Identification of novel acetylcholinesterase inhibitors: Indolopyrazoline derivatives and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2016.05.002

The synthesis of novel indolopyrazoline derivatives (P1-P4 and Q1-Q4) has been characterized and evaluated as potential anti-Alzheimer agents through in vitro Acetylcholinesterase (AChE) inhibition and radical scavenging activity (antioxidant) studies. Sp

Full text of DOI:10.1016/j.bioorg.2016.05.002

Bioorganic Chemistry 67 (2016) 9–17
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification of novel acetylcholinesterase inhibitors: Indolopyrazoline
derivatives and molecular docking studies
b,c,
c,d
Sridevi Chigurupati a, , Manikandan Selvaraj
, Vasudevan Mani
,
⇑
⇑
Kesavanarayanan Krishnan Selvarajan c, Jahidul Islam Mohammad e, Balaji Kaveti a, Hriday Bera f,
Vasanth Raj Palanimuthu f, Lay Kek Teh b,c, Mohd Zaki Salleh b,c
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia
b Integrative Pharmacogenomics Institute (iPROMISE), Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
c Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia
d Department of Pharmacology and Toxicology, College of Pharmacy, Qassim University, Buraidah 51452, Saudi Arabia
e Department of Pharmacology, Faculty of Medicine, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia
f Department of Pharmaceutics, Faculty of Pharmacy, AIMST University, Semeling, 08100 Bedong, Kedah, Malaysia
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of novel indolopyrazoline derivatives (P1-P4 and Q1-Q4) has been characterized and eval-
uated as potential anti-Alzheimer agents through in vitro Acetylcholinesterase (AChE) inhibition and rad-
Received 7 April 2016
Revised 10 May 2016
Accepted 12 May 2016
Available online 13 May 2016
ical scavenging activity (antioxidant) studies. Specifically, Q3 shows AChE inhibition (IC50
0.68 0.13 M) with strong DPPH and ABTS radical scavenging activity (IC50: 13.77 0.25 M and
IC50: 12.59 0.21 M), respectively. While P3 exhibited as the second most potent compound with
AChE inhibition (IC50: 0.74 0.09 M) and with DPPH and ABTS radical scavenging activity (IC50
13.52 0.62 M and IC50: 13.13 0.85 M), respectively. Finally, molecular docking studies provided
:
l
l
l
l
:
Keywords:
Indole
Pyrazoline
Molecular docking
Antioxidant
Acetylcholinesterase
l
l
prospective evidence to identify key interactions between the active inhibitors and the AChE that further-
more led us to the identification of plausible binding mode of novel indolopyrazoline derivatives.
Additionally, in-silico ADME prediction using QikProp shows that these derivatives fulfilled all the prop-
erties of CNS acting drugs. This study confirms the first time reporting of indolopyrazoline derivatives as
potential anti-Alzheimer agents.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
enzyme butyrylcholinesterase also hydrolyses Ach but, AChE
inhibitors are commonly prescribed to improve the cholinergic
Cholinergic dysfunction and increased oxidative stress play an
important role in the pathogenesis of Alzheimer’s disease (AD).
Acetylcholinesterase (AChE) is an enzyme that is responsible for
the termination of cholinergic signalling by hydrolyzing acetyl-
choline (ACh). Therefore, inhibition of both AChE and oxidative
stress could be effective in the treatment and management of AD
[1]. Until recently, reversible AChE inhibitors (e.g. Donepezil,
Rivastigmine and Galantamine) were used to treat the symptoms
caused by cholinergic dysfunction in AD [2]. However, the related
signalling in AD. The use of the above class of drugs for the treat-
ment of AD has been limited by its serious side effects, so the
search for novel compounds remains as an emerging demand for
the treatment of AD. Indole alkaloids and its analogues are well
known for their AChE inhibitory [3–5] and antioxidant effects
[5–7]. Pyrazolines are also well known for various biological activ-
ities including antioxidant [8] and cholinesterase and selective
monoamine oxidase B inhibitors for the treatment of AD [9,10]
and Parkinson’s disease [11]. There are various report which
showed that the hybrid of two active moiety enhance the activity
like hybrids of indole, quinoline, bisindole, benzothiazole, benzim-
idazole, biscoumarin, oxadiazole showed potent activity [12–18].
Both pyrazoline and indole nucleus are potent AChE inhibitors.
Till date, no studies are reported in hybrid of these two nuclei and
investigated either as an AChE inhibitor or antioxidant.
⇑
Corresponding authors at: Integrative Pharmacogenomics Institute (iPROMISE),
Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak
Alam, Selangor Darul Ehsan, Malaysia. Tel.: +60 33258 4652; fax: +60 33258 4658
(S. Manikandan), Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
AIMST University, Semeling 08100, Bedong, Kedah, Malaysia. Tel.: +60 4429
8000x1284 (Ch. Sridevi).
In the present study, the 3-acetylindole moiety is modified into
indolopyrazolines (P1-P4 and Q1-Q4) via indolochalcones as
E-mail addresses: sridevi.phd@gmail.com (S. Chigurupati), manifans@gmail.com
(M. Selvaraj).
http://dx.doi.org/10.1016/j.bioorg.2016.05.002
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

10
S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
intermediate compounds. The synthesized compounds were char-
acterized and evaluated for their possible in vitro antioxidant and
AChE inhibitory potentials.
2. Results and discussion
2.1. Chemistry
Each experimental value is expressed as the mean Standard
error mean and all experiments are carried in triplicates (n = 3).
Statistical analysis was performed using Graph Pad prism 5.0.
