Investigation of indole functionalized pyrazoles and oxadiazoles as anti-inflammatory agents: Synthesis, in-vivo, in-vitro and in-silico analysis

Article abstract of DOI:10.1016/j.bioorg.2021.105068

There are several potential side and adverse effects are found to be associated with the anti-inflammatory drugs in clinical practice. The long-term use of these clinical agents highly unsafe. It encouraged the development of novel heterocyclic compounds with potential anti-inflammatory activity and low to no toxicity. In present investigation, a total of 12 indole functionalized pyrazole and oxadiazole derivatives were designed, synthesized and evaluated for the in-vivo anti-inflammatory and analgesic potential. These compounds displayed comparable anti-inflammatory and analgesic potential to the reference drugs. Finally, molecular docking analysis was performed considering different anti-inflammatory targets to determine the mechanistic target of the designed molecules. Detailed analysis suggested that the molecules inhibit COX-2, preferably over other anti-inflammatory targets. The results suggested that two compounds (15c and 15f) were found promising candidates for the development of novel anti-inflammatory agents.

Full text of DOI:10.1016/j.bioorg.2021.105068

Bioorganic Chemistry 114 (2021) 105068
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Investigation of indole functionalized pyrazoles and oxadiazoles as
anti-inflammatory agents: Synthesis, in-vivo, in-vitro and in-silico analysis
a
,
2
a,
2
a,
1
b
Devendra Kumar , Ravi Ranjan Kumar , Shelly Pathania , Pankaj Kumar Singh ,
c
a,*
Sourav Kalra , Bhupinder Kumar
a
Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Ghal Kalan, Ferozpur G.T. Road MOGA-142001, Punjab, India
Integrative Physiology and Pharmacology, Institute of Biomedicine, Faculty of Medicine, University of Turku, FI-20520 Turku, Finland
Department of Pharmaceutical Technology (Process Chemistry), National Institute of Pharmaceutical Education and Research, SAS Nagar, Sector 67, Mohali, Punjab
60062, India
b
c
1
A R T I C L E I N F O
A B S T R A C T
Keywords:
There are several potential side and adverse effects are found to be associated with the anti-inflammatory drugs
in clinical practice. The long-term use of these clinical agents highly unsafe. It encouraged the development of
novel heterocyclic compounds with potential anti-inflammatory activity and low to no toxicity. In present
investigation, a total of 12 indole functionalized pyrazole and oxadiazole derivatives were designed, synthesized
and evaluated for the in-vivo anti-inflammatory and analgesic potential. These compounds displayed comparable
anti-inflammatory and analgesic potential to the reference drugs. Finally, molecular docking analysis was per-
formed considering different anti-inflammatory targets to determine the mechanistic target of the designed
molecules. Detailed analysis suggested that the molecules inhibit COX-2, preferably over other anti-inflammatory
targets. The results suggested that two compounds (15c and 15f) were found promising candidates for the
development of novel anti-inflammatory agents.
Anti-inflammatory
Antioxidants
Indole derivatives
Oxadiazole derivatives
COX-2 inhibitors
1
. Introduction
Inflammation is a protective mechanism that defends the human
overexpression of hepatic acute-phase proteins such as serum amyloid A
and C-reactive protein. It leads to the leucocytosis and thrombocytosis
processes and causes the synthesis of adhesion molecules in endothelial
cells [12]. Arachidonic acid is another key component present in cell
membranes which causes the activation of other key inflammatory
mediator known as eicosanoids [13,14]. Other inflammatory mediators
also include 5-lipoxygenase, 12- lipoxygenase, and cyclooxygenase
[15,16]. Steroidal anti-inflammatory drugs are the first class of drugs
used for the treatment of inflammation but use of these drugs is asso-
ciated with severe side/adverse effects [17]. These adverse effects are
reduced or minimized by non-steroidal anti-inflammatory drugs
(NSAIDs) but other adverse effects such as gastric ulcers and renal
function impairments mark questions on their use [18–20]. Such prob-
lems associated with NSAIDs were fixed by the discovery of selective
COX-2 inhibitors [21,22] but long-term use of these COX-2 inhibitors
produced serious side effects such as cardiovascular complications
[23,24] due to decreased level of anti-thrombotic prostaglandins
[25,26].
body from any injury or damage to its cells by any harmful foreign
particles or stimuli [1–4]. Pain, heating sensation, swelling, and redness
are major signs of inflammation in any tissue which ultimately alters the
tissue function [5]. During inflammation, various microcirculatory
events take place such as white blood cell recruitment, alteration in
vascular permeability, production and release of pro-inflammatory/
inflammatory mediators, tissue destruction etc. [6,7]. Interleukin (IL)-
1
β is one of the key inflammatory mediators and helps in production of
potent pro-inflammatory cytokines [8,9]. It helps in various acute phase
inflammation reactions in body [10]. In case of acute inflammation,
body can restore tissue homeostasis and its functions [11], but in some
cases, it becomes chronic and continuous inflammatory condition affects
the tissue and body functions of a person to great extent. In cases where
IL-1β level is low, the release of adrenocorticotropic hormone and for-
mation of cytokines such as IL-6 is triggered which leads to
*
Corresponding author.
E-mail addresses: shellpathania91@gmail.com (S. Pathania), bhupinderkumar@isfcp.org (B. Kumar).
Co-corresponding author.
1
2
Both authors have equal contribution.
https://doi.org/10.1016/j.bioorg.2021.105068
Received 22 May 2021; Received in revised form 1 June 2021; Accepted 5 June 2021
Available online 8 June 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
From NSAIDs, Indomethacin is one of the most promising anti-
detailed pharmacodynamics.
inflammatory agents. It is found to possess good potency in the treat-
ment of various inflammatory disorders such as rheumatoid arthritis,
ankylosing spondylitis, osteoarthritis etc. However, it is also associated
with serious gastric ulcer problems due to its acidic nature and high
affinity for COX-1 inhibition. Thus, the development of new potential
anti-inflammatory drug molecules with no or minimal adverse effect is
still a need of the hour. Despite hard work and exhaustive research ef-
forts worldwide a set of enormously important fundamental questions
associated with inflammation mechanism is unresolved [27]. In case of
indomethacin, one strategy to minimize adverse effects is to perform
structural modifications at acid group present at C3 position while
retaining the indole pharmacophore. From literature survey, it is found
that structural modification at C3 position of 2-phenylindole presents a
potent pharmacophore for anti-inflammatory drug development. The
resultant compounds are sought to possess more potent anti-
inflammatory and antioxidant properties and improved LOX inhibition
2. Results and discussion
2.1. Chemistry
For synthesis of pyrazoline derivatives of indole (9a-9c), firstly 2-
phenylindole-3-carboxaldehyde (5) was prepared by reaction of aceto-
phenone and phenylhydrazine which were further reacted with
Vilsmeier-haack reagent to give 5. It was further reacted with suitable
acetophenones to synthesize chalcones (7a, b) which on reaction with
appropriate hydrazine yielded target pyrazoline derivatives of indole
(9a-9c) as per synthetic Scheme 1. Similarly, for synthesis of oxadiazole
derivatives of indole (15a-15i), indole-3-acetic acid (10) was used as
starting material. Indole-3-acetic acid (10) was first esterified with
ethanol which on further treatment with hydrazine hydrate produced
indole-3-acetic hydrazide (13). This indole-3-acetic hydrazide (13) was
[
28]. On other hand, Celecoxib, containing pyrazoline moiety, is a well-
known selective COX-2 inhibitor used for the treatment of inflammation
29,30]. The anti-inflammatory and pain-relieving properties of cele-
reacted with suitable benzoic acids in presence of POCl under suitable
3
conditions to give target oxadiazole derivatives of indole (15a-15i) as
[
per synthetic Scheme 2. The synthesized compounds were characterized
using Mass, IR, and NMR spectroscopic analysis.
coxib result from inhibition of prostaglandin (PG) synthesis by selective
inhibition of PG G/H synthase-2 (encoded by gene PTGS2). Similarly,
1
,3,4-oxadiazole is also found to possess significant anti-inflammatory
3
. In-vivo studies
potential [31,32]. 2-Aryl-5-subtituted-1,3,4-oxadiazole is a key phar-
macophoric component with anti-inflammatory activity [33–35]. Thus,
considering the above-mentioned information and taking forward the
ongoing research efforts related to the design and development of potent
anti-inflammatory agents, we designed and synthesized a library of
indole functionalized pyrazole and oxadiazole derivatives, as described
in Fig. 1. Further, we evaluated them for their anti-inflammatory po-
tential along with analgesic and antioxidant properties. Finally, in-silico
analysis was performed to better understand the binding modes of the
designed molecules in the catalytic domain of various possible anti-
inflammatory targets. Most of the compounds were found to possess
moderate anti-inflammatory and analgesic properties along with a good
antioxidant profile, which warrants further study of these molecules for
3
.1. Anti-inflammatory activity
Carrageenan test is greatly sensitive to clinically useful NSAIDs and
has been widely accepted as
a useful model to measure anti-
inflammatory drugs. Carrageenan injection generated intense inflam-
mation (edema as being the principal symptom) which peaked between
2
and 4 h and is attributed to release of inflammation related mediators,
which is the moment when its maximum effect is demonstrated and the
moment when the anti-inflammatory effect of the test product is best
observed. The pharmacological results listed in Table 1 represents the
mean changes in paw edema volume mL ± SD of animals pretreated with
the reference drugs and test compounds after 1 h, 2 h and 4 h from the
Fig. 1. Designing strategy of indole derivatives as anti-inflammatory agents.
2

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
Scheme 1. Synthetic route for the synthesis of the title compounds (9a-c).
