Design, synthesis, in-silico and biological evaluation of novel donepezil derivatives as multi-target-directed ligands for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2016.09.057

A novel series of donepezil based multi-functional agents “(E)-5,6-dimethoxy-2-(4-(4-substituted piperazin-1-yl)benzylidene)-2,3-dihydro-1H-inden-1-ones” have been designed and synthesized as potential anti-Alzheimer's agents. In-vitro studies revealed th

Full text of DOI:10.1016/j.ejmech.2016.09.057

Accepted Manuscript
Design, synthesis, in-silico and biological evaluation of novel donepezil derivatives as
multi-target-directed ligands for the treatment of Alzheimer's disease
Chandra Bhushan Mishra, Shikha Kumari, Apra Manral, Amresh Prakash, Vikas
Saini, Andrew M. Lynn, Manisha Tiwari
PII:
S0223-5234(16)30793-0
10.1016/j.ejmech.2016.09.057
EJMECH 8923
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 June 2016
Revised Date: 3 September 2016
Accepted Date: 18 September 2016
Please cite this article as: C.B. Mishra, S. Kumari, A. Manral, A. Prakash, V. Saini, A.M. Lynn, M. Tiwari,
Design, synthesis, in-silico and biological evaluation of novel donepezil derivatives as multi-target-
directed ligands for the treatment of Alzheimer's disease, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.09.057.
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Design, Synthesis, in-Silico and Biological Evaluation of Novel Donepezil Derivatives as
Multi-Target-Directed Ligands for the Treatment of Alzheimer’s Disease
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Chandra Bhushan MishraaÎ, Shikha KumariaÎ, Apra ManralaÎ, Amresh PrakashbÎ, Vikas SainiaÎ, Andrew M Lynnb,
Manisha Tiwari *a
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aBio-Organic research laboratory, Dr. B. R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi
110007, India
bSchool of Computational & Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India
Î
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All these authors have contributed equally.
*Correspondence
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Dr. Manisha Tiwari
Dr. B. R. Ambedkar Centre for Biomedical
Research, University of Delhi, Delhi 110007, India
E-mail: mtiwari07@gmail.com
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Abstract
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A novel series of donepezil based multi-functional agents “(E)-5,6-dimethoxy-2-(4-(4-
substituted piperazin-1-yl)benzylidene)-2,3-dihydro-1H-inden-1-ones” have been designed and
synthesized as potential anti-Alzheimer’s agents. In-vitro studies revealed that these compounds
demonstrated moderate to good AChE and Aβ aggregation inhibitory activity. These derivatives
are also endowed with admirable antioxidant activity. Among the entire series compounds IP-9,
IP-13 and IP-15 appeared as most active multi-functional agents and displayed marked AChE
inhibitory, Aβ disaggregation and antioxidant activity. Studies indicate that IP-13 and IP-15
showed better AChE inhibitory activity than the standard drug donepezil and IP-9, IP-13 as well
as IP-15 exhibited better Aβ aggregation inhibitory activity than curcumin. These compounds
(IP-9, IP-13 and IP-15) successfully diminished H2O2 induced oxidative stress in SH-SY5Y
cells and displayed excellent neuroprotective activity against H2O2 as well as Aβ induced
toxicity in SH-SY5Y cells in a concentration dependent manner. Moreover, these derivatives did
not exert any significant toxicity in neuronal SH-SY5Y cells in cytotoxicity assay. To elucidate
the plausible binding mode of the compounds IP-9, IP-13 and IP-15, molecular docking studies
and molecular dynamics (MD) simulation studies were also performed and the results indicate
their significant interactions with the active sites of AChE as well as Aβ1-42 peptide. Thus, the
present study evidently showed that IP-9, IP-13 and IP-15 are potent multi-functional agents
against Alzheimer’s disease and might serve as promising lead candidates for anti-Alzheimer
drug development.
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1. Introduction
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Alzheimer’s Disease (AD) is an age-related fatal neurodegenerative disorder affecting 36 million
people worldwide and it is estimated to get doubled every 20 years, leading to more than 115
million AD patients in 2050 [1]. AD is the most common form of dementia and generally
characterized by memory loss, progressive and irreversible cognitive impairments, linguistic
decline, severe behavioral anomalies and eventual death [2]. Several researches are in
understanding the complex pathogenesis of AD, but its etiology remains elusive. However,
various clinical hallmarks such as low levels of acetylcholine (ACh), β-amyloid (Aβ) deposits,
tau (τ)-protein aggregation, oxidative stress, inflammation and dyshomeostasis of biometals are
considered to play imperative roles in AD pathogenesis [3]. To date, four acetylcholinesterase
inhibitors (AChEIs), named donepezil, tacrine, rivastigmine, galantamine [4] and one N-methyl-
D-aspartate (NMDA) receptor antagonist memantine [5] have been approved for the treatment of
AD. Unfortunately, the number of these anti-Alzheimer drugs are very few and they all provide
only symptomatic relief rather than cure of AD. Since 2003, no new treatment option has entered
the clinical market and more than 200 drug candidates have failed in advanced clinical trials [6].
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AD is a multifactorial disease and to battle with this disease, multi-target strategies ruled over
traditional one protein, one target and one drug strategy. As a consequence, a new horizon in
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drug research involving multi-target-directed ligand (MTDL) strategy has appeared [7].
Designing of multi-target-directed ligands (MTDLs) widely employs synthetic hybrid
compounds, logically designed to integrate into a single molecular framework consisting of at
least two different pharmacophoric moieties [8].
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In this context, we have recently reported novel indandione and diallyldisulphide analogs as
potent MTDLs for the treatment of AD [9-10]. Continuing with our research on various
heterocyclic compound families with their potential application in AD, we herein report the
synthesis and biological evaluation of novel donepezil derivatives for their potential use as anti-
AD agents. Donepezil is a potent AChEIs and has proved to be very effective for the palliative
treatment of AD, but donepezil only maintain the choline levels in the AD brain. The purpose of
this work is to discover novel multi-potent compounds, i.e. a single molecule that can display
more pharmacological properties all in concert, such as the amelioration of the cholinergic
transmission, inhibition of Aβ accumulation and oxidative stress leading to a synergy for
effective management of AD. To achieve this goal, we rationally designed and synthesized a
series of novel (E)-2-(4-(4-(substitued)piperazin-1-yl)benzylidene)-5,6-dimethoxy-2,3-dihydro-
1H-inden-1-one derivatives, which consists of the active part of donepezil, and a curcumin
fragment as well as a piperazine moiety inside one moleule as shown in Figure 1.
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Structure activity relationship (SAR) studies of donepezil showed that its 5,6-dimethoxy-2,3-
dihydro-1H-inden-1-one moiety is essential for exerting biological activity [11]. Besides this,
these designed hybrids also contain curcumin fragment, which is a well-known antioxidant and a
potent anti-amyloidogenic agent [12]. Piperazine and its derivatives represent an important class
of molecules that exhibits a broad spectrum of central nervous system (CNS) activities [13-15].
Several researchers also debated piperazine derivatives as potential neuroprotective agents [16].
For this reason, we have also installed piperazine scaffold in our designed molecular frame,
which may provide the best anti-AD efficacy and neuroprotective ability. In this paper, we
depicted design, synthesis and pharmacological assessment of novel donepezil based multi-
functional compounds as anti-AD agents. The pharmacological evaluation of these novel
compounds includes the in-vitro AChE and butyrylcholinesterase (BuChE) inhibition, Aβ dis-
aggregation, anti-oxidant activity and neuroprotective efficacy. Additionally, in-silico molecular
docking and molecular dynamic (MD) simulation studies were also carried out to elucidate the
possible binding interaction mode of target compounds with AChE and Aβ1-42 peptide.
