1,2,4-Triazole-conjugated 1,3,4-thiadiazole hybrid scaffolds: A potent ameliorant of carrageenan-induced inflammation by lessening proinflammatory mediators

Article abstract of DOI:10.1002/ardp.201900233

Inflammation acts as an alarming signal for the progression of various biological complications. Various reports in the literature have revealed that heterocycle-containing synthetic compounds have a restorative capability against acute and chronic inflammatory stages. In the current study, we synthesized a series of 1,2,4-triazole-conjugated 1,3,4-thiadiazole hybrid scaffolds and evaluated their impacts against carrageenan-induced paw edema and proinflammatory markers in Wistar rats. Further, 3D QSAR study (three-dimensional quantitative structure–activity relationships), ADMET (absorption, distribution, metabolism, and excretion) profiling, and docking studies were performed to determine the possible mechanism of the action of the derivatives. The study shows that the most active derivatives, 13f and 13g, have optimal logP, a higher anti-inflammatory activity score, and poor metabolism at various sites of cytochrome P450. The docking studies recommended that the synthesized compounds have a similar affinity as the ligands A307, 63X, and S58 to interact with tumor necrosis factor-α, COX-1, and COX-2. So, these molecules will definitely hold a promise for the future drug development initiative.

Full text of DOI:10.1002/ardp.201900233

Received: 6 August 2019
Revised: 23 September 2019
Accepted: 2 October 2019
|
|
DOI: 10.1002/ardp.201900233
F U L L P A P E R
1
,2,4‐Triazole‐conjugated 1,3,4‐thiadiazole hybrid scaffolds:
A potent ameliorant of carrageenan‐induced inflammation by
lessening proinflammatory mediators
1
2,3
1
1
Prateek Pathak
|
Parjanya K. Shukla
|
Vladislav Naumovich
|
Maria Grishina
|
2
1
Amita Verma
|
Vladimir Potemkin
1
Laboratory of Computational Modeling of Drugs, Higher Medical and Biological School, South Ural State University, Chelyabinsk, Russia
2
Bioorganic and Medicinal Chemistry Research Laboratory, Department of Pharmaceutical Sciences, Faculty of Health Sciences, Sam Higginbottom University of
Agriculture, Technology and Sciences, Allahabad, India
3
Krishnarpit Institute of Pharmacy, Uttar Pradesh Technical University, Allahabad, India
Correspondence
Abstract
Prateek Pathak, Maria Grishina and Vladimir
Potemkin, Laboratory of Computational
Inflammation acts as an alarming signal for the progression of various biological
Modeling of Drugs, Higher Medical and
complications. Various reports in the literature have revealed that heterocycle‐
containing synthetic compounds have a restorative capability against acute and
chronic inflammatory stages. In the current study, we synthesized a series of 1,2,4‐
triazole‐conjugated 1,3,4‐thiadiazole hybrid scaffolds and evaluated their impacts
against carrageenan‐induced paw edema and proinflammatory markers in Wistar
rats. Further, 3D QSAR study (three‐dimensional quantitative structure–activity
relationships), ADMET (absorption, distribution, metabolism, and excretion) profiling,
and docking studies were performed to determine the possible mechanism of the
action of the derivatives. The study shows that the most active derivatives, 13f and
Biological School, South Ural State University,
Tchaikovsky Street, 20‐A, Chelyabinsk, Russia,
454008.
Email: prateekpharm05@gmail.com (P. P.),
grishinama@susu.ru (M. G.) and potemkinva@
susu.ru (V. P.)
Amita Verma, Bioorganic and Medicinal
Chemistry Research Laboratory, Department
of Pharmaceutical Sciences, Faculty of Health
Sciences, Sam Higginbottom University of
Agriculture, Technology and Sciences,
Allahabad, India.
Email: amitaverma.dr@gmail.com (A. M.)
1
3g, have optimal logP, a higher anti‐inflammatory activity score, and poor
metabolism at various sites of cytochrome P450. The docking studies recommended
that the synthesized compounds have a similar affinity as the ligands A307, 63X, and
S58 to interact with tumor necrosis factor‐α, COX‐1, and COX‐2. So, these molecules
will definitely hold a promise for the future drug development initiative.
Funding information
Ministry of Education and Science of Russia,
Grant/Award Numbers: 4.7279.2017/8.9,
4
.8298.2017/8.9; Government of the Russian
Federation, Grant/Award Number: Contract
2.A03.21.0011; Russian Foundation for Basic
Research, Grant/Award Number:
8‐53‐45015
0
K E Y W O R D S
1
1,3,4‐thiadiazole, 1,2,4‐triazole, ADMET study, anti‐inflammatory activity, docking study
[1]
1
|
INTRODUCTION
characterized by gathering and migration of fluids. This inflammatory
response is influenced by various physiological and immunological
[2]
Inflammation is a sign of various life‐threatening diseases. It acts as a
multifaceted protective mechanism of the body against toxic stimuli.
Inflammation may range from a small area to a widespread response,
mediators. The initial state of inflammation refers to acute inflamma-
tion and concord as an initial response against tissue injury. It is
mediated through the release of some autocoids like serotonin,
Abbrevations: 3D QSAR study, three‐dimensional quantitative structure–activity relationships; ADMET, absorption, distribution, metabolism, and excretion; COX1/COX2, cyclooxygenase‐1/
cyclooxygennase‐2; CYP450, cytochrome‐450; GI abs, gastrointestinal absorption; H Acc and H Don, total no of H donor and acceptor; IL‐10, interleukin‐10; IL‐1β, interleukin‐1β; Lipinski’s
violation, total no of violation of Lipinski rule of five; logP, octanol–water partition coefficient; NF‐κB, nuclear factor κ‐light‐chain‐enhancer of activated B cells; TNF‐α, tumor necrosis factor‐α;
TPSA, total polar surface area.
Arch Pharm Chem Life Sci. 2019;e1900233.
wileyonlinelibrary.com/journal/ardp © 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201900233

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PATHAK ET AL.
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[
3]
thromboxanes, histamine, and leukotrienes.
However, prolonged
indomethacine and other cyclic nucleotide phosphodiesterases inhibitor,
for example, tadalafil, contains an indole ring system. This confirms that
the indole ring system could improve the biological efficacy (anti‐
inflammatory response) of the synthesized compounds. It is also
propounded that in new drug discovery methodology, the development
of hybrid molecules by combining different pharmacophores in one
framework can produce compounds with interesting biological char-
acteristics. Encouraged by these interpretations, in the current study,
we are presenting the synthesis of 1,2,4‐triazole‐conjugated thiadiazole
hybrid scaffolds via an indole ring bridge as a potent anti‐inflammatory
agent. We have also established a plausible possible mechanism by
disparage proinflammatory mediators and docking studies.
acute inflammation provokes a chronic inflammatory condition. It
includes the formation and release of different interleukins (ILs),
[4–6]
interferon, tumor necrosis factor‐α (TNF‐α), and cytokines.
Non-
steroidal anti‐inflammatory agents (NSAIDs) are a specific class of drugs
that comprise analgesic, antipyretic, and anti‐inflammatory potency. The
well‐known members of this group are aspirin, ibuprofen, naproxen,
[7]
diclofenac, and indomethacin. The main site of activity for most
NSAIDs is cyclooxygenase (COX)‐1, COX‐2 attenuated by PGs
(
proinflammatory mediators) and thromboxane. Considerable pharma-
cological research has explained that NSAID‐associated treatment may
[8,9]
increase the risk of stroke.
So, there is a need for concurring
development of new NSAIDs with minimal adverse effects and higher
efficacy. Heterocycle‐bearing nitrogen and sulphur moieties constitute
the core structure of a number of biologically active compounds. Among
them, triazole and thiazole are widely attractive molecules due to their
diverse biological activity. Structurally, 1,2,4‐triazole is a five‐membered
2
2
|
RESULTS AND DISCUSSION
|
Chemistry
.1
[8]
ring structure ruffled of three‐nitrogen and two carbon atoms. The
The designing of the hybrid scaffolds was achieved via modification
of the substituted 1,2,4‐triazole ring system with the 1,3,4‐
thiadiazole skeleton. The indole ring serves as a bridge fragment
between both nucleolus. The reaction scheme was a composite of
eight different steps (Scheme 1). The first step was concordant with
the Fischer esterification, where aromatic carboxylic acid converted
into an aromatic acid ester (2a–c) in the presence of methanol and
sulphuric acid. The sulphuric acid acted as a catalyst in the reaction.
Further, the second step involved the formation of acid
hydrazides (3a–c). The section of the scheme consists of the
attachment of the hydrazide group via replacing the H atom of the
hydroxyl group of an aromatic ester.
1
,2,4‐triazole ring system is acknowledged due to its broad array of
[10]
[11]
biological behavior, for example, anti‐inflammatory,
analgesic,
[16]
[
12,13]
[14]
[15]
antimicrobial,
anticancer,
+2
antitumor,
anti‐tubercular,
an-
[18]
[
17]
ticonvulsant,
Ca ‐activated potassium (Maxi‐K) channel opening,
[19]
and antiviral properties. Thus, 1,2,4‐triazole is markedly an important
[20]
pharmacophore. Ahirwar et al.
illustrated a number of novel 1,2,4‐
triazole analogs on the basis of a stratified based virtual screening
protocol. They identified that their synthesized derivatives showed
7
2.733–89.217% inhibition of acetic acid‐induced writhing reflex and
7.980–49.950% inhibition of carrageenan‐induced paw edema. Out of
2
them, compound 3j and 3b exhibited the most potent activity (49.95%
and 48.27% inhibition, respectively) against carrageenan‐induced paw
edema (Figure 1). Preliminary structure–activity relationships of the
synthesized compounds revealed that the introduction of the 2,4‐
dimethyl group on the benzene ring led to equivalent activity as
reference drug indomethacin. However, the addition of the nitro or the
amino group on the benzene ring had a great effect on the analgesic
potency. This observation was found to be in line with previous studies
The third synthetic protocol contains the formation of substi-
tuted dithiocarbazinic acid salts (4a–c). The reaction was completed
via the addition of carbon disulphide (CS )/KOH with compounds
2
3a–c. This synthetic step needed great attention and was immedi-
ately utilized for a further procedure without purification.
The fourth step of reaction involved the synthesis of the 1,2,4‐triazole
ring system. In this part of the synthesis, substituted dithiocarbazinic acid
salts were converted into the 1,2,4‐triazole ring (5a–c).
[20]
conducted by other research on selective inhibitors.
This was the
reason to select the 1,2,4‐triazole ring as our first parent skeleton. On
the contrary, 1,3,4‐thiadiazoles are also an imperative class of
compounds, exhibiting an extensive range of biological activities such
Further, in the fifth step, N‐(5‐mercapto‐3‐substituted‐1,5‐
dihydro‐1,2,4‐triazol‐4‐yl)acetamide compounds (7a–e) were
synthesized, namely, the electrophilic substitution of chlorine
atom from the acetyl or benzoyl chloride (6) in the presence of a
saturated solution of sodium acetate and acetic acid.
[
21–23]
[24–26]
[6]
[27,28]
as anticancer,
antiviral,
antibacterial,
antioxidant,
and anti‐inflamma-
and so on. So, as a second skeleton, we selected the
,3,4‐thiadiazoles ring for the designing of derivatives. It was also
observed that different types of selective COX‐2 inhibitors like
[29]
[30]
[31,32]
anxiolytic,
antitubercular,
anticonvulsant,
[33,34]
tory activities,
The sixth protocol consists of the synthesis of 5‐m‐substituted‐
phenyl‐[1,3,4]thiadiazol‐2‐ylamine (10a–d). The reaction processed
under reflux where an ethanolic solution of aromatic carboxylic acid
1
FIGURE 1 Some 1,2,4‐triazole
derivatives as potent anti‐inflammatory
agents