Scheme 2. Synthetic route for the synthesis of the title compounds (15a-i).
induction of inflammation, together with the percent inhibition of
induced rat paw edema by the test compounds. Statistical differences of
control, reference and test groups were carried out using F test (ANOVA)
followed by post hoc test. The screened results revealed that strong in-
hibition of edema was observed after 4 h. The tested compound 15f
showed significant anti-inflammatory activity (% edema reduction =
the compounds. A significant reduction of painful sensation due to tail
immersion in warm water was observed following the administration of
the test compounds. The effect was prominently noticed after a latency
period of 2 h. Overall, the reaction time significantly increased in rats
receiving the test compounds 15c and 15f indicating a potent analgesic
effect. The analgesic activity of the compounds was done at the same
dose as used for anti-inflammatory activity. The detailed results of the
test compounds along with the positive control, diclofenac, are shown in
Table 2.
7
4.07%) comparable to that of indomethacin (92.59% edema reduction)
and higher than rest of the molecules. Interestingly, after 1 h of dose
administration 15b is showing the topmost % inhibition amongst all
compounds in paw edema, but after 2 h there is not much improvement
in the % inhibition value which suggests that the compound might have
been inactivated/metabolised. 15b is having unsubstituted aryl ring
which could be more prone to metabolism and probably, therefore it is
showing lower activity after 4 h.
3
.3. In-vitro COX inhibitory activity
Considering the molecular modelling results, selected compounds
(
15a-i) were evaluated for in vitro COX inhibitory activity and their
selectivity using COX inhibitor screening assay kit against ovine COX-1
and human COX-2. The percentage inhibition of enzyme at 10 µM
concentration of compounds is expressed in Table 3 (n = 2). From re-
sults, it was observed that most of the compounds display COX-2
3
.2. Analgesic activity
Tail-flick method was used to determine the analgesic potential of
3

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
Table 1
Table 2
In vivo analgesic activity of tested compounds using tail-flick method.
In vivo anti-inflammatory activity of tested compounds using carrageenan-
induced paw edema in rats.
Compound
Predrug (mean ± ±
SEM) Reaction time
in (sec)
Reaction time in sec (mean ± ±SEM)
Compound
Normal
paw
Mean paw volume ± ±SEM (mL) and % inhibition
3
0 min
1 h
2 h
3 h
Time after carrageenan injection
volume
Vehicle
2.58 ± 0.24
2.98 ± ±0.30
2.35 ± 0.24
2.11 ± 0.33
2.29 ± 0.26
2.17 ± 0.19
2.41 ± 0.24
2.65 ± ±0.29
2.39 ± 0.31
2.24 ± 0.33
2.39 ± ±0.17
2.35 ± 0.15
2.37 ± 0.13
2.57 ± 0.36
2.76 ±
.44
7.50 ± ±
.37
4.92 ±
.31
3.35 ±
.24
4.15 ±
.11
5.28 ±
.28
3.25 ±
.14
4.95 ± ±
.34
4.45 ±
.24
5.10 ±
.44
4.28 ± ±
.37
4.38 ±
.19
3.31 ±
.45
4.39 ±
.28
2.78 ±
0.37
2.85 ±
0.23
2.82 ±
0.41
(
mL)
0 h
1 h
2 h
4 h
0
Diclofenac
7.75 ± ±
0.47
8.91 ± ±
0.27
8.31 ± ±
0.42
Control
0.016 ±
.006
0.013 ±
.004
0.049 ±
0.007
0.049 ±
0.008
0.046 ±
0.004
0.043 ±
0.006
0
0
9
9
9
1
1
1
1
1
1
1
1
1
a
5.30 ±
0.33
5.33 ±
0.29
5.13 ±
0.44
Indomethacin
0.042 ±
0.004
0.036 ±
0.006
0.033 ±
0.004
0.015 ±
0.004
0
0
b
4.74 ±
0.17
5.32 ±
0.22
5.11 ±
0.39
(
12.12%)
(30.30%)
0.038 ±
0.005
(33.34%)
0.031 ±
0.005
(92.59%)
0.027 ±
0.005
0
9
9
9
1
1
1
1
1
1
1
1
1
a
0.012 ±
.003
0.043 ±
c
4.97 ±
0.22
5.34 ±
0.27
5.11 ±
0.38
0
0.004
0
(
6.06%)
(21.21%)
0.039 ±
0.006
(36.67%)
0.035 ±
0.005
(44.44%)
0.026 ±
0.004
5a
5b
5c
5d
5e
5f
5g
5h
5i
4.88 ±
0.17
5.15 ±
0.26
5.07 ±
0.47
b
0.011 ±
.005
0.044 ±
0
0
0.005
4.77 ±
0.37
5.21 ±
0.24
4.76 ±
0.29
(
0.0%)
(15.15%)
0.038 ±
0.004
(20.0%)
0.033 ±
0.006
(44.44%)
0.030 ±
0.007
0
c
0.010 ±
.006
0.046 ±
5.44 ± ±
0.37
7.69 ± ±
0.20
6.39 ± ±
0.37
0
0.005
0
(
-9.09%)
(15.15%)
0.037 ±
0.005
(23.33%)
0.034 ±
0.004
(25.92%)
0.028 ±
0.006
4.77 ±
0.17
5.31 ±
0.29
5.03 ±
0.27
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.012 ±
.005
0.042 ±
0
0
0.006
5.24 ±
0.37
5.71 ±
0.25
5.12 ±
0.17
(
9.09%)
(24.24%)
0.035 ±
0.006
(26.67%)
0.033 ±
0.005
(40.74%)
0.026 ±
0.005
0
0.013 ±
.007
0.045 ±
5.15 ± ±
0.33
6.98 ± ±
0.28
6.08 ± ±
0.47
0
0.005
0
(
3.03%)
(38.88%)
0.036 ± ±
0.005
(33.34%)
0.026 ± ±
0.006
(51.85%)
0.021 ± ±
0.007
4.74 ±
0.27
5.41 ±
0.32
4.97 ±
0.42
0.011 ± ±
.005
0.039 ± ±
0
0
0.005
4.44 ±
0.37
5.21 ±
0.30
4.54 ±
0.27
(
15.15%)
(24.24%)
0.039 ±
0.007
(50.0%)
0.035 ±
0.004
(62.96%)
0.025 ±
0.006
0
0.012 ±
.003
0.041 ±
4.94 ±
0.47
5.47 ±
0.22
5.02 ±
0.16
0
0.006
0
(
12.12%)
(18.18%)
0.037 ±
0.004
(23.33%)
0.034 ±
0.005
(51.85%)
0.024 ±
0.004
0.010 ±
.005
0.043 ±
0
0.004
(
0.0%)
(18.18%)
0.035 ± ±
0.006
(20.0%)
0.024 ± ±
0.005
(48.14%)
0.020 ± ±
0.006
Table 3
In vitro COX % inhibition studies.
0.013 ± ±
.003
0.038 ± ±
0
0.004
(
24.24%)
(33.33%)
0.037 ±
0.007
(63.34%)
0.031 ±
0.003
(74.07%)
0.027 ±
0.003
a
Compound
% COX inhibition
COX-1
SI
0.011 ±
.006
0.040 ±
0
0.005
COX-2
(
12.12%)
(21.21%)
0.038 ±
0.005
(33.34%)
0.034 ±
0.006
(40.74%)
0.029 ±
0.003
1
1
1
1
1
1
1
1
1
5a
5b
5c
5d
5e
5f
15.26
28.21
42.26
26.89
32.27
25.69
17.89
49.18
36.44
36.40
90.64
24.81
55.10
63.23
40.83
35.36
56.31
32.38
27.88
33.81
29.20
48.58
1.62
1.95
1.49
1.52
1.09
2.19
1.81
0.57
0.93
0.80
0.53
0.012 ±
.004
0.041 ±
0
0.005
(
12.12%)
(21.21%)
0.036 ±
0.006
(26.67%)
0.032 ±
0.007
(37.03%)
0.028 ±
0.006
0.013 ±
.005
0.042 ±
0
0.007
(
12.12%)
(30.3%)
(36.67%)
(44.44%)
5g
5h
5i
selectivity except compounds 15h and 15i which were found to be COX-
selective. Compound 15f was found to be most selective COX-2 in-
b
Indomethacin
c
1
Diclofenac
hibitor with selectivity index of 2.19 followed by compound 15b (1.95).
At concentration of 10 µM, compound 15c showed most potent inhibi-
tion of COX-2 with 63.23% with selectivity index of 1.49. While, 15g
although has good SI but still shows low inhibitory potential against
COX-2 and probably therefore it is also showing lower inhibitory po-
tential in animal study. Another observation made is improved %
inhibitory profile of 15d in animal study however, the compound
showed only sub-optimal levels of inhibition in both isoforms of COX.
Limited SAR explicitly indicated the structural features which could be
considered responsible for the compounds which are more active against
COX-2 over COX-1. In general, electron-withdrawing substituent con-
taining compounds showed an improved COX-2 inhibitory profile.
While electron-donating substituent containing compounds showed less
inhibition of COX-2. Compound 15a, a benzyl derivative which does not
retain the planar conformation also showed poor inhibitory potential
than other compounds of the class. Conversely, in case of COX-1, elec-
tron-donating substituent containing compounds (15h & 15i) fared
better than electron-withdrawing substituent containing compounds.
Accordingly, almost all the compounds showed good selectivity amongst
COX-1 and COX-2, except for 15e, which showed similar levels of in-
hibition in both the isoforms.
The enzyme inhibition for COX was calculated at 10 µM concentration for each
compound.
SI (Selectivity index) = COX-1 IC50/COX-2 IC50
.
a
%
inhibition is represented as mean value of two determinations (deviation
less than is < 10%).
b
c
The results are taken from reference [36]. The results are taken from
reference [37] at 10 µg/mL.
