Identification and development of thiazole leads as COX-2/5-LOX inhibitors through in-vitro and in-vivo biological evaluation for anti-inflammatory activity

Article abstract of DOI:10.1016/j.bioorg.2020.103882

Treatment of inflammation using NSAIDs is coupled with a risk of severe gastric adverse events. Development of dual COX-2/5-LOX inhibitors turns out to be an imperative area devoted to safer NSAIDs. A series of thiourea, thiazole, and thiazolidene derivatives were synthesized by green synthetic approach and COX-1, COX-2 and 5-LOX inhibition screening resulted in the identification of a potent compound 6l with IC₅₀ of 5.55 μM, 0.09 μM, and 0.38 μM respectively. Compound 6l made significant decrease (60.82%) in the carrageenan-induced edema in male Wistar rats. qRT-PCR analysis and determination of PGE₂ and LTB₄ in the rat paw tissues indicated that this thiazole based dual inhibitor significantly reduced the expression of COX-2 and 5-LOX genes besides the marked reduction in both PGE₂ and LTB₄ levels. The gastric safety profiling revealed an enhanced gastrointestinal safety of the compound 6l on histopathological examination. Molecular docking studies at COX-2 and 5-LOX active sites were consistent with biological studies by significant protein-ligand interaction. Besides, results of in-vitro PGE₂ and LTB₄ studies on RAW 264.7 cells as well as antioxidant studies were parallel to the dual inhibitory activity. The present investigations identify a promising lead having anti-inflammatory potential with an improved gastric safety profile.

Full text of DOI:10.1016/j.bioorg.2020.103882

Bioorganic Chemistry 100 (2020) 103882
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Identification and development of thiazole leads as COX-2/5-LOX inhibitors
through in-vitro and in-vivo biological evaluation for anti-inflammatory
activity
Jaismy Jacob P, S.L. Manju
Department of Chemistry, School of Advanced Sciences, Vellore Institute of Technology, Vellore, Tamil Nadu, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Treatment of inflammation using NSAIDs is coupled with a risk of severe gastric adverse events. Development of
dual COX-2/5-LOX inhibitors turns out to be an imperative area devoted to safer NSAIDs. A series of thiourea,
thiazole, and thiazolidene derivatives were synthesized by green synthetic approach and COX-1, COX-2 and 5-
LOX inhibition screening resulted in the identification of a potent compound 6l with IC50 of 5.55 µM, 0.09 µM,
and 0.38 µM respectively. Compound 6l made significant decrease (60.82%) in the carrageenan-induced edema
Anti-inflammatory activity
COX
LOX
Thiazole
Thiazolidene
in male Wistar rats. qRT-PCR analysis and determination of PGE
2
and LTB in the rat paw tissues indicated that
4
this thiazole based dual inhibitor significantly reduced the expression of COX-2 and 5-LOX genes besides the
marked reduction in both PGE and LTB levels. The gastric safety profiling revealed an enhanced gastro-
intestinal safety of the compound 6l on histopathological examination. Molecular docking studies at COX-2 and
-LOX active sites were consistent with biological studies by significant protein-ligand interaction. Besides,
2
4
5
results of in-vitro PGE
2
and LTB studies on RAW 264.7 cells as well as antioxidant studies were parallel to the
4
dual inhibitory activity. The present investigations identify a promising lead having anti-inflammatory potential
with an improved gastric safety profile.
1
. Introduction
In today’s world, chronic inflammation and associated complica-
more complex [5,6]. So far, the only approved 5-LOX inhibitor for
clinical use is zileuton. But its use is limited because of poor pharma-
cokinetics and hepatotoxicity [7]. In the investigation for new drugs
devoid of side effects while retaining high anti-inflammation potency,
the unique dual COX-2/5-LOX inhibitory concept has been introduced.
Accordingly, several dual inhibitors have been reported but they are in
different phases of clinical trials. Licofelone, 1 is an interesting dual
COX/LOX inhibitor in phase III clinical trial for osteoarthritis that in-
hibit COXs non-selectively with COX IC50 = 0.21 μM and 5-LOX acti-
vating protein (FLAP) with 5-LOX IC50 = 0.18 μM [8]. Two other
molecules with significant dual activity identified are tepoxalin, 2 ap-
proved for veterinary use [9] and darbufelone, 3 which is in phase III
clinical trial for rheumatoid arthritis [10]. Tenidap, 4 is a cytokine
modulating anti-rheumatic drug with COX/5-LOX inhibition that was
more active than conventional NSAIDs in the clinical treatment of
rheumatoid arthritis, but its development was halted due to kidney and
liver toxicity [11]. Structures of dual COX and LOX inhibitors were
tions such as osteoarthritis and cancer have become the most challen-
ging diseases which need special attention [1]. It is well known that
alteration in the arachidonic acid pathway and overproduction of
prostaglandins (PGs) and leukotrienes (LTs) are the root cause of
chronic inflammation [2,3]. The up-regulation of COX-2 isoenzyme
during inflammation and involvement of leukotrienes and lipoxins
produced by lipoxygenases (LOX) in bronchial asthma pathogenesis and
edema development clearly explains the role of these enzymes in in-
flammation [4]. Non selective inhibition of constitutive form of cy-
clooxygenase, viz COX-1 alone or combined COX-1 and COX-2 inhibi-
tion is responsible for the severe adverse effects of NSAIDs [4]. Gastric
side effects associated with traditional NSAIDs and withdrawal of se-
lective COX-2 inhibitors such as valdecoxib and rofecoxib from medical
use due to cardiovascular events makes the inflammation treatment
Abbreviations: COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; 5-LOX, 5-lipoxygenase; NSAID, non steroidal anti-inflammatory agent; PGE
2
, prostaglandin E ;
2
LTB
4
, leukotriene B ; qRT-PCR, quantitative real-time polymerase chain reaction; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; LPS, Lipopolysaccharide; ip,
4
intraperitoneal; FLAP, 5-lipoxygenase activating protein; ROS, reactive oxygen species; DPPH, 2,2-diphenyl-1-picrylhydrazyl; NO, nitric oxide; PBS, phosphate-
buffered saline
E-mail addresses: jaismyjacobp@gmail.com (J. Jacob P), slmanju@vit.ac.in (S.L. Manju).
https://doi.org/10.1016/j.bioorg.2020.103882
Received 3 January 2020; Received in revised form 13 April 2020; Accepted 22 April 2020
Available online 25 April 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 1. Structures of some dual COX/LOX inhibitors in clinical trials, dual COX-2/5-LOX inhibitors, 5-LOX inhibitors and newly designed molecules. Potential
functional groups for dual COX-2/5-LOX activity and thiazole ring are shown in circles.
2

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Scheme 1. Synthesis of N-(amino-4-
carbonothioyl)thiophene-2-carbox-
amide 4a-c, (2-amino-4-phenylthiazol-
5
-yl)(thiophen-2-yl)methanone 6a-m
and
N-(3-phenyl-4-(4-(substituted)
phenyl)thiazol-2(3H)-ylidene)thio-
phene-2-carboxamide 6n-r, reagents
and reaction conditions: (1) 2-thiophe-
necarbonyl chloride, (2) potassium
thiocyanate, (3) morpholine, dipheny-
lamine, aniline, (a) water, 0.1 equiv
TBAF, room temperature, 1 h, then heat
at 80 °C, 2 h.
shown in Fig. 1. Recently, indole-2-amide derivatives were reported by
Li et al. as dual COX-2/5-LOX inhibitors with marked in-vitro and in-vivo
anti-inflammatory potential [12]. Hybrids of diaryl-1,5-diazoles and
form viz thiourea and designed a series of compounds, 4a-c (Fig. 1).
Further, structural modifications such as N-substitution of secondary
amine and electron releasing and withdrawing substitutions at p-phenyl
are done to optimize the anti-inflammatory efficacy of the molecules,
6a-r.
morpholines with CF
3
and SO
2
NH substituted phenyl rings showed
2
significant in-vitro dual inhibition and anti proliferative activity [13]. In
spite of the intense research in this regard, so far no dual COX-2 and 5-
LOX inhibitor could be accomplished clinically. Hence, the designing of
novel pharmacophores with dual COX-2 and 5-LOX inhibition would
pave a new therapeutic option for anti-inflammatory therapy with an
enhanced safety profile.
This work provided a one-pot multi-component green synthetic
method for thiazole and thiazolidene derivatives. The in-vitro inhibitory
activities of target compounds for COX-1, COX-2, 5-LOX, PGE and
2
LTB have been evaluated. Further, in-vivo anti-inflammatory and anti-
4
ulcer potential were studied. In order to explore the mechanism of se-
lective COX-2 and 5-LOX inhibition, gene expression studies by quan-
titative real-time polymerase chain reaction (qRT-PCR) were performed
successfully on rat paw tissues. Besides, active site binding interactions
of the most active molecule on COX-2 and 5-LOX enzymes were ex-
plored by molecular modeling.
Thiazole is a unique five-membered heterocyclic compound with a
variety of therapeutic utility such as anti-inflammatory, antioxidant
[
14,15], anticancer [16] and anti-HIV [17] properties etc. Moreover, a
variety of drugs such as suphathiazole, niridazole, thiabendazole, fa-
netinole and preferential COX-2 inhibitor meloxicam, 5 (Fig. 1) possess
thiazole ring in their structure. Recently, 1,2-benzisothiazol-3(2H)-one-
1
,1-dioxides were screened for their COX-1/COX-2 inhibition and found
2
. Results and discussion
to possess similar activity to celecoxib. In-vivo anti-inflammatory and
antiulcer property of the above compounds were significant
with ~ 60% inhibition of edema [18]. Sathya et al. designed and syn-
thesized 2, 4-bis(aryl/heteroaryl)-5-acylthiazole, 6 (Fig. 1) as dual in-
hibitors of COX-2 and 5-LOX with COX-2 IC50 = 2.10 μM and 5-LOX
IC50 = 1.37 μM respectively. Furthermore, a combination of thiazoli-
dinone and thiazole rings, compound 7 (Fig. 1) showed 67.3% protec-
tion on in-vivo anti-inflammatory models [19]. Recently, our group
reported substituted 2-amino thiazole derivatives as potent 5-LOX in-
hibitors and effective anti-inflammatory agents. The compounds 8, 9,
and 10 (Fig. 1) have shown considerable inhibition of 5-LOX
2.1. Chemistry
The multi-component one-pot green synthesis of N-(substituted
carbamothioyl)thiophene-2-carboxamide 4a-c [23–29], (2-(sub-
stituted)-4-(4-substituted
phenyl)thiazol-5-yl)(thiophen-2-yl)metha-
none 6a-m and N-(3-phenyl-4-(4-(substituted phenyl)thiazol-2(3H)-
ylidene)thiophene-2-carboxamide 6n-r is accomplished as depicted in
Scheme 1 by a modified green synthetic route. The present synthesis is a
modification of our previous work where cetyltrimethylammonium
bromide (CTAB), a phase transfer catalyst was used for the synthesis of
thiourea from acyl chloride, potassium thiocyanate and amines using
toluene: water system. This study attempted to carry out a simple and
efficient method of one pot green synthesis using tetrabutylammonium
fluoride (TBAF) for N and S heterocyclics in water. Reaction conditions
were optimized by conducting a model reaction using thiophene-2-
carbonyl chloride, potassium thiocyanate, morpholine and phenacyl
bromide. Comparison reactions were performed using similar catalysts
such as tetrabutylammonium bromide (TBAB), tetrabutylammonium
hydrogensulphate (TBAHS) and CTAB (Table 1). Optimal reaction
conditions showed that water acted as a suitable solvent not only in
terms of yield but also in its environmental safety, easy to handle and
cost-effectiveness. The entire reaction was carried out in a single pot
containing water and TBAF. Briefly, 2-thiophenecarbonyl chloride and
potassium thiocyanate were added to aq. solution of 0.1 equiv of TBAF
to afford thiophene-2-carbonylisothiocyanate. These isothiocyanates
were further converted to corresponding N-(substituted carbamothioyl)
thiophene-2-carboxamide by adding secondary amine and stirring for
(
IC50 ~ 10 μM, 0.9 μM and 1.4 μM respectively) and appreciable anti-
inflammatory activities [20,21]. These thiazole analogues showed
equivalent potency with that of zileuton (IC50 ~ 1.5 μM), a selective 5-
LOX inhibitor [22]. In the present work, newly designed molecules
combined the pharmacophore requirements of dual COX-2 and 5-LOX
inhibitor.
As a continuation and modification of our earlier work [20], herein
we report the design and novel green synthesis of 2-(substituted)-4-(4-
substituted phenyl)thiazol-5-yl)(thiophen-2-yl)methanone 6a-m and N-
(
3-phenyl-4-(4-(substituted phenyl)thiazol-2(3H)-ylidene)thiophene-2-
carboxamide 6n-r as dual COX-2/5-LOX inhibitors (Fig. 1) Considering
peculiarly larger COX-2 active site, a 4-phenyl group along with a 5-
thiophene-2-carbonyl group was introduced into the thiazole and
thiazolidene nucleus which can selectively bind to the secondary pocket
and give sufficient steric bulk to block the COX-2 hydrophobic channel.
At 5-LOX enzyme, 2-substituted secondary amine and 5-thiophenyl-2-
carbonyl group in the thiazole ring provides H-bond acceptor and hy-
drophobic centroid features respectively to the molecule. N-hydroxy
urea group of the zileuton is incorporated in the scaffold in its isosteric
1
h at RT. Addition of equivalent amounts of phenacyl bromides to the
reaction mixture containing thiourea and reflux at 80 °C for 2 h resulted
3

