Development of novel: N -(6-methanesulfonyl-benzothiazol-2-yl)-3-(4-substituted-piperazin-1-yl)-propionamides with cholinesterase inhibition, anti-β-amyloid aggregation, neuroprotection and cognition enhancing properties for the therapy of Alzheimer's disease

Article abstract of DOI:10.1039/d0ra00663g

A novel series of benzothiazole-piperazine hybrids were rationally designed, synthesized, and evaluated as multifunctional ligands against Alzheimer's disease (AD). The synthesized hybrid molecules illustrated modest to strong inhibition of acetylcholinesterase (AChE) and Aβ_1-42 aggregation. Compound 12 emerged as the most potent hybrid molecule exhibiting balanced functions with effective, uncompetitive and selective inhibition against AChE (IC₅₀ = 2.31 μM), good copper chelation, Aβ_1-42 aggregation inhibition (53.30%) and disaggregation activities. Confocal laser scanning microscopy and TEM analysis also validate the Aβ fibril inhibition ability of this compound. Furthermore, this compound has also shown low toxicity and is capable of impeding loss of cell viability elicited by H₂O₂ neurotoxicity in SHSY-5Y cells. Notably, compound 12 significantly improved cognition and spatial memory against scopolamine-induced memory deficit in a mouse model. Hence, our results corroborate the multifunctional nature of novel hybrid molecule 12 against AD and it may be a suitable lead for further development as an effective therapeutic agent for therapy in the future.

Full text of DOI:10.1039/d0ra00663g

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Development of novel N-(6-methanesulfonyl-
benzothiazol-2-yl)-3-(4-substituted-piperazin-1-
yl)-propionamides with cholinesterase inhibition,
anti-b-amyloid aggregation, neuroprotection and
cognition enhancing properties for the therapy of
Alzheimer's disease†
Cite this: RSC Adv., 2020, 10, 17602
b
Chandra Bhushan Mishra,‡a Shruti Shalini,‡a Siddharth Gusain,a Amresh Prakash,
Jyoti Kumari,a Shikha Kumari,a Anita Kumari Yadav,a Andrew M. Lynnc
a
*
and Manisha Tiwari
A novel series of benzothiazole–piperazine hybrids were rationally designed, synthesized, and evaluated as
multifunctional ligands against Alzheimer's disease (AD). The synthesized hybrid molecules illustrated
modest to strong inhibition of acetylcholinesterase (AChE) and Ab1-42 aggregation. Compound 12
emerged as the most potent hybrid molecule exhibiting balanced functions with effective, uncompetitive
and selective inhibition against AChE (IC50 ¼ 2.31 mM), good copper chelation, Ab1-42 aggregation
inhibition (53.30%) and disaggregation activities. Confocal laser scanning microscopy and TEM analysis
also validate the Ab fibril inhibition ability of this compound. Furthermore, this compound has also shown
low toxicity and is capable of impeding loss of cell viability elicited by H2O2 neurotoxicity in SHSY-5Y
cells. Notably, compound 12 significantly improved cognition and spatial memory against scopolamine-
induced memory deficit in a mouse model. Hence, our results corroborate the multifunctional nature of
novel hybrid molecule 12 against AD and it may be a suitable lead for further development as an
effective therapeutic agent for therapy in the future.
Received 21st January 2020
Accepted 19th April 2020
DOI: 10.1039/d0ra00663g
rsc.li/rsc-advances
with an average life span of 9 years aer diagnosis.1 AD leaves an
enormous emotional and nancial burden on patients, their
1. Introduction
families and society.
Alzheimer's disease (AD) is a predominant source of irreversible
dementia, resulting in more than 75% of the dementia cases
worldwide. It is known to be a multifactorial neurodegenerative
disorder designated by progressive loss of memory and other
cognitive functions. The main risk factor of AD is aging;
however the mechanism underlying the foundation of AD due
to aging is yet to be rmly elucidated. It is clinically marked by
the progression from episodic memory problems to a slow
global decline of cognitive function. Patients in end-stage AD
become bedridden and are highly dependent on custodial care,
There are approximately 44 million people affected by Alz-
heimer's disease, and it is expected to increase three times by
2050; these perturbing numbers show that Alzheimer's disease
(AD) remains a serious socio-economical problem.2 AD is esti-
mated to have cost the world $604 billion in 2010 alone.3 These
costs are staggering, particularly in light of worldwide increase
in the number of AD cases. AD remains the most prevalent
unmet medical need because of its chronicity, cost, severity, and
lack of mechanism based treatment.4
Extensive research from several years has still not been able
to establish the exact molecular-mechanistic aspects of AD.
Clinically, AD is indicated by widespread neuronal cell death in
the brain, which corresponds to deposition of abundant brillar
plaques, primarily comprising the beta-amyloid (Ab) peptide.5,6
The identied pathological hallmarks of AD are senile plaques
of amyloid beta protein, intracellular neurobrillary tangles
(NFTs), and neuronal degeneration. Accumulation of Ab
peptides encourages conformational changes that lead to
further non-covalent polymerization into a heterogeneous
aDr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, New Delhi
110007, India. E-mail: mtiwari07@gmail.com
bAmity Institute of Integrative Sciences and Health (AIISH), Amity University Haryana,
Amity Education Valley, Gurgaon-122413, India
cSchool of Computational & Integrative Sciences, Jawaharlal Nehru University, New
Delhi 110067, India
† Electronic supplementary information (ESI) available: In silico study data, 1H
and 13C spectra of all synthesized compounds have been kept as supplementary
data. See DOI: 10.1039/d0ra00663g
‡ These authors contributed equally to this work.
17602 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
range of oligomeric class that nally forms amyloid brils.7 used to develop a variety of hybrid compounds having capability
Vulnerability of brain to oxidative stress is due to its high to act simultaneously in diverse biological targets. The complex
demand for oxygen, abundant lipid content and relative scarcity pathophysiology of AD may necessitate combination or multi
of antioxidant enzymes.8–10 Consequences of neuronal loss drug targeted ligand therapy as disease modifying strategy.19
because of oxidation include upregulation of proinammatory MTDLs approach involves molecular hybridization technique in
cytokines and irreparable DNA damage.11,12 Metal dyshomeo- which two or more pharmacophore of bioactive molecule are
stasis in the body, especially in the brain, can be a critical incorporated into single drug molecule. This approach reduces
mechanism for neurodegenerative processes, including oxida- the side effects and lead to a better pharmacokinetic prole of
tive stress generation or amyloid plaques aggregation.13
the MTDL compared to two or more separate individual drug
Despite the fact that Alzheimer's disease (AD) was rst representing respective single pharmacophore.20 This approach
described over 100 years ago, disease-modifying treatment has of discovery of drug candidate which is capable of targeting
not yet been developed for this disease. There is no complete multiple factors involved in AD complex pathogenesis and
cure for AD, but some medicines can ameliorate its clinical having multiple properties like antioxidant, neuroprotective,
symptoms by having an impact on cognitive functions, delaying metal chelation, anti-inammatory, anti-Ab aggregation and
functional decline and improving behavioural changes.14,15 Still, cholinesterase and secretase inhibitory activities, would greatly
their therapeutic interest is limited owing to their side effects facilitate in improving therapeutic strategies.21
and to the fact that they only treat symptoms. There are only
In this context, we have previously developed various
four drugs in the market for the treatment of AD, three multifunctional synthetic molecules against AD such as done-
cholinesterase inhibitors (Donepezil, Rivastigmine, Galant- pezil derivatives, indandione derivatives and diallyl disulphide
amine) and an N-methyl-D-aspartate (NMDA) receptor AD derivatives.22–24 In the present work, we have rationally designed
antagonist (Memantine) thereby limiting its treatment possi- novel N-(6-methanesulfonyl-benzothiazol-2-yl)-3-(4-substituted-
bilities. No new drugs have been approved for the treatment of piperazin-1-yl)-propionamides as potential multifunctional
AD since 2003.16,17 Decades of research in AD pathophysiology agents for AD, which consists of benzothiazole moiety as well as
has led to target identication for new drug development. piperazine moiety.
Nowadays, multi targeted directed ligands (MTDL) approach of
Benzothiazole are compounds with fused benzene and
AD therapy is widely used, where ChE inhibition is joined with thiazole moiety containing nitrogen and sulphur, which has
additional biological properties.18 Drugs with neuroprotective fascinated interest in central nervous system (CNS)-related drug
and disease-modifying properties are of greater research discovery. Various studies have reported that benzothiazole
interest. Multi-target-directed ligands (MTDL) strategy has been derivatives possess a broad spectrum of biological activities
Fig. 1 Designing strategy of multifunctional benzothiazole–piperazine hybrids.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17603

View Article Online
RSC Advances
Paper
including AChE inhibitory action, Ab-binding ability and neu-
roprotective property.25,26 For example, benzothiazole contain-
ing Ab binding agents thioavin-T and PIB are widely used in
detecting amyloid brils in vivo as well in vitro.27 Additionally,
a well-known neuroprotective drug riluzole containing benzo-
thiazole core has shown very potent neuroprotective effect in
various in vitro as well as in vivo models and is widely used
clinically for therapy of amyotrophic lateral sclerosis.28 More-
over, benzothiazole derivative sabeluzole (NMDA antagonist),
has also been reported to impede the clinical progression of
AD.29 Conversely, piperazine is a exible ring system consisting
of a six-member ring with two nitrogen atoms at 1, 4 positions.
Piperazines are reported for various therapeutic uses and
specially, this ring system has been widely used for CNS drug
discovery.30–32 Studies have supported that piperazine deriva-
tives show a very high binding affinity to amyloid, capability to
cross blood brain barrier and in addition they also provide rapid
elimination of non-specic signals from the brain.33 Moreover,
piperazines containing neuroprotective agents VK-28 as well as
SP-04 are well studied as effective neuroprotective agents in
2. Results and discussion
2.1. Synthesis
Compound
number
Compound
number
R-group
R-group
4 (85%)
11 (88%)
5 (88%)
6 (75%)
12 (74%)
13 (81%)
14 (69%)
7 (82%)
8 (84%)
9 (79%)
15 (86%)
16 (75%)
17 (71%)
.
various neurodegenerative models34
A lot of efforts have been made by several research groups to
modify donepezil pharmacophore (standard AChE inhibitor) by
various approaches such as bioisosteric replacement of phar-
macophoric elements, hybridization of two pharmacophore,
and linker optimization. Indeed, some of the modied leads 10 (76%)
have shown potential multifunctional activities for the treat-
ment of AD.35 It is well studied that basic piperidine moiety of
standard AChE inhibitor donepezil actively participates in
Synthesis of designed benzothiazole–piperazine hybrids 4–17
have been carried out according to the conditions described in
Scheme 1. Briey, commercially available 4-methylsulfonyl
aniline was stirred with potassium thiocyanate (KSCN) and Br2
was added slowly to achieve benzothiazole-2-amine 1. Further,
benzothiazole amine was reacted with 3-chloropropionyl chlo-
ride in 1,4-dioxane to give key intermediate 2 in high yield.