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2. Results and Discussion
2.1. Chemistry
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Total 15 new (E)-2-(4-(4-(substitued)piperazin-1-yl)benzylidene)-5,6-dimethoxy-2,3-dihydro-
1H-inden-1-one derivatives were synthesized in good yields. The synthetic pathway of these
novel derivatives is outlined in scheme 1. Briefly, an equimolar ratio of commercially available
substituted piperazine and 4-flourobenzaldehyde was taken and heated at a temperature of 100-
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1300C in dimethylformamide (DMF) to afford key intermediates (17-31). The appearance of
sharp singlet at 9.77-9.88 ppm in 1HNMR spectra confirms the formation of formyl derivatives
(17-31). Finally, the Knoevenagel condensation reaction was employed to link indanone moiety
with various substituted piperazines through methylene linker. To achieve target compounds (32-
46), the formyl intermediate (17-31; 20 mmole) and 5,6-dimethoxy-1-indanone (20 mmole) was
reacted in anhydrous ethanol and KOH was used as a catalyst. All the final compounds were
purified by column chromatography by using chloroform/methanol (98:02) as an eluent to afford
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pure compounds as single E-diastereomers which was confirmed by H NMR and NOESY
experiment (There was no signal observed corresponding to a possible coupling of the olephinic
proton around 7.36 ppm with the indanone 3-proton around 3.95 ppm, supplementary material).
All compounds were fully characterized by 1HNMR, 13CNMR, mass spectroscopy as well as
elemental analysis.
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Reagents and conditions: A: Anhydrous Dimethylformamide, K2CO3/Na2CO3, 100-1300C, 12-14 h. B: Anhydrous
Ethanol, KOH, 12-14 h, RT.
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Scheme 1: Synthetic procedure of Dimethoxy Indanone-Piperazine derivatives (1-46)
2.2. Biological Assays
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2.2.1. In-vitro AChE and BuChE inhibition and structure activity relationship (SAR)
studies
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Novel 5,6-dimethoxy indanone-piperazine derivatives (32-46) were subjected to study their
potential as AChE (from electric eel) and BuChE (from equine serum) inhibitors by following
the spectrophotometric methods which were previously explained by Ellman et al. [17].
Donepezil was taken as reference drug and the results of ChEs inhibition along with their
selectivity index is shown in Table 1. It was observed that all test compounds displayed
moderate to good inhibitory activity towards both ChEs with IC50 values ranging from lower
micromolar to nanomolar. On the basis of these results, following SAR has been derived to
analyze the influence of different substituents at the terminal position of piperazine moiety.
Compound 36 (IC50 = 0.194 ꢀM), 37 (IC50 = 0.119 ꢀM), and 38 (IC50 = 0.112 ꢀM), containing
electron rich p-methyl, 2,4-dimethyl and p-methoxy group, respectively presented better AChE
inhibitory activity as compared to compound 33 and 34 bearing electron withdrawing p-chloro
(IC50 = 0.291ꢀM), and p-fluoro groups (IC50 = 0.528 ꢀM) on the terminal phenyl ring.
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Surprisingly, compound 35 (IC50= 0.153 ꢀM) bearing strong electron withdrawing
trifluoromethyl group presented better activity, which might be due to its favorable conformation
that allows it to fit suitably within the active gorge of the enzyme. Henceforth, the order of
inhibitory potency against AChE was as follows: 2,4-dimethyl> p-methoxy> p-CF3> p-methyl>
p-chloro> p-fluoro. Seeing, compound 32 having an unsubstituted phenyl ring exhibited weaker
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inhibitory activity (11.7 folds) as compared to substituted phenyl derivatives (33-38). The
introduction of a linker between adjacent piperazine moiety and phenyl ring apparently leads to
further reduction in inhibitory potency. For instance, in compounds 41 and 42 the presence of
carbonyl and methylene linker reduces their activity to 15.1 and 23.2 folds, respectively as
compared to compound 32. However, the presence of linker seems to be beneficial for BuChE
activity, as exceptionally compound 42 showed strong inhibitory potency against BuChE (IC50 =
0.088 nM) which was 38 folds more potent than donepezil (IC50 = 3.36 ꢀM). It may be
concluded that the presence of the benzyl group at the terminal position might provide the
necessary flexibility inside the active site of the enzyme. Introduction of bulkier group like
benzhydral (compound 39) and difluorobenzhydral (IP-9) also exhibited strong inhibitory
activity against AChE. IP-9 showed excellent inhibitory potential and selectivity towards AChE
(AChE; IC50= 0.045 ꢀM, BuChE/ AChE; 67)
The replacement of the phenyl ring with several heterocyclic moieties was proved to be more
beneficial for AChE inhibition as well as selectivity profile of the compounds. In this sub-series
compound containing furoyl (IP-13), pyridine (compound 45) and pyrimidine moieties (IP-15)
showed noteworthy AChE inhibition activity. However, compound 46 appeared as most active
and selective derivative for AChE and displayed comparative more potency (AChE; IC50= 0.025
ꢀM, BuChE/ AChE; 97.3) than standard drug donepezil (AChE; IC50= 0.039 ꢀM, AChE/BuChE;
86.1). The overall results indicate that the majority of the tested compounds, excluding
compound 42 (IC50 = 0.088 ꢀM), demonstrated comparatively lower inhibitory potency against
EqBuChE with IC50 values ranging from 0.214 to 12.22 ꢀM indicating their preferable
selectivity towards AChE. This selectivity profile may prove to be beneficial to diminish
peripheral cholinergic side effects. On the basis of the aforementioned results, three compounds
IP-9, IP-13 and IP-15 emerged out as leading ChEs inhibitors among the entire tested series and
therefore, these compounds were picked up to pursue further experiments.
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2.2.2. Kinetic study of the AChE inhibition
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To elucidate the mechanism of AChE inhibition, a kinetic study was carried out with the most
potent AChE inhibitor IP-15, by graphical analysis of their steady state inhibition data. The
Lineweaver-Burk double reciprocal plots were generated (Figure 2) which showed the
interception of the lines above the x-axis with both increased slope (decreased Vmax) and
intercepts (higher Km), at increasing concentrations of the inhibitor. This pattern indicated a
mixed-type inhibition and further verifies that IP-15 might be capable to interact with the
catalytic active site (CAS) as well as the peripheral anionic site (PAS) of AChE.
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2.2.3. Inhibition of self-mediated Aβ1 42 aggregation
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To examine the anti-amyloidogenic activity of these new compounds, standard Thioflavin-T
(ThT) assay was performed to monitor and quantify the aggregation of Aβ1-42 synthetic peptide
into fibrils [18]. In comparison with the reference compound curcumin, the results indicate that
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the majority of the compounds, prevented self-mediated Aβ1-42 aggregation with the percentage
of inhibition ranging from 48.5% to 83.7% at 25 ꢀM (Table 2).
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The compounds 32-38 exhibited significant inhibition of Aβ aggregation (51.5-70.5%) while,
compound 37 bearing p-methoxy substitution at the phenyl ring displayed strongest inhibitory
activity (70.5%) in this series, which was 1.26 fold better than curcumin (55.7%). The p-fluoro
substituted compound 34 showed fairly better activity than compounds with p-chloro (compound
33, 59.5%) or p-methyl substitution (compound 36, 63.5%). Whereas, compound 32 having an
unsubstituted phenyl ring was found as a weak inhibitor (51.5%) among phenyl substituted (32-
38) analogs.