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Table 1
Optimization of the reaction conditions.a
.
Sl no
Solvent
Catalyst
Temperature (°C)
Time (h)
Yield %
1
2
3
4
5
6
7
8
9
Water
Water
Water
Water
Water
Water
Water
Water
Water
DMSO
DMF
–
RT
60
24
24
24
5
–
–
–
–
Reflux
RT
60
10
45
72
91
88
80
80
25
0
TBAF
TBAF
3
TBAF
80
2
TBAB
TBAHS
CTAB
TBAF(0.1)
TBAF(0.1)
TBAF(0.1)
TBAF(0.1)
80
3
80
3.5
3.5
2
80
1
1
1
1
0
1
2
3
80
80
2
Ethanol
Acetonitrile
80
2
80
76
80
2
a
Isolated yields.
in lead molecules 6a-m. This method followed a novel route of [3 + 2]
Hantzsch thiazole synthesis. Interestingly, according to the literature,
the addition of phenacyl bromides to the monosubstituted thiourea, 4c
resulted in thiazolidene derivatives, 6n-r rather than thiazole at the
same reaction conditions (Scheme 1) [30]. But reaction between 4c and
All synthesis proceeded smoothly and obtained derivatives in good
yields (78–92%). The structures of the compounds synthesized are
shown in Table 2. The structures of the molecules were confirmed by
1
13
IR, H NMR, and C NMR. The IR spectrum of 4a-c exhibited a sharp
intense peak of thiourea carbonyl group in the range of
−
1
4
-flourophenacyl bromide followed [3 + 2] thiazole synthesis and
1658–1672 cm
and NH absorption peak in the range of
−1
formed 6m, a thiazole. The thioureas (4a-c) formed as an intermediate
were separated and purified for the purpose of biological screening.
This green synthetic methodology was further extended to various N’-
disubstituted and monosubstituted thioureas and phenacyl bromides
substituted with electron-withdrawing and electron-donating groups.
3263–3278 cm . Upon thiazole/thiazolidene formation the free NH
stretch disappeared accompanied by the appearance of the thiazole/
−1
thiazolidene CeN stretch at ~ 1340 cm , while aromatic C]C
−1
1
stretching was observed at ~ 1500 cm . The H NMR spectra of 4c
showed sharp singlets at 12.392 and ~ 9.005 ppm was ascribed to NH
Table 2
In-vitro inhibitory activities on COX-2 and 5-LOX enzymes,
.
Compound
R
3
IC50
±
SD (μM)
COX-2
–
5-LOX
4
4
4
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
a [23,24]
–
–
–
8.39 ± 1.51
7.66 ± 0.26
8.35 ± 0.16
6.02 ± 0.17
5.07 ± 0.1
6.46 ± 0.28
6.58 ± 0.05
6.61 ± 0.19
6.65 ± 0.385
7.60 ± 0.75
4.20 ± 0.12
0.58 ± 0.03
0.39 ± 0.01
0.57 ± 0.04
0.38 ± 0.01
8.11 ± 0.15
6.15 ± 0.80
0.58 ± 0.04
9.39 ± 1.02
5.73 ± 0.25
4.96 ± 0.75
–
b [25–27]
83.24 ± 3.32
6.23 ± 0.66
60.59 ± 2.9
0.55 ± 0.02
3.77 ± 0.13
3.62 ± 0.51
4.75 ± 0.45
5.88 ± 0.03
3.92 ± 0.25
0.61 ± 0.01
4.03 ± 0.18
0.62 ± 0.01
6.48 ± 0.11
0.09 ± 0.002
6.23 ± 0.36
54.09 ± 0.41
0.77 ± 0.12
0.45 ± 0.04
6.64 ± 0.12
0.89 ± 0.01
0.07 ± 0.007
c [28,29]
a [23]
H
b
c
d
e
f
NO
F
2
CF
CN
CH
H
3
3
g
h
i
NO
2
3
F
j
CF
CN
CH
F
3
k
l
m
n
o
p
q
r
H
NO
2
3
CF
CN
CH
–
3
Etoricoxib
Zileuton
–
0.14 ± 0.01
aIC50 value is the compound concentration required to inhibit 50% of COX-2 or 5-LOX.
4

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 2. (a) ORTEP diagram of compound 6h, CCDC no. 1951604. (b) ORTEP diagram of compound 6p, CCDC no. 1959072. In this structure because of high torsion,
flipping occurs between S
2
and C21 part of the molecule. Disordered F
2
was observed due to rotation.
Table 3
Table 4
In-vitro inhibitory activities of selected compounds on COX-1 and COX-2, IC50
In-vitro PGE
2
and LTB
4
inhibitory activities in LPS-challenged RAW 264.7 cells.a
values and selectivity Indices (SI).
Compound
Compound
IC50
±
SD (μM)
IC50
±
SD (μM)a
Selectivity Index (SI)
PGE
2
LTB
4
COX-1
COX-2
6
b
7.87 ± 0.59
0.36 ± 0.09
0.94 ± 0.03
0.48 ± 0.07
6.32 ± 0.91
4.76 ± 0.47
0.44 ± 0.07
–
0.89 ± 0.02
0.72 ± 0.04
0.36 ± 0.02
0.28 ± 0.02
0.96 ± 0.06
0.58 ± 0.02
–
6
6
6
6
6
6
b
h
j
8.14 ± 0.81
8.05 ± 1.32
6.30 ± 0.40
5.55 ± 0.77
6.42 ± 0.71
6.05 ± 1.30
6.39 ± 0.83
0.55 ± 0.02
0.61 ± 0.01
0.62 ± 0.01
0.09 ± 0.002
0.77 ± 0.12
0.89 ± 0.01
0.07 ± 0.007
14.5
6h
13.19
10.16
61.66
8.34
6j
6l
l
6o
o
r
6r
6.79
Etoricoxib
Zileuton
Etoricoxib
91.28
0.47 ± 0.02
b
a
SI: IC50 (COX-1)/IC50 (COX-2).
IC50 value is the compound concentration required to produce 50% inhibition
of PGE and LTB
a
IC50 value is the compound concentration required to inhibit 50% of COX-1
2
4
.
or COX-2.
released in each well are inversely proportional. Among them, best six
compounds with significant percentage inhibition for COX-2 and 5-LOX
were selected for further COX-1 IC50 determination. Compound 6l
protons. Compounds 6a-f showed two triplets of morpholine hydrogen
at ~ 3.63 and 3.84 ppm respectively with integration of four each.
Aromatic protons were present in the range of 6.765–8.106 ppm con-
showed
excellent
selectivity
towards
COX-2
with
1
3
firming the formation of expected thiazoles/thiazolidenes. The
C
IC50
=
0.09 0.002 μM, whereas IC50 for COX-1 was
±
NMR displayed two prominent peaks at ~ 180 and 170 ppm ascribed to
5.55 ± 0.77 μM. The selectivity index of the most potent compound 6l
(SI = 61.66) was superior to remaining compounds and comparable to
that of etoricoxib (COX-2 IC50 = 0.07 ± 0.007 μM, SI = 91.28), a
selective COX-2 inhibitor (Table 3). Among the synthesized precursor
and target compounds, thiourea derivatives exhibited poor COX-2 in-
hibition. Similarly, thiazoles with no substitution on p-phenyl ring at C-
1
3
carbonyl carbon and C-2 of thiazole ring respectively, whereas in
C
NMR of thiazolidene series, carbonyl carbon and C-2 of thiazolidene
peak were observed at 169 and 168 ppm respectively. In general, when
phenacyl bromides substituted with electron withdrawing groups ex-
hibited fast reaction rate and good yield. The ORTEP diagrams of
compound 6h and 6p with CCDC no. 1951604 and 1959072 are pre-
sented in Fig. 2. The thiazole moiety formed by this reaction was un-
dergoing a rearrangement and instead of an expected phenyl metha-
none substitution at C-5 of thiazole ring, surprisingly, we found
thiophenyl methanone substitution at C-5. Interestingly, this method
offers a specific technique which could be applied to introduce a variety
of substitution at C-5 of thiazole ring using different aryl or heteroaryl
acid chlorides. Alternatively, monosubstituted acylthiourea viz N-
4 of thiazole ring (R ) showed less activity. Among thiazole derivatives,
3
diphenylamino thiazoles have shown better COX-2 inhibition than
morpholine derivatives. In comparison to thiazole compounds thiazo-
lidene derivatives also exhibited significant COX-2 inhibition especially
when R
3
was substituted with electron withdrawing groups (NO and
2
CF
3
). The exceptional selectivity and activity of (2-(diphenylamino)-4-
(p-tolyl)thiazol-5-yl)(thiophen-2-yl)methanone, 6l to COX-2 over COX-
1 could be due to more surrounding aromatic rings on thiazole ring
which afforded bulkiness to the molecule. Though the substituents of
the most active compounds are diverse, the tertiary amino group and p-
phenyl substitution on central thiazole ring play a major role in the
anti-inflammatory activity. Generally, compounds with both electron-
(
phenylcarbamothioyl)thiophene-2-carboxamide (4c) produced thia-
zolidenes consistent with previous reports [24,30].
2
.2. COX-1, COX-2 and 5-LOX enzyme inhibitory activity
withdrawing and donating groups at R showed significant COX-2 in-
3
hibition.
COX-2 and 5-LOX inhibitory screening assays were conducted on all
5
-LOX inhibitory screening was carried out for all the compounds
the compounds, 4a-c and 6a-r. The COX inhibitory activity was even-
tually measured by determining the amount of PG synthesized by the
enzyme in the presence of test compounds at different concentrations.
Herein, the concentration of the inhibitor present and the amount of PG
synthesized. In-vitro 5-LOX screening assay measures the amount of
hydroperoxides produced during the lipoxygenation reaction by pur-
ified 5-lipoxygenase enzyme. It is a sensitive method to detect
5