Molecular docking studies were carried out [15] further to
explore the feasibility of the structural topographies required for
the interaction of the indolopyrazoline derivatives with the AChE
enzyme. It also permits to elucidate the possible key active site
residues involved in the intermolecular interactions with the
ligand [19]. So furthermore, the synthesized compounds were eval-
uated for their possible QikProp prediction of ADME properties for
all the eight indolopyrazoline derivatives [20].
Indolopyrazolines (P1-P4 and Q1-Q4) were obtained by reflux-
ing the indolochalcones (III and V) and acid hydrazides in 1:2 ratio
respectively in the presence of glacial acetic acid (see Schemes 1
and 2). The mechanism involved in the conversion of chalcone to
pyrazole is given in Fig. 1 The indolochalcones were obtained by
Aldol condensation of 3-acetyl indole and substituted benzaldehy-
des in the presence of aqueous NaOH Solution. Solidified crude
products were recrystallized from aqueous ethanol. The indolopy-
razolines were obtained in high yields. The purity of the indolopy-
razoline compounds was checked by the Rf value of TLC (Thin Layer
Scheme 1. Synthesis of indolopyrazolines (P1-P4).
Scheme 2. Synthesis of indolopyrazolines (Q1-Q4).

S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
11
OH
O
C
C
N
C
H
C
H
R'
R
C
H
C
H
R'
R
..
H2N
NH2
H
N
H
Chalcone
H
OH
C
C
C
H
R'
C
N
C
C
H
R'
R
+ H2O
R
H
H
N
..
H
N
H
N
H
H
H
-
H
C
C
N
C
C
H
R'
H2C
R'
+ H+
R
H
C
N
..
R
H
N
H
N
H
H+
H
C
H
C
H
-
H
C
R'
H2C
R'
C
C
N
N
R
R
H
H
N
N
Pyrazole
Fig. 1. The mechanism involved in the conversion of chalcone to pyrazole.
Chromatography) and melting point. Physical data and spectro-
scopic techniques like UV–Visible, 1H NMR, mass spectra and
CHN analysis confirmed the structures and purity of the synthetic
compounds.
This part confirmed the synthesis of a series of eight new indolopy-
razoline compounds.
The kmax for the newly synthesized indolopyrazolines was
found to be in the range of 300–440 nm. The formation of pyrazo-
line was revealed by the presence of doublet between 2.12–
2.16 ppm and a triplet at 4.51–4.58 ppm in proton NMR spectra.
All other aliphatic and aromatic protons were observed within
the expected regions. The compounds were further confirmed by
their characteristic mass fragment spectra. The mass fragment pat-
tern of compound P4 given in Fig. 2, displayed parent ion peak at
399, base peak at 51, and different fragment peaks at 139, 117,
42 and 20. Similarly, all the novel compounds were characterized.
2.2. Antioxidant assay
The in vitro antioxidant potential of synthesized indolopyrazoli-
nes (P1-P4 and Q1-Q4) was evaluated using DPPH (1,1-Diphenyl-
2-picrylhydrazyl) stable free radical, ABTS (2,20-azinobis (3-ethyl
benzothiazoline-6-sulfonic acid) radical cation scavenging assay
and compared to ascorbic acid. The results revealed that all the
indolopyrazolines are good at scavenging free radicals of DPPH
and ABTS when compared with the reference standard, ascorbic
acid at the same concentrations. However, on finding the IC50

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S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
Cl
O
N
C
N
N
C24H18ClN3O
H
+
.
Cl
O
N
C
Fig. 4. ABTS radical cation scavenging assay results of indolopyrazolines.
N
(12.59 6.1), P3 (13.13 2.5) and Q4 (13.38 0.33) and the IC50
values were also comparable to ascorbic acid (11.5 1.9).
N
+
H
C
Parent ion peak: m/z:399
(R.I. = 39%)
2.3. Anticholinesterase enzyme inhibition assay
+
m/z:76
Cl
(R.I = 42%)
In AChE assay, compounds Q3 (0.68 0.13) and P3 (0.74 0.09)
showed better IC50 values compared to that of the positive control,
Donepezil (0.01 0.4). It is notable that out of the eight synthe-
sized compounds, P3 and Q3 performed well in both AChE and
antioxidant assays.
O
C
+
m/z:139
(R.I.= 46%
+
C
N
H
m/z:117
Base peak: m/z: 51
(R.I. = 100%)
(R.I. = 47%)
+
2.4. Docking studies and binding mode analysis
O
C
m/z:28
Molecular modeling studies were accomplished to investigate
the possible binding mode of eight indolopyrazoline derivatives
(P1-P4 and Q1-Q4) targeting the crystal structure of acetyl choli-
nesterase (PDB ID: 1EVE) using Glide docking program, Schrodin-
ger Maestro software (version 6.9; Schrodinger LLC, New York).
All the eight indolopyrazoline derivatives were docked into the
active site of the AChE. The results were analyzed based on the
glide docked scores obtained from GLIDE (Grid-based Ligand Dock-
ing with Energetics) docking [21]. The docked binding mode was
analyzed for the interactions between specific compounds and
AChE. In-depth analysis of the interaction pattern for the most
active compounds P3, P4, Q3 and Q4 are detailed in the following
section. The binding mode of the compound P3 positioned in the
ravine of the AChE active site showed that Phe288 of the AChE
form hydrogen bond with oxygen attached to the methanone
(R.I. = 20%)
+
CH2
CH2
Fig. 2. Mass fragmentation pattern of P4.
group. The two phenyl groups of the P3 form
p-p stacking with
Trp279, while the residues such as Trp84, Phe330, Tyr121, Ile287,
Tyr334 and Phe331 forms hydrophobic interaction with an indole
and pyrazol ring of compound P3 as shown in Fig. 5a.