3.4. Antioxidant activity
The synthesized compounds exhibited the potential to scavenge free
radicals in varying capacities in the DPPH assay. Activity was influenced
by substitutions on aryl ring coupled to the oxadiazole/pyrazole nu-
cleus. Compounds with halogens and methoxy group, emerged as better
antioxidants amongst all the compounds. This superiority may be due to
the presence of electron cloud on the aryl rings which can easily donate
electron to DPPH. The free-radical scavenging activity of the synthesized
compounds, which lack methoxy group is attributable to the presence of
the oxadiazole nucleus itself. Donation of hydrogen leaves the
4

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
compounds in their radical form and their structure gets stabilized
owing to electronic conjugation, thus favoring the reaction to occur.
IC50 of the synthesized derivatives was also calculated and compounds
targets [38], we utilized a detailed molecular docking analysis to un-
derstand the mechanism of action of these anti-inflammatory agents. All
four prominent targets (COX-1, COX-2, LOX-5 and LOX-15) were
therefore deliberated in this analysis against all 12 molecules. The
docking protocol was validated by re-docking the co-crystallized ligand
within the catalytic domain of specific selected target protein. It was
observed that the re-docked pose of co-crystallized ligand retained the
binding conformation to the co-crystallized pose with the RMSD value
of < 1 Å. To identify the anti-inflammatory mechanism of the designed
molecules, key interactions reported in previous studies as essential for
the inhibition of the selected target were considered along with the
obtained docking score for each hit.
1
5c, 15e and 15f were found to be the most potent antioxidant agents,
with IC50 (1.55, 2.48 and 2.51 µg/mL) comparable to ascorbic acid
1.18 µg/mL) (Table 4).
(
3
.5. SAR studies
Careful inspection of correlation between the structures of the
various synthesized molecules and the observed anti-inflammatory,
analgesic and antioxidant activity showed a relationship between the
varied substituents on the oxazole/pyrazole ring and the biological ac-
tivity. However, the first and foremost conclusion drawn from the SAR
observations was that the substitution on the 2nd position of indole
nucleus decreased both the anti-inflammatory and analgesic activity of
the synthesized compounds. Moreover, in case of anti-inflammatory
profile, the compounds such as 15c, 15d, 15e and 15f with electron-
withdrawing substituents on the aryl ring coupled to oxazole nucleus
showed improved activity. While, unsubstituted/benzylated derivatives
along with compounds having electron-donating substituents on the aryl
ring coupled to oxazole nucleus showed decreased anti-inflammatory
activity. In case of analgesic activity, a similar structure-activity pro-
file was observed, with compounds having electron-withdrawing sub-
stituents showed better activity over unsubstituted or electron-donating
group containing compounds. Overall, limited SAR derived from the
developed compounds highlighted the significance of electron-
withdrawing group as beneficial features for anti-inflammatory and
analgesic activity.
The collected data indicated that in the case of COX-1, none of the
analogs maintained the key H-bond interactions in the catalytic domain
as observed in the reported COX-1 inhibitors. However, most of them did
maintain crucial
π
- interaction with Tyr385 (Table 5) and showed good
π
estimated interaction energies (based on Glide docking scores) for the
docked poses, with docking score ranging from ꢀ 10.32 to ꢀ 9.54 Kcal/
mol. Nevertheless, failure to form crucial H-bonds suggests that mech-
anistically, these molecules do act via COX-1 inhibition. Against COX-2,
conversely, these molecules, when optimally docked, maintained the
required key H-bond interactions with Met522, and/or Ser530, along
with other crucial interactions and accordingly showed excellent dock-
ing scores ranging from ꢀ 11.12 to ꢀ 6.65 Kcal/mol. One crucial infor-
mation obtained during molecular docking analysis was that out of the
12 molecules, only 9 molecules belonging to indole-oxadiazole scaffold
could bind to catalytic domain of COXs, while remaining 3 molecules
belonging to indole-pyrazole scaffold were sterically unfavorable to
occupy the binding pocket in both COX-1 and COX-2. Thus, each of these
9
indole-oxadiazole analogs studied are predicted to undergo preferable
and stronger binding interactions with COX-2 over COX-1 binding
domain, while the 3 molecules belonging to indole-pyrazole scaffold are
not expected to act via any of the COX isoforms.
3
.6. Computational study
Considering the designing of the molecules which involves incor-
Extending the analysis to LOXs, the docking analysis with LOX-5
revealed that most of the compounds bind with the catalytic domain
porating different pharmacophoric substructures present in well-
established anti-inflammatory agents acting on different molecular
Table 4
Antioxidant activity of compounds (9a-c), (15a-i).
Compd. Code
Absorbance
Absorbance at 517 nm (% Reduction in absorbance)
g/mL g/mL g/mL
IC50
(μg/mL)
2
μ
4
μ
6
μ
8
μg/mL
10 μg/mL
Control
Abscontrol
Abssample
0.689
0.365
0.689
0.689
0.689
0.689
–
1.18
Ascorbic acid
0.289
0.218
0.189
0.114
(
47.02)
0.473
31.34)
0.448
34.97)
0.429
37.73)
0.415
39.76)
0.469
31.93)
0.401
41.79)
0.404
41.36)
0.439
36.28)
0.429
37.73)
0.439
36.28)
0.481
30.18)
0.449
34.83)
(58.05)
0.421
(68.35)
0.379
(72.56)
0.288
(83.45)
0.260
9
9
9
1
1
1
1
1
1
1
1
1
a
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
Abssample
3.30
2.70
2.57
2.45
3.01
1.55
2.53
2.48
2.51
2.55
2.97
2.61
(
(38.89)
0.406
(44.99)
0.332
(58.02)
0.269
(62.26)
0.201
b
(
(41.07)
0.402
(51.81)
0.309
(60.95)
0.259
(70.82)
0.209
c
(
(41.65)
0.393
(55.15)
0.301
(64.40)
0.252
(69.66)
0.211
5a
5b
5c
5d
5e
5f
5g
5h
5i
(
(42.96)
0.388
(56.31)
0.345
(63.42)
0.285
(69.37)
0.239
(
(43.68)
0.309
(55.15)
0.389
(49.92)
0.239
(65.31)
0.340
(58.63)
0.211
(69.37)
0.265
(65.31)
0.197
(71.40)
0.202
(
(
(43.54)
0.359
(50.65)
0.311
(61.53)
0.261
(70.68)
0.209
(
(47.89)
0.379
(54.86)
0.329
(62.11)
0.241
(69.66)
0.201
(
(44.99)
0.383
(52.24)
0.307
(65.02)
0.247
(70.82)
0.219
(
(44.41)
0.373
(55.44)
0.334
(64.15)
0.283
(68.21)
0.245
(
(45.86)
0.393
(51.52)
0.319
(58.92)
0.231
(64.44)
0.203
(
(42.96)
(53.70)
(66.47)
(70.53)
%
Reduction in absorbance = (Abscontrol ꢀ Abstest)/Abscontrol × 100.
The inhibitory concentration (IC50) value, representing the concentration required to exhibit 50% antioxidant activity. IC50 values were calculated by linear regression
of plots where the abscissa represented the concentration of the compounds (
μ
g/ml) and the ordinate, the average percentage of antioxidant activity.
5

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
Table 5
Results of molecular docking analysis for the indole functionalized pyrazoles/oxadiazoles.
S.
Compound
ID
COX-1 (1PGF)
COX-2 (6COX)
LOX-5 (3 V99)
LOX-15 (1IK3)
No.
Docking
Score
H-
Docking
score
H-bond
Docking
score
H-bond
Docking
core
H-bond
bond
9
9
9
1
a
–
–
–
–
–
–
–
–
–
ꢀ 4.725
ꢀ 4.69
Gln557, Gln363
–
–
b
–
–
–
–
–
–
–
–
c
–
ꢀ 5.857
ꢀ 5.153
–
–
5a
ꢀ 8.983
Leu607, Fe701
ꢀ 8.01
Hie513, Gln514,
His518
15b
15c
15d
15e
ꢀ 9.841
¡9.965
ꢀ 10.296
ꢀ 9.818
–
ꢀ 9.826
¡11.126
ꢀ 6.652
ꢀ 8.649
Met522
Met522
–
ꢀ 4.289
¡3.924
ꢀ 5.081
ꢀ 3.504
Gln363, Fe701
Fe701
ꢀ 5.001
¡7.67
ꢀ 7.279
ꢀ 7.54
Hie513
–±
–
Gln514, His518
His518
Leu607, Fe701
Gln363, Fe701,
Phe610
–
Phe518
Gln514, Arg726
1
5f
¡9.546
–±
¡8.497
Gln192,
Leu352
–
Ser530
Ser530
¡5.344
Asn554
¡7.088
Hie513
1
1
1
5g
5h
5i
ꢀ 10.17
ꢀ 9.65
–
–
–
ꢀ 8.58
ꢀ 8.327
ꢀ 8.27
ꢀ 6.039
ꢀ 4.157
ꢀ 5.534
Asn554, Tyr558
Ala606
ꢀ 6.457
ꢀ 6.513
ꢀ 5.346
–
Hie513, Gln514,
Hie513
ꢀ 10.32
Val671
interacting with Fe2 ion present as the co-factor. Importantly, the 3
molecules belonging to indole-pyrazole scaffold bound well to the cat-
alytic site following a similar pose to the indole-oxadiazole analogs.
Careful inspection disclosed that the nitrogen atoms present in oxadia-
zoles/pyrazoles were involved in chelating/interacting with the Fe.
Overall, the molecules formed the key H-bond interactions with Gln363,
Asn554 and Leu607 and showed good estimated interaction energies
+
Diclofenac and Indomethacin. After 1 h interval in in vivo anti-
inflammatory studies, compound 15b showed maximum % inhibition
amongst all compound in paw edema, however after 4 h it was found
slightly less active which could be attributed to its metabolic liability.
