Synthesis, biological evaluation and docking study of 1,3,4-thiadiazole-thiazolidinone hybrids as anti-inflammatory agents with dual inhibition of COX-2 and 15-LOX

Article abstract of DOI:10.1016/j.bioorg.2018.06.036

Selective inhibition of both cyclooxygenase-2 (COX-2) and 15-lipooxygenase (15-LOX) may provide good strategy for alleviation of inflammatory disorders while minimizing side effects associated with current anti-inflammatory drugs. The present study descri

Full text of DOI:10.1016/j.bioorg.2018.06.036

Accepted Manuscript
Synthesis, biological evaluation and docking study of 1,3,4-thiadiazole-thiazo-
lidinone hybrids as anti-inflammatory agents with dual inhibition of COX-2 and
15-LOX
Yasser M. Omar, Hajjaj H.M. Abdu-Allah, Samia G. Abdel-Moty
PII:
S0045-2068(18)30449-8
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.036
YBIOO 2416
Reference:
Bioorganic Chemistry
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Revised Date:
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5 May 2018
28 June 2018
29 June 2018
Please cite this article as: Y.M. Omar, H.H.M. Abdu-Allah, S.G. Abdel-Moty, Synthesis, biological evaluation and
docking study of 1,3,4-thiadiazole-thiazolidinone hybrids as anti-inflammatory agents with dual inhibition of
COX-2 and 15-LOX, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.036
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Synthesis, biological evaluation and docking study of 1,3,4-
thiadiazole-thiazolidinone hybrids as anti-inflammatory agents with
dual inhibition of COX-2 and 15-LOX
Yasser M. Omara, Hajjaj H. M. Abdu-Allaha*†, Samia G. Abdel-Motya
a Department Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt. E-mail: hajjaj@aun.edu.eg
† Present address: Department of Applied Biochemistry, Tokai University, 4-1-1
Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
1

Abstract
Selective inhibition of both cyclooxygenase-2 (COX-2) and 15-lipooxygenase (15-
LOX) may provide good strategy for alleviation of inflammatory disorders while
minimizing side effects associated with current anti-inflammatory drugs. The present
study describes the synthesis, full characterization and biological evaluation of a
series of thiadiazole-thiazolidinone hybrids bearing 5-alk/arylidene as dual inhibitors
of these enzymes. Our design was based on merging pharmacophores that exhibit
portent anti-inflammatory activities in one molecular frame. 5-(4-hydroxyphenyl)-
1,3,4-thiadiazol-2-amine (3) was efficiently synthesized, chloroacetylated and
cyclized to give the key 4-thiazolidinone (5). Knovenagel condensation of 5 with
different aldehydes afforded the final compounds 6a-m, 7, 8 and 9. These compounds
were subjected to in vitro COX-1/COX-2, 15-LOX inhibition assays. Compounds (6a,
6f, 6i, 6l, 6m and 9) with promising potency (IC50 = 70-100 nM) and selectivity index
(SI = 220-55) were further tested for in vivo anti-inflammatory activity and effect on
gastric mucosa. The most promising compound (6l) inhibits COX-2 enzyme at a
nanomolar concentration (IC50 = 70 nM, SI = 220) with simultaneous inhibition of 15-
LOX (IC50 = 11 µM). These results are comparable to the potency and selectivity of
the standard drugs of both enzymes; celecoxib (COX-2 IC50 = 49 nM, SI = 308) and
zileuton (15-LOX IC50 = 15 µM) in one construct. Interestingly three compounds (6a,
6l and 9) exhibited equivalent to or even higher than that of celecoxib in vivo anti-
inflammatory activity at 3ꢀh interval with good GIT safety profile. Molecular docking
study conferred binding sites of these compounds on COX-2 and 15-LOX. Such type
of compounds would represent valuable leads for further investigation and
derivatization.
Keywords: 1,3,4-thiadizole, 4-thiazolidinone, cyclooxygenase, lipoxygenase, anti-
inflammatory.
2