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 3. A, D, G, J- Histology of liver, kidney, stomach, and intestine of control (400×). B, E, H, K- Histology of liver, kidney, stomach, and intestine at a dose of
00 mg/kg after compound 6l treatment (400×). C, F, I, L- Histology of liver, kidney, stomach, and intestine at a dose of 2000 mg/kg after compound 6l treatment
400×).
5
(
hydroperoxide at different positions within the fatty acid of any carbon
length. Results demonstrated appreciable 5-LOX inhibition for com-
pound 6l (IC50 = 0.38 ± 0.01 μM) and 6j (IC50 = 0.39 ± 0.01 μM).
The standard drug zileuton showed IC50 = 0.14
Thiazolidene derivative 6o, with NO group at R showed significant 5-
LOX inhibition with IC50 = 0.58 ± 0.04 μM. The thiazole derivatives
±
0.01 μM.
2
3
6

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
rats, in which rats were subjected to antiulcer study and anti-in-
flammatory study subsequently.
Carrageenan induced rat paw edema bioassay is a standard method
for studying anti-inflammatory properties of the test molecule. After 1 h
of oral administration of test and standard compounds, acute in-
flammation was induced on hind paws by intraplantar injection of 1%
w/v carrageenan. The paw thickness was measured and anti-in-
flammatory activity was displayed as percentage inhibition of in-
flammation at time intervals 1, 2, 4 and 6 h, results are displayed in
Fig. 4. Anti-inflammatory study of compound 6l revealed significant
inhibition of edema (60.82 ± 1.96%) in comparison to control and
indomethacin (53.21 ± 0.76%) after 6 h. The test compound showed a
gradual and remarkable dose-depended increase in anti-inflammatory
activity during the study period. But, when the dose of 6l increased
from 10 to 20 mg/kg there was a trivial improvement in the observed
inhibitory activity pointing to the saturation of receptors at given doses.
Gross observation of the gastrointestinal mucosa following oral
administration of compound 6l at 10 and 20 mg/kg illustrated a normal
stomach texture whereas indomethacin 10 mg/kg treatment produced
redness and ulceration in the gastric mucosa. In addition, histopatho-
logical examination of the treated rat stomachs was carried out to assess
the degree of inflammatory response produced in the gastric layers. The
results are displayed in Fig. 5. The histopathological examination de-
monstrated a normal histology for the compound 6l which ensured its
gastrointestinal safety and potential medicinal value, whereas rats
treated with indomethacin developed severe mucosal sloughing, gran-
ulation tissue and lymphocytic infiltrate showing its potential for ul-
ceration.
Fig. 4. Effect of compound 6l on carrageenan-induced inflammation model in
rat paw tissue. All values are expressed as the mean ± SEM (n = 5). One way
ANOVA was used to calculate statistical significance. *p < 0.050 vs control
group.
with diphenylamine substitution at C-2 of thiazole ring have better
biological profile compared to other compounds in the series. The re-
sults are displayed in Table 2.
2
.3. PGE
2
and LTB inhibitory activity in LPS challenged RAW 264.7 cell
4
lines
The levels of PGE
challenged macrophage RAW 264.7 cells. After LPS induction, PGE
and LTB levels in the wells were increased from 0.04 to 1.28 pg/ml and
.03 to 0.92 pg/ml respectively. All the tested compounds inhibited
both PGE and LTB in a concentration-depended manner. Compound
l inhibited PGE synthesis with IC50 = 0.48 ± 0.07 μM and LTB
synthesis at IC50 = 0.28 ± 0.02 μM, whereas, selective COX-2 in-
hibitor, etoricoxib exhibited PGE inhibition at IC50 = 0.44 ± 0.07 μM
and 5-LOX inhibitor, zileuton showed LTB inhibition at
IC50 = 0.47 ± 0.02 μM (see Table 4). These inhibitory studies dis-
played the inhibitory potency of synthesized compounds towards PGE
2
and LTB
4
were considerably up-regulated in LPS-
2.5. PGE
2
and LTB inhibitory activity in rat paw tissues during
4
carrageenan-induced edema
2
4
0
Besides inhibition of rat paw swelling, the anti-inflammatory ac-
tivity of compound 6l was confirmed by a variety of biochemical assays.
The carrageenan-induced rat paw edema was coupled with a marked
2
4
6
2
4
increase in PGE
rageenan, the PGE
10 fold increase over 0 to 6 h. The LTB
but lesser (6 fold) compared to PGE
treatment with compound 6l showed a remarkable reduction in both
PGE and LTB levels in the hind paw. Compound 6l at 10 mg/kg
proved to be a more potent inhibitor of PGE and LTB synthesis than
indomethacin 10 mg/kg. Results are depicted in Fig. 6.
2
and LTB
4
levels. After intraplantar injection of car-
2
levels in the hind paws of control animals showed a
levels also increased markedly
at the same time interval. Pre-
2
4
4
2
2
and LTB
4
beyond any doubt.
2
4
Compound 6l has shown promising anti-inflammatory potential
2
4
among the synthesized compounds with significant inhibition of COX-2,
5
-LOX, PGE
2
and LTB . Hence compound 6l was chosen for further
4
biological studies.
2.6. qRT-PCR studies
2
.4. In-vivo studies
Gene expression studies were performed on rat paw tissues fol-
lowing to anti-inflammatory screening to explore the gene expression
level mechanism of the anti-inflammatory activity of compound 6l.
Gene expressions of all the target genes were normalized with an en-
dogenous control (GAPDH); subsequently, the expression of COX/LOX
genes in the treated groups was normalized with that in the control
group. COX-1, COX-2, and 5-LOX genes up-regulated their expression
significantly (p < 0.050) at 6.6, 4.8, and 9.0 fold respectively as
shown in Fig. 7. However, the expression of all the studied genes was
significantly reduced in treated rats. Interestingly, a concentration-de-
pendent activity was observed with compound 6l as 20 mg/kg was
more efficient in controlling the gene expression compared to lower
dose treatment, 10 mg/kg. In addition, compound 6l significantly re-
duced the expression of genes in comparison to indomethacin 10 mg/kg
especially against COX-2, where it was down-regulated (p < 0.050).
COX-1 gene expression was not affected greatly with compound 6l
treatment whereas 2.7 fold increases was observed with indomethacin.
Thus, these molecules could be used as promising leads for anti-in-
flammatory therapy.
2.4.1. Acute toxicity studies
In-vivo acute toxicity study of compound 6l was performed on male
Wistar rats as per OECD guidelines to scrutinize the toxic effects. The
animals were administered with compound 6l at oral doses of 50, 500
and 2000 mg/kg. The animals were observed carefully and con-
tinuously for the first four hours for any signs of toxicity and thereafter
at regular intervals on the first day. Afterwards, the animals were ex-
amined daily once for fourteen days. The animals were forfeited after
1
4 days and histological examination of kidney, liver, stomach, and
intestine did not show any significant structural variation in compar-
ison to control (Fig. 3).
2.4.2. In-vivo anti-inflammatory activity and gastric safety profiling
Gastric ulceration and bleeding are the most important side effects
of NSAIDs. So compound 6l exhibiting promising in-vitro COX-2/5-LOX
inhibition was further studied for in-vivo anti-inflammatory activity and
its ulcerogenic potential. A rational study was designed on male Wistar
7

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 5. 1. Histology of gastric mucosa of control
rats (A) showing normal stomach mucosa
(
100x), (B) normal stomach architecture
showing the surface mucous cells extending into
gastric pits (arrow) (200×), (C) normal adjacent
gastric mucosa, (400×). 2. Histology of gastric
mucosa of rats treated with 6l at 20 mg/kg (D)
showing normal stomach mucosa (100×), (E)
normal stomach architecture showing the sur-
face mucous cells extending into gastric pits
(
arrow) (200×), (F) normal adjacent gastric
mucosa, no evidence of gastric ulceration,
400×). 3. Histology of gastric mucosa of rats
(
treated with indomethacin 10 mg/kg (G) Gastric
mucosa with ulceration (a), sloughing of mu-
cosal surface (b), granulation tissue and mucosal
acute on chronic inflammatory infiltrate (c) (ar-
rows), (100×), (H) mucosal lymphocytic in-
filtrates in the background of developing chronic
gastritis, (arrows) (400×), (I) acute on chronic
inflammatory granulation tissue with ulceration,
(
400×).
2.7. Antioxidant activity
[32,33].
Reactive oxygen species (ROS) is one of the root causes of in-
2
.7.2. H
Compound 6h showed the maximum percentage of H
scavenging (70.33 1.97%) followed by 6l which showed
2.94 ± 1.75% scavenging activity at 20 μM concentration. Etoricoxib
and indomethacin had 51.93 ± 1.25% and 49.45 ± 0.22% of H
scavenging activity respectively. Ascorbic acid showed 21.86 ± 0.68%
2
O
2
radical scavenging assay
flammation and cancer. Antioxidants play a crucial role in interfering
the production as well as inactivation of ROS. Mostly free radical sca-
vengers and antioxidants facilitate anti-inflammatory therapy.[31]
Results of the antioxidant study are duly presented in Fig. 8.
2
O radical
2
±
6
O
2 2
2
.7.1. DPPH radical scavenging assay
H
2
O scavenging activity. In this study, molecules act either as electron
2
Radical scavenging activity and anti-inflammatory efficacy have
or proton donors to reduce H
2
O
2
to H O [34].
2
been proved to have a burly correlation. 2,2-Diphenyl-1-picrylhydrazyl
(
DPPH) radical scavenging activity of all the compounds at a con-
centration of 20 μM was evaluated. Majority of the compounds showed
less antioxidant activity in comparison to standard, ascorbic acid
2.7.3. Iron chelating assay
The iron chelating activity of all the synthesized compounds was
screened at 20 μM concentration. Among the compounds, N-(sub-
stituted carbamothioyl)thiophene-2-carboxamide derivatives (4a-c)
showed high iron-binding capability (~62%) compared to thiazole and
thiazolidene derivatives. Compound 6l exhibited moderate iron che-
lating activity (44.50 ± 1.06%). Even though the majority of the 5-
(
85.04 ± 1.06%) except for compound 4a and 6m, which manifested
significant free radical scavenging as that of standard, with
6.47 1.55% and 83.55 1.38% scavenging respectively.
Whereas, most active dual-acting inhibitor, 6l exhibited relatively
8
±
±
lower DPPH scavenging activity (48.85
±
0.27%) similar to in-
2
+
domethacin (55.49 ± 0.93%) and etoricoxib (57.19 ± 0.27%) in-
dicating that these molecules possibly proceeds through a non-redox
mechanism. In this study, strangely enough, we couldn’t find any lin-
earity between anti-inflammatory potency and antioxidant activity
because of exceptionally variant free radical scavenging activity
LOX inhibitors act by inhibiting oxidation of ferrous (Fe ) to ferric
3+
(Fe ) we couldn’t establish linearity between 5-LOX activity and iron
chelating activity for compounds with 5-LOX inhibition. The iron
binding activity of 4a-c could be because of the thiourea group present
in the molecule [35].
8