15b is having unsubstituted aryl ring which could be more prone to
metabolism and probably, therefore it is showing lower activity after 4
h. While, 15g although has good SI but still shows low inhibitory po-
tential against COX-2 and probably therefore it is showing lower
inhibitory potential in animal study. Further, exhaustive structure-based
in-silico analysis disclosed that one class of molecules i.e., indole-
oxadiazole derivatives may act by selectively inhibiting COX-2, while
another class of molecules i.e., indole-pyrazole derivatives may act by
blocking LOX-5. Overall, the results suggested that two compounds, 15c
and 15f, can be further explored and developed as potent anti-
inflammatory agents.
(
based on Glide docking scores) for the docked poses, with docking score
ranging from ꢀ 6.03 to ꢀ 3.50 Kcal/mol. Further, the docking analysis
with LOX-15 revealed that most of the molecules make H-bond inter-
action with the histidine residues chelating with the co-factor. Also,
some of the molecules formed the crucial H-bond interaction with
Gln514 and showed good binding score (Glide score) ranging from
ꢀ
8.01 to ꢀ 5.00 Kcal/mol. It is also worth mentioning that although
many compounds showed better and preferable binding towards LOX-15
over LOX-5, the 3 molecules belonging to indole-pyrazole scaffold were
again sterically unfavorable to occupy the binding pocket in LOX-15 and
therefore can only be expected to act via LOX-5 as an anti-inflammatory
agent. The detailed results of docking scores for each of the indole-
oxadiazole analogs interacting with each of the anti-inflammatory tar-
gets are summarized in Table 5, and the 3D interaction diagrams of top
hits with all four targets are shown in Fig. 2.
5. Experimental
5.1. Chemistry
The chemicals were purchased from Sigma-Aldrich, SRL and Avra
and were used further without procurement. The solvents used in re-
actions were either dried or freshly distilled as per requirements and
procedures. The progress of reactions was monitored using pre-coated
silica TLC plates (Merck Silica-gel F254) using different mobile phases
like hexane-ethyl acetate or chloroform-methanol for TLC development.
The developed TLCs were visualized either under UV light at wave-
lengths of 254 nm and 360 nm or in iodine chamber. The melting points
of synthesized compounds were recorded using Veego melting point
apparatus and are uncorrected. 1H NMR and C NMR spectra were
recorded on Bruker Advance II instrument at 400 MHz frequency, in
Finally, in order to assess the drug-like characteristics of these
compounds, various physicochemical parameters of were evaluated
[
39]. Almost all the indole-oxadiazole based compounds showed good
drug-like properties and follow Lipinski’s rule of five, while indole-
pyrazole violated one of the lipinski’s rules (Table 6). The logP value
of most of the compounds was found to be less than five and hydrogen
bond donor and acceptor atoms are also less than five. Except a few, all
the compounds showed 100% human oral absorption in Qikprop
studies. The QP PCaco values were also in the optimum range and hence
these compounds are expected to possess optimum cell membrane
permeability. Thus, it can be concluded that most of the compounds
have an optimum drug-like profile and can be developed as potential
drug molecule.
13
CDCl
3
or DMSO and TMS (δ = 0) as an internal standard at PU (Punjab
University), Chandigarh. Chemical shifts have been expressed in δ
values downfield from TMS. The multiplicity of NMR signals is desig-
nated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) and
bs (broad singlet). Coupling constant (J) values are reported in hertz. IR
spectra were recorded on Thermofischer FT-IR spectrophotometer using
KBr pellets at ISF College of Pharmacy Moga.
4
. Conclusion
In summary, 12 molecules pertaining to indole-oxadiazole and
indole-pyrazole scaffolds were designed, synthesized and evaluated for
their in-vivo anti-inflammatory, analgesic and anti-oxidant activities. To
our delight, compounds belonging indole-oxadiazole scaffold demon-
strated moderate to excellent anti-inflammatory activities in vivo,
especially compounds 15c and 15f presented more promising and
comprehensive NSAID-like profile in carrageenan-induced paw edema
and tail flick experiments, in comparison to FDA-approved NSAID,
5.1.1. General procedure for the preparation of hydrazone (3)
A mixture of acetophenone (5 g, 41.61 mmol, 1) and phenyl-
hydrazine (5 g, 46.23 mmol, 2) were dissolved in ethanol (10 mL), taken
in a well dried round bottom flask and 5–7 drops of glacial acetic acid
was added into the reaction mixture. Then reaction mixture was stirred
for 1–2 hrs at room temperature. The reaction progress was monitored
by TLC. After completion of reaction, ethanol (4 mL) was added to the
6

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
Fig. 2. 3-D interaction diagrams for the indole functionalized pyrazoles/oxadiazoles within the binding cavity of target enzymes a) 15c in COX-1b) 15c in COX-2c)
1
5c in LOX-5 d) 15c in LOX-15 e) 15f in COX-1f) 15f in COX-2 g) 15f in LOX-5 h) 15f in LOX-15.
7

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
Table 6
Physicochemical properties of the indole functionalized pyrazoles/oxadiazoles.
S. No.
Compound ID
Mol. MW
QP logPo/w
HB donor
HB acceptor
QP PCaco
QP logKhsa
Percent Oral Absorption
Rule of Five
1
2
3
4
5
6
7
8
9
1
1
1
.
9a
379.46
5.688
6.265
7.868
4.248
3.742
4.291
4.237
3.049
2.968
3.838
2.754
3.852
1
3.5
3.5
1
3003.737
2998.352
6482.755
1720.586
1536.841
1840.676
1536.17
1.256
1.402
2.072
0.619
0.503
0.627
0.622
0.467
0.307
0.52
100
1
1
1
0
0
0
0
0
0
0
0
0
.
9b
458.356
413.521
289.336
275.309
354.205
309.754
320.307
291.309
305.335
290.324
305.335
1
100
.
9c
1
100
.
15a
15b
15c
15d
15e
15f
15g
15h
15i
1
2.5
2.5
2.5
2.5
3.5
3.25
3.25
3.5
3.25
100
.
1
100
.
1
100
.
1
100
.
1
182.956
85.289
92.079
100
.
2
465.831
0.
1.
2.
1
1568.368
399.854
2.5
1
0.238
0.523
89.638
100
1559.614
reaction mixture. The precipitated solid was then filtered, washed with
cold hexane and dried to afford the acetophenone phenylhydazone (3)
in good yield.
C6), 7.21 (dt, 1H, J
1
= 8 Hz, J
2
= 4 Hz, Indole-C5), 6.78 (t, 1H, J = 4
= 4 Hz, J = 12 Hz, Pyrazoline-H), 3.45
Hz, Pyrazoline), 3.87 (dd, 1H, J
(dd, 1H, J
= 4 Hz, J
NMR (175 MHz, DMSO‑d
1
2
1
3
1
2
= 12 Hz, Pyrazoline-H), 2.27 (s, 3H, CH
3
);
C
6
) δ(ppm): 169.37, 151.49, 138.78, 135.34,
5
.1.2. General procedure for the preparation of 2-Phenylindole (4)
132.23, 131.97, 129.65, 128.32, 128.11, 127.98, 127.87, 126. 85,
Compound (3) (2.5 g, 11.90 mmol) and 10 g of polyphosphoric acid
125.96, 124.13, 120.93, 120.04, 115.91, 110.86, 55.33, 40.45, 21.96;
þ
was heated for 1–2 hrs with continuous stirring. The reaction progress
was monitored by TLC. After completion of reaction, 160 mL of cold
water was added with well stirring. Precipitated solid was filtered under
the vacuum, washed with 2–3 times of cold water and cold ethanol,
dried to afford the 2-phenyl indole (4) in good yield.
MS: m/z [M] for C25
H
21
N
3
O, calculated 379.16; observed: 379.
5.1.5.2. 1-(3-(4-bromophenyl)-5-(2-phenyl-1H-indol-3-yl)-4,5-dihydro-
1H-pyrazol-1-yl)ethan-1-one (9b). Dark brown solid, Yield: 52%, mp:
188–190 ◦C, Rf 0.20, IR (KBr)
ν
max (cm- ): 3444 (N
1
H stretching),
–
–
H stretching), 2305(C
–
N stretching), 1638 (C–
O),
–
3
050 (aromatic C
1470 (C
–
C), 1209 (C
–
N), 665 (C-Br). 1H NMR (500 MHz, DMSO‑d )
6
5
.1.3. General procedure for the preparation of 2-Phenylindole-3-carbox-
aldehyde (5)
Compound 4 (5.5 mmol, 1 eq) was added to the Vilsmeier-haack
δ(ppm): 8.52 (s, 1H, NH), 7.85 (d, 1H, J = 8 Hz, Indole-C4), 7.51 (d, 1H,
J = 8 Hz, Indole-C7), 7.76 (d, 2H, J = 8 Hz, Ar-H attached to pyrazoline),
7.62 (d, 2H, J = 8 Hz, Ar-H attached to pyrazoline), 7.49–7.41 (5H, m,
Ar-H), 7.31 (dt, 1H, J = 8 Hz, J = 4 Hz, Indole-C6), 7.23 (dt, 1H, J = 8
reagent prepared by the addition of POCl
3
(3.8 eq) in DMF (5.1 mmol
◦
◦
of 4) at 0 C. The mixture was stirred at 0 C for 2 hrs to ensure the
complete consumption of starting material by TLC monitoring. Then
reaction was quenched slowly by adding ice and neutralized the reaction
mixture by adding the 37% of NaOH solution. The resulting precipitate
was filtered and dried to afford the 2-Phenylindole-3-carboxaldehyde
1
2
1
Hz, J = 4 Hz, Indole-C5), 6.75 (t, 1H, J = 4 Hz, Pyrazoline), 3.79 (dd,
2
1H, J = 4 Hz, J = 12 Hz, Pyrazoline-H), 3.38 (dd, 1H, J = 4 Hz, J =
1
2
1
2
1
3
12 Hz, Pyrazoline-H), 2.26 (s, 3H, CH ); C NMR (175 MHz, DMSO‑d )
3
6
δ(ppm): 169.42, 151.56, 138.91, 135.46, 132.41, 132.16, 129.83,
(
5) in good yield.