Graphical abstract.
3

1. Introduction
Inflammation is involved in many diseases ranging from microbial infections, neural
disorders to cancer metastasis [1]. A key step of inflammation is the activation of a
cyclooxygenases (COX) and lipoxygenases (LOX) responsible for production of
several inflammatory mediators from arachidonic acid [2]. There are two isoforms of
cyclooxygenase enzyme: COX-1 and COX-2 [3]. While COX-1 is involved in the
synthesis of prostaglandins responsible for maintaining normal body function in
kidney, GIT and other organs, COX-2 is mainly induced during inflammation [4].
Classical non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, diclofenac
and indomethacin inhibit both COX isoforms leading to effective anti-inflammatory
response but with high risk of gastric ulceration and kidney damage upon prolonged
use[5]. Efforts to design selective COX-2 inhibitors were crowned by approval of two
products in the late nineties: Celebrex® (celecoxib)[6] and Vioxx® (rofecoxib)[7].
These selective COX-2 inhibitors demonstrate good anti-inflammatory activity with
good gastric and kidney safety profile [5]. However, recent reports have been
published correlating the new selective COX-2 inhibitors with cardiovascular
complications leading to the withdrawal of the rofecoxib on 2004 [8]. On the other
hand, overexpression of 15-LOX is associated with several inflammatory diseases
such as osteoarthritis [9] and asthma [10-12]. For instance, 15-LOX is responsible for
the production of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and eoxins which
contribute to airway inflammation and injury in asthmatic patient [13]. Prolonged use
of COX inhibitors shunts the metabolism of arachidonic acid towards the LOX
pathway and worsen the asthmatic patient condition as in aspirin-intolerant asthma [4,
14]. Zileuton is the only approved selective LOX inhibitor and was used in the
treatment of chronic asthma [15]. Therefore, development of selective COX-
2/COX-1 inhibitors with LOX inhibitory properties seems to deliver compounds with
safe gastrointestinal and cardiovascular profiles.
Accordingly, we envisage selective inhibition of both COX-2 and 15-LOX may
provide a good strategy for alleviation of inflammatory disorders while minimizing
side effects with potential application in asthmatic patients.
Recent trials to develop dual inhibitors of COX and LOX led to compounds I-VI (Fig.
1) which demonstrated moderate selectivity towards COX-2 at micromole level. for
example, compound I incorporating quinazoline and triazole rings inhibits COX-2
and 15-LOX with IC50 = 0.16 and 5.21 µM, respectively [16] and selectivity index
4

(SI) = 32. Similarly, quinoline derivative II [17] and celecoxib analogue III [18] were
found to inhibit both COX-2 and 15-LOX with five to seven-fold selectivity towards
COX-2 at low micromole potency. A hybrid molecule IV [19] containing 4-
thiazolidinone and thiazole rings has been discovered using computer aided drug
design as inhibitor of both COX-1 and 15-LOX without inhibitory activity on COX-2
till 200 µM. A very related compound V [20] was separately published containing 4-
thiazolidinone with methoxyphenyl group showed better inhibitory activity against
15-LOX (IC50 = 17.11 µM) than quercetin.
Br
OH
N
O
N
Cl
CH3
N
N
N
Cl
S
N
O
N
N
II
I
COX-2 IC50 = 1.13 µM
SI = 7.61
15-LOX IC50 = 5.91 µM
COX-2 IC50 = 0.16 µM
SI = 32.93
15-LOX IC50 = 5.21 µM
O
CH3
O
O
S
N
N
S
S
NH
N
N
S
Cl
N
IV
COX-1 IC50= 10 µM
Cl
CH3
OCH3
15-LOX IC50 = 143 µM
III
COX-2 IC50 = 1.14 µM
SI = 5.08
15-LOX IC50 = 4.71 µM
O
O
NH
S
N
O
N
S
NH
HN
H
N
OCH3
VI
COX-2 IC50= 0.11 µM
N
V
S
SI = 454.5
15-LOX IC50= 17.11 µM
S
H3CO
NH
N
Fig. 1. Chemical structure of certain reported COX and/or 15-LOX inhibitors.
5

On the other hand, a fused system containing 1,3,4-thiadiazole ring VI [21] exhibited
good inhibitory activity against COX-2 (IC50 = 0.11 µM) with high selectivity (SI =
454.5). These data encouraged us to design a new series of compounds combining
both 4-thiazolidinone and 1,3,4-thiadiazole in order to achieve selectivity and dual
targeting of both COX-2 and 15-LOX.
2. Result and discussion
2.1. Design
In continue of our interest in developing thiazolidinone hybrids [22], anti-
inflammatories with dual inhibition of COX-2 and LOX [23] and based on the above-
mentioned examples (Fig. 1), we surmised that the hybridization of the
pharmacophores 1,3,4-Thiadiazole and 4-thiazolidinone in one molecular frame could
show highly effective anti-inflammatory with broad spectrum and minimum side
effects. Combining both scaffolds was expected to inhibit both COX-2 (1,3,4-
thiadiazole), LOX (4-thiazolidinone) and provide better selectivity towards COX-2
over COX-1 enzyme due to their large volume which will not fit in the smaller COX-
1 binding pocket [24]. To test these hypotheses, we have synthesized compounds 1, 3
and 6a (Fig. 2). The new hybrid compound incorporating both 4-thiazolidinone and
1,3,4-thiadiazole 6a was compared with its building blocks; 4-thiazolidinone (1) and
1,3,4-thiadiazole (3) in terms of molecular volume, potency and selectivity against
both COX-2 and 15-LOX enzymes (Fig. 2). Molecular volume was calculated using
MOE program vsurf_D1 descriptor. The hybrid 6a was about two-folds larger, three-
folds more potent and two-folds more selective than 3 against COX-2. The hybrid 6a
also demonstrates better inhibitory activity against 15-LOX (about 1.5 folds more
potent than 1). Based on these results, we designed a library of new compounds 6-9
to elaborate other structural features of the hybrid molecules. The arylidene moiety
attached to the 4-thiazolidinone ring is varied as a fine tuner to optimise both
selectivity and potency against COX-2 and 15-LOX enzymes.
6