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 6. Effects of compound 6l on PGE
2
and LTB
4
levels in paw tissues at 6 h after injection of carrageenan in the left hind paw. Animals were pre-treated with
indomethacin and compound 6l 1 h before the carrageenan injection. The animals were sacrificed 6 h later and concentration of PGE
2
(Graph A) and LTB (Graph B)
4
was determined in the supernatant prepared from paws collected. Control group was treated only with saline. Pre-treatment was not given to carrageenan group. The
###
values were expressed as the mean ± SEM.
compared with the carrageenan alone group.
p < 0.001 carrageenan group compared to control group. *p < 0.05, **p < 0.01, and ***p < 0.001 were
amino group was residing in a hydrophobic pocket created by His90,
Thr94, Pro514, Asp515, Pro191, Tyr355, Gly354, Gln192, and Ser 353
residues. The thiophenyl ring appeared in hydrophobic interaction with
Ser581. Similarly, the 4-methyl phenyl ring was interacting and en-
closed in a hydrophobic cavity created by His356 and Gln350. The
larger volume of COX-2 active site accommodates the bulky aromatic
rings of compound 6l to attain the best binding conformation. Docking
simulations afforded the binding interactions of compound 6l and 5-
LOX active site. Sulphur atoms of thiazole and thiophene ring of com-
pound 6l formed H bonds with Trp147 and Gln417 respectively.
Binding energy of compound 6l at 5-LOX active site was −6.99 Kcal/
mol. While the reference drug zileuton showed a binding score of
−
6.43 Kcal/mol and two H bonds with Asn425 and Gln363. The 6l
molecule was surrounded by Ala157, Thr40, Asn148 and Glu412. The
compound 6l formed pi-sulfur interaction with Met145. Interactions of
6
l at COX-2 and 5-LOX are duly depicted in Fig. 9.
Fig. 7. Effect of compound 6l on COX-1, COX-2 and 5-LOX gene expression
induced by carrageenan in the rat paw at 6 h. GAPDH was used as an internal
control. Relative COX-1, COX-2 and 5-LOX gene expression was calculated at
3. Conclusions
The present study described the green synthesis of thiazole and
thiazolidene derivatives as potential dual COX-2/5-LOX inhibitors.
Synthesized derivatives included 3 thioureas (4a-c), 13 substituted
thiazoles (6a-m) and 5 substituted thiazolidenes (6n-r). These mole-
cules were tested for both in-vitro and in-vivo anti-inflammatory assays.
In-vitro assays demonstrated that the compound 6l with diphenylamino
group and 4-tolyl group on thiazole core was the most effective COX-2
and 5-LOX dual inhibitor with IC values of 0.09 µM and 0.38 µM
*
p < 0.050. The values were expressed as the mean ± SEM for experiments
performed in triplicates.
2
.7.4. Nitric oxide (NO) scavenging assay
In the NO scavenging assay, the synthesized compounds were able
to forage NO moderately at 20 μM concentration. Compound 6o and 6h
showed the highest activity with 57.30 ± 0.97% and 54.23 ± 0.75%
respectively. Compound 6l showed 42.47 ± 0.80% NO scavenging
activity which was similar to etoricoxib and 2 fold higher than in-
domethacin and ascorbic acid.
5
0
respectively. Interestingly, compound 6l showed high selectivity to-
wards COX-2 with a selectivity index of 61.66 which was comparable to
that of etoricoxib which has SI of 91.28. Selected compounds showed a
significant inhibition of PGE
2
and LTB on LPS challenged RAW 264.7
4
2.8. Molecular docking study
cells. Meanwhile, in-vivo acute toxicity study was performed on rats and
there was no evidence of toxicity. In-vivo anti-inflammatory assay re-
sults of compound 6l showed significant reduction in edema in male
Wistar rats (60.82%). Furthermore, the compound 6l has better gastric
safety profile than indomethacin on ulcerogenic potential screening.
Histopathological examination of rat stomach treated with 6l displayed
a normal gastric mucosa with no ulceration. Subsequently, Rat paw
tissues obtained followed to anti-inflammatory study, were subjected to
Experimental studies showed the potential of compound 6l as a dual
inhibitor of COX-2 and 5-LOX enzymes. To visualize the possible in-
teractions of compound 6l at COX-2 and 5-LOX active sites, molecular
docking studies were performed using AutoDockTools-1.5.6 software.
The compound 6l proved the best binding pose in the COX-2 active site
with binding energy −7.54 Kcal/mol. The oxygen atom of carbonyl
group formed an H-bond with His351 amino acid residue. The diphenyl
qRT-PCR studies, PGE
2
, and LTB inhibitory studies. The qRT-PCR
4
9

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 8. Effect of synthesized compounds on DPPH scavenging, H
2
O scavenging, NO scavenging, and Iron chelating activity.
2
studies depicted a significant inhibition of 6l on up-regulation of COX-2
and 5-LOX genes which was well matched with the obtained results.
The compound 6l significantly reduced the up-regulation of COX-2 and
compounds was > 95%. The elemental analysis values were found to
be within ± 0.4% of the calculated values (Perkin Elmer 2400 CHNS
analyzer).
5
-LOX sparing its effect on COX-1. Similarly, PGE
2
and LTB
4
levels in
General method of preparation of compounds (4a-c, 6a-r).
1.6 mmol of Potassium thiocyanate was added to a solution of 15 ml
water and 0.1 equiv tetrabutylammonium fluoride (TBAF). To the
above mixture, 1.5 mmol thiophene-2-carbonyl chloride was added and
stirred for 30 min at RT. Further, 1.5 mmol Aromatic or alkyl amines
were added and continued stirring until the reaction was completed
(30–60 min), monitored by TLC (Compounds 4a-c were separated at
this stage). Then substituted phenacyl bromide (1.5 mmol) was added
and refluxed with stirring at 80 °C for 2 h. The solid obtained was fil-
tered and column chromatography was performed to achieve pure
products (hexane:ethylacetate).
the treated rat paws were very less compared to control. These stepwise
investigations of in-vitro, in-vivo and qRT-PCR results indicated the
discovery of a new ligand with high potency and selectivity against
COX-2/5-LOX. The ligand 6l could of course be further studied for the
development of a potent anti-inflammatory drug with improved gastric
safety profile.
4
. Experimental section
4.1. Chemistry
N-(morpholine-4-carbonothioyl)thiophene-2-carboxamide (4a). Off
−1
white solid, 89%, mp 162–164 °C. IR (KBr, cm ) 3278 (NH str), 2970
All the chemicals were purchased from Sigma-Aldrich, Merck, TCI
(
aromatic CeH str), 1658 (amide C]O str), 1442 (aromatic C]C str),
and Alfa Aesar and were used without further purification. Millipore
water was used as a solvent for all reaction. Progression of the reaction
was monitored by thin-layer chromatography on Merck silica gel 60
F254 pre-coated plates with ethyl acetate: hexane as mobile phase and
visualization of the chromatogram was done by UV light and iodine
chamber. Melting points were determined using the Guna melting point
apparatus with open capillary tubes and were uncorrected. FTIR spectra
of the synthesized compounds were recorded on (ATR-FTIR Jasco-
1
261 (morpholine CeN), 1111 (morpholine CeOeC), 731 (aromatic
1
CeH bend). H NMR (400 MHz, DMSOd
6
) δ: 3.57 (t, J = 26.2 Hz, 6H,
morpholine-H), 4.15 (s, 2H, morpholine-H), 7.20 (t, J = 4.5 Hz, 1H,
Thiophene-H), 7.91 (d, J = 4.9 Hz, 1H, Thiophene-H), 8.09 (d,
13
J = 3.7 Hz, 1H, Thiophene-H), 10.89 (s, 1H, NH). C NMR (100 MHz,
DMSOd
6
) δ: 50.9, 51.7, 66.1, 128.8, 131.7, 133.9, 138.2, 158.6, 179.5.
+
ESI-MS m/z for C10
H
12
2
N O
2
S
2
[M + H] calcd 257.0340, found 257.
Elemental analysis (%) calcd for C10
H
12
N
2
O S2, C 46.86, H 4.72, N
2
1
13
4
100) with DRS technology. H and C NMR spectra were recorded on
Bruker advance DMX 400 MHz NMR spectrometer using CDCl and
DMSO‑d containing internal standard tetramethylsilane (TMS). In
1
0.93, S 25.01; found: C 46.54, H 4.37, N 10.71, S 25.07.
3
N-(diphenylcarbamothioyl)thiophene-2-carboxamide (4b). Yellow
6
−1
solid, 88%, mp 132–134 °C. IR (KBr, cm ) 3263 (NH str), 3070
NMR spectra, chemical shifts (δ) are expressed in parts per million
(
aromatic CeH str), 1672 (amide C]O str), 1485 (aromatic C]C str),
(
(
ppm) and coupling constants (J) are expressed in hertz (Hz). HRMS
High-resolution mass spectra) spectra were acquired using JOEL HR
1
6
94 (aromatic CeH bend). H NMR (400 MHz, CDCl
3
) δ: 6.90 (t,
J = 7.4 Hz, 1H, Thiophene-H), 7.02 (t, J = 4.8 Hz, 1H, Ar-H), 7.06 (d,
J = 8.6 Hz, 2H, Ar-H), 7.24 (t, J = 6.0 Hz, 2H, Ar-H), 7.32–7.37 (m,
mass spectrometer. The chromatographic analysis was carried out by
UPLC-PDA equipped with a pump quaternary solvent manager auto
sampler- sample manager FTN, and PDA-E-LAMBDA detector. The used
analytical column was Acquity UPLC BEH C 18 (150 mm × 2.1 mm i.d.,
6
H, Ar-H), 7.41 (d, J = 2.8 Hz, 1H, Ar-H), 7.51 (d, J = 4.9 Hz, 1H, Ar-
13
H), 8.54 (s, 1H, Ar-H). C NMR (100 MHz, CDCl
3
) δ: 117.8, 121.0,
1
1
26.9, 127.5, 127.9, 128.7, 129.3, 129.4, 130.3, 132.5, 135.8, 136.4,
1
30 Å, 1.7 µm) at 37℃. The mobile phase was composed of 0.01 M
+
43.2, 156.4, 181.5. ESI-MS m/z for C18
H
14
N
2
OS
2
[M + H] calcd
Disodium phosphate and Methanol (10:50 v/v). Purity of all the tested
10