Finally, substituted piperazine was coupled with key interme-
diate 2 by using a catalytic amount of NaHCO3 and NaI to yield
target compounds N-(6-methanesulfonyl-benzothiazol-2-yl)-3-
(4-substituted-piperazin-1-yl)-propionamide 4–17. Synthesized
interaction with the AChE active site and this basic moiety is
considered as an essential pharmacophoric element to produce
inhibitory action.36
Therefore, by considering these evidences benzothiazole–
piperazine hybrid molecules have been designed to develop
effective multifunctional agents against AD. In due course,
benzothiazole and piperazine pharmacophore have been
hybridized by using a exible propanamide linker. We have
made bioisosteric replacement of the piperidine ring of done-
pezil with piperazine ring and it was thought that this bio-
isosteric replacement may provide better pharmacological
action against AD. Additionally, it is also assumed that the
exible propanamide linker will provide enough exibility to
hybrid pharmacophore for adequate adjustment at CAS as well
as the PAS binding site of AChE active site. Thus, this drug
designing strategy may confer a series of benzothiazole–piper-
azine hybrid molecules as potential multifunctional agents that
1
molecules were completely characterized by HNMR, 13C NMR
and mass spectroscopy. Purity of target compounds were
examined by reverse phase HPLC.
2.2. In vitro studies
2.2.1. Cholinesterase inhibitory activity and SAR discus-
might display polypharmacological action all in concert, such sion. The inhibitory potencies of synthesized hybrid molecules
as AChE inhibition, Ab disaggregation, and neuroprotection 4–17 were assessed against AChE and BuChE by following the
(Fig. 1). Synthesized compounds have been evaluated for their well standard Ellman's method,37 with donepezil as the refer-
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) ence drug. The inhibitory prole of these hybrid molecules for
inhibitory action, Ab disaggregation activity, metal chelation AChE and BuChE is presented in Table 1. As shown in Table 1,
potency and neuroprotective capability. Additionally, molecular all target compounds exhibited moderate to good inhibition
docking and molecular dynamic (MD) simulation have also toward ChEs. On the basis of obtained inhibitory prole,
been carried out to explore molecular level interaction of these following structure activity relationship has been observed.
molecules with AChE and Ab peptide. Moreover, in vivo efficacy Compound 4, containing unsubstituted phenyl showed
of the most active compound was illustrated in the scopolamine moderate inhibition towards AChE (IC50 ¼ 24.14 mM). Hybrid
induced mouse model of dementia.
molecules 5 (IC50 ¼ 18.31 mM), 6 (IC50 ¼ 13.05 mM) and 7 (IC50 ¼
17604 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Scheme 1 Reagents and conditions: (A) KSCN, Br2, glacial acetic acid, 0–5 ꢀC, 6 h; (B) 3-Chloropropionyl chloride, TEA, 1,4-dioxane, reflux, 8 h;
(C) Substituted piperazines, NaHCO3, NaI, dried acetonitrile, reflux, 8–12 h.
Table 1 Inhibition of AChE, BuChE, and Ab1-42 aggregation by the synthesized compounds
Compounds
IC50 ꢁ SD (mM) AChEa
IC50 ꢁ SD (mM) BuChEb
Self Ab1-42 aggregation inhibitionc (%)
4
5
6
7
8
9
10
11
12
13
14
15
16
17
24.14 ꢁ 0.721
18.31 ꢁ 0.777
13.05 ꢁ 0.813
11.25 ꢁ 1.803
20.18 ꢁ 1.435
14.45 ꢁ 1.032
7.834 ꢁ 0.810
4.548 ꢁ 0.984
2.319 ꢁ 0.410
10.87 ꢁ 0.657
14.59 ꢁ 0.438
19.48 ꢁ 0.452
5.244 ꢁ 0.690
26.43 ꢁ 0.551
4.45 ꢁ 0.37
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54.29502 ꢁ 0.265
55.53029 ꢁ 0.134
51.74112 ꢁ 0.289
54.34428 ꢁ 0.427
54.43522 ꢁ 0.159
52.31708 ꢁ 0.114
53.40836 ꢁ 0.322
52.22614 ꢁ 0.513
53.30605 ꢁ 0.541
53.6736 ꢁ 0.228
54.48827 ꢁ 0.885
52.44591 ꢁ 1.203
45.3109 ꢁ 0.241
53.40836 ꢁ 0.805
n.ad
Rivastigmine
Galantamine
Tacrine
Donepezil
Curcumin
1.32 ꢁ 0.09
3.12 ꢁ 0.44
0.054 ꢁ 0.85
8.71 ꢁ 1.36
n.ad
3.12 ꢁ 0.44
n.ad
0.13 ꢁ 0.21
n.ad
0.049 ꢁ 0.05
n.ad
n.ad
50.23 ꢁ 1.201
a
Half maximal inhibitory concentration of AChE from electric eel (means ꢁ SD of three experiments). b Half maximal inhibitory concentration of
BuChE from equine serum (means ꢁ SD of three experiments). c Self-induced Ab1-42 aggregation inhibition (50 mM) by tested inhibitors at 50 mM by
thioavin-T based uorescence method (means ꢁ SD of three experiments). d na-no activity.
11.25 mM), bearing electron withdrawing group F, Cl, NO2 benzylic carbon (compound 11) promisingly enhanced inhibi-
respectively displayed better inhibitory actions as compared to tory action and this bulkier group containing compound
compound 4 which contains unsubstituted phenyl at the showed an IC50 value of 4.54 mM for AChE. Introduction of one
terminal end. Thus, substitution with electron withdrawing nitrogen as heteroatom to terminal benzene ring (compound
groups bestowed better inhibitory action as compared to 12) provided most active AChE inhibitor of the series with an
unsubstituted counterpart, although this trend was not IC50 value in the low micromolar range (2.31 mM). This
observed for compound 17 (IC50 ¼ 26.43 mM) which has electron compound appeared as a most potent AChE inhibitor in the
withdrawing group CF3. It was detected that derivatives 8 and 9, entire series, although, two nitrogen containing pyrimidine
bearing electron donating group, methyl as well as methoxy derivative 13 failed to give promising inhibitory action. Thus, it
respectively showed slightly higher IC50 value as compared to may be hypothesized that 2-pyridine at the terminal end of this
unsubstituted phenyl as well as with compounds having elec- pharmacophore is well tolerated inside the active site of AChE.
tron withdrawing group. However, the introduction of the Molecular docking study also conrmed adequate binding of
exible benzyl group (compound 10) in this pharmacophore this molecule in the active site of AChE. At the terminal end
produced effective inhibitory action against AChE (IC50 ¼ 7.83 furoyl (compound 14) as well as benzyl ester (compound 15) was
mM). Noticeably, introduction of one more phenyl moiety on also not favourable to exert effective inhibition against AChE. A
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17605

View Article Online
RSC Advances
Paper
g-secretases and b-secretases. Ab1-42 folds into b-sheets, self-
assembling into toxic brillar aggregates. These brillar
aggregates (plaques) play a crucial role in initiating the patho-
physiology of AD. Inhibition of the formation of brillar
aggregates is one of the most prominent approaches to develop
effective therapeutic agents for AD.44 To study the effect of
compounds 4–17 on the aggregation propensity of Ab1-42
,
a Thioavin-T (ThT) assay was performed, curcumin was used
as a reference (Table 1, Fig. 3a). Most of the compounds
exhibited strong inhibitory activity against Ab1-42 aggregation
and it was better than standard curcumin. These compounds
have shown the percentage of inhibition ranging from 45.31%
to 55.53% at the concentration of 50 mM. Hybrid molecule 12
which was very effective AChE inhibitor, also exhibited signi-
cant inhibition of Ab1-42 aggregation compared to the standard
(Fig. 3b). Thus, these molecules are capable in inhibiting Ab1-42
aggregation effectively, being an effective inhibitor of AChE.
2.2.4. Confocal microscopy of compound 12 on Ab1-42
aggregation inhibition. The clearance of amyloid beta brils is
an effective therapeutic strategy for AD treatment. Hence, the
drug designed to combat AD should have the potential of
removing Ab1-42 mature brils. The Ab1-42 aggregation activity of
the most promising compound 12 was monitored by Confocal
Laser Scanning Microscopy (CSLM) in order to complement the
Fig. 2 Lineweaver–Burk reciprocal plot of AChE initial velocity with
increasing substrate concentrations showing kinetic study of AChE
inhibition mechanism with compound 12.
moderate IC50 value 10.87 and 14.59 mM were noticed for
compound 14 and compound 15, respectively. Markedly,
compound having piperonyl group (compound 16) showed low
micromolar range inhibition (IC50 ¼ 5.24 mM) against AChE.
Thus, it may be hypothesized that exible bulkier side group
like piperonyl is also favourable in inhibiting AChE very effec-
tively. Inhibition data for BuChE clearly indicate that none of
the compounds showed inhibition to BuChE (IC50 value of >100
mM), indicating their high selectivity towards AChE. Such type of
selective inhibition for AChE may be benecial in reducing
peripheral cholinergic side effects. The level of AChE is higher
in the brain whereas the level of BuChE is higher in the blood.
Thus, inhibition of BuChE will result into lower blood cholin-
esterase levels thus amplifying the risk of cholinergic distress by
enhancing peripheral cholinergic side effects.38,39 BuChE has
also been reported to be allied with metabolism of drugs and
lipoproteins. Therefore, selective inhibition of AChE over
BuChE is an important outlook for discovery of safe anti-
alzheimer agents.40,41 For instance, AChE inhibitors used for
the therapy of AD; donepezil, galantamine and rivastigmine,
former two are selective AChE inhibitors and show better
curative effects in contrast to the third which is non selective
inhibitor. Also, tacrine, owing to its low selectivity for AChE
showed extreme side effects,42 and ultimately has been with-
drawn from market.