Fig. 2. Diagram represents the new design of synthetic dual COX-2/15-LOX inhibitors.
2.2. Chemistry
Compound 1 (Fig. 2) which is (5Z)-5-benzylidene-2-(4-hydroxyanilino)-1,3-thiazol-
4(5H)-one was prepared using a previously reported procedure [25]. The new
intermediate 5 was prepared via 4 steps with overall yield 50%, (Scheme 1). Firstly,
4-acetoxybenzoyl chloride was condensed with thiosemicarbazide in dry THF to
produce 2, which was simultaneously deacetylated and cyclized to give 3 upon reflux
with phosphorus oxychloride. This was confirmed by disappearance of the ester and
amidic carbonyl groups of 2 (1749 and 1623 cm-1) in the IR spectrum after cyclization
to give 3. Additionally, the methyl protons (δ 2.3 ppm) and the two NH protons of the
1
acylated thiosemicarbazide 2 at δ 9.3 and 10.3 ppm disappear in the H-NMR
spectrum of 3. Chloroacetylation of 3 was achieved by controlled addition of
chloroacetyl chloride over 5 h to yield 4. The chloroacetamido group of 4 was proved
by the appearance of amidic carbonyl band at 1693 cm-1 and the appearance of CH2
1
protons singlet signal at δ 4.35 in the IR and H-NMR spectrum of 4, respectively.
Uncontrolled addition of chloroacetyl chloride in this reaction resulted in partial
esterification of the phenolic hydroxyl group and difficulty in purification.
Heterocyclization to yield 5 was performed by reflux of 4 with ammonium
thiocyanate in ethanol. Formation of 4-thiazolidinone ring was confirmed by the shift
of amidic carbonyl band to a higher frequency (1720 cm-1) in the IR spectrum and the
7

1
CH2 upfield shift to δ 4.11 in the H-NMR spectrum. The purity of all synthesized
compounds was checked by elemental analyses. Efficient synthesis of intermediate 5
enabled us to explore the Knoevenagel condensation reaction of various aldehydes
with the active methylene of the 4-thiazolidinone ring, (Scheme 2). This reaction was
catalysed by the addition of piperidine to the ethanolic solution of 5 and the
appropriate aldehyde to give the ylidene derivatives 6-9 in fairly good yields 50-83%.
All products were characterized by IR, NMR, mass spectrometry and elemental
analysis.
O
Cl
N N
HN NH
O S
S
NH2
b
O
+
NH2
2 H2N
a
S
3
N
NH2
O
HO
H
CH3
O
CH3
2
O
O
O
N
N
HN
N
4
N
d
Cl
c
HO
S
NH
S
S
N
HO
5
Scheme 1. Synthesis of 1,3,4-thiadiazole-thiazolidinone hybrids. Reagents and conditions: (a) dry THF, rt, 24 h,
89%yield; (b) (i) POCl3, reflux, 2 h, (ii) H2O, reflux, 4 h, (ii) NH4OH, 73%yield in 3 steps; (c) ClCH2COCl,
benzene, reflux, 3-5 h, 91.8% yield; (d) NH4SCN, EtOH, reflux, 3-5 h, 83% yield.
O
N
HN
N
HO
S
S
N
6 a-m
O
R
O
N
HN
N
N
a
5
HN
N
HO
S
b
S
N
c
HO
S
S
N
7
8
N
d
O
S
HN
N
N
N
9
S
HO
Scheme 2. Synthesis of target compounds 6a-m and 7-9. Reagents and conditions: Appropriate aldehyde (a:
substituted benzaldehyde, b: pyridine-3-carboxaldehyde, c: cyclohexanecarboxaldehyde, d: cinnamaldehyde),
Piperidine, EtOH, reflux, 24 h, 50-83% yield.
8

