Synthesis, biological evaluation and molecular docking studies of N-acylheteroaryl hydrazone derivatives as antioxidant and anti-inflammatory agents

Article abstract of DOI:10.1007/s11164-015-2176-1

In search of new therapeutics with greater potency, three new series of 3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide derivatives have been synthesized and evaluated for their in vitro antioxidant and anti-inflammatory activities. The hydrazones bearing a core pyrazole, chromone and tetrazolo[1,5-a]quinoline scaffold showed promising activities. Interestingly, compounds 3a (EC₅₀ = 06.00 ± 2.36) and 5c (EC₅₀ = 07.21 ± 0.67) showed the most potent antioxidant activity, while compounds 3a (EC₅₀ = 10.25 ± 1.08), 7b (EC₅₀ = 10.50 ± 0.99) and 7c (EC₅₀ = 11.18 ± 0.15) showed significant anti-inflammatory activity. Furthermore, molecular docking studies also revealed a significant correlation between the binding score and biological activity for these compounds to describe the molecular basis for the structure activity relationship (SAR) results. As these compounds are good cyclooxygenase inhibitors, isoenzyme inhibitory potency studies are warranted.

Full text of DOI:10.1007/s11164-015-2176-1

Res Chem Intermed
DOI 10.1007/s11164-015-2176-1
Synthesis, biological evaluation and molecular docking
studies of N-acylheteroaryl hydrazone derivatives
as antioxidant and anti-inflammatory agents
1
1
•
Pravin S. Mahajan
Vijay M. Khedkar
•
Mukesh D. Nikam
3
•
2
•
Prakash C. Jha
Charansingh H. Gill
2
1
Dhiman Sarkar
•
Received: 15 April 2015 / Accepted: 13 July 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract In search of new therapeutics with greater potency, three new series of
-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide derivatives have
3
been synthesized and evaluated for their in vitro antioxidant and anti-inflammatory
activities. The hydrazones bearing a core pyrazole, chromone and tetrazolo[1,5-
a]quinoline scaffold showed promising activities. Interestingly, compounds 3a
(
EC50 = 06.00 ± 2.36) and 5c (EC50 = 07.21 ± 0.67) showed the most potent
antioxidant activity, while compounds 3a (EC50 = 10.25 ± 1.08), 7b (EC50
0.50 ± 0.99) and 7c (EC50 = 11.18 ± 0.15) showed significant anti-inflammatory
=
1
activity. Furthermore, molecular docking studies also revealed a significant corre-
lation between the binding score and biological activity for these compounds to
describe the molecular basis for the structure activity relationship (SAR) results. As
these compounds are good cyclooxygenase inhibitors, isoenzyme inhibitory potency
studies are warranted.
Keywords N-Acylhydrazones Á Anti-inflammatory Á Antioxidant Á Docking study Á
Structure–activity relationship
&
Charansingh H. Gill
chgill16@gmail.com
1
Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad,
Maharashtra 431 004, India
2
3
Combi-Chem Resource Centre, CSIR-National Chemical Laboratory, Pune 411008, India
School of Chemical Sciences, Central University of Gujarat, Sector-30,
Gandhinagar 38200, Gujarat, India
123

P. S. Mahajan et al.
Introduction
The main aim of therapeutics is to relieve pain and minimize side effects. In this
respect, antioxidants are important to maintain health and to cure diseases [1]. In
various pathophysiological conditions, excessive generation of reactive oxygen
species (ROSs) results in oxidative stress. Antioxidants attract much attention as
aging, cancer, atherosclerosis, and some other serious diseases have been confirmed
to correlate with low density lipoprotein (LDL), cell membranes, and DNA exposed
to oxidative stress [2, 3].
Nonsteroidal anti-inflammatory drugs (NSAIDs) relieve pain and inflammation
[
4] by inhibiting the enzyme prostaglandin endoperoxidase, popularly known as
cyclooxygenase-2 (COX-2) [5], thereby blocking the biosynthesis of prostaglandin
6]. It is well known that NSAIDs are associated with several side effects such as
[
gastrointestinal mucosal damage, bleeding, intolerance, renal toxicity [7] and
dyspepsia [8]. Several attempts were made to improve the safety of existing
NSAIDs, including the use of coating or encapsulation, and the use of rectal or
topical formulations [9–11], but an ideal solution to the problem was not achieved.
Therefore, current research focuses on production of new, improved non-opioid
analgesics similar to opioids but without their side effects, and with improved
activity.
ROSs produced by phagocytic leukocytes (e.g., neutrophils, monocytes,
macrophages, eosinophils) during the inflammatory process invade tissues [12].
Neutrophils play a key role in inflammatory response and also generate a toxic
oxygen metabolite including ROSs by various pathways [13, 14] and it is well
known that free radicals play an important role in inflammatory action [15]. Thus,
the agents that can scavenge these ROSs can be of assistance in the treatment of
inflammatory disorders.
N-acylhydrazones (NAHs) are stable imine equivalents which provides a suitable
template for the chelation of Lewis acids [16], and are expanded to the chemical and
pharmaceutical industries [17]. The pyrazole ring is a predominant pharmacophore
in many drugs such as celecoxib, pyrazofurin and, notably, rimonabant, having a
pyrazole core with N-acylhydrazone. The bioactive system containing thieno[2,3-
c]pyrazole derivatives are a class of fused heterocyclic compounds which have
attracted interest in medicinal chemistry owing to their antitumor [18–22], antiviral
[23], anti-inflammatory [24] activities. Several other compounds containing the
thieno[2,3-c]pyrazole scaffold were described as inhibitory for phosphodiesterase
7
A (PDE7A) [25] and as potassium channel inhibitors [26].
A literature survey reveals that some pharmacophores like chromones and their
hydrazone derivatives possess potential antioxidant and anti-inflammatory activities
27, 28]. The tetrazolo[1,5-a]quinoline nucleus with hydrazones [29] and a few
[
pyrazole carbohydrazide hydrazone derivatives serve as new scaffolds for anti-
inflammatory activity [30–32]. However, there have been no reports on the
synthesis and biological evaluation of 3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-
5
-carbohydrazide hydrazone derivatives.
1
23

Synthesis, biological evaluation and molecular docking…
With the hope of greater potency, additional pharmacokinetic advantages and our
continuous attention towards synthesis of biologically active heterocyclic com-
pounds [33–35], we thought of preparing new hydrazone derivatives in order to
screen them for antioxidant and anti-inflammatory activities. Anti-inflammatory
activities shown by the compounds investigated herein motivated us to evaluate
whether they are a good ligand to the known target protein related to inflammation,
i.e., cyclooxygenase-2. We have tried to understand the interactions involved in the
binding of these compounds to cyclooxygenase-2 via computational docking
methods for gaining insights into the experimentally observed inhibition pattern.
Experimental
Materials and methods
The melting points of all synthesized compounds were determined by an open
capillary method and were uncorrected. All chemicals were analytical grade and
used without further purification. Precoated plates (silica gel 60 F254) were used for
analytical thin-layer chromatography (TLC) for monitoring the reaction progress,
and spots were visualized with ultraviolet (UV) light and iodine. Infrared (IR)
spectra (KBr disc) were recorded on a Jasco Fourier transform-infrared (FTIR-4100)
1
spectrometer (Japan). Proton nuclear magnetic resonance ( H NMR) spectra were
recorded on a Bruker Advance II 400 MHz spectrometer in deuterated dimethyl-
silane (DMSO-d ; d in ppm relative to tetramethylsilane). Carbon nuclear magnetic
6
1
resonance ( C NMR) spectra were recorded on a Bruker Advance II 100 MHz
3
spectrometer in DMSO-d . Mass spectra were recorded on a Q-Tof micro mass
6
spectrometer (Waters Corporation) by the electro-spray (ES) method. Elemental
analysis was performed on Perkin-Elmer EAL-240 elemental analyzer.
Substituted tetrazolo[1,5-a]quinoline-4-carbaldehyde (2a–2g), 1,3-diphenyl-1H-
pyrazole-4-carbaldehyde (4a–h) and 3-formylchromone (6a–d) were prepared in
accordance with Refs. [8, 36, 37], respectively.
General procedure for the synthesis of hydrazones (3a–g)
The carbohydrazide 1 (1 mmol) and substituted tetrazolo[1,5-a]quinoline-4-car-
baldehyde (2a-g) (1 mmol) were added in methanol with a few drops of acetic acid
with stirring for 15-30 min. (monitored by TLC) at room temperature. After
completion of the reaction, the reaction mixture was poured on crushed ice. The
resulting crude product was purified by column chromatography on silica gel by
EtOAc:petroleum ether (9:1) as an eluent to afford the pure title compounds 3a–g.
0
(
E)-N -((7-ethoxytetrazolo[1,5-a]quinolin-4-yl)methylene)-3-methyl-1-phenyl-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (3a) Yellow solid, m.p. 240–242 °C,
Yield: 75 %. IR (KBr, cm ): 3444 (NH), 3063, 2927 (Ar–CH), 1636 (amide C=O),
-
1
1
1
614 (C=N). H NMR (400 MHz, DMSO-d , d, ppm): 1.32 (t, 3H, CH ), 2.54 (s,
6
3
3
H, CH ), 4.06 (q, 2H, OCH ), 7.12 (s, 1H), 7.21-7.78 (m, 5H, Ar–H), 7.54 (d, 1H,
3
2
123