PATHAK ET AL.
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2a–c
3a–c
1
6
5
a–c
4a–c
7
a–e
13a–g
1
2a–d
11
10a–d
9
8a–d
SCHEME 1 Scheme for the synthesis of the desired products
and an aqueous solution of thiosemicarbazide conjugated in the
presence of sulphuric acid.
we conclude that in the current study, Mannich bases were formed by
condensing the acidic imino group of isatin instead of the of N‐(5‐
mercapto‐3‐substituted‐1,5‐dihydro‐1,2,4‐triazol‐4‐yl)acetamide (7a–e)
NH proton with formaldehyde and piperidine. The reaction scheme is
illustrated in Scheme 1. However, the substitution is showed in Table 1.
During the seventh step, the 5‐substituted‐phenyl‐1,3,4‐
thiadiazol‐2‐amine analogues (10a–d) were condensed with isatin
(
11) compounds in the presence of a few drops of glacial acetic
acid as a catalyst to produce Schiff bases (Z)‐3‐(5‐p‐susituted‐
,3,4‐thiadiazol‐2‐ylimino)‐3,3a‐dihydro‐1H‐indol‐2(7aH)‐one
1
2
.2
|
Three‐dimensional quantitative
(
12a–d) in rather good yields.
structure–activity relationships (3D QSAR) and
The eighth synthetic protocol formed the final products (13a–g).
absorption, distribution, metabolism, and excretion
The reaction involved addition of analogues N‐(5‐mercapto‐3‐sub-
situted‐1,5‐dihydro‐1,2,4‐triazol‐4‐yl)acetamide (7a–e) with (Z)‐3‐(5‐p‐
substituted‐1,3,4‐thiadiazol‐2‐ylimino)‐3,3a‐dihydro‐1H‐indol‐2(7aH)‐
one (12a–d). It is notable that Mannich base formation can take place
at both the isatin compound and triazole compound NH protons, but it
is known that the isatin compound (12a–d) NH proton is more active
(ADME) prognosis
The 3D QSAR has a great role in the ADME profile of any drug. So,
during the designing of the new molecule, this needs great attention.
The present segment of our research emphasized on QSAR and ADME
profile evaluation of synthesized analogous. The 3D QSAR and ADME
prognosis results are illustrated in Table 2. The physicochemical result
showed that the entire series of the compounds consisted of an
optimal logP value (nearly 5) and TPSA (total polar surface area; range
[34]
than that of the triazole compound (7a–e).
In addition, after the
1
Mannich reactions, the H NMR spectra of the compounds show an NH
proton signal at δ 07.01 ppm, which corresponds to a triazole NH, so

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TABLE 1 The groups R, R1, and R2 used in the synthesis of the
derivatives 13a–g
stated that the entire series do not have the tendency to metabolize at
the site of CYP1A2 and CYP2C19 (except 13a and 13e at the site of
cytochrome P450, CYP2C19). However, the compounds 13b and 13g
were not metabolized at the site of cytochrome P450 (CYP2C9). It was
also found that these compounds (13a, 13c, 13e, and 13f) have an
optimal metabolism score at the site of 2D6 and 3A4. The overall
metabolism profile revealed that 13c, 13e, 13f, and 13g have better
potential against various metabolism site of cytochrome P450. The
toxicity prediction showed that the entire series has no toxic effect.
This made these molecules more attractive for further modification
and development. The intact series of the derivatives were explored,
which showed that the hydrogen atom attached to the 1,2,4‐triazole
and 1,3,4‐thiadiazole has great contribution toward pharmacophoric
capacity. However, the carbon atom within the indole ring acts as an
antipharmacophore. The pharmacophore, antipharmacophore, and
ballast components of the entire series were displayed in Figure 2.
The prognosis study concluded that maximizing the number of
hydrogen atoms has potential against inflammation and this may be
selective subject to further designing the molecule. Therefore, on the
basis of 3D QSAR and ADMET properties, the derivatives 13f and 13g
were identified as the most promising molecules due to their optimal
logP, lower metabolism probability, and minimal toxicity. The
structural modification suggested replacing the indole ring with any
Derivatives
R
R1
CH
R2
1
3a
3
1
3b
CH
CH
CH
3
1
1
1
1
1
3c
3d
3e
3f
3
3
other heterocyclic ring system toward finding
a more potent
compound. These minor structural changes can enhance the physico-
chemical characters as well as pharmacophores through minimizing
the antipharmacophore space of the derivatives.
Benzene
2
.3
|
Anti‐inflammatory pharmacology
2
.3.1 | In vitro COX inhibition assay
The targeted 1,2,4‐triazole‐conjugated 1,3,4‐thiadiazole hybrid scaf-
folds (13a–g) were evaluated in vitro for cyclooxygenase (COX)
enzyme inhibition at 1.0 μM concentration. The activity was reported
in terms of % inhibition and selectivity index. The measurement of
the activity was obtained via comparison against the reference drug
celecoxib. The enzymes inhibition result is illustrated in Table 3.
However, the graphical presentation against COX‐1 and COX‐2 is
displayed in Figure 3. The assay showed that the targeted derivatives
have excellent inhibitory activity against COX‐1 and moderate
inhibitory activity against the CoX‐2 enzyme. The derivatives 13c,
Benzene
3g
CH
3
13f, and 13g were found to be the most prominent inhibitors within
the series. The derivative 13c expressed 52.10 ± 1.62% inhibition
against COX‐1 and 67.50 ± 1.11% inhibition against COX‐2. How-
ever, the derivative 13f displayed 53.10 ± 0.99% and 71.12 ± 1.28%
inhibition against COX‐1 and COX‐2 enzyme. Furthermore, a slight
decline in the inhibition activity was observed in 13g (52.50 ± 1.10%
against COX‐1 and 69.10 ± 1.75% against COX‐2, respectively). Apart
from this, significant drops in inhibition were observed by the
derivatives 13a, 13b, 13d, and 13e. The result showed that derivative
13a has the lowest inhibition potency in the series. However,
derivative 13f was found to be most significant in the entire series of
compounds.
of 119.3–139.5). However, the 3D QSAR and ADME study showed
that the entire series of derivatives have some deviation from
[
9]
Lipinski’s rule in terms of H donor and acceptor and need to be
optimized in future research. The synthesized compounds showed
lower gastrointestinal (GI) absorption, which certainly played an
important role in lowering bioavailability and pharmacological re-
sponse. However, the compounds 13c, 13f, and 13g displayed a better
pharmacological response. It was showed that these three compounds
have slightly better oral absorption in the series. The metabolism study

