Environmentally benign synthesis of substituted pyrazoles as potent antioxidant agents, characterization and docking studies

Article abstract of DOI:10.1007/s13738-020-02042-6

Oxidative stress is an important cause of neurogenerative diseases; treatment with a reliable antioxidant with no side effects is a potential tool?to overcome with such diseases. The aim of this study is to explore new and effective antioxidants and their

Full text of DOI:10.1007/s13738-020-02042-6

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-020-02042-6
ORIGINAL PAPER
Environmentally benign synthesis of substituted pyrazoles as potent
antioxidant agents, characterization and docking studies
Channa Basappa Vagish1 · Achutha Dileep Kumar1 · Karthik Kumara2,5 · Hamse Kameshwar Vivek3 ·
Nagamallu Renuka4 · Neratur Krishnappagowda Lokanath2 · Kariyappa Ajay Kumar1
Received: 27 April 2020 / Accepted: 14 August 2020
© Iranian Chemical Society 2020
Abstract
Oxidative stress is an important cause of neurogenerative diseases; treatment with a reliable antioxidant with no side efects
is a potential tool to overcome with such diseases. The aim of this study is to explore new and efective antioxidants and their
mechanism; in this context, the manuscript demonstrates the greener approach for the synthesis of series of novel thiophene-
pyrazole hybrids, 5(a–m) through [3+2] annulation of chalcones, 3(a–g) and phenylhydrazine hydrochloride, 4(a–b) in
citrus extract medium in the presence of tetrabutylammonium bromide. The structures were confrmed by spectroscopic
and crystallographic studies and further screened in vitro for their free radical scavenging activities. Preliminary assessment
results show that, among the synthesized series, compounds 5c, 5d, 5f, 5i, 5j, 5l, and 5m have potent antioxidant activities.
Docking studies reveal that the ligands 5e, 5f, 5m, and 5q bind to superoxide dismutase at the Cu–Zn domain and thereby
increase its activity and reduce reactive oxygen species; therefore, these ligands might be better antioxidant molecules which
could down and regulate the oxidation stress within the body.
Keywords Annulation · Chalcones · Condensation · Hydroxyl · Radical · Scavenging
Abbreviations
TBAB
DNA
IL-6
Interleukin 6
Tetrabutylammonium bromide
Deoxyribonucleic acid
AAPH-2,2’ Azobis(2-amidinopropane) dihydrochloride
DPPH
TLC
2,2-Diphenyl-1-picrylhydrazyl
Thin-layer chromatography
Nuclear magnetic resonance
Electrospray
ROS
Reactive oxygen species
PC12
Pheochromocytoma 12 cell lines
Nuclear factor erythroid 2-related factor 2
Tumour necrosis factor alpha
NMR
ESI
Nrf2
TNF-α
APCI
UPLC
Atmospheric pressure chemical ionization
Ultimate performance liquid
chromatography
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-020-02042-6) contains
supplementary material, which is available to authorized users.
CCD
Charge couple device
CCDC
OPLS
Cambridge Crystallographic Data Centre
Optimized potentials for liquid simulations
* Kariyappa Ajay Kumar
Cu–Zn SOD Cu–Zn superoxide dismutase
ajaykumar@ycm.uni-mysore.ac.in
PDB
Protein Data Bank
1
Department of Chemistry, Yuvaraja College, University
of Mysore, Mysuru, India
2
Department of Studies in Physics, University of Mysore,
Introduction
Manasagangotri, Mysuru, India
3
Research and Innovation Center, BGSIT Campus,
Design and synthesis of novel potent molecules with antioxi-
dant efects are propelling research to prevent the deleterious
efects caused by free radicals. The synthetic and biologi-
cal potency and fexibility of pyrazole analogues have made
them popular targets. The pyrazoles were frst isolated from
watermelon seeds and are the chief secondary metabolites
Adichunchanagiri University, B.G. Nagara, Mandya, India
4
Department of Chemistry, GSSS Institute of Engineering
and Technology for Women, Mysuru, India
5
Present Address: Department of Physics, School
of Sciences, Jain University (Deemed to be University),
Bengaluru 560 011, India
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
of certain marine organisms [1]. Pyrazoles were prepared
by a protocols like the reaction of chalcones with phenylhy-
drazine in acetic acid medium under refux conditions [2],
1,3-dipolar cycloaddition of nitrile imines to alkenes [3],
reaction of 1,3-diketones with hydrazines [4], triphenylphos-
phine–potassium carbonate promoted the [3+2] annulation
to access spirocyclopentane pyrazolones with high diastere-
oselectivities [5], via Vilsmeier–Haack reaction [6], addition
of diazomethane to α, β-unsaturated divinylsulfone [7], and
by one-pot synthesis from enones, hydrazides, and halides
[8], etc.
[20]. A few pyrazole core-containing drugs with signifcant
biological activities are shown in Fig. 1 [21–23].
In sight of an outsized range of synthetic and biological
applications of pyrazoles, and also in search of new antioxi-
dants, as drug candidates, the present study demonstrates
the utility of green solvent citrus extract for the synthesis
of series of new triaryl-pyrazoles and the results of their
in vitro DPPH and hydroxyl radical scavenging activity.
Experimental
Pyrazoles are ubiquitous scafolds and thought to be
promising molecules with potential applications in medici-
nal chemistry. For instance, the molecules with pyrazole
core can protect DNA against AAPH-induced oxidation [2]
and inhibit cyclooxygenases [9], antifungal [10], antidiabetic
[11], anti-angiogenic [12], anti-convulsant [13] activities.
Synthesized series of 3,5-bis(substituted)pyrazoles show
potent antibacterial and antioxidant activities [14], pyra-
zole α-aminophosphonates inhibit acetylcholinesterase
against Alzheimer’s disease [15], curcumin pyrazole deriva-
tives alleviate oxidative stress-induced PC12 neuronal dam-
age, reduce intracellular ROS, and activate Nrf2 signalling
pathway [16], 1-(2,4-dimethoxyphenyl)-3-(1,3-diphenyl-
1H-pyrazol-4-yl)-propenones inhibit TNF-α and IL-6 [17],
pyrazole-tethered chitosan Schif bases show promising
antimicrobial activities [18], pyrazole–acetamide deriva-
tives and their cobalt and copper coordination complexes
show signifcant antioxidant activity [19], etc.
Melting points were determined by an open capillary tub-
ing method. Progress of the reactions was checked on thin-
layer chromatography (TLC) plates pre-coated with silica gel
using solvent system hexane: ethyl acetate (1:4), and spots
1
were visualized under UV light. H NMR (400 MHz) and
13C NMR (100 MHz) spectra were recorded on Agilent NMR
spectrometer. Mass spectra were obtained on ESI/APCI-
Hybrid Quadrupole, Synapt G2 HDMSACQUITY UPLC
model spectrometer, and CHN analyses with a Thermo
Finnigan Flash EA 1112 CHN analyser. The X-ray intensity
data were collected at a temperature of 293(2) K on a Bruker
Proteum2 CCD difractometer equipped with an X-ray gen-
erator operating at 45 kV and 10 mA, using CuKα radiation
of wavelength 1.54178 Å. Data were collected for 24 frames
per set with diferent settings of φ (0° and 90°), keeping the
scan width of 0.5°, exposure time of 3 s, and the sample to
detector distance of 50 mm.
The molecular manipulation of a compound continues to
be heavy line of approach for the invention of new drugs.