P. S. Mahajan et al.
Ar–H), 8.12 (s, 1H, thiophene-H), 8.38 (d, 1H), 8.58 (s, 1H), 9.02 (s. 1H, CH=N),
1
3
1
1
1
1
1.89 (s, 1H, NH). C NMR (100 MHz, DMSO-d , d, ppm): 12.90, 14.22, 61.63,
6
07.13, 116.20, 116.50, 123.51, 124.21, 125.12, 129.60, 130.40, 130.72, 131.13,
32.90, 135.25, 137.29, 138.11, 141.05, 142.91, 144.13, 147.89, 149.01, 159.41,
?
62.50. High resolution mass spectroscopy (HRMS) (ESI?) m/z: (M ? H) calcd.
for C H N O S 497.1504, found 497.1488. Anal. calcd. for C H N O S: C,
25 20 8 2
2
5
20
8
2
6
0.47; H, 4.06; N, 22.57; S, 6.46; found: C, 60.22; H, 3.90; N, 22.87; S, 6.28.
0
(
E)-3-methyl-1-phenyl-N -(tetrazolo[1,5-a]quinolin-4-ylmethylene)-1H-thieno[2,3-
c]pyrazole-5-carbohydrazide (3b) Yellow solid, m.p. 261–264 °C, Yield: 77 %.
IR (KBr, cm ): 3446 (NH), 3076, 2929 (Ar–CH), 1630 (amide C=O), 1606 (C=N).
-
1
1
H NMR (400 MHz, DMSO-d , d, ppm): 2.51 (s, 3H, CH ), 7.07–7.80 (m, 5H, Ar–
6
3
H), 8.01 (dd, 1H), 8.07 (dd, 1H), 8.16 (s, 1H, thiophene-H), 8.26 (d, 1H), 8.46 (d,
1
1
H), 8.51 (s, 1H), 9.00 (s. 1H, CH=N), 11.91 (s, 1H, NH). C NMR (100 MHz,
3
DMSO-d , d, ppm): 12.96, 116.22, 116.60, 116.91, 123.39, 125.05, 125.08, 127.51,
6
1
1
4
2
28.03, 129.06, 129.09, 130.08, 133.31, 135.08, 138.70, 141.42, 143.63, 146.01,
?
47.07, 151.31, 161.90. HRMS (ESI?) m/z: (M ? H) calcd. for C H N OS
2
3 16 8
53.1619, found 453.1616. Anal. calcd. for C H N OS: C, 61.05; H, 3.56; N,
3 16 8
2
4.76; S, 7.09; found: C, 61.10; H, 3.72; N, 24.61; S, 7.13.
0
(
E)-N -((8-methoxytetrazolo[1,5-a]quinolin-4-yl)methylene)-3-methyl-1-phenyl-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (3c) Yellow solid, m.p. 242–244 °C,
Yield: 72 %. IR (KBr, cm ): 3458 (NH), 3066, 2921 (Ar–CH), 1630 (amide
-
1
1
C=O), 1612 (C=N). H NMR (400 MHz, DMSO-d , d, ppm): 2.56 (s, 3H, CH ),
6
3
3
H), 8.18 (d, 1H), 8.55 (s, 1H), 9.02 (s. 1H, CH=N), 11.91 (s, 1H, NH). C NMR
.74 (s, 3H, OCH ), 7.22-7.84 (m, 5H, Ar–H), 7.30 (s, 1H, Ar–H), 7.42 (d, 1H, Ar–
3
1
3
(
100 MHz, DMSO-d , d, ppm): 13.00, 55.61, 109.09, 116.25, 116.91, 119.21,
6
1
1
22.41, 125.92, 126.11, 129.88, 130.39, 131.10, 135.58, 137.61, 138.02, 141.89,
?
43.18, 144.51, 150.02, 152.08, 158.61, 163.11. HRMS (ESI?) m/z: (M ? H)
calcd for C H N OS 483.1347, found 483.1327. Anal. calcd. for C H N OS: C,
24 18 8
2
4
18
8
5
9.74; H, 3.76; N, 23.22; S, 6.65; found: C, 59.63; H, 3.80; N, 23.48; S, 6.87.
0
(
E)-3-methyl-N -((7-methyltetrazolo[1,5-a]quinolin-4-yl)methylene)-1-phenyl-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (3d) Yellow solid, m.p. 206–208 °C,
Yield: 79 %. IR (KBr, cm ): 3463 (NH), 3084, 2928 (Ar–CH), 1646 (amide C=O),
-
1
1
1
610 (C=N). H NMR (400 MHz, DMSO-d , d, ppm): 2.34 (s, 3H, CH ), 2.48 (s,
6
3
3
H, CH ), 7.21–7.74 (m, 5H, Ar–H), 7.66 (s, 1H, Ar–H), 7.98 (d, 1H, Ar–H), 8.12
3
(
s, 1H, thiophene-H), 8.40 (d, 1H), 8.58 (s, 1H), 9.00 (s. 1H, CH=N), 11.91 (s, 1H,
1
3
NH). C NMR (100 MHz, DMSO-d , d, ppm): 13.02, 21.78, 116.08, 116.71,
6
1
25.09, 125.28, 127.19, 128.51, 129.38, 129.91, 130.88, 132.49, 135.41, 136.53,
38.02, 138.79, 141.58, 143.92, 144.73, 148.77, 150.78, 162.29. HRMS (ESI?) m/z:
1
?
M ? H) calcd. for C H N OS 467.1398, found 467.1381. Anal. calcd. for
(
C H N OS: C, 61.79; H, 3.89; N, 24.02; S, 6.87; found: C, 61.95; H, 3.91; N,
2
4 18 8
2
4 18 8
2
3.90; S, 6.94.
0
(
E)-3-methyl-N -((8-methyltetrazolo[1,5-a]quinolin-4-yl)methylene)-1-phenyl-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (3e) Yellow solid, m.p. 224–226 °C,
1
23