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TABLE 2 Physiochemical properties (ADME) calculation of synthesized derivatives (13a–g)
Lipinski
Der
LogP TPSA
vio
H Acc H Don
GI Abs CYP1A2
CYP2C19
Yes
CYP2C9
Yes
CYP2D6
0.38
CYP3A4
0.62
Tox
Ph Score
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
4.6
5.3
5.0
5.5
5.5
5.1
4.9
119.3
198
2
7
1
1
1
1
1
1
2
Low
Low
Low
Low
Low
Low
Low
No
No
No
No
No
No
No
0.00 0.00
0.00 0.91
0.08 0.91
0.00 0.00
0.00 0.91
0.00 0.91
0.00 0.00
2
10
9
No
No
0.73
0.97
161.4
115.6
128.5
124.9
139.5
2
No
Yes
0.86
0.71
2
7
No
Yes
0.86
0.98
2
8
Yes
Yes
0.53
0.00
3
8
No
Yes
0.79
0.00
3g
2
8
No
No
0.62
0.98
Note: logP is the logarithm of the octanol–water partition coefficient; 3A4 is the probability of metabolism at CYP450 3A4; 2D6 is the probability of
metabolism at CYP450 2D6. Columns CYP1A2, CYP2C19 CYP2C9, CYP2D6, and CYP3A4 showed the metabolism on different site of cytochrome‐P450.
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; GI abs, gastrointestinal absorption; H Acc, H acceptor; H don, H donor;
Lipinski’s vio, total number of violation of Lipinski rule of five; Ph Score, pharmacophoric score calculated by the Cinderella shoe method; Tox, probability
of maximum toxicity of the compounds; TPSA, total polar surface area.
2
.3.2 | In vivo anti‐inflammatory activity
benzamide on the azanylidene‐indole fragment. However, the
compound 13f has only minor differences between 13e, that is
the methoxy‐Ph ring was replaced by pyridine at the 1,3,4‐thiadiazole
skeleton. These minor changes in the structure made the molecule
most active within the series of compounds. Careful comparison with
standard emphasized that the compound is similarly active (50%
inhibition at 1 hr, 52.63% inhibition at 3 hr, and 57.14% inhibition at
We examined the in vivo anti‐inflammatory activity of synthesized
compounds (13a–g) via carrageenan‐induced paw edema model. The
percentage inhibition of the synthesized compounds was evaluated
by comparing with standard (diclofenac) at regular interval of time,
that is, 0, 1, 3, and 6 hr. The mean difference in the paw volume
is given in Table 4. However, the percentage inhibition of paw edema
is illustrated in Table 5. The graphical illustration of the activity
is displayed in Figures 4 and 5.
6
hr, respectively) as diclofenac. The final synthesized compound 13g
has a similar substitution pattern like 13a with a minor change, that
is, attachment of phenol via the substitution of Cl‐Ph on the 1,3,4‐
thiadiazole ring. This change potentially increases the anti‐inflam-
matory response of the compound (50% inhibition at 1 hr, 52.63%
inhibition at 3 hr, and 46.42% inhibition at 6 hr, respectively). The
standard drug showed 50.00% inhibition against inflammation at
intervals of 1 hr, 57.89% at 3 hr, and 60.71% at 6 hr. The study
revealed that the derivatives 13c, 13f, and 13g are the most
prominent derivatives in the entire series. The SAR studies conclude
The entire compounds in the series 13a–g exhibited the anti‐
inflammatory activity in the range of 20.00–50.00% after 1 hr,
1
0.53–52.63% after 3 hr, and 03.57–57.14% after 6 hr (shown in
Table 4).
The compound 13a constructed by the addition of 4‐chlor-
obenzene on 1,3,4‐thiadiazole, piperidine on the 1,2,4‐triazole
ring, showed mild anti‐inflammation potency. However, compound
1
2
3b having two NO ‐Ph groups, that is, attached to 1,3,4‐
that methoxyphenyl, benzene, and NO ‐phenyl substitutions are
2
thiadiazole in place of 4‐chlorobenzene and the 1,2,4‐triazole ring
in place of piperidine, showed little improvement in anti‐
inflammatory response but could not be considered as a significant
change.
important for anti‐inflammatory activity.
2
.4
|
Effect on proinflammatory cytokines and
In the compound 13c, the NO ‐Ph group of 1,3,4‐thiadiazole was
2
inflammatory mediators
replaced with methoxy‐Ph. To our surprise, the compound showed
great anti‐inflammatory activity (50% inhibition at 1 hr, 47.36%
inhibition at 3 hr, and 46.42% inhibition at 6 hr, respectively). The
result showed that the methoxy‐benzene group plays a key role in
anti‐inflammatory activity. Toward the search of the new lead
molecule, in the compound 13d, we replaced 1,3,4‐thiadiazole
methoxy‐Ph with Cl‐Ph and nitro‐Ph of 1,2,4‐triazole ring
with methoxy‐Ph. The synthesized compound showed poor
anti‐inflammatory response than 13c. The compound 13e showed a
slight improvement in anti‐inflammatory activity (42.10% at 3 hr and
To determine the effect against proinflammatory cytokine and
inflammatory mediators, the blood samples were evaluated according
to the manufacturer’s instructions. The compounds displayed significant
change (p < 0.001) against proinflammatory cytokine and inflammatory
mediators (Figure 6). Table 6 shows the impact of the proinflammatory
cytokines on the normal control group, carrageenan‐induced, and
treated groups. The result showed that in the entire series, the
compound 13f most significantly lowered the tested proinflammatory
mediators (TNF‐α: 55.87 ± 2.82 ng/ml; NF‐κB: 150.45 ± 4.20 ng/ml; IL‐6:
102.10 ± 3.10 ng/ml; IL‐1β: 35.10 ± 2.5 ng/ml) as compared to Control.
The carrageenan‐induced group of animals had an expanded content of
proinflammatory cytokines, which was lessened by the treatment of the
synthesized derivatives as compared to normal control rats.
46.42% at 6 hr, respectively) as compared to compound 13d at
delayed hours. The compound consists of three distinguished
changes in the substitution pattern, that is, methoxy‐Ph on the
1,2,4‐triazole ring, pyridine on 1,3,4‐thiadiazole, and methoxy‐

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FIGURE 2 Pharmacophore (red), antipharmacophore (blue), and ballast (white) parts of the derivatives 13a–g against inflammation (L to R)
2
.5 Docking study
|
TABLE 3 In vitro cyclooxygenase (COX) % inhibition of synthe-
sized compounds 13a–g
Mesmerized by the notable anti‐inflammatory activity shown by
objected molecules against inflammation where TNF‐α, COX‐1, and
COX‐2 receptors are overexpressed, we used binding information of
our receptor against TNF‐α, COX‐1, and COX‐2 inhibitors for
validation of our results. The present study was based on a genetic
algorithm and in silico approaches. The details of ligand docking
scores of synthesized compounds are displayed in Tables 7–9. The
study utilized these to comprehend the binding competence of
compounds with a crystal structure of TNF‐α, COX‐1, and COX‐2
receptors. The methodology also validates the binding mode and
participating amino acid group with synthesized compounds at the
site of the active conformation of the cocrystal ligand A307 (for
TNF‐α), 63X (for COX‐1), and S58 (for COX‐2). In the docking
calculations, the LF rank score showed the virtual screening score
and used to recognize the correct ligand pose. The negative value of
the LF rank seems suitable for the selection of pose docked in protein
Deriva-
tives
COX‐1 (10 µl,
i.e., 1.0 μM)
COX‐2 (10 µl,
i.e., 1.0 μM)
Si (COX‐
2/COX‐1)
13a
13b
13c
0.30.25 ± 1.00***
0.30.60 ± 1.85***
0.52.10 ± 1.62*
0.40.20 ± 1.15***
0.40.10 ± 1.25**
0.53.10 ± 0.99*
0.52.50 ± 1.10*
0.52.80 ± 1.20
42.12 ± 1.24***
44.20 ± 1.25***
67.50 ± 1.11**
50.12 ± 1.08***
50.12 ± 1.50***
71.12 ± 1.28 (ns)
69.10 ± 1.75 (ns)
72.15 ± 1.00
1.39
1.44
1.29
1.24
1.24
1.22
1.31
1.36
1
1
1
1
3d
3e
3f
3g
Celecoxib
Note: % inhibition showed the means of three determinations using an
ovine COX‐2/COX‐1 assay kit.
Abbreviations: ANOVA, analysis of variance; SEM, standard error of the
mean; Si, selectivity index (COX‐2/COX‐1)
*p < 0.05, **p < 0.001, ***p < 0.0001 (significant).

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FIGURE 3 Images (a) and (b) show the in vitro COX‐1/COX‐2 enzyme inhibition assay of synthesized derivatives
active sites. Whereas LFdG, that is, ΔG‐score designed for the
estimation of free energy of ligand–protein complex in kcal/mol. LFV
score refers to the virtual screening docking score, this shows the
score for the virtual screening of active ligand to produce maximum
efficiency. LFLE refers to an estimated ligands efficiency computed
from the lead finder G‐score.
13e, and 13f showed the lowest bind energy (−11.148, −11.725, and
−12.37 kcal/mol, respectively). The receptor interaction, lower ΔG
docking score, and better LFLE score showed that compounds 13c,
13f, and 13g are most promising inhibitors of TNF‐α receptor. The
interaction of synthesized compounds with TNF‐α domain is
illustrated in Figure 6. In detail, the most pharmacologically active
compound 13f has the tendency to interact by steric clashes, that is,
C3‐LEU120 (2.9 Å); S15‐GLY121 (3.0 Å). The compound showed LF
rank score, −10.745 kcal/mol; ΔG docking score, −11.598 kcal/mol;
LFV score, −12.37 kcal/mol; LFLE score, −0.242. However, the second
most promising derivative 13g showed H‐bond: O40‐LYS98 (2.0 Å)
and steric clashes: O21‐LEU120 (2.8 and 3.0 Å). The interaction
scores were measured as: LF rank score −8.63 kcal/mol, ΔG docking
score −9.551 kcal/mol; LFV score, −10.3 kcal/mol: LFLE score,
−0.239. Results indicated that the whole series intensively showed
interaction with receptor protein through H‐bond and steric clashes.
The compounds had longer and broader structures when compared
with the cocrystal ligand and the protein binding pocket size. This
was an extensive cause for minor discrepancies in the binding
2
.5.1 | Binding analysis of inhibitors docked to
TNF‐α with a small molecule inhibitor A307 domain
The result showed that each of the derivatives has an affinity toward
proinflammatory receptors. The derivatives 13b, 13c, and 13g
showed both H‐bond interaction and steric clashes with the protein.
However, the compounds 13a, 13d, 13e, and 13f have the tendency
to interact through steric clashes only. We observed that cocrystal
ligand A307 and synthesized compounds 13a, 13c, 13e, and 13f
showed steric interaction with GLY121 residue. However, steric
interaction with SER60 were found to be common between
compounds 13a and cocrystal ligand A307. The compounds 13d,
TABLE 4 Anti‐inflammatory activity of the synthesized compounds 13a–g on carrageenan‐induced acute paw edema
Mean difference in paw volume (ml) after drug treatment (mean ± SEM)
Compound code
Control drug
0 min
1 hr
3 hr
6 hr
0.38 ± 0.01
0.27 ± 0.02*
0.34 ± 0.01*
0.36 ± 0.01*
0.30 ± 0.01*
0.29 ± 0.01*
0.29 ± 0.01*
0.27 ± 0.01*
0.30 ± 0.01*
0.48 ± 0.01
0.32 ± 0.01*
0.42 ± 0.01*
0.44 ± 0.02*
0.35 ± 0.02*
0.35 ± 0.01*
0.35 ± 0.01*
0.32 ± 0.01*
0.35 ± 0.01*
0.57 ± 0.02
0.35 ± 0.01*
0.51 ± 0.01*
0.53 ± 0.02*
0.40 ± 0.02*
0.41 ± 0.01*
0.40 ± 0.02*
0.36 ± 0.02*
0.39 ± 0.02*
0.66 ± 0.02
0.38 ± 0.01*
0.61 ± 0.01*
0.60 ± 0.02*
0.45 ± 0.02*
0.46 ± 0.02*
Standard drug
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
*
*
0.44 ± 0.02
0.39 ± 0.01*
0.45 ± 0.02*
3g
Note: Data were expressed as mean ± standard deviation.
Abbreviation: SEM, standard error of the means.
*
p < 0.0001 versus left hind paw two‐way analysis of variance followed by Bonferroni posttest as compared with Control.