2.2.2. Kinetic characterization of AChE inhibition. The
most potent compound 12 was further studied to get an insight
into the mechanism of AChE inhibition by enzyme kinetics
studies. The molecular docking studies revealed that the
compound 12 was binding to both the CAS and PAS site, giving
a mixed type of inhibition. However, the inverse of the reaction
rates versus the inverse of substrate at different concentrations
of the compound 12, Lineweaver–Burk reciprocal plots were
formed (Fig. 2). Their Lineweaver–Burk reciprocal plots exhibi-
ted a decrease in Vmax as well as Km with increasing inhibitor
(compound 12) concentration. The result revealed that
compound 12 exhibited an uncompetitive type of inhibition.43
2.2.3. Modulation of self- induced Ab1-42 aggregation. A
Fig. 3 (a) Inhibition of Ab42 aggregates studied by thioflavin (ThT)
assay. Ab42 (50 mM) was incubated alone and independently with
compounds and their influence on Ab aggregation was quantified by
measuring ThT fluorescence intensity, represented as normalized
fluorescence intensity (NFI) at 485 nm for a given time point (48 h); (b)
ThT emission fluorescence spectra (range 450–550 nm) of Ab1-42
alone in blue and in the presence of compound 12 (50 mM) in red.
major hallmark of AD is the formation of amyloidb (Ab1-42
)
peptides from amyloid precursor protein (APP) aer cleavage by
17606 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 4 Assessment of the effect of compound 12 on anti-aggregation property of amyloid beta peptide (Ab1-42). Confocal laser scanning
microscopic analysis of the deposited amyloid beta aggregates as assessed in 48 h. The fluorescence intensity of confocal microscopic image
represents a vision of Ab1-42 amyloid aggregates, with representative Ab1-42 peptide (control) co-incubated with the presence of compound 12.
Fig. 5 TEM analysis of Ab1-42 aggregation in the presence of compound 12: (a) Ab1-42 alone (50 mM), incubated at 37 ꢀC for 48 h. (b) Ab1-42 (50 mM)
and curcumin (50 mM) were incubated at 37 ꢀC for 48 h. (c) Ab1-42 (50 mM) and 12 (50 mM) were incubated at 37 ꢀC for 48 h.
results of ThT binding assay, (Fig. 4). The higher uorescence in
confocal laser scanning microscopy images validated the
noticeable brillar formation in Ab1-42 alone (control) group,
and further, decrease in the uorescence showed thatAb1-42
aggregation were inhibited when Ab1–42 was incubated with
compound 12. The results conrm that compound 12 pro-
ciently prevents Ab1-42 bril formation and has potential of Ab1-
2.2.6. Disaggregation of self-induced Ab1-42 aggregation.
The most active multipotent compound 12 displaying higher
potential to inhibit self-mediated Ab1-42 aggregation was further
evaluated for its ability to disaggregate self-induced Ab1-42
aggregation by ThT assay. At 37 C for 24 h, fresh Ab peptides
were incubated to assist the formation of brils. Compound 12
ꢀ
was then added to the sample mixture and incubated for
aggregation inhibition property.
42
2.2.5. TEM study of compound 12 on inhibition of Ab1-42
bril formation. In line with the ThT binding assay and CSLM,
most potent Ab1-42 aggregation inhibitor 12 was subjected to
TEM analysis for studying its effect on Ab aggregation process.
At 0 h time point, samples with Ab1-42 showed no aggregation
(data not shown). While aer incubating for 48 h at 37 ꢀC,
sample with Ab1-42 alone had typically aggregated into mature,
bulkier and dense amyloid brils (Fig. 5a). In comparison,
electron micrographs of Ab samples incubated with curcumin
and compound 12 respectively under similar experimental
conditions showed small dissociated aggregates and no char-
acteristic brils of Ab1-42 (Fig. 5b and c). The results of TEM were
in concordance with the results of ThT studies and CSLM
studies, proving the effectiveness of compound 12 in slowing
down the rate of Ab1-42 brils formation in vitro.
Fig. 6 ThT emission fluorescence spectra (range 450–550 nm) of Ab1-
42 alone in blue and in the presence of compound 12 (50 mM) in red for
disaggregation studies.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17607

View Article Online
RSC Advances
Paper
2.2.7. Metal chelating properties of compound 12. The
ability of our synthesized compound 12 to chelate bio-metals
like Cu(II) and Fe(II) was performed by UV-vis spectroscopy.23
On the addition of CuSO4 into reaction mixture with compound
12, it was observed that there was a visible bathochromic shi
in the maximum absorption wavelength from 239 nm to 246 nm
and 284 nm to 293 nm, and the absorbance also dropped
noticeably, signifying the formation of a compound 12–Cu(II)
complex (Fig. 7a). The compound did not show any activity with
ferrous ion. Job's method was employed to calculate the stoi-
chiometry of the 12–Cu(II) complex45 by preparing a series of
reaction mixture by varying the proportion of the metal ion and
compound and keeping the total concentration constant. The
absorbance of the complex of CuSO4 and compound 12 at
different concentrations were determined using UV spectros-
copy. As shown in Fig. 7b, the absorbance change at 333 nm
were plotted, the two straight lines intersected at a mole frac-
tion of 1 implying a 1 : 1, 12–Cu(II) complex.
2.2.8. Molecular docking studies. Additionally, to explore
the interaction mode of hybrid molecule 12 with AChE (PDB
code: 1EVE) and Ab1-42 (PDB code: 1IYT), a molecular docking
simulation study was performed using the Autodock 4.2 so-
ware.22 Compound 12 showed good interaction within the active
site of AChE. As illustrated in Fig. 8, this compound was binding
simultaneously to both the enzymatic catalytic active site (CAS)
and the peripheral anionic site (PAS). It was visualized that
benzothiazole core oriented towards CAS site and pyridine
piperazine moiety engaged with PAS site. It was also noticed
that propanamide linker have provided suitable length between
CAS binding group as well as PAS binding group. Additionally,
amide group of propanamide linker is also being involved in
hydrogen bond interaction, which may provide stability of
ligand within active site of AChE. His440, Phe288, Ser286 are
involved in H-bond interaction with hybrid molecule 12 (Fig. 8).
In mid-gorge sites, a hydrogen bond with the residue his440
and a cation–p interaction with ser200 and glu199 were
Fig.
7 Metal chelation study: (a) UV absorbance spectrum of
compound 12 (60 mM) alone in blue or in the presence of CuSO4 (60
mM) in red, FeSO4 (60 mM) in green in buffer (20 mM HEPES, 150 mM
NaCl, pH 7.4); (b) determination of the stoichiometry of complex
Cu2+–12 by Job's method.
another 24 h. Disaggregation studies were assessed by ThT
assay, where uorescence intensities indicate the degree of Ab
brillar aggregation. Compound 12 showed 48.64% disaggre-
gation (Fig. 6), which implies that it can prociently inhibit the
already formed amyloid beta plaque.
Fig. 8 Proposed binding mode of compound 12 in the active site of AChE (PDB: 1EVE). The black dashed lines represent hydrogen bonds.
17608 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 9 Proposed binding mode of compound 12 in the active site of Ab1-42 (PDB: 1IYT). The blue dashed lines represent hydrogen bonds.
observed and could enhance the interaction with AChE. Trp279 increase in RMSD of 0.05 nm during 0–10 ns, the consequences
and Tyr334 at PAS are involved in p–p stacking interaction with of small dris of ꢂ0.02–0.04 nm can be seen at 10–25 ns and the
pyridine moiety of compound 12. Additionally, compound 12 quite stable trajectory during 25–100 ns. The initial dris in plot
suitably interacts with CAS site and their benzothiazole moiety represented the structural perturbation arises due to protein–
imparts hydrophobic interaction with CAS site residue Trp84.
ligand interactions which is nally settled around 25 ns and we
Assessment of Fig. 9 to understand the binding mode of can observe the stable conformational dynamics of AChE–12
compound 12 with Ab1-42 shows that compound 12 interacts complex till the simulation end at 100 ns.
well with Ab1-42. Compound 12 is transversely tted along the
Another effective parameter, radius of gyration (Rg) is
major helix, and Lys28 is involved in H-bond interaction with applied to investigate the conformational stability and integrity
a methylsulfonyl group of compound 12. In addition, prop- of protein and protein–ligand complex46 (Fig. 10b). The Rg plot
anamide and pyridine piperazine fragments display hydro- of all Ca-atoms shows that the structural composition of AChE
phobic interactions with residues Lys16, Leu17, Val18, Phe20, is retained in water, having Rg value 2.30 ꢁ 0.02 nm. However,
Ala21, Asp23, Val24, ser26, Ile31, Leu34 and Met35 resulting in the structural integrity of AChE–12 complex remains stable
stabilized compound 12–Ab1-42 complex very nicely. These around Rg value 2.27 ꢁ 0.02 nm. This slight decrease in Rg value
results indicate that the hydrophobic interactions and the indicates the stable molecular interaction of protein–ligand
hydrogen bonding interaction play crucial roles in the effective complex.
inhibition of Ab1-42 aggregation.
To determine the structural stability of AChE and AChE–12
2.2.9. Molecular dynamics (MD) simulation studies with complex we also monitored the solvent accessible surface area
AChE and Ab1-42. We have completed molecular dynamics (MD) (SASA) as shown in Fig. 10c. We observed that the structure of
simulation for 100 ns to examine the structural stability and both, AChE and AChE–12 complex having almost similar
and average SASA value ꢂ310 nm2. This result suggested that the
22
molecular interaction of protein (AChE and Ab1-42
)
complex with compound 12. To examine the structural stability ligand is nicely tted at the active site of AChE, thus, the
of AChE and AChE–12 complex, we computed the time evolu- conformational dynamics analyses RMSD, Rg and SASA results
tion plot of all Ca-atom RMSD (Fig. 10a). It can be seen that the were consistent, indicating the stable interaction of AChE–12
conformational dynamics of AChE remains stable around complex.
RMSD value 0.15 nm during the simulation of 100 ns in water at
To investigate conformational uctuations, and the effect of
300 K. The RMSD trajectory archives equilibrium ꢂ20 ns and local dynamics on the structural complex of AChE–12, and
the stable equilibrium is observed up to ꢂ65 ns. The small dri residues involved in molecular interaction, we calculated the
of 0.05 nm arises ꢂ75 ns, however, the equilibrium is regained average uctuation of each amino acids of AChE. The RMSF plot
ꢂ85 ns, suggesting the stable structural dynamics of protein in of all Ca-atoms is shown in Fig. 10d. The RMSF results clearly
water. The RMSD plot of AChE–12 complex shows initial indicate that the active site residues which were involved in
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17609

View Article Online
RSC Advances
Paper
Fig. 10 Molecular dynamic simulation studies with AChE (a) The equilibrium average of Ca RMSD of AChE (black), AChE–12 docked complex
(red) in water at 300 K; (b) time evolution plot of radius of gyration (Rg) plot of AChE (black), AChE–12 complex (red) in water at 300 K; (c) time
evolution plot of solvent accessible surface area (SASA) plot of AChE (black), AChE–12 complex (red) in water at 300 K; (d) root mean square
fluctuation (RMSF) plot of AChE (black), AChE–12 complex (red) in water at 300 K; (e) structural snapshot of AChE–12 complex at the time interval
of 10 ns, during the simulation in water at 300 K.
