Design and synthesis of some acridine-piperazine hybrids for the improvement of cognitive dysfunction

Article abstract of DOI:10.1111/cbdd.13017

A novel series of hybrid molecules (5a–5m) was designed, synthesized and evaluated as multifunctional cholinesterase (ChE) inhibitors against cognitive dysfunction. Heterocyclic moieties acridine and piperazine were conjugated with suitable linkers in a single scaffold, and the structures of the target compounds were confirmed by IR, ¹H NMR, ¹³C NMR, and LC-MS analysis. The pharmacological activity of synthesized compounds was evaluated using behavioral models of amnesia viz. step-down passive avoidance and elevated plus maze at a dose 0.5?mg/kg as compared to standard rivastigmine. In vitro acetylcholinesterase (AChE) inhibition studies using brain homogenate of mice as the enzyme source revealed that most of the compounds exhibited a significant ability to inhibit the enzyme cholinesterase with compound 5c being the most potent (IC₅₀ 0.33?μm). Biochemical estimation of oxidative stress markers viz. plasma nitrite, thiobarbituric acid reactive substances, catalase, superoxide dismutase, and glutathione has been carried out using the respective assays to see the effect of the synthesized compounds on the scopolamine-induced oxidative damage. The molecular docking studies indicated the binding mode of the compounds to the catalytic site, peripheral site, and mid-gorge of AChE simultaneously. The calculated absorption, distribution, metabolism and excretion properties ensured the drug-likeness of the target compounds. The synthesized compounds were found to be potential cognitive enhancers, which were able to interfere with the scopolamine-induced oxidative stress also.

Full text of DOI:10.1111/cbdd.13017

PROF. POONAM PIPLANI (Orcid ID : 0000-0003-4144-5164)
Article type : Research Article
Design and synthesis of some acridine-piperazine hybrids for the improvement of cognitive
dysfunction.
Anuradha Sharma and Poonam Piplani*
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014
*Corresponding Author
Prof. Poonam Piplani, Ph.D.
Professor (Pharmaceutical Chemistry)
University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh-160014
INDIA
Phone : +91-172-2534111
Fax : +91-172-2543101,
Mobile : +91-9357036068
mail id:- ppvohra28in@yahoo.co.in
Funding
The financial support provided by the University Grant Commission (UGC), New Delhi, India,
under Research Fellowship in Science for meritorious Student (RFSMS) Scheme is gratefully
acknowledged.
Design and synthesis of some acridine-piperazine hybrids for the improvement of cognitive
dysfunction.
Abstract
A novel series of hybrid molecules (5a-5m) was designed, synthesized and evaluated as
multifunctional cholinesterase (ChE) inhibitors against cognitive dysfunction. Heterocyclic
moieties acridine and piperazine were conjugated with suitable linkers in a single scaffold and
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13017
This article is protected by copyright. All rights reserved.

1
13
the structures of the target compounds were confirmed by IR, H NMR, C NMR and LC-MS
analysis. The pharmacological activity of synthesized compounds was evaluated using
behavioral models of amnesia viz. step down passive avoidance and elevated plus maze at a dose
0.5mg/kg as compared to standard rivastigmine. In vitro acetylcholinesterase (AChE) inhibition
studies using brain homogenate of mice as the enzyme source revealed that most of the
compounds exhibited a significant ability to inhibit the enzyme cholinesterase with compound 5c
being the most potent (IC50 0.33 µM). Biochemical estimation of oxidative stress markers viz.
plasma nitrite, thiobarbituric acid reactive substances, catalase, superoxide dismutase and
glutathione has been carried out using the respective assays to see the effect of the synthesized
compounds on the scopolamine induced oxidative damage. The molecular docking studies
indicated the binding mode of the compounds to the catalytic site, peripheral site and mid-gorge
of AChE simultaneously. The calculated ADME properties ensured the drug-likeness of the
target compounds. The synthesized compounds were found to be potential cognitive enhancers,
which were able to interfere with the scopolamine-induced oxidative stress also.
Keywords: Acridine; Antioxidant, Cognition Enhancer, Docking, Piperazine, Synthesis.
Graphical Abstract
HIGHLIGHTS
 A series of acridine-piperazine hybrids was designed and synthesized as potential
cognition enhancers.
 The synthesized compounds were evaluated for cognitive potential using step down
passive avoidance and elevated plus maze models supported by biochemical estimation
for acetylcholinesterase.
This article is protected by copyright. All rights reserved.

 Biochemical estimation of oxidative stress markers viz. plasma nitrite, thiobarbituric acid
reactive substances, catalase, superoxide dismutase and glutathione has been carried out.
 The IC50 of the most potent compound 5c against acetylcholinesterase was found to be
0.33 µM.
 Docking studies of the target compounds 5a-5m were performed using Glide and VLife
softwares to explore the binding modes within the enzyme.

