showed reduced
escape latency than the control. However, there was a greater
decrease in escape latency in compound 4 (20 and 30 mg/Kg;
p.o.) treated groups compared to compound 4 (10 mg/Kg; p.o.)
treated group.
Table 4. Effect of compound 4 on acetylcholinesterase
activity in brain regions of the rat
Treatment
‘n’ moles of substrate
hydrolyzed/min/mg protein
[Dose(mg/kg)]
Control
46.56 ± 0.66
33.78 ± 0.94*
30.85 ± 0.47*
28.24 ± 0.76*
Compound 4 (10.0)
Compound 4 (30.0)
Donepezil (10.0)
Data are expressed as means± SEM (n = 3). Data were statistically
analyzed by one-way ANOVA. *p < 0.05 compared to the control.
2.7.2
In-vivo Evaluation
2.7.2.1. Learning and memory
The most active compound 4 was further evaluated for
Spatial Reference Memory (SRM) and Spatial Working Memory
(SWM) and observations were expressed as mean ± SEM. Spatial
reference memory data analyzed by repeated measure two-way
ANOVA revealed that there were significant differences among
treatment [F (4, 60) = 199.4, p < 0.05] and time [F (2, 60) =
1759, p < 0.05]. Post-hoc analysis showed that there was no
significant difference among the groups on day one. Further,
there was a significant interaction [F (8, 60) = 55.73, p < 0.05]
between treatment and time. However, on day two,
a
considerable decrease was observed in escape latency in
donepezil and compound 4 (20 and 30 mg/Kg; p.o.) treated
groups compared to the control. There was no significant
difference between compound 4, 20mg/Kg; p.o. and, 30mg/Kg;
p.o. treated groups (Figure 6A). To test whether this memory
trace was retained and consolidated for future navigation, the
probe trial was repeated after the twenty-four hours spatial
reference memory task. One-way ANOVA revealed significant
B
A
150
150
Ascorbic Acid 10 ug/ml
Ascorbic Acid 10 ug/ml
10ug/ml comp 4
10ug/ml comp 4
100ug/ml comp 4
100ug/ml comp 4
10ug/ml Donepezil
100ug/ml Donepezil
100
100
10ug/ml Donepezil
a,b
100ug/ml Donepezil
a
a,b
50
50
a,b,c,d
a
a,b,c,d
a,b,c
a,b,c
0
0
l
l
4
4
i
l
l
i
4
4
zil
epe
m
zil
epe
p
p
m
p
p
pez
one
D
m
co
pez
one
D
l
m
co
om
c
om
c
0 ug/
0 ug/
1
l
m
l
1
l
m
l
Don
d
m
i
/
l
l
Don
d
m
/
c
g/
0u
10
i
l
m
m
g/
0u
c
g/
0u
10
ug
10
/
m
m
g/
0u
ug
10
/
c A
i
ug
10
c A
b
i
ug
10
r
b
10
r
co
10
co
As
As
Figure 5. Graphical comparison of the in-vitro antioxidant effect of compound 4 and Donepezil (A) DPPH assay (B) H2O2 free radical scavenging assay. Values
a
c
d
are expressed as the mean ± SEM (n=3). p< 0.05, bp < 0.05 p< 0.05 and p < 0.05 compared to the control, compound 4 (10µg/ml), compound 4 (100µg/ml)
and donepezil (10µg/ml) respectively (one-way ANOVA followed by Newman-Keuls test).
Table 5. Nootropic effect of Compound 4 in rats on ‘escape latency’ in Acquisition phase of SWM
Treatment
Escape Latency (s)
Dose mg/Kg; p.o.
Control
Day 1
Day 2
Day 3
Day 4
17.45 ± 0.62
16.88 ± 0.61
17.57 ± 0.56
16.95 ± 0.73
16.87 ± 0.70
16.97 ± 0.80
14.38 ± 0.41a
15.45 ± 0.38a
15.69 ± 0.64a
15.23 ± 0.99a
17.38 ± 0.79
7.78 ± 0.27a
15.12 ± 0.80a,b
12.23 ± 0.63a,b,c
10.37 ± 0.46a,b,c
14.12 ± 0.78
7.33 ± 0.31a
10.20 ± 0.50a,b
8.87 ± 0.48a
8.92 ± 0.43a
Donepezil
10mg/Kg 4
20mg/Kg 4
30mg/Kg 4
Values are expressed in the mean ± SEM (n=6), ap< 0.05, bp< 0.05 and cp< 0.05, as compared to the control, donepezil and compound 4, (dose- 10mg/Kgp.o)
respectively (two way ANOVA followed by Bonferronipost tests).