17610 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 11 Molecular dynamic simulation with Ab1-42; (a) the equilibrium average of Ca RMSD of Ab1-42 (magenta), Ab1-42–12 docked complex
(green) in water at 300 K; (b) time evolution plot of radius of gyration (Rg) plot of Ab1-42 (magenta), Ab1-42–12 docked complex (green) in water at
300 K; (c) time evolution plot of solvent accessible surface area (SASA) plot of Ab1-42 (magenta), Ab1-42–12 docked complex (green) in water at
300 K; (d) root mean square fluctuation (RMSF) plot of Ab1-42 (magenta), Ab1-42–12 docked complex (green) in water at 300 K; (e) structural
snapshot of Ab1-42–12 complex at the time interval of 10 ns, during the simulation in water at 300 K.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17611

View Article Online
RSC Advances
Paper
Fig. 12 Principal component (PC) analysis of protein and protein–ligand complex: (A) the collective motion of AChE (black), AChE–12 complex
(red) using projections of MD trajectories on two eigenvectors corresponding to the first two PCs. (B) Ab1-42 (green) and Ab1-42–12 docked
complex (magenta).
interaction with compound 12 having lower average uctuation. understand the contribution of active site residues on confor-
Furthermore, the almost complete overlapping RMSF plots of mational stability of Ab1-42–12 complex. RMSF plot shows that
AChE and AChE–12 complex signies no conformational the residues belonging to Ab1-42 having average uctuation
changes in native structure of protein upon the binding of #0.4 nm, whereas, the average uctuation of Ab1-42 is slightly
ligand. Thus, to conrm the results we also measured the increases upon binding with 12. The secondary structure anal-
evolution plot of secondary structure (ESI Fig. S1 and S2†) and ysis shows that the helical structure of Ab1-42 remains consistent
the structural snapshots of AChE–12 (Fig. 10e) at time interval with crystal structure, however, we observed the propensity to
of 10 ns. Both analyses show consistent results with RMSF form turn like structure for the residues belonging to N-and C-
which again conrm the favourable interaction of 12 at active terminal of Ab (ESI Fig. S3 and S4†). The binding of 12 disrupted
site of AChE.
the salt bridge between the Asp23–Lys28 which prevent the self-
The conformational stability of Ab1-42–12 complex was aggregation of Ab1-42, thus, we observed turn like structure at C-
measured using structural order parameters, RMSD, Rg and terminal of Ab1-42–12 complex, however, the helical structure of
SASA. Fig. 11a shows that RMSD plot of Ab1-42 remains stable Ab at N-terminal remains consistent with crystal structure. The
around 0.5 nm in water at 300 K. The consecutive dris in structural snapshot of Ab1-42–12 complex at the time interval of
trajectory, during the initial 0–20 ns represents the exible 10 ns (Fig. 11e) provide the clear evidence that binding inter-
nature of Ab1-42, however, it attains equilibrium at ꢂ20 ns and action of 12 is largely localized at the C-terminal and the bulky
the stable structure can be seen for 20–100 ns of simulation. side chain of 12 create structural hindrance for salt bridge
However, the plot shows lower RMSD value upon binding with associated, thus prevent the protein aggregation the patholog-
12, during initial 0–25 ns of simulation. The trajectory of Ab1-42
–
ical state of Alzheimer disease.47
12 complex nds stable at 25–40 ns, followed by continuous
To further explore the dynamic properties of the investigated
increase in RMSD ꢂ40–55 ns. With the dri of ꢂ0.5 nm which structures in our simulations, the essential dynamics (ED)
suggested the conformational ipping, it attains stable equi- analysis on the Ca atoms was performed.48 ED actually reects
librium at ꢂ55–95 ns and during the last 5 ns, it dropped down the overall conformational space of protein and protein–12
to the conformational state observed for ligand unbound Ab1-42
.
complex during simulations (Fig. 12A).The projection of
The comparative structural stability and integrity of Ab1-42
and Ab1-42–12 complex examined by Rg plot (Fig. 11b) shows
initially exible Rg trajectory for Ab1-42 which attains equilib-
rium ꢂ20 ns and structural dynamics remains stable with
average Rg value 1.8 ꢁ 0.02 nm for the period of 20–100 ns.
Differently, the Rg plot of Ab1-42–12 complex shows initial uc-
tuation around Rg value 1.8 nm which is dropped down to
1.3 nm at ꢂ20 ns, thereaer, with the dri of 0.3 the trajectory
remains uctuating around Rg value 1.6 which can be observed
during 24–40 ns. We nd subsequent decrease in Rg value ꢂ48
ns and the stable conformation of protein–ligand complex with
Rg value 1.1 nm is observed during the period of 50–100 ns of
simulation. Thus, the Rg analysis also anticipated the two major
conformational states of Ab1-42–12 complex. However, the SASA
plot (Fig. 11c) of Ab1-42 remains stable ꢂ26 nm2, whereas, we
nd slightly lower SASA value (ꢂ25 nm2) as compared protein
alone in water. This observation suggested the stable associa-
tion of 12 with Ab1-42. We also examine RMSF plot (Fig. 11d) to
Fig. 13 Effects of various concentrations of compound 12 on cell
viability in SHSY5Y cells after treatment for 24 h. Cell viability was
measured by MTT assay. Data were shown as mean ꢁ SD of three
independent experiments and each included triplicate sets. ***p <
0.001 vs. control, statistical analysis was performed.
17612 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 14 A: Protective effects of compound 12 on H2O2-induced cell death in SHSY5Y cells. Cells were incubated with different concentration of
12 for 3 h and then treated with 200 mM H2O2 for 24 h. Cell viability was measured by MTT assay. Data were shown as mean ꢁ SD of three
independent experiments and each included triplicate sets. ###p < 0.001 compared to control, ***p < 0.001 compared to H2O2 treated cells; B:
phase-contrast micrographs showing morphological changes in H2O2-induced neurotoxicity and neuroprotection of compound 12 in SH-SY5Y
cells. (a) Cells without treatment showed healthy shapes. (b) H2O2 alone (200 mM) induced neurotoxicity. (c) Co-treatment of compound 12 (5
mM) for 24 h with H2O2 (200 mM) and showed neuroprotection.
trajectories onto the rst two principal components (PC1, PC2) 24 h, and cell viability assay was performed using the 3-(4,5-
shows the motion of investigated system: AChE and AChE–12 dimethyl dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT).
complex in phase space and is illustrated in Fig. 12A.
As given in Fig. 13, no cytotoxicity was displayed by the
In the ED analysis shows, both AChE and AChE–12 complex compound 12 at concentrations of 1 and 5 mM aer incubation
show similar phase space behaviour, however, the ligand 12 for 24 h. When the concentration was increased up to 10 mM,
decreases the exibility in the AChE protein which is suggesting compound 12 displayed more than 67% cell viability. Hence,
the normal function of the protein upon complex formation these results suggest that compound 12 exhibits very low
with 12.
Differently, the ED analysis of Ab1-42 and Ab1-42–12 complex
toxicity to SHSY5Y cells in the range of tested concentrations.
2.2.11. Neuroprotection against H2O2-induced death in
shows that the protein–12 complex has not explored the same SH-SY5Y cells. The ability of compound 12 to provide neuro-
conformational space as the native protein, Ab1-42 (Fig. 12B). protection against H2O2-induced damage in SH-SY5Y cells were
Instead, it is restricted to small excursions slightly away from its assessed using a cell viability assay at two concentrations: 1 and
initial conformation. In this Fig. 12B, we can see irregular 5 mM. It was observed in the Fig. 14A and B that exposing SH-
stretches or clusters of line in the ED plot of Ab. Two features SY5Y cells with H2O2 for 24 h resulted in a considerable
are very apparent from these plots. Firstly, the clusters are well decrease in cell viability (54.90%, ###p < 0.001). However, when
dened in Ab1-42–12 complex structure than the protein Ab1-42 the cells were treated with the test compound 12 along with
alone. Secondly, the Ab–12 complex covers a smaller region on H2O2, it was found that the compound 12 imparted signicant
the plot particularly along PC1 plane compared to that covered neuroprotection at concentration 5 mM. Hybrid molecule 12
by native Ab1-42. It is also clear from all the plots that the exi- induced marked cellular recovery at this concentration (86.96%,
bility of Ab1-42 decreases upon binding to 12, suggesting that 12 ***p < 0.01), as compared to the H2O2-treated cells. Thus, it
can efficiently prevent the self-aggregation of Ab1-42, the path- proves our hypothesis that the tested hybrid molecule display
ological state of Alzheimer disease.49,50 Our observation thus neuroprotection against H2O2 induced neurotoxicity through its
corroborates with the idea of higher exibility of Ab1-42 at 300 K. ability to block H2O2 mediated oxidative stress and damage.
2.2.10. In vitro cytotoxicity assay. Compound 12 possessing
AChE inhibition, metal ions chelation and Ab1-42 aggregation
inhibition was further evaluated for cytotoxicity studies in the
2.3. In vivo studies
human neuronal cell line SHSY5Y. SH-SY5Y cells were exposed
to compound 12 at different concentrations (1, 5 and 10 mM) for
The clinical manifestation of dementia is the impairment of
learning as well as memory and is considered as a main
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17613

View Article Online
RSC Advances
Paper
symptom of AD. Scopolamine is a known muscarinic receptor Compound 12 at both the doses showed signicant reductions of
antagonist that interferes with transmission of acetylcholine in transfer latencies (Table 2), which indicates its anti-dementia and
the central nervous system51 inducing dysregulation in cholin- nootropic potential against scopolamine induced memory decit.
ergic signaling pathway causing severe damage in learning, and
2.3.2. Effect of compound 12 in social recognition test. To
short term maintenance of spatial memory tasks. Therefore, study the ability of compound 12 in enhancing cognition in
anti-dementia and nootropic effect compound 12 was evaluated scopolamine induced mice, well distinguished social recogni-
against scopolamine-induced mice model of dementia.