Introduction
Cognition (derived from the Latin word “cognitio”) may be defined as the process of amassing
knowledge and understanding through thought, experience, and the senses (1-3). Cognitive
deterioration characterized by deficit cholinergic neurotransmission and aggregation of β-
amyloid protein is the main testimonial evidence in various dementing disorders mainly in
Alzheimer disease (AD), Parkinson’s disease, stroke, schizophrenia, seizure disorders and
traumatic brain injury etc (4-5). Acetylcholine (ACh) neurotransmitter plays a major role in
memory and dementia. The brains of persons with severe cognition dysfunction show
consistently depleted ACh levels. The deficiency of ACh at the post-synaptic receptor site is
attributed to the acetate and choline due to the hydrolysis of ACh by enzyme acetylcholinesterase
(AChE) (6). AChE inhibition belongs to the cholinergic replacement strategy designed for the
compensation of primary selective degeneration of the cholinergic neurons. AChE inhibitors
delay the inter synaptic degeneration of ACh by AChE, which leads to an increase in
accessibility of ACh at the synaptic gap junction (7).
AChE has two binding sites i.e. catalytic active site (CAS) at the bottom and peripheral active
site (PAS) at the entrance of gorge (8). Selectively, ligands can bind to either the CAS or the
PAS and it has been hypothesized that formation of β-amyloid plaques and tangles is aggravated
through interaction of ligands with PAS. However, literature reports have revealed that dual
binding site AChE inhibitors facilitate cholinergic neurotransmission as well as interfere with the
aggregation of toxic β-amyloid (9). Till now, the approval of only four AChE inhibitors viz.
tacrine, donepezil, rivastigmine and galanthamine have been granted for the treatment of
moderate to severe AD by the European and United State regulatory authorities (10). The
narrowness of the current therapeutics makes treatment of AD the current biggest unmet medical
need in cognitive disorders.
This article is protected by copyright. All rights reserved.

Literature evidences have suggested that accumulation of plaques and tangles in the brain
facilitates the generation of free-radicals scavengers leading to an increase in the oxidative stress
markers, which damage lipid and protein content of the neurons. This oxidative stress is
responsible for neurodegeneration, nerve cell structural and functional damage and neuronal
apoptosis in patients leading to cognitive dysfunction. Hence many antioxidants have been
introduced both clinically and pre-clinically for the treatment of cognitive dysfunction and
consideration has also been given to oxidative stress induced in cognitive deficits (11-12).
At this juncture, acridine absorbed our attention as a chemically versatile scaffold exhibiting
diverse biological properties (13-14). Tacrine (acridine containing drug) was the first FDA
approved non-selective AChE inhibitor for the treatment of AD (10). It remains a reference
structure in developing AChE inhibitors, although the lack of selectivity and liver toxicity has
limited its clinical applicability (15). In this respect, lot of efforts have been made to design and
synthesize tetrahydoacridine (tacrine) based agents and its hybrids as AChE inhibitors with lesser
side-effects and a better pharmacokinetic profile. The potential activity of acridine derivatives
during random screening as AChE inhibitors (16-18) motivated us to explore this nucleus and
pursue our research in this direction so as to minimize the side effects caused by it.
Literature further reveals the significance of piperazino moiety for its high potency, selectivity of
action and excellent therapeutic profile in cognitive dysfunction, which has caught the attention
of many medicinal chemists and pharmacologists to further explore this group (19-20). Easy
functionalization of piperazine makes it an alluring building block for designing and synthesizing
new compounds of therapeutic utility (21). This instigated the present investigator to develop
acridine-piperazine hybrids devoid of unwanted side effects.
Therefore, in the present work, a series of acridine-piperazine hybrids has been synthesized and
studied as potential cognition enhancers using scopolamine induced dementia, biochemical
estimations of AChE and oxidative stress markers. Moreover, to further investigate the
mechanism of action involving AChE, in silico studies have been performed.
Experimental Section
Melting points are uncorrected and were measured in open capillary tubes, using a Veego
1
13
melting point apparatus. H and C NMR spectra were obtained using a Bruker Avance II 400
MHz spectrometer. Elemental analyses were done on a Thermo scientific CHNSO elemental
This article is protected by copyright. All rights reserved.

analyser (Model: Flash 2000) at Central Instrumental Laboratory, Panjab University, Chandigarh
and results were within 0.4% of the theoretical values. Furthermore, all the compounds were
checked by thin layer chromatography (TLC) and showed single spots. Taken together, both
features ensure ≥ 95% of purities. The LC-MS spectra (ESI positive mode) were obtained on
Waters, Q-TOF Micromass spectrometer. The FTIR spectra were obtained from a PerkinElmer
Spectrum Version 10.03.08 using KBr disc method for the preparation of the sample.
Chemistry
General Procedure for the synthesis of substituted anthranilic acids (1a-1g). A suspension of o-
bromobenzoic acid (1 g, 85 mmoL), substituted anilines (25.5 mmoL) and copper powder (0.1 g)
o
was refluxed in ethanol (20 mL) at 80 C for 12-18 h in the presence of anhydrous potassium
carbonate (1 g) (22). The reaction was monitored by TLC. The suspension was cooled to 20-25
oC and was poured into ice-cold water. The contents were further acidified with concentrated
hydrochloric acid to yield the precipitates, which were washed with water and dissolved in 5%
sodium hydroxide solution (50 mL). The resulting solution was boiled after addition of activated
charcoal. The hot solution was then filtered and concentrated hydrochloric acid was added to the
filtrate to obtain the crude product which was filtered, dried and recrystallized from ethanol to
yield the corresponding intermediates 1a-1g.
General Procedure of substituted acridones (2a-2g). A mixture of various substituted anthranilic
acid 1a-1g (1 g, 4.03 mmoL) and concentrated sulphuric acid (5 mL) was heated at 100 oC for 8-
11 h. The completion of reaction was monitored by TLC. The reaction mixture was cooled to
room temperature and crushed ice was further added to the obtained viscous mass to yield the
crude product. It was filtered and washed with 5% aqueous solution of sodium carbonate. The
precipitate obtained was filtered, dried and crystallized from ethanol. It was recrystallized with
ethanol-methanol to obtain the corresponding substituted acridones 2a-2g.
General Procedure of substituted 9-chloroacridines 3a-3g. Substituted acridone 2a-2g (4.0
mmoL) was added to phosphorus oxychloride (POCl3) (1 mL, 8.0 mmoL) and was refluxed at
110 oC for 7-13 h. The resulting suspension was cooled to room temperature and crushed ice was
added to the viscous mass. The solid so obtained was filtered, dried and recrystallized from
ethanol to obtain 3a-3g.
General Procedure of acridine-piperazine hybrids 5a-5m. A mixture of substituted piperazine
(8.32 mmoL), substituted 9-chloroacridine (4.03 mmoL) and sodium iodide (0.5 g) was refluxed
This article is protected by copyright. All rights reserved.