Synthesis, biological evaluation and molecular docking…
-
Yield: 75 %. IR (KBr, cm ): 3448 (NH), 3078, 2928 (Ar–CH), 1638 (amide C=O),
1
1
1
611 (C=N). H NMR (400 MHz, DMSO-d , d, ppm): 2.34 (s, 3H, CH ), 2.48 (s,
6
3
3
H, CH ), 7.19–7.74 (m, 5H, Ar–H), 7.82 (d, 1H, Ar–H), 7.99 (dd, 1H, Ar–H), 8.10
3
(
s, 1H, thiophene-H), 8.22 (d, 1H), 8.57 (s, 1H), 9.01 (s. 1H, CH=N), 11.90 (s, 1H,
1
3
NH). C NMR (100 MHz, DMSO-d , d, ppm): 13.00, 21.78, 115.91, 116.88,
6
1
25.21, 125.42, 127.01, 128.77, 129.19, 130.03, 130.81, 132.09, 135.48, 136.69,
38.31, 138.61, 141.92, 143.59, 144.92, 149.01, 151.00, 161.99. HRMS (ESI?) m/z:
1
?
M ? H) calcd. for C H N OS 467.1398, found 467.1381. Anal. calcd. for
(
C H N OS: C, 61.79; H, 3.89; N, 24.02; S, 6.87; found: C, 61.95; H, 3.91; N,
2
4 18 8
2
4 18 8
2
3.90; S, 6.94.
0
E)-3-methyl-N -((9-methyltetrazolo[1,5-a]quinolin-4-yl)methylene)-1-phenyl-1H-
(
thieno[2,3-c]pyrazole-5-carbohydrazide (3f) Yellow solid, m.p. 230–232 °C,
Yield: 78 %. IR (KBr, cm ): 3450 (NH), 1633 (amide C=O), 1611 (C=N). H
-
1
1
NMR (400 MHz, DMSO-d , d, ppm): 2.32 (s, 3H, CH ), 2.51 (s, 3H, CH ),
6
3
3
7
.24–7.74 (m, 5H, Ar–H), 7.90 (dd, 1H, Ar–H), 8.01 (s, 1H, thiophene-H), 8.07 (d,
1
H), 8.14 (d, 1H), 8.55 (s, 1H), 8.97 (s. 1H, CH=N), 11.92 (s, 1H, NH). C NMR
3
1
(
100 MHz, DMSO-d , d, ppm): 13.02, 21.79, 116.03, 117.08, 125.29, 125.71,
6
1
27.00, 128.57, 129.11, 129.67, 131.10, 132.19, 135.58, 136.13, 138.22, 138.90,
?
1
calcd. for C H N OS 467.1398, found 467.1381. Anal. calcd. for C H N OS: C,
42.00, 143.52, 144.08, 148.88, 150.91, 162.08. HRMS (ESI?) m/z: (M ? H)
2
4
18
8
24 18 8
6
1.79; H, 3.89; N, 24.02; S, 6.87; found: C, 61.95; H, 3.91; N, 23.90; S, 6.94.
0
(
E)-N -((7-methoxytetrazolo[1,5-a]quinolin-4-yl)methylene)-3-methyl-1-phenyl-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (3g) Yellow solid, m.p. 180–182 °C,
Yield: 67 %. IR (KBr, cm ): 3456 (NH), 3081, 2929 (Ar–CH), 1635 (amide
-
1
1
C=O), 1611 (C=N). H NMR (400 MHz, DMSO-d , d, ppm): 2.56 (s, 3H, CH ),
6
3
3
H), 8.14 (s, 1H, thiophene-H), 8.35 (d, 1H), 8.60 (s, 1H), 9.02 (s. 1H, CH=N), 11.91
.84 (s, 3H, OCH ), 7.16 (s, 1H, Ar–H), 7.25–7.84 (m, 5H, Ar–H), 7.44 (d, 1H, Ar–
3
1
3
(
s, 1H, NH). C NMR (100 MHz, DMSO-d , d, ppm): 13.01, 55.85, 108.91,
6
1
16.42, 117.13, 119.02, 122.08, 125.82, 126.29, 130.01, 130.60, 131.32, 135.51,
37.91, 138.23, 142.15, 143.44, 144.41, 150.19, 152.20, 158.69, 163.02. HRMS
1
?
ESI?) m/z: (M ? H) calcd. for C H N O S 483.1347, found 483.1327. Anal.
(
calcd. for C H N O S: C, 59.74; H, 3.76; N, 23.22; S, 6.65; found: C, 59.63; H,
2
5 20 4 3
2
5 20 4 3
3
.80; N, 23.48; S, 6.87.
General procedure for the synthesis of hydrazones (5a–h)
To a stirred solution of carbohydrazide 1 (1 mmol) in methanol, was added an
equimolar amount of the appropriate 1,3-diphenyl-1H-pyrazole-4-carbaldehyde
derivatives (4a–h) with a few drops of acetic acid. The reaction mixture was stirred
at room temperature, until TLC indicated the end of reaction (15–30 min). After
completion of the reaction, the reaction mixture was poured on crushed ice. The
resulting crude product was purified by column chromatography on silica gel by
EtOAc:petroleum ether (8:2) as an eluent to afford the pure title compounds 5a–h.
123

P. S. Mahajan et al.
0
E)-N -((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phenyl-1H-thieno[2,3-
(
c]pyrazole-5-carbohydrazide (5a) Yellow solid, m.p. 210–212 °C, Yield: 65 %.
IR (KBr, cm ): 3260 (NH), 2924 (Aromatic C–H), 1650 (C=O), 1608 (C=N). H
-
1
1
NMR (400 MHz, DMSO-d , d, ppm): 2.48 (s, 3H, CH ), 7.17–7.72 (m, 15H, Ar–H),
6
3
8
.18 (s, 1H, thiophene-H), 8.38 (s, 1H), 9.36 (s, 1H, CH=N), 11.35 (brs, 1H, NH).
C NMR (100 MHz, DMSO-d , d, ppm): 12.98, 114.60, 116.64, 116.89, 120.70,
1
3
6
1
1
25.52, 127.80, 128.38, 129.01, 129.77, 129.95, 130.30, 130.85, 131.10, 132.83,
35.83, 138.41, 138.67, 141.43, 143.62, 144.50, 152.21, 163.05. HRMS (ESI?) m/z:
?
M ? H) calcd. for C H N OS 503.1652, found 503.1656. Anal. calcd. for
(
C H N OS: C, 69.30; H, 4.41; N, 16.72; S, 6.38; found: C, 69.52; H, 4.44; N,
2
9 22 6
2
9 22 6
1
6.51; S, 6.08.
0
(E)-3-methyl-N -((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-1-phe-
nyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide (5b) Yellow solid, m.p.
-
1
2
1
5
83–285 °C, Yield: 72 %. IR (KBr, cm ): 3258 (NH), 2924 (Aromatic C–H),
650 (C=O), 1605 (C=N), 1526 (NO , anti), 1507, 1393 (NO , sym), 1252, 748,
1
09. H NMR (400 MHz, DMSO-d , d, ppm): 2.39 (s, 3H, CH ), 7.18-7.22(dd, 2H,
2
2
6
3
Ar–H), 7.46–7.84 (m, 10H, Ar–H), 8.02–8.05 (dd, 2H, Ar–H), 8.22 (s, 1H,
1
thiophene-H), 8.35 (s, 1H), 9.41 (s, 1H, CH=N), 11.45 (brs, 1H, NH). C NMR
3
(
100 MHz, DMSO-d , d, ppm): 12.99, 114.70, 116.61, 116.93, 120.89, 125.52,
6
1
1
25.88, 126.80, 127.89, 129.94, 130.09, 130.88, 131.28, 135.81, 138.18, 138.73,
40.21, 141.44, 143.62, 144.40, 146.11, 152.29, 163.01. HRMS (ESI?) m/z:
?
M ? H) calcd. for C H N O S 548.1503, found 548.1509. Anal. calcd. for
(
C H N O S: C, 63.61; H, 3.87; N, 17.91; S, 5.86; found: C, 63.33; H, 4.01; N,
2
9 21 7 3
2
9 21 7 3
1
8.16; S, 5.50.
0
(E)-N -((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-
phenyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide (5c) Yellow solid, m.p.
-
1
2
46–248 °C, Yield: 59 %. IR (KBr, cm ): 3246 (NH), 3043, 2929 (Ar–CH),
1
646 (amide C=O), 1599, 1504, 1383, 1338, 858, 743, 665. H NMR (400 MHz,
1
DMSO-d , d, ppm): 2.51 (s, 3H, CH ), 3.78 (s, 3H, OCH ), 7.04 (d, 2H, Ar–H), 7.20
6
3
3
(
d, 2H, Ar–H), 7.40–7.81 (m, 10H, Ar–H), 8.21 (s, 1H, thiophene-H), 8.44 (s, 1H),
1
3
9
1
1
1
.20 (s, 1H, CH=N), 11.78 (brs, 1H, NH). C NMR (100 MHz, DMSO-d , d, ppm):
6
3.00, 60.47, 114.28, 115.30, 116.61, 116.92, 120.41, 125.55, 126.09, 127.70,
29.23, 129.95, 130.19, 130.84, 131.02, 135.82, 138.30, 138.71, 141.42, 143.62,
?
44.38, 152.20, 162.09, 163.05. HRMS (ESI?) m/z: (M ? H) calcd for
C H N O S 533.1759, found 533.1739. Anal. calcd. for C H N O S: C,
0
3
24
6
2
30 24 6 2
6
7.65; H, 4.54; N, 15.78; S, 6.02; found: C, 67.49; H, 4.58; N, 15.47; S, 6.18.
0
(
nyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide
E)-N -((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phe-
(5d) Yellow
solid.
m.p.
-
1
2
51–254 °C, Yield: 65 %. IR (KBr, cm ): 3263 (NH), 1640 (C=O), 1610(C=N).
1
H NMR (400 MHz, DMSO-d , d, ppm): 2.44 (s, 3H, CH ), 7.21–8.05 (m, 14H, Ar–
6
3
H), 8.22 (s, 1H, thiophene-H), 8.32 (s, 1H), 9.46 (s, 1H, NH), 11.44 (brs, 1H, NH).
1
3
C NMR (100 MHz, DMSO-d , d, ppm): 12.98, 114.52, 116.63, 116.91, 117.13,
6
1
1
20.39, 125.50, 128.07, 129.27, 129.96, 130.10, 130.86, 131.19, 131.38, 135.82,
38.30, 138.68, 141.40, 143.61, 144.10, 152.00, 163.05, 164.00. HRMS (ESI?) m/z:
1
23