2.3.1. Effect of compound 12 on transfer latencies by EPM decrease in the T2/T1 ratio (second interaction trial/rst
test.
tion test was performed.53 The results indicated a signicant
For the study of learning and memory in rodents which relies on interaction trial ratio) aer treatment with compound 12 as
the intrinsic tendency of mice to stay in closed spaces, EPM compared to the scopolamine treated group. This suggests that
model is used widely.52 Therefore, compound 12 was evaluated compound 12 signicantly enhanced the short-term memory.
for anti-amnesic activities in this model. During the training Our results indicated that compound 12 at 20 mg kgꢃ1 have
trial, our result showed no signicant difference in the transfer shown a T2/T1 ratio 0.377 ꢁ 0.00072 as compared to scopol-
latencies among the treatment groups in EPM i.e. all the mice of amine (3 mg kgꢃ1) and standard drug donepezil (5 mg kgꢃ1
)
the different groups came into the closed arm of the maze treated group which displayed a T2/T1 ratio 1.103 ꢁ 0.037 and
within stipulated time period. Nonetheless, in the scopolamine 0.555 ꢁ 0.133 respectively (Fig. 15). Thus, these results evidently
treated group (3 mg kgꢃ1), the transfer latency time increased support cognition enhancing ability of benzothiazole–pipera-
signicantly when compared to the control group, which indi- zine hybrid molecule 12.
cated the memory impairment during the retention trial
(Table 2). In comparison, there were signicant reduction in the
transfer latency in the donepezil (5 mg kgꢃ1) treated group that
showcased its potential in reversing memory impairment
induced by scopolamine.
Similarly, animals treated with compound 12 at 10 mg kgꢃ1 and
20 mg kgꢃ1 signicantly reversed the effects of scopolamine.
Table 2 Treatment effect of test compound 12 on the transfer latency
(TL) by EPM (elevated plus maze) test in scopolamine induced mice
model of dementia
Transfer latency (TL)
Groups
in secondsa
Group 1 vehicle
Group 2 scopolamine
Group 3 scopolamine + donepezil
Group 4 scopolamine + 12 (10 mg kgꢃ1
Group 5 scopolamine + 12 (20 mg kgꢃ1
9.82 ꢁ 1.42
22.00 ꢁ 0.81
15.08 ꢁ 1
Fig. 15 Treatment effect of test compound 12 at 20 mg kgꢃ1 on the
T2/T1 (second interaction trial/first interaction trial ratio) in social
recognition test in scopolamine induced model. Data is expressed as
mean SEM of 6 animals. ###p < 0.001 compared to vehicle, ***p <
0.001 and **p < 0.01 compared to scopolamine treated groups.
)
)
12.81 ꢁ 2.08
12.62 ꢁ 2.60
a
Data is expressed as SEM of n ¼ 6.
17614 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
10 ml glacial acetic acid) was added dropwise by keeping
temperature 0–5 ꢀC. Aer addition of bromine, reaction was
continued for 6 h at RT. Salt appeared in reaction mixture was
ltered and dissolved in water. Salt solution was treated with
ammonium hydroxide to give benzothiazole-2-amine in good
yield.
4.2.1. 6-Methanesulfonyl-benzothiazol-2-yl amine (1). Yellow
solid; yield 75% (1.71 g); mp 216–218 ꢀC. 1H-NMR (400 MHz,
DMSO-D6) d 8.21 (s, 1H), 7.95 (s, 2H), 7.67 (d, J ¼ 10.4 Hz, 1H), 7.43
(d, J ¼ 8.5 Hz, 1H), 3.13 (s, 3H); LC-MS: m/z; 229 (M + 1).
3. Conclusion
In conclusion, a series of multifunctional benzothiazole–
piperazine hybrids (4–17) were developed to target multifaceted
nature of Alzheimer's disease. The synthesized derivatives
potentially and selectively inhibited AChE over BuChE with IC50
in micromolar range. Furthermore, the derivatives inhibited
Ab1-42 aggregation efficiently. The results obtained from
numerous in vitro and in vivo experiments established the
multimodal mechanism of action of most active compound 12.
In molecular docking studies, compound 12 adequately inter-
acts with AChE and Ab peptide. Further, we performed MD
simulation for compound 12 which reveals its procient
binding with AChE and Ab and steady interaction with the vital
active site residual during 100 ns simulation. Compound 12 was
non-cytotoxic to neuronal cells SHSY-5Y. This compound could
efficiently reduce death induced by H2O2 treatment in the SHSY-
5Y cells. It potentially attenuated scopolamine-induced cogni-
tive impairments in mice model in the EPM and social recog-
nition behavioral assays. Therefore, these properties together
proved the multifunctional nature of 12, and its ability to alle-
viate multifaceted nature of AD. Furthermore, there is an
enormous possibility for further development of this best lead
molecule. Efforts in enhancing the efficiency of compound 12
through various chemical modications have already been
instigated by this research group.
4.3. Synthesis of 3-chloro-N-(6-methanesulfonyl-
benzothiazol-2-yl)-propionamide (3)
An equimolar amount of benzothiazole amine 1 (10 millimole),
3-chloropropionyl chloride (10 millimole) and TEA (catalytic
amount) were reuxed in 1,4-dioxane. Reaction mixture was
poured in chilled water and obtained precipitate was ltered to
give
key
intermediate
3-chloro-N-(6-methanesulfonyl-
benzothiazol-2-yl)-propionamide (2).
4.3.1. 3-Chloro-N-(6-methanesulfonyl-benzothiazol-2-yl)-pro-
ꢀ
pionamide (2). White solid; yield 72% (2.28 g); mp 198–199 C;
1H-NMR (400 MHz, DMSO-D6) d 12.77 (s, 1H), 8.62 (s, 1H), 7.91 (s,
2H), 3.89 (t, J ¼ 6.2 Hz, 2H), 3.03 (t, J ¼ 6.3 Hz, 2H); LC-MS: m/z;
319 (M + 1).
4.4. Synthesis of N-(6-methanesulfonyl-benzothiazol-2-yl)-3-
(4-substituted-piperazin-1-yl)-propionamide (4–17)
4. Experimental methods
4.1. General
An equimolar ratio of key intermediate 3 and substituted
piperazines were reuxed in acetonitrile with catalytic amount
of NaHCO3 and NaI for 8–14 h. Reaction mixture was diluted
with water and crude product was extracted with ethyl acetate,
dried with anhydrous sodium sulphate. Evaporation of ethyl
acetate under reduced pressure was provided crude products.
Crude products were puried with column chromatography
using chloroform and methanol (90 : 5) to give pure target
compounds 4–17 in very good yield.
All the reagents and dried organic solvents were obtained from
Sigma Aldrich (St. Louis, MO, USA.), TCI Chemicals (Tokyo,
Japan), and Merck (Darmstadt, Germany). 1H and 13C NMR
spectra were acquired with a Jeol NMR spectrophotometer USA
at 400 and 100 MHz, respectively. Chemical shis are shown in
parts per million (ppm) proportional to tetramethylsilane
(TMS). The coupling constants (J) are presented in hertz (Hz),
and the splitting patterns are designated by using
abbreviations: s (singlet), d (doublet), t (triplet), m (multiplet),
brs (broad singlet), and dd (double doublet). LC/MS data were
recorded with an Agilent 6310 Ion trap LC/MS system and
elemental analysis (C, H and N) was carried out on Elementar
analysensysteme. Melting points were taken in open capillaries
by using KSPII melting point apparatus (KRUSS, Germany).
Purity of all target compounds were analysed by using Shi-
madzu reverse phase HPLC system (Kyoto, Japan) coupled with
a photodiode array detector (PDA) and C-18 column. Acetoni-
trile and methanol (80 : 20) were used as a mobile phase,
keeping ow rate 1 ml minꢃ1. All target compounds displayed
>97% purity.
4.4.1. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-(4-phenyl-
piperazin-1-yl)-propionamide (4). White solid; yield 85% (205.6
1
ꢀ
mg); mp 201–202 C. H-NMR (400 MHz, DMSO-D6) d 8.59 (s,
1H), 7.89 (s, 2H), 7.16 (t, J ¼ 7.6 Hz, 2H), 6.88 (d, J ¼ 8.2 Hz, 2H),
6.72 (t, J ¼ 7.3 Hz, 1H), 3.20 (s, 3H), 3.08 (s, 4H), 2.71 (s, 4H), 2.55
(t, J ¼ 4.1 Hz, 4H); 13C-NMR (100 MHz, DMSO-D6) d 172.5, 162.1,
152.7, 151.1, 136.1, 132.0, 129.4, 125.7, 122.8, 121.3, 119.3,
115.2, 53.6, 52.3, 44.0, 33.3; LC-MS: m/z; 445 (M + 1); anal. calcd
for: C21H24N4O3S2; C, 56.73; H, 5.44; N, 12.60; found: C, 56.99;
H, 5.68; N, 12.42.
4.4.2. 3-[4-(4-Fluoro-phenyl)-piperazin-1-yl]-N-(6-methane-
sulfonyl-benzothiazol-2-yl)-propionamide (5). White solid; yield
88% (196.8 mg); mp 200–202 ꢀC. 1H-NMR (400 MHz, DMSO-D6)
d 8.59 (s, 1H), 7.88 (s, 2H), 6.88–7.01 (m, 4H), 3.21 (s, 3H), 3.03
(s, 4H), 2.71 (s, 4H), 2.54 (s, 4H); 13C-NMR (100 MHz, DMSO-D6)
d 172.7, 161.9, 152.6, 148.2, 136.5, 132.6, 125.3, 122.5, 121.2,
4.2. Synthesis of 6-methanesulfonyl-benzothiazol-2-yl amine
(1)
4-Methylsulfony aniline (10 millimole) and potassium thiocya- 117.3, 115.6, 53.2, 52.8, 49.7, 44.0, 33.3; LC-MS: m/z; 463 (M + 1);
nate (40 millimole) were stirred in glacial acetic acid at room anal. calcd for: C21H23FN4O3S2; C, 54.53; H, 5.01; N, 12.11;
temperature (RT) for 1 h. Aer that, bromine (10 millimole in found: C, 54.31; H, 5.19; N, 12.39.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17615

View Article Online
RSC Advances
Paper
C28H30N4O3S2; C, 62.90; H, 5.66; N, 10.48; found: C, 63.12; H,
5.89; N, 10.23.