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Further, the replacement of substituted phenyl ring with other heterocyclic or non-heterocyclic
substitutions (compound 39-46), with the exception of compound 42 featuring a benzyl group
(48.5%), exhibited significantly enhanced inhibitory activity. Among this sub-series, compounds
39, IP-9, IP-13 and IP-15 have shown excellent inhibition of Aβ aggregation with percentage
inhibition 83.7%, 78.2%, 80.4% and 81.6%, respectively. Therefore, these four inhibitors and
curcumin were selected to evaluate their IC50 values. All the compounds possess significantly
lower IC50 values than curcumin. IP-9, bearing difluorobenzhydryl piperazine achieved lowest
IC50 value of 7.4 ꢀM, which was 2.86 fold more potent than curcumin. Interestingly, the obtained
result of Aβ anti-aggregation activity is consistent with AChE inhibition results.
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2.2.4. Inhibition of Aβ1 42 fibril formation monitored by Transmission Electron Microscopy
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(TEM)
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To complement the results of the ThT binding assay, the anti- Aβ1−42 aggregation activity of two
most promising compounds IP-9 and IP-15 was monitored by transmission electron microscopy
(TEM). (Figure 3). The inhibitory activity of curcumin was taken as positive control. After
incubation at 37°C for 48 h, Aβ1-42 alone (50 µM) get aggregated into mature, thick and denser
aggregates (Figure 3b) as compared to unaggregated Aβ1-42 alone (50 µM) (Figure 3a) which,
was kept at 0°C. However, disaggregation effects were sharply noticed upon addition of IP-9 and
IP-15 (25 µM each) as few slender Aβ fibrils were observed (Figure 3d and 3e) as compared to
curcumin (Figure 3c). Hence, the obtained TEM results are consistent with ThT binding study
and collectively suggested that IP-9 and IP-15 could inhibit Aβ aggregation markedly through
direct interaction or by blocking the PAS site of AChE.
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2.2.5. Molecular docking with AChE
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Molecular docking study was performed using AutoDock 4.2 to analyze the interaction mode of
compounds with the reported crystal structure of AChE (PDB ID: 1EVE). Compound IP-15 and
IP-9 were predicted to show excellent interaction with AChE (Figure 4a and 4b). The result
shows residues: Tyr130 and Ser286 are involved in H-bond interaction (Figure 4). Trp279 and
Tyr334 at PAS are involved in π-π interaction with a pyrimidine moiety of IP-15 and a diphenyl
moiety of IP-9. Additionally, both derivatives suitably interact with CAS site and their indanone
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moiety impart hydrophobic interaction with CAS site residue Trp84. Both representative ligands
binds suitably in the active site of AChE and nicely interact with CAS as well as PAS site.
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2.2.6. Molecular docking studies with Aβ1-42
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To understand the binding mode and interaction of compounds with Aβ1-42, a docking study has
been performed with X-ray crystal structure of human Aβ1-42 (PDB ID: 1YT). Docking results
showed that most active derivative IP-15 nicely binds with Aβ1-42. Compound IP-15 is
transversely fitted along the major helix, and His14 is involved in H-bond interaction with a
methoxy group of indanone moiety (Figure 5). Pyrimidine ring as well as indanone moiety
of IP15 imparted hydrophobic interaction with residues Leu17, Val18, Phe19, Phe20, Val24,
Ile31, Leu34 and Met35 resulting stabilized IP-15-Aβ1-42 complex. These interactions are found
similar as standard Aβ1-42 inhibitor curcumin where 3-hydroxy and 4-methoxy group of curcumin
involved in H-bond interaction with His14 and imparted hydrophobic interactions with Phe, Val,
Ile and Val [19]. Our docking results reveal that IP-15 bind with Aβ1-42 very similar to curcumin
in terms of H-bond interaction, hydrophobic interaction as well as sitting pattern (Figure 5) [19].
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2.2.7. Molecular Dynamics (MD) Simulation Studies with AChE
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We have performed molecular dynamics (MD) simulation for 40 ns to determine the structural
stability and molecular interaction of protein (AChE and Aβ1-42) and complex with IP-15. To
study this, we first measured the root mean square deviation (RMSD) to determine the global
structural stability of acetylcholinesterase-IP-15 complex in water up to 40 ns of simulation. Plot
of time versus RMSD for all Cα atoms is shown in Figure 6A. Results showed that (i) in the
water RMSD trajectory (black) achieved the stable equilibrium in ~7 ns and is maintained up to
40 ns of simulation (ii) Trajectory obtained for acetylcholinesterase-IP-15 complex, achieved the
equilibrium at 0.22 nm in few nanoseconds and remains stable during the simulation. A gradual
increase in RMSD (~0.06 nm) is recorded in between 20-30 ns, which quickly attains the stable
equilibrium. The change in RMSD ~0.16 of backbone atoms with respect to native suggested
that IP-15 was bound tightly in the active region. And, the ligand is stabilized with hydrogen
and hydrophobic interactions with the surrounding residues during the simulation. Radius of
gyration (Rg) is an effective tool to determine the structural compactness of protein. It can be
defined as the mass weighted root mean square distance of a collection of atoms from their
common center of mass. We carried Rg analysis to examine the conformational changes and
dynamic stability of AChE and acetylcholinesterase-IP-15 complex in water at 300K. A time
evolution plot of Rg for Cα atoms is shown in Figure 6B. From this figure, we can see that
secondary and tertiary structure of both AChE and complex with IP-15 remains stable during the
entire simulation time. The average Rg value for complex remain low (~2.23 nm), while the
native remains stable at an average Rg value ~2.67 nm during the simulation. This result suggests
that the structural integrity of AChE is maintained even after the formation of complex with IP-
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We have also measured the solvent accessible surface area (SASA) of AChE and
acetylcholinesterase-IP-15 complex. Higher SASA is an evidence of an unfolding of structure
and residues which are exposed to solvent. The time evolution plot of the solvent accessible
surface area for each residue of AChE and complex is shown in Figure 7A. In this figure we can
see that residues of complex (red picks) having either similar SASA value or lower to native
protein residues (black). The lower SASA picks (red) obtained from complex indicate that IP-15
on binding to AChE which may lead to tight packing of hydrophobic residues aligned at the core
of the active site. It makes the pocket inaccessible to the solvent molecules and thereby
optimizing van der Waals interactions. This SASA analysis is found to be in support of Rg result.
We examine the time-average RMSF plot of Cα atoms for local change in structure (Figure 7B).
Results showed that residues have average residual fluctuations < 0.07 nm which belongs to
stable conformation (α-helices and β-sheets). Peaks showing displacements >0.09 nm belong to
loop region. The result of RMSF plot revealed that overall flexibility of IP-15 bounded AChE
was increased (0.03 nm) as compared to native, which was oblivious. However, no any
conformational changes are recorded during the simulation. This result is found to be consistent
with secondary structure analysis at DSSP (Figure 8A & 8B) and therefore, it provides clear
evidence that secondary and tertiary content of AChE bounded with IP-15 was remained stable
during the simulation. The potential energy was analyzed during the 40 ns, and MD simulations
to measure the system stability. The average of potential energy plot (Figure 7C) for AChE in
water (black) shows that the protein was stabilized at approximately ~1.327×106 kJ/mol, while
the potential energy of IP-15 bounded AChE is stabilized at approximately ~1.354×106 kJ/mol.
This result indicates that acetylcholinesterase-IP-15 complex, on an average remained stable
during the simulation.