Synthesis, biological evaluation and molecular docking…
?
M ? H) calcd. for C H FN OS 521.1558, found 521.1561. Anal. calcd. for
(
C H FN OS: C, 66.91; H, 4.07; N, 16.14; S, 6.16; found: C, 67.12; H, 4.10; N,
2
9
21
6
2
9
21
6
1
5.75; S, 6.02.
0
(
E)-N -((3-(4-hydroxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phenyl-
1
H-thieno[2,3-c]pyrazole-5-carbohydrazide (5e) Yellow solid, m.p. 270–272 °C,
-1 1
Yield: 67 %. IR (KBr, cm ): 3480 (OH). 3300 (NH), 1670 (C=O), 1600 (C=N). H
NMR (400 MHz, DMSO-d , d, ppm): 2.48 (s, 3H, CH ), 6.86 (d, 2H, Ar–H),
6
3
7
.44–7.98 (m, 12H, Ar–H), 8.20 (s, 1H, thiophene-H), 8.43 (s, 1H), 9.25 (s. 1H,
1
3
CH=N), 11.30 (brs, 1H, NH), 14.02 (s, 1H, OH). C NMR (100 MHz, DMSO-d , d,
6
ppm): 13.01, 114.48, 116.60, 116.90, 117.19, 120.40, 125.52, 126.10, 127.98,
1
1
29.30, 129.95, 130.07, 130.87, 130.90, 135.82, 138.25, 138.70, 141.42, 143.61,
?
44.06, 152.05, 159.90, 163.01. HRMS (ESI ?) m/z: (M ? H) calcd. for
C H N O S 519.1601, found 519.1605. Anal. calcd. for C H N O S: C,
2
9
22
6
2
29 22 6 2
6
7.17; H, 4.28; N, 16.21; S, 6.18; found: C, 66.91; H, 4.26; N, 16.50; S, 6.27.
0
(
E)-N -((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phenyl-
1
Yield: 70 %. IR (KBr, cm ): 3210 (NH), 1665 (C=O), 1600 (C=N). H NMR
H-thieno[2,3-c]pyrazole-5-carbohydrazide (5f) Yellow solid, m.p. 295–297 °C,
-1 1
(
400 MHz, DMSO-d , d, ppm): 2.51 (s, 3H, CH ), 6.91 (d, 2H, Ar–H), 7.41–7.79
6
3
(
m, 10H, Ar–H), 7.88 (d, 2H, ArH), 8.22 (s, 1H, thiophene-H), 8.44 (s, 1H), 9.40 (s.
1
3
1
1
1
1
H, CH=N), 11.81 (brs, 1H. NH). C NMR (100 MHz, DMSO-d , d, ppm): 13.00,
6
14.45, 116.61, 116.93, 120.45, 125.53, 127.02, 129.32, 129.94, 130.01, 130.82,
30.86, 131.02, 132.21, 135.82, 136.00, 138.28, 138.72, 141.44, 143.61, 144.04,
?
52.03, 163.02. HRMS (ESI?) m/z: (M ? H) calcd. for C H ClN OS 537.1262,
2
9
21
6
found 537.1271. Anal. calcd for C H ClN OS: C, 64.86; H, 3.94; N, 15.65; S,
2
9
21
6
5
.97; found: C, 64.57; H, 4.00; N, 15.75; S, 6.13.
0
(
E)-3-methyl-1-phenyl-N -((1-phenyl-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (5g) Yellow solid, m.p. 290–291 °C,
Yield: 72 %. IR (KBr, cm ): 3268 (NH), 1645 (C=O), 1618 (C=N). H NMR
-
1
1
(
400 MHz, DMSO-d , d, ppm): 2.40 (s, 3H, CH ), 2.61 (s, 3H, CH ), 7.30–7.82 (m,
6
3
3
1
4H, Ar–H), 8.21 (s, 1H, thiophene-H), 8.41 (s, 1H), 9.27 (s. 1H, CH=N), 11.30
1
3
(
brs, 1H, NH). C NMR (100 MHz, DMSO-d , d, ppm): 13.00, 21.20, 114.41,
6
1
1
1
5
16.63, 116.91, 120.46, 125.54, 127.05, 129.01, 129.35, 129.93, 130.02, 130.82,
30.85, 131.04, 132.23, 135.83, 138.24, 138.71, 141.45, 143.62, 144.6, 152.01,
?
63.01. HRMS (ESI?) m/z: (M ? H) calcd. for C H N OS 517.1808, found
3
0 24 6
17.1822. Anal. calcd. for C H N OS: C, 69.75; H, 4.68; N, 16.27; S, 6.21; found:
3
0 24 6
C, 69.88; H, 4.73; N, 16.09; S, 6.00.
0
(
nyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide
E)-N -((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-3-methyl-1-phe-
(5h) Yellow
solid,
m.p.
-
1
2
95–297 °C, Yield: 60 %, IR (KBr, cm ): 3252 (NH), 1675 (C=O), 1610
1
(
C=N). H NMR (400 MHz, DMSO-d , d, ppm): 2.43 (s, 3H, CH ), 7.38–7.88 (m,
6
3
1
4H, Ar–H), 8.20 (s, 1H, thiophene-H), 8.40 (s, 1H), 9.40 (s. 1H, CH=N), 11.40
1
3
(
brs, 1H, NH). C NMR (100 MHz, DMSO-d , d, ppm): 13.00, 114.46, 116.63,
6
1
16.90, 121.40, 124.31, 125.56, 127.09, 128.98, 129.92, 130.06, 130.87, 131.02,
123

P. S. Mahajan et al.
1
HRMS (ESI?) m/z: (M ? H) calcd. for C H BrN OS 581.0757, found
32.56, 132.89, 135.82, 138.21, 138.71, 141.46, 143.65, 144.50, 152.48, 163.01.
?
29
21
6
581.0780. Anal. calcd. for C H BrN OS: C, 59.90; H, 3.64; N, 14.45; S, 5.51;
29 21 6
found: C, 60.15; H, 3.63; N, 14.70; S, 5.37.
General procedure for the synthesis of hydrazones (7a–h)
To a solution of carbohydrazide 1 and substituted 3-formylchromone (6a–d) in
methanol, was added a few drops of acetic acid with stirring at room temperature
with the reaction monitored by TLC (15–30 min). After completion of reaction, the
reaction mixture was poured on crushed ice. The resulting crude product was
purified by column chromatography on silica gel by EtOAc:petroleum ether (9:1) as
an eluent to afford the pure title compounds 7a–h.
0
(
E)-N -((7-chloro-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-1H-thieno[2,3-
c]pyrazole-5-carbohydrazide (7a) Yellow solid, m.p. 188–190 °C, Yield: 78 %. IR
-
KBr, cm ): 3448 (NH), 3050 (Ar–H), 2922, 1704 (amide C=O), 1642 (pyrone
1
(
1
C=O). H NMR (400 MHz, DMSO-d , d, ppm): 2.45 (s, 3H, CH ), 7.26-7.68 (m,
6
3
5
H, Ar–H), 7.48 (d, 1H), 7.65 (dd, 1H), 8.02 (d, 1H), 8.11 (s, 1H, thiophene-H), 8.25
1
3
(s, 1H), 8.60 (s. 1H, CH=N), 10.56 (s, 1H, NH). C NMR (100 MHz, DMSO-d , d,
ppm): 13.10, 106.08, 116.10, 116.72, 118.03, 123.03, 124.60, 125.15, 125.43,
6
1
1
4
29.56, 130.46, 135.04, 138.12, 141.47, 143.42, 145.01, 155.05, 157.03, 162.04,
?
63.44, 176.90. HRMS (ESI?) m/z: (M ? H) calcd. for C H ClN O S
2
3
15
4 3
63.0629, found 463.0621. Anal. calcd. for C H ClN O S: C, 59.68; H, 3.27;
2
3
15
4 3
N, 12.10; S, 6.93; found: C, 59.64; H, 3.48; N, 11.98; S, 6.84.
0
(
E)-N -((7-bromo-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-
H-thieno[2,3-c]pyrazole-5-carbohydrazide (7b) Yellow solid, m.p.
1
-
1
2
14–216 °C, Yield: 81 %. IR (KBr, cm ): 3442 (NH), 1690 (amide C=O), 1634
1
(
pyrone C=O). H NMR (400 MHz, DMSO-d , d, ppm): 2.45 (s, 3H, CH ),
6
3
7
thiophene-H), 8.21 (s, 1H), 8.62 (s. 1H, CH=N), 10.62 (s, 1H, NH). C NMR
.32–7.76 (m, 5H, Ar–H), 7.55 (d, 1H), 7.71 (dd, 1H), 8.04 (d, 1H), 8.10 (s, 1H,
1
3
(
100 MHz, DMSO-d , d, ppm): 13.12, 105.98, 116.08, 116.78, 117.98, 123.01,
6
1
1
24.62, 125.11, 125.45, 129.60, 130.50, 134.97, 138.14, 139.70, 141.50, 143.45,
?
45.05, 154.98, 157.0, 161.95, 163.48, 176.91. HRMS (ESI?) m/z: (M ? H)
calcd. for C H BrN O S 507.0124, found 507.0115. Anal. calcd. for C H
23 15-
2
3
15
4
3
BrN O S: C, 54.45; H, 2.98; N, 11.04; S, 6.32; found: C, 54.66; H, 3.09; N, 10.95;
3
4
S, 6.53.
0
(
E)-N -((6-chloro-7-methyl-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-
1
Yield: 80 %. IR (KBr, cm ): 3451 (NH), 1698 (amide C=O), 1629 (pyrone C=O).
H-thieno[2,3-c]pyrazole-5-carbohydrazide (7c) Yellow solid, m.p. 177–179 °C,
-1
1
H NMR (400 MHz, DMSO-d , d, ppm): 2.33 (s, 3H, CH ), 2.47 (s, 3H, CH ), 7.21
6
3
3
(
s, 1H), 7.29–7.88 (m, 5H, Ar–H), 8.00 (s, 1H), 8.12 (s, 1H, thiophene-H), 8.23 (s,
1
3
1
ppm): 13.01, 18.29, 106.03, 115.90, 116.22, 116.69, 118.08, 125.33, 128.94, 129.61,
H), 8.63 (s. 1H, CH=N), 10.48 (s, 1H, NH). C NMR (100 MHz, DMSO-d , d,
6
1
30.55, 131.55, 133.80, 135.04, 138.10, 139.75, 143.42, 155.00, 161.97, 163.13,
1
23