The manipulation might involve the eforts to bring two
or more diferent molecules with identical activity in one
molecule through synthetic means to get lead compound.
For instance, thiazolidinedione clubbed pyrazoles enhanced
antidiabetic, anti-infammatory, and antioxidant activity
compared with the parent thiazolidinedione and pyrazoles
Synthesis of chalcones, 3(a–g): To a solution mixture
of 2,4-dichlorobenzaldehyde, 1 (10 mmol) and substituted
acetophenone, 2(a–g) (10 mmol) in methyl alcohol, potas-
sium hydroxide solution (40%, 2 mL) was added. Then, the
mixture was stirred at room temperature for 2–3 h. After
the completion, the reaction mixture was poured into ice
cold water and kept in a refrigerator overnight. The sol-
ids separated were fltered, washed successively with cold
Fig. 1 Pyrazole core-containing
drugs with their biological
activities
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Journal of the Iranian Chemical Society
hydrochloric acid (5%), and cold water, and were recrystal-
lized from methyl alcohol to get the compounds 3(a–g).
Extraction of juice Orange lemons were obtained from the
locally grown lemon trees. Lemons were cut and squeezed
to get the pulp juice (100 mL) into a beaker. The pulp juice
was transferred to a jar and was diluted with water (50 mL).
The mixture was well agitated to a fne solution with the
help of mechanical stirrer. Then, the solution was warmed
for 30 min at 45–50 °C and fltered to get fne juice.
acetophenones, 2(a–g) by Claisen–Schmidt reaction. Then,
a series of novel triaryl-pyrazoles 5(a–m) were synthesized
by [3+2] annulation reaction of chalcones, 3(a–g) and phe-
nylhydrazine hydrochloride, 4(a–b) in citrus extract medium
in the presence of phase transfer catalyst TBAB under refux
conditions (Fig. 2).
Molecular docking
General procedure for the synthesis of 1,5-diaryl-3-(2,4-
dichlorophenyl)-4,5-dihydro-1H-pyrazoles, 5(a–m): To a
solution of chalcones, 3(a–g) (10 mmol) and phenylhydra-
zine hydrochloride, 4(a–b) (10 mmol) in freshly prepared
lemon juice (30 mL), tetrabutylammonium bromide (TBAB)
(0.001 mol) was added. Then, the mixture was refuxed on
a water bath for 2–3 h. The progress of the reaction was
monitored by TLC. After the completion, the reaction mix-
ture was poured into crushed ice; the solids separated were
fltered and washed successively with dilute NaHCO3 and
ice cold water. The crude solids were purifed by column
chromatography using solvent system hexane: ethylacetate
(4:1) to get target compounds 5(a–m).
The coordinates of catalase and Cu–Zn SOD were obtained
from the Brookhaven Protein Data Bank, whose PDB ids are
2CAG [24] and 1CB4 [25], respectively. Ligands 5(a–m) were
drawn using two-dimensional sketcher, and energy minimiza-
tion was computed by OPLS 2005. Proteins were prepared by
retrieving into workspace. Protein structure was corrected, by
using protein preparation wizard module to correct the miss-
ing loops and in the protein. Water molecules from proteins
were removed beyond 5 Å from the heteroatom, respectively.
Water molecules which are thought to be important in aiding
the interaction between the receptor were optimized during
protein preparation wizard. Automated necessary bonds, bond
orders, hybridization, explicit hydrogens, and charges were
assigned. OPLS 2005 force feld was applied to the protein
to restrained minimization, and RMSD of 0.30 Å was set to
In anticipation of potent antioxidants, initially, the
1-(2,4-dichlorophenyl)-3-arylprop-2-en-1-ones, 3(a–g)
were prepared from 2,4-dichlorobenzaldehyde, 1 and
Fig. 2 Schematic diagram for the synthesis of pyrazoles, 5(a–m)
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Journal of the Iranian Chemical Society
converge heavy atoms during the pre-processing of protein
before starting docking. Extra-precision docking was employed
for each compound into the receptor grid of radii 30 Å, and
the docking calculation was judged based on the docking score
and glide energy [26].
3-(Benzo[d][1,3]dioxol-5-yl)-1-(2,4-dichlorophenyl)
prop-2-en-1-one, 3g: Yield 66%; m.p. 145–147 °C. 1H NMR
(CDCl3, δ ppm): 6.062 (s, 2H, OCH2O), 6.976–7.112 (m,
3H), 7.138 (d, 1H, J=11.8 Hz, COCH=), 7.661–7.733 (m,
3H), 8.025 (d, 1H, J = 13.8 Hz, C=CH). Anal. Calcd. for
C16H10Cl2O3 (%): C, 59.84; H, 3.14; Found: C, 59.77; H,
3.06.
Results and discussion
5-(2,4-Dichlorophenyl)-1,3-diphenyl-4,5-dihydro-
1
1H-pyrazole, 5a: Yield 79%; m.p. 125–127 °C. H NMR
1-(2,4-Dichlorophenyl)-3-phenylprop-2-en-1-one, 3a:
(CDCl3, δ ppm): 3.039 (dd, 1H, J=10.7, 23.6 Hz, C4–Ha),
3.812 (dd, 1H, J = 4.6, 28.8 Hz, C4–Hb), 5.215 (dd, 1H,
J = 6.1, 19.2 Hz, C5–H), 6.710–7.006 (m, 5H, Ar–H),
7.018–7.083 (m, 6H, Ar–H); 13C NMR (CDCl3, δ ppm):
43.68 (1C, C-4), 64.20 (1C, C-5), 111.35 (1C), 114.26 (1C),
125.16 (1C), 125.73 (1C), 125.98 (1C), 126.74 (1C), 128.53
(1C), 128.79 (1C), 129.37 (1C), 129.42 (1C), 129.59 (1C),
130.06 (1C), 131.70 (1C), 134.20 (1C), 134.68 (1C), 134.90
(1C), 141.40 (1C), 142.80 (1C), 145.32 (1C, C-3). MS m/z:
368.07 (M + 2, 34), 366.07 (M + , 100); Anal. Calcd. for
C21H16Cl2N2 (%): C, 68.68; H, 4.39; N, 7.63; Found: C,
68.61; H, 4.35; N, 7.6.
1
Yield 75%; m.p. 143–145 °C. H NMR (CDCl3, δ ppm):
7.243 (d, 1H, J=10.4 Hz, COCH=), 7.427–7.516 (m, 5H,
Ar–H), 7.566–7.640 (m, 2H, Ar–H), 7.989–8.011 (m, 2H,
Ar–H), 8.148 (d, 1H, J=16.0 Hz, C=CH). Anal. Calcd. for
C15H10Cl2O (%): C, 65.01; H, 3.64; Found: C, 64.90; H,
3.58.
3-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)prop-2-en-1-
1
one, 3b: Yield 83%; m.p. 143–145 °C. H NMR (CDCl3,
δ ppm): 6.872 (s, 1H, Ar–H), 7.145 (d, 1H, J = 12.8 Hz,
COCH=), 7.213–7.436 (m, 3H, Ar–H), 7.911–7.929 (d, 2H,
Ar–H), 8.015 (d, 1H, J=15.2 Hz, C=CH). MS m/z: 316.09
(M+6, 3), 314.09 (M+4, 32), 312.13 (M+2, 95), 310.09
(M+, 100); Anal. Calcd. for C15H9Cl3O (%): C, 57.82; H,
2.91; Found: C, 57.68; H, 2.81.