Figure 6. Graphical comparison of nootropic effect (A) in terms of ‘the escape latency’ among various groups in SRM test days 1-3. Values are expressed as
a
b
c
the mean ± SEM (n=6). p< 0.05, p < 0.05 and p < 0.05 compared to the control, donepezil and compound 4 , dose 1 (10mg/Kg p.o) respectively (two-way
ANOVA followed by Bonferroni post-tests). (B) in terms of ‘the mean time spent in the target quadrant’ among various groups in ‘Probe Trial’ on day 4. Values
a
b
are expressed as the mean ± SEM (n=6). p< 0.05 and p < 0.05 compared to the control and donepezil respectively (one-way ANOVA followed by Newman-
Keuls test).
An overall positive effect of compound 4 was found
consistently throughout behavioral tests where it showed
significantly attenuated disruption of long-term spatial navigation
in both the SRM and SWM assays. Escape latency was found
same on the first day in all the groups including control and
standard but a significant difference observed on second day
onwards. The physicochemical parameters and appropriate
blood-brain barrier permeability reasons for its comparable drug
efficacy on day 2nd in the Spatial Reference Memory (SRM) and
Spatial Working Memory (SWM) of behavior test. The results
suggested that the escape latency of the compounds 4 treated
group was significantly (p<0.05) more than that of the control
.indicating an increased efficacy to the learning and memory
abilities. In conclusion, the compound 4 was found to be a
promising active molecule for uplifting the learning and memory
in rats.
In conclusion, from the above studies, we have successfully
identified a new class of potent cognition-enhancing chemical
entity. Among the identified compounds, compound 4 deserves
further studies which can lead to a discovery of a new lead
having a potent cognition-enhancing property.
30
control positive
a
Compound 4
b
Donepezil
20
10
0
l
i
4
z
ive
d
e
it
n
p
s
u
e
o
n
2.7.2.2. Reduced GSH estimation
po
l
Do
mp
Co
o
r
t
n
o
GSH is a low molecular weight tripeptide which plays a crucial
role in counterattacking ROS.32 Severe depletion of GSH level in
the brain due to the oral administration of aspartame was
observed.33 The method illustrated by Ellman34 was used for
determination of antioxidant activity. Moderate increase in GSH
level was evident among groups treated with compound 4 (24.52
±0.26 GSH (µg/mg protein)) compared to control, reflecting its
antioxidant nature, whereas the standard donepezil-treated group,
the GSH level was found 19.12±1.40 GSH (µg/mg protein),
which was insignificant compared to control.(Figure 7)
c
Figure 7. Graphical comparison of in-vivo Reduced GSH estimation.of
compound 4 and Donepezil. Values are expressed as the mean ± SEM (n=3).
ap< 0.05 and bp < 0.05 compared to the control and compound 4 respectively
(one-way ANOVA followed by Newman-Keuls test).
4.
Material and methods
4.1.
General
All reagents and solvents used in this study were of analytical
grade purity and were procured from Sigma-Aldrich (India).
Donepezil was obtained as a gift from Cipla Ltd. (Maharashtra,
India). The melting points of the compounds were determined in
open capillary tubes using a BI 9300 Bumstead/Electrothermal
Stuart (SMPIO) melting point apparatus and are uncorrected. The
reaction progression was monitored by thin layer
chromatography with ethyl acetate: hexane (3:7) as the mobile
phase on TLC silica gel 60 F254 aluminum sheets (Merck,
India). UV spectral analysis was performed using a JASCO
(Model 7800) UV-VIS spectrophotometer. FT-IR spectra were
recorded on a Shimadzu FT-IR 8400S spectrophotometer at the
3.
