Investigation of antioxidant and anti-nociceptive potential of isoxazolone, pyrazolone derivatives, and their molecular docking studies

Article abstract of DOI:10.1002/ddr.21711

A series of new isoxazolone (3a–d) and pyrazolone (4a–d) derivatives were synthesized and assessed for their antioxidant and analgesic activity. Among synthesized compounds, 3b and 4b having nitro (NO₂) group show high analgesic activity at a dose of 6 mg/kg. Analgesic activity was further proceeded to explore the contribution of opioidergic mechanisms in the mediation of analgesic effects. Animals were administered with naloxone, a nonselective opioid inverse agonist, at the dose of 0.5 mg/kg. The results obtained suggested that the analgesic effects of the synthesized compounds were not reversed by naloxone, specifying that the compounds 3b and 4b do not follow the opioidergic pathway in order to relieve pain in animal models. Further, the binding interactions of compounds 3b and 4b were analyzed by docking them against nonopioid receptors COX-1 (3N8X) and COX-2 (3LN1). The results demonstrate the analgesic potential of isoxazolone and pyrazolone derivatives, especially compounds 3b and 4b can be considered promising lead molecules for further investigation and development into potent analgesic drugs. In addition, the antioxidant potential of compounds was also found to be related to better analgesic activity, thus providing an insight into the role of oxidative stress in the mediation of analgesia.

Full text of DOI:10.1002/ddr.21711

Received: 26 November 2019
DOI: 10.1002/ddr.21711
Revised: 16 May 2020
Accepted: 11 June 2020
R E S E A R C H A R T I C L E
Investigation of antioxidant and anti-nociceptive potential of
isoxazolone, pyrazolone derivatives, and their molecular
docking studies
Tayyaba Anwar1 | Humaira Nadeem1
| Sadia Sarwar2 | Humaira Naureen2
|
Safia Ahmed3 | ArifUllah Khan4
| Muazzam Arif1
1Department of Pharmaceutical Chemistry,
Abstract
Riphah Institute of Pharmaceutical Sciences,
Riphah International University, Islamabad,
Pakistan
A series of new isoxazolone (3a–d) and pyrazolone (4a–d) derivatives were synthe-
sized and assessed for their antioxidant and analgesic activity. Among synthesized
compounds, 3b and 4b having nitro (NO2) group show high analgesic activity at a
dose of 6 mg/kg. Analgesic activity was further proceeded to explore the contribu-
tion of opioidergic mechanisms in the mediation of analgesic effects. Animals were
administered with naloxone, a nonselective opioid inverse agonist, at the dose of
0.5 mg/kg. The results obtained suggested that the analgesic effects of the synthe-
sized compounds were not reversed by naloxone, specifying that the compounds 3b
and 4b do not follow the opioidergic pathway in order to relieve pain in animal
models. Further, the binding interactions of compounds 3b and 4b were analyzed by
docking them against nonopioid receptors COX-1 (3N8X) and COX-2 (3LN1). The
results demonstrate the analgesic potential of isoxazolone and pyrazolone deriva-
tives, especially compounds 3b and 4b can be considered promising lead molecules
for further investigation and development into potent analgesic drugs. In addition,
the antioxidant potential of compounds was also found to be related to better anal-
gesic activity, thus providing an insight into the role of oxidative stress in the media-
tion of analgesia.
2Department of Pharmacognosy, Riphah
Institute of Pharmaceutical Sciences, Riphah
International University, Islamabad, Pakistan
3Department of Microbiology, Faculty of
Biological Science, Quaid e Azam University,
Islamabad, Pakistan
4Department of Pharmacology, Riphah
Institute of Pharmaceutical Sciences, Riphah
International University, Islamabad, Pakistan
Correspondence
Humaira Nadeem, Department of
Pharmaceutical Chemistry, Riphah Institute of
Pharmaceutical Sciences, Riphah International
University, Islamabad, Pakistan.
Email: humaira.nadeem@riphah.edu.pk
K E Y W O R D S
analgesic, antioxidant, docking, isoxazolone, pyrazolone
1
|
INTRODUCTION
radical, hydrogen peroxide, and superoxide radical anion, are regulated
by endogenous antioxidants such as glutathione peroxidase, superox-
Pain is a typical sensory experience related to unpleasant awareness
of a noxious stimulus or bodily harm. The initiation of pain is stimu-
lated by nociceptors present in the peripheral nervous system
because of malfunctioning or damage to the central or peripheral ner-
vous system. One of the key factors involved in malfunctioning of the
nervous system as well as the cellular injury is oxidative stress that
has been associated with a number of diseases, including pain and
inflammation. The oxidative effects of reactive oxygen species (ROS),
produced in metabolic and biological processes, such as hydroxyl
ide dismutase, uric acid and bilirubin, and also by exogenous antioxi-
dants such as vitamins C and E (Erel, 2004). Oxidative stress results
from the imbalance of these two mechanisms; the extent of the
body's antioxidants and the production of hazardous free radicals.
Aging process, accelerated by excessive free radicals that result in oxi-
dative stress, damages the human body at multiple levels causing
numerous disorders such as pain and inflammation (Melov et al., 2000;
Rajendran et al., 2014). It has been reported that antioxidants can
elicit analgesic effects via several mechanisms, such as preventing the
Drug Dev Res. 2020;1–11.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals, LLC.