Fig. 5b shows the docking orientation of compound P4, where
the Phe288 of the AChE forms hydrogen bond with methanone
oxygen, while the phenyl group forms hydrophobic interactions
with Phe290, Tyr334 and Tyr121 while a chlorophenyl ring of
the P4 forms hydrophobic interaction with Ile287. Additionally,
the indole ring forms hydrophobic contact with Trp84, Phe330
and Phe331.
The compound Q3 as shown in Fig. 6a quartered in the active
site of AChE form stable network of hydrogen bonding between
the indole NH with His440 and the acetic acid oxygens forming
hydrogen bonds with Arg289 and Phe288, respectively. Specifi-
Fig. 3. DPPH radical scavenging assay results of indolopyrazolines.
values of eight compounds, P3 and Q3 were proved to be the best
compounds in scavenging free radicals (see Figs. 3 and 4).
In DPPH assay, the IC50 values obtained with the compounds
(P1-P4; Q1-Q4) showed varying degree of activities in terms of
the IC50 value (see Table 1). The IC50 values obtained with three
compounds, namely, P3 (13.52 6.2), Q3 (13.77 2.5) and Q4
(14.86 2.4) were comparable with that of the positive control,
ascorbic acid (12.79 1.9). Similarly, in ABTS assay, these com-
pounds also showed better IC50 values with compounds Q3
cally, the indole ring also forms stable
and His440, respectively.
p-p stacking with Trp84
The complex is also stabilized by the hydrophobic interaction
established between the benzene ring of Q3 with Tyr70 and

S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
13
Table 1
IC50 values of indolopyrazolines.
Compound code
Chemical structure
IC50
(lM
SEMa)
DPPH radical scavenging assay
25.18 0.94
ABTS radical cation scavenging assay
25.52 0.64
AChE inhibition assay
10.58 0.34
P1
P2
P3
17.43 0.44
13.52 0.62
16.12 0.73
13.13 0.85
60.82 0.15
0.74 0.09
P4
Q1
15.42 0.98
15.48 1.11
9.72 0.29
25.45 1.4
24.25 0.9
86.56 0.97
Q2
Q3
15.99 0.92
16.16 0.34
27.13 0.31
13.77 0.25
12.59 0.21
0.68 0.13
Q4
14.86 0.14
13.38 0.13
8.67 0.33
Ascorbic acid
Donepezil
12.79 0.99
11.5 0.89
–
O
–
–
0.01 0.40
N
O
O
a
Standard mean error using Graph Pad prism 5 (n = 3).
Tyr121. Additionally, pyrazole phenoxy ring forms hydrophobic
interactions with Ile287, Tyr334, Phe331 and Phe330.
In the case of Q4 the orientation of ligands forms a stable net-
work of hydrogen bonding between the indole NH with His440
and the acetic acid oxygen’s forming hydrogen bonds with
Arg289 and Phe288, respectively. In addition, the indole ring also
interactions with Phe330 and Tyr130. Moreover, the chloroben-
zene ring forms hydrophobic interactions with Tyr121, Tyr70,
Val71 and Pro86. Likewise, the phenoxy group also forms
hydrophobic interactions with Ile287, Tyr334 and Phe331 (Fig. 6b).
AChE inhibitory activities for compound Q1-Q4 varies due to
the presence of different substituted aryl groups at ‘R’ position.
Existence of electron withdrawing groups in the aryl group
forms stable
p-p stacking with Trp84, His440 and hydrophobic

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S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
Fig. 5. Shows the binding mode of the compound in stick (a) compound P3 and (b) compound P4 in the AChE active site. Key interacting residues are shown in line, and the
hydrogen bonds are represented by a yellow dashed line.
Fig. 6. Shows the binding mode of the compound in stick (a) compound Q3 and (b) compound Q4 in the AChE active site. Key interacting residues are shown in line, and the
hydrogen bonds are represented as a dashed yellow line.
decreases the activity. Through docking studies it’s evident that the
existence of AChE hydrophobic residues at the site of ‘R’ group
interacting site (aryl group) does not prefer electron withdrawing
groups, which eventually decrease the activity. Hence the activity
profile of compound Q1-Q4 varies. In the case of compound Q4
the presence of Cl has the tendency to donate electron, so its activ-
ity is better compared to nitro group (compound Q2) and pyridine
(compound Q1) which are electron withdrawing groups. In turn
compound Q3 with a simple aryl group such as benzene without
any electron withdrawing group ranks top in the activity profile,
thus establishing possible hydrophobic interactions with the active
site hydrophobic residues of AChE.
tion, transport, and ultimately bioavailability. Eight of our
indolopyrazoline derivatives solubility values were within the
range [22]. Further, the predicted values for the blood–brain bar-
rier (BBB) penetration of all the compounds seems to be in the
optimum penetrate range (–2 to +2 scale) and consequently are
measured as active compounds on the CNS. Finally, the Lipinski’s
rule of five and Qikprop rule of three were all within the range
for the indolopyrazoline derivatives and thus making these deriva-
tives as suitable drug candidates.
3. Conclusions
In conclusion, eight indolopyrazoline derivatives were synthe-
sized and assessed for AChE inhibition and antioxidant activity
agents against AD. The biological activity assay confirms that most
of the indolopyrazoline derivatives were potent inhibitors of AChE.