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2.2.8. Molecular Dynamics (MD) Simulation studies with Aβ1-42
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We examined the all Cα-atoms RMSD to determine the conformational stability of Aβ and Aβ-
IP-15 complex in water (Figure 9A). From this figure, we found that trajectory obtained for Aβ,
achieves equilibrium at 5 ns and a stable conformation of Aβ is seen up to 18 ns. A drift of ~0.2
nm was seen at 20 ns, which quickly dropped at 22 ns to attain the equilibrium. Thereafter,
almost steady equilibrium was maintained up to 40 ns. However, in case of Aβ-IP-15 complex,
trajectory shows the sharp transition in initial few nanoseconds and dropped to attain
equilibrium at 5 ns. And, trajectory remains steady at ~0.6 up to the end of the simulation. This
result suggested that IP-15 was bound tightly in the active site region of Aβ and was stabilized
with H-bond and hydrophobic interactions with the surrounding residues as like well studied
inhibitor curcumin [19].
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Another, effective parameter to determine the conformational stability and structural integrity of
a protein is radius of gyration (Rg). We have also examined the time evolution plot of Rg to
define the dynamic adaptability of Aβ and Aβ-IP-15 complex. A time evolution plot of Rg of all
backbone atoms is shown in Figure 9B. Rg plot obtained for Aβ, shows average fluctuation of
0.2 nm during the initial 0-20 ns and an equilibrium is achieved at 23 ns, which was maintained
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up to the end of the simulation. The Rg trajectory of Aβ-IP-15 complex quickly achieves the
equilibrium and was continued up to approx 7 ns. At 7-20 ns, trajectory drops to the lowest Rg
value ~1.4. Thereafter, a stable equilibrium was observed up to the end of the simulation. This
result suggests that secondary and tertiary structure of Aβ-IP-15 has remained intact during the
simulation.
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We have performed RMSF of Cα atoms from its time averaged position to establish the
conformational fluctuations of Aβ1-42 and Aβ-IP-15 complex at the residual level (Figure 10A).
From this figure, we can see that the average residual fluctuation of all residues in the case of
Aβ-IP15 complex is lower than Aβ. Results suggest that α-helical structure of Aβ is more
precisely maintained due to binding of IP-15. We have further validated this result at VMD
(timeline). A time series secondary structural analysis at VMD also suggested that Aβ-IP-15
complex having the highest percentage of α-helix content as compared to Aβ. We also carried
salt-bridge analysis at VMD and found that there was no signal for the salt bridge formation
between the residues Asp23 and Lys28 in Aβ-IP-15 complex. Additionally, we also examine the
solvent accessible surface area (SASA) to apprehend the dynamic progression in hydrophobic
residues. SASA plot shown in Figure 10B, provides a clear evidence that binding
of IP15 influences the hydrophobic domain of Aβ residues, which may inhibit the fibril
formation. Furthermore, to examine the stability of docked complex (Aβ-IP-15), we have
calculated the potential energy with respect to the starting conformation, as a function of time
(Figure 10C). The average of the potential energy plot shows that the potential energy for Aβ-
IP15 complex remains more negative (approximately
(approximately
2.33×105 kJ/mol). All these observations suggested that Aβ in the presence of
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5.73×105 kJ/mol) as compared to Aβ
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ligand showed more stable conformation and IP-15 is largely stabilized with hydrophobic
interaction similar to standard inhibitor curcumin [19].
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2.2.9. In vitro antioxidant activity Oxygen radical absorbance capacity (ORAC-FL) assay
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Several evidences in literature have underlined that oxidative stress is an early feature of AD
pathology [20], for this reason, reduction of the oxidative stress is a crucial aspect in designing agents
for the treatment of AD. In this context, we have evaluated preliminary antioxidant activities of target
compounds 32-46 by following the well-established ORAC-FL method. Trolox, a water-soluble
vitamin E analog, was used as a standard The antioxidant capacity of compounds 32-46 were
.
determined by their competition with fluorescein in the radical capture process, using a fluorescence
microplate reader (Table 1). The ability of compounds 32-46 to scavenge radicals at concentrations
between 1-25 ꢀM were compared with that of the highly potent compound Trolox and were expressed
as their Trolox equivalent. These compounds exhibited significant radical-capture capacity and almost
all the target compounds demonstrated moderate to good antioxidant capacities ranging from 0.64 to
3.89 times the Trolox value. Compounds 37, IP-9, 43 and IP-13 showed the most potent antioxidant
activity within the series with ORAC-FL values of 3.39, 2.98, 3.35 and 3.89 trolox equivalents,
respectively. The presence of oxygen atom in various substituted moieties appears to be crucial to the
compound’s radical scavenging ability.
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2.2.10. Cytotoxicity of synthetic compounds in SH-SY5Y cells
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To determine the potential cytotoxic effects of synthesized novel compounds on neuronal cell
line SH-SY5Y, three most potent compounds IP-9, IP-13 and IP-15 were selected for treatment,
with different concentrations ranging from 1-100 ꢀM. After exposing the cells to these
compounds for 48 h, the cell viability was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium (MTT) assay [21-22]. The Figure 11 shows a dose-dependent effect of
tested compounds on the viability of SH-SY5Y cells, for instance, IP-9 (1 ꢀM: 97.1 ± 3.5%; 5
ꢀM: 95.4 ± 3.6%; 10 ꢀM: 95.7 ± 5.4%; 25 ꢀM: 92.8 ± 2.5%; 50 ꢀM: 88.6 ± 3.1%; 75 ꢀM: 84.3 ±
2.6% and 100 ꢀM: 80.3 ± 2.5%), IP-13 (1 ꢀM: 96.0 ± 4.5%; 5 ꢀM: 94.4 ± 4.6%; 10 ꢀM: 92.3 ±
5.4%; 25 ꢀM: 90.1 ± 4.5%; 50 ꢀM: 86.9 ± 2.9%; 75 ꢀM: 83.7 ± 3.2%; 100 ꢀM: 81.1 ± 3.3%)
and IP-15 (1 ꢀM: 98.5 ± 2.5%; 5 ꢀM: 96.1 ± 3.2%; 10 ꢀM: 94.7 ± 5.4%; 25 ꢀM: 92.8 ± 1.5%;
50 ꢀM: 89.3 ± 3.2%; 75 ꢀM: 85.7 ± 2.5% and 100 ꢀM: 82.4 ± 2.2%) display cytotoxicity. The
results demonstrated that these newly synthesized compounds did not display the cytotoxic effect
on the cells in the range of tested concentrations
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2.2.11. Intracellular antioxidant Potential
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The reduction of oxidative stress plays a crucial step in designing multi-functional agents for AD
disease [23]. Consequently, the intracellular antioxidant capacity of selected compounds IP-9,
IP-13 and IP-15 (5, 10 and 20 ꢀM) against H2O2-induced ROS in SH-SY5Y cells were
examined using DCFH-DA fluorescent assay. As illustrated in Figure 12, exposure of SH-SY5Y
cells to H2O2 (200 ꢀM) increased the intracellular ROS accumulation by 1.55 folds, p < 0.001, in
24 h. Upon treatment with compound IP-13, a dose dependent inhibitory effect was seen (5 ꢀM,
p < 0.05; 10 ꢀM, p < 0.01 and 20 ꢀM, p < 0.001) on ROS accumulation without any change in
basal levels of ROS. On the other hand, compounds IP-9 and IP-15 considerably reduced
oxidative stress at higher dose, i.e., at 10 ꢀM, 20 ꢀM, p < 0.05 and 10 ꢀM, p < 0.05; 20 ꢀM, p <
0.01, respectively. Therefore, the present study indicates that IP-9, IP-13 and IP-15 have the
enormous capability to protect neuronal cells against oxidative stress and further supports the
potential of IP-9, IP-13 and IP-15 as good anti-oxidant agents.
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2.2.12. Neuroprotective effect against H2O2-induced oxidative cell damage in SH-SY5Y
cells
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The neuroprotective potential of the compounds on H2O2-induced oxidative stress in SH-SY5Y
cells were assessed by cell viability assay at three different concentrations i.e, 5, 10 and 20 µM.