8
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PATHAK ET AL.
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TABLE 5 Percentage inhibition of paw edema of synthesized
compounds 13a–g in carrageenan‐induced paws edema model
energies while comparing with experimental data. The binding mode
between TNF‐α domain target and the molecules 13a to 13g
including A307 and the structures in complex derived from the best
dock results cluster pose are given in Figure 7.
Percentage inhibition of paw edema
Drug code
1 hr
3 hr
6 hr
Standard drug
50
57.89****
10.52****
10.52****
47.36****
36.84****
42.10****
52.63**
60.71****
3.57****
14.28****
46.42****
39.28****
46.42****
57.14*
2
.5.2 | Binding analysis of inhibitors docked to
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
20****
20****
50 (ns)
40***
40****
50*
COX‐1 with a small molecule inhibitor 63X domain
The docking calculation at the site (grid) of domain ligand 63X showed
that each of the derivatives has an excellent affinity to interact with
the COX‐1 receptor. Except for 13d, the entire synthesized series
showed H‐bond interaction and steric clashes with the selected COX‐1
protein. We observed a common interaction with TYR355 between
the cocrystal ligand 63X and synthesized compounds 13a, 13b, 13c,
and 13g. The compounds 13a, 13e, 13f, and 13g showed the lowest
binding energy (−12.919, −13.957, −12.15, and −12.854 kcal/mol,
respectively). The docking calculations showed that except for
3g
50*
52.63**
46.42****
Note: % inhibition values were calculated by one‐way analysis of variance
Dunnett’s test).
p < 0.05; **p < 0.001; ****p < 0.0001 (significant) compared with the
standard drug.
(
*
FIGURE 4 Images (a–c) show the graphical bar of in vivo anti‐inflammatory activity of the derivatives (% inhibition of paw edema volume) at
, 3, and 6 hr against carrageenan‐induced rat paw edema
1

PATHAK ET AL.
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FIGURE 5 Graphs (a) and (b) showed carrageenan‐induced paw edema activity of the synthesized derivatives (difference in paw edema
volume) at 1, 3, and 6 hr
compound 13d, all the derivatives have a significant ΔG docking score.
The interaction results on the selected site of COX‐1 were illustrated
in Figure 7. Briefly, the most pharmacologically significant compound
(2.9 Å). Further, the docking score was found as, LF rank score,
−9.911 kcal/mol; ΔG docking score, −11.314 kcal/mol; LFV score,
−12.854 kcal/mol; and LFLE score, −0.283. The docking calculation
indicated that the compounds have intensive potential to bind with
selected COX‐1 protein. The best dock result cluster pose is displayed
in Figure 8.
13f, N16 and N20 atom showed H‐bond interaction with ARG120
(1.7 Å each) and steric clashes, that is, N16‐VAL116 (2.0 Å); S15‐
TYR353 (2.9 Å); C35 and C40 with GLU524 (2.9 Å each). The
ligand–protein complex showed −9.003 kcal/mol LF rank score,
−
9.759 kcal/mol ΔG docking score, −12.15 kcal/mol LFV score, and
0.203 LFLE score. However, the second lead compound 13g also
2
.5.3 | Binding analysis of inhibitors docked to
COX‐2 with a small molecule inhibitor S58 domain
−
showed multiple interactions, that is, H‐bond: N39, O12, and N16‐
ARG120 (2.3, 2.0, and 2.2 Å, respectively); N16‐TYR355 (2.4 Å); steric
Clashes: N20‐ARG120 (2.8 Å); S15‐TYR355 (3.0 Å), and H51‐VAL119
The docking calculation at the site (grid) of domain ligand S58
expressed that the entire series has mild to moderate affinity
ng/ml
ng/ml
ng/ml
ng/ml
FIGURE 6 Images (a–d) show the effect of the compounds against proinflammatory cytokines and inflammatory mediators. IL‐6,
interleukin‐6; IL‐1β, interleukin‐1β; NF‐κB, nuclear factor κ‐light‐chain‐enhancer of activated B cells; TNF‐α, tumor necrosis factor‐α

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TABLE 6 Effect of derivatives 13a–g on proinflammatory cytokines and inflammatory mediators
Treatment
TNF‐α
NF‐κB
IL‐6
IL‐1β
Control
40.22 ± 2.09
120.10 ± 4.18**
63.20 ± 2.65**
85.15 ± 2.23**
79.48 ± 3.03**
84.65 ± 4.45**
74.12 ± 2.2**
55.87 ± 2.82**
81.21 ± 3.52**
142.52 ± 4.09
242.25 ± 8.98**
164.30 ± 4.35**
210.10 ± 3.45**
198.10 ± 6.25**
202.18 ± 1.25**
182.62 ± 2.54**
150.45 ± 4.20*
198.86 ± 6.21**
102.50 ± 4.29
301.22 ± 4.60**
106.20 ± 4.21*
156.10 ± 3.25**
142.20 ± 4.50
148.65 ± 1.20
138.50 ± 2.58
102.10 ± 3.10*
144.26 ± 3.72
21.82 ± 1.35
95.95 ± 1.25**
40.2 ± 1.10**
72.10 ± 2.5**
70.52 ± 2.1**
72.0 ± 1.14**
69.85 ± 2.87**
35.10 ± 2.5**
70.92 ± 2.6**
Carrageenan‐induced
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
3g
Data are presented as the mean ± SEM (n = 6) and analyzed by one‐way analysis of variance Dunnett test as statistically significantly.
Abbreviations: IL‐6, interleukin‐6; IL‐1β, interleukin‐1β; NF‐κB, nuclear factor κ‐light‐chain‐enhancer of activated B cells; SEM, standard error of the
mean; TNF‐α, tumor necrosis factor‐α.
*
p < 0.05, p < 0.001 (ns) compared with the Control.
toward COX‐2 receptor. The derivatives 13a, 13b, 13e, and 13f
showed both H‐bond interaction and steric clashes with the
protein. However, the derivative 13g had the potential for
forming H‐bonds with selected proteins. The derivatives 13c
and 13d have a tendency to interact through steric clashes with
the receptor protein only. It was observed that cocrystal ligand
S58 and synthesized compounds 13b, 13e, 13f, and 13g interact
with the GLY121 residue, which showed similarity in binding
characteristics. However, steric interaction with LEU352 was
found common between compound 13d and cocrystal ligand S58.
The compounds 13a, 13e, and 13f showed the lowest bind energy
COX‐2. All the docking results of the synthesized compounds
with COX‐2 domains are illustrated in Figure 8. In detail, the most
pharmacologically significant compound 13f showed multiple
interactions, for example, H‐bond: H58‐GLN192 (2.0 Å); N28‐
GLN192 (2.6 Å), and steric clashes C31‐GLN192 (2.9 Å); O45‐
ASP347 (2.5 Å); C48‐GLN350 (2.9 Å); O46‐HIS356 (2.9 Å). The
compound showed −7.642 kcal/mol LF rank score, −5.052 kcal/
mol ΔG docking score, and −8.57 kcal/mol LFV score. However,
another potent derivative 13g showed H‐bond O12‐GLN192
(2.4 Å); H61‐GLU346 (2.5 Å) and −7.059 kcal/mol LF rank score,
−5.539 kcal/mol ΔG docking score, and −7.694 kcal/mol LFV
score. The docking calculation indicated that the entire set of
derivatives possessed mild to moderate capability to interact
with the selected receptor protein. The structural variations were
(
−8.596, −7.886, and −8.57 kcal/mol, respectively). The receptor
interaction, lower ΔG docking score, and better LFLE score result
showed that compounds 13e and 13f are most promising against
TABLE 7 Docking study of synthesized derivatives 13a–g against 2AZY (TNF‐α) protein–ligand complex
LF rank score
(kcal/mol)
ΔG docking score LFV score
Derivatives
H‐bond interactions
(kcal/mol)
(kcal/mol)
LFLE score
1
1
1
1
1
1
1
3a
3b
3c
3d
3e
3f
Steric clashes: S15‐GLY121 (2.9 Å); C3‐LEU120
−8.709
−10.895
−9.97
−9.532
−10.681
−0.238
(
3.0 Å); C1‐SER60 (2.9 Å)
H‐bond: H58‐LEU120 (2.1 Å); O43‐LYS11 (2.0 Å);
O43‐LYS98 (1.9 Å); O42‐ASP10 (1.8 Å)
−9.407
−8.939
−9.975
−10.281
−11.598
−9.551
−8.376
−10.94
−10.699
−11.148
−11.725
−12.37
−10.3
−0.209
−0.203
−0.238
−0.224
−0.242
−0.239
−0.209
H‐bond: O44‐TYR119 (2.1 Å); O43‐LYS98 (1.9 Å)
Steric clashes: O40‐GLY121 (2.8 Å)
Steric clashes: N26‐TYR59 (3.0 Å); H56‐TYR151
−9.176
−9.207
−10.745
−8.63
(
2.9 Å)
Steric clashes: S15‐GLY121 (2.9 Å); C3‐LEU120
2.8 Å); S31‐LEU120 (2.8 Å)
Steric clashes: C3‐LEU120 (2.9 Å); S15‐GLY121
3.0 Å).
(
(
3g
H‐bond: O40‐LYS98 (2.0 Å)
Steric clashes: O21‐LEU120 (2.8 Å, 3.0 Å)
Reference ligand
A307
Steric clashes: O35‐GLY121 (2.8 Å); C8‐SER60
(2.9 Å); C7‐TYR151 (2.9 Å); F4‐TYR151 (2.7 Å);
F4‐GLN 61 (2.7 Å).
−7.179
−10.336
Abbreviations: LF, lead finder; LFLE, ligands efficiency computed from the lead finder G‐score; LFV score, virtual screening docking score; TNF‐α, tumor
necrosis factor‐α.