Particularly compound Q3 possessing phenyl moiety was found to
2.5. Theoretical ADME prediction of indolopyrazoline derivatives
Theoretical calculations of the ADME (Absorption, Distribution,
Metabolism and Excretion) properties of indolopyrazoline deriva-
tives (P1-P4 and Q1-Q4) were done using QikProp [20]. Drug kinet-
ics and exposure of tissues to drug influences the pharmacological
activity and the performance of a drug, which is ultimately deter-
mined by its ADME properties. Nearly 12 physically significant
descriptors and pharmacologically relevant properties of indolopy-
razoline derivatives were predicted and analyzed (Supplementary
Material, Table S1).
be the best potent AChE inhibitor (IC50: 0.68 0.13
strong DPPH and ABTS radical scavenging activity with IC50:
13.77 0.25 M and IC50: 12.59 0.21 M, respectively. While
compound P3 exhibited as the second most potent compound
(IC50: 0.74 0.09 M) with DPPH and ABTS radical scavenging
activity IC50: 13.52 0.62 M and IC50:13.13 0.85 M, respec-
lM) with
l
l
l
l
l
tively. In this case, Q3 and P3 are the most potent compounds
bearing phenyl moiety among all the indolopyrazoline derivatives.
Besides, the above mentioned potent compounds, Q4 and P4 are
Aqueous solubility (QPlogS) of organic compounds plays a key
impact on many ADME associated properties like uptake, distribu-

S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
15
the second level compounds in the activity profile as shown in
Table 1. Interestingly, both Q4 and P4 bears chlorophenyl moiety
that establishes an interaction with the hydrophobic residue in
the active site of AChE.
Docking studies and the inhibition assay revealed the capability
of indolopyrazoline derivatives to bind the AChE and induces
strong inhibitory effect. Qikprop prediction of ADME properties
indicates that compounds are CNS active and fulfilled the Lipinski’s
rule of five. Collectively, the results propose that the indolopyrazo-
line derivatives, specifically compound Q3, P3, Q4 and P4 could be
deliberated as the potential drug candidates for AD.
Ar-H4, H5, H6, and H7), 8.01 (2H, d, J = 8.5 Hz, Ar-H300, H500), 9.04
(2H, d, J = 8.5 Hz, Ar-H200, H600), 9.53 (1H, brs, NH group of indole
ring); Anal. Calcd for C23H18N4O: C = 75.39, H = 4.95, N = 15.29.
Found: C = 75.29, H = 4.94, N = 15.27; MS (m/z, (relative abun-
dance, %)): 366 (M+), 116, 106, 78, 76, 51, 28, 27, 26; UV–vis
(MeOH) (kmax/nm): 295.
4.3.2. (3-(1H-indol-3-yl)-5-phenyl-4,5-dihydropyrazol-1-yl)
(2,4dinitrophenyl) methanone (P2)
Yield: 86%, mp: 200–202 °C, Rf (TLC): 0.62, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.16 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.54
(1H, t, J = 7.0 Hz, CH of pyrazoline), 6.22 (1H, d, J = 7.2 Hz, @CH of
indole), 6.85–7.07 (5H, m, Ar-H20, H30, H40, H50, H60), 7.16–7.48
(4H, m, Ar-H4, H5, H6, and H7), 8.56–9.25 (3H, m, Ar-H200, H500,
H600), 9.54 (1H, brs, NH group of indole ring); Anal. Calcd for
4. Experimental
4.1. General
C
23H17N5O4: C = 64.03, H = 4.01, N = 16.39. Found: C = 64.01,
H = 4.00, N = 16.37; MS (m/z, (relative abundance, %)): 427 (M+),
167, 116, 76, 51, 46, 30, 27, 26; UV–vis (MeOH) (kmax/nm): 333.
All the solvents and chemicals used were of analytical grade and
obtained from Sigma-Aldrich and Merck Pvt. Ltd., and were used
without further purification. The melting points of the compounds
were determined on a Thoshniwal electric melting point apparatus
and the values were uncorrected. The purity of all the compounds
was routinely checked by TLC on Silica gel-GF 254 (Merck) coated
plates. Spots of TLC were identified by iodine chamber. The UV–
Visible spectra of the compounds were recorded on double beam
Shimadzu UV1800 spectrophotometer. 1H NMR spectra were
recorded on Bruker UX-NMR Instrument, using TMS as an internal
standard, CDCl3 as solvent; and chemical shift values were
expressed in d ppm. Elemental analyses of synthesized compounds
were done over Perkin Elmer 240 CHN Analyzer.
4.3.3. (3-(1H-indol-3-yl)-5-phenyl-4,5-dihydropyrazol-1-yl) (phenyl)
methanone (P3):
Yield: 86%, mp: 142–144 °C, Rf (TLC): 0.58, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.14 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.52
(1H, t, J = 7.0 Hz, CH of pyrazoline), 6.24 (1H, d, J = 7.2 Hz, @CH of
indole), 6.83–7.04 (5H, m, Ar-H20, H30, H40, H50, H60), 7.15–7.46
(4H, m, Ar-H4, H5, H6, and H7), 7.7–8.01 (6H, m, Ar-H200, H300,
H400, H500, H600), 9.51 (1H, brs, NH group of indole ring); Anal. Calcd
for C24H19N3O: C = 78.88, H = 5.24, N = 11.50. Found: C = 78.84,
H = 5.22, N = 11.51; MS (m/z, (relative abundance, %)): 365 (M+),
116, 111, 76, 51, 28, 27, 26; UV–vis (MeOH) (kmax/nm): 312.
4.2. General procedure for synthesis of indolochalcones (III and V)
4.3.4. (3-(1H-indol-3-yl)-5-phenyl-4,5-dihydropyrazol-1-yl) (4-
chlorophenyl) methanone (P4)
Aldol condensation was carried out by the following method, a
solution of NaOH (8 mL, 10% in water) was added drop wise to a
well stirred solution of 0.01 M of 3-acetyl indole and 0.01 M of
benzaldehyde and p-formyl phenoxy acetic acid respectively, in
20 mL ethanol at cold temperature [23,24]. The reaction mixture
was then stirred for 24 h in ice bath. The reaction was monitored
by TLC. The mixture was then diluted with ice water and acidified
with concentrated HCl. The product was filtered and dried. The
product was recrystallized with aqueous ethanol. The purity of
the compound was checked by the Rf value of TLC and melting
point.