Conclusion
The methoxy-substituted compound 4 demonstrated potent
cognition-enhancing. In the docking studies, compound 4
confirmed its interaction with the important active-site gorge
residues Phe 330 and Trp279 responsible for its high affinity
towards AChE. The compound 4 showed a significant selectivity
index in comparison to standard donepezil in in-vitro studies. The
ex vivo study also confirmed the ability of compound 4 to cross
the blood brain barrier and selective AChE inhibition at
respective brain site owing to its appropriate physicochemical
parameter e.g. partition coefficient (log P), Caco-2 cell
permeability (PPCaco), brain/blood partition coefficient (log
BB), and MDCK cell permeability (PPMDCK), that elicited a
considerable decrease in escape latency in SRM and SWM in-
vivo models. Further, the in-vitro DPPH (2, 2-diphenyl -1-picryl -
hydrazyl) and hydrogen peroxide radical scavenging assay and
in-vivo reduced glutathione estimation supported that compound
4 would be a promising drug candidate to treat the learning and
memory disorders due to its antioxidant and selective AChE
inhibition activity.
1
scanning range 400–4000 cm-1. H spectra were recorded on a
JEOL AL 300 FT-NMR spectrometer in DMSO-d6.
Tetramethylsilane (TMS) was used as an internal standard.
Chemical shift values were expressed in parts per million (δ).
The purity of the final compounds (>95%) was determined by
elemental analysis. Elemental analysis was performed using an
Exeter CE-440 elemental analyzer.
4.2.
Chemistry

4.2.1.
General procedure for the synthesis of 1-12
4-(benzylideneamino)benzoic acid (7)
λmax (nm): 274. IR (KBr, cm-1): 3066-2854 (Carboxylic -OH str);
1681 (C=O str); 1595 (C=N str); 1429 (aromatic C=C str).1H-
NMR (300 Hz, δ H, DMSO):; 7.52-8.12 (m, 9H, aromatic); 8.41
(s, 1H, N=CH); 12.00 (s, 1H, COOH). Elemental analysis, for,
C14H11NO2, calculated (%): C 74.65, H 4.92, N 6.22; found (%):
C 74.36, H 4.97, N 6.90.
0.1M of para-aminobenzoic acid (Series 1) or 3,5-
dimethoxyaniline (Series 2) was dissolved in 10 mL of methanol
followed by addition of equimolar quantities of various
substituted aldehydes or ketones with slow and constant stirring.
Few drops of glacial acetic acid were added as an acid catalyst.
The reaction mixture was refluxed at 40˚C for 4-6 h*. After the
completion of the reaction, the solution was allowed to cool to
room temperature. The solution was then poured into a petri dish
and allowed to stay overnight for crystallization. The crystals
were collected, washed with acetone and recrystallized with
methanol.
4-(2-nitrobenzylideneamino)benzoic acid (8)
λmax (nm): 269. IR (KBr, cm-1): 3408 (Carboxylic –OH str); 1703
(C=O str); 1624 (C=N str); 1348 & 1288 (Nitro –NO str); 1519
(aromatic C=C str).1H-NMR (300 Hz, δ H, DMSO): 8.88 (s, 1H,
N=CH); 7.33-8.12 (m, 8H, aromatic); 12.01 (s, 1H, COOH).
Elemental analysis, for, C14H10N2O4, calculated (%): C 62.22, H
3.73, N 10.37; found (%): C 64.90, H 4.04, N 9.65.
* For Nitro and ketone compounds, the reaction was continued
for 16 h to 18 h.
2-(3,5-dimethoxyphenylimino)methyl)phenol (1)
4-(4-nitrobenzylideneamino)benzoic acid (9)
λmax (nm): 266. IR (KBr, cm-1): 1606 (C=N str); 3460 (Phenolic –
OH str); 1141 & 1207 (aryl alkyl ether –CO str); 1573 (aromatic
C=C str).1H-NMR (300 Hz, δ H, CDCl3): 3.83 (s, 6H, OCH3);
6.40-7.40 (m, 7H, aromatic); 8.60 (s, 1H, N=CH); 13.14 (s, 1H,
OH);. Anal. calcd (%) for C15H15NO3: C 70.02, H 5.88, N 5.44;
found (%): C 70.21, H 5.16, N 5.50.
λmax (nm): 277 . IR (KBr, cm-1): 3383 (Carboxylic –OH str); 1683
(C=O str); 1600 (C=N str); 1521 (aromatic C=C str); 1342 &
1294 (Nitro -NO str). 1H-NMR (300 Hz, δ H, DMSO): 7.52-8.25
(m, 8H, aromatic); 8.84 (s, 1H, N=CH); 12.10 (s, 1H, COOH).