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
Fig. 9. Binding modes of compound 6l with amino acid residues of COX-2 (pdb ID 5IKT) and 5-LOX (pdb ID 3O8Y), (generated in Discovery Studio Visualizer) (A)
Docked pose at COX-2 active site (B) 2-Dimensional representation at COX-2 active site (C) Docked pose at 5-LOX active site (D) 2-Dimensional representation at 5-
LOX active site. Dashed green lines represent H-bonds. Colours depicted are Ligand: green, active site amino acid: orange, nitrogen: blue, oxygen: red, sulfur: yellow.
3
39.0548, found 339. Elemental analysis (%) calcd for C18
H
14
N
2
OS2,
C
CDCl ) δ: 3.59 (t, J = 4.7 Hz, 4H, morpholine-H), 3.80 (t, J = 5.2 Hz,
3
6
3.88, H 4.17, N 8.28, S 18.95; found: C 63.62, H 4.20, N 8.62, S 19.04.
4H, morpholine-H), 6.79 (t, J = 3.9 Hz, 1H, Thiophene-H), 7.28 (t,
N-(phenylcarbamothioyl)thiophene-2-carboxamide (4c). Off White
J = 6.4 Hz, 3H, Ar-H), 7.40 (d, J = 7.4 Hz, 1H, Ar-H), 7.44 (d,
−
1
13
solid, 93%, mp 134–136 °C. IR (KBr, cm ) 3273 (NH str), 2964
J = 3.8 Hz, 1H, Ar-H), 7.66 (d, J = 7.1 Hz, 2H, Ar-H), C NMR
(
aromatic CeH str), 1658 (amide C]O str), 1483 (aromatic C]C str),
(100 MHz, CDCl ) δ: 48.0, 66.1, 119.9, 127.3, 128.1, 128.4, 128.8,
3
1
7
21 (aromatic CeH bend). H NMR (400 MHz, CDCl
3
) δ: 7.18 (t,
130.7, 131.8, 137.5, 139.6, 151.7, 170.8, 187.7. HRMS (ESI-MS) m/z
+
J = 4.8 Hz, 1H, Ar-H), 7.28 (d, J = 7.5 Hz, 1H, Ar-H), 7.40 (t,
J = 8.0 Hz 2H, Ar-H), 7.68 (d, J = 7.8 Hz, 2H, Ar-H), 7.72 (t,
for C18
H
16
N
2
O
2
S
2
[M + H] calcd 357.0653, found 357.0648.
Elemental analysis (%) calcd for C18
H
16
N
2
O S2, C 60.65, H 4.52, N
2
1
3
J = 5.2 Hz, 2H, Ar-H), 9.00 (s, 1H, NH), 12.39 (s, 1H, NH). C NMR
7.86, S 17.99; found: C 60.27, H 4.21, N 7.56, S 17.83.
(
100 MHz, CDCl3) δ: 124.2, 126.9, 128.6, 128.9, 130.8, 134.4, 135.9,
(2-Morpholino-4-(4-nitrophenyl)thiazol-5-yl)(thiophen-2-yl)methanone
+
−1
1
2
5
2
37.6, 161.2, 178.1. ESI-MS m/z for C12
H
10
N
2
OS
2
[M + H] calcd
(6b). Fluorescent yellow solid, 80%, mp 144–146 °C. IR (KBr, cm
)
63.0235, found 263. Elemental analysis (%) calcd for C12
H
10
N
2
OS2,
C
3070 (aromatic CeH str), 1609 (C]O str), 1512 (NO
2
str), 1444
4.94, H 3.84, N 10.68, S 24.44; found: C 54.89, H 3.92, N 10.88, S
4.56.
(aromatic C]C str), 1344 (thiazole CeN), 1298 (morpholine CeN),
1
1111 (morpholine CeOeC), 712 (aromatic CeH bend). H NMR
(
2-Morpholino-4-phenylthiazol-5-yl)(thiophen-2-yl)methanone (6a).
(400 MHz, CDCl ) δ: 3.63 (t, J = 4.8 Hz, 4H, morpholine-H), 3.84 (t,
3
−
1
Yellow solid, 91%, mp 124–126 °C. IR (KBr, cm ) 2991 (aromatic
J = 4.8 Hz, 4H, morpholine-H), 6.87 (t, J = 4.0 Hz, 1H, Ar-H), 7.40 (d,
CeH str), 1732 (C]O str), 1610 (C]N str) 1490 (aromatic C]C str),
J = 3.2 Hz, 1H, Ar-H), 7.55 (d, J = 4.8 Hz, 1H, Ar-H), 7.71 (d,
1
3
1
417 (thiazole CeN), 1288 (morpholine CeN), 1112 (morpholine
J = 8.4 Hz, 2H, Ar-H), 8.10 (d, J = 8.4 Hz, 2H, Ar-H), C NMR
1
CeOeC), 694 (aromatic CeH bend), 638 (CeS str). H NMR (400 MHz,
(100 MHz, CDCl ) δ: 48.2, 66.0, 123.1, 127.6, 130.5, 133.7, 133.9,
3
11