4.4.3. 3-[4-(4-Chloro-phenyl)-piperazin-1-yl]-N-(6-methane-
sulfonyl-benzothiazol-2-yl)-propionamide (6). White solid; yield
75% (204.0 mg); mp 210–212 ꢀC.$1H-NMR (400 MHz, DMSO-D6)
d 8.59 (s, 1H), 7.89 (s, 2H), 7.17 (d, J ¼ 8.7 Hz, 2H), 6.89 (d, J ¼
8.7 Hz, 2H), 3.21 (s, 3H), 3.08 (s, 4H), 2.71 (s, 4H), 2.53 (s, 4H);
13C-NMR (100 MHz, DMSO-D6), 172.2, 162.3, 152.6, 150.2,
135.9, 132.4, 128.9, 125.3, 122.8, 122.6, 121.3, 117.3, 53.5, 52.7,
4.4.9. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-(4-pyridin-2-
yl-piperazin-1-yl)-propionamide (12). White solid; yield 74% (199.8
mg); mp 201–203 ꢀC.$1H-NMR (400 MHz, DMSO-D6) d 8.59 (s, 1H),
8.05 (s, 1H), 7.89 (s, 2H), 7.47 (t, J ¼ 7.6 Hz, 1H), 6.77 (d, J ¼ 8.7 Hz,
1H), 6.58 (t, J ¼ 6.0 Hz, 1H), 3.42 (s, 4H), 3.21 (s, 3H), 2.71 (s, 4H),
2.48 (s, 4H); 13C-NMR (100 MHz, DMSO-D6) d 172.5, 162.3, 159.2,
152.6, 147.9, 138.0, 136.0, 132.5, 125.3, 122.5, 121.3, 113.5, 107.6,
53.6, 52.7, 45.1, 44.5, 33.4; LC-MS: m/z; 446 (M + 1); anal. calcd for:
C20H23N5O3S2; C, 53.91; H, 5.20; N, 15.72; found: C, 53.91; H,
5.20; N, 15.72; found: C, 53.69; H, 5.02; N, 15.95.
4.4.10. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-(4-pyri-
midin-2-yl-piperazin-1-yl)-propionamide (13). White solid;
yield 81% (204.2 mg); mp 174–176 ꢀC. 1H-NMR (400 MHz,
DMSO-D6) d 12.21 (s, 1H), 8.59 (s, 1H), 8.30 (d, J ¼ 4.7 Hz, 2H),
7.89 (s, 2H), 6.57 (t, J ¼ 4.7 Hz, 1H), 3.66–3.68 (m, 4H), 3.21 (s,
3H), 2.70 (s, 4H), 2.45–2.47 (m, 4H); 13C-NMR (100 MHz,
DMSO-D6) d 172.3, 162.5, 161.7, 158.4, 152.5, 135.8, 132.5,
125.3, 122.5, 121.3, 110.6, 53.6, 52.7, 44.5, 43.6, 33.2; LC-MS:
m/z; 447 (M + 1); anal. calcd for: C19H22N6O3S2; C, 51.10; H,
4.97; N, 18.82; found: C, 50.85; H, 5.09; N, 18.55.
4.4.11. 3-[4-(Furan-2-carbonyl)-piperazin-1-yl]-N-(6-methane-
sulfonyl-benzothiazol-2-yl)-propionamide (14). White solid; yield
69% (156.8 mg); mp 190–192; ꢀC.$1H-NMR (400 MHz, DMSO-D6)
d 8.59 (s, 1H), 7.88 (s, 2H), 6.88–7.01 (m, 4H), 3.21 (s, 3H), 3.03
(s, 4H), 2.71 (s, 4H), 2.54 (s, 4H); 13C-NMR (100 MHz, DMSO-D6)
d 172.2, 162.5, 158.6, 152.5, 147.1, 145.2, 136.1, 132.4, 124.6,
122.8, 121.4, 115.9, 111.1, 53.4, 53.0, 44.5, 33.4; LC-MS: m/z; 463
(M+1); anal. calcd for: C20H22N4O5S2; C, 51.93; H, 4.79; N, 12.11;
found: C, 51.71; H, 4.99; N, 12.32.
4.4.12. 4-[2-(6-Methanesulfonyl-benzothiazol-2-yl carbamoyl)-
ethyl]-piperazine-1-carboxylic acid benzyl ester (15). White solid;
yield 86% (226.7 mg); mp 159–161 ꢀC. 1H-NMR (400 MHz, DMSO-
D6) d 8.59 (s, 1H), 7.89 (s, 2H), 7.29 (d, J ¼ 17.4 Hz, 5H), 5.03 (s, 2H),
3.32 (d, 4H), 3.21 (s, 3H), 2.67 (s, 4H), 2.37 (s, 4H); 13C-NMR (100
MHz, DMSO-D6) d 172.4, 162.4, 154.9, 152.2, 137.5, 135.7, 132.4,
128.8, 128.3, 127.9, 125.1, 122.5, 121.0, 66.7, 53.5, 52.6, 44.4, 43.9,
33.4; LC-MS: m/z; 503 (M + 1); anal. calcd for: C23H26N4O5S2; C,
54.96; H, 5.21; N, 11.15; found: C, 55.20; H, 5.02; N, 11.32.
4.4.13. 3-(4-Benzo[1,3]dioxol-5-yl methyl-piperazin-1-yl)-N-
48.5, 44.4, 33.3; d LC-MS: m/z; 480 (M + 1); anal. calcd for: C21
-
H23ClN4O3S2; C, 52.65; H, 4.84; N, 11.70; found: C, 52.44; H,
5.07; N, 11.98.
4.4.4. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-[4-(4-nitro-
phenyl)-piperazin-1-yl]-propionamide (7). White solid; yield 82%
(212.7 mg); mp 209–211 ꢀC.$1H-NMR (400 MHz, DMSO-D6) d 8.59
(s, 1H), 8.00 (d, J ¼ 9.2 Hz, 2H), 7.89 (s, 2H), 6.99 (d, J ¼ 9.2 Hz,
2H), 3.41 (s, 4H), 3.21 (s, 3H), 2.71 (s, 4H), 2.52 (s, 4H); 13C-NMR
(100 MHz, DMSO-D6) d 172.3, 162.4, 155.2, 152.5, 137.3, 135.5,
132.5, 126.2, 125.4, 123.1, 122.6, 121.1, 113.1, 53.5, 52.2, 46.7,
44.5, 33.5; LC-MS: m/z; 490 (M + 1); anal. calcd for: C21H23N5O5S2;
C, 51.52; H, 4.74; N, 14.31; found: C, 51.29; H, 4.55; N, 14.58.
4.4.5. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-(4-p-tolyl-
piperazin-1-yl)-propionamide (8). White solid; yield 84% (224.1
1
ꢀ
mg); mp 197–198 C. H-NMR (400 MHz, DMSO-D6) d 8.59 (s,
1H), 7.89 (s, 2H), 6.97 (d, J ¼ 8.2 Hz, 2H), 6.78 (d, J ¼ 8.2 Hz, 2H),
3.21 (s, 3H), 3.02 (s, 4H), 2.71 (s, 4H), 2.54 (s, 4H), 2.15 (s, 3H)
13C-NMR (100 MHz, DMSO-D6) d 172.4, 161.7, 152.3, 149.2,
136.3, 132.6, 130.2, 127.9, 125.0, 122.8, 121.6, 115.8, 53.8, 52.6,
49.1, 44.6, 32.9, 20.4; LC-MS: m/z; 459 (M + 1); anal. calcd for:
C
22H26N4O3S2; C, 57.62; H, 5.71; N, 12.22; found: C, 57.37; H,
5.94; N, 12.02.
4.4.6. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-[4-(4-
methoxy-phenyl)-piperazin-1-yl]-propionamide (9). White solid;
yield 79% (202.8 mg); mp 177–179 ꢀC. 1H-NMR (400 MHz,
DMSO-D6) d 8.59 (s, 1H), 7.89 (s, 2H), 6.83 (d, J ¼ 9.2 Hz, 2H),
6.76 (d, J ¼ 9.2 Hz, 2H), 3.63 (s, 3H), 3.21 (s, 3H), 2.96 (d, J ¼
4.1 Hz, 4H), 2.71 (s, 4H), 2.54 (t, J ¼ 4.1 Hz, 4H); 13C-NMR (100
MHz, DMSO-D6) d 172.1, 162.3, 152.9, 152.3, 149.1, 145.8, 135.8,
131.9, 130.8, 127.8, 124.8, 120.5, 117.0, 114.4, 55.6, 53.5, 52.9,
50.1, 44.5, 33.3; LC-MS: m/z; 475 (M + 1); anal. calcd for:
C
22H26N4O4S2; C, 55.68; H, 5.52; N, 11.81; found: C, 55.44; H,
5.71; N, 11.60.
4.4.7. 3-(4-Benzyl-piperazin-1-yl)-N-(6-methanesulfonyl-benzo-
thiazol-2-yl)-propionamide (10). White solid; yield 76% (207.9 mg);
mp 147–149 ꢀC. 1H-NMR (400 MHz, DMSO-D6) d 8.59 (s, 1H), 7.89
(s, 2H), 7.24 (dt, J ¼ 23.5, 7.2 Hz, 5H), 3.40 (s, 2H), 3.21 (s, 3H), 2.65
(s, 4H), 2.34–2.46 (m, 8H); 13C-NMR (100 MHz, DMSO-D6) d 172.5,
162.6, 152.3, 140.4, 138.9, 135.6, 132.6, 128.6, 127.3, 125.5, 122.7,
121.4, 62.5, 53.0, 52.8, 44.5, 33.3; LC-MS: m/z; 459 (M + 1); anal.
calcd for: C22H26N4O3S2; C, 57.62; H, 5.71; N, 12.22; found: C, 57.81;
H, 5.49; N, 12.48.
4.4.8. 3-(4-Benzhydryl-piperazin-1-yl)-N-(6-methanesulfonyl-
benzothiazol-2-yl)-propionamide (11). White solid; yield 88%
(216.8 mg); mp 170–172 ꢀC. 1H-NMR (400 MHz, DMSO-D6) d 8.59
(s, 1H), 7.89 (s, 2H), 7.37 (d, J ¼ 7.8 Hz, 4H), 7.24 (t, J ¼ 7.6 Hz,
4H), 7.13 (t, J ¼ 7.3 Hz, 2H), 4.21 (s, 1H), 3.21 (s, 3H), 2.64 (s, 4H),
2.26–2.46 (m, 8H); LC-MS: m/z; 535 (M + 1); anal. calcd for:
(6-methanesulfonyl-benzothiazol-2-yl)-propionamide
(16).
White solid; yield 75% (201.4 mg); mp 206–208 ꢀC. 1H-NMR (400
MHz, DMSO-D6) d 12.70 (s, 1H), 8.59 (s, 1H), 7.89 (s, 2H), 6.78
(d, J ¼ 6.7 Hz, 2H), 6.68 (d, J ¼ 8.9 Hz, 1H), 5.93 (s, 2H), 3.330 (s,
2H), 3.21 (s, 3H), 2.64 (s, 4H), 2.31–2.46 (m, 8H); 13C-NMR (100
MHz, DMSO-D6) d 172.3, 162.2, 152.4, 143.4, 135.7, 132.4, 129.0,
128.0, 127.3, 125.2, 122.7, 121.2, 75.6, 53.5, 53.0, 51.9, 44.4, 33.3;
LC-MS: m/z; 503 (M + 1); anal. calcd for: C23H26N4O5S2; C, 54.96;
H, 5.21; N, 11.15; found: C, 54.71; H, 4.99; N, 10.90.