128.51, 128.28, 128.09, 128.01, 126.99, 126.16, 124.28, 121.07,
þ
1
20.21, 116.03, 110.97, 55.52, 40.57, 22.12. MS: m/z [M] for
5
.1.4. General procedure for the synthesis of Chalcone derivatives (7)
A mixture of 2-Phenylindole-3-carboxaldehyde (1 g, 4.51 mmol, 5),
3
C25H20BrN O, calculated 457.07; observed: 457.
acetophenone (0.5 mL, 4.51 mmol, 6) and piperidine (1.8 mL) was taken
in a well-dried round bottom flask and heated for 2–4 hrs, then ethanol
5
.1.5.3. 3-(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)-2-phenyl-1H-
◦
indole (9c). Brown solid, Yield: 65%, mp: 195–197 C, Rf 0.43, IR
1
(
7 mL), glacial acetic acid and water (1:1) were added to resulting red
max (cm- ): 3342 (N H stretching), 3050 (aromatic C
–H
–
(
KBr)
stretching), 2316 (C
500 MHz, DMSO‑d
ν
–
–
C), 1230 (C–
–
N stretching), 1565 (C N). H NMR
1
solution until first appearance cloudiness. The progress of the reaction is
monitored by TLC. The resulting product was filtered off and washed
with water, dried to afford the desired product (7).
–
6
(
) δ(ppm): 8.45 (s, 1H, NH), 7.83 (d, 1H, J = 8 Hz,
Indole-C4), 7.54 (d, 1H, J = 8 Hz, Indole-C7), 7.67 (d, 2H, J = 8 Hz, Ar-
H), 7.54–7.39 (m, 9H, Ar-H), 7.31–7.16 (m, 4H, Ar-H, Indole),
5
.1.5. General procedure for the synthesis of pyrazoline derivatives (9a-c)
6
1
1
1
1
1
4
.99–6.96 (m, 2H, Ar-H), 6.23 (t, 1H, J = 4 Hz, Pyrazoline), 3.83 (dd,
A mixture of Chalcone (500 mg, 1.54 mmol, 7a-b), hydrazine hy-
H, J
1
= 4 Hz, J
2
= 12 Hz, Pyrazoline-H), 3.47 (dd, 1H, J
1
= 4 Hz, J
2
=
1
3
drate (2.62 mmol, 1.7 eq, 8) or phenylhydrazine (3.09 mmol, 2 eq, 8a)
and glacial acetic acid (6 mL) were refluxed for 5–7 hrs continuously.
The reaction progress was monitored by TLC continuously using solvent
system 30% ethyl acetate and hexane (3:7). On completion of the re-
action (TLC monitoring) the resulting solution was cooled and neutral-
ized with 20% KOH solution. After adding the crushed ice into resulting
solution then precipitate was formed, filtered off, washed with cold
water and dried to afford the final product in good yield.
2 Hz, Pyrazoline-H); C NMR (175 MHz, DMSO‑d
6
) δ(ppm): 154.21,
45.19, 140.89, 137.11, 133.98, 132.87, 131.54, 129.13, 128.82,
28.11, 128.08, 127.46, 125.39, 122.03, 121.79, 121.37, 117.84,
þ
15.75, 112.18, 60.18, 40.88; MS: m/z [M] for C29
H
23
N
3
, calculated
13.19; observed: 413.
5
.1.6. General procedure for the synthesis of Indole ester (11)
Compound 10, Indole-3-acetic acid (5 g, 28.54 mmol), was taken in a
well dried round bottom flask containing ethanol (15 mL) and 10–12
drops of conc. sulfuric acid were added. The contents were refluxed for
2–3 hrs. The reaction progress was monitored by TLC. After completion,
the reaction mixture was cooled at room temperature and sodium
metasulphite was added to consume the excess acid. Then mixture was
filtered off and collected the filtrate. The excess solvent was evaporated
from the mixture to afford indole-3-acetic ester (11) in good yield and
used for further reaction without purification.
5
1
0
.1.5.1. 1-(3-phenyl-5-(2-phenyl-1H-indol-3-yl)-4,5-dihydro-1H-pyrazol-
◦
-yl)ethanone (9a). Light brown solid, Yield: 54%, mp: 190–192 C, Rf
1
.32, IR (KBr)
ν
max (cm- ): 3444 (N
–
H stretching), 3049 (aromatic
–
H stretching), 2350 (C
–
N stretching), 1620 (C
N). H NMR (500 MHz, DMSO‑d ) δ(ppm): 8.43 (s, 1H, NH),
.85 (d, 1H, J = 8 Hz, Indole-C4), 7.56 (d, 1H, J = 8 Hz, Indole-C7),
.47–7.39 (10H, m, Ar-H),7.28 (dt, 1H, J = 8 Hz, J = 4 Hz, Indole-
–
–
O), 1447 (C
–
–
C),
C
1
1
7
7
209 (C
–
6
1
2
8

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
5
.1.7. General procedure for the preparation of Indole-3-acetic hydrazide
13)
A mixture of Indole-3-acetic ester (5 g, 24.60 mmol, 11) and hy-
126.81, 125.40, 123.20, 122.51, 121.59, 119.94, 118.81, 111.35,
þ
(
108.17, 22.26. MS: m/z [M] for C17
H12BrN
3
O, calculated 353.01;
observed: 353.
drazine hydrate (147.60 mmol, 6 eq, 12) was taken in a well-dried round
bottom flask and refluxed for 2–3 hrs. The reaction progress was
monitored by TLC continuously using solvent system 30% ethyl acetate
and hexane (3:7). On completion of the reaction (TLC monitoring) the
resulting solution was cooled at RT and excess solvent was removed by
Rota evaporator. The resulting mixture was kept in the refrigerator for
5.1.8.4. 2-((1H-indol-3-yl)methyl)-5-(4-chlorophenyl)-1,3,4-oxadiazole
◦
(15d). Brown solid, Yield: 70%, mp: 180–182 C, Rf 0.56, IR (KBr)
1
ν
max (cm- ): 3447 (N
–
H stretching), 3226 (aromatic C
–
H stretching),
N), 1653 (aromatic C C),
O), 780 (C-Cl). 1NMR (500 MHz,
–
2991 (aliphatic C
–
H stretching), 2391 (C
–
–
1289 (C N stretching). 1135 (C
–
3
–4 hrs and then a crystal of ice was added and it initiated the crystal-
DMSO‑d ) δ(ppm): 8.43 (s, 1H, NH), 8.19 (s, 1H, Indole-C2), 8.03 (d, 2H,
6
lization of our product. The pure crystals of required product were
separated, dried to afford the Indole-3-acetic hydrazide (13) in high
yield and used for the further reaction.
J = 8 Hz, Ar-H), 7.85 (d, 1H, J = 8 Hz, Indole-C4), 7.74 (d, 1H, J = 8 Hz,
Indole-C7), 7.48 (d, 2H, J = 8 Hz, Ar-H), 7.21–7.17 (m, 2H, Indole-C5
1
3
and C6), 4.47 (s, 2H, CH ), C NMR (175 MHz, DMSO‑d ) δ(ppm):
2
6
1
65.87, 163.38, 136.18, 134.01, 133.93, 129.43, 128.05, 126.27,
5
.1.8. General procedure for the preparation of oxadiazole derivatives
15a-i)
A mixture of Indole-3-acetic hydrazide (1 g, 5.28 mmol, 13),
124.51, 123.70, 121.65, 120.43, 119.44, 111.45, 107.86, 23.34. MS: m/
þ
z [M] for C17H12ClN O, calculated 309.06; observed: 309.
3
(
different aromatic acids (5.28 mmol, 1 eq, 13) and phosphorus oxy-
5
.1.8.5. 2-((1H-indol-3-yl)methyl)-5-(4-nitrophenyl)-1,3,4-oxadiazole
chloride (5 mL) were taken in a well-dried round bottom flask and
◦
(
15e). Brown solid, Yield: 60%, mp: 198–200 C, Rf 0.7, IR (KBr)
νmax
◦
1
heated for 5–6 hrs at 60 C. The reaction progress was monitored by TLC
cm- ): 3404 (N H stretching), 3108 (aromatic C H stretching), 2849
– –
(
continuously using solvent system 20% acetone and hexane (2:8). On
completion of the reaction (TLC monitoring) the resulting mixture was
cooled at room temperature. The resulting mixture was neutralized by
adding the aqueous solution of sodium bicarbonate and mixture was
cooled in refrigerator to form the precipitate of required product. The
precipitate was filtered off, washed with cold water, dried and recrys-
tallized from aqueous ethanol to afford the final product in good yield.