at 80 oC for 13-19 h in ethyl methyl ketone in the presence of anhydrous potassium carbonate
(1 g). The completion of the reaction was monitored by TLC. The crude product obtained was
filtered, washed repeatedly with water to remove unreacted amine. It was dried and crystallized
from ethanol to yield 5a-5m. The detailed spectral information of the compounds is given in the
supplementary file.
Pharmacology
Animals. Albino mice (female, Laca strain) weighing 25-30 g, were purchased from Central
Animal House, Panjab University, Chandigarh, India. All the research protocols were followed
by the Institutional Animal Ethical Committee, Panjab University, Chandigarh, India.
Drugs. Rivastigmine (0.5 mg/kg, obtained from Sun Pharmaceutical Industries Ltd, Silvassa,
India) was used as the standard drug. Scopolamine (0.5 mg/kg i.p.) was used as an inducer of
cognition deficits, which is qualitatively similar to that occurring in senile dementic subjects
(24). The test compounds were dissolved in sterile distilled water/0.1% DMSO and administered
intraperitoneally (i.p.) to animals.
Step-down passive avoidance test. Rodent memory evaluator (IMCORP, Ambala Cantt, India)
was used to carry out the behavioral test (25).
Elevated plus maze. The time taken by the experimental animal to enter the enclosed arm in the
acquisition trial (first) was noted, so called as initial transfer latency (ITL). The retention
(second) trial was performed 24 hours after the acquisition trial, so called retention transfer
latency (RTL). It was expressed as the percentage of retention (26).
Biochemical Parameters Estimation
Brain tissue preparation. After behavioral assessment, animals were sacrificed by spinal
dislocation and the whole brains were quickly removed, rinsed with ice-cold isotonic saline. A
brain homogenate (10% w/v) was prepared using 0.03 M sodium phosphate buffer (pH 7.4) and
centrifuged using an Ultra-Turrax T25 (USA) homogenizer at 10000 rpm for 15 minutes. The
aliquots of supernatant were used for the biochemical estimation. Biuret method was employed
to determine protein content using standard bovine serum albumin (BSA) (1 mg/mL) (27).
Acetylcholinesterase assay. The cholinergic dysfunction was assessed by estimation of the
cholinergic marker, AChE in the whole brain according to the Ellman method (23).
This article is protected by copyright. All rights reserved.

Assay for the estimation of oxidation stress parameters. Green et al., was first to develop the
assay of nitrite estimation. The cytosolic fractions of different brain regions were served as an
indicator of nitric oxide production (28). Malondialdehyde (MDA) content is a measure of lipid
peroxidation assay and the procedure was based on the original work of Kobayashi et al (29).
The protocol of the catalase assay was laid by Claiborne et al (30). Kono et al., developed the
method to perform SOD assay and the auto-oxidation of hydroxylamine was observed by
measuring the change in optical density at 560 nm for 2 min at 30-60 s intervals (31). The
reduced glutathione assay has been developed by Jollow et al., that is based on Ellman method
(32). All statistical analyses were performed using GraphPad Prism 7 software (GraphPad
Software, San Diego, CA, USA).
In Silico Studies
ADME property prediction was carried out using QikProp module to evaluate DrugLikeness of
the synthesized compounds (33).
Molecular docking studies were performed using Glide module of Schrodinger (34) and
Biopredicta module of VLife MDS (version 4.6.28102016) (35).
Results and Discussion
Molecular Design. As reported previously in the introduction, acridine and piperazine scaffolds
have emerged as good AChE inhibitors as these interact with catalytic anionic site (CAS) and
peripheral active site (PAS) of AChE respectively through hydrophobic, vander wal and π-π
interactions. Therefore, our research group hypothesized that amalgamating both moieties might
afford more effective multifunctional molecules which could not only inhibit AChE but also
exhibit neuroprotective effect. The interactions of the designed compound have been analysed
using molecular docking tools by placing the compound into the binding cavity of the enzyme in
a non-covalent fashion. The selection of the compounds to be synthesized has been done on the
basis of high binding interactions and low binding energies. The preliminary docking studies
indicated acrdine and piperazine as suitable templates to design novel cognition enhancers.
Chemistry. In the light of above considerations, the designed hybrids were synthesized as shown
in Scheme 1 and 2.
This article is protected by copyright. All rights reserved.