Synthesis, biological evaluation and molecular docking…
?
76.90. HRMS (ESI?) m/z: (M ? H) calcd. for C H ClN O S 477.0786, found
1
2
4
17
4 3
4
found: C, 60.74; H, 3.57; N, 11.97; S, 6.84.
77.0770. Anal. calcd. for C H ClN O S: C, 60.44; H, 3.59; N, 11.75; S, 6.72;
2
4
17
4 3
0
(
E)-N -((6,7-dimethyl-4-oxo-4H-chromen-3-yl)methylene)-3-methyl-1-phenyl-1H-
thieno[2,3-c]pyrazole-5-carbohydrazide (7d) Yellow solid, m.p. 154–156 °C,
Yield: 78 %, IR (KBr, cm ): 3476 (NH), 1688 (amide C=O), 1644 (pyrone C=O).
-
1
1
H NMR (400 MHz, DMSO-d , d, ppm):, 2.27 (s, 3H, CH ), 2.31 (s, 3H, CH ), 2.45
6
3
3
(
thiophene-H), 8.29 (s, 1H), 8.65 (s. 1H, CH=N), 10.42 (s, 1H, NH). C NMR
s, 3H, CH ), 7.16 (s, 1H), 7.64 (s, 1H), 7.32-7.74 (m, 5H, Ar–H), 8.12 (s, 1H,
3
1
3
(
100 MHz, DMSO-d , d, ppm): 13.00, 17.21, 17.72, 106.05, 115.92, 116.68, 118.05,
6
1
1
24.01, 125.36, 129.55, 130.57, 131.58, 133.82, 135.05, 138.06, 141.21, 143.44,
?
49.31, 152.31, 154.66, 162.06, 163.11, 176.92. HRMS (ESI?) m/z: (M ? H)
calcd. for C H N O S 457.1332, found 457.1318. Anal. calcd. for C H N O S:
25 20 4 3
2
5
20
4
3
C, 65.77; H, 4.42; N, 12.27; S, 7.02; found: C, 65.72; H, 4.45; N, 12.14; S, 6.91.
Biological methods
Antioxidant activity
2
mL of solution containing 0.2-mM freshly prepared 1,1-diphenyl-2-picryl-
hydrazil (DPPH) in methanol and different concentrations of synthesized
compounds (10–100 lg/mL) were prepared to perform the reaction. After
incubation at room temperature for 30 min, the absorbance at 517 nm was
measured spectrophotometrically [38]. A graph pad prism was used to calculate
the concentration required to obtain a 50-% antioxidant effect (EC ) and is
5
0
compared with the butylated hydroxy toluene (BHT) standard, by performing
the experiment in triplicate. DPPH radical scavenging capacity was measured
by:
%
scavenging activity ¼ AcontrolÀ Atest=Acontrol  100
A
¼ Absorbance of the control; A ¼ Absorbance of the test compounds
test
control
Anti-inflammatory activity
The reaction mixture consisting of 0.5 mL of 10-% human red blood cell (HRBC)
suspension, 1 mL hypo saline, 1 mL of 0.2-M phosphate buffer, and 1 mL of test
sample at different concentrations in normal saline was incubated at 37 °C for
3
0 min and centrifuged at 3000 rpm for 30 min. The hemoglobin content of the
supernatant solution was estimated at 560 nm spectrophotometrically. Each
experiment was performed in triplicate and distilled water used as the control in
this study. EC50 values were determined for each compound. Where the blood
control represents zero percent stability or 100 % lysis, the percentage of HRBC
heamolysis was calculated by:
123

P. S. Mahajan et al.
%
of Heamolysis ¼ ðO.D. of Control À O.D. of Test sample=O.D. of ControlÞ
 100
Molecular docking
Molecular docking studies were performed using the grid-based ligand docking with
energetics (Glide) [39, 40] module integrated in the Schr o¨ dinger molecular
modeling package (Schr o¨ dinger, Inc., USA, version-2014–14) installed on a
Windows workstation with an Intel (R) Xenon 2.8-GHz processor and 32 GB of
physical memory. It is an interactive molecular graphics program for docking
calculations, studying ligand-receptor interactions and identifying possible binding
sites of biomolecules. The X-ray crystal structure of cyclooxygenase-2 (COX-2) in
complex with its inhibitor diclofenac was retrieved from the Protein Data Bank
(
www.rcsb.org; PDB ID: 1PXX).
First, the crystal structure was pre-processed using the Protein Preparation
Wizard by deleting the crystallographically observed water molecules since no
water molecule was observed to be conserved, correcting the mistakes in the PDB
structure and optimizing the hydrogen bonds. Following this, hydrogen atoms were
added to the crystal structure corresponding to pH 7.0, considering the appropriate
ionization states for the acidic and basic amino acid residues. The most likely
positions were selected for the –OH and –SH hydrogen atoms, protonation states
and tautomers of His residues and Chi ‘flip’ assignments were made for Asn, Gln
and His amino acid residues. Following the assignment of charge and protonation
state, energy minimization with a root mean square deviation (RMSD) value of
˚
0
.30 A was carried out using an OPLS-2005 force field to relieve the steric clashes
among the residues due to addition of hydrogen atoms.
The three-dimensional (3D) structures of N-acylheteroaryl hydrazone (NAH)
derivatives were built using the builder panel in Maestro followed by ligand
preparation using the Ligprep module which performs addition of hydrogens,
adjusting realistic bond lengths and angles, correcting chiralities, ionization states,
tautomers, stereo chemistries and ring conformations. Geometry minimization was
then performed on all the structures by means of the OPLS 2005 force field using a
default setting (Macro Model, Schr o¨ dinger, LLC, USA) till it reached an RMSD
˚
cutoff of 0.01 A and the resulting structures were then used for carrying out the
docking study.
The receptor grid was generated to define the active site of COX-2 for docking
using the Receptor Grid Generation panel in Glide. It uses two cubical boxes having
a common centroid to organize the calculations: a larger enclosure and a smaller
˚
binding box. A Grid file was generated by defining a 14 9 14 9 14 A box centered
on the geometric centroid of the COX-2 structure to explore a large region of the
enzyme structure. Default values were maintained for the van der Waals scaling
while partial charges were assigned from the input structure, rather than from the
force field, by selecting the use input partial charges option. All the N-
acylheteroaryl hydrazone (NAH) derivatives were docked into the binding site
defined using the Grid generation protocol utilizing the extra precision (XP) Glide
1
23