Elemental analysis, for, C14H10N2O4, calculated (%): C 62.22, H
3.73, N 10.37; found (%): C 63.46, H 3.82, N 10.32.
N-(4-nitrobenzylidene)-3,5-dimethoxybenzenamine (2)
4-(3,4,5-trimethoxybenzylideneamino)benzoic acid (10)
λmax (nm): 267. IR (KBr, cm-1): 1593 (-C=N str); 1518 (aromatic
–C=C str); 1153 & 1205 (Nitro –NO str); 1468 (=C-H str).1H-
NMR (300 Hz, δ H, CDCl3): 3.83 (s, 6H, OCH3); 6.42 (m, 3H,
aromatic); 7.60- 7.77 (m, 2H, aromatic-NO2); 7.08 (d, J= 7.8Hz,
1H, aromatic-NO2); 8.28 (d, J = 7.5 Hz, 1H, aromatic-NO2); 8.93
(s, 1H, N=CH); Anal. calcd (%) for C15H14N2O4: C 62.93, H 4.93,
N 9.79; found (%): C 62.89, H 4.86, N 9.77.
λmax (nm): 280. IR (KBr, cm-1): 3070-2881 (Carboxylic –OH str);
1681 (C=O str); 1597 (C=N str); 1579 (aromatic C=C str); 1421
(-OH bending); 1286 & 1126 (aryl alkyl ether –CO str).1H-NMR
(300 Hz, δ H, DMSO): 3.71 (s, 9H, OCH3); 6.61-8.15 (m, 6H,
aromatic); 8.42 (s, 1H, N=CH); 12.0 (s, 1H, COOH). Elemental
analysis, for, C17H17NO5, calculated (%): C 64.75, H 5.43, N
4.44; found (%): C 65.05, H 5.41, N 4.25.
3,5-dimethoxy-N-((4-
methoxyphenyl)(phenyl)methylene)benzenamine (3)
4-((4-methoxyphenyl)(phenyl)methyleneamino)benzoic acid (11)
λmax (nm): 285. IR (KBr, cm-1): 2924-2777 (Carboxylic –OH str);
1662 (C=O str); 1600 (C=N str); 1174 & 1238 (Ether C-O str);
1319 (Carboxylic C-O str); 1495 (aromatic C=C str).1H-NMR
(300 Hz, δ H, DMSO): 3.85 (s, 3H, -OCH3); 5.85 (s, 2H,
aromatic); 6.53 (d, J = 8.1, 2H, aromatic); 7.08 (s, J = 8.4, 2H,
aromatic); 7.53-7.75 (m, 7H, aromatic); 11.96 (s, 1H, -COOH).
Elemental analysis, for, C21H17NO3 calculated (%): C 76.12, N
5.17, H 4.23; found (%) C 76.73, H 5.76, N 4.99.
λmax (nm): 316. IR (KBr, cm-1): 1124 & 1153 (aryl alkyl ether –
CO str); 1593 (C=N str); 1504 (aromatic –C=C str); 1325-1467
(aromatic multiple peaks of –CH str).1H-NMR (300 Hz, δ H,
CDCl3): 3.74 (s, 9H, OCH3); δ 6.39-7.98 (m, 12H, aromatic).
Anal. calcd (%) for C22H21NO3: C 76.06, H 6.09, N 4.03; found
(%): C 76.20, H 6.03, N 4.14.
N-(3,4,5-trimethoxybenzylidene)-3,5-dimethoxybenzenamine (4)
λmax (nm): 313. IR (KBr, cm-1): 1591 (C=N str); 1124 & 1153
(aryl alkyl ether –CO str); 1504 (aromatic C=C str); 1487 (=C-H
str).1H-NMR (300 Hz, δ H, CDCl3): 3.82 (s, 6H, OCH3); 3.93 (s,
9H, OCH3); 8.33 (s, 1H, N=CH); 6.30 (s, 3H, aromatic); 7.14 (s,
2H, aromatic). Anal. calcd (%) for C18H21NO5: C 65.24, H 6.39,
N 4.23; found (%): C 65.32, H 6.44, N 4.39.