1-(3-Chlorophenyl)-5-(2,4-dichlorophenyl)-3-phenyl-
4,5-dihydro-1H-pyrazole, 5b: Yield 79%; m.p. 154–155 °C.
1H NMR (CDCl3, δ ppm): 3.036 (dd, 1H, J=6.8, 16.8 Hz,
C4–Ha), 3.769 (dd, 1H, J=12.4, 17.2 Hz, C4–Hb), 5.213 (dd,
1H, J=6.4, 12.4 Hz, C5–H), 6.702–6.818 (m, 5H, Ar–H),
6.998–7.063 (m, 5H, Ar–H), 7.152–7.245 (m, 2H, Ar–H);
13C NMR (CDCl3, δ ppm): 43.66 (1C, C-4), 64.37 (1C, C-5),
111.33 (1C), 113.54 (1C), 125.26 (1C), 125.70 (1C), 126.46
(1C), 126.66 (1C), 127.94 (1C), 128.34 (1C), 128.42 (1C),
128.57 (1C), 128.68 (1C), 128.85 (1C), 129.84 (1C), 131.61
(1C), 134.72 (1C), 134.92 (1C), 141.44 (1C), 142.96 (1C),
145.26 (1C, C-3). MS m/z: 406.02 (M+6, 4), 404.02 (M+4,
32), 402.05 (M + 2, 95), 400.04 (M + , 100); Anal. Calcd.
for C21H15Cl3N2 (%): C, 62.79; H, 3.76; N, 6.97; Found: C,
62.73; H, 3.70; N, 6.90.
1-(2,4-Dichlorophenyl)-3-(p-tolyl)prop-2-en-1-one, 3c:
1
Yield 73%; m.p. 130–132 °C. H NMR (CDCl3, δ ppm):
2.435 (s, 3H, CH3), 7.241 (d, 1H, J = 9.2 Hz, COCH=),
7.268–7.312 (m, 2H), 7.425–7.505 (m, 2H, Ar–H),
7.621–7.644 (m, 1H, Ar–H), 7.910–7.930 (m, 2H, Ar–H),
8.142 (d, 1H, J = 15.2 Hz, –C=CH). Anal. Calcd. for
C16H12Cl2O (%): C, 66.00; H, 4.15; Found: C, 65.92; H,
4.07.
1-(2,4-Dichlorophenyl)-3-(4-methoxyphenyl)prop-2-en-
1-one, 3d: Yield 80%; m.p. 136–137 °C. 1H NMR (CDCl3,
δ ppm): 3.870 (s, 3H, OCH3), 6.955–6.998 (m, 2H, Ar–H),
7.225 (d, 1H, J = 16 Hz, COCH=), 7.421–7.478 (m, 2H,
Ar–H), 7.600–7.621 (m, 1H, Ar–H), 7.624–8.023 (m, 2H,
Ar–H), 8.116 (d, 1H, j=16 Hz, –C=CH). C16H12Cl2O2 (%):
C, 62.56; H, 3.94; Found: C, 62.50; H, 3.87.
3-(4-Chlorophenyl)-5-(2,4-dichlorophenyl)-1-phenyl-4,5-
dihydro-1H-pyrazole, 5c: Yield 60%; m.p. 113–115 °C. 1H
NMR (CDCl3, δ ppm): 3.088 (dd, 1H, J = 7.6, 16.80 Hz,
C4–Ha), 3.749 (dd, 1H, J=13.2, 17.0 Hz, C4–Hb), 5.190 (dd,
1H, J=6.9, 12.0 Hz, C5–H), 6.798–6.811 (m, 5H, Ar–H),
7.105–7.255 (m, 7H, Ar–H); 13C NMR (CDCl3, δ ppm):
43.70 (1C, C-4), 64.18 (1C, C-5), 111.27 (1C), 113.52 (1C),
119.10 (1C), 125.23 (1C), 125.61 (1C), 127.37 (2C), 128.56
(1C), 129.01 (1C), 129.81 (1C), 129.94 (2C), 131.54 (1C),
134.71 (1C), 135.02 (1C), 137.67 (1C), 138.46 (1C), 142.95
(1C), 145.29 (1C, C-3). MS (m/z): 406.03 (M+6, 3), 400.03
(M+4, 31), 400.03 (M+2, 95), 400.03 (M+, 100); Anal.
Calcd. for C21H15Cl3N2 (%): C, 62.79; H, 3.76; N, 6.97;
Found: C, 62.73; H, 3.71; N, 6.90.
1-(2,4-Dichlorophenyl)-3-(2-methoxyphenyl)prop-2-en-
1-one, 3e: Yield 70%; m.p. 109–111 °C. 1H NMR (CDCl3,
δ ppm): 3.810 (s, 3H, OCH3), 7.072 (d, 1H, J = 16.4 Hz,
–COCH=), 6.998–7.190 (m, 4H, Ar–H), 7.845 (d, 1H,
J = 12.0 Hz, –C=CH), 7.465–7.534 (m, 3H, Ar–H). Anal.
Calcd. for C16H12Cl2O2 (%): C, 62.56; H, 3.94; Found: C,
62.51; H, 3.85.
1-(2,4-Dichlorophenyl)-3-(3,4-dimethoxyphenyl)prop-
1
2-en-1-one, 3f: Yield 66%; m.p. 153–155 °C. H NMR
(CDCl3, δ ppm): 3.836 (s, 3H, OCH3), 3.860 (s, 3H, OCH3)
7.060–7.231 (m, 3H), 7.376 (d, 1H, J=11.6 Hz, COCH=),
7.565–6.730 (m, 3H), 8.081 (d, 1H J = 12.6 Hz, C=CH).
Anal. Calcd. for C17H14Cl2O3 (%): C, 60.55; H, 4.18; Found:
C, 60.45; H, 4.12.
1-(3-Chlorophenyl)-3-(4-chlorophenyl)-5-(2,4-dichlo-
rophenyl)-4,5-dihydro-1H-pyrazole, 5d: Yield 65%; m.p.
1
134–136 °C. H NMR (CDCl3, δ ppm): 3.044 (dd, 1H,
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Journal of the Iranian Chemical Society
J=6.8, 16.8 Hz, C4–Ha), 3.749 (dd, 1H, J=11.6, 16.8 Hz,
C4–Hb), 5.192 (dd, 1H, J=6.8, 12.0 Hz, C5–H), 6.605–6.807
(m, 4H, Ar–H), 7.015–7.249 (m, 7H, Ar–H); 13C NMR
(CDCl3, δ ppm): 43.77 (1C, C-4), 64.23 (1C, C-5), 111.32
(1C), 113.50 (1C), 119.07 (1C), 125.25 (1C), 125.60 (1C),
127.52 (2C), 128.53 (1C), 129.05 (1C), 129.83 (1C), 129.91
(2C), 131.52 (1C), 134.77 (1C), 135.10 (1C), 137.65 (1C),
138.42 (1C), 142.96 (1C), 145.32 (1C, C-3). Anal. Calcd. for
C21H14Cl4N2 (MW=434) (%): C, 57.83; H, 3.24; N, 6.42;
Found: C, 57.76; H, 3.17; N, 6.38.
127.68 (1C), 129.06 (1C), 133.31 (1C), 133.71 (1C), 139.81
(1C), 142.39 (1C), 155.55 (1C, C-3). MS (m/z): 400.04
(M+4, 12), 398.08 (M+2, 35), 396.08 (M+, 100); Anal.