4.4.14. N-(6-Methanesulfonyl-benzothiazol-2-yl)-3-[4-(4-tri-
uoromethyl-phenyl)-piperazin-1-yl]-propionamide (17). White
solid; yield 71% (208.8 mg); mp 197–199 ꢀC. 1H-NMR (400 MHz,
DMSO-D6) d 8.62–8.54 (1H), 7.89 (s, 2H), 7.45 (d, J ¼ 8.8 Hz, 2H),
7.02 (d, J ¼ 8.8 Hz, 2H), 3.22 (dd, J ¼ 9.6, 4.4 Hz, 7H), 2.71 (s, 4H),
2.53 (t, J ¼ 4.8 Hz, 4H); 13C-NMR (100 MHz, DMSO-D6) d 172.8,
161.9, 154.3, 152.3, 135.3, 132.8, 126.6, 124.8, 122.4, 121.2, 113.8;
17616 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
LC-MS: m/z; 513 (M + 1); anal. calcd for: C22H23F3N4O3S2; C, 51.55; for 1 min at RT. Specimen was further transferred for imaging
H, 4.52; N, 10.93; found: C, 51.29; H, 4.28; N, 11.21.
with transmission electron microscopy (JEOL JEM-1400).55
4.5. Inhibition of AChE and BuChE by synthesized
derivatives
4.11. Metal chelation studies
The ability of compounds to chelate metal ions was performed
To determine the compound's ability to inhibit AChE and by UV-spectroscopy. Compound 12 (60 mM) was dissolved in
BuChE revised Ellman methods were performed. Concisely, HEPES buffer with and without the metal ions. UV absorption
stock solution of test compounds (10 mM) were prepared in spectra were recorded with wavelength ranging from 200 to
100% DMSO and diluted using autoclaved water to attain a nal 600 nm. The stoichiometry of the complex 12–Cu2+ was deter-
concentration range between (0.01–100) mM. Six different range mined by Job's method. The tested compound and copper ion
of drug concentration was used to perform the assay. Enzyme were incubated at different molar ratios and UV absorption was
dilutions and substrate dilutions were prepared according to plotted.55
the protocol. This assay was performed as reported by our lab
previously.22,54
4.12. Molecular docking stdies
Atomic coordinates of AChE (PDB ID: 1EVE) and Ab1-42 (PDB ID:
1YT) were taken from the Protein Data Bank (www.rcsb.org).
4.6. Kinetic characterization of AChE inhibition by
compound 12
Autodock 4.2 with standard protocol was used to dock the
16
To study the kinetics of AChE inhibition, three concentrations synthesized compound 12 with AChE and Ab1-42
.
Protein–
of compound 12 (2 mM, 6 mM and 8 mM) were selected. A ligands interactions were assessed by applying Lamarckian
reciprocal plot of 1/velocity versus 1/substrate was plotted at low genetic algorithm (LGA).The most encouraging free binding
substrate concentrations by using modied Ellman's method.54 energy and docking orientations lying within the range of 2.0 A
ꢀ
in root-mean square deviation (RMSD) tolerance were used to
cluster the molecule and ranked accordingly. PyMOL was
employed to visualize molecular interactions. All calculations
were carried out on workstation machines running Linux x86 as
operating systems.56
4.7. Inhibition of self-mediated Ab1-42 aggregation assay
Marketed amyloid beta peptides were obtained and dissolved in
hexauoroisopropanol (HFIP) at 8 mg mlꢃ1. The HFIP was
evaporated and then Ab1-42 stocks were prepared in DMSO,
nally diluted in reaction buffer (50 mM phosphate buffer (pH
7.4)). For the inhibition of self-mediated Ab1-42 aggregation
4.13. Molecular dynamic (MD) simulation studies
experiment, ThT assay was performed as reported by our MD simulations were carried out with GROMACS 4.6.5 suite of
research group.24
programs, using charm force-eld and spc216 water molecules.
The trajectory les were analyzed using GROMACS utilities:
g_gyrate, g_rmsd and g_rmsf to obtain the radius of gyration
(Rg), root mean square deviation (RMSD) and root mean square
uctuation (RMSF). The coordinates of AChE and Ab1-42 were
4.8. Disaggregation of self-induced Ab1-42 aggregation
For the study of disaggregation of self-induꢀced Ab1-42 aggrega-
tion, peptide (100 mM) was incubated at 37 C for 48 h to form
amyloid beta brils and further the compounds were added and
incubated again at 37 ꢀC for 24 h. ThT method was used to
quantify amyloid bril formation.54
minimized with steepest descent, up to
a tolerance of
100 kJ molꢃ1 to remove all bad contacts. Two independent MD
simulations were carried out with standard protocol in water for
AChE and AChE–12 complex, respectively at 300 K to evaluate
the structural changes, binding affinity and stability in course of
simulation. Similarly, another set of MD simulations were
performed for Ab1-42 and Ab1-42–12complex. The SHAKE algo-
4.9. Inhibition of self-mediated Ab1-42 aggregation by ThT
assay using confocal microscopy
Peptide (10 ml) and compounds (10 ml) were incubated for 48 h rithm was used to restrain the covalent bonds containing
at 37 ꢀC. 5 ml from incubation mixture was diluted with 10 mM of hydrogen atoms at their equilibrium distances and periodic
ThT (1 : 2) i.e., 10 ml of ThT in glycine–NaOH and placed on boundary conditions were applied. Particle Mesh Ewald (PME)
glass slide. Fluorescence signal of samples were analyzed at method was employed for computing the long-range electro-
488 nm by confocal laser scanning microscope.
static interactions. A 1.4 nm cut-off for van der Waals interac-
tions and a 1.0 nm cut-off for Coulombs interaction were
adopted in the simulation, respectively. Analytical graphs were
plotted using Xmgrace, VMD and PyMol were used for structure
and molecular interaction analysis.22,53
4.10. Inhibition of self-mediated Ab1-42 aggregation using
TEM
Solution of Ab stock was diluted with 50 mM phosphate buffer
(pH 7.4), and further incubated in the presence and absence of
compound 12 at 37 C for 48 h. The nal concentrations of Ab
peptide and 12 were 50 mM, respectively. Aliquots (10 ml) of the
4.14. Cytotoxicity of synthetic compounds on SHSY-5Y cell
line
ꢀ
samples were put on carbon-coated copper grid. Each grid was To study the cytotoxicity of the compound 12 on neuronal cells,
negatively stained with 0.5% phosphotungstate (PTA) solution MTT assay was performed. 1 ꢄ 104 cells per well were seeded in
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17617

View Article Online
RSC Advances
Paper
96-well plates. The medium was removed aer 24 h and (EPM) task and social recognition test were performed for each
compound 12 at different concentrations (5 mM, 10 mM and 20 animal in each group.
ꢃ1
ꢀ
mM) for 24 h at 37 C were added. Aer 24 h, MTT (5 mg ml
stock) solution (20 ml) per well were added at 37 ꢀC for 4 h. MTT
was removed thereaer and formazon crystals were then dis-
solved in DMSO. The absorbance at 570 nm was recorded.
Results have been shown as the mean ꢁ SD of three indepen-
dent experiments.22
4.18. Elevated plus maze test (EPM)
There are two open arms (50 cm ꢄ 10 cm) and two closed arms
(50 cm ꢄ 10 cm ꢄ 30 cm) in the elevated plus maze. There is
a central platform elevated to a height of 50 cm from the oor.
The experiments were performed in the light phase. At one edge
of the arm, we placed the mice facing the opposite side and
recorded the time taken by animals to enter the closed arm
which was documented as transfer of latency (TL).52
4.15. Neuroprotection against H2O2 induced neurotoxicity
To study the neuroprotective effects of the compound against
hydrogen peroxide (H2O2) induced neurotoxicity, MTT assay
was performed. 2 ꢄ 104 per well SH-SY5Y cells were seeded in
96-well plates. Serum free medium was added aer 24 h of
incubation, containing different concentrations of the
compounds 12 followed by 200 mM H2O2 aer an interval of 3
4.19. Social recognition test
The behavioural assay was performed according to the protocol
given by Timmermann et al.57 The adult mice and the juvenile
(8–12 g) were kept together to make acquainted to themselves,
for 1 h in the test room. Thereaer, the adult mice were brought
back to their respective cages. The rst interactive trial (T1) of
the adult mice with the juvenile mice began aer 30 min of
habituation period. The adult mice were placed with the juve-
nile and the interaction time (T1) was recorded and observed
simultaneously for 5 min. This was subsequently repeated for
all the groups. The second trial began aer 120 min in the same
test cage pursued by recording the time in seconds of adult mice
interacting with the juvenile mice. The obtained results were
given in the form of recognition ratios (T2/T1). Index of
improved short term recognition memory is the decrease in
T2 : T1.
ꢀ
hours at 37 C for 24 h. Cells without treatment were used as
healthy controls and cells treated with H2O2 as control group
were used and the results were evaluated as percentage of
control. Photomicrographs were also captured using camera
attached to the microscope (Olympus, Japan) aer 24 h of
treatment to assess morphological changes in SH-SY5Y cells.24
4.16. Animals
Swiss albino male mice (25–30 g, 2–3 weeks; n ¼ 6 each group)
was procured from animal house facility of Dr. B. R. Ambedkar
Center for Biomedical Research, University of Delhi. Delhi,
India. Polypropylene cages were used for housing the animals
and optimum conditions (temperature 25 ꢁ 2 ꢀC, 12 h light/12 h
dark cycle, 50 ꢁ 5% relative humidity and free access to food
and water ad libitum) were maintained under the standard
laboratory environmental conditions. Thus, all animal proce-
dures were performed in accordance with the guidelines for
care and use of laboratory animals of Institutional Animal
Ethics Committee (IAEC), Dr. B.R. Ambedkar Center for
Biomedical Research, Delhi University and approved by the
animal ethics Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), Government
of India.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Author C. B. M would like to acknowledge the nancial support
from DHR for Young Scientist fellowship. Author S. S is
thankful to Ministry of AYUSH for the nancial support
(JRF). M. T wishes to acknowledge University of Delhi, UGC-SAP
and DST-PURSE for providing research funds to carry out the
study. The authors gratefully acknowledge animal facility of
ACBR and NMR facility of USIC and TEM facility of SAIF, AIIMS.