Yield: 86%, mp: 156–158 °C, Rf (TLC): 0.65, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.14 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.54
(1H, t, J = 7, CH of pyrazoline), 6.22 (1H, d, J = 7.2 Hz, @CH of
indole), 6.87–7.08 (5H, m, Ar-H20, H30, H40, H50, H60), 7.12–7.44
(4H, m, Ar-H4, H5, H6 and H7), 7.62 (2H, d, J = 8.5 Hz, Ar-H200,
H600), 7.91 (2H, d, J = 8.5 Hz, Ar-H300, H500), 9.51 (1H, brs, NH group
of indole ring); Anal. Calcd for C24H18ClN3O: C = 72.09, H = 4.54,
N = 10.51. Found: C = 72.11, H = 4.56, N = 10.54; MS (m/z, (relative
abundance, %)): 399 (M+), 116, 76, 51, 36, 28, 27, 26; UV–vis
(MeOH) (kmax/nm): 325.
4.3.5. 2-(4-(3-(1H-indol-3-yl)-1-isonicotinoyl-4,5-dihydro-1H-
pyrazol-5-yl) phenoxy) acetic acid (Q1)
4.3. General procedure for synthesis of indolopyrazolines (P1-P4 and
Q1-Q4)
Yield: 68%, mp: 152–154 °C, Rf (TLC): 0.54, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.14 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.54
(1H, t, J = 7.0 Hz, CH of pyrazoline), 4.89 (2H, s, OCH2), 6.21 (1H,
d, J = 7.2 Hz, @CH of indole), 6.65 (2H, d, J = 8.4 Hz, Ar-H30, H5),
6.98 (2H, d, J = 8.4 Hz, Ar-H20, H6), 7.14–7.48 (4H, m, Ar-H4, H5,
H6, and H7), 7.91 (2H, d, J = 8.5 Hz, Ar-H300, H500), 9.04 (2H, d,
J = 8.5 Hz, Ar-H200, H600), 9.51 (1H, brs, NH group of indole ring),
11.25 (1H, brs, OH carboxylic acid group); Anal. Calcd for
0.01 M of chalcone and 0.02 M of four different benzoic acid
hydrazides (isonicotino hydrazide, 2,4 dinitrophenyl hydrazine,
benzohydrazide, 4-chlorobenzohydrazide) were taken with 20 mL
glacial acetic acid and refluxed for 10 h above 130 °C separately.
The reaction mixtures were then concentrated and poured into
300 mL of ice-cold water [25,26]. The formed products were fil-
tered and dried. The products were recrystallized with aqueous
ethanol (see Schemes 1 and 2). The purity of the indolopyrazoline
compounds was checked by the Rf value of TLC and melting point.
The structures of indolopyrazolines (P1-P4 and Q1-Q4) were char-
acterized by spectral data.
C25H22N4O4: C = 67.86, H = 5.01, N = 12.66. Found: C = 67.84,
H = 5.03, N = 12.64; MS (m/z, (relative abundance, %)): 442 (M+),
151, 116, 76, 51, 44, 42, 43, 28, 26, 18, 27; UV–vis (MeOH)
(kmax/nm): 320.
4.3.6. 2-(4-(1-(2,4-dinitrobenzoyl)-3-(1H-indol-3-yl)-4,5-dihydro-
1H-pyrazol-5-yl) phenoxy) acetic acid (Q2)
4.3.1. (3-(1H-indol-3-yl)-5-phenyl-4,5-dihydropyrazol-1-yl) (pyridin-
4-yl) methanone (P1)
Yield: 78%, mp: 152–154 °C, Rf (TLC): 0.76, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.12 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.58
(1H, t, J = 7.0 Hz, CAH of pyrazoline), 4.82 (2H, s, OCH2), 6.22
(1H, d, J = 7.2 Hz, @CAH of indole), 6.63 (2H, d, J = 8.4 Hz, Ar-H30,
H50), 6.94 (2H, d, J = 8.4 Hz, Ar-H20, H60), 7.32–7.59 (4H, m,
Yield: 86%, mp: 170–172 °C, Rf (TLC): 0.57, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.12 (2H, d, J = 7, CH2 of pyrazoline), 4.56 (1H, t,
J = 7, CH of pyrazoline), 6.23 (1H, d, J = 7.2 Hz, @CH of indole),
6.84–7.06 (5H, m, Ar-H20, H30, H40, H50, H60) 7.12–7.45 (4H, m,

16
S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
Ar-H4, H5, H6, and H7), 7.96 (2H, d, J = 8.5, Ar-H300, H500), 8.98 (2H,
d, J = 8.5 Hz, Ar-H200, H600), 9.51 (1H, brs, NH group of indole ring),
11.24 (1H, brs, OH carboxylic acid group); Anal. Calcd for
mL, 25 lg/mL, 10 lg/mL concentrations) were incubated with the
ABTS+Å solution for 30 min. The absorbance was measured at
734 nm, and the % inhibition was calculated using the formula
described in Eq. (1) and the IC50 was calculated. Ascorbic acid
was used as the positive control.
C25H22N4O4: C = 59.64, H = 4.20, N = 13.91. Found: C = 59.62,
H = 4.19, N = 13.89; MS (m/z, (relative abundance, %)): 503 (M+),
151, 116, 76, 51, 46, 44, 42, 43, 30, 26, 27, 18; UV–vis (MeOH)
(kmax/nm): 302.