1

2
ANWAR ET AL.
NaNO2 /HCl
antioxidative/oxidative imbalance (Verri Jr et al., 2012). Normally,
analgesics act on peripheral and Central nervious system (CNS) pain
mediators resulting in pain relief without altering consciousness.
Among the most widely used drugs in the treatment of chronic and
acute pain and inflammation, nonsteroidal anti-inflammatory drugs
(NSAIDs) are considered the backbone of the therapy. NSAIDs are
known to block prostaglandin synthesis through COX 1 and COX 2
inhibition, and their use is associated with gastrointestinal mucosal
damage. Target specific potent analgesic agents are, therefore,
needed to impede COX associated side effects. Synthesis of mole-
cules capable of relieving pain as well as reducing oxidative stress can
provide beneficial therapy to address pain and inflammation caused
by an imbalance of ROS.
R
N+
N
Cl-
R
NH2
1
tate
ace
eto
ylac
Eth
H3C
O
R
N
R
N
CH3
NH2OH.HCl
N
O
N
N
HC
O
OC2H5
3(a-d)
O
2(a-d)
NH2-NH2.H2O
R
H C
3
a = OCH3
R
N
b = NO2
c = COOH
d = CI
Pyrazolones and isoxazolones are nitrogen-containing five-mem-
bered heterocycles forming a key pharmacophore in various pharma-
ceutical products. These moieties are associated with a wide range of
pharmacological effects including antimicrobial (Bondock, Rabie,
Etman, & Fadda, 2008; Kömürcü, Rollas, Yilmaz, & Çevıkbas¸, 1995;
N
N
NH
O
4(a-d)
SCHEME 1 Synthesis of isoxazolone and pyrazolone derivatives
H3C
H3C
N
NH
N
N
N
O
H3CO
N
H3CO
N
O
O
3a
3b
3c
3d
4a
H3C
H3C
N
NH
N
O
N
N
O2N
N
O2N
N
O
O
4b
4c
4d
H3C
H3C
N
O
N
N
N
O
O
NH
N
N
HO
HO
O
O
H3C
H3C
N
N
O
N
N
NH
Cl
N
Cl
N
O
O
FIGURE 1 Structures of synthesized
compounds

ANWAR ET AL.
3
H3C
C
TABLE 1 Antioxidant activity of synthesized compounds
O2N
N
X
Sample
% scavenging activity
N
Methanol
0
NO2
R
N
N
+
N
O
3a
29.11
33.88
25.51
34.88
30.22
29.61
27.39
3.54
O2N
3b
3c
Oxidized DPPH
Compound Radical
3d
N
X
4a
H3C
CH3
4b
O
N
4c
N
4d
N
NO2
N
Ascorbic acid
80.31
O2N
NO2
respective diazonium salts, which were coupled with ethyl
acetoacetate to get intermediate hydrazono derivatives 2(a–d).
Finally, the cyclocondensation of hydrazono derivatives with hydrox-
ylamine hydrochloride and hydrazine monohydrate afforded the tar-
get isoxazolone 3(a–d) and pyrazolone derivatives 4(a–d),
respectively, in good yield (Figure 1). The purity of all the compounds
was confirmed with the help of thin-layer chromatography (TLC) and
elemental analysis data. FTIR, 1HNMR, and 13C NMR data were used
to verify the structure of synthesized compounds. The IR spectra of
Stable Product
FIGURE 2 Proposed mechanism free radical scavenging
compounds in 2,2-diphenyl-l-picryl-hydrazyl assay
Mazimba, Wale, Loeto,
& Kwape, 2014; Padmavathi, Subbaiah,
Mahesh, & Lakshmi, 2007; Ragavan, Vijayakumar, & Kumari, 2009),
antioxidant (Manojkumar, Ravi, & Gopalakrishnan, 2009), anti-inflam-
matory (El-Hawash, Badawey, & El-Ashmawey, 2006; Ismail, Ammar,
El-Zahaby, Eisa, & El-Sayed Barakat, 2007; Laughlin et al., 2005), anal-
all compounds showed characteristic peaks of
N N at 1,537–
1,576 cm−1 (for 3a–d) and 1,530–1,590 cm−1 (for 4a–d), whereas
stretchings for C O appeared at 1,745–1,759 cm−1 (for 3a–d) and
1,665–1,680 cm−1 (for 4a–d).
gesic (CechinelFilho et al., 1998; Changtam, Hongmanee,
&
Suksamrarn, 2010; Gökçe, Utku, & Küpeli, 2009), and anticancer activ-
ities (Brana et al., 2006; Panathur et al., 2015).
In the 1HNMR spectra, methine proton of ring adjacent to azo
group resonated downfield at 3.32–3.34 ppm. Singlet of methyl group
appeared at 2.11–2.24 ppm, and aromatic protons resonated in the
expected region.
Some of the initial antipyretic and anti-inflammatory drugs have
pyrazolone as a basic nucleus such as in antipyrine, phenylbutazone,
remifenazone, muzolimine, and oxyphenylbutazone. Isoxazolone
nuclei also play an important role in the design and development of
different dyes such as merocyanine dyes along with applications in
optical research.