Synthesis of substituted anthranilic acids 1a-1g was carried out by copper catalysed N-alkylation
(22) of different substituted anilines with o-bromobenzoic acid (85 mmoL) in the presence of
anhydrous potassium carbonate and ethanol as the solvent respectively. A mixture of compounds
o
1a-1g (4.03 mmoL) and concentrated sulphuric acid (5 mL) was heated at 100 C for 8-11 h to
obtain compounds 2a-2g respectively which were chlorinated by refluxing them with phosphorus
o
oxychloride (POCl3) (1 mL, 8.0 mmoL) at 110 C for 7-13 h to yield the corresponding
compounds 3a-3g (Table 1) respectively.
Subsequently, the target compounds 5a-5m have been synthesized (Table 1) by refluxing the
substituted chloroacridines 3a-3g with substituted piperazines 4i-4vi in ethyl methyl ketone in
the presence of sodium iodide and anhydrous potassium carbonate respectively (Scheme 2).
R1
4i
4ii
4-Fluorophenyl
2-Fluorophenyl
4iii -Phenyl
4iv -Ethyl
4v
-Methyl
4vi 3-Chlorophenyl
This article is protected by copyright. All rights reserved.

Table 1: The physicochemical data of the compounds 3a-3g and 5a-5m.
Molecular
Formulae
C13H8ClN
C13H7Cl2N
C13H7ClFN
C13H7ClFN
C13H7Cl2N
C14H9ClN
C15H14ClN
Molecular
weight
213.66
248.11
231.66
231.66
248.11
227.69
243.74
Percentage
Yield
Melting Point
(oC)
R
R1
Code
3a
3b
3c
3d
3e
3f
H
4-Cl
4-F
2-F
2-Cl
-
-
-
-
-
-
-
77.98
73.05
85.19
76.86
73.15
82.72
82.56
140-142
148-150
202-204
280-282
141-143
123-125
110-112
4-CH3
2,4-CH3
3g
H
C23H20FN3
C23H20ClN3
C23H19ClFN3
357.43
373.88
391.87
77.84
69.54
70.06
180-182
165-168
146-150
5a
5b
5c
4-Cl
4-Cl
4-Cl
C23H19ClFN3
391.13
63.06
280-282
5d
4-Cl
4-Cl
-C2H5
-CH3
C19H20ClN3
C18H18ClN3
325.84
311.81
73.51
93.65
191-193
205-207
5e
5f
4-F
2-F
C23H19ClFN3
C23H19ClFN3
391.87
391.87
70.06
54.14
146-150
196-199
5g
5h
2-Cl
C23H19ClFN3
C24H22FN3
C25H24FN3
391.87
371.45
385.49
76.43
76.68
88.60
268-270
278-280
282-284
5i
5j
4-CH3
2,4-CH3
5k
2,4-CH3
2,4-CH3
C25H24FN3
C21H25N3
385.49
319.45
88.60
73.79
286-288
248-250
5l
-C2H5
5m
Pharmacology
In vitro acetylcholinesterase inhibition. In vitro evaluation of the synthesised acridine-piperazine
hybrids 5a-5m was carried out using the brain supernatants of fresh or untreated mice as a source
of enzyme by Ellman method (23). The concentration vs. percentage inhibition graph was plotted
This article is protected by copyright. All rights reserved.

by taking five different concentrations (1, 5, 10, 25, and 50 μL) of target compounds (5a-5m)
and the standard rivastigmine according to optimal correlation coefficient to determine IC50
(Table S1). Behavioral studies were performed on scopolamine-induced amnesic mice using step
down passive avoidance and elevated plus maze test for cognitive enhancing potential of the
synthesized hybrids.
Behavioral studies on mice by step-down passive avoidance. Three basal readings (basal latency)
were recorded for the training period. After 1 h the latency was noted down and the number of
mistakes made by the animals was counted for 15 minutes. Results of the basal latency and
memory parameters (latency and mistakes) are shown in Table 2 and Figure 1, indicating that
compounds 5a-5m show remarkable improvement in cognitive deficit as compared to both
standard rivastigmine and scopolamine treated groups.
Figure 1. (a) Divergence of basal latency and latency in various groups treated by control, scopolamine, rivastigmine and target
compounds 5a-5m; (b) Graphical representation of the number of mistakes in the various treated groups made by the mice; (c)
Comparison of % retention in various treated groups 5a-5m; (*) p < 0.0001 as compared to control; (#) p < 0.0001 as compared
to scopolamine (n = 5). The intergroup variation was found out using tukey’s test and values were considered statistically
significant (0.0001).
Table 2: The basal latency, latency and the no. of mistakes using passive avoidance protocol and % retention
in the elevated plus maze test of the compounds 5a-5m.
Basal Latency
45.67±12.75
44.08±16.50
41.34±6.80
59.25±17.01
35.50±1.71
29.08±3.30
46.17±11.58
34.75±13.05
63.25±5.91
36.92±5.17
63.58±15.05
29.50±2.60
34.67±1.89
63.58±15.05
Latency
42.75±0.85
87.00±0.92
15.00±1.47
9.00±2.89
9.05±0.65
8.00±1.73
11.00±2.16
7.00±0.91
49.25±3.838
18.25±2.02
16.00±0.41
8.25±1.37
17.25±0.85
14.75±1.11
No. of Mistakes
14.00±0.58
26.00±2.31
8.00±0.58
3.00±0.92
2.25±0.48
2.00±0.41
7.25±0.48
7.25±2.02
7.50±1.55
8.00±0.41
3.25±0.48
4.50±0.87
8.50±2.02
3.00±1.23
% Retention
23.02±0.55
11.44±0.60
55.94±1.21
55.39±1.12
55.52±0.82
78.39±3.38
51.72±1.26
47.46±0.89
52.25±1.96
77.06±1.95
56.10±1.75
59.55±1.74
58.56±1.98
52.27±1.88
Control
Scopolamine
Rivastigmine
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
26.42±3.90
29.50±2.60
10.25±1.65
7.50±1.32
8.25±0.63
4.50±0.65
64.49±1.05
64.24±0.89
5l
5m
This article is protected by copyright. All rights reserved.