4.6. Anticholinesterase enzyme inhibition assay
4.3.7. 2-(4-(1-benzoyl-3-(1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-5-
yl) phenoxy) acetic acid (Q3)
The in vitro AChE inhibitory activity was measured using
the methods described earlier [31]. Briefly, stock solutions
(1 mg/mL) of test compounds were prepared using DMSO.
Yield: 58%, mp: 142–144 °C, Rf (TLC): 0.63, 1H NMR (500 MHz,
CDCl3-d): d/ppm 2.15 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.55
(1H, t, J = 7.0 Hz, CAH of pyrazoline), 4.82 (2H, s, OCH2), 6.23 (1H,
d, J = 7.2 Hz, @CAH of indole), 6.62 (2H, d, J = 8.4 Hz, Ar-H30, H50),
6.95 (2H, d, J = 8.4 Hz, Ar-H20, H60), 7.32–7.59 (4H, m, Ar-H4, H5,
H6, and H7), 7.62–7.96 (5H, m, Ar-H200, H300, H400, H500, H600), 9.54
(1H, brs, NH group of indole ring), 11.21 (1H, brs, OH carboxylic
Working solutions (0.01–100
dilutions. The various concentrations of test compounds (10
were pre-incubated with sodium phosphate buffer (0.1 M; pH
8.0; 150 L), and AChE solution (0.1 U/mL; 20 L) for 15 min at
25 °C. The reaction was initiated by addition of DTNB (10 mM;
10 L) and ATChI (14 mM; 10 L). The reaction mixture was
lg/mL) were prepared by serial
lL)
l
l
l
l
acid group); Anal. Calcd for
C26H23N3O4: C = 70.73, H = 5.25,
mixed using a cyclomixer and incubated for 10 min at room
temperature. The absorbance was measured using a microplate
reader at 410 nm wavelength against the blank reading contain-
N = 9.52. Found: C = 70.70, H = 5.23, N = 9.50; MS (m/z, (relative
abundance, %)): 441 (M+), 151, 116, 76, 51, 44, 42, 43, 30, 28, 26,
27, 18, 51; UV–vis (MeOH) (kmax/nm): 359.
ing 10
lL DMSO instead of test compound. The % inhibition
was calculated using the formula described in Eq. (1) and the
4.3.8. 2-(4-(1-(4-chlorobenzoyl)-3-(1H-indol-3-yl)-4,5-dihydro-1H-
pyrazol-5-yl) phenoxy) acetic acid (Q4)
IC50 was calculated. Donepezil (0.01–100
the positive control.
lg/mL) was used as
Yield: 72%, mp: 122–124 °C, Rf (TLC): 0.59, 1H NMR (500.1 MHz,
CDCl3-d): d/ppm 2.12 (2H, d, J = 7.0 Hz, CH2 of pyrazoline), 4.51
(1H, t, J = 7.0 Hz, CAH of pyrazoline), 4.87 (2H, s, OCH2), 6.27 (1H,
d, J = 7.2 Hz, @CAH of indole), 6.69 (2H, d, J = 8.4 Hz, Ar-H30, H50),
6.98 (2H, d, J = 8.4 Hz, Ar-H20, H60), 7.32–7.58 (4H, m, Ar-H4, H5,
H6, and H7), 7.84 (2H, d, J = 8.5, Ar-H200, H600), 7.98 (2H, d,
J = 8.5 Hz, Ar-H300, H500), 9.54 (1H, brs, NH group of indole ring),
11.24 (1H, brs, OH carboxylic acid group); Anal. Calcd for
4.7. Molecular modeling, Docking and ADME study
In order to reveal the binding modes of our eight synthesized
indolopyrazoline derivatives, docking simulation was performed
targeting the crystal structure acetyl cholinesterase (PDB ID:
1EVE) [32]. Prior to docking, the crystal structure of the AChE
enzyme was prepared using protein preparation wizard. The
crystal structure was retrieved from the protein data bank
(PDB), and the structure was optimized by removing the water
molecules, hetero atoms and co-factors. Hydrogen, missing atoms,
bonds and charges were computed through Maestro [33]. The
eight synthesized indolopyrazoline derivatives used for docking
were ligand prepared and optimized using built and LigPrep
module implemented in Schrodinger Maestro. Ligands prepara-
tion includes generating various tautomers, assigning bond
orders, ring conformations and stereo chemistries. All the
conformations generated were minimized using OPLS2005 force
field prior to docking study. Further, a receptor grid was gener-
ated around the AChE enzyme active site by choosing centroid
of AChE enzyme complexed ligand (Aricept). Grid box size was
set to 20 Å radius, using receptor grid generation implemented
in Glide [33].
Docking calculations were accomplished using Glide: a com-
plete solution for ligand-receptor docking implemented in small
molecule drug discovery suite [33] and as well following the meth-
ods suggested by Taha et al. [15]. All docking calculations were
performed using Standard Precision (SP) mode and Extra Precision
(XP) mode. The Glide docking score was used to determine the best
docked structure from the output. The interactions of these docked
complexes were further analyzed and imaged using PyMOL [34].
Additional, Qikprop prediction of ADME properties were done for
all the eight indolopyrazoline derivatives [20].
C26H22ClN3O4: C = 65.62, H = 4.66, N = 8.83. Found: C = 65.60,
H = 4.63, N = 8.82; MS (m/z, (relative abundance, %)): 475 (M+),
151, 116, 76, 44, 42, 43, 30, 28, 26, 36, 27, 18, 51; UV–vis (MeOH)
(kmax/nm): 299.