2.2
|
Antioxidant activity of synthesized
compounds
Other important biological activities of isoxazolone derivatives
are herbicidal (Hess, 2000), reverse transcriptase inhibitors (Deng
et al., 2006), lipase inhibitors (Lowe et al., 2004), tumor necrosis fac-
tor-alpha (TNF-α) inhibitors (Laughlin et al., 2005), and anti-androgens
(Ishioka, Tanatani, Nagasawa, & Hashimoto, 2003).
All the synthesized compounds were evaluated for their antioxidant
activity through 2,2-diphenyl-l-picryl-hydrazyl (DPPH) free radical scav-
enging assay. The concentration used for evaluating the antioxidant
potential of the synthesized compounds was 10 mg/ml. Ascorbic acid
and methanol were used as positive and negative controls, respectively.
The synthesized compounds showed moderate activity as com-
pared to reference ascorbic acid except for compound 4d, which
showed the lowest activity (Figure 2). Compounds 3b and 3d showed
maximum activity with 33 and 34% scavenging activity (Table 1).
Based on the diverse pharmacological potential of these nitrogen-
containing heterocycles, the current study was designed to synthesize
azo derivatives of isoxazolone and pyrazolone to investigate their
analgesic and antioxidant potential.
2
|
RESULTS AND DISCUSSION
Chemistry
2.3
|
Assessment of analgesic activity of
2.1
|
synthesized compounds
The synthesis of isoxazolone and pyrazolone derivatives was carried
out as per Scheme 1. Substituted anilines were diazotized to
Analgesic potential of synthesized compounds was examined by means
of the hot plate method. The test compounds, as well as standard, were

4
ANWAR ET AL.
TABLE 2 Analgesic activity of synthesized compounds
0 min
30 min
60 min
90 min
7.02 0.54
120 min
Saline
3a
6.76 0.58
7.66 0.88
6.66 0.88
9.66 0.88
8.33 0.88
8.33 1.45
8.33 0.88
6.33 0.88
8.66 0.33
7.24 0.72
6.84 0.58
10.33 0.67
16.33 0.88
17.00 1.73
11.33 0.88
13.00 0.57
16.67 0.67
14.33 1.76
12.00 0.57
12.72 1.51
6.86 0.71
7.67 0.33
18.77 0.57
13.67 0.88
12.00 1.53
10.00 0.57
20.77 6.56
11.67 1.20
10.00 1.15
14.68 0.77
7.14 0.56
12.33 2.67
24.33 2.33
11.00 0.57
9.00 0.577
9.00 1.00
30.33 3.48
8.67 0.33
10.67 1.20
17.74 0.58
14.00 3.05
20.72 1.20
12.000 0.57
11.67 1.45
8.00 1.00
3b
3c
3d
4a
4b
23.67 2.02
10.00 0.57
12.67 1.20
16.24 0.68
4c
4d
Tramadol
Note: Data are expressed as mean SEM. p < .05.
TABLE 3 Analgesic activity of compounds 3b and 4b
0 min 30 min
60 min
90 min
120 min
Normal saline
3b (3 mg/kg)
Naloxone + 3b
4b (3 mg/kg)
Naloxone + 4b
Tramadol
+
5.20 0.57
8.66 0.57
10.00 0.67
9.33 0.88
11.33 0.88
14.66 0.33
633 0.33
10.33 0.66
11.77 1.56
11.05 0.57
13.00 1.33
16.33 0.66
7.00 0.57
13.66 0.66
15.67 1.02
13.00 0.57
15.72 1.2
19.66 0.33
7.66 0.33
15.68 0.33
16.33 1.48
14.66 0.33
16.77 0.57
21.66 0.66
5.66 0.33
6.83 0.88
6.33 0.33
7.66 0.88
8.66 0.66
Note: Data are expressed as mean SEM. p < .05.
administered at a dose level of 6 mg/kg, and the response was recorded
at 0, 30, 60, 90, and 120 min. Tramadol and Normal saline were used as
a positive and negative control, respectively.
All the synthesized compounds, 3a–d, and 4a–d showed signifi-
cant analgesic effect as compared to baseline reading. Interestingly,
compounds 3b and 4b showed longer latency time as compared to
the standard, tramadol at a dose level of 6 mg/kg (Table 2). In the case
of 3b and 4b, latency time continued to increase with time, while in
other compounds maximum latency time was observed after 60 or
90 min then decreased afterward.
2.4
|
Mechanism of analgesic effect
To investigate the mechanism of analgesic effect of 3b and 4b, animals
were administered with naloxone, a nonselective opioid inverse agonist,
at the dose of 0.5 mg/kg to investigate the involvement of opioidergic
mechanism. The results obtained suggested that the analgesic effects of
the synthesized compounds were not reversed by naloxone, specifying
that the compounds 3b and 4b do not follow the opioidergic pathway in
order to relieve pain in animal models (Table 3).
FIGURE 3 Effect of synthesized compounds on latency time in a
hot plate assay. Values are expressed as Mean SEM, n = 3. *p < .05
and **p < .01 vs. saline group, one-way analysis of variance with post
hoc Tukey's test
The latency time of compound 3b (3 mg/kg) treated group at 0, 30,
60, 90, and 120 min. Was 5.66 0.33, 8.60 0.57, 10.33 0.66,

ANWAR ET AL.
5
13.66 0.66, and 15.66 0.33 s (p < .05 vs. saline group), respectively
(Figure 3).
potential, thereby signifying the involvement of ROS in the progression
of nociception. Another derivative 3c having good antioxidant potential
also showed increased latency time initially, which later on decreased
with time. This may be accredited to the presence of a polar carboxylic
group rendering the molecule less available for absorption.