Treatment of cells with H2O2 (200 µM) for 24 h, resulted in a marked decrease in cell viability
(46%, p < 0.001) as compared to untreated control. The co-treatment of cells with compounds
diminished H2O2-induced neurotoxicity. As can be seen in Figure 13, the selected compounds
exhibited neuroprotective effects at concentrations ranging from 5 to 20 µM, with compounds
IP-9 and IP-13 showing the highest protective capability at 10 µM (p < 0.01) while IP-15
showed similar protective ability at a relatively higher concentration i.e, 20 µM (p < 0.01).
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Furthermore, microscopic observations displayed that a large amount of H2O2-treated cells
became rounded, vacuolated and floated, which clearly indicated apoptosis or necrosis in these
cells. All these morphological alterations clearly indicated H2O2 induced neurotoxicity in SH-
SY5Y cells (Figure 14). However, the treatment (20 ꢀM) with IP-9, IP-13 and IP-15
successfully blocked these morphological transformations and significantly prohibited the cell
death provoked by H2O2 toxicity in these cells (Figure 14c, 14d and 14e). These results imply
that the neuroprotective action of the synthesized compounds might come from radical
scavenging action.
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2.2.13. Neuroprotection against Aβ1–42 -induced toxicity
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Aβ-induced apoptotic neuronal cell death is considered as a decisive episode in the pathology of
AD [24]. To consolidate the results obtained from Aβ1-42 inhibition studies, further experiments
were conducted to assess the potential of IP-9, IP-13 and IP-15 against Aβ1–42 induced
neurodegeneration in SH-SY5Y neuroblastoma cells, using a cell viability assay (Figure 15).
Three different concentrations i.e, 5, 10 and 20 µM were used in this experiment. A marked
decline in cell viability (45.3%, p < 0.001) was observed on treatment of cells with Aβ1–42 (25
µM) for 48 h, as compared to untreated control. It was evident from the results that the selected
compounds imparted marked neuroprotection at the given concentrations (5 to 20 µM).
Compound IP-15 showed the highest protective capacity at 10 µM (p < 0.001), while
compounds IP-9 and IP-13 revealed a similar effect at a higher concentration i.e, 20 µM (p <
0.001), when compared with the only Aβ1–42 (25 µM) treated control.
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Furthermore, phase contrast micrographs revealed a marked change in the morphology of Aβ1–42
treated cells as compared to control cells. After Aβ1–42 treatment, cells were fewer in number
with visibly shrinked cell body, fading synapses, loss of adherence and fragmented or rounded
floating cells which signifies the onset of apoptosis or necrosis (Figure 16). However,
compounds (20 µM) treatment significantly restored these alterations in SH-SY5Y cells. Thus, it
seems reasonable to propose that neuroprotection against Aβ1–42 induced neurotoxicity shown by
these compounds is due to their anti-Aβ aggregation action.
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3. Conclusion
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In conclusion, our study focused on the synthesis, in-silico and pharmacological studies of novel
designed donepezil derivatives as multi-functional agent for therapy AD. These compounds
showed promising AChE inhibitory activity, Aβ disaggregation potential, antioxidant and
neuroprotective activity in numerous in-vitro studies. Among all, compound IP-9, IP-13 and IP-
15 appeared as most active and selective AChE inhibitors,which also endowed with good Aβ
disaggregation, antioxidant and neuroprotective potential. Besides their multifunctional activity
compound IP-13 and IP-15 showed better AChE inhibitory activity as compared to standard
drug donepezil. Derivatives IP-9, IP-13 and IP-15 significantly reduced H2O2 induced oxidative
stress in SH-SY5Y cells. Furthermore, these derivatives exert a potential neuroprotective effect
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against H2O2 and Aβ induced cytotoxicity in SH-SY5Y cell line. In molecular docking studies,
these derivative nicely interact with AChE and Aβ peptide. MD simulation studies of compound
IP-15 reveal its efficient binding with AChE as well as Aβ and consistent interaction with the
essential active site residual during 40ns simulation. Additionally, most active derivatives IP-9,
IP-13 and IP-15 came out as non toxic chemical entities in MTT assay which was performed by
using SH-SY5Y cells. Therefore, we may conclude that IP-9, IP-13 and IP-15 emerged as
potent and safe multifunctional agents for AD and might be used as lead molecules for further
development.
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4. Experimental Protocols
4.1. Chemistry
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All the chemicals and reagents were obtained from Sigma Aldrich (St. Louis, MO, USA), Alfa
Aesar (Massachusetts), S.D Fine Chemicals (India) and Merck (Darmstadt, Germany) and
solvents for reaction medium were dried by standard methods. Melting points were taken in open
capillaries using model KSPII, KRUSS, (Germany). The nuclear magnetic resonance (NMR)
spectra were recorded on high resolution Jeol-400 MHz NMR spectrophotometer (USA). Mass
spectra were recorded on an Agilent 6310 Ion trap LC/MS and elemental analysis (C, H and N)
was carried on Elementar analysensysteme.
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General Synthetic procedure of 4-(4-substituted piperazin-1-yl) benzaldehyde (17-31)
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Intermediates (17-31) were synthesized according to our previously reported method and the
spectral data were matched with our previous publication [9].
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General Procedure for the Synthesis of (E)-2-(4-(4-(substituted) piperazin-1-yl)
benzylidene)-5,6-dimethoxy-2,3-dihydro-1H-inden-1-one (32-46)
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Equimolar ratios (1 molar each) of 5,6-dimethoxyindanone and respective piperazine aldehyde
were reacted in anhydrous ethanol in the presence of 5 ml ethanolic solution of KOH (4g KOH
in 100 ml). The reaction mixture was stirred overnight, then, on the next day the appeared solid
product was filtered and washed twice with ethanol and water. The crude products were dried in
oven and purified by column chromatography using chloroform/methanol (97:03) as eluent. The
compounds were fully characterized by using H NMR, C NMR and Mass spectroscopy. The
spectral characterization of all final compounds except 40, 44 and 46 are given in the
supplementary information.
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(E)-2-(4-(4-(Bis(4-fluorophenyl)methyl)piperazin-1-yl)benzylidene)-5,6-dimethoxy-2,3-
dihydro-1H-inden-1-one (40)
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Light yellow solid; yield 59%; mp: 198-2000C; H NMR (CDCl3, 400 MHz): δ 2.53 (s, 4H,
piperazine), 3.31 (s, 3H, piperazine), 3.90-4.00 (m, 8H, CH2, 2X CH3), 4.25 (s, 1H, CH), 6.88-
7.02 (m, 7H, Ar), 7.36-7.40 (m, 5H, Ar+CH), 7.55 (t, 4H, Ar, J= 8.7 Hz); C NMR (CDCl3, 100
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MHz): δ 32.2, 47.7, 51.4, 56.0, 56.1, 74.3, 104.9, 107.1, 114.6, 115.3, 115.5, 125.9, 129.1, 131.4,
131.9, 132.1, 132.6, 137.9, 144.4, 149.4, 151.4, 154.9, 160.6, 163.0, 193.1; LC-MS: 567 (M+1);
Anal. Calcd for C35H32F2N2O3: C, 74.19; H, 5.69; N, 4.94; Found: C, 74.42; H, 5.94; N, 4.67.