(aliphatic C–
–
–
–
N), 1571 (aromatic C C), 1518
H stretching), 2316 (C
–
–O), 1190 (C
–
N stretching), 1030 (C
–O). 1
NMR (500
and 1340 (N
MHz, DMSO‑d
H), 8.14 (d, 2H, J = 8 Hz, Ar-H), 7.87 (d, 1H, J = 8 Hz, Indole-C4),
.76 (d, 1H, J = 8 Hz, Indole-C7), 7.16 (dd, 1H, J = 8 Hz, J = 4 Hz,
Indole-C6), 7.09 (dd, 1H, J = 4 Hz, Indole-C5), 4.49 (s, 2H,
= 8 Hz, J
), C NMR (175 MHz, DMSO‑d ) δ(ppm): 165.97, 163.48, 148.73,
36.60, 129.65, 127.47, 126.68, 125.29, 123.01, 122.35, 121.39,
6
) δ(ppm): 8.47 (s, 1H, NH), 8.21 (m, 3H, Indole-C2, Ar-
7
1
2
1
2
1
3
CH
2
6
1
þ
5
.1.8.1. 2-((1H-indol-3-yl)methyl)-5-benzyl-1,3,4-oxadiazole
(15a).
max
H stretching), 2950
111.23, 108.06, 23.78. MS: m/z [M] for C17H12N O , calculated
4 3
◦
Dark brown solid, Yield: 54%, mp: 163–165 C, Rf 0.32, IR (KBr)
ν
320.09; observed: 320.
1
(
(
(
cm- ): 3280 (N
aliphatic C H stretching), 2357 (C
N stretching), 1235 (C
–
H stretching), 3053 (aromatic C
–
–
–
N), 1574 (aromatic C
–
C), 1358
5
.1.8.6. 4-(5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)phenol
1
C
–
–
O). H NMR (500 MHz, DMSO‑d
6
) δ(ppm):
◦
(
15f). Dark brown solid, Yield: 75%, mp: 165–167 C, Rf 0.8, IR (KBr)
1
8
.34 (s, 1H, NH), 7.79 (s, 1H, indole-C2), 7.61 (d, 1H, J = 8 Hz, Indole-
max (cm- ): 3404 (O H stretching), 3209 (N H stretching), 3068
– –
ν
C4), 7.38 (d, 1H, J = 8 Hz, Indole-C7), 7.30–7.25 (m, 4H, Ar-H),
aromatic C H stretching), 2849 (aliphatic C H stretching), 2348
– –
(
7
.21–7.16 (m, 3H, Ar-H, indole), 4.85 (s, 2H, CH
2
), 4.03 (s, 2H, CH
2
),
–
–
–
C), 1085 (C–N stretching), 1030 (C–
–
(
C
N), 1610 (aromatic C
O).
1
3
1
C NMR (175 MHz, DMSO‑d
6
) δ(ppm): 166.38, 163.95, 136.23,
NMR (500 MHz, DMSO‑d
6
) δ(ppm): 11.06 (s, 1H, OH), 8.43 (s, 1H,
1
1
34.52, 127.87, 127.53, 127.34, 126.33, 121.67, 119.32, 119.10,
NH), 7.79 (d, 1H, J = 8 Hz, Indole-C4), 7.58 (d, 1H, J = 8 Hz, Indole-C7),
þ
10.97, 108.45, 33.54, 26.26. MS: m/z [M] for C18
H
15
N
3
O, calcu-
7
1
.46–7.24 (m, 3H, Ar-H), 7.09 (t, 1H, Ar-H, J = 8 Hz, Indole-C6), 7.01 (t,
H, Ar-H, J = 8 Hz, Indole-C5), 6.91 (d, 2H, J = 8 Hz, Ar-H), 4.38 (s, 2H,
lated 289.12; observed: 289.
1
3
CH2), C NMR (125 MHz, CDCl3): 165.05, 164.04, 160.53, 136.10,
5
.1.8.2. 2-((1H-indol-3-yl)methyl)-5-phenyl-1,3,4-oxadiazole
(15b).
128.09, 126.53, 123.97, 121.17, 118.62, 118.11, 116.00, 114.07,
◦
þ
Blackish brown solid, Yield: 58%, mp: 172–174 C, Rf 0.20, IR (KBr)
111.48, 106.84, 21.43. MS: m/z [M] for C17H13N O , calculated
3
2
1
ν
max (cm- ): 3310 (N
–
H stretching), 3057 (aromatic C
–
H stretching),
291.10; observed: 291.
–
H stretching), 2316 (C
–
N), 1574 (aromatic C
–
–
C),
2
952 (aliphatic C
1
1
340 (C N stretching), 1220 (C
–
–
O). H NMR (500 MHz, DMSO‑d )
6
5
.1.8.7. 2-((1H-indol-3-yl)methyl)-5-(4-methoxyphenyl)-1,3,4-oxadia-
δ(ppm): 8.28 (s, 1H, NH), 7.74 (s, 1H, indole-C2), 7.57 (d, 1H, J = 8 Hz,
◦
zole (15g). Yellowish brown solid, Yield: 80%, mp: 145–147 C, Rf 0.8,
1
Indole-C4), 7.35 (d, 1H, J = 8 Hz, Indole-C7), 7.59–7.51 (m, 3H, Ar-H),
max (cm- ): 3309 (N H stretching), 3068 (aromatic C H
–
–
IR (KBr)
–
–
stretching), 2919 (aliphatic C H stretching), 2316 (C N), 1610 (aro-
ν
7
7
.41–7.35 (m, 2H, Ar-H), 7.27 (dd, 1H, J
1
= 8 Hz, J
2
= 4 Hz, Indole-C6),
–
1
3
–
–
C), 1176 (C–
–O). 1
NMR (500 MHz,
.18 (dd, 1H, J
1
= 8 Hz, J
2
= 4 Hz, Indole-C5), 4.82 (s, 2H, CH
) δ(ppm): 165.99, 163.78, 136.11, 132.82,
28.79, 127.28, 127.02, 125.34, 123.23, 121.78, 120.76, 119.12,
2
),
C
matic C
N stretching), 1130 (C
NMR (175 MHz, DMSO‑d
6
DMSO‑d
6
) δ(ppm): 8.45 (s, 1H, NH), 8.02 (s, 1H, Indole-C2), 7.87 (d, 2H,
1
1
2
Ar-H, J = 8 Hz), 7.81 (d, 1H, J = 8 Hz, Indole-C4), 7.68 (d, 1H, J = 8 Hz,
Indole-C7), 7.29 (dt, 1H, J = 4 Hz, Indole-C6), 7.21 (dt, 1H,
= 8 Hz, J
= 8 Hz, J = 4 Hz, Indole-C5), 6.97 (d, 2H, Ar-H, J = 8 Hz), 4.49 (s,
H, CH ), 3.83 (s, 3H, OCH
þ
13 3
10.79, 107.82, 26.26. MS: m/z [M] for C17H N O, calculated
1
2
75.10; observed: 275.
J
1
2
1
3
2
2
3 6
), C NMR (175 MHz, DMSO‑d ) δ(ppm):
5
.1.8.3. 2-((1H-indol-3-yl)methyl)-5-(2-bromophenyl)-1,3,4-oxadiazole
166.52, 164.03, 163.07, 136.98, 127.04, 126.78, 123.97, 121.77,
◦
(
15c). Yellowish brown solid, Yield: 60%, mp: 198–200 C, Rf 0.43, IR
120.52, 119.23, 118.83, 115.27, 112.02, 107.54, 54.33, 23.12. MS: m/z
1
þ
(
KBr)
matic C
358 (C
ν
max (cm- )
ν
max (cm-1): 3265 (N
–
H stretching), 3056 (aro-
[M] for C18H15N O , calculated 305.11; observed: 305.
3
2
–
–
H stretching), 2924 (aliphatic C
–
H stretching), 1574 (C
–
1
1
N), 1235 (C
–
O), 670 (C-Br). H NMR (500 MHz, DMSO‑d )
6
5
.1.8.8. 4-(5-((1H-indol-3-yl)methyl)-1,3,4-oxadiazol-2-yl)aniline
δ(ppm): 8.29 (s, 1H, NH), 8.17 (s, 1H, Indole-C2), 7.83 (d, 1H, J = 8 Hz,
Indole-C4), 7.70 (d, 1H, J = 8 Hz, Indole-C7), 7.68 (d, 1H, Ar-H, J = 8
Hz), 7.40–7.31 (m, 3H, Ar-H), 7.24–7.20 (m, 1H, Ar-H, indole), 7.14 (t,
◦
(
15h). Light brown solid, Yield: 78%, mp: 193–195 C, Rf 0.6, IR (KBr)
1
ν
2
max (cm- ): 3354 (N H stretching), 3056 (aromatic C H stretching),
–
–
–
–
–
1
926 (aliphatic C H stretching), 2374 (C
–
N), 1631 (aromatic C
C),
O). NMR
) δ(ppm): 8.47 (s, 1H, NH), 8.03 (s, 1H, Indole-C2),
1
3
1
H, Ar-H, J = 8 Hz), 4.45 (s, 2H, CH
2
), C NMR (175 MHz, DMSO‑d
6
)
1608 (N–
H bending), 1246 (C
(500 MHz, DMSO‑d
–N stretching), 1090 (C–
δ(ppm): 166.31, 163.90, 136.18, 134.44, 132.36, 131.60, 127.53,
6
9

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
7
1
7
.83 (d, 1H, J = 8 Hz, Indole-C4), 7.77 (d, 2H, Ar-H, J = 8 Hz), 7.70 (d,
COX-2 inhibitory activities using COX inhibitor screening assay kit
(Cayman chemical company) [43]. The compounds were screened at
one concentration (10 µM) against ovine COX-1 and human COX-2 for
determining inhibition and their selectivity as per protocol described by
manufacturer. The inhibitors for COX-1/COX-2 were taken in 950 µL
buffer (Co-solvated in DMSO) and added 10 µL heme followed by 10 µL
of respective enzyme. For background inactivated enzyme was used in
reaction. Reaction was initiated by adding 10 µL arachidonic acid and
H, J = 8 Hz, Indole-C7), 7.31 (dt, 1H, J
1
= 8 Hz, J = 4 Hz, Indole-C6),
2
.23 (dt, 1H, J
1
= 8 Hz, J
2
= 4 Hz, Indole-C5), 6.82 (d, 2H, Ar-H, J = 8
1
3
Hz), 4.53 (s, 2H, CH
DMSO‑d
23.35, 121.48, 120.51, 119.23, 118.67, 115.97, 111.53, 107.98, 24.76.