4.4. DPPH radical scavenging assay
Preparation of DPPH solution was adopted from Molyneux [27]
and Blois [28] with minor modification. All the test compounds
were dissolved in 95% ethanol. Briefly, 0.5 mL of test compounds
were added (0 - blank control, 10, 25, 50, 100, 250, 500 and
1000 lg/mL) to 0.5 mL of DPPH (2 lM in 95% ethanol) and the
mixture was incubated at room temperature for 30 min. The
absorbance was measured at 517 nm [8], and the percentage
inhibition of test compounds was calculated using the following
equation using Microsoft Excel software (version 2010). Ascorbic
acid was used as the positive control [29].
% Inhibition ¼ ð1 ꢀ absorbance sample=absorbance controlÞ ꢁ 100
ð1Þ
The IC50 (half maximal inhibitory concentration) was calculated
by constructing a non-linear regression graph between % inhibition
vs concentration, using Graph Pad prism software (version 5).
4.5. ABTS free-radical cation scavenging assay
The ABTS free radical cation scavenging ability of the synthe-
sized compounds was determined according to the procedure
described earlier [30]. ABTS was dissolved in distilled water
(7 ꢁ 10ꢀ3 M) and potassium persulphate (2.45 ꢁ 10ꢀ3 M) was
added. This reaction mixture was left overnight (12–16 h) in the
dark, at room temperature. Various concentrations of test sub-
Acknowledgment
The authors are thankful to AIMST University, Malaysia for pro-
viding the facilities for this project and the Ministry of Higher Edu-
cation, Malaysia (MOHE) for funding the computational part
(Software and Workstation) through the ‘‘TRGS” (grant no. 600-
RMI/TRGS 5/3 (1/2014)-3).
stances (1000 lg/mL 500 lg/mL, 250 lg/mL, 100 lg/mL, 50 lg/

S. Chigurupati et al. / Bioorganic Chemistry 67 (2016) 9–17
17
inhibitors of [small beta]-glucuronidase, DFT, and in silico SAR intimations,
RSC Adv. 6 (4) (2016) 3276–3289.
Appendix A. Supplementary material
[15] M. Taha, N.H. Ismail, S. Imran, M. Selvaraj, F. Rahim, Synthesis of novel
inhibitors of [small beta]-glucuronidase based on the benzothiazole skeleton
and their molecular docking studies, RSC Adv. 6 (4) (2016) 3003–3012.
[16] N.K. Zawawi, M. Taha, N. Ahmat, A. Wadood, N.H. Ismail, F. Rahim, M. Ali, N.
Abdullah, K.M. Khan, Novel 2,5-disubtituted-1,3,4-oxadiazoles with
benzimidazole backbone: a new class of beta-glucuronidase inhibitors and
in silico studies, Bioorg. Med. Chem. 23 (13) (2015) 3119–3125.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.05.
002.
[17] N.K.N.A. Zawawi, M. Taha, N. Ahmat, N.H. Ismail, A. Wadood, F. Rahim, A.U.
Rehman, Synthesis, in vitro evaluation and molecular docking studies of
References
biscoumarin thiourea as a new inhibitor of
(2015) 36–44.
[18] M. Taha, N.H. Ismail, S. Imran, M. Selvaraj, A. Rahim, M. Ali, S. Siddiqui, F.
Rahim, K.M. Khan, Synthesis of novel benzohydrazone-oxadiazole hybrids as
beta-glucuronidase inhibitors and molecular modeling studies, Bioorg. Med.
Chem. 23 (23) (2015) 7394–7404.
[19] G. Brahmachari, C. Choo, P. Ambure, K. Roy, In vitro evaluation and in silico
screening of synthetic acetylcholinesterase inhibitors bearing functionalized
piperidine pharmacophores, Bioorg. Med. Chem. 23 (15) (2015) 4567–4575.
[20] A.D.M.E. Rapid, QikProp, Schrödinger LLC, New York, 2012.
[21] R.A. Friesner, J.L. Banks, R.B. Murphy, T.A. Halgren, J.J. Klicic, D.T. Mainz, M.P.
Repasky, E.H. Knoll, M. Shelley, J.K. Perry, D.E. Shaw, P. Francis, P.S. Shenkin,
Glide: a new approach for rapid, accurate docking and scoring. 1. Method and
assessment of docking accuracy, J. Med. Chem. 47 (7) (2004) 1739–1749.
[22] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 46 (1–3) (2001) 3–
26.
[23] R. Suthakaran, G. Somasekhar, C. Sridevi, M. Marikannan, K. Suganthi, G.
Nagarajan, Synthesis, antiinflammatory, antioxidant and antibacterial
activities of 7-methoxy benzofuran pyrazoline derivatives, Asian J. Chem. 19
(5) (2007) 3353–3362.
[24] V.H. Babu, C. Sridevi, A. Joseph, K.K. Srinivasan, Synthesis and biological
evaluation of some novel pyrazolines, Indian J. Pharm. Sci. 69 (3) (2007) 470–
473.
[25] C. Sridevi, K. Balaji, A. Naidu, R. Sudhakaran, Synthesis of some phenylpyrazolo
benzimidazolo quinoxaline derivatives as potent antihistaminic agents, E – J.
Chem. 7 (1) (2010) 234–238.
a-glucosidases, Bioorg. Chem. 63
[1] M. Moniruzzaman, M. Asaduzzaman, M.S. Hossain, J. Sarker, S.M. Rahman, M.
Rashid, M.M. Rahman, In vitro antioxidant and cholinesterase inhibitory
activities of methanolic fruit extract of Phyllanthus acidus, BMC Complement.
Altern. Med. 15 (2015) 403.
[2] R.M. Lane, M. Kivipelto, N.H. Greig, Acetylcholinesterase and its inhibition in
Alzheimer disease, Clin. Neuropharmacol. 27 (3) (2004) 141–149.