Calcd. for C22H18Cl2N2O (%): C, 66.51; H, 4.57; N, 7.05;
Found: C, 66.42; H, 4.50; N, 6.95.
1-(3-Chlorophenyl)-5-(2,4-dichlorophenyl)-3-(4-meth-
oxyphenyl)-4,5-dihydro-1H-pyrazole, 5h: Yield 62%; m.p.
1
110–113 °C. H NMR (CDCl3, δ ppm): 2.976 (dd, 1H,
J = 6.8, 16.8 Hz, C4–Ha), 3.799 (s, 3H, –OCH3), 3.879
(dd, 1H, J=11.8, 17.0 Hz, C4–Hb), 5.539 (dd, 1H, J=7.0,
17.0 Hz, C5–H), 6.714–6.824 (m, 1H, Ar–H), 6.904–7.025
(m, 4H, Ar–H), 7.122–7.234 (m, 4H, Ar–H), 7.436–7.765
(m, 3H, Ar–H); 13C NMR (CDCl3, δ ppm): 37.37 (1C, C-4),
50.57 (1C, C-5), 56.09 (1C, -OCH3), 108.92 (1C), 111.35
(1C), 120.20 (1C), 120.05 (1C), 122.62 (1C), 122.82 (1C),
124.18 (1C), 124.21 (1C), 124.92 (1C), 125.06 (1C), 125.64
(1C), 126.37 (1C), 127.63 (1C), 130.26 (1C), 133.53 (1C),
134.21 (1C), 139.98 (1C), 142.53 (1C), 155.65 (1C, C-3)MS
(m/z): 436.03 (M+6, 3), 434.03 (M+4, 32), 432.04 (M+2,
98), 430.02 (M+, 100); Anal. Calcd. for C22H17Cl3N2O (%):
C, 61.20; H, 3.97; N, 6.49; Found: C, 61.12; H, 3.91; N,
6.44.
5-(2,4-Dichlorophenyl)-1-phenyl-3-(p-tolyl)-4,5-dihydro-
1
1H-pyrazole, 5e: Yield 60%; m.p. 183–185 °C. H NMR
(CDCl3, δ ppm): 2.372 (s, 1H, CH3), 3.001 (dd, 1H, J=6.0,
17.2 Hz, C4–Ha), 3.933 (dd, 1H, J=12.8, 17.2 Hz, C4–Hb),
5.527 (dd, 1H, J = 6.2, 12.0 Hz, C5–H), 6.617 (m, 1H,
Ar–H), 6.744–6.778 (m, 1H, Ar–H), 7.043–7.268 (m, 7H,
Ar–H), 7.463 (m, 1H, Ar–H), 7.593–7.806 (m, 2H, Ar–H);
13C NMR (CDCl3, δ ppm): 21.43 (1C, –CH3), 42.01 (1C,
C-4), 60.50 (1C, C-5), 110.72 (1C), 113.29 (1C), 119.05
(1C), 125.74 (1C), 125.90 (1C), 127.37 (1C), 128.0 (1C),
128.17 (1C), 129.18 (1C), 129.35 (1C), 129.82 (1C), 130.03
(1C), 132.42 (1C), 134.07 (1C), 134.97 (1C), 137.33 (1C),
139.42 (1C), 145.24 (1C), 148.31 (1C, C-3). MS (m/z):
384.03 (M+4, 10) 382.089 (M+2, 65), 380.08 (M+, 100);
Anal. Calcd. for C22H18Cl2N2 (%): C, 69.30; H, 4.76; N,
7.35; Found: C, 69.23; H, 4.70; N, 7.29.
5-(2,4-Dichlorophenyl)-3-(2-methoxyphenyl)-1-phenyl-
4,5-dihydro-1H-pyrazole, 5i: Yield 65%; m.p. 117–119 °C.
1H NMR (CDCl3, δ ppm): 3.176 (dd, 1H, J=7.2, 17.2 Hz,
C4–Ha), 3.803 (dd, 1H, J = 12.0, 16.8 Hz, C4–Hb), 3.903
(s, 3H, –OCH3), 5.403 (dd, 1H, J = 7.2, 12.0 Hz, C5–H),
6.750–6.851 (m, 6H, Ar–H), 7.055–7.481 (m, 6H, Ar–H);
13C NMR (CDCl3, δ ppm): 43.14 (1C, C-4), 55.96 (1C,
–OCH3), 59.10 (1C, C-5), 108.29 (1C), 110.64 (1C), 111.33
(1C), 113.85 (1C), 119.26 (2C), 119.39 (2C), 124.01 (1C),
124.90 (1C), 129.85 (1C), 130.34 (1C), 132.70 (1C), 134.67
(1C), 138.92 (1C), 146.24 (1C), 148.25 (1C), 149.18 (1C),
150.26 (1C, C-3). MS (m/z): 400.02 (M + 2, 16), 399.02
(52), 398.02 (M+2, 82), 396.02 (M+, 100); Anal. Calcd.
for C22H18Cl2N2O (%): C, 66.51; H, 4.57; N, 7.05; Found:
C, 66.45; H, 4.54; N, 7.00.
1-(3-Chlorophenyl)-5-(2,4-dichlorophenyl)-3-(p-tolyl)-
4,5-dihydro-1H-pyrazole, 5f: Yield 68%; m.p. 173–174 °C.
1H NMR (CDCl3, δ ppm): 2.356 (s, 3H, CH3), 2.955 (dd, 1H,
J=6.1, 17.2 Hz, C4–Ha), 3.965 (dd, 1H, J=12.8, 17.0 Hz,
C4–Hb), 5.572 (dd, 1H, J=6.0, 12.2 Hz, C5–H), 6.655–6.671
(m, 1H, Ar–H), 6.690–6.754 (m, 1H, Ar–H), 7.132–7.319
(m, 7H, Ar–H), 7.623–7.840 (m, 2H, Ar–H); 13C NMR
(CDCl3, δ ppm): 21.39 (1C, –CH3), 42.05 (1C, C-4), 61.35
(1C, C-5), 110.32 (1C), 113.33 (1C), 120.15 (1C), 125.72
(1C), 125.94 (1C), 126.97 (1C), 128.09 (1C), 128.23 (1C),
129.21 (1C), 129.23 (1C), 129.83 (1C), 131.20 (1C), 132.61
(1C), 135.02 (1C), 135.06 (1C), 137.63 (1C), 139.52 (1C),
143.20 (1C), 148.36 (1C, C-3). MS (m/z): 419.06 (M + 4,
19), 417.06 (M+2, 94), 415.05 (MH+, 100); Anal. Calcd.
for C22H17Cl3N2 (%): C, 63.56; H, 4.12; N, 6.74; Found: C,
63.48; H, 4.05; N, 6.67.
1-(3-Chlorophenyl)-5-(2,4-dichlorophenyl)-3-(2-meth-
oxyphenyl)-4,5-dihydro-1H-pyrazole, 5j: Yield 68%; m.p.