2
), 4.48 (bs, 2H, Ar-NH
2
), C NMR (175 MHz,
6
) δ(ppm): 165.87, 163.38, 151.37, 135.76, 127.08, 126.31,
1
þ
MS: m/z [M] for C17
14 4
H N O, calculated 290.11; observed: 290.
◦
5
.1.8.9. 2-((1H-indol-3-yl)methyl)-5-(3-methoxyphenyl)-1,3,4-oxadia-
incubated at 37 C for 2 min. The reaction was stopped by adding 50 µL
◦
zole (15i). Brown solid, Yield: 65%, mp: 178–180 C, Rf 0.5, IR (KBr)
HCl after which 100 µL of SnCl
2 2
was added which reduce PGH initially
1
ν
max (cm- ): 3301 (N
–
H stretching), 3045 (aromatic C
–
H stretching),
formed to PGF
2
α. The amount of PGF
2
α
formed was determined in 96
–
H stretching), 2316 (C
–
N), 1531 (aromatic C
–
–
C),
well plate using ELISA reader at 406 nm wavelength. The results are
expressed as percentage inhibition of respective enzymes at 10 µM
concentration.
2
943 (aliphatic C
1
1
608, 1276 (C
–
N
stretching), 1190 (C
O). NMR (500 MHz,
6
DMSO‑d ) δ(ppm): 8.28 (s, 1H, NH), 8.17 (s, 1H, Indole-C2), 7.85 (d, 1H,
J = 8 Hz, Indole-C4), 7.75 (d, 1H, J = 8 Hz, Indole-C7), 7.70 (d, 1H, Ar-
H, J = 8 Hz), 7.41–7.35 (m, 3H, indole-C7, Ar-H), 7.34 (dt, 1H, J
1
= 8
5
.4. Antioxidant activity
Hz, J
2
= 4 Hz, Indole-C6), 7.26 (dt, 1H, J
1
= 8 Hz, J
2
= 4 Hz, Indole-C5),
1
3
6
.98 (d, 1H, Ar-H, J = 8 Hz), 4.48 (s, 2H, CH
2
), 3.81 (s, 3H, OCH
3
),
C
The antioxidant activity of synthesized compounds was evaluated
NMR (175 MHz, DMSO‑d ) δ(ppm): 165.46, 163.65, 162.87, 136.95,
6
using DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging
assay with ascorbic acid as standard. DPPH solution was prepared by
dissolving 0.1 mM DPPH in methanol and kept in dark for 2 h. After 2 h,
1
29.48, 126.84, 125.78, 123.87, 121.65, 121.60, 120.70, 120.52,
þ
1
18.95, 111.87, 111.75, 107.42, 56.13, 23.34. MS: m/z [M] for
, calculated 305.11; observed: 305.
18 15 3 2
C H N O
2
mL of this DPPH solution was taken, added 2 mL of different con-
centrations of test samples (2, 4, 6, 8 and 10 g/mL) and mixed thor-
μ
5
5
.2. In-vivo studies
oughly. The resulting mixture was incubated for 30 min at room
temperature and absorbance was recorded at wavelength of 517 nm
.2.1. Anti-inflammatory activity
[
44,45]. The assay was performed in triplicate and values were
The synthesized compounds were evaluated for their anti-
expressed as mean ± standard deviation. The percentage of free radical
inflammatory activity
t using carrageenan-induced paw oedema
scavenging activity was calculated by using following:
method in Wistar rats of either sex, weighing 160–180 g (n = 6) [40].
Briefly, the rats were administered suspension of test compounds (50
mg/kg, orally) in PEG 400, 30 min before carrageenan injection (0.1 mL
saline, containing 1% carrageenan). The rats of control group received
the same volume of vehicle. Indomethacin was used as standard anti-
inflammatory agents at concentrations of 5, 7.5 and 10 mg/kg. The
volume of the paw was measured by a plethysmometer immediately
after the injection. Subsequent readings of the volume of the same paw
were recorded at 1, 2, 4 h intervals. The percent anti-inflammatory ac-
tivity was calculated according to the following formula:
Percentage scavenging = (A ꢀ B)/A × 100
Where, A = absorbance of control (DPPH); B = absorbance of sample
5.5. In-Silico studies
Total 12 indole functionalized pyrazole and oxadiazole derivatives
were sketched and cleaned by utilizing the builder tool option in the
Schr o¨ dinger 2020–4 molecular modeling platform (Schr o¨ dinger Inc,
USA). The molecules were optimized with OPLS3e force field [46] using
the Ligprep module of Schr o¨ dinger at pH range of 7.0 ± 0.5. For the
molecular docking analysis in different anti-inflammatory targets
including COX-1, COX-2, LOX-5 and LOX-15, the co-crystallised struc-
ture 1PGF, 6COX, 3 V99 and 1IK3, respectively, were obtained from the
Protein Data Bank (PDB; www.rcsb.org). These structures were chosen
on the basis of their resolution and considering the co-crystallized li-
gands. Protein Preparation Wizard in the Schr o¨ dinger platform was used
to add the hydrogen atoms and partial charges, and to check the bond
orders. Water molecules in all the system were removed and energy was
minimized using an OPLS3e force field. Glide module in Schr o¨ dinger,
which is based on the OPLS3e force field, was used to perform the mo-
lecular docking analysis with high accuracy using the XP (extra preci-
sion) mode where further elimination of false positives is accomplished
by more extensive sampling and advanced scoring, resulting in even
higher enrichment. The position of glide grid was defined based on the
co-crystallized ligands in the already prepared (both X-ray solved and
homology modelled) proteins. Post-docking minimization was executed
to optimize the ligand geometries [47]. Further, Qikprop module of
Schr o¨ dinger was used to predict the drug-like and ADME properties of
the compounds [48].
Percentage inhibition of edem a = (1 ꢀ Vt/Vc) × 100
Where, Vt and Vc are volumes of edema in drug treated and control
groups, respectively.
5
.2.2. Analgesic activity
The analgesic activity of synthesized compounds at a dose of 50 mg/
kg was evaluated using tail flick method in Wistar rats of either sex,
weighing 160–180 g (n = 6). The analgesic potential was measured by
using Dynamic Plantar Aesthesiometer [41]. In this method, first the rats
were habituated for two hours before performing the experiment to
familiarize them. The mechanical threshold was measured by an elec-
tronic von Frey tip and calculated the force applied by withdrawal of
paw. The test compounds were administered orally at mentioned dose
while the Declofenac was used as standard drug at dose of 40 mg/kg.
The tip of the tail was placed on heat source and basal reaction time to
heat stimulus was noted. The tail was withdrawn by the animal and this
was considered the endpoint [42]. The reaction time of tail was noted at
interval of 30 min, 1, 2, 3 h intervals after dose administration. Each test
was repeated five times at average cut off 20 sec. The maximum and
minimum values are excluded, and the average of remained three values
was used for mechanical threshold. The values are expressed as mean ±
SD.
Declaration of Competing Interest
5
.3. In-vitro COX inhibitory study
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
The synthesized compounds were screened for in vitro COX-1 and
1
0

D. Kumar et al.
Bioorganic Chemistry 114 (2021) 105068
Acknowledgements
[26] J. Martinez-Gonzalez, L. Badimon, Mechanisms underlying the cardiovascular
effects of COX-inhibition: benefits and risks, Curr. Pharm. Design 13 (2007)
2
215–2227.
The authors are also thankful to Mr. Praveen Garg, Chairman and
Prof. (Dr) G. D. Gupta, Director cum Principal, ISF College of Pharmacy,
Moga, Punjab for his continuous support and encouragement. PKS
would like to thank TCSMT (University of Turku) for research funding.
[
[
27] P. Rao, E.E. Knaus, Evolution of nonsteroidal anti-inflammatory drugs (NSAIDs):
cyclooxygenase (COX) inhibition and beyond, J. Pharm. Pharm. Sci. 11 (2008)
8
1–110s.
28] H.S. ElBordiny, M.M. El-Miligy, S.E. Kassab, H. Daabees, W.A.M. Ali, S.A.M. El-
Hawash, Design, synthesis, biological evaluation and docking studies of new 3-(4,
5
-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole derivatives as potent
antioxidants and 15-lipoxygenase inhibitors, Eur. J. Med. Chem. 145 (2018)
94–605.
29] J. Fort, Celecoxib, a COX-2–specific inhibitor: the clinical data, Am. J. Orthop.
Belle Mead, NJ) 28 (1999) 13-18.
30] L. Gong, C.F. Thorn, M.M. Bertagnolli, T. Grosser, R.B. Altman, T.E. Klein,
Celecoxib pathways: pharmacokinetics and pharmacodynamics, Pharmacogenet.
Genomics 22 (2012) 310.
References
5
[
[
[
[
1] S.I. Grivennikov, F.R. Greten, M. Karin, Immunity, inflammation, and cancer, Cell
(
1
40 (2010) 883–899.
2] S. Sharma, D. Kumar, G. Singh, V. Monga, B. Kumar, Recent advancements in the
development of heterocyclic anti-inflammatory agents, Eur. J. Med. Chem. 112438
(
2020).
[
[
31] J. Bostr o¨ m, A. Hogner, A. Llin `a s, E. Wellner, A.T. Plowright, Oxadiazoles in
medicinal chemistry, J. Med. Chem. 55 (2012) 1817–1830.
[
3] S. Steinmann, M. Schoedsack, F. Heinrich, P.C. Breda, A. Ochel, G. Tiegs,
K. Neumann, Hepatic ILC2 activity is regulated by liver inflammation-induced
cytokines and effector CD4+ T cells, Sci. Rep. 10 (1) (2020) 1–13.