4-(1-(4-hydroxy-3-methoxyphenyl)ethylideneamino)benzoic acid
(12)
λmax (nm): 278. IR (KBr, cm-1): 3363 (Phenolic –OH str); 2847
(Carboxylic –OH str); 1662 (C=O str); 1578 (C=N str); 1602
(aromatic C=C str); 1209 and 1168 (aryl alkyl ether –CO str).1H-
NMR (300 Hz, δ H, DMSO): 3.33 (s, 3H, -CH3); 3.84 (s, 3H, -
OCH3); 5.86 (s, 2H, aromatic); 6.53 (d, J = 8.7, 2H, aromatic);
6.86 (s, J = 8.1, 1H, -OH); 7.43-7.62 (m, 3H, aromatic); 11.93 (s,
1H, -COOH). Elemental analysis, for,C16H15NO4, calculated (%):
C 66.41, H 4.83, N 5.16; found (%): C 65.12, H 4.20, N 5.55.
N-(2-nitrobenzylidene)-3,5-dimethoxybenzenamine (5)
λmax (nm): 270. IR (KBr, cm-1): 1597 (-C=N str); 1521 (aromatic
–C=C str); 1203 & 1344 (Nitro –NO str).1H-NMR (300 Hz, δ H,
CDCl3): 3.79 (s, 6H, OCH3); 6.33-8.21 (m, 7H, aromatic); 8.41
(s, 1H, N=CH). Anal. calcd (%) for C15H14N2O4: C 62.93, H 4.93,
N 9.79; found (%): C 62.84, H 4.83, N 9.85.
4.3.
Estimation of cholinesterase activity (in-vitro)
The effectiveness of the tested compounds to inhibit
acetylcholinesterase from the electric eel (E.C. 3.1.1.7) and
butyrylcholinesterase from human serum (E.C. 3.1.1.8) was
examined with IC50 values. Ellman’s spectrophotometric
analysis35 was used to determine IC50 values by recording the rate
of increase in the absorbance at 412 nm for 5 min. A stock
solution of AChE was prepared by dissolving AChE in 0.1 M
phosphate buffer (pH 8.0), and a stock solution of BChE was
prepared by dissolving the lyophilized powder in an aqueous
solution of gelatin 0.1%. The final assay solution consisted of 0.1
4-(2-hydroxybenzylideneamino)benzoic acid (6)
λmax (nm): 236. IR (KBr, cm-1): 3423 (Phenolic –OH str); 3070-
2856 (Carboxylic –OH str); 1689 (C=O str); 1610 (C=N str);
1572 (aromatic C=C str).1H-NMR (300 Hz, δ H, DMSO): 5.86
(s, 1H, -OH); 7.53-7.73 (m, 8H, aromatic); 8.12 (s, 1H, N=CH);
12.01 (s, 1H, COOH). Elemental analysis, for, C14H11NO3,
calculated (%): C 69.70, H 4.60, N 5.81; found (%): C 70.51, H
4.42, N 6.19.

M phosphate buffer (pH 8.0) with 340 mM 5,5’-dithiobis(2-
nitrobenzoic acid), 0.02 unit/ml of AChE or BChE and 550 mM
carboxamide (3F9) (PDB Code: 4TPK) , and AChE complexed
with E2020 (donepezil) (PDB Code: 1EVE) was chosen as the
target protein.38 The structures were prepared using the protein
preparation wizard in Maestro 10.5.014. by adding hydrogen,
assigning partial charges using the OPLS-2005 force field,
assigning protonation states, and determining restrained and
partial energy minimization. The ligand was removed, and the
ligand binding site was defined. The default settings were used
for all other parameters. The extra precision (GLIDE-XP)
implemented in GLIDE was used for docking.
of
substrate
(acetylthiocholine
iodide,
ATCh
or
butyrylthiocholine iodide, BTCh, respectively). Different
concentrations of test compounds between 20 and 80% were
selected to obtain inhibition of the enzymatic activity. From
aliquots (50 µL), increasing concentrations of the compounds
were added to the assay solution after preincubation for 20 min at
37 °C with the enzyme followed by the addition of substrate. The
blank assay consisted of all components except AChE or BChE
to account for the non-enzymatic reaction. The reaction rates
were compared, and the percent inhibition due to the increasing
concentrations of the compound was calculated. The
concentration of each test compound was recorded in triplicate,
and their IC50 values were determined graphically from percent
inhibition curves.(36a, 36b)
4.6.2.