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TABLE 8 Docking study of the synthesized derivatives 13a–g against 5WBE (COX‐1) protein–ligand complex
LF rank score
(kcal/mol)
ΔG docking
LFV score
(kcal/mol)
Derivatives
H‐bond interactions
score (kcal/mol)
LFLE score
1
3a
H‐bond: N16‐TYR355 (2.5 Å); O12‐ARG120 (2.5 Å); H51‐
GLU524 (1.8 Å)
−9.89
−12.062
−5.54
−12.919
−0.302
1
3b
H‐bond: N20‐ARG120 (1.6 Å); N16‐ARG120 (2.4 Å)
−4.487
−8.759
−0.123
Steric clashes: C36‐GLU524 (2.8 Å); O42‐LEU352 (2.9 Å);
O42‐VAL349 (2.7 Å); O44, C33, and N26 with ILE89 (3.0, 3.0,
and 2.9 Å, respectively); O44‐TYR355 (2.8 Å); C30‐ALA527
(2.9 Å)
1
3c
H‐bond: O12‐ARG120 (1.6 Å); N16‐ARG120 (2.4 Å)
Steric clashes: N28 and C24‐GLU524 (2.9 and 3.0 Å,
respectively); C30‐ALA527 (2.9 Å); C3‐VAL116 (2.9 Å); C24‐
ARG120 (2.9 Å); C13, N10, and C7 with TYR355 (2.9, 2.7,
and 2.7 Å, respectively)
−7.267
−7.141
−9.892
−0.162
1
3d
3e
Steric clashes: C3, N28 and C4 with ILE523 (2.7, 2.6, and
−4.065
−9.338
−3.941
−4.944
−0.094
−0.255
2.8 Å, respectively); C42‐SER530 (2.6 Å); C2 and C4 with
LEU352 (2.6 Å)
1
H‐bond: N16 and N20 with ARG120 (21.9 and 2.0 Å,
respectively)
−11.719
−13.957
Steric clashes: O21 and C27 with ARG120 (2.7 and 2.9 Å,
respectively); C34‐VAL119 (2.8 Å)
1
3f
H‐bond: N16 and N20 with ARG120 (1.7 Å each)
Steric clashes: N16‐VAL116 (2 Å); S15‐TYR353 (2.9 Å); C35
and C40 with GLU524 (2.9 Å each)
−9.003
−9.911
−9.759
−12.15
−0.203
−0.283
1
3g
H‐bond: N39, O12, and N16‐ARG120 (2.3, 2.0, and 2.2 Å,
respectively); N16‐TYR355 (2.4 Å)
−11.314
−12.854
Steric clashes: N20‐ARG120 (2.8 Å); S15‐TYR355 (3.0 Å),
H51‐VAL119 (2.9 Å)
Reference
ligand 63X
H‐bond: O25‐TYR355 (3.0 Å)
Steric clashes: O25‐TYR355 (3.0 Å)
−8.938
−8.473
−9.946
−0.339
Abbreviations: LF, lead finder; LFLE, ligands efficiency computed from the lead finder G‐score; LFV score, virtual screening docking score.
TABLE 9 Docking study of the synthesized derivatives 13a–g against 1CX2 (COX2) protein–ligand complex
LF rank score
kcal/mol
ΔG docking
LFV score
kcal/mol
Derivatives
H‐bond interactions
score kcal/mol
LFLE score
13a
13b
13c
H‐bond: O21‐ASP515 (2.10 Å); C33‐HIS95 (2.2 Å)
Steric clashes: C1‐PRO191 (3.0 Å); O12‐GLY354 (2.9 Å)
−6.232
−5.887
−4.307
−7.716
−4.471
−4.742
−8.596
−6.983
−7.57
−0.193
H‐bond: O44‐SER353 (2.4 Å)
Steric clashes: O21‐HIS351 (2.6 Å), C22‐GLN192 (2.8 Å)
−0.099
−0.108
Steric clashes: O21‐ASP515 (2.6 Å); C36‐ALA516 (3.1 Å);
C39‐HIS90 (2.9 Å); O43‐ALA516 (2.9 Å); O43‐ARG513
(
2.9 Å)
1
1
3d
3e
Steric clashes: C30‐LEU93 (2.7 Å); N20‐LEU93 (2.7 Å);
N16‐TYR355 (2.9 Å); C30‐LEU352 (3.0 Å)
−4.323
−6.111
−4.775
−5.438
−7.621
−7.886
−0.126
H‐bond: O45‐HIS95 (2.0 Å)
0.105
Steric clashes: C38‐HIS95 (3.0 Å); C11‐GLN192 (3.0 Å);
O21‐HIS351 (2.7 Å)
1
3f
H‐bond: H58‐GLN192 (2.0 Å); N28‐GLN192 (2.6 Å)
Steric clashes: C31‐ GLN192 (2.9 Å); O45‐ASP347 (2.5 Å);
C48‐GLN350 (2.9 Å); O46‐HIS356 (2.9 Å)
−7.642
−5.052
−8.57
−0.305
1
3g
H‐bond: O12‐GLN192 (2.4 Å); H61‐GLU346 (2.5 (2.5 Å)
−7.059
−10.03
−5.539
−9.233
−7.694
−0.238
−0.355
Reference ligand S58
H‐bond: H37‐GLN192 (2.0 Å), O21‐PHE518 (2.3 Å)
Steric clashes: C9‐LEU352 (2.9 Å); F24‐TYR355 (2.9 Å)
−11.817
Abbreviations: LF, lead finder; LFLE, ligands efficiency computed from the lead finder G‐score; LFV score, virtual screening docking score.

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FIGURE 7 Images (a) and (b) of the synthesized derivatives (13f and 13g) into the active site of the tumor necrosis factor‐α (TNF‐α)
receptor (PDB: 2AZY) showed the interaction side chain of amino acid, green color dotted line showed the H‐bond interaction with
amino acid residue. However, protein TNF‐α complex with 13a−g (stick) compounds including the A307 cocrystal ligand (stick)
found in terms of geometry and atomic basin as compared to
standard cocrystal ligand S58 and this may be the cause of the
narrowing of the binding grid of the protein pocket. The
interaction between synthesized ligands and the COX‐2 domain
is illustrated in Figure 9.
protein as the reference drug and can interact as a similar blueprint
and approach.
3
|
CONCLUSION
In summary, we observed that these multiple interactions (with
TNF‐α, COX‐1, and COX‐2) with the parent skeleton proved that
synthesized derivatives have notable potency to fit with similar
In this communication, we delivered a series of 1,2,4‐triazole‐
conjugated 1,3,4‐thiadiazole hybrid scaffolds via three components,

PATHAK ET AL.
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FIGURE 8 Images (a) and (b) of the synthesized derivatives (13f and 13g) into the active site of COX‐1 receptor (PDB: 5WBE) showed the
interaction side chain of amino acids, green color dotted line showed the H‐bond interaction with amino acid residue. However, protein COX‐1
complex with 13a–g (stick) compounds including the A307 cocrystal ligand (stick)
followed by physicochemical, 3D QSAR analysis, docking, and proved
them as promising anti‐inflammatory agents. The study revealed that
compounds 13a, 13e, 13f, and 13g have an optimal logP value, which
advocates that the compounds have a superior predisposition in
transport through physiological barriers. Furthermore, in vitro and in
vivo anti‐inflammatory activity assay confirms that the derivatives
13c, 13f, and 13g have a prominent response against inflammation.
The measurement of proinflammatory mediators in the treated

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FIGURE 9 Images (a) and (b) of the synthesized derivatives (13f and 13g) into the active site of COX‐2 receptor (PDB: 1CX2) showed the
interaction side chain of amino acids, green color dotted line showed the H‐bond interaction with amino acid residue. However, COX‐2 complex
with 13a−g (stick) compounds including the A307 cocrystal ligand (stick)