32] A. Rathore, R. Sudhakar, M.J. Ahsan, A. Ali, N. Subbarao, S.S. Jadav, S. Umar, M.
S. Yar, In vivo anti-inflammatory activity and docking study of newly synthesized
benzimidazole derivatives bearing oxadiazole and morpholine rings, Bioorg. Chem.
[
[
[
[
4] V. Menkin, Inflammation: a protective mechanism, Arch. Intern. Med. 48 (1931)
2
49–261.
5] O. Takeuchi, S. Akira, Pattern recognition receptors and inflammation, Cell 140
2010) 805–820.
6] R. Medzhitov, Inflammation 2010: new adventures of an old flame, Cell 140 (2010)
7
0 (2017) 107–117.
[
[
33] S.A. Khan, K. Saleem, Z. Khan, Synthesis, characterization and in vitro antibacterial
activity of new steroidal thiazolo quinoxalines, Eur. J. Med. Chem. 42 (2007)
(
1
03–108.
(
2010) 771–776.
34] S. Wagle, A.V. Adhikari, N.S. Kumari, Synthesis of some new 2-(3-methyl-7-
substituted-2-oxoquinoxalinyl)-5-(aryl)-1, 3, 4-oxadiazoles as potential non-
steroidal anti-inflammatory and analgesic agents, Indian J. Chem. 47 (2008)
7] O. Chertov, D. Yang, O.Z. Howard, J.J. Oppenheim, Leukocyte granule proteins
mobilize innate host defenses and adaptive immune responses, Immunol. Rev. 177
(
2000) 68–78.
4
39–448.
[
[
8] S.M. Opal, V.A. DePalo, Anti-inflammatory cytokines, Chest 117 (2000)
[
35] S.V. Bhandari, K.G. Bothara, M.K. Raut, A.A. Patil, A.P. Sarkate, V.J. Mokale,
Design, synthesis and evaluation of antiinflammatory, analgesic and ulcerogenicity
studies of novel S-substituted phenacyl-1, 3, 4-oxadiazole-2-thiol and Schiff bases
of diclofenac acid as nonulcerogenic derivatives, Bioorg. Med. Chem. 16 (2008)
1
162–1172.
9] C.A. Feghali, T.M. Wright, Cytokines in acute and chronic inflammation, Front.
Biosci. 2 (1997) d12–d26.
[
[
10] G. Arango Duque, A. Descoteaux, Macrophage cytokines: involvement in immunity
and infectious diseases, Front. Immunol. 5 (2014) 491.
1
822–1831.
[
[
36] D. Raffa, B. Maggio, F. Plescia, S. Cascioferro, M.V. Raimondi, S. Plescia, M.
G. Cusimano, Pyrazolo [3, 4-d] pyrimidine Derivatives as COX-2 Selective
Inhibitors: Synthesis and Molecular Modelling Studies, Arch. Pharm. 342 (2009)
11] Y. Zhou, Y. Hong, H. Huang, Triptolide attenuates inflammatory response in
membranous glomerulo-nephritis rat via downregulation of NF-κB signaling
pathway, Kidney Blood Pressure Res. 41 (2016) 901–910.
3
21–326.
[
[
[
12] J.H. Fritz, S.E. Girardin, How Toll-like receptors and Nod-like receptors contribute
to innate immunity in mammals, J. Endotoxin Res. 11 (2005) 390–394.
13] V.S. Hanna, E.A.A. Hafez, Synopsis of arachidonic acid metabolism: A review,
J. Adv. Res. 11 (2018) 23–32.
37] M.B. Palkar, A.S. Singhai, P.M. Ronad, A. Vishwanathswamy, T.S. Boreddy, V.
P. Veerapur, M.S. Shaikh, R.A. Rane, R. Karpoormath, Synthesis, pharmacological
screening and in silico studies of new class of Diclofenac analogues as a promising
anti-inflammatory agents, Bioorg. Med. Chem. 22 (2014) 2855–2866.
14] Z. Ye, Y. Shen, K. Jin, J. Qiu, B. Hu, R.R. Jadhav, K. Sheth, C.M. Weyand, J.
J. Goronzy, Arachidonic acid-regulated calcium signaling in T cells from patients
with rheumatoid arthritis promotes synovial inflammation, Nat. Commun. 12 (1)
[
[
[
[
38] P.K. Singh, O. Silakari, Multitargeting Heterocycles: Improved and Rational
Chemical Probes for Multifactorial Diseases, Key Heterocycle Cores for Designing
Multitargeting Molecules, Elsevier2018, pp. 1-29.
(
2021) 1–17.
39] S. Pathania, P.K. Singh, Analyzing FDA-approved drugs for compliance of
pharmacokinetic principles: should there be a critical screening parameter in drug
designing protocols? Expert Opin. Drug Metab. Toxicol. 1–4 (2020).
[
[
15] A. Gaddi, A.F. Cicero, E.J. Pedro, Clinical perspectives of anti-inflammatory
therapy in the elderly: the lipoxigenase (LOX)/cycloxigenase (COX) inhibition
concept, Arch. Gerontol. Geriatr. 38 (2004) 201–212.
40] C.A. Winter, E.A. Risley, G.W. Nuss, Carrageenin-induced edema in hind paw of the
rat as an assay for antiinflammatory drugs, Proc. Soc. Exp. Biol. Med. 111 (1962)
16] X. Gao, R. Zhu, J. Du, W. Zhang, W. Gao, L. Yang, Inhibition of LOX-1 prevents
inflammation and photoreceptor cell death in retinal degeneration, Int.
Immunopharmacol. 80 (2020), 106190.
5
44–547.
41] D. Pc, Studies on central nervous system depressants. (III). Influence of some
tranquillizing agents on morphine analgesia, Arch. Int. Pharmacodyn. Ther. 141
[
[
[
17] D.M. Poetker, D.D. Reh, A comprehensive review of the adverse effects of systemic
corticosteroids, Otolaryngol. Clin. North Am. 43 (2010) 753–768.
18] R.O. Day, G.G. Graham, Non-steroidal anti-inflammatory drugs (NSAIDs), BMJ 346
(
1963) 223–232.
[
[
42] O. Davies, J. Raventos, A. Walpole, A method for the evaluation of analgesic
activity using rats, Br. J. Pharmacol. Chemother. 1 (1946) 255.
(
2013), f3195.
19] P. Chawla, S. Kalra, R. Kumar, R. Singh, S.K. Saraf, Novel 2-(substituted phenyl
Imino)-5-benzylidene-4-thiazolidinones as possible non-ulcerogenic tri-action drug
candidates: synthesis, characterization, biological evaluation And docking studies,
Med. Chem. Res. 28 (3) (2019) 340–359.
43] E.M. Ahmed, M.S. Hassan, A.A. El-Malah, A.E. Kassab, New pyridazine derivatives
as selective COX-2 inhibitors and potential anti-inflammatory agents; design,
synthesis and biological evaluation, Bioorg. Chem. 95 (2020), 103497.
44] M. Djukic, M. Fesatidou, I. Xenikakis, A. Geronikaki, V.T. Angelova, V. Savic,
M. Pasic, B. Krilovic, D. Djukic, B. Gobeljic, In vitro antioxidant activity of
thiazolidinone derivatives of 1, 3-thiazole and 1, 3, 4-thiadiazole, Chem.-Biol.
Interact. 286 (2018) 119–131.
[
[
[
20] D. Kumar, S. Sharma, S. Kalra, G. Singh, V. Monga, B. Kumar, Medicinal
Perspective of Indole Derivatives: Recent Developments and Structure-Activity
Relationship Studies, Curr. Drug Targets 21 (9) (2020) 864–891.
[
[
[
21] R.J. Flower, The development of COX2 inhibitors, Nat. Rev. Drug Discov. 2 (2003)
45] G. Bansal, S. Singh, V. Monga, P.V. Thanikachalam, P. Chawla, Synthesis and
biological evaluation of thiazolidine-2, 4-dione-pyrazole conjugates as
antidiabetic, anti-inflammatory and antioxidant agents, Bioorg. Chem. 92 (2019),
1
79–191.
22] D.O. Stichtenoth, J.C. Fr o¨ lich, The second generation of COX-2 inhibitors, Drugs 63
(
2003) 33–45.
1
03271.
23] B. Nehra, S. Rulhania, S. Jaiswal, B. Kumar, G. Singh, V. Monga, Recent
advancements in the development of bioactive pyrazoline derivatives, Eur. J. Med.
Chem. 112666 (2020).
[
[
46] E. Harder, W. Damm, J. Maple, C. Wu, M. Reboul, J.Y. Xiang, L. Wang, D. Lupyan,
M.K. Dahlgren, J.L. Knight, OPLS3: a force field providing broad coverage of drug-
like small molecules and proteins, J. Chem. Theory Comput. 12 (2016) 281–296.
47] R.A. Friesner, R.B. Murphy, M.P. Repasky, L.L. Frye, J.R. Greenwood, T.A. Halgren,
P.C. Sanschagrin, D.T. Mainz, Extra precision glide: Docking and scoring
incorporating a model of hydrophobic enclosure for proteinꢀ ligand complexes,
J. Med. Chem. 49 (2006) 6177–6196.
[
[
24] M. Arora, S. Choudhary, P.K. Singh, B. Sapra, O. Silakari, Structural investigation
on the selective COX-2 inhibitors mediated cardiotoxicity: A review, Life Sci. 251
(
2020), 117631.
25] J. Kirchrath, K. Schrr, Cyclooxygenase-2 inhibition and side-effects of non-steroidal
anti-inflammatory drugs in the gastrointestinal tract, Curr. Med. Chem. 7 (2000)
[
48] L. Schr o¨ dinger, QikProp, version 3.5, New York, NY (2012).
1
121–1129.
1
1