2.3. Biology
2.3.1 In vitro cyclooxygenase lipoxygenase inhibitory activity.
All compounds were tested for inhibitory activity of ovine COX-1 and human
recombinant COX-2. The concentration of the tested compounds causing 50%
inhibition (IC50, µM) and selectivity index (COX-1 IC50/COX-2 IC50) were calculated
(Table 1).
Table 1.
In vitro COX-1, COX-2 and 15-LOX inhibitory results
Compounds
COX-1
IC50 (µM)
-
COX-2
IC50 (µM)
-
Selectivity
index
-
15-LOX
IC50 (µM)
8.24
R
No.
-
1
-
8.94
0.33
0.41
0.085
0.11
0.18
0.12
0.14
0.11
0.11
0.10
0.09
0.10
0.18
0.07
0.10
0.41
0.22
0.08
0.049
27.09
16.86
54.82
94.73
54.50
85.08
76.71
111.91
103.82
146.20
179.00
135.20
63.00
220.29
141.10
20.66
35.86
190.38
308.16
-
3
-
H
6.48
5
3.11
4.66
5.74
6a
4-Methyl
4-Propyl
4-Isopropyl
4-Hydroxy
2-hydroxy
4-Methoxy
3,4-Dimethoxy
3,4,5-Trimethoxy
4-Dimethylamino
4-Chloro
3,4-Dichloro
4-Bromo
-
10.42
9.81
8.96
6b
7.63
6c
10.21
10.74
12.31
11.42
14.62
16.11
13.52
11.34
15.42
14.11
8.47
8.78
6d
9.74
6e
9.87
6f
9.74
6g
12.67
11.74
12.87
10.52
11.87
10.84
7.22
6h
6i
6j
6k
6l
6m
7
8
-
7.89
5.97
-
15.23
15.1
13.2
9
-
-
Celecoxib
Diclofenac
Sod.
-
-
5.29
-
0.3
-
17.63
-
-
Zileuton
15.6
9

The hybrid 6a combining scaffolds targeting both COX-2 and 15-LOX exhibited
better selectivity and potency in comparison with analogues containing each scaffold
alone (compounds 1 and 3). COX-2 IC50 of 6a is 0.085 µM (IC50 of 3 = 0.33 µM) with
selectivity index of 54 for COX-2 enzyme (SI of 3 = 27). 15-LOX IC50 of 6a is 5.74
µM (IC50 of 1 = 8.24 µM). Encouraged by these subtle differences, we envisaged
further modification of the arylidene moiety of 6a may be a fine tuner of the activity
of hybrids towards COX-2 and 15-LOX enzyme. We have introduced different
modifications including ring substitutions on the arylidene moiety or its replacement
with pyridene (7) or cyclohexyl (8). This resulted in significant changes in the
potency and selectivity for COX-2 enzyme and the potency towards 15-LOX enzyme.
For instance, arylidene ring substitution with 3,4-dichloro atoms in 6l led to a little
increase in enzyme inhibition (IC50 = 0.070 μM) with a marked effect on selectivity
(COX-2 SI = 220). Compound 6l is four folds more selective than the parent hybrid
6a. In contrast, compound 6k with only 4-chloro substitution on the arylidene moiety
showed marked decrease in potency and selectivity. On the other hand, among the
arylidene ring substitution with methoxy groups (6g-i) was tolerated. Therefore, 6i
which has three methoxy groups is 3 folds more selective than the parent hybrid 6a. In
general, introducing aliphatic substituent on the phenyl ring of the arylidene moiety
(6b-d) led to decrease in potency while increasing selectivity in comparison with the
parent hybrid 6a. Methyl derivative 6b showed higher potency and selectivity than
propyl or isopropyl. Furthermore, hydroxyl substitution (6e and 6f) enhanced the
selectivity of the hybrid molecule. Compound 6f with 2-hydroxy group was more
potent and selective than 6e with 4-hydroxy one. The absence of the arylidene moiety
(5) or its replacement with pyridyl 7 or cyclohexyl moiety (8) decreased both potency
and selectivity. The lower activity of compounds 6f with 2-hydroxyphenyl, 6e with 4-
hydroxyphenyl and 7 with pyridyl than 6a with phenyl suggests that the interaction at
phenyl binding site is hydrophobic in nature. An interesting observation was that
insertion of ethylene spacer in compound 9 had a slight effect on enzyme inhibitory
activity (IC50 = 0.080 μM) and 3.5 folds increase in selectivity than the parent hybrid
6a. Most compounds have similar potency but significantly different selectivity.
Therefore, the preliminary structure activity relationship could be concluded: the
arylidene moiety is required for high potency and selectivity, substitution on arylidene
ring affect the selectivity more than potency through hydrophobic and electronic
interactions.
10