All results are expressed in the form of mean ± SEM (n=5). Significance for the basal latency and latency was determined by
two-way ANOVA by Tukey’s test and for number of mistakes and %retention was determined by one-way ANOVA followed by
Tukey’s test.
Behavioral studies on mice using elevated plus maze. Retention and memory acquisition of all
the compounds was screened for antiamnesic activity. Amongst the synthesised compounds,
compound 5c was found to be an active compound and significantly improved learning and
cognition with % retention of 78.39 at a dose of 0.5 mg/kg in comparison to rivastigmine (Table
2 and Figure 1).
Biochemical estimation
Biochemical estimation of the acetylcholinesterase enzyme. Biochemical studies were carried out
to figure out the mechanism of action of the enzyme inhibition by all the synthesized compounds
using Ellman method (23). The AChE inhibitory profile of the synthesized compounds in
comparison to the standard and the inducer is depicted in Figure 2 graphically. The results of
biochemical studies were found to be in agreement with the behavioral studies. The active
compounds (5c and 5d) have shown a lower value of the μmoles of AChE (Table 3).
Table 3: Biochemical estimation of the AChE enzyme and oxidative stress parameters.
Plasma
Lipid
Peroxidationc
0.57±0.05
3.98±0.20
3.13±0.51
1.93±0.28
1.88±0.27
1.80±0.06
1.96±0.20
1.81±0.26
1.85±0.32
1.98±0.29
2.01±0.13
1.94±0.20
1.97±0.29
1.84±0.36
Superoxide
Dismutasee
10.91±0.51
1.28±0.11
2.75±0.06
5.61±0.71
3.77±0.29
5.32±0.30
4.62±0.23
3.32±0.29
4.68±0.60
4.43±0.45
5.13±0.24
3.48±0.40
5.21±0.40
3.61±0.58
Glutathione
Reductasef
S. No.
Acetylcholinesterasea
Catalased
nitriteb
Control
Scopolamine
Rivastigmine
0.00203±0.00024
0.00358±0.00016
0.00182±0.00038
0.00172±0.00028
0.00164±0.00005
0.00159±0.00009
0.00154±0.00012
0.00153±0.00035
0.00172±0.00028
0.00171±0.00028
0.00175±0.00001
0.00172±0.00003
0.00157±0.00038
0.00158±0.00014
240.17±6.57
714.83±18.47
357.83±10.16
477.5±5.89
7.53±0.33
1.53±0.45
3.66±0.58
4.95±0.52
4.85±0.74
5.14±0.38
5.22±0.32
5.61±0.41
5.56±0.42
5.32±0.12
5.43±0.34
5.08±0.46
5.55±0.25
5.33±0.37
0.0337±0.0016
0.0104±0.0020
0.0159±0.0009
0.0201±0.0029
0.0196±0.0028
0.0187±0.0006
0.0204±0.0021
0.0189±0.0027
0.0193±0.0034
0.0206±0.0030
0.0209±0.0014
0.0190±0.0022
0.0187±0.0032
0.0192±0.0037
5a
5b
5c
5d
5e
5f
5g
5h
5i
333.50±10.16
290.83±10.54
345.17±12.17
336.83±8.60
408.50±10.76
425.83±13.64
478.50±7.76
408.17±11.73
363.50±11.40
408.50±11.26
5j
5k
0.00159±0.00007
0.00172±0.00022
348.83±0.01
471.83±10.26
1.96±0.21
1.87±0.21
5.59±0.29
5.67±0.28
4.00±0.38
4.47±0.33
0.0184±0.0023
0.0195±0.0022
5l
5m
All the results are expressed in the form of mean ± standard error mean (SEM). One-way ANOVA followed by Tukey’s test was
a
employed to determine significance. μmoles of AchE/min/mg pr, bNitrite conc. μg/mL, cn moles of MDA/mg protein, dCatalase
activity/min, eSOD units/mg protein and fμmoles of GSH/mg protein
Biochemical estimation of the parameters of oxidative stress: plasma nitrite, lipid peroxidation
thiobarbituric acid reactive substances, catalase, superoxide dismutase and glutathione.
Oxidative stress is the most important factor which aggravates neurodegeneration by producing
free radicals and reactive oxygen species that ultimately result in oxidative stress and damage to
This article is protected by copyright. All rights reserved.