Synthesis, biological evaluation and molecular docking…
scoring function to rank the docking poses and to estimate the protein–ligand
binding affinities. The Glide algorithm carries out a systematic search of positions,
orientations and conformations for the ligand in the active site of the enzyme using a
funnel-type approach. The output files were analyzed using Maestro’s pose viewer
utility.
Results and discussion
Chemistry
The synthesis of new 3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-acylhydrazone
derivatives (Scheme 2) was initiated from the 3-methyl-1-phenyl-1H-thieno[2,3-
c]pyrazole-5-carbohydrazide 1 prepared in good yield, according to the literature
procedure [41] (Scheme 1). The aldehyde function of P was converted to the
corresponding methyl ester Q by cyclocondensation between 5-chloro-4-carbalde-
hyde P and methyl thioglycolate in 85 % yield. 3-Methyl-1-phenyl-1H-thieno[2,3-
c]pyrazole-5-carboxylate Q reacted with hydrazine hydrate in methanol at reflux
afforded corresponding carbohydrazide 1 in 80 % yield (Scheme 1). Finally, the
new stable solid NAH compounds 3a–g, 5a–h, and 7a–d were prepared in good
yields by condensing key intermediate 1 with the suitable heterocyclic aldehydes in
methanol using acetic acid as a catalyst (Scheme 2). All the compounds were
1
13
characterized by FTIR, H NMR, C NMR, elemental analysis and HRMS.
1
The H NMR spectrum of compound 3a displayed a broad singlet at d 11.89 ppm
(
NH) and at d 9.02 ppm (CH=N). The IR spectrum of compound 3a showed a
-1
significant absorption bands at 3444 and 1636 cm assigned to –NH and amide
CONH), respectively. Its HRMS spectrum showed a molecular ion peak at
97.1504, consistent with its molecular formula C H N O S. In the IR spectrum
(
4
2
5 20 8 2
-
1
of compounds 5a and 7a, the absorption bands at 3260 and 3448 cm for NH
-
stretching and at 1650 and 1704 cm
1
for amide stretching were observed,
1
respectively. The H NMR of compound 5a showed the characteristic –NH signal at
d 11.35 ppm and the –CH=N proton at d 9.36 ppm. Similarly, 7a exhibited a singlet
peak at d 10.56 ppm for an NH proton and at d 8.60 ppm related to –CH=N. The
mass spectra of compounds 5a and 7a detected expected molecular ion peaks at
5
03.1656 and 463.0621, corresponding to molecular formula C H N OS and
22
2
9
6
O
O
O
H C
OCH
3
H C
H C
3
3
3
HS
O
OCH3 NH NH . H O
NHNH
2
N
Cl
N
S
2
2
2
N
S
N
N
N
K CO , DMF
CH OH, Reflux
3
2
3
8
0 °C
P
Q
1
Scheme 1 3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide
123

P. S. Mahajan et al.
Scheme 2 Synthesis of N-acylhydrazone derivatives
C H ClN O S, respectively. All other derivatives were well proven by the
2
3
15
4 3
spectral techniques.
Biological evaluation and in silico study
In vitro antioxidant activity
Due to the lower electron density of C=N bonds [42], hydrazones are superior
radical acceptors compared to imines. So, it was thought that N-acylhydrazones
could show better antioxidant activity. A DPPH radical scavenging method was
used to evaluate the antioxidant potential of all synthesized compounds [43, 44]
(
Table 1). During the study of antioxidant activity of all compounds (Table 1), it
has been observed that compounds 3a (EC50 = 6.00 ± 2.36 lg/mL) and 5c
EC50 = 7.21 ± 0.67 lg/mL) showed the most potent activity as compared to all
other hydrazone series compounds and also compared to the standard drug BHT
EC50 = 8.25 ± 0.34 lg/mL), while compounds 3c, 3d, 5b, 5e and 7c showed good
(
(
activity (ranging 9.32 ± 1.77–15.57 ± 4.21) as compared to other compounds of
the series and less comparable to BHT.
1
23

Synthesis, biological evaluation and molecular docking…
Table 1 In vitro antioxidant activity (DPPH radical scavenging method) and anti-inflammatory activity
(HRBC membrane stabilization method) of compounds 3a–3g, 5a–5h and 7a–d
Compound R Antioxidant activity Anti-inflammatory activity
4
R
1
R
2
R
3
(
EC50 ± SD)
lg/mL)
(EC50 ± SD) (lg/mL)
(
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
7
7
7
7
a
b
c
d
e
f
–H –OCH
2
3
CH
3
–H
–H
–H
–H
–H
–H
–H
–CH
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–
06.00 ± 2.36
47.00 ± 2.79
15.00 ± 1.32
12.00 ± 0.97
59.99 ± 1.54
51.00 ± 0.76
56.63 ± 1.66
[100
77.31 ± 2.34
10.25 ± 1.08
45.54 ± 1.46
49.64 ± 4.22
11.26 ± 2.00
19.36 ± 0.97
66.23 ± 1.45
13.23 ± 2.36
54.00 ± 2.13
16.00 ± 2.19
52.32 ± 1.33
50.13 ± 2.03
49.99 ± 1.39
14.00 ± 1.26
38.09 ± 0.99
33.38 ± 0.31
10.50 ± 0.99
11.18 ± 0.15
23.00 ± 0.75
–
–H –H
–H
–H –OCH
–H –H
–H
–OCH
3
–H –CH
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –H
–H –Cl
–H –CH
3
–H
–CH
–H
3
g
a
b
c
d
e
f
3
–H
–NO
2
12.32 ± 1.33
07.21 ± 0.67
[100
–OCH
3
–F
–OH
–Cl
09.32 ± 1.77
76.09 ± 3.33
57.77 ± 1.65
81.34 ± 1.99
[ 100
g
h
a
b
c
d
–CH
–Br
–Cl
–Br
–CH
–CH
–
3
19.90 ± 3.48
15.57 ± 4.21
97.00 ± 2.64
8.25 ± 0.34
–
3
3
3
BHT
DFS
–
–
–
–
–
–
11.70 ± 0.98
Values are expressed as mean ± standard deviation (n = 3)
BHT Butylated hydroxy toluene, DFS Diclofenac sodium, SD standard deviation
In vitro-anti-inflammatory activity
Anti-inflammatory agents act by either inhibiting lysosomal enzymes or by
stabilizing lysosomal membranes, and HRBC membranes are similar to these
lysosomal membrane components [45]. Hence, lysis of an HRBC membrane is
taken as a measure of anti-inflammatory activity. In vitro anti-inflammatory activity
was studied via the HRBC membrane stabilization method against the standard drug
diclofenac sodium (DFS) [46] (Table 1).
As shown in Table 1, compounds 3b (EC50 = 10.25 ± 1.08), 7b (EC50
0.50 ± 0.99) and 7c (EC50 = 11.18 ± 0.15) exhibited the most potent anti-
=
1
inflammatory activity as compared to all other hydrazone series compounds and
standard drug DFS (EC50 = 11.70 ± 0.98), while compounds 3e, 3f, 5a, 5c and 5g
were found to be active as compared to all other compounds and less comparable to
DFS. The anti-inflammatory activity of hydrazones could be due to their binding
123

P. S. Mahajan et al.
onto the erythrocyte membranes with subsequent alteration of the charges on the
surface of the cells. This might have prevented physical interaction with aggregating
agents or promote dispersal by mutual repulsion of like charges involved in the
haemolysis of red blood cells [47].
Molecular docking
A molecular docking study was carried out to gain insight into molecular
interactions that govern the binding of NAH derivatives with the target enzyme
COX-2. Structures of NAH derivatives as well as the enzyme were kept flexible to
obtain different binding conformations and the best docked complex obtained from
it was analyzed in detail. The molecular docking protocol utilized in the current
study was validated by extracting the native ligand diclofenac from the binding site
and re-docking it to the binding site of COX-2. The RMSD between the original
conformation of diclofenac and the conformation obtained from its re-docking into
˚
COX-2 structure was found to be less than 1 A, validating the reliability and
reproducibility of the docking procedure (Fig. 1).
Results of the molecular docking study have been analyzed on the basis of four
main parameters- Glide score, Glide energy, non-bonded interactions (van der
Waals and coulombic) and H-bonds. Considering these parameters, the binding
affinity of the NAH derivatives towards COX-2 has been discussed. Ten different
binding conformations were retained for each compound from docking simulations.
Table 2 presents the intermolecular interaction energy values obtained from the
docking calculation.
The ligands have been ranked on the basis of their Glide score and the percentage
enzyme activity. Analysis of the docking poses showed that NAH derivatives got
docked into the binding site of COX-2 at the same site as was observed for
diclofenac complexed with COX-2 and, hence, could serve as a pertinent starting
point for the rational design of potent COX-2 inhibitors. All the compounds
Fig. 1 Overlay of the X-ray conformation of diclofenac (blue back-bone) over its best docked
conformation (green back-bone). (Color figure online)
1
23