1
145–147 °C. H NMR (CDCl3, δ ppm): 3.052 (dd, 1H,
J = 5.6, 12.4 Hz, C4–Ha), 3.886 (s, 3H, –OCH3), 3.934
(dd, 1H, J=12.4, 17.2 Hz, C4–Hb), 5.565 (dd, 1H, J=5.6,
11.6 Hz, C5–H), 6.657–6.82 (m, 2H, Ar–H), 7.093–7.513
(m, 6H, Ar–H), 7.644–7.806 (m, 3H, Ar–H); 13C NMR
(CDCl3, δ ppm): 45.15 (1C, C-4), 55.41 (1C, -OCH3), 60.36
(1C, C-5), 110.75 (1C), 111.50 (1C), 113.30 (1C), 118.93
(1C), 120.89 (1C), 121.21 (1C), 127.95 (1C), 128.24 (1C),
128.85 (1C), 129.75 (1C), 130.00 (1C), 130.54 (1C), 132.43
(1C), 133.86 (1C), 134.95 (1C), 137.72 (1C), 145.37 (1C),
148.13 (1C, C-3), 157.59 (1C). MS (m/z): 436.03 (M+6, 3),
434.03 (M+4, 30), 432.03 (M+2, 95), 430.04 (M+, 100);
Anal. Calcd. for C22H17Cl3N2O (%): C, 61.20; H, 3.97; N,
6.49; Found: C, 61.12; H, 3.91; N, 6.43.
5-(2,4-Dichlorophenyl)-3-(4-methoxyphenyl)-1-phenyl-
4,5-dihydro-1H-pyrazole, 5g: Yield 65%; m.p. 123–125 °C.
1H NMR (CDCl3, δ ppm): 2.982 (dd, 1H, J=6.8, 16.8 Hz,
C4–Ha), 3.832 (s, 3H, –OCH3), 3.917 (dd, 1H, J = 12.0,
16.8 Hz, C4–Hb), 5.532 (dd, 1H, J = 7.2, 17.2 Hz, C5–H),
6.791–6.828 (m, 1H, Ar–H), 6.903–7.001 (m, 4H, Ar–H),
7.116–7.230 (m, 4H, Ar–H), 7.459–7.674 (m, 3H, Ar–H);
13C NMR (CDCl3, δ ppm): 37.26 (1C, C-4), 60.67 (1C, C-5),
56.10 (1C, -OCH3), 109.24 (1C), 111.42 (1C), 120.19 (1C),
120.40 (1C), 122.55 (1C), 122.75 (1C), 123.18 (1C), 123.71
(1C), 124.32 (1C), 124.92 (1C), 125.34 (1C), 126.22 (1C),
1 3

Journal of the Iranian Chemical Society
5-(2,4-Dichlorophenyl)-3-(3,4-dimethoxyphenyl)-1-
phenyl-4,5-dihydro-1H-pyrazole, 5k: Yield 63%; m.p.
protocol. In this context, a series of pyrazole analogues,
5(a–m) were synthesized by the reaction of chalcones,
3(a–g) and phenylhydrazine hydrochloride, 4(a–b) in citrus
extract medium supported by phase transfer catalyst. It was
observed during the course of reaction, a slight turbidity
appeared was turned to homogeneity after the addition of
tetrabutylammonium bromide as phase transfer catalyst [28].
1
154–145 °C. H NMR (CDCl3; δ ppm): 2.994 (dd, 1H,
C4–Ha, J=6.1, 16.4 Hz, C4–Ha), 3.893 (s, 3H, OCH3), 3.939
(dd, 1H, J = 6.2, 16.6 Hz, C4–Hb), 3.975 (s, 3H, OCH3),
5.515 (dd, 1H, J=6.6, 17.6 Hz, C5–H), 6.607–6.743 (d, 1H,
Ar–H), 6.745–6.834 (m, 2H Ar–H), 6.963 (d, 1H, Ar–H),
7.026–7.248 (m, 5H, Ar–H) 7.455 (s, 2H, Ar–H); 13CNMR
(CDCl3; δ ppm): 43.14 (1C, C-4), 55.93 (1C, –OCH3),
55.93 (1C, –OCH3), 59.10 (1C, C-5), 108.29 (1C), 110.62
(1C) 111.33 (1C), 113.85 (1C), 119.26 (1C), 119.39 (2C),
123.61 (1C), 125.20 (1C), 129.81 (2C), 130.34 (1C), 132.79
(1C), 134.66 (1C), 139.10 (1C), 146.32 (1C), 148.29 (1C),
149.18 (1C), 150.26 (1C, C-3). MS (m/z): 430.22 (M + 4,
11), 428.21 (M + 2, 66), 426.21 (M + , 100); Anal. Calcd.
for C23H20Cl2N2O2 (%): C, 64.65; H, 4.72; N, 6.56; Found:
C, 60.60; H, 4.57; N, 6.49.
1
The H NMR spectra of 5(a–m) did not show the doublet
appearing for alkenyl protons of 3(a–g) confrms the (3+2)
annulation to form the target compounds.
In 1H NMR spectra of compounds 5(a–m), the
–CH2– protons, C4–Ha, and C4–Hb of pyrazole are diaste-
reotopic. The C4–Ha proton appears as a doublet of doublet
at δ 2.955–3.176 (J=5.6–10.7 & 16.8–23.6 Hz) ppm; C4–Hb
proton appears as a doublet of doublet at δ 3.749–3.965
(J = 4.6–13.2, & 16.8–28.8 Hz) ppm; and C5–H appears
as a doublet of doublet at δ 5.190–5.572 (J = 5.6–7.2 &
11.6–19.2 Hz) ppm. The signals that appear in the region
δ 6.605–7.840 ppm were attributed to aromatic protons.
Other than these, 5e, 5f show singlets at δ 2.372, 2.356 ppm
for CH3; 5g, 5h, 5i, 5j show singlets at δ 3.832, 3.799,
3.903, 3.886 ppm for OCH3; 5k shows singlet at δ 3.893,
3.975 ppm for OCH3; and 5l shows a singlet at δ 5.994 ppm
for OCH2O protons.
3-(Benzo[d][1,3]dioxol-5-yl)-5-(2,4-dichlorophenyl)-
1-phenyl-4,5-dihydro-1H-pyrazole, 5l: Yield 71% yield;
m.p. 182–183 °C. 1H NMR (CDCl3, δ ppm): 2.967 (dd, 1H,
J=6.4, 17.2 Hz, C4–Ha), 3.900 (dd, 1H, J=12.0, 17.2 Hz,
C4–Hb), 5.517 (dd, 1H, J=6.4, 12.4 Hz, C5–H), 5.994 (s, 2H,
OCH2O), 6.592–6.614 (m, 1H, Ar–H), 6.742–6.805 (m, 2H,
Ar–H), 7.002–7.255 (m, 5H, Ar–H), 6.592–6.614 (m, 1H,
Ar–H), 7.465 (m, 1H, Ar–H); 13C NMR (CDCl3, δ ppm):
42.19 (1C, C-4), 60.65 (1C, C-5), 101.30 (1C, OCH2O),
111.80 (1C) 114.14 (1C), 115.08 (1C), 115.20 (1C), 119.02
(1C), 120.43 (1C), 121.73 (1C), 126.28 (1C), 127.83 (1C),
128.05 (2C), 130.17 (1C), 133.04 (1C), 143.23 (1C), 144.04
(1C), 148.66 (1C), 148.78 (1C, C-3), 150.81 (1C), 151.01
(1C). MS (m/z): 414.01 (M + 4, 11) 412.01 (M + 4, 64),
410.01 (M+, 100); Anal. Calcd. for C22H16Cl2N2O2 (%): C,
64.25; H, 3.92; N, 6.82; Found: C, 64.19; H, 3.87; N, 6.77.
3-(Benzo[d][1,3]dioxol-5-yl)-1-(3-chlorophenyl)-5-(2,4-
dichlorophenyl)-4,5-dihydro-1H-pyrazole, 5m: We have
reported the synthesis, spectroscopic and crystallographic
characterization earlier [27].