the neuronal cells leading to cognitive impairments. The potential of cognition enhancers as
antioxidants is an important aspect of neuroprotection which has been properly investigated by
determination of biochemical markers of oxidation stress in scopolamine induced amnesia.
Oxidative stress and ROS are proposed to be the major biochemical manifestations of the aging
process. Scopolamine causes a significant increase in the oxidative stress markers as compared to
the control group indicating a state of oxidation stress and oxidative damage.
In the present work, biochemical estimation of the parameters such as plasma nitrite,
thiobarbituric acid reactive substances, catalase, superoxide dismutase and glutathione in the
mice brain supernatants was carried out. A significant increase in AChE, plasma nitrite,
malondialdehyde along with a remarkable decrease in activity of catalase, superoxide and
glutathione reductase was observed in group of mice treated with scopolamine as shown in
Figure 2 and Table 3.
Figure 2. Effect of the synthesized compounds 5a-5m treatment on (a) AChE, (b) nitrite, (c) malondialdehyde (MDA), (d)
catalase, (e) superoxide dismutase (SOD) and (f) reduced glutathione (GSH) on condition of cholinergic deficit and oxidative
stress induced by scopolamine. (*) p < 0.0001 as compared to control; (#) p < 0.0001 in comparison to scopolamine (n = 5).
The intergroup variation was evaluated by employing Tukey’s test and values were considered statistically significant (0.0001).
The compounds 5c and 5e were promising in decreasing the levels of plasma nitrite with the
nitrite concentrations 290.83 μg/mL and 336.83 μg/mL as compared to 714.83 μg/mL of the
scopolamine treated group. Thiobarbituric acid reactive substances (TBARS) assay is performed
to estimate the lipid peroxidation products quantitatively in the mice brain homogenate sample.
The absorbance was measured at 532 nm to estimate the MDA reactive products. The groups
treated with compounds 5c and 5e exhibited lower level of MDA as compared to those treated
This article is protected by copyright. All rights reserved.

with scopolamine (inducer) and rivastigmine (standard). The hydrogen peroxide breakdown is
measured for the biochemical estimation of catalase. As compared with the scopolamine-treated
group compounds 5c, 5e and 5g significantly averted the loss of catalase activity with
catalase/min value of 5.16, 5.61 and 5.32 respectively. Lower value of SOD units/mg of the
protein for compounds 5c and 5e (5.32 and 3.32) proposed the possible role of the synthesised
compounds in reversal of scopolamine induced oxidative damage.
GSH is a well-known antioxidant, present in higher concentrations in the mitochondrial matrix
that is synthesized in the cytoplasm. The increased oxidative stress leads to decrease the value of
reduced glutathione. It is observed that the group treated with scopolamine depicted lowest value
of GSH as compared to the control. GSH levels were found to be higher (0.0187 μ moles of
GSH/mg protein) in the groups treated with compound 5c, as compared to the inducer
scopolamine (0.0104 μ moles of GSH/mg protein) and standard rivastigmine (0.0159 μ moles of
GSH/mg protein) treated groups.
The synthesised compounds exhibited better activity as compared to the rivastigmine treated
group, thus indicating that besides improving cognition, the compounds were found to be better
than rivastigmine in terms of managing the scopolamine induced oxidative stress. Thus by
regulating the cholinergic deficit through AChE inhibition and oxidative stress, the target
compounds can be used as dual functional molecules, displaying promising role in treatment of
cognitive dysfunction.
In Silico Studies
Forecasting ADME Property. The acceptability of the compounds was estimated on the basis of
the Lipinski’s rule of five (36). The ADME properties of the most potent compound 5c are shown
in Table SII.
Molecular Docking Studies. Molecular docking studies were performed to support the data
obtained from in vitro, in vivo evaluations and ADME screening for determination of the best in
silico conformation of the most potent molecule. The PDB crystal structure of recombinant
human AChE (rhAChE) complexed with E2020 (37) (Figure 3(a)) is taken from the protein data
bank for analysis of the crucial key interactions. Figure 3(b) clearly shows the binding pocket in
the form of mesh structure with the cocrystal. All the ligands were found to interact with key
amino acids of the enzyme and the hypothetical modes of all the synthesised compounds
showing the crucial interactions in the defined grid are depicted in Figure 3c-d. For analyzing the
This article is protected by copyright. All rights reserved.