Synthesis, biological evaluation and molecular docking…
Table 2 Quantitative analysis of the docking results- Glide score, Glide energy and non-bonded inter-
actions (van der Waals (VDW) and electrostatic)
Compound
Glide score
Glide energy
VDW energy
Coulombic energy
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
7
7
7
7
a
b
c
d
e
f
-6.002
-8.925
-7.255
-7.134
-8.115
-7.75
-36.273
-59.304
-48.732
-47.392
-54.895
-50.177
-43.658
-53.584
-44.706
-52.442
-45.886
-46.615
-47.278
-52.674
-49.13
-34.989
-54.256
-45.793
-45.149
-50.175
-47.046
-41.814
-50.445
-42.607
-48.993
-43.792
-44.327
-45.093
-49.375
-46.861
-47.775
-52.808
-51.101
-47.027
-50.324
-1.285
-5.048
-2.939
-2.244
-4.721
-3.131
-1.844
-3.139
-2.099
-3.449
-2.094
-2.289
-2.185
-3.299
-2.269
-2.156
-5.551
-4.704
-3.056
-4.268
g
a
b
c
d
e
f
-6.294
-8.048
-6.703
-7.865
-6.933
-7.008
-7.020
-7.95
g
h
a
b
c
d
-7.359
-7.583
-8.869
-8.197
-7.656
-8.077
-49.931
-58.359
-55.806
-50.083
-54.592
DFS
exhibited good binding affinity towards COX-2 with an average docking score of
7.05. The docking score of these ligands varied from –6.00 to -8.95, while the
docking score for the native ligand diclofenac was found to be -8.07.
-
A plot of the anti-inflammatory activity expressed as EC50 values versus the
Glide docking score of NAH derivatives is shown in Fig. 6. A significant correlation
was observed between the docking scores of the docked ligands and EC50 values,
validating that the binding site of NAH derivatives is similar to that of diclofenac. A
per-residue interaction analysis between the protein and the NAH derivatives was
performed to investigate detailed interpretations of the molecular mechanisms
involved in their anti-inflammatory activity. From the ensuing docked structures, it
is clear that all the NAH derivatives snugly fit into the active site of COX-2,
resulting in various close contacts with the amino acid residues lining the active site.
The per residue interaction analysis could also provides an explanation for the
observed difference in binding affinity for these molecules. For the sake of brevity,
the per-residue interaction analysis has been discussed in detail only for the most
active compounds 3b, 3e, 7b and 7c (Fig. 1).
Docking of the most active compound 3b into the active site of COX-2 showed
that the inhibitor binds at the same site as the native ligand diclofenac with a
123

P. S. Mahajan et al.
significantly higher binding affinity (Fig. 2). Even the Glide score for 3b was found
to be higher (-8.925) than that of diclofenac (-8.077). The per-residue interaction
analysis showed that the van der Waals interactions (-54.256 kcal/mol) surpassed
the electrostatic contribution (-5.048 kcal/mol) in the overall binding affinity. The
overall binding energy of compound 3b was also found to be -59.304 kcal/mol as
compared to -54.592 kcal/mol observed for diclofenac. The higher binding affinity
observed for 3b can be explained in terms of the specific bonded and non-bonded
per residue interactions with the residues comprising the active site. The compound
is stabilized within the active site through extensive van der Waals contacts with
Ala527 (-1.655 kcal/mol), Ile517 (-1.492 kcal/mol), Ala516 (-2.778 kcal/mol),
Asp515 (-2.993 kcal/mol), Arg513 (-2.317 kcal/mol), Trp387 (-1.274 kcal/mol),
Tyr385 (-1.184 kcal/mol), Tyr355 (-2.291 kcal/mol), Gly354 (-2.505 kcal/mol),
Val349 (-2.395 kcal/mol), Gln192 (-4.413 kcal/mol), Thr94 (-3.642 kcal/mol)
and His90 (-1.185 kcal/mol) residues. The enhanced binding affinity of 3b can also
be attributed to favorable electrostatic contacts with Arg513 (-3.031 kcal/mol),
Arg433 (-1.507 kcal/mol) and Ser353 (-2.222 kcal/mol) residues lining the active
site. The enzyme-inhibitor complex was further stabilized by crucial hydrogen
bonding interactions observed with Ser353, Leu352 and Tyr115. These hydrogen
bonds may act as ‘‘anchors’’, guiding the 3D orientation of the ligand in its active
site, thereby facilitating the steric and electrostatic interactions.
The results obtained from molecular docking for the second most active
compound 7b showed that it binds to the target at the same site (Fig. 3) as occupied
by diclofenac and 3b, indicating that even this compound is mediating its activity
via inhibiting COX-2. The Glide score was observed to be -8.869 with an overall
binding energy of -58.359 kcal/mol. The binding affinity of 7b was, like 3b, found
to be better than diclofenac. Inspection of the van der Waals and electrostatic
interaction energies revealed that the compound shares higher values of van der
Waals interaction energy (-52.808 kcal/mol) over the electrostatic component
(
-5.551 kcal/mol) in the overall binding energy. The per residue ligand interaction
Fig. 2 Model of compound 3b docked into the binding site of COX-2 (image shows the interactions
˚
observed with the residues within 5 A distance of the ligand)
1
23

Synthesis, biological evaluation and molecular docking…
Fig. 3 Model of compound 7b docked into the binding site of COX-2
energy distribution shows that it forms favorable van der Waals interactions with
His388 (-4.793 kcal/mol), Trp387 (-1.194 kcal/mol), His386 (-4.634 kcal/mol),
Tyr385 (-1.553 kcal/mol), Asn382 (-1.872 kcal/mol), Phe210 (-1.84 kcal/mol),
His207 (-2.709 kcal/mol), Thr206 (-1.358 kcal/mol), Gln203 (-7.636 kcal/mol)
and Ala202 (-1.714 kcal/mol). It also showed significant electrostatic interactions
with His388 (-1.831 kcal/mol), His207 (-1.613 kcal/mol), Thr206 (-1.937 kcal/-
mol) and Gln203 (-1.98 kcal/mol) along with a crucial hydrogen bonding
interactions via Thr206 to anchor the ligand at its active site in the enzyme.
The Glide score for 7c (Fig. 4) was -8.197 with an overall binding energy of
-
55.806 kcal/mol. Wherein the contribution from van der Waals interaction was
seen to be -51.101 kcal/mol while coulombic interaction contributed -4.704 kcal/-
mol, which is slightly better than diclofenac. Significant van der Waals interactions
were observed with His388 (-3.139 kcal/mol), Trp387 (-1.188 kcal/mol), His386
(
(
-2.365 kcal/mol), Tyr385 (-1.066 kcal/mol), Asn382 (-1.188 kcal/mol), Phe210
-1.069 kcal/mol), His207 (-1.091 kcal/mol), Thr206 (-1.041 kcal/mol) and
Fig. 4 Model of compound 7c docked into the binding site of COX-2
123