2.3.2. In vitro lipoxygenase inhibitory activity.
All compounds were tested for 15-LOX enzyme of soybean. The new synthesized
compounds are more potent than the reference drug zileuton against 15-LOX enzyme.
The most active compound was 5 which lacks arylidene moiety. Compound 5 has an
IC50 of 3.11 μM. Among arylidene containing compounds, 6a with unsubstituted
phenyl ring was the most potent derivative (IC50 of 5.74 μM). All trialled substitutions
on the phenyl ring led to a decrease in potency. Replacement of the phenyl group 6a
with a cyclohexyl moiety 8 retain activity. Compound 9 with ethylene spacer showed
marked decrease in potency.
2.3.3. In vivo anti-inflammatory activity.
Compounds (6a, 6f, 6i, 6l, 6m and 9) were selected for in vivo study by carrageenan
induced paw edema method in rats at 28 µmol/kg. Celecoxib and diclofenac sodium
were used as reference drugs. The results are presented as a percentage of edema
inhibition at time interval of 1, 2, 3, 4 h (Table 2). All tested compounds showed a
gradual increase of the anti-inflammatory activity up to its maximum after 3 h.
Compounds (6a, 6l and 9) were the most active derivatives and exhibited comparable
inhibitory activity to the two reference drugs (Fig. 3).
Table 2.
% of edema inhibition of the synthesized compounds.
Compounds
% Inhibition of Edema
2 h 3 h
No.
1h
4 h
18.18 ± 6.63
22.73 ± 6.63a
40.91 ± 8.35 d
25.00 ± 2.78 a
13.64 ± 5.79
29.55 ± 6.63 b
31.82 ± 5.08 c
38.63 ± 2.78 d
41.51 ± 3.53d 42.11 ± 7.13 d 40.35 ± 4.29 d
41.51 ± 4.62 d 40.35 ± 6.45 d 33.33 ± 5.95 d
47.17 ± 6.40 d 52.63 ± 5.95 d 36.84 ± 4.30 d
37.74 ± 3.77 d 38.60 ± 3.92 d 17.54 ± 3.51 a
33.96 ± 5.97 d 47.37 ± 6.20 d 29.83 ± 4.80 d
52.83 ± 6.67 d 54.39 ± 1.75 d 35.09 ± 2.15 d
45.28 ± 6.26 d 45.61 ± 5.12 d 35.09 ± 6.56 d
64.15 ± 1.89 d 50.88 ± 5.95 d 40.35 ± 4.30 d
Celecoxib
Diclofenac Sod.
6a
6f
6i
6l
6m
9
a. Significant difference at P < 0.05, b. Significant difference at P < 0.01, c.
Significant difference at P < 0.001, d. Significant difference at P < 0.0001.
11

C elecoxib
6a
6l
9
75
60
45
30
15
0
1h
2h
3h
4h
T im e
Fig. 3. % Edema inhibition of tested compounds (6a, 6l and 9) in comparison with celecoxib.
2.3.4. Ulcerogenic liability.
Gastrointestinal (GI) complications are the most serious side effects of NSAIDs.
Microscopic examination of gastrointestinal mucosa for the presence of lesions
following oral administration of a single dose of tested compound as well as the
reference drug has been taken as an indication for the ulcerogenic effect. Compounds
(6f, 6l, 6m and 9) were selected for testing of their ulcerogenic effect at a dose of 20
mg/kg which is double the dose of in vivo anti-inflammatory test. Increasing the
amount of tested compounds was made to ensure the safety at a higher dose.
Indomethacin was the reference drug at a dose of 10 mg/kg (as a positive control).
Microscopic examination of gastric mucosa following administration of tested
compounds and control group showed normal glandular architecture and gastric pits
(Fig. 4 and 5) in comparison with indomethacin which induced desquamation of
surface epithelial cells, marked loss of normal glandular architecture, edema and
dilation of blood vessel (Fig. 4 and 5).
12

Fig. 4. Light microscopic pictures of gastric mucosa of the control group (Fig. A), tested compounds
(6l (Fig. D) and 6f (Fig. E)) showed normal glandular architecture and gastric pits. The indomethacin
group (Fig. B and C) showed induced desquamation of surface epithelial cells edema and dilation of
blood vessel. Magnification 4x; scale bar 200 µm.
13