The latency time of compound 4b (3 mg/kg) at 0, 30, 60, 90, and
120 min. Was 6.33 0.33, 9.33 0.88, 11.05 0.57, 13.00 0.57,
and 14.66 0.33 s (p < .05 vs. saline group), respectively (Figure 4).
2.5
|
Antioxidant potential and analgesic activity of
2.6
|
Docking studies
target compounds
2.6.1
|
Structural assessment of target proteins
As evident from results, the highest analgesic activity was exhibited by
3b and 4b, both bearing para nitrophenyl moiety in the skeleton. Both
these derivatives were also found to possess higher antioxidant
Prostaglandin-endoperoxide synthase (PTGS) or cyclooxygenase
(COX) enzyme regulates the production of prostanoids that is, prosta-
glandins (such as prostacyclin) and thromboxane, from arachidonic
acid. The molecular biology of both COX-1 and COX-2 reveals that
both the isozymes form dimmers having nearly identical catalytic sites,
similar molecular weights (approximately 70 and 72 kDa, respectively),
and 65% homology in amino acid sequence (Nina, Bernèche, &
Roux, 2000).
The COX-1 (PDB ID: 3N8X) protein selected as the target in this
research is comprised of 1,104 amino acid residues and the structure
architecture manifested that the protein consisted of 49% coils (542
residues), 41% helices (454 residues), 14% turns (156 residues), and
9% β sheets (110 residues). The resolution of the selected protein was
2.75 Å, as confirmed by X-ray diffraction studies, with an R-value of
0.181. The dimensions of length and angles of coordinates were also
observed, and the values of unit cell length were a = 181.709,
b = 181.709, and c = 103.454 with angles 90ꢀ, 90ꢀ, and 120ꢀ for all α,
β, and γ dimensions, respectively. Most of the amino acid residues
were present in the favored region that is, 97.55%, as shown by the
Ramachandran graph, representing good accuracy of phi (φ) and psi
(ψ) angles among the coordinates of protein.
FIGURE 4 Effect of synthesized compounds on latency time in a
hot plate assay. Values are expressed as Mean SEM, n = 3. **p < .01
vs. saline group, one-way analysis of variance with post hoc
Tukey's test
The protein selected as the target for the docking of synthesized
compounds with COX-2 (PDB ID: 3LN1), consisted of 41% helices (827
residues), 7% β sheets (156 residues), 50% coils (1,011 residues), 6% turns
TABLE 4 Chemoinformatic properties of synthesized compounds
Properties
MW (g/mol)
HBA
3a
3b
3c
3d
4a
4b
4c
4d
233
6
248
8
247
7
2,378
5
232
6
247
8
246
7
237
5
HBD
—
—
1
—
1
1
2
1
LogP
2.21
72.63
60.56
1.32
46.4
24.00
175.3
Yes
2.11
109.22
60.40
1.56
65.7
23.94
159.0
Yes
2.06
100.70
61.06
1.50
60.0
24.20
164.3
Yes
2.83
63.40
59.34
1.45
51.4
23.52
162.9
Yes
1.45
75.42
62.22
1.33
48.5
24.66
173.5
Yes
1.36
112.01
62.07
1.57
69.1
24.60
157.2
Yes
1.31
103.49
62.72
1.51
63.0
24.86
162.5
Yes
2.08
66.19
61.01
1.46
54.0
24.18
161.1
Yes
PSA (Å2)
MR (cm3)
Den. (g/cm3)
ST (dyne/cm)
PZ (cm−3
)
MV (cm3)
Lipinski rule
Abbreviations: Den., density; HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; MR, molar refractivity; MV, molar volume; MW, molecular
weight; PSA, polar surface area; PZ, polarizability; ST, surface tension.

6
ANWAR ET AL.
(124 residues), and a total of 2,208 amino acid residues. The R-value of
selected protein appeared to be 0.232 and the resolution was 2.4 Å. Unit
cell dimensions for the lengths were observed to be; a = 180.942,
b = 135.377, and c = 124.079 with angles 90ꢀ, 90ꢀ, and 90ꢀ for all α, β,
and γ, respectively. Ramachandran plot confirmed that 98.32% amino
acids were in the allowed regions for the phi (φ) and psi (ψ) angles.
The hydrophobicity and Ramachandran plots for the target pro-
teins are given in Figures S13–S16.
synthesized compounds (348.19 g/mol and 247.21) were much better
than the standard (<5,000 g/mol). As suggested by Lipinski's RO5, the
molecules with more than 5 HBD, MWT over 500, logP over 5, and
more than 10 HBA would most probably have poor absorption. Over-
all, the predicted values justified the importance of compounds 3b
and 4b as promising drug candidates (Table 4).
2.6.3
|
Docking
2.6.2
|
Chemo-informatics and Lipinski's rule
The affinity among the protein targets and the ligands can be investi-
gated using molecular docking. In this AutoDockVina (Trott
&
The computational results showed that 3b and 4b possessed 8 HBA
(each), 0 and 1 HBD, and 2.11 and 1.36 LogP values, respectively, jus-
tifying their drug-like behavior. Further, the molecular weights of the
Olson, 2010) program was used for the docking analysis through PyRx
(Dallakyan & Olson, 2015) user interface. The protocol used for
molecular docking was validated by re-docking the co-crystallized
inhibitors back into respective enzymes (Figure 5).