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
1
41.4, 143.8, 147.6, 155.4, 178.8. HRMS (ESI-MS) m/z for
7.48 (t, J = 8.1 Hz, 5H, Ar-H), 7.56 (d, J = 8.1 Hz, 4H, Ar-H), 7.86 (d,
+
13
C
18
H
15
N
3
O
4
S
2
[M + H] calcd 402.0504, found 402.0501. Elemental
J = 4.9 Hz, 1H, Ar-H). C NMR (100 MHz, CDCl
3
) δ: 122.5, 126.1,
analysis (%) calcd for C18
found: C 53.57, H 3.43, N 9.99, S 16.0.
4-(4-Fluorophenyl)-2-morpholinothiazol-5-yl)(thiophen-2-yl)metha-
H
15
N
3
O
4
S
2, C 53.85, H 3.77, N 10.47, S 15.97;
126.8, 127.4, 127.9, 128.8, 133.2, 133.9, 134.9, 143.7, 144.2, 156.9,
+
169.8, 180.0. HRMS (ESI-MS) m/z for C26
H
18
N
2
OS
2
[M + H] calcd
(
439.0861, found 439.0855. Elemental analysis (%) calcd for
−1
none (6c). Yellow solid, 86%, mp 110–112 °C. IR (KBr, cm ) 2962
C
26
H
18
N OS2, C 71.21, H 4.14, N 6.39, S 14.62; found: C 70.90, H 4.60,
2
(
aromatic CeH str), 1614 (C]O str), 1595 (C]N str), 1442 (aromatic
N 6.79, S 14.39.
C]C str), 1365 (thiazole CeN), 1259 (morpholine CeN), 1111 (mor-
(2-(Diphenylamino)-4-(4-nitrophenyl)thiazol-5-yl)(thiophen-2-yl)me-
thanone (6 h). Fluorescent yellow solid, 92%, mp 158–160 °C. IR (KBr,
pholine CeOeC), 1029 (C-F str), 719 (aromatic CeH bend), 646 (CeS
1
−1
str). H NMR (400 MHz, CDCl
3
) δ: 3.60 (t, J = 3.9 Hz, 4H, morpholine-
cm ) 3076 (aromatic CeH str), 1604 (C]O str), 1510 (aromatic C]C
1
H), 3.81 (t, J = 4.3 Hz, 4H, morpholine-H), 6.80 (t, J = 4.2 Hz, 1H, Ar-
H), 6.94 (t, J = 8.2 Hz, 2H, Ar-H), 7.29 (d, J = 5.2 Hz, 2H, Ar-H), 7.67
str), 1340 (thiazole CeN), 788 (aromatic CeH bend), 698 (CeS str). H
NMR (400 MHz, CDCl ) δ: 6.87 (t, J = 4.8 Hz, 1H, Ar-H), 7.30 (t,
3
1
3
(
t, J = 6.0 Hz, 2H, Ar-H). C NMR (100 MHz, CDCl
3
) δ: 48.0, 66.1,
J = 6.7 Hz, 2H, Ar-H), 7.44–7.48 (m, 9H, Ar-H), 7.55 (d, J = 4.9, 1H,
13
1
1
15.1, 115.3, 119.9, 127.3, 128.5, 130.8, 131.4, 131.5, 131.6, 135.5,
Ar-H), 7.71 (d, J = 8.8, 2H, Ar-H), 8.08 (d, J = 8.1 Hz, 2H, Ar-H).
C
35.6, 137.2, 151.7, 163.6, 166.1, 170.9, 186.4. HRMS (ESI-MS) m/z
NMR (100 MHz, CDCl ) δ: 123.2, 126.1, 127.2, 127.8, 129.9, 130.5,
3
+
for C18
H15FN
2
O
S
2 2
[M + H] calcd 375.0559, found 375.0552.
134.1, 141.1, 143.8, 144.0, 147.5, 154.2, 170.0, 179.1. HRMS (ESI-MS)
+
Elemental analysis (%) calcd for C18
H15FN
2
O
2
S
2, C 57.74, H 4.04, N
m/z for C26
H
17
N
3
O
S
3 2
[M + H] calcd 484.0711, found 484.0706.
7
.48; S 17.12 found: C 57.76, H 4.02, N 7.83, S 17.02.
Elemental analysis (%) calcd for C26
H
17
N
3
O S2, C 64.58, H 3.54, N
3
(
2-Morpholino-4-(4-(trifluoromethyl)phenyl)thiazol-5-yl)(thiophen-2-
8.69, S 13.26; found: C 64.15, H 3.58, N 8.59, S 13.30.
−1
yl)methanone (6d). Yellow solid, 84%, mp 92–94 °C. IR (KBr, cm
)
(2-(Diphenylamino)-4-(4-fluorophenyl)thiazol-5-yl)(thiophen-2-yl)me-
−
1
3
076 (aromatic CeH str), 1718 (C]O str), 1508 (C]N str) 1473
thanone (6i). Yellow solid, 86%, mp 130–132 °C. IR (KBr, cm ), 3061
(aromatic CeH str), 1608 (C]O str), 1442 (aromatic C]C str), 1332
(
aromatic C]C str), 1327 (thiazole CeN), 1228 (morpholine CeN),
1
1
099 (morpholine CeOeC), 804 (aromatic CH bend), 630 (CeS str). H
(thiazole CeN), 1151 (C-F str) 750 (aromatic CeH bend), 684 (CeS
1
NMR (400 MHz, CDCl
3
) δ: 3.62 (t, J = 4.6 Hz, 4H, morpholine-H), 3.83
t, J = 5.1 Hz, 4H, morpholine-H), 6.83 (t, J = 4.7 Hz, 1H, Ar-H), 7.32
d, J = 3.0 Hz, 1H, Ar-H), 7.48 (t, J = 8.2 Hz, 3H, Ar-H), 7.63 (d,
str). H NMR (400 MHz, CDCl
3
) δ: 6.82 (t, J = 4.8 Hz, 1H, Ar-H), 6.88
(
(
(t, J = 8.8 Hz, 2H, Ar-H), 7.28–7.32 (m, 3H, Ar-H), 7.41–7.50 (m, 9H,
13
Ar-H), 7.51–7.55 (m, 2H, Ar-H). C NMR (100 MHz, CDCl ) δ: 114.9,
3
1
3
J = 8.1 Hz, 2H, Ar-H). C NMR (100 MHz, CDCl
3
) δ: 48.2, 66.1, 121.2,
115.1, 122.0, 126.2, 126.9, 127.5, 129.8, 131.1, 131.1, 131.6, 131.7,
1
24.9, 124.9, 127.5, 129.9, 133.6, 138.6, 143.7, 156.6, 171.4, 179.1.
133.4, 133.9, 143.8, 144.2, 155.9, 161.8, 164.2, 169.9, 179.7. HRMS
+
+
HRMS (ESI-MS) m/z for C19
H
F
15 3
2
N O
2
S
2
[M + H] calcd 425.0527,
(ESI-MS) m/z for C26
H
17FN
2
OS
2
[M + H] calcd 457.0766, found
found 425.0524. Elemental analysis (%) calcd for C19
H
15
F N
3 2
O
S
2 2,
C
457.0761. Elemental analysis (%) calcd for C26
H
17FN OS2, C 68.40, H
2
5
3.77, H 3.56, N 6.60, S 15.11 found: C 53.77, H 3.67, N 6.62, S 15.19.
-(2-Morpholino-5-(thiophene-2-carbonyl)thiazol-4-yl)benzonitrile
3.75, N 6.14, S, 14.04; found: C 68.38, H 3.92, N 6.29, S 14.32.
(2-(Diphenylamino)-4-(4-(trifluoromethyl)phenyl)thiazol-5-yl)(thio-
phen-2-yl)methanone (6j). Yellow solid, 88%, mp 128–130 °C. IR (KBr,
4
−1
(
6e). Bright yellow solid, 89%, mp 158–160 °C. IR (KBr, cm ) 2984
−1
(
aromatic CeH str), 2218 (CN str), 1598 (C]O str), 1510 (C]N str)
cm ) 3062 (aromatic CeH str), 1598 (C]O str), 1498 (aromatic C]C
1
1
473 (aromatic C]C str), 1413 (thiazole CeN), 1292 (morpholine
str), 1317 (thiazole CeN), 754 (aromatic CeH bend), 690 (CeS str). H
CeN), 1111 (morpholine CeOeC), 794 (aromatic CeH bend), 642
NMR (400 MHz, CDCl ) δ: 6.84 (t, J = 4.1 Hz, 1H, Ar-H), 7.29 (t,
3
1
(
CeS str). H NMR (400 MHz, CDCl
3
) δ: 3.63 (t, J = 4.6 Hz, 4H,
J = 7.0 Hz, 2H, Ar-H), 7.36 (d, J = 3.0 Hz, 1H, Ar-H), 7.42 (m, 11H,
morpholine-H), 3.82 (t, J = 5.2 Hz, 4H, morpholine-H), 6.76 (t,
J = 5.0 Hz, 1H, Ar-H), 7.27 (d, J = 2.9 Hz, 1H, Ar-H), 7.32 (d,
J = 5.0 Hz, 1H, Ar-H), 7.54 (d, J = 8.4 Hz, 2H, Ar-H), 7.66 (d,
Ar-H), 7.71 (d, J = 8.0 Hz, 1H, Ar-H), 8.08 (d, J = 8.2 Hz, 1H, Ar-H).
1
3
C NMR (100 MHz, CDCl ) δ: 122.7, 124.8, 124.9, 124.9, 126.1, 127.1,
3
127.6, 129.9, 130.3, 130.6, 133.8, 134.0, 138.4, 143.8, 144.1, 155.4,
1
3
+
J = 8.4 Hz, 2H, Ar-H). C NMR (100 MHz, CDCl
3
) δ: 48.1, 66.0, 114.6,
170.0, 179.4. HRMS (ESI-MS) m/z for C27
H
17
F
3
N
2
OS
2
[M + H] calcd
1
1
3
18.1, 120.0, 127.3, 129.1, 129.2, 131.3, 131.8, 136.8, 143.3, 153.1,
507.0734, found 507.0731. Elemental analysis (%) calcd for
+
71.4, 185.8. HRMS (ESI-MS) m/z for C19
H
15
N
3
O
S
2 2
[M + H] calcd
C
27
H
17
F
3
N OS2, C 64.02, H 3.38, N 5.53, S, 12.66; found: C 64.05, H
2
82.0606, found 382.0603. Elemental analysis (%) calcd for
3.57, N 5.50, S 12.69.
C
19
H
15
N
3
O
2
S
2, C 59.82, H 3.96 N 11.02, S, 16.81; found: C 59.89, H
.96, N 11.02, S 15.91.
2-Morpholino-4-(p-tolyl)thiazol-5-yl)(thiophen-2-yl)methanone (6f).
4-(2-(Diphenylamino)-5-(thiophene-2-carbonyl)thiazol-4-yl)benzoni-
−
1
3
trile (6 k). Yellow solid, 85%, mp 188–190 °C. IR (KBr, cm ) 3095
(aromatic CeH str), 2222 (CN str), 1600 (C]O str), 1487 (aromatic
(
−
1
Pale yellow solid, 81%, mp 106–108 °C. IR (KBr, cm ) 2978 (aromatic
CeH str), 2856 (C-CH str), 1597 (C]O str), 1508 (aromatic C]C str),
481 (thiazole CeN), 1286 (morpholine CeN), 1112 (morpholine
C]C str), 1338 (thiazole CeN), 758 (aromatic CeH bend), 699 (CeS
1
3
str). H NMR (400 MHz, DMSOd
6
) δ: 6.99 (t, J = 4.2 Hz, 1H, Ar-H),
1
7.35 (t, J = 7.2 Hz, 2H, Ar-H), 7.46 (d, J = 3.7 Hz, 1H, Ar-H), 7.48 (t,
J = 8.0, 4H, Ar-H), 7.56 (d, J = 7.9, 4H, Ar-H), 7.61 (d, J = 8.3, 2H,
Ar-H), 7.74 (d, J = 8.2 Hz, 2H, Ar-H), 7.93 (d, J = 4.9 Hz, 1H, Ar-H).
1
CeOeC), 786 (aromatic CeH bend), 651 (CeS str). H NMR (400 MHz,
CDCl
3
) δ: 2.28 (s, 3H, CH ), 3.61 (t, J = 4.7 Hz, 4H, morpholine-H),
3
1
3
3
7
.81 (t, J = 5.1 Hz, 4H, morpholine-H), 6.77 (t, J = 4.9 Hz, 1H, Ar-H),
C NMR (100 MHz, DMSOd ) δ: 111.6, 119.0, 122.4, 126.9, 127.9,
6
.01 (d, J = 7.9 Hz, 2H, Ar-H), 7.24 (d, J = 4.8 Hz, 1H, Ar-H), 7.39 (d,
128.7, 130.5, 130.6, 132.4, 135.0, 135.8, 139.5, 143.5, 144.2, 155.0,
1
3
+
J = 8.1 Hz, 2H, Ar-H), 7.44 (d, J = 5.9 Hz, 1H, Ar-H). C NMR
170.1, 178.7. HRMS (ESI-MS) m/z for C27
H
17
N
OS
3 2
[M + H] calcd
(
100 MHz, CDCl
3
) δ: 21.4, 48.1, 66.1, 120.4, 127.3, 128.7, 129.6,
464.0813, found 464.0811. Elemental analysis (%) calcd for
1
32.7, 133.5, 138.9, 143.8, 158.3, 171.2, 179.7. HRMS (ESI-MS) m/z
C
27
H
17
N OS2, C 69.96, H 3.70, N 9.06, S 13.83; found: C 69.61, H 4.00,
3
+
for C19
H
18
2
N O
2
S
2
[M + H] calcd 371.0810, found 371.0807.
N 9.59, S 14.48.
Elemental analysis (%) calcd for C19
H
18
N
2
O
2
S
2, C 61.60, H 4.90, N
(2-(Diphenylamino)-4-(p-tolyl)thiazol-5-yl)(thiophen-2-yl)methanone
−
1
7
.56, S, 17.31; found: C 60.98, H 4.89, N 7.39, S 17.29.
2-(Diphenylamino)-4-phenylthiazol-5-yl)(thiophen-2-yl)methanone
(6l). Yellow solid, 86%, mp 142–144 °C. IR (KBr, cm ) 3059 (aromatic
CeH str), 1583 (C]O str), 1446 (aromatic C]C str), 1328 (thiazole
(
−
1
1
(
6 g). Pale yellow solid, 86%, mp 201–204 °C. IR (KBr, cm ) 3076
CeN), 736 (aromatic CeH bend), 692 (CeS str). H NMR (400 MHz,
(
(
(
aromatic CeH str), 1597 (C]O str), 1490 (aromatic C]C str), 1332
CDCl
3
) δ: 2.33 (s, 3H, CH ) 6.83 (t, J = 3.9 Hz, 1H, Thiophene-H), 7.09
3
1
thiazole CeN), 792 (aromatic CeH bend), 690 (CeS str). H NMR
(d, J = 8.0 Hz, 2H, Ar-H), 7.25–7.29 (m, 3H, Ar-H), 7.39–7.47 (m, 8H,
Ar-H), 7.49 (d, J = 3.8 Hz, 1H, Ar-H), 7.62 (d, J = 8.0 Hz, 2H, Ar-H).
400 MHz, DMSO ) δ: 6.91 (t, J = 4.0 Hz, 1H, Ar-H), 7.26 (d, J = 6.2,
6
1
3
2
H, Ar-H), 7.34 (t, J = 6.9, 3H, Ar-H), 7.41 (d, J = 7.9 Hz, 2H, Ar-H),
C NMR (100 MHz, CDCl ) δ: 21.6, 120.8, 126.1, 126.8, 127.3, 128.2,
3
12