In their 13C NMR spectra, compounds 5(a–m) show
signals for C-4, C-5, and C-3 carbons at δ 37.26–45.15,
50.50–64.37, and 145.26–155.65 ppm, respectively. The
array of signal appears in the region δ 108.29–157.59 ppm
for aromatic carbons. Furthermore, 5e, 5f show signals at δ
21.43, 21.39 ppm for CH3; 5g, 5h, 5i, 5j, 5k show signals
at δ 55.41 –56.10 ppm for OCH3; and 5l shows a signal at
δ 101.30 ppm for OCH2O carbons. All compounds show a
base peak for M+ion comparable to their molecular masses,
and M+2, M+4 and M+6 isotopic peaks in their respec-
tive mass spectra, and also an agreeing match for elemental
analysis data with theoretically calculated values. Further-
more, among the series, the structure of 5f and 5m [27] was
confrmed by single-crystal X-ray difraction studies.
The compound 1-(3-chlorophenyl)-5-(2,4-
dichlorophenyl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazole 5f
(CCDC#: 1557563) was crystallized from methanol: ace-
tonitrile (7:1 v/v) to present straw crystals suitable for single-
crystal difraction (crystal size 0.35×0.27×0.24 mm) with
the following crystallographic parameters: a=8.9975(11)
Å, b = 9.4884(18) Å, c = 11.8674(16) Å, α = 84.30(2)°,
β = 80.39(2)o, γ = 86.26(2)°, V = 992.8(3) Å3, Z = 2. The
crystal structure is triclinic with a space group of P − 1.
Complete crystallographic data refnement of a compound,
5f is given in Table S1. The ORTEP of the molecule with
thermal ellipsoids drawn at 50% probability is shown in
Fig. 3.
Spectroscopic, elemental, and X-ray difraction stud-
ies provide the structure proofs of new compounds. For
1
instance, in H NMR spectra, compounds 3(a–g) show
doublets at δ 7.072–7.376 (J = 10.4–16.4 Hz) ppm and at
δ 7.845–8.148 (J=12.2–16.0 Hz) ppm for alkenyl α-proton
(–CO–CH=) and β-proton (C=CH), respectively. An array
of signals appears as multiplet at δ 6.872–8.011 ppm which
was attributed to aromatic protons. Furthermore, compound
3c shows a singlet at δ 2.435 ppm for CH3, and 3d and 3e
show a singlet at δ 3.870 ppm and δ 3.810 ppm, respec-
tively, for OCH3 protons. Compound 3f shows singlets at
δ 3.836 ppm, and δ 3.860 ppm for OCH3, and 3g shows a
singlet at δ 6.062 ppm for OCH2O protons.
Pyrazole is an important pharmacophore in the devel-
opment of antioxidants; our strategic aim was to transform
the chalcones to pyrazoles by an environmentally benign
Antioxidant activity Oxidative stress ends up in increase
in various physical and physiological disorders, and hence,
1 3

Journal of the Iranian Chemical Society
Table 1 DPPH radical scavenging activity of the compounds 5(a–m)
Compound % Radical scavenging activitya (% I)
25 (µg/mL) 50 (µg/mL) 75 (µg/mL) 100 (µg/mL)
5a
5b
5c
5d
5e
5f
12.03 0.25 15.96 0.40 19.35 0.59 22.22 0.18
11.28 0.45 15.60 0.56 19.45 0.44 22.43 0.65
20.76 0.25 27.90 0.46 37.79 0.52 45.14 0.35
23.91 0.24 26.94 0.44 35.79 0.53 46.16 0.35
10.20 0.70 13.40 0.83 15.76 0.66 18.05 0.52
15.23 0.70 18.41 0.84 23.76 0.66 26.02 0.50
9.22 0.43 11.52 0.51 13.36 0.33 16.09 0.30
10.30 0.52 13.92 0.58 17.18 0.34 20.89 0.32
15.65 0.50 17.72 0.56 22.78 0.38 25.49 0.53
22.50 0.67 27.75 0.45 35.28 0.34 42.48 0.36
10.80 0.45 12.50 0.37 15.63 0.34 17.30 0.45
15.50 0.43 18.71 0.76 22.39 0.36 25.8 0.10
20.35 0.50 29.71 0.56 36.03 0.53 44.45 0.10
15.08 0.52 16.87 0.89 21.98 0.31 24.25 0.22
5g
5h
5i
5j
5k
5l
5m
bAA
aValues are mean SD (n=3); bascorbic acid used as standard
Fig. 3 ORTEP diagram of molecule 5f with thermal ellipsoids drawn
at 50% probability
compounds 1-(3-chlorophenyl)-5-(2,4-dichlorophenyl)-3-(p-
tolyl)-4,5-dihydro-1H-pyrazole 5f, 5-(2,4-dichlorophenyl)-
3-(2-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazole 5i,
and 3-(benzo[d] [1,3] dioxol-5-yl)-5-(2,4-dichlorophenyl)-1-
phenyl-4,5-dihydro-1H-pyrazole 5l show promising activity
comparable to the respective standards. Rest of the com-
pounds of the synthesized series have shown either moder-
ate or poor radical scavenging activities. Interestingly, the
compounds 5c, 5d, 5j, and 5m show the better antioxidant
there is an urgent need for exploring a novel antioxidant
drug with specifcity for targets. Hydroxyl radical is a potent
reactive oxygen species in biological system, which reacts
with fatty acid moieties of cell membrane, and causes dam-
age to the cell leading to carcinogenesis, mutagenesis, and
cytotoxicity. In recent times, the synthesis of compounds
with antioxidant potential has increased and a vast num-
ber of structurally diverse compounds have been reported
[29]. Keeping this visible, the series of newly synthesized
pyrazoles, 5(a–m) were screened for antioxidant properties
by in vitro DPPH and hydroxyl radical scavenging activity
by Blois method [30, 31] and a known procedure [32, 33].
The experiments were conducted in triplicate; the results
were taken as a mean variance (SD) and are summarized
in Tables 1 and 2, respectively.