E-value (kcal/mol) was used to assess the affinity of protein and
the best-docked pose complex. It provides a prediction of binding free
energy and binding constant for docked ligands (Table 5).
The docking results of synthesized compounds with COX-1 (PDB
ID: 3N8X) revealed good binding affinity of compounds 3b and 4b
with binding energies of −8.0 and −7.9 kcal/mol, respectively.
Although, 3b formed five strong hydrogen bonds with different amino
acids of the active site and 4b formed only three hydrogen bonds, the
difference in their binding energies was minimal. This could probably
be due to one unfavorable acceptor-acceptor interaction that 3b
formed with GLY-B: 533 residue of the active site (Figure 6, S1-S4).
When docked with COX-2 (PDB ID: 3LN1), 3b and 4b showed
the binding energies of −8.2 and −8.4 kcal/mol, respectively.
Although 3b formed four hydrogen bonds with different amino acid
residues present in the active site whereas 4b formed one hydrogen
bonds, but the binding energy of 4b was lower than the binding
energy of 3b. This might be due to the fact that hydrogen bonds are
effective at a distance of less than 3.5 Å. Hydrogen bonds with bond
distances of more than 3.5 Å should be considered as interactions. In
the case of 3b, the bond distances of four hydrogen bonds range from
4.34 to 6.23 Å whereas the hydrogen bond distance in the case of 4b
was 3.34 Å (refer to supplementary file for bond distances details).
FIGURE 5 Validation of the molecular docking protocol using co-
crystallized ligands
TABLE 5 Binding energies of the compounds 3b, 4b and the
standard (Tramadol)
Binding energy (kcal/mol)
Target proteins
3b
4b
Tramadol
−7.3
COX-1 (PDB ID: 3N8X)
COX-2 (PDB ID: 3NL1)
−8.0
−8.2
−7.9
−8.4
−7.8
FIGURE 6 3D binding interactions of compounds 3b (a) and 4b (b) with amino acid residues of target protein COX-1 (PDB ID: 3N8X)

ANWAR ET AL.
7
FIGURE 7 3D binding interactions of compounds 3b (a) and 4b (b) with amino acid residues of target protein COX-2 (PDB ID: 3LN1)
FIGURE 8 3D binding interactions of reference compound (Tramadol) with amino acid residues of target protein A; COX-1 (PDB ID: 3N8X)
and B; COX-2 (PDB ID: 3LN1)
Nonetheless, both the compounds showed reasonable binding ener-
gies (Figure 7, S7-10).
and 34% scavenging activity, whereas compound 4a and 4b showed 30
and 29% activity. All the synthesized derivatives exhibited significant
anti-nociceptive potential when compared to negative control due to
the presence of azole moieties. Among synthesized derivatives, 3b and
4b containing the nitro group showed excellent analgesic activity
greater than the standard drug, Tramadol. Since both these derivatives
also possess anti-oxidant potential, it can be concluded that molecules
with good antioxidant potential prove to be better drug candidates for
analgesic activity. Furthermore, molecular docking results predicted
promising binding affinities of compounds 3b and 4b against COX-1
and COX-2. Henceforth, it is concluded that the synthesized derivatives
of isoxazolone and pyrazolone can be considered lead molecules in the
development of analgesic and antioxidant drugs having lesser adverse
effects as compared to the conventional NSAIDs.
The binding affinity of the reference drug, in the case of both
COX-1 and COX-2, was lower than the synthesized compounds 3b
and 4b (Table 5). As tramadol follow opioidergic pathway for its anal-
gesic effect, the binding pose analysis also reveals that tramadol does
not form reasonable interactions with the amino acid residues in the
active sites of both COX-1 and COX-2 with 1 and 0 hydrogen bonds,
respectively (Figures 8, S5, S6, S11, S12).
3
|
DATA ANALYSIS
Data were presented as mean SEM and were analyzed through one-
way analysis of variance followed by Tukey's post hoc test using
Graph Pad Prism 6.0. A p < .05 will be considered significant.
5
|
EXPERIMENTAL
| Chemistry
4
|
CONCLUSIONS
5.1
All the synthesized compounds 3 (a–d) and 4 (a–d) exhibited moderate
antioxidant activity compared to ascorbic acid. 3b and 3d showed 33
All the chemicals and solvents used were acquired from DAEJUNG,
Sigma-Aldrich, and Alfa Aesar. Synthesized compounds were purified

8
ANWAR ET AL.
by recrystallization using ethanol, and purity was assessed using TLC
in the solvent system (ethyl acetate: pet-ether 3:1, chloroform: metha-
nol 9:1). Merck TLC Silica gel 60F254 was used. Stuart melting point
apparatus was used for recording melting points.
7.04 (m, 4H, Aryl H), 3.61 (s, 3H, OCH3), 3.34 (s, 1H, CH), 2.07 (s,
3H, CH3);13CNMR (DMSO-d6, 100 MHz, δ ppm): 151.2, 149.0,
127.5, 127.2, 116.3, 116.0 (Ar C), 161.5, 150.2, 54.3 (isoxazolone-C),
53.4 (OCH3), 11.6 (isoxazole-CH3); Anal. Calcd. For C11H11N3O3: C,
56.59; H, 4.71; N, 18; Found: C, 55.98; H, 4.24; N, 17.55.