[3] M. Khoobi, F. Ghanoni, H. Nadri, A. Moradi, M. Pirali Hamedani, F. Homayouni
Moghadam, S. Emami, M. Vosooghi, R. Zadmard, A. Foroumadi, A. Shafiee, New
tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: efficient
synthesis, biological assessment and docking simulation study, Eur. J. Med.
Chem. 89 (2015) 296–303.
[4] N. Mishra, D. Sasmal, Additional acetyl cholinesterase inhibitory property of
diaryl pyrazoline derivatives, Bioorg. Med. Chem. Lett. 23 (3) (2013) 702–705.
[5] M. Atanasova, G. Stavrakov, I. Philipova, D. Zheleva, N. Yordanov, I.
Doytchinova, Galantamine derivatives with indole moiety: docking, design,
synthesis and acetylcholinesterase inhibitory activity, Bioorg. Med. Chem. 23
(17) (2015) 5382–5389.
[6] C.L. Cardoso, I. Castro-Gamboa, D.H. Silva, M. Furlan, A. Epifanio Rde, C. Pinto
Ada, C. Moraes de Rezende, J.A. Lima, S. Bolzani Vda, Indole glucoalkaloids from
Chimarrhis turbinata and their evaluation as antioxidant agents and
acetylcholinesterase inhibitors, J. Nat. Prod. 67 (11) (2004) 1882–1885.
[7] L.C. Klein Jr., C.S. Passos, A.P. Moraes, V.G. Wakui, E.L. Konrath, A. Nurisso, P.A.
Carrupt, C.M. de Oliveira, L. Kato, A.T. Henriques, Indole alkaloids and
semisynthetic indole derivatives as multifunctional scaffolds aiming the
inhibition of enzymes related to neurodegenerative diseases – a focus on
Psychotria L. Genus, Curr. Top. Med. Chem. 14 (8) (2014) 1056–1075.
[8] C. Sridevi, K. Balaji, A. Naidu, R. Sudhakaran, Antioxidant and antiinflammatory
activity of some phenylpyrazolo benzimidazolo quinoxaline derivatives, J.
Pharm. Res. 2 (9) (2009) 1370–1372.
ˇ
[26] A. Voskiene, V. Mickevicius, Cyclization of chalcones to isoxazole and pyrazole
˙
derivatives, Chem. Heterocycl. Compd. 45 (12) (2010) 1485–1488.
[27] P. Molyneux, The use of the stable free radical diphenylpicrylhydrazyl (DPPH)
for estimating antioxidant activity, Songklanakarin J. Sci. Technol. 26 (2)
(2004) 211–219.
[28] M.S. Blois, Antioxidant determinations by the use of a stable free radical,
Nature 181 (4617) (1958) 1199–1200.
[29] C. Sridevi, K. Balaji, A. Naidu, R. Sudhakaran, Antioxidant, antiinflammatory
and antihistaminic activities of some phenylpyrazolo benzothiazolo
quinoxaline derivatives, Int. J. Chem. Sci. 7 (2) (2009) 1117–1126.
[30] R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans,
Antioxidant activity applying an improved ABTS radical cation decolorization
assay, Free Radical Biol. Med. 26 (9–10) (1999) 1231–1237.
[31] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Feather-Stone, A new and rapid
colorimetric determination of acetylcholinesterase activity, Biochem.
Pharmacol. 7 (1961) 88–95.
[9] A. Aliabadi, A. Foroumadi, A. Mohammadi-Farani, M. Garmsiri Mahvar,
Synthesis and evaluation of anti-acetylcholinesterase activity of 2-(2-(4-(2-
Oxo-2-phenylethyl)piperazin-1-yl) ethyl)Isoindoline-1,3-dione derivatives
with potential anti-alzheimer effects, Iran. J. Basic Med. Sci. 16 (10) (2013)
1049–1054.
[10] N. Khorana, K. Changwichit, K. Ingkaninan, M. Utsintong, Prospective
acetylcholinesterase inhibitory activity of indole and its analogs, Bioorg.
Med. Chem. Lett. 22 (8) (2012) 2885–2888.
[11] G. Ucar, N. Gokhan, A. Yesilada, A.A. Bilgin, 1-N-Substituted thiocarbamoyl-3-
phenyl-5-thienyl-2-pyrazolines:
a
novel cholinesterase and selective
monoamine oxidase inhibitors for the treatment of Parkinson’s and
B
Alzheimer’s diseases, Neurosci. Lett. 382 (3) (2005) 327–331.
[12] M. Taha, N.H. Ismail, A. Khan, S.A.A. Shah, A. Anwar, S.A. Halim, M.Q. Fatmi, S.
Imran, F. Rahim, K.M. Khan, Synthesis of novel derivatives of oxindole, their
urease inhibition and molecular docking studies, Bioorg. Med. Chem. Lett. 25
(16) (2015) 3285–3289.
[13] M. Taha, N.H. Ismail, S. Imran, A. Wadood, F. Rahim, M. Ali, A.U. Rehman, Novel
quinoline derivatives as potent in vitro [small alpha]-glucosidase inhibitors: in
silico studies and SAR predictions, MedChemComm 6 (10) (2015) 1826–1836.
[14] M. Taha, N.H. Ismail, S. Imran, E.H. Anouar, M. Ali, W. Jamil, N. Uddin, S.M.
Kashif, Identification of bisindolylmethane-hydrazone hybrids as novel
[32] G. Kryger, I. Silman, J.L. Sussman, Structure of acetylcholinesterase complexed
with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs,
Structure (London, England: 1993) 7 (3) (1999) 297–307.
[33] Schrodinger Release 2015-1: Maestro, version 10.1, Schrodinger, LLC, New
York, 2015.
[34] PyMOL Molecular Graphics System, Schrodinger LCC., NY, USA, 2010.