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(E)-2-(4-(4-(Furan-3-carbonyl)piperazin-1-yl)benzylidene)-5,6-dimethoxy-2,3-dihydro-1H-
inden-1-one (44)
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Light yellow solid; yield 53%; mp: 218-2200C; H NMR (CDCl3, 400 MHz): δ 3.39 (t, 4H,
piperazine, J= 5.3 Hz), 3.92-3.99 (m, 12H, piperazine, CH3X 2, CH2), 6.51 (t, 1H, Ar, J=1.5 Hz),
6.94-6.98 (m, 3H, Ar), 7.06 (d, 1H, Ar, J= 3.0 Hz), 7.33 (s, 1H, CH), 7.50 (d, 2H, Ar, J=10.0
Hz), 7.59 (d, 2H, Ar, J=8.4 Hz); 13C NMR (CDCl3, 100 MHz): δ 32.1, 47.9, 56.0, 56.1, 104.8,
107.1, 111.3, 115.1, 116.8, 126.6, 131.2, 132.1, 133.3, 143.8, 144.4, 147.6, 149.3, 150.9, 154.9,
158.9, 193.0; LC-MS: 459 (M+1); Anal. Calcd for C27H26N2O5: C, 70.73; H, 5.72; N, 6.11; Found:
C, 70.97; H, 5.93; N, 5.89.
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(E)-5,6-Dimethoxy-2-(4-(4-(pyrimidin-2-yl)piperazin-1-yl)benzylidene)-2,3-dihydro-1H-
inden-1-one (46)
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Pale yellow solid; yield 86%; mp: 178-1800C; H NMR (CDCl3, 400 MHz): δ 3.41 (t, 4H,
piperazine, J= 5.3 Hz), 3.93-4.01 (m, 12H, piperazine, CH3X 2, CH2), 6.54 (t, 1H, Ar, J= 4.9
Hz), 6.97-7.00 (m, 3H, Ar), 7.34 (s, 1H, CH), 7.55(s, 1H, Ar), 7.60 (d, 2H, Ar, J= 8.4 Hz), 8.34-
8.35 (m, 2H, Ar); 13C NMR (CDCl3, 100 MHz): δ 32.2, 43.2, 47.6, 56.0, 56.1, 104.8, 107.1, 110.2,
115.0, 126.2, 131.3, 132.0, 132.1, 132.5, 144.4, 149.3, 151.4, 154.9, 157.7, 161.4, 193.2; LC-MS:
443 (M+1); Anal. Calcd for C26H26N4O3: C, 70.57; H, 5.92; N, 12.66; Found: C, 70.81; H, 6.13; N,
12.91.
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4.2. Biological Evaluation
4.2.1 Inhibition of AChE and BuChE
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AChE and BuChE inhibitory activities of the synthesized compounds were assessed by the Ellman’s
method with minor modification [17]. In brief, a stock solution of the test compounds (10 mM) was
prepared in ethanol and further diluted using 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) to provide a
final concentration (0.01-100 ꢀM). The enzyme solutions were freshly prepared by dissolving
lyophilized powder in double-distilled water. The assay solution included of 1 mL of 0.1 M
phosphate buffer KH2PO4/K2HPO4, 25ꢀL of AChE (0.22 U/mL, E.C. 3.1.1.7, from electric eel) or 25
ꢀL of BuChE (0.06 U/mL, E.C. 3.1.1.8, from equine serum) and 100 ꢀL of various concentrations of
synthesized compounds which was kept for 5 min before addition of 100 ꢀL of 0.01 M DTNB.
Donepezil was used as positive control in the same range of concentrations. The reaction was
initiated by the addition of 20 ꢀL of the 0.075 M substrate solution (acetylthiocholine/
butyrylthiocholine) and 2 min after substrate was added and the absorption was measured at 25 °C at
412 nm. The non-enzymatic hydrolysis of acetylthiocholine/ butyrylthiocholine iodide was also
deliberated in enzyme-free assay systems, and the results were used as blank. Inhibitor-free assay
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systems were utilized to measure the full activity. Finally, the percent inhibition was calculated by
using the following expression: (1 - Ai/Ac) x 100, where Ai and Ac are the absorbance obtained for
AChE in the presence and absence of the inhibitors, respectively, after subtracting the respective
background. Each experiment was performed in triplicate, and results are presented as mean ±
standard deviation. IC50 of the inhibitors was calculated by nonlinear regression analysis, using the
Graph Pad Prism 5 program.
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4.2.2. AChE inhibition: A Kinetic Study
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The kinetic study of AChE was executed by utilizing the earlier described method [10]. The test
compound was added into the assay solution and pre-incubated with the enzyme at 37°C for 15 min,
followed by the addition of substrate. Then after, kinetic characterization of the hydrolysis of ATC
catalyzed by AChE was carried out spectrometrically at 412 nm. A parallel control was made with the
assay solution of no inhibitor for each time. The plots were assessed by a weighted least square
analysis, which assumed that the variance of V to be a constant percentage of V for the whole data
set. Slopes of these reciprocal plots were then plotted against the concentration of the inhibitors in a
weighted analysis and Ki was determined as the ratio of the plot intercept to the plot slope.
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4.2.3. Thioflavin T (ThT) assay to study Aβ aggregation
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Aβ1–42 peptides were first treated with hexafluoroisopropanol (HFIP) at 5mg/ml to avoid self-
aggregation. The clear solution consisting the dissolved peptide was then aliquoted in micro
centrifuge tube. The HFIP was evaporated under a stream of nitrogen until a clear film remained in
the test tube. The pre-treated Aβ1-42 samples were then dissolved in DMSO to make a stable stock
solution (Aβ = 1mM). A mixture of the peptide (10 ꢀL, 25 ꢀM, final concentration) with or without
the tested compound (25 ꢀM) was incubated at 37°C for 48 h. To quantify the amyloid fibril
formation, the thioflavin-T fluorescence method was utilized. Blanks consisting 50mM phosphate
buffer (pH 7.4) instead of Aβ with or without inhibitors were also carried out. After incubation,
samples were diluted to make a final volume of 200 ꢀL with 50 mM glycine–NaOH buffer (pH 8.0)
containing thioflavin-T (5 ꢀM). Afterward the fluorescence intensities were measured five minutes
later (excitation, 450 nm; emission, 485 nm). The percent inhibition of aggregation was calculated by
the expression (1-IFi/IFc) × 100% where, IFi and IFc are the fluorescence intensities obtained for Aβ
in the presence and absence of inhibitors after subtracting the background, respectively. Each
measurement was carried out in triplicate [9-10].
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4.2.4. Transmission electron microscopy (TEM) assay
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Prior to use Aβ1–42 peptide (Sigma) stock was diluted with 20 mM phosphate buffer (pH 7.4) at
4°C to 40 mM. Aβ1–42 was incubated within the presence and absence of compound IP-9, IP-15
and Curcumin at 37°C for 48 h. The final concentration of Aβ1–42 and test compounds were
50μM and 25μM respectively. Aliquots (10 μL) of the each sample were placed on a carbon
coated copper/rhodium grid for 2 min at room temperature. Each grid staining was performed
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with uranyl acetate (1%) for 2 min. The excess staining solution was discarded and the specimen
was transferred for imaging with transmission electron microscopy (JEOL JEM-1400).
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4.2.5. Molecular docking studies
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Atomic coordinates of AChE (PDB ID: 3V7X) and Aβ1-42(PDB ID: 1YT) were taken from the
Protein Data Bank (www.rcsb.org) [25]. Steepest descent method from Gromacs 4.6.5 was
utilized to optimize the coordinates and AChE as well as Aβ1-42 were saved in pdbqt format to
conduct docking analysis. AutoDock 4.2 with standard protocol was used to dock the
synthesized compounds with AChE and Aβ1-42. Protein-ligands interactions was assessed by
applying Lamarckian genetic algorithm (LGA) [26].The most encouraging free binding energy
and docking orientations lying within the range of 2.0 Å in root-mean square deviation (RMSD)
tolerance were used to cluster the molecule and ranked accordingly. PyMOL was employ to
visualize molecular interactions. All calculations were carried out on workstation machines
running Linux x 86 as operating systems.