The synthesis of isoxazolone and pyrazolone derivatives was car-
ried out as per Scheme 1. The purity of all the compounds was con-
firmed with the help of TLC and elemental analysis data (LECO-183
CHNS analyzer). FTIR (Bruker FTIR Spectrometer Alpha II), 1HNMR,
and 13C NMR (Bruker Spectrophotometer DRX400, TMS as standard)
data were used to verify the structure of synthesized compounds.
5.1.5
|
3-methyl-4-[(Z)-(4-nitrophenyl) diazenyl]-1,
2-oxazol-5(4H)-one (3b)
Yield: 59.2%; m.p: 225–230 ꢀC; Rf: 0.74 (ethylacetate: pet-ether 3:1),
0.57 (chloroform: methanol 9:1); IR cm−1:1,745 (C O), 1,628 (C N),
1,592 (C C), 1,551 (N N); 1H-NMR (DMSO, 400 MHz, δ ppm): 7.90–
7.27 (m, 4H, Aryl H), 3.80 (s, 1H, CH), 2.13 (s, 3H, CH3);13CNMR
(DMSO-d6, 100 MHz, δ ppm): 152.5, 140.5, 124.0, 124.0, 122.0,
122.0 (Ar C), 165.2,149.9, 53.2 (isoxazolone-C), 15.9 (isoxazole-
CH3); Anal. Calcd. For C10H8N4O4: C, 48.3; H, 3.22; N, 22.56; Found:
C, 47.98; H, 3.09; N, 21.55.
5.1.1
|
General procedure for the synthesis of
substituted azo ethyl acetoacetate compounds
Substituted aniline (0.01 mol) was dissolved in concentrated HCI
(10 ml) and cooled to 0–5 ꢀC. To this solution, 0.01 mol of sodium
nitrite dissolved in 10 ml water was added and stirred vigorously for
an hour to prepare diazonium salt. The clear solution of diazonium salt
was added dropwise to an ice-cooled solution of ethyl acetoacetate
(0.01 mol) and anhydrous sodium acetate (2 g in 10 ml distilled water)
in ethanol (20 ml). The stirring was continued for further 3 hr at room
temperature until the solid separated, which was filtered, dried, and
recrystallized from ethanol to get the corresponding hydrazono deriv-
atives 2 a–d (Goda, Maarouf, & El-Bendary, 2003).
5.1.6
|
4-[(Z)-(3-methyl-5-oxo-4, 5-dihydro-1, 2-
oxazol-4-yl) diazenyl] benzoic acid (3c)
Yield: 68%; m.p: 198–201 ꢀC; Rf: 0.75 (ethylacetate: pet-ether 3:1),
0.59 (chloroform: methanol 9:1); IR cm−1: 1,759 (C O), 1,633 (C N),
1,598 (C C), 1,560 (N N); 1H-NMR (DMSO, 400 MHz, δ ppm): 7.68–
7.14 (m, 4H, Aryl H), 3.24 (s,1H, CH), 2.03 (s, 3H, CH3);13CNMR
5.1.2
|
General procedure for preparation of
(DMSO-d6,
100 MHz,
δ
ppm):169.0
(COOH),
149.0,
substituted phenyl azo isoxazol-5-ones 3(a–d)
134.0,134.0,120.4, 120.4, 119.5 (Ar C), 167.5, 150.2, 55.5 (iso-
xazolone-C), 13.5 (isoxazole-CH3); Anal. Calcd. For C11H9N3O4: C,
53.39; H, 3.64; N, 16.99; Found: C, 52.99; H, 3.09; N, 16.50.
To a suspension of compound 2 (0.01 mol) in ethanol, 0.01 mol of
hydroxylammonium chloride and anhydrous sodium acetate (2 g) were
added. The reaction mixture was refluxed for 4–5 hr until the reaction
completed. Cooling to room temperature furnished solid, which was
collected by filtration, dried, and recrystallized from ethanol (Goda
et al., 2003).
5.1.7
|
4-[(Z)-(4-chlorophenyl) diazenyl]-3-methyl-
1, 2-oxazol-5(4H)-one (3d)
Yield: 78%; m.p: 202–205 ꢀC; Rf: 0.83 (ethylacetate: pet-ether 3:1),
0.61 (chloroform: methanol 9:1); IR cm−1: 1,756 (C O), 1,624 (C N),
1,604 (C C), 1,576 (N N); 1H-NMR (DMSO, 400 MHz, δ ppm): 7.14–
6.66 (m, 4H, Aryl H), 3.16 (s,1H, CH), 2.13 (s, 3H, CH3);13CNMR
(DMSO-d6, 100 MHz, δ ppm): 146.5, 132.5, 122.3, 122.3, 120.5,
120.5 (Ar C), 166.2, 150.9, 60.0 (isoxazolone-C), 13.5 (isoxazole-
CH3); Anal. Calcd. For C10H8CIN3O2: C, 50.49; H, 7.57; N, 17.67;
Found: C, 49.99; H, 7.07; N, 17.50.
5.1.3
|
General procedure for preparation of
substituted phenyl azo pyrozoline-5-ones (4a–d)
Hydrazine monohydrate (0.01 mol) was added to a suspension of
compound 2 (0.01 mol) in ethanol (30 ml). The reaction mixture was
heated under reflux for 5 hr. The formed solid was filtered off, dried,
and purified by recrystallization (Scheme 1) (Goda et al., 2003).