PATHAK ET AL.
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|
groups showed that they are downregulated. The docking studies
recommended that synthesized compounds have moderate to
significant potential to interact with ligand TNF‐α, COX‐1, and
COX‐2 proteins. The result recommends that these molecules will
definitely hold a promise for the future drug development initiative.
after the completion of the reaction, the mixture was concentrated
under reduced pressure. The resulting crude solid was filtered,
washed with water, and recrystallized from aqueous ethanol.
4
.1.4 | General procedure for the synthesis of the
potassium salts of the substituted dithiocarbazinic
4
|
EXPERIMENTAL
acids (4a–c)
0
.01 mol of compounds 3a–c were dissolved in KOH (0.02 mol)
4
4
.1 Chemistry
|
containing absolute alcohol (50 ml). Further, carbon disulphide
(0.013 mol) was added and the mixture was stirred at room
temperature for 16 hr. The mixture was diluted with ether (30 ml)
and stirred further for 1 hr. The potassium salt was used immediately
for the next stage without further purification.
.1.1 | General
The experiment was carried out using commercially available
analytical grade solvents and reagents without further purification.
The melting points of the synthesized compounds were determined
in an open capillary tube Hicon melting point apparatus and were not
corrected. Thin‐layer chromatography (TLC) was performed on silica
gel G‐coated plates to detect the completion of the reaction. The
diverse mobile phase was selected in a different proportion according
to the assumed polarity of the products. The spots were visualized by
exposure to the iodine vapor. Infrared (IR) spectra were recorded in
Perkin Elmer RX‐I spectrophotometer using KBr pellets and the
4
1
.1.5 | General procedure for the synthesis of the
,2,4‐triazoles (5a–c)
Hydrazine hydrate (99%; 0.02 mol) was gradually added to the
dithiocarbazinic acids (potassium salt; 0.01 mol) (4a–c) dissolved in
water (20 ml) with stirring and the mixture was refluxed gently for
3 hr, during which hydrogen sulphide evolved and the color of the
reaction mixture changed to dark green. When the evolution of
hydrogen sulfide ceased (lead acetate test), it was then cooled to 5°C
and acidified with conc. HCl to pH 1.00. A yellow solid separated out
which was filtered, washed with water, and recrystallized from
ethanol to make the 1,2,4‐triazole skeleton.
−
1 1
reported wave numbers are given in cm
. H NMR spectra were
recorded in dimethyl sulfoxide (DMSO) on Cryomagnet spectrometer
1
3
4
3
00 MHz (Bruker). However, C NMR was recorded in CDCl using
Bruker Avance II 100 NMR spectrometer, respectively. Chemical
shifts were reported as δ (ppm) relative to tetramethylsilane (TMS) as
an internal standard. Mass spectra were recorded on Q‐ToF Micro
+
Waters spectrometer and reported as (M+H) . However, elemental
analysis was carried out on a Vario EL‐III CHNOS elemental analyzer.
The original NMR spectra are provided as Supporting Information
as are the InChI codes of the investigated compounds together with
some biological activity data.
4
.1.6 | General procedure for the synthesis of
N‐(5‐mercapto‐3‐subsituted‐1,5‐dihydro‐1,2,4‐
triazol‐4‐yl)acetamides (7a–e)
Substituted 1,2,4‐triazoles (0.5 mol) (5a–c) were dissolved in glacial
acetic acid (25 ml) containing 25 ml of saturated solution of sodium
acetate. In case the substance did not dissolve completely, the
mixture was warmed and the solution was cooled in ice bath with
stirring. To this, acetyl chloride or benzoyl chloride (0.06 mol) (6) was
added drop‐wise so that vigorous reaction did not take place. After
half an hour, a white product was separated and filtered. The product
was washed with 50% aqueous acetic acid and finally with water. It
was purified by recrystallization from absolute alcohol.
4
.1.2 | General procedure for the synthesis of the
acid esters (2a–c)
The corresponding carboxylic acid (0.1 mol) (1a–c) was dissolved in
2 4
methanol (50 ml). Conc. H SO (0.02 mol) was added in the reaction
mixture in a drop‐wise manner and refluxed for 8–10 hr. The
completion of reaction monitored by TLC (n‐hexane/ethyl acetate
7
:3) and an excess amount of methanol was removed. The reaction
mixture was poured into water, neutralized with sodium carbonate
and extracted with diethyl ether (3 × 50 ml). The combined organic
layers were dried over anhydrous sodium sulphate.
4
5
.1.7 | General procedure for the synthesis of
‐m‐subsituted‐phenyl‐[1,3,4]thiadiazol‐2‐ylamines
(10a–d)
4
.1.3 | General procedure for the synthesis of the
acid hydrazides (3a–c)
An ethanolic solution of aromatic carboxylic acid (0.05 mol) (8a–d)
was added to an aqueous solution of thiosemicarbazide (0.05 mol) (9)
with constant stirring, and a few drops of conc. sulphuric acid was
added and heated for 4 hr at 80–90°C, after completion of reaction
The aryl ester (0.02 mol) (2a–c) was dissolved in methanol (30 ml).
Now (80%, 0.04 mol) hydrazine hydrate was slowly added with
stirring and the reaction mixture was heated under reflux. The
reaction was monitored by TLC (petroleum ether/acetone 8:2) and
2 3
(TLC), cool and poured to ice‐cold water, basified with 10% Na CO
solution, filter, dried, and recrystallized from suitable solvent.

16 of 20
PATHAK ET AL.
|
4
.1.8 | General procedure for the synthesis of the
Schiff bases (Z)‐3‐(5‐p‐substituted‐1,3,4‐thiadiazol‐2‐
ylimino)‐3,3a‐dihydro‐1H‐indol‐2(7aH)‐one (12a–d)
(Z)‐N‐(5‐Mercapto‐3‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐
yl)‐N‐((3‐((5‐(4‐nitrophenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐2‐oxoindo-
lin‐1‐yl)methyl)acetamide (13b)
Physical state: Yellowish brown powder; Yield: 74%; Mol wt: 644.64;
Equimolar quantities (0.01 mol) of isatin (11) and the corresponding
f
M.p. 191–193°C; R , 0.81; solubility in ethanol, acetone, chloroform,
1
,3,4‐thiadiazole compound (10a–d) were dissolved in warm ethanol
−
1
DMSO; FTIR spectra (KBr) Vmax cm : 3,077 Ar (C–Hstr), 1,595 Ar
(
50 ml) containing glacial acetic acid (0.5 ml). The reaction mixture
(
C═Cstr), 1,686 (C═Ostr), 1,322 Ar (C–Nstr), 1,414 (N–Nstr), 1,158
C–Nstr), 1,125 (CH3 str), 1,215 (N–Nstr), 727 (C–S–C thiadizolestr),
was refluxed for 4 hr and then kept at room temperature overnight.
The resultant solid was washed with dilute ethanol, dried, and
recrystallized from ethanol–water (1:2) mixture to afford the
compounds.
(
1,492 (–C═N ring stretching), 1,613 Ar (C═C ring stretching), 874 Ar
(
C–Hdef), 1,692 (C═Ostr), 1,296 Ar (C–NO2 str), 1,377 Ar (C–NO2 str),
1
1
,091 (C–Nstr), and 752 (C–Sstr); H NMR (400 MHz, DMSO, TMS) δ
ppm: 1.52 (s, 1H, SH), 2.08 (s, 3H, CH
s, 2H, CH ), 7.34 (t, 1H, CH, isatin), 7.60 (t, 1H, CH, isatin), 7.73 (d,
H, CH, isatin), 7.91 (d, 1H, CH, isatin), 8.03 (d, 1H, 2 × CH, NO ‐Ph),
‐Ph), and 9.13
) δ ppm: 22.8, 68.1, 70.2,
3
), 5.02 (s, 1H, CH, triazole), 5.54
(
2
4
.1.9 | General procedure for the synthesis of
1
8
2
N‐(5‐mercapto‐3‐substituted‐1,5‐dihydro‐1,2,4‐
triazol‐4‐yl)‐N‐((2‐oxo‐3‐(5‐substituted‐1,3,4‐
thiadiazol‐2‐ylimino)‐3,3a‐dihydro‐2H‐indol‐
.27 (d, 1H, 2× CH, NO
2
‐Ph), 8.42 (d, 1H, 2 × CH, NO
2
13
(
s, 1H, NH); C NMR (100 MHz, CDCl
3
1
1
15.8, 117.7, 124.0, 128.6, 129.7, 131.4, 134.9, 139.6, 147.4, 149.6,
1
(7aH)‐yl)substituted)acetamide (13a–g)
+
63.9, 171.10, and 17.9; Mass spectra: 644.10 (M+H) ; Elemental
, Calculated: C, 50.31; H, 3.13; N, 21.73;
Found: C, 51.12; H, 3.42; N, 21.30.
20 10 6 2
analysis for C27H N O S
The corresponding Schiff bases (7a–e and 12a–d) (0.002 mol)
were dissolved in absolute DMSO (30 ml). Then formaldehyde
(
37%, 0.5 ml) and piperidine (0.2 mmol) were added drop‐wise
(
Z)‐N‐(5‐Mercapto‐3‐(4‐nitrophenyl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐
with vigorous stirring. After combining all reagents, the reaction
mixture was stirred at room temperature for 5–6 hr. The mixture
was cooled; the solid product was filtered and washed with
petroleum ether. The solid that separated was recrystallized from
ethanoldioxane (1:2) to yield the title compounds 13a–g. The
reaction mixture was stirred at the ambient temperature for 1 hr
and left overnight. The solid separated was collected by filtration,
washed with aqueous ethanol, dried, and crystallized from
ethanol.
yl)‐N‐((3‐((5‐(4‐methoxyphenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐2‐oxoin-
dolin‐1‐yl)methyl)acetamide (13c)
Physical state: Yellowish red powder; Yield: 70%; Mol wt: 629.67;
M.p. 260–262°C; R
f
, 0.66; solubility in ethanol, acetone, chloroform,
−
1
DMSO; FTIR spectra (KBr) Vmax cm : 3,116 Ar (C–Hstr), 1,570 Ar
(
C═Cstr), 1,751 (C═Ostr), 1,380 Ar (C–Nstr), 1,428 (N–Nstr), 1,224
C–Nstr), 1,169 (CH3 str), 1,234 (N–Nstr), 730 (C–S–C thiadizolestr),
(
1
,481 (–C═N ring stretching), 1,622 Ar (C═C ring stretching), 894 Ar
(C–Hdef), 1,601 (C═Ostr), 1,296 Ar (C–NO2 str), 1,099 (Ar–OCH3 str),
1
1
,085 (C–Nstr), and 698 (C–Sstr); H NMR (400 MHz, DMSO, TMS) δ
(
Z)‐N‐((3‐((5‐(4‐Chlorophenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐2‐oxoin-
dolin‐1‐yl)methyl)‐N‐(5‐mercapto‐3‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐
,2,4‐triazol‐4‐yl)acetamide (13a)
Physical state: Yellowish white powder; Yield: 73%; Mol wt: 590.08;
M.p. 178–180°C; R , 0.86; solubility in ethanol, acetone, chloroform,
DMSO; Fourier‐transform infrared (FTIR) spectra (KBr) Vmax cm
ppm: 1.52 (s, 1H, SH), 2.08 (s, 3H, CH
(s, 1H, CH, triazole), 5.54 (s, 2H, CH ), 7.03 (d, 1H, 2× CH, methoxy‐
Ph), 7.34 (t, 1H, CH, isatin), 7.60 (t, 1H, CH, isatin), 7.73 (d, 1H, CH,
isatin), 7.77 (d, 1H, 2× CH, methoxy‐Ph), 8.11 (d, 1H, 2× CH, NO ‐Ph),
C NMR
) δ ppm: 22.8, 55.8, 68.1, 70.2, 114.9, 115.6, 117.7,
3 3
), 3.81 (s, 3H, OCH ), 5.02
2
1
2
13
f
8.42 (d, 1H, 2× CH, NO
2
‐Ph), and 9.13 (s, 1H, NH);
−
1
:
(100 MHz, CDCl
3
3,080 Ar (C−Hstr), 1,614 Ar (C═Cstr), 885 Ar (C−Hdef), 1,701 (C═Ostr),
1,349 Ar (C−Nstr), 1,513 (N−Nstr), 1,281 (C−Nstr), 1,405 (C−Hstr),
1,239 (CH3 str), 1,196 (N−Nstr), 794 (C−S−C thiadizolestr), 735
125.1, 126.2, 128.5, 129.4, 131.9, 135.1, 147.1, 148.2, 160.6, 163.5,
+
170.7, and 175.1; Mass spectra: 629.13 (M+H) ; Elemental analysis
23 9 5 2
for C28H N O S , Calculated: C, 53.41; H, 3.68; N, 20.02; Found: C,
(C−Clstr), 1,576 Ar (C═C ring stretching), 1,436 (–C═N ring
53.10; H, 3.62; N, 19.06.
1
stretching), 1,349 (C═Cstr), and 669 (C–Sstr); H NMR (400 MHz,
DMSO, TMS) δ ppm: 1.52 (s, 1H, SH), 2.08 (s, 3H, CH
CH, triazole), 5.54 (s, 2H, CH ), 7.34 (t, 1H, CH, isatin), 7.53 (d, 1H,
× CH, Cl‐Ph), 7.60 (t, 1H, CH, isatin), 7.73 (d, 1H, CH, isatin), 7.90 (d,
H, CH, isatin), 8.02 (d, 1H, 2 × CH, pyridine), 8.68 (d, 1H, 2× CH,
3
), 5.02 (s, 1H,
(Z)‐N‐((3‐((5‐(4‐Chlorophenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐2‐oxoin-
dolin‐1‐yl)methyl)‐N‐(5‐mercapto‐3‐(4‐methoxyphenyl)‐1,5‐dihydro‐
4H‐1,2,4‐triazol‐4‐yl)acetamide (13d)
2
2
1
Physical state: Yellowish brown powder; Yield: 79%; Mol wt: 619.12;
1
3
pyridine), and 9.13 (s, 1H, NH); C NMR (100 MHz, CDCl
3
) δ ppm:
f
M.p. 231–233°C; R , 0.86; solubility in ethanol, acetone, chloroform,
−
1
2
1
2.8, 68.1, 70.2, 115.8, 117.7, 122.2, 124.4, 128.9, 131.6, 134.3,
DMSO; FTIR spectra (KBr) Vmax cm : 3,080 Ar (C–Hstr), 1,614 Ar
(C═Cstr), 885 Ar (C–Hdef), 1,701 (C═Ostr), 1,349 Ar (C–Nstr), 1,513
(N–Nstr), 1,281 (C–Nstr), 1,405 (C–Hstr), 1,239 (Ar–OCH3 str), 1,196
(N–Nstr), 794 (C–S–C thiadizolestr), 735 (C–Clstr), 1,576 Ar (C═C ring
36.6, 147.4, 149.6, 163.8, 170.7, and 174.1; Mass spectra: 589.09
+
(
M+H) ; Elemental analysis for
26
C H
20ClN
9
O S
2 2
,
Calculated:
C, 52.92; H, 3.42; N, 21.36; Found: C, 52.02; H, 3.12; N, 21.19.