P. S. Mahajan et al.
Gln203 (-3.927 kcal/mol) residues in the active site. Further, it also formed
favorable electrostatic interactions with Gln203 (-1.127), Phe210 (-1.185) and
His207 (-1.114) residues in the active site. However, no significant hydrogen
bonding interactions were observed.
Finally, docking of the fourth most active compound 3e (Fig. 5) into COX-2
produced a relatively lower Glide score (-8.115) compared to compound 3b, 7b
and 7c but better than diclofenac, which is in harmony with experimentally
observed activity for these compounds. The overall binding energy of the compound
was found to be -54.895 kcal/mol wherein the contribution of van der Waals
components was found to be more prevalent (-50.175) than the electrostatic
interaction energy (-4.721 kcal/mol). It forms an extensive chain of favorable van
der Waals interactions with Ser530 (-1.119 kcal/mol), Ala527 (-1.189 kcal/mol),
Leu525 (-1.23 kcal/mol), Val523 (-1.89 kcal/mol), Phe518 (-3.757 kcal/mol),
Ile517 (-1.334 kcal/mol), Asp515 (-1.476 kcal/mol), Arg513 (-2.166 kcal/mol),
Tyr385 (-1.077 kcal/mol), Tyr355 (-2.03 kcal/mol), His351(-1.00 kcal/mol),
Val349 (-2.202 kcal/mol) and Thr94 (-3.272 kcal/mol) residues in the active site
of COX-2. Additionally, favorable electrostatic interactions were also observed with
Arg513 (-1.253 kcal/mol), Leu352 (-2.607 kcal/mol), Asp190 (-1.705 kcal/mol)
and Arg120 (-3.441 kcal/mol) along with a H-bond interaction with the active site
via Leu352 contributing to stabilization of the ligand–enzyme complex.
Overall, the molecular docking results obtained for the NAH derivatives were
found to be in agreement with the observed anti-inflammatory activity. The per-
residue ligand-receptor interaction analysis showed that the primary driving force
for mechanical interlocking was the steric complementarity between the docked
ligands and the active site of the enzyme, as evidenced from the relatively higher
contribution of van der Waals interaction over other components contributing to the
overall binding scores. The binding pattern predicted by docking complemented
with a detailed per residue interaction analysis may facilitate the rational design of
anti-inflammatory leads (Fig. 6).
Fig. 5 Model of compound 3e docked into the binding site of COX-2
1
23

Synthesis, biological evaluation and molecular docking…
Fig. 6 Correlation plot of anti-inflammatory (EC50) versus the Glide docking score for NAH derivatives
Table 3 Pharmacokinetic properties important for good oral bioavailability of compounds 3a–3g, 5a–5h
and 7a–d
Compound
%
ABS (A )
Volume TPSA NROTB HBA HBD Log Lipinski’s Drug likeness
3
2
(A )
P
Violations model score
Rule
–
–
–
–
6
4
5
4
4
4
5
6
7
7
6
6
6
6
6
4
4
4
4
\10 \5
B5
B1
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
7
7
7
7
a
b
c
d
e
f
34.93 417.2
20.30 374.8
29.13 400.4
26.03 391.4
26.03 391.4
26.03 391.4
29.13 400.4
42.11 438.0
50.14 461.3
50.90 463.5
43.80 442.9
44.87 446.0
46.76 451.5
47.80 454.5
48.25 455.8
19.34 372.0
20.85 376.4
25.06 388.6
26.10 391.6
111.6
102.3
111.6
102.3
102.3
102.3
111.6
77.11
122.9
86.3
77.1
97.3
77.1
77.1
77.1
89.4
89.4
89.4
89.4
10
9
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
4.4
0
0
0
0
0
0
0
2
2
2
2
2
2
2
2
0
1
1
0
0.22
0.24
0.26
0.28
0.03
0.04
0.29
0.26
0.18
0.51
0.69
0.79
0.81
0.40
0.51
0.27
0.08
0.02
-0.05
3.9
4.0
4.4
4.4
4.3
4.0
5.6
5.6
5.7
5.8
5.1
6.3
6.1
6.4
4.8
4.9
5.1
4.9
10
9
9
9
g
a
b
c
d
e
f
10
7
10
8
7
8
7
g
h
a
b
c
d
7
7
7
7
7
7
%
ABS percentage of absorption; MW molecular weight; TPSA topological polar surface area; NROTB
number of rotatable bonds; Log P logarithm of compound partition coefficient between N-octanol and
water; HBA number of hydrogen bond acceptors; HBD number of hydrogen bond donors
123

P. S. Mahajan et al.
In silico absorption, distribution, metabolism, excretion (ADME) and drug
likeness analysis
A computational study of all the synthesized compounds was performed for the
prediction of absorption, distribution, metabolism, and excretion (ADME) proper-
ties using a Molinspiration online property calculation toolkit [48], and the values
are presented in Table 3. Some compounds showed good absorption (% ABS) [49],
in the range of 42.11–50.90 %. Furthermore, compounds 3a, 3d, 3e, 3f, 7a, 7b and
7
d are in good agreement with Lipinski’s rule of five and, thus, show good drug-like
properties [50, 51]. A molecule likely to be developed as an orally active drug
candidate should show no more than one violation of the above shown criteria.
Compounds 3a, 3d, 3e, 3f, 7a, 7b and 7d followed the criteria for an orally active
drug and, therefore, these compounds may have a good potential for eventual
development as oral agents. An overall drug-likeness was studied using molsoft
online and from the data, most of the compounds have shown a good model score.
Hence, these molecules showed good drug-like properties.
Structure activity relationship (SAR)
The results of the antioxidant and anti-inflammatory screening confirmed some
specific and remarkable facts about the structure activity relationship (SAR) of the
synthesized molecules, which could help us to understand a possible pharma-
cophoric benefactor for these series. The variation and dependence of the activity
profile of molecules have shown the dependence on structural variations in
molecules. The important thing is to determine the SARs.
Effect of absence of the substituent on the quinoline ring (3a–3g) The presence of
the quinoline ring 3b without any substitution does not make the molecule
specifically active towards antioxidant activity, but it has shown the highest potent
anti-inflammatory activity (10.25 ± 1.08 lg/mL) of all the studied compounds.
This indicates the effect of lack of substitution on tetrazolo[1,5-a]quinoline.
Effect of an electron donating alkoxy and alkyl group on the phenyl ring (3a–
3
g) The methoxy and ethoxy group and its position to quinoline ring have an
essential role in determined the antioxidant and anti-inflammatory activities.
Substitution of the ethoxy group on quinoline ring 3a make the molecule
exclusively active towards potent DPPH radical scavenging activity. While
substitution of methoxy group 3c and 3d makes the molecule active for antioxidant
activity. This clearly indicates that these two groups are favorable regarding the
development of broad spectrum molecule active against antioxidant activity. In the
next, the screening data clearly reveals impact of the position of methyl group such
that the substitution of methyl group at 7, 8 position (3e and 3f resp.) has shown
good anti-inflammatory activity and at 9 position (3g) does not show any response
to the activity.
Effect of the substituent at the para-position on the phenyl ring (5a–5h) The
compound without any substitution on the phenyl ring (5a) has shown good anti-
1
23

Synthesis, biological evaluation and molecular docking…
inflammatory activity, which points out again the effect of the absence of the
substitution on the phenyl ring. The substitution of an electron-withdrawing nitro
group (5b) and electron-donating groups (5c–5h) at the para position on the phenyl
ring clearly show that the electron donating methoxy group 5c (active for
antioxidant and anti-inflammatory activity), 5e (active against DPPH radicals), 5g
(
active for anti-inflammatory activity) and an electron withdrawing group (active
against DPPH radicals) may be good for the development of a broad spectrum of
antioxidant and anti-inflammatory molecules.
Combined effect of a halo and a methyl group (7a–d) From the obtained data,
compound 7c with a bromo- and methyl group showed more potency compared to
other compounds of the series. This indicates that such a modification increases the
molecule’s potency against oxidants and inflammatory agents.
Conclusions
Three new series of 3-methyl-1-phenyl-1H-thieno[2,3-c]pyrazole-5-carbohydrazide
derivatives with remarkable antioxidant and anti-inflammatory activities have been
prepared. Compounds 3a and 5c showed the most significant antioxidant activity
among the series with them being comparable to the BHT standard, while
compounds 3a, 7b, 7c and 3e showed promising anti-inflammatory activity
comparable to DFS. This observed anti-inflammatory activity trend was further
confirmed by molecular docking studies with respect to the binding energy of these
NAH derivatives towards the COX-2 enzyme. The binding mode predicted for these
compounds could establish a link between their binding affinity and the observed
biological activity, thereby providing insights into specific bonded and non-bonded
interactions governing the activity. Furthermore, ADME parameters predicted for
these compounds showed good drug-like properties and can be developed as an oral
drug candidate. In conclusion, 3a, 3d, 3e, 3f, 7a, 7b and 7d compounds from these
present series can be further optimized and developed as lead molecules. Further
biological evaluation to define the mode of action as well as study of the isoenzyme
inhibitory potency to assess the full potential of hydrazones is warranted.
Acknowledgments The author P. S. M. thanks the University Grants Commission, New Delhi, India,
and M. D. N. thanks the Council of Scientific and Industrial Research, New Delhi, India for providing a
senior research fellowship, and also thanks the Department Head of the Department of Chemistry, Dr.
Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra for providing laboratory
facilities. We are also thankful to the Haffkine Research Institute, Mumbai for providing biological
activities.
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