5.1.8
|
4-[(Z)-(4-methoxyphenyl) diazenyl]-5-
5.1.4
|
4-[(Z)-(4-methoxyphenyl) diazenyl]-3-
methyl-2, 4-dihydro-3H-pyrazol-3-one (4a)
methyl-1, 2-oxazol-5(4H)-one (3a)
Yield: 63%; m.p: 225–230 ꢀC; Rf: 0.81 (ethylacetate: pet-ether 3:1),
0.62 (chloroform: methanol 9:1); IR cm−1: 1,670 (C O), 1,636 (C N),
1,625 (N H), 1,596 (C C), 1,530 (N N); 1H-NMR (DMSO, 400 MHz,
δ ppm):11.48 (s,1H, NH), 7.50–6.99 (m, 4H, Aryl H), 3.77 (s, 3H,
Yield: 65%; m.p: 195–197 ꢀC; Rf: 0.62 (ethylacetate: pet-ether 3:1),
0.51 (chloroform: methanol 9:1); IR cm−1:1,755 (C O), 1,632 (C N),
1,551 (C C), 1,537 (N N); 1H-NMR (DMSO, 400 MHz, δ ppm): 7.52–

ANWAR ET AL.
9
OCH3), 3.34 (s,1H, CH), 2.14 (s, 3H, CH3);13CNMR (DMSO-d6,
100 MHz, δ ppm): 156.2, 153.2, 120.9, 120.9, 110.5, 110.5 (Ar C),
165.0, 155.2, 56.9 (isoxazolone-C), 57.2 (OCH3), 14.2 (isoxazole-CH3);
Anal. Calcd. For C11H12N4O2: C, 56.84; H, 5.16; N, 24.11; Found: C,
55.96; H, 5.07; N, 24.00.
test compounds (10 mg) and incubated in darkness for 30 min at room
temperature. The radical scavenging potential of the compounds was
determined by checking the absorbance at 517 nm using a UV spec-
trophotometer. The decrease in absorbance with
a subsequent
increase in the concentration of the test compound results in an
increase in free radical scavenging. The conversion of the blue color
to yellowish-orange color showed scavenging of the free radicals. Per-
centage scavenging activity was calculated by the following equation;
5.1.9
|
5-methyl-4-[(Z)-(4-nitrophenyl) diazenyl]-2,
4-dihydro-3H-pyrazol-3-one (4b)
ðA−BÞ
%Scavenging =
× 100
A
Yield: 75%; m.p: 195–198 ꢀC; Rf: 0.68 (ethylacetate: pet-ether 3:1),
0.53 (chloroform: methanol 9:1); IR cm−1: 1,665 (C O), 1,628 (N H),
1,627 (C N), 1,599 (C C), 1,590 (N N); 1H-NMR (DMSO, 400 MHz,
δ ppm): 10.79 (s,1H, NH), 7.86–7.37 (m, 4H, Aryl H), 3.70 (s,1H,
CH), 2.29 (s, 3H, CH3);13CNMR (DMSO-d6, 100 MHz, δ ppm):
152.1, 145.5, 122.3, 122.3, 120.5, 120.5 (Ar C), 169.1, 155.0, 56.2
(isoxazolone-C), 14.5 (isoxazole-CH3); Anal. Calcd. For C10H9N5O3: C,
48.54; H, 3.64; N, 28.31; Found: C, 47.69; H, 3.00; N, 28.00.
where A = absorbance of negative control, B = absorbance of test
sample.
5.3
|
Analgesic activity
Central anti-nociceptive activity of compounds 3(a–d) and 4(a–d) was
assessed by hot plate assay. Mice were separated into three groups
with Group 1 used as a negative control (saline group, 10 ml/kg, i.p.),
group 2 received dose of test compounds (6 mg/kg, i.p.), while group
3 served as a positive control (tramadol, 6 mg/kg, i.p.). Mice were
administered with test compounds and standard drugs, respectively.
Thirty minutes after injecting the drugs, mice were shifted to a hot
plate (TWCLB2180, Henan Aibote) maintained at 55 ꢀC and potential
to a pain reaction (flicking of rear paw, licking or jumping) was noted
in seconds, evaluating their reaction to a thermal stimulus. To avoid
thermal paw injury cut off time was set as 30 s. The observations
were taken at 0, 30, 60, and 120 min after drug administration. The
latency time in seconds was then compared with standard drug-
treated animals (Mathew, Keshavayya, Vaidya, & Giles, 2007).
5.1.10
|
4-[(Z)-(3-methyl-5-oxo-4, 5-dihydro1H
pyrazol -4-yl) diazenyl] 6benzoic acid (4c)
Yield: 60%; m.p: 270–274 ꢀC; Rf: 0.64 (ethylacetate: pet-ether 3:1),
0.50 (chloroform: methanol 9:1); IR cm-1: 1,675 (C O), 1,622 (C N),
1,620 (N H), 1,597 (C C), 1,565 (N N); 1H-NMR (DMSO, 400 MHz,
δ ppm): 11.09 (s,1H, NH), 7.86–7.37 (m, 4H, Aryl H), 4.17 (s,1H,
CH), 1.90 (s, 3H, CH3); 13CNMR (DMSO-d6, 100 MHz, δ ppm):
169.0 (COOH), 149.0, 134.0, 134.0, 120.4, 120.4, 119.5 (Ar C),
167.5, 150.2, 55.5 (isoxazolone-C), 13.5 (isoxazole-CH3); Anal. Calcd.