interactions, the compound 5c has been taken into consideration because all the compounds
interacted in the similar manner.
The most potent compound 5c exhibited excellent AChE inhibitory activity. Its conformation
fitted suitably in the defined grid covering the CAS and PAS of the crystal structure, acting as a
dual binding site inhibitor. The acridine moiety of compound 5c interacted with the key amino
acids (Trp86, Gly121, Tyr341 and Phe338) present at the bottom of the gorge and the
fluoropiperazine ring showed crucial interactions at the entrance of the gorge (Trp286 and
Phe297) as depicted in the 3D and 2D-ligand interaction diagram (Figure 3e and 3f). Fluorine
attached to the ring formed the hydrogen bond of distance 1.486 Å with Phe299. Both the
moieties showed interactions with Tyr124 amino acid present at the mid gorge site of the
enzyme. It is relevant to mention here that the hybrids may follow the same mode of action as
the various dual binding site inhibitors such as donepezil. In all the cases, it showed interactions
with the CAS and PAS, hence it might inhibit the amyloid formation.
Figure 3. (a) The crystal structure of rhAChE (PDB ID 6EY7) with its cocrystal E2020 (marked in the box), (b) The key amino
acids surrounding the cocrystal in the binding pocket (marked as the meshed shape) in the defined grid, (c) The hypothetical
binding motifs of all the ligands in the enzyme pocket by Glide, (d) The hypothetical binding modes of all the ligands by VLife,
(e) 3D ligand interaction diagram showing the binding orientation of the active comp 5c with the key amino acids present in CAS
and PAS of the binding pocket and (f) 2D-diagram showing the probable binding mode of the active comp 5c within the enzyme
pocket.
All the synthesized ligands showed Gscore ranging from -13.10 to -08.90 and dock score ranging
from -4.88 to -1.17 (Table SIII) theoretically, which are comparable to the pharmacological
activity. Overall, electron withdrawing substituents (like Cl and F) were found to be favorable
for AChE inhibitory activity and vice-versa. The above findings suggested that the substitution
of a bulky group at ninth position of tricyclic ring system might influence the electronic changes
to affect the interaction with enzyme, thus influence their potency.
This article is protected by copyright. All rights reserved.

Conclusion
A series of novel acridine-piperazine hybrids has been designed, synthesized and evaluated as
potential cognition enhancers. The results of in vitro AChE inhibition assay were also in
agreement with the results obtained from the behavioral studies and in silico data. The role of
these compounds in the management of cognitive dysfunction may be attributed to oxidative
stress as illustrated by estimation of oxidative stress parameters. Based on the results of the
behavioral, biochemical and in silico studies, compounds 5c and 5e have displayed appreciable
AChE inhibitory activity as compared to the standard rivastigmine.
The molecular docking studies identified these active compounds as the dual binding site
inhibitors displaying significant binding interactions at CAS and PAS within the binding pocket
of the AChE enzyme. The present study concluded that acridine-piperazine hybrids can serve as
the analogues having promising role in the management of cognitive dementia and the most
potent compound 5c can serve as promising lead in this arena of research for any further pursuit.
References
1. Bradford, E. E., Jentzsch, I., and Gomez, J. C. (2015). From self to social cognition:
theory of mind mechanisms and their relation to executive functioning. Cognition, 138,
21-34.
2. World Health Organization. (1993). The ICD-10 classification of mental and behavioural
disorders: diagnostic criteria for research.
3. Peña-Casanova, J., Sánchez-Benavides, G., de Sola, S., Manero-Borrás, R. M., and
Casals-Coll, M. (2012). Neuropsychology of Alzheimer's disease. Archives of Medical
Research, 43(8), 686-693.
4. Sachdeva, A., Kumar, K., and Anand, K. S. (2015). Non Pharmacological Cognitive
Enhancers-Current Perspectives. Journal of Clinical and Diagnostic Research: JCDR,
9(7), VE01.
5. Sahakian, B. J., Bruhl, A. B., Cook, J., Killikelly, C., Savulich, G., Piercy, T., Hafizi, S.,
Perez, J., Fernandez-Egea, E., Suckling, J., and Jones, P. B. (2015). The impact of
neuroscience on society: cognitive enhancement in neuropsychiatric disorders and in
healthy people. Phil. Trans. R. Soc. B, 370(1677), 20140214.
This article is protected by copyright. All rights reserved.

6. Soreq, H., and Seidman, S. (2001). Acetylcholinesterase-new roles for an old actor.
Nature Reviews Neuroscience, 2(4), 294-302.
7. Roesler, R., & Schröder, N. (2011). Cognitive enhancers: focus on modulatory signaling
influencing memory consolidation. Pharmacology Biochemistry and Behavior, 99(2),
155-163.
8. Craig, L. A., Hong, N. S., and McDonald, R. J. (2011). Revisiting the cholinergic
hypothesis in the development of Alzheimer's disease. Neuroscience & Biobehavioral
Reviews, 35(6), 1397-1409.
9. Bredesen, D. E. (2014). Reversal of cognitive decline: a novel therapeutic program.
Aging (Albany NY), 6(9), 707-717.
10. Schneider, L. S. (2000). A critical review of cholinesterase inhibitors as a treatment
modality in Alzheimer's disease. Dialogues in Clinical Neuroscience, 2(2), 111.
11. Cotman, C. W., Head, E., Muggenburg, B. A., Zicker, S., and Milgram, N. W. (2002).
Brain aging in the canine: a diet enriched in antioxidants reduces cognitive dysfunction.
Neurobiology of Aging, 23(5), 809-818.
12. Deak, F., Freeman, W. M., Ungvari, Z., Csiszar, A., and Sonntag, W. E. (2016). Recent
developments in understanding brain aging: Implications for Alzheimer’s disease and
vascular cognitive impairment. The Journals of Gerontology Series A: Biological
Sciences and Medical Sciences, 71(1), 13-20.
13. Kumar, R., Kaur, M., and Kumari, M. (2012). Acridine: A versatile heterocyclic nucleus.
Acta Pol. Pharm, 69(1), 3-9.
14. AN de Aquino, R., V Modolo, L., B Alves, R., and de Fátima, Â. (2013). Design of new
drugs for the treatment of Alzheimer's disease based on tacrine structure. Current drug
Targets, 14(3), 378-397.
15. Lleó, A. (2007). Current therapeutic options for Alzheimer's disease. Current Genomics,
8(8), 550-558.
16. Hamulakova, S., Janovec, L., Hrabinova, M., Kristian, P., Kuca, K., Banasova, M., and
Imrich, J. (2012). Synthesis, design and biological evaluation of novel highly potent
tacrine congeners for the treatment of Alzheimer's disease. European Journal of
Medicinal Chemistry, 55, 23-31.
This article is protected by copyright. All rights reserved.