Table 2 Hydroxyl radical scavenging activity of the compounds 5(a–
m)
Compound % Radical scavenging activitya (% I)
25 (µg/mL) 50 (µg/mL) 75 (µg/mL) 100 (µg/mL)
5a
5b
5c
5d
5e
5f
10.03 0.27 13.96 0.42 19.35 0.57 22.22 0.19
10.23 0.26 12.69 0.60 15.32 0.26 20.22 0.29
19.46 0.64 26.23 0.52 34.89 0.26 43.56 0.33
20.46 0.64 28.63 0.45 37.77 0.91 45.07 0.25
11.23 0.45 14.19 0.24 20.36 0.53 26.01 0.25
13.45 0.32 18.21 0.41 26.34 0.36 33.08 0.56
7.50 0.54 12.30 0.56 21.27 0.31 26.57 0.42
10.67 0.39 15.30 0.23 20.72 0.18 25.46 0.60
14.75 0.36 18.54 0.18 25.94 0.46 32.38 0.53
20.55 0.66 28.41 0.17 36.34 0.36 42.80 0.36
10.55 0.28 14.34 0.47 20.08 0.37 24.48 0.17
12.53 0.25 18.83 0.40 26.40 0.27 32.64 0.37
19.53 0.52 27.83 0.40 35.40 0.27 44.84 0.46
12.02 0.05 17.95 0.12 25.58 0.20 32.03 0.32
The synthesized series of compounds 5(a–m)
showed modest to good antioxidant activities. Pre-
liminary assessment result shows that compounds
3-(4-chlorophenyl)-5-(2,4-dichlorophenyl)-1-phe-
nyl-4,5-dihydro-1H-pyrazole, 5c (20.76–45.14% and
19.46–43.56%), 1-(3-chlorophenyl)-3-(4-chlorophenyl)-
5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole, 5d
(23.91-46.16% and 20.46-45.07%), 1-(3-chlorophenyl)-5-
(2,4-dichlorophenyl)-3-(2-methoxyphenyl)-4,5-dihydro-
1H-pyrazole, 5j (22.50-42.48% and 20.55-42.80%)
and 3-(benzo[d] [1,3] dioxol-5-yl)-1-(3-chlorophenyl)-
5-(2,4-dichlorophenyl)-4,5-dihydro-1H-pyrazole, 5m
(20.35–44.45% and 19.53–44.84%) show the excellent
activities in both DPPH and hydroxyl radical scavenging
assay comparable with ascorbic acid and BHA and even with
the reported structurally related compounds [34, 35]. The
5g
5h
5i
5j
5k
5l
5m
bBHA
b
aValues are mean SD of three replicates (n=3); BHA was used as
a standard
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Journal of the Iranian Chemical Society
activities for both radical scavenging methods in compari-
son with the similar structurally related pyrazole molecules
[36, 37].
Ala1 (Fig. 9). Thus, the ligands, 5c and 5d, bind to Cu–Zn
SOD at the Cu–Zn domain and thereby increase its activity
and reduce ROS [36, 37, 39]; thus, these ligands might be
better antioxidant molecules which could downregulate the
oxidants so generated in the body. Based on the molecular
docking results, it is concluded that the ligands 5d and 5c
interact more efciently with catalase rather than Cu–Zn
SOD; these results are based on the docking score which
depict the ligand efciency to bind frmly towards the puta-
tive bind site of the targeted protein.
Molecular docking studies Molecular docking is virtual
aiding techniques which hold great promise within the feld
of drug discovery and computational biology to discover
novel ligands for receptors of a known target. Hence, we
docked all the ligands 5(a–m) to test the virtual geometrical
binding with the targeted Cu–Zn SOD and catalase enzymes
which are one among the key players in downregulating free
radicals generated during various physiological conditions
and also in certain diseases like diabetes, CVD, various
forms of cancer, etc. [38]. Active site (His48, His46, His61,
His63, and His120) of Cu–Zn SOD contains Cu–Zn domain,
binding to this domain which, in turn, increases the catalytic
activities of Cu–Zn SOD. In-silico docking details for the
compounds 5(a–m) are tabulated in Table 3.
Conclusion
To sum up, a series of novel triaryl-pyrazoles synthesized by
an environmentally benign protocol and the new compounds
were characterized by spectroscopic and crystallographic
studies. Preliminary results of biological activity screening
show that compounds 5c, 5d, 5f, 5i, 5j, 5l, and 5m possess
potent antioxidant activities in both DPPH and hydroxyl
radical scavenging assays. Molecular docking proves that
the ligands 5c, 5d, 5j, and 5m bind to SOD at the Cu–Zn
domain and thereby increase its activity and reduce ROS;
thus, these ligands could be better antioxidant molecules
which could downregulate the oxidants so generated in
the body. Further, using detailed structural modelling and
docking eforts we show possible structural and chemical
features on both the small molecules and the protein that
might contribute to the binding and inhibition. The dem-
onstrated green synthesis paves the way for future eforts at
Docking results depicted via glide dock determined the
optimal orientation of the docked inhibitor, exactly to the
active site. Ligand 5c interacting with catalase occupies the
better pose at the putative binding site (Fig. 4) with dock-
ing score of − 4.76 kcal/mol (Table 3), whereas ligand 5d
forms π–π stacking with Phe335 (Fig. 5). These results
were compared with standard ascorbic acid, which forms
salt bridge with protoporphyrin IX containing Fe (HEME)
(Fig. 6). In the case of Cu–Zn SOD, ligand 5c shows two
interactions, which are π-cation and hydrogen bonding with
Ala1 (Fig. 7). Ligand 5d forms π-cation interaction with
Ala1 (Fig. 8), whereas the standard ascorbic acid forms
hydrogen bond with Gly106, Ser109 and salt bridge with
Table 3 Docking score result of synthesized compounds 5(a–m)
Protein
Ligand
Catalase (PDB id: 2CAG)
Cu–Zn SOD (PDB id: 1CB4)
Docking
Gevdw (kcal/
Genergy (kcal/
mol)
Gemodel (kcal/
mol)
Docking
Gevdw (kcal/
Genergy (kcal/
mol)
Gemodel (kcal/
mol)
score (kcal/ mol)
mol)
score (kcal/ mol)
mol)
5a
5b
5c
5d
5e
5f
−4.23
−3.81
−4.76
−3.93
−3.87
−4.00
−4.99
−5.01
−5.65
−5.55
−4.90
−4.17
−3.76
−20.64
−20.27
−28.05
−33.57
−32.92
−37.59
−35.71
−37.92
−46.30
−40.29
−45.34
−46.10
−35.68
−39.47
−26.24
−33.62
−41.66
−39.42
−42.96
−43.18
−48.96
−46.82
−52.80
−54.38
−60.34
−54.20
−52.88
−51.65
−40.39
−0.58
−0.28
0.03
−25.39
−28.35
−30.95
−28.84
−30.19
−32.39
−32.06
−34.27
−33.46
−31.18
−31.21
−37.03
−33.98
−35.77
−24.88
−37.20
−39.74
−37.67
−40.52
−41.21
−43.04
−44.96
−43.54
−42.85
−42.95
−45.45
−43.50
−44.28
−27.76
−29.90
−34.23
−34.06
−39.25
−37.33
−37.56
−42.52
−38.16
−41.07
−43.35
−36.51
−37.94
−17.30
−29.68
−27.28
−24.44
−29.24
−29.69
−29.24
−30.39
−26.43
−30.24
−31.31
−29.51
−32.52
−15.15
1.93
−1.22
−1.35
1.69
5g
5h
5i
2.01
−1.73
−0.98
1.85
1.94
2.08
5j
5k
5l
5m
Ascorbic acid −8.16
−4.89
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Journal of the Iranian Chemical Society
Fig. 4 Molecular interactions of catalase with ligand 5c. Ligand is represented as pink transparent cloud, and catalytically amino acids within 4Å
from the binding site are represented as grey transparent cloud
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Journal of the Iranian Chemical Society
Fig. 5 Molecular interactions of catalase with ligand 5d. Ligand is represented as pink transparent cloud, and catalytically amino acids within
4Å from the binding site are represented as grey transparent cloud
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Journal of the Iranian Chemical Society
Fig. 6 Molecular interactions of catalase with standard ascorbic acid. Ascorbic acid is represented as pink transparent cloud, and catalytically
amino acids within 4Å from the binding site are represented as grey transparent cloud
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Journal of the Iranian Chemical Society
Fig. 7 Molecular interactions of Cu–Zn SOD with ligand 5c. Ligand is represented as pink transparent cloud, and catalytically amino acids
within 4Å from the binding site are represented as grey transparent cloud
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Journal of the Iranian Chemical Society
Fig. 8 Molecular interactions of Cu–Zn SOD with ligand 5d. Ligand is represented as pink transparent cloud, and catalytically amino acids
within 4Å from the binding site are represented as grey transparent cloud
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Journal of the Iranian Chemical Society
Fig. 9 Molecular interactions of Cu–Zn SOD with standard ascorbic acid. Ascorbic acid is represented as pink transparent cloud, and catalyti-
cally amino acids within 4Å from the binding site are represented as grey transparent cloud
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Journal of the Iranian Chemical Society
14. E.A. Musad, R. Mohamed, B.A. Saeed, B.S. Vishwanath, K.M.
Lokanatha Rai, Bioorg. Med. Chem. Lett. 21, 3536 (2011)
15. S. Shaikh, P. Dhavan, G. Pavale, M.M.V. Ramana, B.L. Jadhav,
Bioorg. Chem. 96, 103589 (2020)
synthesizing pyrazole analogues which may fnd widespread
applications in medicinal chemistry.