4.17. Drugs and experimental design
Scopolamine hydrobromide was procured from Sigma-Aldrich
(MO, USA).Scopolamine was dissolved in saline and given
intraperitoneally (i.p.) in a dose of 3 mg kgꢃ1. Five groups with n
¼ 6 were designed and mice were randomly allocated in each
group. Animals in group I were given scopolamine 3 mg kgꢃ1
References
1 M. Citron, Nat. Neurosci., 2002, 5, 1055–1057.
2 B. Klimova, P. Maresova and K. Kuca, Curr. Alzheimer Res.,
2016, 13, 1249–1258.
.
b. wt per day, i.p51,52 Group II animals were treated together
with scopolamine 3 mg kgꢃ1 b. wt per day, i.p. and standard
drug Donepezil (5 mg kgꢃ1 b. wt per day; i.p). In groups III & IV,
scopolamine followed by treatment with compound 12 (10 and
20 mg kgꢃ1 b. wt per day, i.p.) were given. Animals in group V
were used as vehicle controls and they were administered saline
(daily, i.p). For 7 consecutive days, all the drugs including
scopolamine were given daily. Behavioural studies were carried
out, at the end of treatment schedules. Elevated plus maze
ˇ
´
´
ˇ
ˇ
3 P. Maresova, H. Mohelska, J. Dolejs and K. Kuca, Curr.
Alzheimer Res., 2015, 12, 903–911.
4 H. C. Beerens, C. Sutcliffe, A. Renom-Guiteras, M. E. Soto,
R. Suhonen, A. Zabalegui, C. Bokberg, K. Saks and
J. P. Hamers, J. Am. Med. Dir. Assoc., 2014, 15, 54–61.
5 C. Haass and D. J. Selkoe, Nat. Rev. Mol. Cell Biol., 2007, 8,
101–112.
¨
17618 | RSC Adv., 2020, 10, 17602–17619
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
6 T. P. Knowles, M. Vendruscolo and C. M. Dobson, Nat. Rev. 35 A. K. Rathi, R. Syed, H. S. Shin and R. V. Patel, Expert Opin.
Mol. Cell Biol., 2014, 15, 384–396. Ther. Pat., 2016, 26(7), 777–797.
7 S. Ayala, P. Genevaux, C. Hureau and P. Faller, ACS Chem. 36 L. Peauger, R. Azzouz, V. Gembus, M. L. T¸ın¸ta¸s,
ˆ
´
¨
Neurosci., 2019, 10, 3366–3374.
J. S. O. Sopkova-de, P. Bohn, C. Papamicael and
8 C. Cheignon, M. Tomas, D. Bonnefont-Rousselot, P. Faller,
C. Hureau and F. Collin, Redox Biol., 2018, 14, 450–464.
9 G. Nesi, S. Sestito, M. Digiacomo and S. Rapposelli, Curr.
Top. Med. Chem., 2017, 17(27), 3062–3079.
10 C. Cheignon, M. Tomas, D. Bonnefont-Rousselot, P. Faller,
C. Hureau and F. Collin, Redox Biol., 2018, 14, 450–464.
V. Levacher, J. Med. Chem., 2017, 60, 5909–5926.
37 G. L. Ellman, K. D. Courtney, V. Andres Jr and
R. M. Featherstone, Biochem. Pharmacol., 1961, 7, 88–95.
38 D. R. Liston, et al., Pharmacology of selective
acetylcholinesterase inhibitors: implications for use in
Alzheimer's disease, Eur. J. Pharmacol., 2004, 486, 9–17.
11 G. Caruso, S. F. Spampinato, V. Cardaci, F. Caraci, 39 Li Luo, et al., Design, synthesis and evaluation of phthalide
M. A. Sortino and S. Merlo, Curr. Pharm. Des., 2019, 25(45),
4771–4781.
12 K. A. Jellinger, J. Cell. Mol. Med., 2010, 14, 457–487.
13 A. I. Bush, J. Alzheimer's Dis., 2013, 33, S277–S281.
alkyl
tertiary
amine
derivatives
as
promising
acetylcholinesterase inhibitors with high potency and
selectivity against Alzheimer's disease, Bioorg. Med. Chem.,
2020, 115400.
14 M. L. Seux, J. Rotrou and A. S. Rigaud, PSN, 2008, 6, 82–90. 40 A. Kumar, A. Singh and Ekavali, Pharmacol. Rep., 2015, 67(2),
15 K. Blennow, M. J. de Leon and H. Zetterberg, Lancet, 2006,
368, 387–403.
16 K. G. Yiannopoulou and S. G. Papageorgiou, Ther. Adv.
Neurol. Disord., 2013, 6, 19–33.
17 R. Anand, K. D. Gill and A. A. Mahdi, Neuropharmacology,
2014, 76, 27–50.
18 R. Ali, I. A. Sheikha, N. R. Jabirb and M. A. Kamal, Am. J.
Neuroprot. Neuroregener., 2012, 4, 136–144.
195–203, DOI: 10.1016/j.pharep.2014.09.004.
41 H. Aoyama and T. Doura, Bioorg. Med. Chem. Lett., 2020, 30,
126888.
42 G. A. de Souza, et al., J. Enzyme Inhib. Med. Chem., 2019, 34,
631–637.
43 N. A. Vyas, S. B. Singh, A. S. Kumbhar, D. S. Ranade,
G. R. Walke, P. P. Kulkarni, V. Jani, U. B. Sonavane,
R. R. Joshi and S. Rapole, Inorg. Chem., 2018, 57, 7524–7535.
19 S. Sestito, S. Wang, Q. Chen, et al., Eur. J. Med. Chem., 2019, 44 J. Hardy and D. J. Selkoe, Science, 2002, 297, 353–356.
174, 216–225.
45 H. Du, X. Liu, J. Xie and F. Ma, ACS Chem. Neurosci., 2019, 10,
2397–2407.
46 O. Crescenzi, S. Tomaselli, R. Guerrini, S. Salvadori,
A. M. D'Ursi, P. A. Temussi and D. Picone, Eur. J. Biochem.,
2002, 269, 5642–5648.
20 M. Maspero, D. Volpato, D. Cirillo, et al., Bioorg. Chem., 2020,
96, 103633.
´
21 F. J. Perez-Areales, A. L. Turcu, M. Barniol-Xicota, et al., Eur.
J. Med. Chem., 2019, 180, 613–626.
22 C. B. Mishra, S. Kumari, A. Manral, A. Prakash, V. Saini, A. M 47 S. Vilasi, R. Carrotta, C. Ricci, G. C. Rappa, F. Librizzi,
Lynn and M. Tiwari, Eur. J. Med. Chem., 2017, 125, 736–750.
23 A. Manral, V. Saini, P. Meena and M. Tiwari, Bioorg. Med.
Chem., 2015, 23, 6389–6403.
24 C. B. Mishra, A. Manral, S. Kumari, V. Saini and M. Tiwari,
Bioorg. Med. Chem., 2016, 24, 3829–3841.
V. Martorana and M. R. Mangione, ACS Chem. Neurosci.,
2019, 10(8), 3565–3574.
48 A. Prakash, G. Dixit, N. K. Meena, R. Singh, P. Vishwakarma,
S. Mishra and A. M. Lynn, J. Biomol. Struct. Dyn., 2018, 36(9),
2391–2406.
25 J. M. Song, A. M. Di Battista, Y. M. Sung, J. M. Ahn, 49 L. Vugmeyster, M. A. Clark, I. B. Falconer, D. Ostrovsky,
R. S. Turner, J. Yang, D. T. Pak, H. K. Lee and H. S. Hoe,
Exp. Neurol., 2014, 252, 105–113.
D. Gantz, W. Qiang and G. L. Hoatson, J. Biol. Chem., 2016,
291(35), 18484–18495.
26 R. Ali and N. Siddiqui, J. Chem., 2013, 1–12.
50 J. Hardy and D. J. Selkoe, Science, 2002, 297, 353–356.
27 C. A. Mathis, N. S. Mason, B. J. Lopresti and W. E. Klunk, 51 A. Blokland, Scopolamine Rev., 2005, 1, 1–76.
Semin. Nucl. Med., 2012, 42, 423–432.
28 C. B Mishra, S. Kumari and M. Tiwari, Eur. J. Med. Chem.,
2015, 6, 1–34.
29 N. Guzior, A. Wieckowska, D. Panek and B. Malawska, Curr.
Med. Chem., 2015, 22(3), 373–404.
52 A. C. Sharma and S. K. Kulkarni, Prog. Neuro-
Psychopharmacol. Biol. Psychiatry, 1992, 16, 117–125.
53 C. B. Mishra, S. Kumari, A. Prakash, R. Yadav, A. K. Tiwari,
P. Pandey and M. Tiwari, Eur. J. Med. Chem., 2018, 151,
520–532.
30 C. B Mishra, S. Kumari, A. Angeli, S. M. Monti, M. Buonanno, 54 T. Umar, S. Shalini, M. K. Raza, S. Gusain, J. Kumar, P. Seth,
M. Tiwari and C. T. Supuran, J. Med. Chem., 2017, 60, 2456–
2469.
31 C. B. Mishra, S. Kumari, A. Angeli, S. Bua, M. Tiwari and
C. T. Supuran, J. Med. Chem., 2018, 61, 3151–3165.
M. Tiwari and N. Hoda, Eur. J. Med. Chem., 2019, 175, 2–19.
55 T. Umar, S. Shalini, M. K. Raza, S. Gusain, J. Kumar,
W. Ahmed, M. Tiwari and N. Hoda, MedChemComm, 2018,
9, 1891–1904.
32 S. Kumari, C. B. Mishra and M. Tiwari, Pharmacol. Rep., 56 A. C. Wallace, R. A. Laskowski and J. M. Thornton, Protein
2016, 68, 250–258. Eng., Des. Sel., 1995, 8(2), 127–134.
33 D. Ozkay Umide, C. O. Devrim, Y. Ozkay and Y. Ozturk, 57 D. B. Timmermann, K. Sandager-Nielsen, T. Dyhring,
¨
¨
¨
Pharmacol. Rep., 2012, 64, 834–847.
34 S. Stayte and B. Vissel, Front. Neurosci., 2014, 8, 113.
M. Smith, A. M. Jacobsen, E. Ø. Nielsen, M. Grunnet,
J. K. Christensen, D. Peters, K. Kohlhaas, G. M. Olsen and
P. K. Ahring, Br. J. Pharmacol., 2012, 167, 164–182.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 17602–17619 | 17619