PATHAK ET AL.
17 of 20
|
stretching), 1,436 (–C═N ring stretching), 1,349 (CH3 str), and 669
28 8 4 2
for C34H N O S , Calculated: C, 60.34; H, 4.17; N, 16.56; Found: C,
1
(C–Sstr); H NMR (400 MHz, DMSO, TMS) δ ppm: 1.12 (s, 1H, SH),
60.42; H, 4.51; N, 16.10.
2
.08 (s, 3H, CH
s, 2H, CH ), 7.05 (d, 1H, 2 × CH, methoxyl‐Ph), 7.34 (d, 1H, 2 × CH,
methoxy‐Ph), 7.53 (d, 1H, 2 × CH, Cl‐Ph), 7.60 (t, 1H, CH, isatin), 7.73
3 3
), 3.81 (s, 3H, OCH ), 5.02 (s, 1H, CH, triazole), 5.54
(
2
(Z)‐N‐((3‐((5‐(4‐Hydroxyphenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐2‐oxoin-
dolin‐1‐yl)methyl)‐N‐(5‐mercapto‐3‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐
1,2,4‐triazol‐4‐yl)acetamide (13g)
(
d, 1H, CH, isatin), 7.94 (d, 1H, CH, isatin), 8.02 (d, 1H, 2 × CH, Cl‐Ph),
13
8
.42 (d, 1H, 2 × CH
2 2
, NO ‐Ph), and 9.13 (s, 1H, NH); C NMR
(100 MHz, CDCl
3
) δ ppm: 22.8, 55.8, 68.1, 70.2, 114.9, 115.6, 117.7,
Physical state: Yellowish brown powder; Yield: 74%; Mol wt: 571.63;
1
1
25.1, 126.2, 128.5, 129.4, 131.9, 135.1, 147.1, 148.2, 160.6, 163.6,
M.p. 219–221°C; R
f
, 0.79; solubility in ethanol, acetone, chloroform,
+
−1
70.7, and 175.1; Mass spectra: 618.10 (M+H) ; Elemental analysis
DMSO; FTIR spectra (KBr) Vmax cm : 3,082 Ar (C–Hstr), 886 Ar
(C–Hdef), 1,699 (C═Ostr), 1,341 Ar (C–Nstr), 1,526 Ar (C–NO2 str),
1,432 (C–Hstr), 1,177 Ar (OHstr), 1,110 (N–Nstr), 751 (C–S–C
thiadizolestr), 1,629 Ar (C═C ring stretching), 1,598 (–C═N ring
for C28
8 3 2
H23ClN O S , Calculated: C, 54.32; H, 3.74; N, 18.10; Found:
C, 54.02; H, 3.86; N, 18.34.
1
(
Z)‐N‐(5‐Mercapto‐3‐(pyridin‐4‐yl)‐1,5‐dihydro‐4H‐1,2,4‐triazol‐4‐
yl)‐N‐((3‐((5‐(4‐methoxyphenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐
‐oxoindolin‐1‐yl)methyl)benzamide (13e)
Physical state: Yellowish brown powder; Yield: 68%; Mol wt: 647.73;
M.p. 216–218°C; R , 0.84; solubility in ethanol, acetone, chloroform,
DMSO; FTIR spectra (KBr) Vmax cm : 3,118 Ar (C–Hstr), 833 Ar
stretching), 1,304 (C═Cstr), and 668 (C–Sstr); H NMR (400 MHz,
3
DMSO, TMS) δ ppm: 1.54 (s, 1H, SH), 3.81 (s, 3H, OCH ), 5.02 (s, 1H,
2
CH, triazole), 6.86 (d, 1H, 2 × CH, hydroxyl‐Ph), 7.34 (d, 1H, CH,
isatin), 7.60 (t, 1H, CH, isatin), 7.73 (d, 1H, CH, isatin), 7.91 (d, 1H, CH,
f
isatin), 8.03 (s, 1H, 2 × CH, pyridine), 8.66 (d, H, 2 × CH, pyridine),
13
−1
3
9.13 (s, 1H, NH), and 9.67 (s, 1H, OH); C NMR (100 MHz, CDCl ) δ
(
C–Hdef), 1,756 (C═Ostr), 1,373 Ar (C–Nstr), 1,491 (N–Nstr), 1,235
C–Nstr), 1,413 (C–Hstr), 1,123 (N–Nstr), 788 (C–S–C thiadizolestr),
ppm: 22.8, 65.1, 70.2, 114.2, 115.2, 117.2, 122.2, 124.2, 126.2, 128.9,
(
131.2, 136.6, 144.9, 146.2, 158.5, 163.2, 165.2, and 168.9; Mass
+
1
,595 Ar (C═C ring stretching), 1,215 (Ar–OCH3 str), 1,379 (–C═N
spectra: 571.12 (M+H) ; Elemental analysis for C26
H
21
9 3
N O S
2
,
1
ring stretching), 1,293 (C═Cstr), and 725 (C–Sstr); H NMR (400 MHz,
Calculated: C, 54.63; H, 3.70; N, 22.05; Found: C, 54.42; H, 3.98; N,
21.60.
DMSO, TMS) δ ppm: 1.24 (s, 1H, SH), 3.81 (s, 3H, OCH
CH, triazole), 5.54 (s, 2H, CH ), 7.03 (d, 1H, 2 × CH, methoxy‐Ph),
.34 (t, 1H, CH, isatin), 7.54 (d, 1H, 2 × CH, Ph), 7.60 (t, 1H, CH,
isatin), 7.62 (d, 1H, CH, Ph), 7.73 (d, 1H, CH, isatin), 7.91 (d, 1H, CH,
isatin), 7.95 (d, 1H, 2 × CH, Ph), 8.66 (d, 1H, 2 × CH , pyridine), and
) δ ppm: 55.8, 68.9, 69.7,
3
), 5.02 (s, 1H,
2
7
4.2
|
Pharmacology
2
.2.1 | In vitro COX inhibition assay[9,35]
4
13
9
1
1
.13 (s, 1H, NH); C NMR (100 MHz, CDCl
3
14.8, 115.2, 117.9, 122.2, 123.1, 124.4, 127.5, 128.1, 128.9, 131.2,
All the synthesized derivatives (13a–g) were evaluated against
COX‐1 and COX‐2 enzymes. The analysis was done by using
colorimetric enzyme assay kit (Catalog No. 760131; Cayman
Chemicals Inc., Ann Arbor, MI). The determination of peroxidase
activity is evaluated by a colorimetric method at 590 nm. During
assay, the appearance of oxidized N,N,N,N‐tetramethyl‐p‐phenyle-
nediamine (TMPD) was measured. Stock solutions of test deriva-
tives (10 µl, i.e., 1 µg/ml) were prepared by dissolving in DMSO. All
the derivatives are mixed with the series of supplied reagents,
buffer solution (0.1 M Tris‐HCl, pH 8), heme and either COX‐1 or
COX‐2 enzyme. These solutions were incubated for a period of
5 min at 25°C, after which colorimetric substrate TMPD and
arachidonic acid were added quickly. The solutions were shaken
for a few seconds and then incubated at 25°C for 2 min. The
reaction produced a blue color that absorbs at 590 nm. The
intensity of the blue color was determined by a microplate reader
(PowerWave XS2). A blank solution was prepared by addition of all
the assay components, except the substrate. By removing the
blank control, we obtained the corrected activity. Celecoxib (1 μM)
was used as a standard drug. The percent COX inhibition was
calculated using the following equation:
31.9, 136.6, 137.4, 147.4, 149.2, 160.6, 163.5, 172.0, and 176.1;
+
Mass spectra: 647.15 (M+H) ; Elemental analysis for C32
25
H N
9 3
O S
2
,
Calculated: C, 59.34; H, 3.89; N, 19.46; Found: C, 58.95; H, 4.19; N,
0.10.
2
(
Z)‐N‐(5‐Mercapto‐3‐(4‐methoxyphenyl)‐1,5‐dihydro‐4H‐1,2,4‐tria-
zol‐4‐yl)‐N‐((3‐((5‐(4‐methoxyphenyl)‐1,3,4‐thiadiazol‐2‐yl)imino)‐2‐
oxoindolin‐1‐yl)methyl)benzamide (13f)
Physical state: Yellowish powder; Yield: 76%; Mol wt: 676.77; M.p.
2
f
21–213°C; R , 0.87; solubility in acetone, chloroform, DMSO; FTIR
−1
spectra (KBr) Vmax cm : 3,096 Ar (C–Hstr), 1,599 Ar (C═Cstr), 1,696
(
C═Ostr), 1,380 Ar (C–Nstr), 1,433 (N–Nstr), 1,239 (C–Nstr), 1,301
N–Nstr), 734 (C–S–C thiadizolestr), 1,526 (–C═N ring stretching),
(
1
,638 Ar (C═C ring stretching), 883 Ar (C–Hdef), 1,566 (C═Ostr),
1
,347 (Ar–OCH3 str), 1,088 (Ar–OCH3 str), 1,012 (C–Nstr), and 667
1
(C–Sstr); H NMR (400 MHz, DMSO, TMS) δ ppm: 1.54 (s, 1H, SH),
3
3 2
.81 (s, 3H, OCH ), 5.02 (s, 1H, CH, triazole), 5.54 (s, 2H, CH ), 7.05
(d, 1H, 2 × CH, methoxy‐Ph), 7.34 (d, 1H, 2 × CH, methoxyl‐Ph), 7.54
(d, 1H, 2 × CH, Ph), 7.60 (t, 1H, CH, isatin), 7.73 (d, 1H, CH, isatin),
1
3
7
.91 (d, 1H, 2 × CH, Ph), and 9.13 (s, 1H, NH); C NMR (100 MHz,
) δ ppm: 55.8, 68.9, 79.1, 114.5, 115.1, 117.7, 120.1, 124.4,
25.1, 128.8, 129.6, 130.1, 132.6, 137.6, 147.4, 160.6, 161.2, 163.2,
CDCl
3
COX inhibition activity % = 1 A
where A is the absorbance of the inhibitor at 590 nm and C is the
absorbance of the 100% initial activity without inhibitor at 590 nm.
I
/C × 100,
1
1
I
+
72.0, and 175.1; Mass spectra: 676.18 (M+H) ; Elemental analysis