Molecular docking
The crystallographic and trajectory water molecules, ions and
ligand compounds were removed from developed protein. The
ligands were built using Maestro 10.5.014 build panel and
prepared by Lig Prep 3.9 (Schrödinger, LLC, USA, 2016-1)
application that uses OPLS 2005 force field. OPLS stands for
optimized potential liquid simulations, and it gave the
corresponding energy minima 3D conformers of the ligands. The
proteins were prepared using Schrodinger software, Maestro, and
Glide. The Glide XP algorithm was employed, using a grid box
volume of 10_10_10 Å. All the structures were fitted into the
binding pocket, and the lowest energy pose for each docking run
was retained.39
4.4.
Enzyme kinetics study
Ellman’s spectrophotometric analysis was used to identify the
type of inhibition. ATCh and BTCh were used as substrates in
different concentrations, both below and above Km, in an
alternative sequence in a phosphate buffer at pH 8, with a fixed
concentration of cholinesterase, in the absence or presence of
different compounds. The compound concentration was
maintained close to the IC50 value of enzyme inhibition. The
inhibitory kinetics were evaluated by the Lineweaver and Burk
method.37
4.7.
Determination of partition coefficient
The lipophilic constant of all of the compounds (1-12) was
determined in n-octanol and buffer (pH 7.4) by the shake flask
method40. The log P was calculated by correlating the absorbance
with the concentration using a standard plot.
4.5.
Free radical scavenging activity
4.5.1.
DPPH (2, 2-diphenyl -1-picryl -hydrazyl) radical
scavenging activity
4.8.
Determination ADME properties using Qikprop
The free radical scavenging activity of the synthesized
The ADME properties were assessed for the ligands obtained
from LigPrep. LigPrep generated ligand with the desired
structural and chemical features for further computational
analyses. The compounds (1-12) along with donepezil were
subjected to QikProp module of the Schrödinger suite to
calculate the ADME properties. The newly identified ligands
checked for octanol/water partition coefficient, Lipinski's rule of
five and Jorgensen's rule of three, gut-blood barrier, binding to
human serum albumin, aqueous solubility, brain/blood partition
coefficient, skin permeability, and percent of human oral
absorption.
compound 4 was examined using DPPH (2, 2-diphenyl -1-picryl -
hydrazyl)
method.30
Ascorbic
acid
((5R)-[(1S)-1,2-
Dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one) was used as
standard. 1 ml solution of each compound at different
concentration (10, 100, and 500 µg/ml) in methanol was mixed
with 2 ml of DPPH solution (0.5 mM). The mixture was shaken
vigorously and allowed to stand in dark place at room
temperature for 30 min, and the absorbance was read at 517 nm.
The antiradical activity was calculated in percentage as per the
following formula: Free radical scavenging activity (%) =
(absorbance of control – absorbance sample/absorbance of
control) × 100.
4.9.
Biological studies
4.5.2.
Hydrogen peroxide radical scavenging activity
4.9.1. Animals
Charles Foster rats of the albino strain (4 to 5 months old and
150 to 200 g) of either sex were procured from the Central
Animal House, Institute of Medical Sciences, Banaras Hindu
University, Varanasi. They had access to water adlibitum and
were fed a semi-synthetic balanced diet, with an occasional
supply of green vegetables (salad leaves). Six rats were housed
per cage at 22 ºC ± 3 ºC and 45-55% relative humidity. Twelve-
hour of light and dark cycles was strictly followed in a fully
ventilated room. Prior permission was obtained from the
Institutional Animal Ethical Committee (Registration No:
542/02/ab/CPCSEA).
The scavenging ability of the hydrogen peroxide radical of the
compound 4 was measured by H2O2 radical scavenging assay.31
A solution of hydrogen peroxide (40 mM) was prepared in
phosphate buffer saline (pH-7.4) at 20ºC. Hydrogen peroxide
solution (0.6mL) was added to 1mL of standard ascorbic acid (10
µg/mL) and different concentration of compound 4 (10, 100 and
500 µg/mL). The absorbance of hydrogen peroxide was
determined at 230 nm in UV/visible spectrophotometer after 10
minutes against a blank solution containing phosphate buffer
saline. The assay was performed in triplicate. The hydrogen
peroxide radical scavenging activity was measured by the
following equation. H2O2 activity (%) = (Absorbance of control -
Absorbance of sample / Absorbance of control) × 100.