Fig. 4. Light microscopic pictures of gastric mucosa of the control group (Fig. A), tested compounds
(6l (Fig. D) and 6f (Fig. E)) showed normal glandular architecture and gastric pits. The indomethacin
group (Fig. B and C) showed induced desquamation of surface epithelial cells edema and dilation of
blood vessel. Magnification 10x; scale bar 100 µm.
2.3.5. Molecular modelling.
2.3.5.1. Docking study on COX-2.
Volume, LogP and docking scores (Table 3) were calculated using MOE 2016.09
software and the human COX-2 enzyme (5KIR) was used for the docking study. The
docking scores ranged from -7.018 to -7.948 showing rough correlation with the IC50
of the synthesized compounds. As mentioned earlier, the size of COX-2 pocket is
bigger than COX-1 which allows selective binding of larger molecules. The most
selective compound 6l showed the largest calculated volume among the series (Fig. 6)
and the least selective compound 5 showed the least calculated volume. Additionally,
6l possesses the highest LogP which may also have contributed to its high in vivo
anti-inflammatory activity.
14

Table 3.
Volume, LogP and docking score of the new synthesis compounds.
Compound No.
Volume a
672.4
LogP b
2.433
4.981
5.280
6.197
6.125
4.674
4.672
4.938
4.680
4.423
4.897
5.574
6.201
5.780
3.749
4.997
5.626
-
ΔG COX-2 c
-7.232
ΔG 15-LOX c
-5.269
-5.038
-5.083
-5.016
-5.334
-5.206
-4.794
-4.481
-5.257
-5.542
-4.695
-4.747
-4.478
-4.698
-5.052
-4.898
-3.672
-
5
972.0
-7.018
6a
1005.1
1090.4
1077.1
950.8
-7.221
6b
-7.948
6c
-7.416
6d
-7.162
6e
939.6
-7.239
6f
1007.0
1058.9
1026.4
1090.9
1058.4
1118.6
1098.9
919.3
-7.359
6g
-7.886
6h
-7.911
6i
-7.404
6j
-7.762
6k
7.612
6l
-7.134
6m
-7.112
7
957.6
-7.371
8
9
1087.5
-
-7.815
-8.375
Rofecoxib
a. Volume calculated using vsurf_D1.
b. LogP (o/w).
c. ΔG (Kcal/mol)
Fig. 5. Compound 6l (showed in green) binds effectively to the pocket of COX-2 enzyme (Fig. A) in
compare with COX-1 (Fig. B). The co-crystalized ligand rofecoxib (showed in black Fig. A) and
Ibuprofen® (showed in red Fig. B) are also explored.
In terms of binding and interaction, the three most potent compounds (6l, 9 and 6i)
explore good binding with COX-2 enzyme. For instance, compound 6l (Fig.7) form
15

three hydrogen bonds with Arg513 (2.62 Å), Phe518 (2.05 Å) and Ile517 (2.42 Å) and
one hydrophobic interaction with Ala527. Also, 9 (Fig. 8) forms two hydrogen bonds
with Arg513 (3.52Å) and Phe518 (2.51 Å). Lastly, 6i (Fig. 9) forms two hydrogen
bonds with Arg513 (2.79Å) and Leu352 (1.92 Å) and one hydrophobic interaction
with Ser353.
Fig. 6. Interaction of 6l with COX-2 enzyme.
Fig. 7. Interaction of 9 with COX-2 enzyme.
16

Fig. 8. Interaction of 9 with COX-2 enzyme.
2.3.5.2. Docking study on 15-LOX.
Human 15-LOX enzyme (4NRE) was used for the docking study. The docking scores
ranged from -3.672 to -5.542 showing rough correlation with the IC50 of the
synthesized compounds. There is a good correlation between the molecular volume
and IC50 of the synthesized compounds where smaller molecules have higher potency
than larger one (Table 3).
In terms of binding and interaction, the three most potent compounds (5, 6a and 8)
form a hydrogen bond with Glu 613 (Fig. 10 to 12) with a distance of (2.26 Å, 2.25 Å
and 2.28 Å, respectively). Compound 8 form additional hydrogen bond with Lys 612
(2.36 Å)
17

Fig. 9. Interaction of 5 with 15-LOX enzyme.
Fig. 10. Interaction of 6a with 15-LOX enzyme.
18