18 of 20
PATHAK ET AL.
|
[
9,35–37]
4
.2.2 | In vivo anti‐inflammatory activity
The anti‐inflammatory results were analyzed for statistical
significance (expressed as mean ± standard error of the mean [SEM])
between the vehicle control and the treated groups using one‐way
ANOVA followed by Dunnett’s test using GraphPad Instant software.
The blood samples were collected from all the groups of rats via
puncturing the retro‐orbital sinus plexus and were kept in a labeled
centrifuge tube.
Animals
Swiss albino Wistar rats of both sexes, weighing between 150 and
00 g, were used for the present study. The animals were kept in the
animal house of Adina Institute of Pharmaceutical Sciences, Sagar
M.P.), India at 25°C, 45–55% humidity and with 12‐hr light/dark
2
(
cycles under standard atmosphere. The animals were fed with
commercially available standard pellets and water was given ad
libitum. All the protocols for conducting the animal study were
reviewed and approved according to the Adina Institute of
Pharmaceutical Sciences, Animal Ethics Committee Guidelines and
as per CSCPEA regulations with reference number 1546/PO/E/S/11/
CPCSEA.
4.2.3 | Estimation of proinflammatory cytokines
and inflammatory mediators
Proinflammatory cytokines and inflammatory mediators, namely
TNF‐α, IL‐1β, IL‐10, and NF‐κb, were determined using standard kits
as per the manufacturer’s instructions.
Oral toxicity study
According to OECD‐423 guidelines for acute toxicity study, adult
and healthy Wistar rats of both sexes weighing 150–200 g were
used in this experiment. The animals were divided into three
groups and were starved for at least 24 hr before the experiment.
The control group received a 1% solution of sodium carboxymethyl
cellulose (CMC). The test group received the synthesized com-
pounds in sodium CMC (1%) in doses of 5, 10, 20, or 30 mg/kg
body weight and administered orally. The animals were monitored
at least once during the first 2 hr, and then again at 24 hr, for any
behavioral changes and in case of death. All the Wistar rats
survived till the acute toxicity study period and no major changes
were found.
4.3 Computational methodology
|
All the computational studies were carried out on a 4 CPU (Intel
Core2 Quad CPU Q9550@2.83 GHz) ACPI x64 workstation with
Windows 8.1 operating system. The protein was prepared, docked
and its interaction with different ligands was studied using Cresset
[38]
Discovery Services Flare.
4.3.1 | Receptor preparation
The cocrystal structure of TNF‐α (protein id: 2AZY), COX‐1 (protein id:
5WBE), and COX‐2 (protein id: 1CX2) were downloaded from the
Protein Data Bank (https://www.rcsb.org/) into Flare and prepared using
the Build Model10 tool from BioMol Tech, 11 to add hydrogen atoms,
optimize hydrogen bonds, remove atomic clashes and assign optimal
protonation states to the protein structure. The protein chain‐A was
covered as part of the protein preparation. Evaluation of correct
protonation state of ligands, amino acid side chains and reoptimize water
orientations of suboptimal water hydrogen bonding framework on the
binding site was confirmed visually. As many of the modeled binding
modes clash with the flexible side chain of amino acid residue, the side
chain atoms were minimized with the XED force field 12 for each ligand.
Treatment regimen
Six groups of rats were used and each group contained six animals to
study the impact of the synthesized compound on carrageenan‐
induced acute paw edema, as represented in Figure 2.
Group I (control group): The animals were treated with 1 ml of
0.5% sodium CMC after 30 min of induction of inflammation.
Groups II (standard group): After the induction of inflamma-
tion, the animals were treated with a dose of 20 mg/kg of the
suspension of diclofenac (standard) after 30 min of induction of
inflammation.
Group III (test group): The animals were treated with an
equimolar dose of the test suspension after 30 min of induction of
inflammation.
4.3.2 | Ligand setup
The structures of compounds 13–g were prepared using Marvin
[39]
Sketch 5.5.0.1 software.
Hydrogen atoms were added in the
The paw volume was measured at 1, 3, and 6 hr, using
a plethysmometer.
structures. All the atom MMFF94s force field parameterization was
assigned and then energy minimized through transformation
obtained at pH 7. Gasteiger partial charges were added to the
ligands. Nonpolar hydrogen atoms were merged and rotatable bonds
were defined.
Paw edema volume was compared with the vehicle control group
and the percentage reduction was calculated as
%
Edema inhibition = [(Vt–Vc)control − (Vt − Vc)tested
(Vt − Vc)control] × 100,
/
4
.3.3 | Docking
where V
interval and V
interval, respectively.
t
is the difference in volume of edema at a specific time
c
was the difference in volume of edema at zero time
Docking, a major computational reproduction study used to predict
the ligand binding with receptor by site difference and the

PATHAK ET AL.
19 of 20
|
inflexibility of ligands was optimized due to receptor. Ligand Fit
protocol of Cresset Flare was used and previously defined affinity
Amita Verma
http://orcid.org/0000-0001-6653-7335
Vladimir Potemkin
http://orcid.org/0000-0002-5244-8718
(grid) was used for the calculation.
.3.4 | Theoretical calculations[40–42]
REFERENCES
4
[
43–46]
The 3D QSAR calculations concord via “Cinderella Shoe”
[1] T. M. Cunha, M. M. Barsante, A. T. Guerrero, W. A. Verri, S. H.
Ferreira, F. M. Coelho, R. Bertini, C. Di Giacinto, M. Allegretti, F. Q.
Cunha, M. M. Teixeira, Br. J. Pharmacol. 2008, 154, 460.
[
45,47]
method and within the “CoMIn algorithm.”
The pharmaco-
phore part (in red), antipharmacophore skeleton (blue in color),
and inactive parts (white color) of the most promising synthe-
sized compounds (13a–g) are displayed in Figure 2. The white
part of the ring is not part in the pharmacological rejoinder but
liable for structure configuration and matrix function to provide
the necessary mutual arrangement of pharmacophoric fragments.
The ADME computations for example, logP, toxicity, metabolism
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Maria Grishina
http://orcid.org/0000-0002-6160-9755
http://orcid.org/0000-0002-2573-4831

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