For C11H10N4O3: C, 53.61; H, 4.06; N, 22.76; Found: C, 52.69; H,
3.90; N, 22.00.
5.1.11
|
4-[(Z)-(4-chlorophenyl) diazenyl]-5-
5.4
|
Docking studies
methyl-2, 4-dihydro-3H-pyrazol-3-one (4d)
5.4.1
|
Chemo-informatics
Yield: 70%; m.p: 225–227 ꢀC; Rf: 0.77 (ethylacetate: pet-ether 3:1),
0.55 (chloroform: methanol 9:1); IR cm-1: 1,680 (C O), 1,631 (C N),
1,602 (C C), 1,595 (N H), 1,556 (N N); 1H-NMR (DMSO, 400 MHz,
δ ppm): 12.14 (s, 1H, NH), 7.62–7.18 (m, 4H, Aryl H), 3.73 (s,1H,
CH), 2.12 (s, 3H, CH3);13CNMR (DMSO-d6, 100 MHz, δ ppm):
152.5, 126.5, 125.3, 125.3, 114.0,114.0 (Ar C), 165.3, 150.2, 56.2
(isoxazolone-C), 15.3 (isoxazole-CH3); Anal. Calcd. For C10H9CIN4O:
C, 50.70; H, 3.80; N, 23.66; Found: C, 49.69; H, 3.00; N, 23.00.
ChemSketch
and
Molinspiration
Cheminformatics
(Che-
minformatics, 2013), an online freeware tool, were used for the
chemo-informatic analysis of the synthesized compounds. Different
parameters including molecular formula, molecular weight, number of
hydrogen bond donors and acceptors, LogP value, PSA, molar refrac-
tivity, density, surface tension, polarizability, molar volume, and
Lipinski rule validation were calculated and reported. A structure of a
single-molecule is first uploaded to this online tool in mol format,
which generates an output in the form of these parameters.
5.2
|
Antioxidant assay
Antioxidant activity of newly synthesized compounds was performed
using DPPH free radical scavenging assay (Molyneux, 2004). Ascorbic
acid served as a positive control, and methanol was taken as a nega-
tive control. 1 ml of DPPH solution (1 mM) was added to each of the
5.4.2
|
Retrieval of protein structure from PDB
The crystal structures of human cyclooxygenases, COX-1 (PDBID:
3N8X), and COX-2 (PDB ID: 3NL1) were retrieved from the Protein

10
ANWAR ET AL.
Data Bank (PDB) (Berman et al., 2000). The hydrophobicity and Ram-
achandran graphs were generated by Discovery Studio 4.1 Client (Dis-
covery Studio Visualizer Software, 2012). The protein architecture
and statistical percentage values of helices, beta-sheets, coils, and
turns were accessed by VADAR 1.8 (Willard et al., 2003).
saved in pdb format using PyMol Software (DeLano, 2002). The struc-
tures of these ligands were redrawn separately using Discovery Studio
Visualizer and saved in pdb format. These drawn structures of ligands
were docked with their respective protein using identical parameters as
other synthesized compounds and the poses obtained were overlapped
with the extracted co-crystallized poses of the ligands.
5.4.3
|
Preparation of protein
5.4.7
|
Docking
Water molecules, co-crystallized inhibitor, and other co-crystallized
molecules were deleted from the structures of COX-1 (PDB code:
3N8X) and COX-2 (PDB code: 3LN1) obtained from the protein
databank, using Discovery Studio Visualizer (Discovery Studio Visual-
izer Software, 2012). These structures were protonated using the
AutoDock Tools program (Morris et al., 2009), and thereafter the
obtained structures were energy minimized and saved in pdbqt
format.
The synthesized ligands, 3a, and 4b, were docked separately against
COX-1 and COX-2 with exhaustiveness value 50 using
=
AutoDockVina Software (Trott & Olson, 2010). The predicted docked
complexes were evaluated based on the lowest binding energy (kcal/
mol) values, and the three-dimensional graphical depictions of all the
docked complexes were accomplished by Discovery Studio (2.1.0)
(Cheminformatics, 2013).
CONFLICT OF INTEREST
5.4.4
|
Prepation of ligand
The authors declare no potential conflict of interest.
The synthesized compounds (3b, 4b) and the reference (Tramadol)
were drawn and energy minimized in Discovery Studio Visualizer soft-
ware (Discovery Studio Visualizer Software, 2012) and saved in pdb
format. For each compound, the most stable conformation was uti-
lized in docking calculation, and the AutoDock Tools program was
used to generate the docking input files in pdbqt format (Morris
et al., 2009).
ORCID
Humaira Nadeem
ArifUllah Khan
https://orcid.org/0000-0002-0169-0811
https://orcid.org/0000-0003-2959-0414
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How to cite this article: T Anwar, H Nadeem, S Sarwar, et al.
Investigation of antioxidant and anti-nociceptive potential of
isoxazolone, pyrazolone derivatives, and their molecular
docking studies. Drug Dev Res. 2020;1–11. https://doi.org/10.
1002/ddr.21711