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
+
1
1
4
28.9, 129.2, 129.7, 130.2, 136.7, 137.7, 142.9, 144.0, 150.4, 169.1,
168.8, 169.1. HRMS (ESI-MS) m/z for C21
H
13
N
3
OS
2
[M + H] calcd
+
87.6. HRMS (ESI-MS) m/z for C27
H
20
N
2
OS
2
[M + H] calcd
388.0500 found 388.0492. Elemental analysis (%) calcd for
53.1017, found 453.1013. Elemental analysis (%) calcd for
OS2, C 71.65, H 4.45, N 6.19, S 14.17; found: C 71.62, H 4.84,
C
21
H
13
N OS2, C 65.10, H 3.38, N 10.84, S 16.55; found: C 65.20, H
3
C
27
H
20
N
2
3.42, N 10.64, S 16.62.
N 5.98, S 14.29.
N-(3-phenyl-4-(p-tolyl)thiazol-2(3H)-ylidene)thiophene-2-carboxamide
−
1
(
4-(4-Fluorophenyl)-2-(phenylamino)thiazol-5-yl)(thiophen-2-yl)me-
(6r). Pale yellow solid, 84%, mp 208–210 °C. IR (KBr, cm ) 3108
(aromatic CeH str), 1583 (C]O str), 1468 (aromatic C]C str), 1366
−1
thanone (6 m). Off white solid, 78%, mp 120–122 °C. IR (KBr, cm
)
1
3
277 (NeH str), 3074 (aromatic CeH str), 1660 (C]O str), 1408
(thiazole CeN), 741 (aromatic CeH bend), 699 (CeS str). H NMR
(
aromatic C]C str), 1354 (thiazole CeN), 1141 (C-F str) 723 (aromatic
(400 MHz, DMSOd
6
) δ: 2.24 (s, 3H, CH ), 7.06–7.11(m, 5H, Ar-H) 7.15
3
1
CeH bend) 646 (CeS str). H NMR (400 MHz, DMSOd
6
) δ: 6.98 (t,
(s, 1H, Ar-H), 7.32 (d, J = 6.64 Hz, 2H, Ar-H), 7.41–7.46 (m, 3H, Ar-H),
13
J = 4.2 Hz, 1H, Ar-H), 7.05 (t, J = 8.7, 1H, Ar-H), 7.13 (t, J = 8.8, 2H,
Ar-H), 7.37 (t, J = 8.1, 2H, Ar-H), 7.44 (d, J = 3.7 Hz, 1H, Ar-H), 7.57
7.51 (d, J = 3.6 Hz, 1H, Ar-H), 7. 65 (d, J = 4.9 Hz, 1H, Ar-H).
C
NMR (100 MHz, DMSOd ) δ: 21.2, 107.8, 127.8, 128.4, 129.1, 129.2,
6
(
t, J = 5.6 Hz, 2H, Ar-H), 7.67 (d, J = 7.9 Hz, 2H, Ar-H), 7.91 (d,
129.3, 129.4, 131.1, 132.3, 137.8, 138.9, 139.1, 143.2, 168.7, 169.1.
1
3
+
J = 4.8 Hz, 1H, Ar-H), 10.91 (s, 1H, NH). C NMR (100 MHz, MHz,
HRMS (ESI-MS) m/z for C21
H
16
N
2
OS
2
[M + H] calcd 377.0704, found
DMSOd
6
) δ: 115.3, 115.5, 115.9, 118.6, 119.4, 123.3, 128.5, 129.7,
377.0701. Elemental analysis (%) calcd for C21
H
16
N OS2, C 67.00, H
2
1
1
31.7, 131.8, 132.0, 132.1, 134.7, 135.1, 140.5, 143.7, 155.8, 161.5,
4.28, N 7.44, S, 17.03; found: C 67.02, H 4.03, N 7.25, S 17.09.
+
63.9, 165.2, 179.3. HRMS (ESI-MS) m/z for C20
H
13FN
2
OS [M + H]
2
calcd 381.0453, found 381.0448. Elemental analysis (%) calcd for
4.2. Biological activity
C
20
H
13FN OS2, C 66.27, H 3.89, N 7.73, S 17.69; found: C 67.19, H
2
3
.51, 7.38, S 17.88.
4.2.1. In-vitro COX-1 and COX-2 enzyme inhibition assay
N-(3,4-diphenylthiazol-2(3H)-ylidene)thiophene-2-carboxamide (6n).
COX-1 and COX-2 enzyme inhibition were screened by COX-1
(human) inhibitor screening assay kit (Catalogue no. 701070 Cayman
chemical, USA) and COX-2 (human) inhibitory screening assay kit
(Catalogue no. 701,080 Cayman chemical, USA) according to manu-
−
1
Off white solid, 85%, mp 198–200 °C. IR (KBr, cm ) 3100 (aromatic
CeH str), 1576 (C]O str), 1467 (aromatic C]C str), 1364 (thiazole
1
CeN), 754 (aromatic CeH bend) 691 (CeS str). H NMR (400 MHz,
DMSO
6
) δ: 7.06 (t, J = 3.7 Hz, 1H, Ar-H), 7.20–7.23 (m, 3H, Ar-H),
facturer’s instructions. Briefly, SnCl
2
reduces COX derived PGH pro-
2
7
.25–7.30 (m, 3H, Ar-H), 7.33–7.36 (m, 2H, Ar-H), 7.40–7.45 (m, 3H,
duced during the COX reaction to PGF2α and was measured directly.
100% initial activity tubes of enzyme were prepared by the addition of
160 μl reaction buffer (0.1 M Tris-HCl of pH 8.0, containing 2 mM
phenol and 5 mM EDTA), 10 μl Heme and 10 μl COX-1/COX-2 enzymes
into particular tubes. Similarly, COX-1 and COX-2 inhibitor tubes were
prepared by adding 10 μl of inhibitor besides above components in each
tube. Background tubes consist of inactivated enzymes obtained by
placing the enzyme tubes in boiling water for 3 min. Then tubes were
incubated at 37 °C for 10 min and addition of 10 μl of arachidonic acid
initiated the reaction in every tube followed by incubation for exactly
2 min at 37 °C. Enzyme catalysis was stopped by the addition of 30 μl of
stannous chloride. PGs produced in each reaction tube were quantified
by ELISA method. All the compounds were screened against COX-2 at
0.01, 0.1, 1.0, 10.0 and 100.0 μM in duplicate and percentage inhibi-
tion was determined. Compounds with promising activity were further
studied against COX-1 at 0.1, 1.0, and 10.0 μM in duplicate and IC50
values were determined from dose-response curve [1].
Ar-H), 7.52 (d, J = 4.8 Hz, 1H, Ar-H), 7.66 (d, J = 6.1 Hz, 1H, Ar-H).
1
3
C NMR (100 MHz, DMSO ) δ: 108.4, 128.4, 128.7, 129.2, 129.2,
6
1
29.3, 129.5, 130.7, 131.1, 132.3, 137.7, 139.1, 143.2, 168.7, 169.0.
+
HRMS (ESI-MS) m/z for C20
H
14
N
2
OS
2
[M + H] calcd 363.0548, found
3
63.0545. Elemental analysis (%) calcd for C20
H
14
N OS2, C 58.96, H
2
3
.22, N 10.31, S 15.74; found: C 59.19, H 3.12, N 10.64, S 15.55.
N-(4-(4-nitrophenyl)-3-phenylthiazol-2(3H)-ylidene)thiophene-2-car-
−1
boxamide (6o). Yellow solid, 87%, mp 248–250 °C. IR (KBr, cm ) 3110
(
(
(
aromatic CeH str), 1605 (C]O str), 1460 (aromatic C]C str), 1362
1
thiazole CeN), 748 (aromatic CeH bend), 697 (CeS str). H NMR
400 MHz, DMSO ) δ: 7.08 (t, J = 4.9, 1H, Ar-H), 7.40–7.42 (m, 2H,
6
Ar-H), 7.44–7.51 (m, 6H, Ar-H), 7.55 (d, J = 3.6 Hz, 1H, Ar-H), 7.69 (d,
1
3
J = 3.7 Hz, 1H, Ar-H), 8.12 (d, J = 8.8 Hz, 2H, Ar-H). C NMR
(
100 MHz, DMSO
6
) δ: 111.2, 123.8, 128.5, 129.1, 129.4, 130.7, 131.3,
1
32.6, 137.0, 137.0, 137.4, 142.9, 147.7, 168.8, 169.1. HRMS (ESI-MS)
+
m/z for C20
H
13
3
N O
3
S
2
[M + H] calcd 408.0398, found 408.0391.
Elemental analysis (%) calcd for C20
H
13
N
3
O S2, C 63.14, H 3.44, N
3
7
.36, S 16.85; found: C 63.34, H 3.61, N 7.30, S 16.78.
N-(3-phenyl-4-(4-(trifluoromethyl)phenyl)thiazol-2(3H)-ylidene)thio-
phene-2-carboxamide (6p). Off white solid, 86%, mp 228–230 °C. IR
4.2.2. In-vitro 5-LOX inhibitory assay
5-LOX enzyme inhibition was studied by Lipoxygenase inhibitor
screening assay kit (Catalogue no. 760700 Cayman chemical, USA)
together with 5-lipoxygenase (potato) screening enzyme (Catalogue
no.60401, Cayman chemical, USA) according to manufacturer’s in-
structions. Briefly, blank (100 μl assay buffer), positive control wells
(90 μl of 5-LOX enzyme and 10 μl assay buffer) 100% initial activity
wells (90 μl 5-LOX enzyme and 10 μl inhibitor vehicle) and inhibitor
wells (90 μl 5-LOX enzyme and 10 μl inhibitor) were incubated for
5 min at RT. 10 μl linoleic acid was added to initiate the reaction. After
incubation, addition of 100 μl chromogen stopped enzyme catalysis and
developed the reaction. The plate was shaken for 5 min and absorbance
was read at 495 nm using the 96 well plate reader. All compounds were
screened at 0.1, 1.0 and 10.0 μM in duplicate and determined IC50
values [1].
−
1
(
KBr, cm ) 3107 (aromatic CeH str), 1576 (C]O str), 1464 (aromatic
C]C str), 1322 (thiazole CeN), 759 (aromatic CeH bend), 689 (CeS
1
str). H NMR (400 MHz, DMSOd
6
) δ: 7.08 (t, J = 4.9 Hz, 1H, Ar-H),
7
1
.38–7.41 (m, 3H, Ar-H), 7.43–7.47 (m, 5H, Ar-H), 7.54 (d, J = 2.4 Hz,
13
H, Ar-H), 7.65 (s, 1H, Ar-H), 7.68–7.69 (m, 2H, Ar-H). C NMR
(
100 MHz, DMSOd
6
) δ: 110.1, 123.0, 125.6, 125.66, 125.7, 128.5,
1
1
29.1, 129.3, 129.3, 129.6, 130.3, 131.3, 132.5, 134.8, 137.4, 137.5,
+
43.0, 168.8, 169.1 HRMS (ESI-MS) m/z for C21
H
13
3
F N
2
OS [M + H]
2
calcd 431.0421, found 431.0416. Elemental analysis (%) calcd for
C
21
H
13
F
3
N OS2, C 58.60, H 3.04, N 6.51, S 14.90 found: C 58.66, H
2
3
.13, N 6.87, S 13.98.
N-(4-(4-cyanophenyl)-3-phenylthiazol-2(3H)-ylidene)thiophene-2-car-
−1
boxamide (6q). Pale yellow solid, 88%, mp 260–262 °C. IR (KBr, cm
)
3
108 (aromatic CeH str), 2227 (CN str), 1575 (C]O str), 1447 (aro-
4.2.3. Measurement of PGE
264.7 cells
2
and LTB levels in LPS- stimulated RAW
4
matic C]C str), 1362 (thiazole CeN), 754 (aromatic CeH bend), 693
1
(
CeS str). H NMR (400 MHz, DMSOd
6
) δ: 7.07 (t, J = 4.0 Hz, 1H, Ar-
To investigate the anti-inflammatory effect of compounds, the pro-
H), 7.37–7.42 (m, 5H, Ar-H), 7.43–7.48 (m, 3H, Ar-H), 7.54 (d, J = 3.6,
duction of PGE
2
and LTB in LPS- stimulated RAW 264.7 cells were
4
1
H, Ar-H), 7.67 (d, J = 4.9 Hz, 1H, Ar-H), 7.75 (d, J = 8.2 Hz, 2H, Ar-
examined. Briefly, RAW 264.7 cells were seeded on 96 well plate at a
1
3
4
H). C NMR (100 MHz, DMSOd
6
) δ: 110.6, 111.8, 118.7, 128.5, 129.1,
density of 1 × 10 cells per well and incubated for 18 h. Cells were pre-
1
29.4, 129.4, 130.3, 131.3, 132.5, 132.6, 135.2, 137.4, 137.4, 142.9,
treated with aspirin (500 μM) for 3 h to inactivate the effect of COX-1
13