Fig. 11. Interaction of 6a with 15-LOX enzyme.
2.3. Conclusion
The current study proposed a new rational design of dual inhibitors of COX-2 and 15-
LOX based on combining 2-amino-1,3,4-thiadiazole as a COX inhibitor
pharmacophore and 4-thiazolodine as a 15-LOX inhibitor pharmacophore into one
construct. The obtained results clearly revealed that the hybrids are much more potent
and selective COX-2 and 15-LOX than their individual components. Moreover,
compounds 6a, 6f, 6i, 6l, 6m and 9 showed anti-inflammatory activity equivalent to
or even higher than that of celecoxib. In vitro COX-1/COX-2 inhibition study
revealed that among the synthesized compounds, compound 6l showed the highest
inhibitory activity against COX-2 with an IC50 values of 70 nM and selectivity index
220. Additionally, the active compounds showed significant in vitro LOX inhibitory
activity higher than that of zileuton and good GIT safety profile and are well tolerated
by experimental animals. The docking experiments attempted to investigate the
binding mode of the most active compounds in the binding site of COX-2 and 15-
LOX and confirmed the high selectivity binding towards COX-2 enzyme over COX-
1. Consequently, The in vitro and in vivo anti-inflammatory profiles of the
synthesized compounds can be a base for developing new anti-inflammatory drugs.
19

3. Experimental.
3.1. Chemistry
All solvents and reagents used for the synthesis of target compounds were of
commercial grade without further purification before use except THF dried by
benzophenone/sodium metal method and benzene dried over sodium metal. Melting
points were determined on an electrothermal melting point apparatus [Cole-Parmer -
Electrothermal IA9100, UK], and were uncorrected. Pre-coated silica gel plates
(TLC) (kieselgel 0.25 mm, 60G F254, Merck, Germany) were used for monitoring of
the chemical reactions. Spots were detected by using ultraviolet lamp at 254 nm
wavelength (Spectroline, model CM-10, USA). (IR) spectra (KBr discs) were
recorded on thermo scientific nicolet IS10 FT IR spectrometer (thermo Fischer
scientific, USA) at Faculty of science, Assiut University, Assiut, Egypt. Most of 1H-
NMR and 13C-NMR spectra were scanned on AVANCE-III High Performance FT-
NMR spectrometer (400 MHz), (Brucker) at Faculty of Science, Sohag University,
Sohag, Egypt. While that of compounds (2, 3 and 4) were performed on a Varian EM-
390 NMR spectrometer (90 MHz, Varian, CA, USA) at Faculty of Science, Assiut
University, Assiut, Egypt. Mass spectra were carried out on Direct Probe Controller
Inlet Part TO Single Quadropole mass analyzer in Thermo Scientific GCMS model
ISQ LT using Thermo X-Calibur software at the Regional Center for Mycology and
Biotechnology (RCMB), Faculty of Science, Al-Azhar University, Nasr city, Cairo,
Egypt. Elemental microanalyses were performed on elemental analyzer model flash
2000 thermo fisher at the Regional Center for Mycology and Biotechnology (RCMB),
Faculty of Science, Al-Azhar University, Nasr city, Cairo, Egypt.
3.1.1. Synthesis of 4-(2-thiocarbamoylhydrazinocarbonyl) phenyl acetate (2).
4-Acetoxy benzoyl chloride (11 g, 55 mmol) was dissolved in dry THF (70 mL) and
the obtained solution was poured to a stirred suspension of thiosemicarbazide (10 g,
109.8 mmol, 2 equv) in dry THF (240 mL). The reaction mixture was stirred at room
temperature for 24 h. The THF was evaporated, distilled water (50 mL) was added to
the dry solid and vigorously stirred for 5 min. The product was filtered to give white
solid. The product was used in the next step without further purification as white
solid; Yield 89%; mp 205-207 °C; IR (cm-1, KBr): 3353, 3296 (NH2, NH), 1749
1
(C=O ester) 1623 (C=O amidic); H-NMR (90 MHz, δ ppm DMSO- d6): 2.3 (s, 3H,
CH3), 7.15,7.9 (dd, J= 9 Hz, 4H, Ar-H), 7.65 (br. s, 2H, NH2), 9.3 (br. s, 1H, NH),
20