17. Hamulakova, S., Imrich, J., Janovec, L., Kristian, P., Danihel, I., Holas, O., Pohanka, M.;
Böhm, S.; Kozurkova, M., and Kuca, K. (2014). Novel tacrine/acridine anticholinesterase
inhibitors with piperazine and thiourea linkers. International Journal of Biological
Macromolecules, 70, 435-439.
18. Li, S. Y., Wang, X. B., Xie, S. S., Jiang, N., Wang, K. D., Yao, H. Q., Sun, H.B., and
Kong, L. Y. (2013). Multifunctional tacrine-flavonoid hybrids with cholinergic, β-
amyloid-reducing, and metal chelating properties for the treatment of Alzheimer's
disease. European Journal of Medicinal Chemistry, 69, 632-646.
19. Rathi, A. K., Syed, R., Shin, H. S., and Patel, R. V. (2016). Piperazine derivatives for
therapeutic use: a patent review (2010-present). Expert Opinion on Therapeutic Patents.
http://dx.doi.org/10.1080/13543776.2016.1189902
20. Piplani, P., and Danta, C. C. (2015). Design and synthesis of newer potential 4-(N-
acetylamino) phenol derived piperazine derivatives as potential cognition enhancers.
Bioorganic Chemistry, 60, 64-73.
21. Martini, E., Ghelardini, C., Dei, S., Guandalini, L., Manetti, D., Melchiorre, M., Norcini,
M.; Scapecchi, S., Teodoria, E., and Romanelli, M. N. (2008). Design, synthesis and
preliminary pharmacological evaluation of new piperidine and piperazine derivatives as
cognition-enhancers. Bioorganic & Medicinal Chemistry, 16(3), 1431-1443.
22. Ullmann, F. (1903). Ueber eine neue bildungsweise von diphenylaminderivaten. Berichte
der deutschen chemischen Gesellschaft, 36(2), 2382-2384.
23. Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961). A new and
rapid colorimetric determination of acetylcholinesterase activity. Biochemical
Pharmacology, 7(2), 88-95.
24. Dimitrova, D., and Getova, D. (2016). Study of the effects of galantamine and memantine
on scopolamine model of cognitive dysfunction. European Neuropsychopharmacology,
26, S648-S649.
25. Ader, R., Weijnen, J. A. W. M., and Moleman, P. (1972). Retention of a passive
avoidance response as a function of the intensity and duration of electric shock.
Psychonomic Science, 26(3), 125-128.
This article is protected by copyright. All rights reserved.

26. Itoh, J., Nabeshima, T., and Kameyama, T. (1990). Utility of an elevated plus-maze for
the evaluation of memory in mice: effects of nootropics, scopolamine and
electroconvulsive shock. Psychopharmacology, 101(1), 27-33.
27. Smith, P., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M.,
Fujimoto, E.K., Goeke, N.M., Olson, B.J., and Klenk, D. C. (1985). Measurement of
protein using bicinchoninic acid. Analytical Biochemistry, 150(1), 76-85.
28. Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S., &
Tannenbaum, S. R. (1982). Analysis of nitrate, nitrite, and [15 N] nitrate in biological
fluids. Analytical Biochemistry, 126(1), 131-138.
29. Wills, E. D. (1966). Mechanisms of lipid peroxide formation in animal tissues.
Biochemical Journal, 99(3), 667.
30. Thomas, D. W. (1988). Handbook of Methods for Oxygen Radical Research. Journal of
Pediatric Gastroenterology and Nutrition, 7(2), 314-316.
31. Kono, Y. (1978). Generation of superoxide radical during autoxidation of hydroxylamine
and an assay for superoxide dismutase. Archives of Biochemistry and Biophysics, 186(1),
189-195.
32. Jollow, D. J., Mitchell, J. R., Zampaglione, N. A., & Gillette, J. R. (1974).
Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,
4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology, 11(3), 151-169.
33. Schrödinger LL. QikProp, version 3.5. New York, NY. 2012.
34. GLIDE, Maestro, version 9.2 (2012) Schrodinger, LLC, New York.
35. VLife MDS 4.6, version 4.6.28102016, Molecular Design Suite, Vlife Sciences
Technologies Pvt. Ltd, Pune. 2013, India, www.vlifesciences.com.
36. Lipinski, C. A. (2004). Lead-and drug-like compounds: the rule-of-five revolution. Drug
Discovery Today: Technologies, 1(4), 337-341.
37. Cheung, J., Rudolph, M. J., Burshteyn, F., Cassidy, M. S., Gary, E. N., Love, J., Franklin,
M.C., and Height, J. J. (2012). Structures of human acetylcholinesterase in complex with
pharmacologically important ligands. Journal of Medicinal Chemistry, 55(22), 10282-
10286.
This article is protected by copyright. All rights reserved.