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4.2.6. Molecular dynamic (MD) simulation studies
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MD simulations were carried out with GROMACS 4.6.5 suite of programs, using charm force-
field and spc216 water molecules. The trajectory files 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 fluctuation (RMSF). The coordinates of AChE and Aβ1-42 were
minimized with steepest descent, up to a tolerance of 100 kJ mol-1 to remove all bad contacts.
Two independent MD simulations were carried out with standard protocol in water for AChE
and AChE-IP-15 complex, respectively at 300K to evaluate the structural changes, binding
affinity and stability in course of simulation. Similarly, another set of MD simulations were
performed for Aβ1-42 and Aβ1-42 – IP-15 complex. The SHAKE algorithm was used to restrain
the covalent bonds containing hydrogen atoms at their equilibrium distances and periodic
boundary conditions were applied. Particle Mesh Ewald (PME) method was employed for
computing the long-range electrostatic interactions. A 1.4 nm cut-off for van der Waals
interactions 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 [27].
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4.2.7. Oxygen radical absorbance capacity (ORAC-FL) assay
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The antioxidant activity of all target compounds was determined by using standard oxygen radical
absorbance capacity (ORAC-FL) assay [9-10]. Trolox ((±)-6-hydroxy-2,5,7,8-tetramethylchromane-
2-carboxylic acid), fluorescein (FL) and AAPH (2,2’-Azo bis-(amidinopropane) dihydrochloride was
procured from Sigma Aldrich. Antioxidant (20 ꢀL) and FL (120 ꢀL; 70mM, final concentration)
solutions were placed in a black 96-well microplate (96F untreat, Nunc). The mixture was
preincubated for 15 min at 37 °C, and then after AAPH solution (60 ꢀL, 12 mM,) was added by using
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a multichannel pipette. The microplate was placed in the microplate reader and the fluorescence was
recorded every minute for 80 min (excitation, 485 nm; emission, 520 nm). Various concentrations of
samples were measured ranging from1-25ꢀM. A blank (FL + AAPH) was also used which consisted
phosphate buffer and eight calibration solutions using Trolox (1-25 ꢀM) as antioxidant were also
carried out in each assay. Every reaction mixtures were prepared in triplicate, and at least three
independent runs were performed for each sample. The antioxidant curves (fluorescence versus time)
were normalized to the curve of the blank. The area under the fluorescence decay curve (AUC) was
calculated using the following equation:
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i = 80
AUC = 1 + Σ (fi/f0)
i = 1
where, f0 is the initial fluorescence reading at 0 min and fi is the fluorescence reading at the time i.
The net AUC was calculated by the following equation: AUCsample - AUCblank. The regression
equations between the net AUC and the Trolox concentrations were calculated. The ORAC-FL value
for each sample was calculated using the standard curve, and the ORAC-FL value of each tested
compound is thus expressed as Trolox equivalents.
4.2.8. Cytotoxicity assay
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The toxic effect of IP-9, IP-13 and IP-15 on human neuroblastoma cells (SH-SY5Y) cells were
examined according to the previous method [9-10]. Briefly, the cells were plated in 96-well plate
at density 2000 cells/well and cultured for 24 h. Further, cells were exposed to the tested
compounds with various concentrations (1-100 μM) for 48 h. A stock solution of tested
compound was prepared in DMSO and then diluted in complete medium to achieve final
concentrations. Cytotoxicity assay was carried out after 48 h incubation with test compounds in
triplicates. The MTT reagent was added to each well and incubated for additional 4 h followed
by solubilization of formazan crystals in DMSO. The absorbance of each well was measured
using a microculture plate reader with wavelength of 570 nm and a reference wavelength of 630
nm. Results are expressed as the mean ± SD of three independent experiments.
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4.2.9. ROS measurements under H2O2 - induced cellular stress
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This assay was performed in SH-SY5Y cells, according to our previous reported method [9-10].
Briefly, intracellular ROS production was measured from SH-SY5Y cells by treating with
DCFH-DA for 20 min after 24 h of H2O2 (200 µM) treatment in the presence or absence of
compounds IP-9, IP-13 and IP-15 (5, 10 and 20 µM). The total fluorescence was measured by
using an ELISA plate reader (Tecan infinity 20) at an emission wavelength of 488 nm and an
excitation wavelength of 524 nm.
4.2.10. Neuroprotection against H2O2 induced neurotoxicity
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A density of 2 x 104 SH-SY5Y cells were seeded per well in 96-well plates. After 24 h of
incubation, the medium was replaced with serum-free DMEM consisting 200 ꢀM H2O2 and
various concentrations of the compounds at 37ºC for 24 h. SH-SY5Y cells were also cultured
without any treatment as control group and the results were indicated by percentage of control.
Cell viability was quantified using the standard MTT assay. Photomicrographs were also taken
with the help of a camera attached to a microscope (Olympus, Japan) after 24 h of treatment to
elucidate morphological alterations in SH-SY5Y cells.
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4.2.11. Aβ1-42 -induced cytotoxicity
Peptides were dissolved in hexafluoroisopropanol (1mg/ml), sonicated and incubated at room
temperature for 24 h followed by lyophilization. SH-SY5Y cells were harvested from flasks and
plated in 96-well polystyrene plates with approximately 2 x 104 cells per well and incubated at
37°C for 24 h to allow cells to attach. Aβ1-42 with or without test compounds were diluted with
medium and added into individual wells. Then after, plates were again incubated for an
additional 48 h at 37°C. Cell viability was determined using standard MTT assay protocol, and
expressed as a percentage of control cells.
Acknowledgment
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The author Chandra Bhushan Mishra is thankful to the University Grants Commission (UGC),
Delhi, India for the award of Dr. D.S. Kothari postdoctoral fellowship. Authors Shikha Kumari,
Apra Manral, and Vikas Saini are thankful to UGC for the financial support. Author Amresh
Prakash is thankful to department of science and technology (DST), Delhi, India for the young
scientist award. Manisha Tiwari is thankful to the University of Delhi for sanctioning research
funds to carry out this study. The University Science Instrumentation Center (USIC) is deeply
acknowledged for providing NMR spectral analysis of the synthesized compounds.
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Figure legends
Figure 1: Molecular framework of designed novel hybrid derivatives.
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Figure 2: Lineweaver-Burk plots resulting from the subvelocity curve of AChE activity with different
substrate concentrations (0.05-0.50 mM) in the absence and presence of IP-15.
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Figure 3: TEM images for Aβ-induced aggregation and test compound induced Aβ
disaggregation. The incubations were carried out with following reagents: (a) 50 ꢀM Aβ1–42
alone at 0 h (b) 50 ꢀM Aβ1–42 alone at 24 h, 37°C (c) 50 ꢀM Aβ1–42 and 25 ꢀM Curcumin (d) 50
ꢀM Aβ1–42 and 25 ꢀM compound IP-9 (e) 50 ꢀM Aβ1–42 and 25 ꢀM compound IP-15.
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Figure 4: A; IP-15 Docked with 1EVE, B; IP-9 Docked with 1EVE.
Figure 5: Molecular docking of IP-15 with Aβ peptide.
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Figure 6: A. RMSD: Backbone RMSD plots of 1EVE (black) and complex with IP15 (red) in
water at 300 K during 40 ns MD simulation B. Radius of gyration (Rg)
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Figure 7: (A) The time evolution plot of the solvent accessible surface area (SASA) for each
residue of acetylcholinesterase and complex. (B) Time-average RMSF plot of
acetylcholinesterase and complex. (C) Energy verses time plot for acetylcholinesterase (black)
and complex (red).