Acknowledgements The authors are grateful to the National Single
Crystal Difractometer Facility, Department of Studies in Physics, and
IOE, University of Mysore for recording, spectra and X-ray difraction
studies.
16. L. Liao, J. Shi, C. Jiang, L. Zhang, L. Feng, J. Liu, J. Zhang,
Neurochem. Int. 125, 82 (2019)
17. B.P. Bandgar, S.S. Gawande, R.G. Bodade, N.M. Gawande, C.N.
Khobragade, Bioorg. Med. Chem. 17, 8168 (2009)
18. S.M. Anush, B. Vishalakshi, B. Kalluraya, N. Manju, Int. J. Biol.
Macromol. 119, 446 (2018)
Authors’ contributions All authors are equally contributed
19. K. Chkirate, S. Fettach, K. Karrouchi, N.K. Sebbar, E.M. Essassi,
J.T. Mague, S. Radi, M.E.A. Faouzi, N.N. Adarsh, Y. Garcia, J.
Inorg. Biochem. 191, 21 (2019)
Funding The authors declare that no funding was received from any
of the funding agencies for this work.
20. G. Bansal, S. Singh, V. Monga, P.V. Thanikachalam, P. Chawla,
Bioorg. Chem. 92, 103271 (2019)
Availability of data and material Parameters in CIF format are avail-
able as Electronic Supplementary Publication from Cambridge Crystal-
lographic Data Centre.
21. K. Vinod, K. Kamalneet, K.G. Girish, K.S. Anil, Eur. J. Med.
Chem. 69, 735 (2013)
22. M. Kishor, R.M. Anthony, T. Loic, S. Michael, J.V. Jean, Angew.
Chem. Int. Ed. 49, 3196 (2010)
Compliance with ethical standards
23. U. Naoto, H. Shigematsu, T. Akihisa, R. Eyanagi, S. Kitamura, O.
Shigeru, J. Med. Chem. 53, 8727 (2010)
24. P. Gouet, H.M. Jouve, P.A. Williams, I. Andersson, P. Andreoletti,
L. Nussaume, J. Hajdu. Nat. Struct. Biol. 3, 951 (1996)
25. M.A. Hough, S.S. Hasnain, J. Mol. Biol. 287, 579 (1999)
26. V.H. Kameshwar, J.R. Kumar, B.S. Priya, S.N. Swamy, Mol. Cell.
Biochem. 426, 161 (2016)
Conflict of interest All authors declare no confict of interest including
fnancial, personal, or other relationships with other people or organi-
zations for this article.
Code availability Not applicable.
27. S. Naveen, K. Kumara, A.D. Kumar, K.A. Kumar, N.K. Lokanath,
Chem. Data Collect. 15–16, 89 (2018)
28. M.G. Prabhudeva, S. Bharath, A.D. Kumar, S. Naveen, N.K.
Lokanath, B.N. Mylarappa, K.A. Kumar, Bioorg. Chem. 73, 109
(2017)
References
29. V.L.M. Silva, J. Elguero, A.M.S. Silva, Eur. J. Med. Chem. 156,
394 (2018)
1. P.S. Parameswaran, C.G. Naik, R.H. Vinod, J. Nat. Prod. 60, 802
(1997)
30. M.S. Blois, Nature 181, 1199 (1958)
2. Y.-F. Li, Z.-Q. Liu, Free Rad. Biol. Med. 52, 103 (2012)
3. J. Prabhashankar, V.K. Govindappa, A.K. Kariyappa, Turk. J.
Chem. 37, 853 (2013)
31. J. Jayabharathi, V. Thanikachalam, N. Rajendraprasath, K. Sara-
vanan, M.V. Perumal, Med. Chem. Res. 21, 1850 (2012)
32. E. Kunchandy, M.N.A. Rao, Int. J. Pharm. 58, 237 (1990)
33. N. Renuka, H.K. Vivek, G. Pavithra, K. Ajay Kumar, Russ. J.
Bioorg. Chem. 43, 197 (2017)
4. H. Hunan, G. Changhua, D. Lisheng, Z. Anjiiang, Molecules 15,
7472 (2010)
5. W. Luo, B. Shao, J. Li, D. Song, X. Yi, F. Ling, W. Zhong, Tetra-
hedron Lett. 60, 151206 (2019)
34. O.S. Aff, O.G. Shaaban, H.A. Abd El Razik, S.E.-D.A. Shams
El-Dine, F.A. Ashour, A.A. El-Tombary, M.M. Abu-Serie, Bioorg.
Chem. 87, 821 (2019)
6. R. Nagamallu, A.K. Kariyappa, Bioorg. Med. Chem. Lett. 23,
6406 (2013)
35. J. Rangaswamy, H.V. Kumar, S.T. Harini, N. Naik, Bioorg. Med.
Chem. Lett. 22, 4773 (2012)
7. P.C. Hiberty, Y.S. Jean, J. Am. Chem. Soc. 101, 2538 (1979)
8. T. Meng, Z. Fu-Min, Tetrahedron 69, 1427 (2013)
9. R. Sribalan, G. Banupriya, M. Kirubavathi, A. Jayachitra, V. Pad-
mini, Bioorg. Med. Chem. Lett. 26, 5624 (2016)
10. Z. Jin, T. Da-Jin, W. Tao, J.Y.K. Si-Si, Z. Zun-Ting, J. Mol. Struct.
1149, 235 (2017)
36. E.M. Flefel, W.I. El-Sofany, M. El-Shahat, A. Naqvi, E. Assirey,
Molecules 23, 2548 (2018)
37. S.A. Ali, S.M. Awad, A.M. Said, S. Mahgoub, H. Taha, N.M.
Ahmed, J. Enz. Inhibit. Med. Chem. 35, 847 (2020)
38. V.K. Shalini, L. Srinivas, Mol. Cell. Biochem. 77, 3 (1987)
39. K.R. Raghavendra, N. Renuka, V.H. Kameshwar, B. Srinivasan,
K. Ajay Kumar, S. Shashikanth, Bioorg. Med. Chem. Lett. 26,
3621 (2016)
11. O. Zuhal, K.H. Burak, G.X. Bulent, C. Unsal, B.A. Altan, Eur. J.
Med. Chem. 42, 373 (2007)
12. A.K. Dileep, S. Bharath, N.D. Rekha, S. Naveen, N.K. Lokanath,
A.K. Kariyappa, Res. Chem. Intermed. 44, 5635 (2018)
13. R. Alam, D. Wahi, R. Singh, D. Sinha, V. Tandon, A. Grover,
Bioorg. Chem. 69, 77 (2016)
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