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
(
(
endogenous cyclooxygenase-1). Then cells were washed with PBS
phosphate buffered saline) twice. Cells were subsequently pre-treated
at 1, 2, 4, and 6 h after injection. The anti-inflammatory effect was
determined as the difference in the reduction of paw volume between
the groups received carrageenan alone and carrageenan with treatment.
After 6 h, the animals were sacrificed by cervical dislocation. Hind paws
were collected below the ankle and stored at −80 °C until assayed. The
stomach was removed and an incision was made longitudinally along
the greater curvature and cleaned with cooled saline. The gastric mu-
cosa was inspected for any evidence of ulcer under the magnifying lens.
Besides, histological studies were performed using hematoxylin and
eosin [40–42].
with various concentrations of test compounds, etoricoxib, and zileuton
(
0.1, 1.0, 10.0 μM) for 2 h before further incubation for 16 h in fresh
Dulbecco’s Modified Eagles medium (DMEM) with or without LPS
1 μg/ml). After incubation, supernatant was collected by centrifuga-
tion.[36] PGE and LTB levels were measured by ELISA technique
using prostaglandin E and leukotriene B parameter Kits according to
(
2
4
2
4
manufacturer’s instruction. (Catalogue No. KGE004B & KGE006B, R&D
Systems, Inc. USA).
4
.2.4. In-vivo biological studies
4.2.7. Determination of PGE
2
and LTB levels in inflamed paws
4
Acute toxicity, in-vivo anti-inflammatory, and gastric safety profiling
Hind paws stored at −80 °C were brought to RT and tissues were
separated by degloving the bone. Then the tissues were homogenized in
ice-cold saline (5 ml) and suspended in acetone for 10 min at RT. After
incubation, tissue homogenates were subsequently centrifuged at 2000g
at 4 °C for 10 min. The aliquots of supernatant were subjected for the
were performed using male Wistar rats. Animals were maintained at
2
3 ± 2 °C in 12 h light/dark cycles with free access to water and food
in the animal house, Vellore Institute of Technology, Vellore, Tamil
Nadu. All the experimental procedures have been duly approved by
CPSCEA (Committee for the Purpose of Control and Supervision of
Experiments on Animals) Institutional Animal Ethics Committee
determination of PGE
2
and LTB
4
levels by ELISA technique using
parameter Kits according to man-
prostaglandin E
2
and leukotriene B
4
(
Approval no. VIT/IEAC/14/Nov5/35).
ufacturer’s instruction [36,43]. (Catalog No. KGE004B & KGE006B, R&
D systems, Inc. USA).
4.2.5. Studies for checking toxicity
Compound 6l was studied for its acute toxicity effect on Wistar rats
4.2.8. Total RNA isolation and cDNA conversion
as per OECD recommendations. Briefly, the animals were separated into
four sets each consisting of three animals. Animals were fasted over-
night before treatment with water ad libitum and 4 h after the treat-
ment. Group I was control received vehicle, and compound 6l at 50 mg/
kg, 500 mg/kg and 2000 mg/kg were administered orally to Group II,
III and IV respectively in a single dose. In the first 4 h, animals were
continuously monitored and then periodically for 24 h. Animals were
sacrificed after 14 days and gross assessment of kidney, liver, stomach,
and intestine was performed by histopathological studies of paraffin-
embedded samples stained with hematoxylin and eosin [37].
Paw tissues for qRT-PCR were stabilized by RNA later technology
(Qiagen, USA). Total RNA was extracted from control, test and standard
paw tissues using Trizol Reagent (Invitrogen, USA) according to man-
ufactures guidelines. RNA was quantified by measuring its absorption at
A260/A280 nm by Nanodrop BioSpectrometer (Eppendorf
BioSpectrometer®, USA). 2 µg of total RNA from all the samples were
reverse transcribed using the Omniscript Reverse Transcription kit
(Qiagen, USA) according to manufactures guidelines. In brief, the total
reaction volume was 20 μl containing 2 μg total RNA, 10x buffer RT,
5 mM dNTP mix, 10 μM oligo dT primer (Qiagen), RNase inhibitor,
Omniscript reverse transcriptase and RNase free water. The reaction
was conducted at 37 °C for 60 min.
4
.2.6. Anti-inflammatory studies and gastric safety profiling
Male Wistar rats weighing 150–180 g were subjected to gastric
safety profiling and carrageenan induced rat paw edema studies sub-
sequently on the same set of rats. The ulcerogenic potential of com-
pound 6l was studied according to Ganguly and Bhatnagar [38] with
minor modification. Animals were grouped to five sets, comprising five
animals each. Group I, control, consists of animals treated with vehicle.
Indomethacin 10 mg/kg was administered to Group II. Test compound
4.2.9. Gene expression analysis in Wistar rat
Expression of three target gene and one internal control (GAPDH)
were measured by qPCR. Primer set for each gene (Table 5) was de-
signed using NCBI primer BLAST software. All primers were procured
from Eurofins Genomics India Pvt. Ltd. (Bangalore, India). cDNA re-
versely transcribed from rats treated with control, test and standard
were used as the template for analyzing the expression of COX-1, COX-2
and 5-LOX genes and GAPDH. The amplifications were carried out in
0.2 ml qPCR 8-strips tubes with optical caps (Gunster Biotech Co., Ltd,
Taiwan) using CFX96 Real-Time System (BIO-RAD, USA). The real-time
PCR reactions were carried out in 25 μl reaction systems with TB Green
Premix Ex Taq II, 12.5 μl (Takara Bio Inc., Japan), 200 ng forward
primer (10 μM), 200 ng reverse primer (10 μM), and 2 μl cDNA tem-
plate. Each reaction was performed in triplicate. Thermal cycling con-
ditions were; initial denaturation 95 °C for 30 s, subsequently 40 cycles
of denaturation at 95 °C for 10 s, annealing at 60 °C for 1 min, and a
final extension at 95 °C for 10 s. A PCR product melt curve analysis was
6
l was administered to Group III, and group IV at 10, and 20 mg/kg
doses respectively. Group V served as carrageenan group for anti-in-
flammatory study. Both test and reference compounds were suspended
in 0.1% w/v CMC-Na (carboxymethyl cellulose sodium) in normal
saline and administered to fasting rats by daily oral dose for 7 succes-
sive days. On the 7th day anti-inflammatory activity of the compound
6
l was assessed by classical carrageenan-induced paw edema model
[
5,39]. 1 h after administration of the doses, subplantar injection of
freshly prepared 1% w/v carrageenan solution (150 μl) in normal saline
induced acute inflammation in the hind paw. The paw volume was
measured using vernier caliper prior to carrageenan injection and then
Table 5
Primers used for the qRT-PCR study.
Sl No
Primer code
Primer Sequence (5′-3′)
Product (bp)
Accession number
NM_017043.4
1
2
3
4
COX-1-F
COX-1-R
COX-2 -F
COX-2 -R
5-LOX-F
GTACCAGGTGCTGGATGGAGA
GGTGGGTAGCGCATCAACAC
CCCACTTCAAGGGAGTCTGG
GTGATCTGGACGTCAACACG
CCTGAGGGATGGATGTGCAA
TTCCAGTTCTTTCCGCCTGT
GGCCACGCTAATCTGACTTTC
ATACGGCCAAATCCGTTCAC
72
87
79
85
NM_017232.3
XM_006237140.2
XM_ 017592435.1
5
-LOX -R
GAPDH-F
GAPDH-R
14

J. Jacob P and S.L. Manju
Bioorganic Chemistry 100 (2020) 103882
done by heating from 65 to 95 °C with increments of 0.5 s. Data analysis
to predefined active sites [20], where polar hydrogens and partial
charges were added to protein and ligand by AutoDockTools. The Au-
togrid program executes an efficient grid-based algorithm which pro-
vides an exhaustive search inside the dimensions of the binding site
cavity. Grid maps of 60 Å × 60 Å × 60 Å points were centered on the
protein active sites. Ligand conformational search was performed by
Lamarckian Genetic Algorithm. The genetic algorithm (GA) population
size was fixed to 150, the number of GA evaluation as 2500000, and GA
docking runs were set to 100. Each ligand was docked separately with
the enzymes in order to achieve best binding conformation. After
docking, the binding energies of the ligands at different enzyme active
sites were analyzed. The H-bonding interaction, hydrophobic interac-
tion and van der Waal’s interaction were studied and explored by
measuring the distance between the protein and ligand.
TM
was performed with the BIORAD CFX Maestro
4.1.2 (Bio-Rad). The
quantification of COX-1, COX-2, and 5-LOX gene expression was per-
−
ΔΔCt
formed using the housekeeping gene GAPDH. The 2
method
(
Livak and Schmittgen, 2001) was used for gene expression ratio cal-
culation [44–46]. Relative amounts of all target genes were expressed
by normalizing to GAPDH and control genes levels.
4
.2.10. Determination of antioxidant activity
4
.2.10.1. DPPH free radical scavenging assay. The free radical
scavenging properties of all the test compounds have been studied as
described.[20,35] Briefly, equal volumes of freshly prepared DPPH
(
0.1 mM) and test compounds (20 μM) in methanol were mixed well
and allowed to stand in dark at RT for 30 min. The absorbance was
measured on UV–Visible spectrophotometer (Shimadzu, Japan) at
5
17 nm. Ascorbic acid served as the reference standard and triplicate
4.4. Statistical analysis
experiments were carried out. The percent DPPH radical scavenging
was calculated by the formula:
In this study, the experimental results were presented as the
mean ± SD of three parallel experiments or otherwise mentioned in
the experimental procedure. Animal experiment data were expressed as
mean standard error ( ± SEM). Statistical evaluation was carried out by
one way analysis of variance (ANOVA). Statistical significance is ex-
pressed as *p < 0.05, **p < 0.01, and ***p < 0.001.
(
A
0
s
A )
%
inhibition =
× 100
A
0
(1)
where A
0
= absorbance of the control and A
s
= absorbance of the test
or standard sample
4
.2.10.2. H
compounds to scavenge H
previously reported method with slight modification [35]. Briefly,
0 mM H solution was prepared in PBS of pH 7.4. 20 μM of test
compounds dissolved in 1 ml DMSO was added to 1 ml of H and
incubated at RT for 10 min. The concentration of H was determined
spectrophotometrically at 230 nm against blank solution of PBS without
. Triplicate experiments were conducted and results are displayed
as mean SD. The percentage of H scavenged by the test
compounds and standard was determined by the formula (1).
2
O
2
radical scavenging assay. The capability of the test
Declaration of Competing Interest
O
2 2
radical was calculated according to
The authors declare that they have no conflict of interest in the
publication.
4
O
2 2
O
2 2
O
2 2
Acknowledgments
H
O
2 2
The authors sincerely acknowledge the financial support of the VIT
Seed Grant that facilitated us to carry out this work and to get it
completed successfully in due course of time. The financial support to
Jaismy Jacob P from the CSIR, Govt. of India, through Senior Research
Fellowship is gratefully acknowledged.
±
O
2 2
4
.2.10.3. Iron chelating assay. Iron (II) chelating ability of the
synthesized compounds was assessed according to Chew et al. [47]
Briefly, 1 ml 0.1 mM ferrous sulphate, was added to 1 ml of test
compound (20 μM), followed by 1 ml 0.25 mM ferrozine. The
thoroughly mixed reaction mixture was allowed to stand for 10 min
and absorbance was observed at 562 nm. All the reading were taken in
triplicate and reported as mean ± SD. The iron (II) chelating property
of the compounds was calculated by the formula (1). Where, control
consists of 1 ml each of 75% methanol, ferrozine and ferrous sulphate.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.103882.
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