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Figure 8: Secondary structure analysis plot for (A) acetylcholinesterase and (B)
acetylcholinesterase – IP-15 complex.
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Figure 9: (A). Root-mean square deviation (RMSD) plot of all Cα-atoms of Aβ1-42, (B) Time
evolution of radius of gyration (Rg) values during 40,000 ps (40 ns) of MD simulation.
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Figure 10: (A) RMSF plot of all Cα atoms of Aβ 1-42 in water at 300 K (black) and Aβ-IP15
complex (red). (B) The time evolution plot of the solvent accessible surface area (SASA) for
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each residue of Aβ
(black) and Aβ-IP15 complex.
(black) and complex (red). (C) Potential energy (kJ/mol) plot of Aβ
1-42
1-42
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Figure 11: Effect of compounds IP-9, IP-13 and IP-15 on the viability of SH-SY5Y cells. The
cells were incubated with the indicated concentrations of the tested compounds for 48 h. The cell
viability was assessed by MTT assay. Percentages of the cell viability are presented as mean ±
SD from 3 independent experiments.
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Figure 12: Compounds IP-9, IP-13 and IP-15 significantly reduced H2O2 induced oxidative stress
in SH-SY5Y cells. All data were expressed as mean ±SD of three experiments and each
included triplicate sets. #p < 0.05, ##p < 0.01, ###p < 0.001 vs control; *p < 0.05, **p < 0.01, ***p
< 0.001 vs H2O2 alone. Statistical analysis was performed using one way ANOVA followed by
Bonferroni test.
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Figure 13: Determination of the viability of SH-SY5Y cells by the MTT assay after treatment
with H2O2 (200 µM) in the absence or presence of the indicated concentrations of (5, 10 and 20
µM) compounds IP-9, IP-13 and IP-15. All data are expressed as mean ± SEM of three
experiments and each included triplicate sets. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control; *p <
0.05, **p < 0.01, ***p < 0.001 vs. Aβ1–42 alone.
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Figure 14: Phase-contrast micrographs showing morphological changes in H2O2-induced
neurotoxicity and neuroprotection of IP-9, IP-13 and IP-15 in SH-SY5Y cells. (a) Cells without
treatment showed healthy shapes. (b) H2O2 alone (200 ꢀM) induced neurotoxicity. (c)
Compound IP-9 (20 ꢀM) was given for 24 h with H2O2 (200 ꢀM) at 37° C. (d) Compound IP-13
(20 ꢀM) was given for 24 h with H2O2 (200 ꢀM) at 37° C and (e) Compound IP-15 (20 ꢀM) was
given for 24 h with H2O2 (200 ꢀM) at 37° C and co-treatment showed neuroprotection.
Figure 15: Neuroprotection against Aβ-induced toxicity. Compounds IP-9, IP-13 and IP-15 were
tested for neuroprotective activity against Aβ toxicity in SH-SY5Y neuroblastoma cell cultures.
Results are expressed as percent viability compared to untreated control. All data were expressed
##
as mean ±SD of three experiments and each included triplicate sets. #p < 0.05, p < 0.01, ###p <
0.001 vs control; *p < 0.05, **p < 0.01, ***p < 0.001 vs Aβ1–42 alone. Statistical analysis was
performed using one way ANOVA followed by Bonferroni test.
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Figure 16: Phase-contrast micrographs showing Aβ-induced neurotoxicity and neuroprotection
of IP-9, IP-13 and IP-15 in SH-SY5Y cells. (a) Cells without treatment showed healthy shapes.
(b) Aβ alone (25 ꢀM) induced neurotoxicity. (c) Compound IP-9 (20 ꢀM) was given for 24 h
with Aβ (25 ꢀM) at 37° C. (d) Compound IP-13 (20 ꢀM) was given for 24 h with Aβ (25 ꢀM) at
37° C and (e) Compound IP-15 (20 ꢀM) was given for 24 h with Aβ (25 ꢀM) at 37° C.
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Tables
Table 1. In-vitro inhibition of AChE and BuChE, selectivity index and oxygen radical
absorbance capacity (ORAC, Trolox equivalents) of 32-46 and donepezil
IC50 ± SD (ꢀM)
AChEa
BuChEb
Test
SIc
ORACd
compounds
(BuChE/AChE)
1.314 ± 0.07
5.98 ± 0.09
4.5
7.6
0.64 ± 0.03
1.87 ± 0.04
32
33
0.291 ± 0.03 2.219 ± 0.15
0.528 ± 0.04 3.245 ± 0.13
0.153 ± 0.01 0.214 ± 0.005
6.1
1.4
1.93 ± 0.08
1.72 ± 0.05
34
35
0.194 ± 0.02 1.849 ± 0.09
9.5
1.30 ± 0.29
36
0.119±0.003 0.603 ± 0.07
0.112 ± 0.02 5.132 ± 0.007
5.06
45.8
59.6
3.39 ± 0.11
1.35 ± 0.14
1.41 ± 0.16
37
38
39
0.082 ±
0.004
4.894 ± 0.07

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Tables
40 (IP-9)
0.045 ±
0.005
3.015 ± 0.08
67
2.98 ± 0.17
1.701 ± 0.08 0.365 ± 0.16
0.2
2.31 ± 0.05
1.69 ± 0.09
41
42
2+.618 ±
0.06
0.088 ± 0.21
0.03
1.858 ± 0.09 12.22 ± 0.18
6.5
3.35 ± 0.14
43
44 (IP-13)
0.034 ±
0.002
1.969 ± 0.06
1.735 ± 0.04
2.434 ± 0.03
3.36 ± 0.2
28.5
3.89 ± 0.01
0.105 ±
0.006
16.5
97.3
86.1
2.08 ± 0.05
2.34 ± 0.03
n.t.
45
46 (IP-15)
Donepezil
0.025 ±
0.001
0.039 ±
0.002
a 50% inhibitory concentration (means ± SD of three experiments) of AChE from electric eel.
b 50% inhibitory concentration (means ± SD of three experiments) of BuChE from equine serum.
cSI selectivity index for AChE; IC50 eeBuChE/eqIC50 AChE.
dData are expressed as
ꢀ
mol of Trolox equivalent/
n.t. - Not tested
ꢀmol of tested compound.

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Tables
Table 2. Inhibition of self-induced Aβ1–42 aggregation by target compounds 32-46 and reference
compound and curcumin
Test compound
A
β1-42 aggregation
Inhibition of Aβ1-42
aggregation
Inhibition (%) a
IC50 (ꢀ
M)b
51.1 ± 0.68
n.t
n.t
n.t
n.t
n.t
n.t
n.t
32
33
59.5 ± 0.73
66.8 ± 0.59
64.2 ± 1.13
63.5 ± 0.43
70.5 ± 0.61
65.7 ± 0.44
78.2 ± 0.82
83.7 ± 1.13
61.4 ± 0.66
34
35
36
37
38
12.2 ± 0.15
7.4 ± 0.10
n.t
39
40 (IP-9)
41

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Tables
48.5 ± 0.33
64.5 ± 0.54
80.4 ± 1.08
73.3 ± 0.87
81.6± 0.78
55.7 ± 0.56
n.t
n.t
42
43
44 (IP-13)
45
10.9 ± 0.17
n.t
45 (IP-15)
Curcumin
9.9 ± 0.14
21.81 ± 0.16
a Inhibition of self-induced Aβ1–42 aggregation (25
fluorescence method (means ± SD of three experiments).b The concentration (
μ
M) by testing inhibitors at 25
μ
M by thioflavin-T based
ꢀ
M) required for 50%
inhibition was determined from dose-response curves. Data are expressed as means ± SD of three
independent experiments; n.t.-Not tested.

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