4.9.2.
Ex vivo AChE inhibition
The ex vivo assay was performed to assess the AChE
inhibition of compound 4. Test drug was given orally (p.o.) to
rats at doses of 10 mg/kg and 30 mg/kg. After one hour, the
animals were sacrificed by decapitation and the brain was
instantly dissected, cleaned with cold saline and kept in
4.6.
Molecular docking
4.6.1.
Preparation of the protein
The BChE complexed with N- [(3R)-1-(2,3-dihydro-1H-inden-
2-yl)piperidin-3-yl]methyl-N-(2methoxyethyl)naphthalene- 2-

phosphate buffer (pH 8.0). Entire brain was then homogenate and
centrifuged to get the supernatant. The collected supernatant was
used for assay following Ellman method to calculate the
percentage of brain AChE Inhibition.
4.9.5.1. Drug Treatment
The rats were divided into 4 groups with each group having 3
rats. Group 1 was considered as normal control saline whereas
group 2 was positive control and treated with aspartame orally at
dose level of (75 mg/kg.weight.body),33 group 3 was treated with
aspartame plus compound 4 (10 mg/kg.weight.body) and group 4
4.9.3.
Drug treatment
The rats were divided into 5 groups with each group having 6
rats. Group 1 was considered as control whereas 2, 3, and 4 were
the test, and group 5 was standard. The compound 4 was
administered as an oral dose of 10, 20 and 30mg/Kg in 0.3%
CMC suspension respectively in the test groups. Donepezil was
administered as an oral dose of 10mg/Kg in the standard group.41
The treatment schedule is mentioned in the supplementary table
2.
was
treated
with
aspartame
plus
donepezil
(10
mg/kg.weight.body). Brain Sample was obtained as per ex vivo
method (Section 4.9.2.).
4.9.5.2.. GSH estimation
Estimation of glutathione (GSH) in the brain tissue
homogenate was performed by the method of Ellman, using 5,5'-
Disulfanediylbis(2-nitrobenzoic acid) (DTNB). In this method
when DTNB was added to the sample containing sulfhydryl
groups that give yellow color and estimated at 412 nm. The
concentration of GSH was calculated from standard GSH curve
and expressed in µmol/mg of protein.
4.9.4.
Morris water maze (MWM) task
The Morris water maze is a fabricated metal semi-circular
pool (120 cm in diameter, 45 cm deep) filled with water to a
depth of approximately 25 cm. The pool was divided into four
equal quadrants, and a platform (15 cm in diameter) was
submerged 0.5 cm below the opaque surface in the center of one
of the quadrants. The pool was located in the center of a small
test room and was surrounded by many extra-maze cues on the
walls of the room and two on the rim of the pool to provide both
proximal and distal visual cues. Individual rats from all groups
were placed by hand into the water facing the wall of the pool
and were then given 60 sec to find the hidden platform. When
successful, the rat was then given 20 sec on the platform to watch
the spatial cues. If the animal failed to find the platform in this
time, it was guided there. Escape latency, swim distance and time
spent in the target quadrant were scored by ANY- Maze video
tracking software.42,43
Acknowledgment
The authors gratefully acknowledge the Ministry of Human
Resource and Development (MHRD), New Delhi, India for the
financial support to Mr. T V R Upendra. Grant No. R / Dev. / IX-
Sch. / (SRF-JRF) Pharm./15402. We would also like to thank
Indian Institute of Technology (BHU) for providing financial
assistance through Institute Research Project (IRP) scheme and
Design Innovation Center (DIC) scheme. Grant No. IIT (BHU) /
R&D/IRP/2015-16/3471/L & Grant No. DIC-IIT(BHU)/L-11
respectively.
Supplementary Material
4.9.4.1. Habituation
Detailed experimental procedures with analytical data for
compounds including 1H NMR. (PDF)
All the rats used in the study were habituated to the pool by
allowing them to perform a 60 sec swim without the platform 24
h before starting the training.
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