10.3 (br. s, 1H, NH); Elemental analysis for C10H11N3O3S (253.28):
Calculated/Found: 47.42/47.69 (%C), 4.38/4.61 (%H), 16.59/16.75 (%N) and
12.66/12.49 (%S).
3.1.2. Synthesis of 2-amino-5-(4-hydroxyphenyl)-1,3,4-thiadiazole (3).
Compound 2 (10 g, 39.5 mmol) and phosphorus oxychloride (39.5 mL, 157.1 mmol, 4
equv.) were refluxed for 1 h. The excess of phosphorus oxychloride was evaporated
under vacuum. cold water (50 mL) was added and the reaction mixture was stirred
under reflux overnight. After cooling to room temperature, the solution was basified
by conc. ammonium hydroxide solution (20%) and the separated solid was filtered
and washed with cooled water to obtain a yellow solid; Yield 73%; mp 209-211 °C;
1
IR (cm-1, KBr): 3396, 3242 (OH, NH2), 1568 (C=N); H-NMR (90 MHz, δ ppm
DMSO-d6): 6.85,7.55 (dd, J= 9 Hz, 4H, Ar-H), 7.2 (br. s, 2H, NH2), 9.9 (br. s, 1H,
OH); EI-MS [m/z (%)]: 192.89 (M+, 75.3%), 119.96 (100% base peak); Elemental
analysis for C8H7N3OS (193.23): Calculated/Found: 49.73/49.58 (%C), 3.65/3.82
%H), 21.75/21.93 (%N) and 16.59/16.72 (%S).
3.1.3. Synthesis of 2-chloro-N-[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]acetamide
(4).
Compound 3 (2.69 g, 10 mmol) was stirred in dry benzene (15 mL). Chloroacetyl
chloride (0.88 mL, 11 mmol, 1.1 equv.) in dry benzene (10 mL) was divided into
three equal parts and added within 30 min time interval. After 1 h from the addition of
the third part, additional amounts of chloroacetyl chloride (0.08 mL, 1 mmol, 0.1
equv.) were added every 1 h until the start consume. The reaction mixture was filtered
while hot and washed with cold methanol. The product recrystalized from dioxane/
water as white solid; Yield 91.8%; mp 256-258 °C; IR (cm-1, KBr): 3396 (NH), 1693
1
(C=O), 1595,1574 (C=N); H-NMR (90 MHz, δ ppm DMSO- d6): 4.35 (s, 2H, CH2),
6.79,7.09 (dd, J= 9 Hz, 4H, Ar-H), 9.8 (br. s, 1H, OH). EI-MS [m/z (%)]:270.92
(M++2, 36.93%), 268.93 (M+, base beak); Elemental analysis for C10H8ClN3O2S
(269.71): Calculated/Found: 44.53/44.81 (%C), 2.99/3.08 (%H), 15.58/15.7 (%N) and
11.89/11.97 (%S).
3.1.4. 2-[(5-(4-Hydroxyphenyl)-1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one (5)
Compound 4 (10 mmol) and ammonium thiocyanate (1.5 g, 20 mmol, 2 equv.) were
stirred in absolute ethanol (20 ml) under reflux for 3 h. the product was filtered while
hot, washed with cooled water and recrystallize from from DMSO/water. The product
21

was brownish solid; Yield 83%; mp >300 °C; IR (cm-1, KBr): broad band at 3409
1
(NH, OH), 1720 (C=O), 1574 (C=N); H-NMR (400 MHz, δ ppm DMSO- d6): 4.11
(s, 2H, CH2), 6.9 ,7.75 (dd, J= 8.56 Hz, 4H, Ar-H), 10.15 (s, 1H, OH), 12.31 (br. s,
1H, NH); 13C-NMR (100 MHz, δ ppm DMSO- d6): 36.06 (CH2),116.59, 121.68,
129.27,160.54 (Ar-C), 174.34(C=O); EI-MS [m/z (%)]:294.82 (M++2, 9.70%), 292.07
(M+, 70.84%), 274.06 (96.58%), 271.28 (77.25%), 58.98 (100% base beak);
Elemental analysis for C11H8N4O2S2 (292.34): Calculated/Found: 45.19/45.42 (%C),
2.76/2.79 (%H), 19.17/19.35 (%N) and 21.94/22.18 (%S).
3.1.5. General procedure for Knoevenagel condensation of compound (5) with
aldehydes to give compounds (6a-m and 7-9).
Compound 5 (0.29 g, 1 mmol) and the appropriate aldehyde (1.5 mmol, 1.5 equv.)
were stirred in absolute ethanol (10 mL). Piperidine (8 drops) was added and the
reaction mixture was refluxed for 16-18 h, cooled and acidified with glacial acetic
acid. The solid product was collected by filtration and washed with cold water.
3.1.5.1. (5Z)-5-benzylidene-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-1,3-
thiazolidin-4-one (6a).
Orange solid; Yield 58.4%; mp >300 °C; IR (cm-1, KBr): broad band at 3409 (NH,
1
OH), 3021 (sp2C-H), 1721 (C=O), 1606 (C=N); H-NMR (400 MHz, δ ppm DMSO-
d6): 6.90 (d, J = 8.44 Hz, 2H, Ar-H), 7.50 (d, J = 6.72 Hz, 1H, Ar-H), 7.57 (t, J = 6.97
Hz, 2H, Ar-H), 7.61-7.80 (m, 5H, Ar-H), 10.13 (br. s, 1H, OH), 12.89 (br. s, 1H,
NH); 13C-NMR (100 MHz, δ ppm DMSO- d6): 116.61, 121.49, 124.58, 129.35,
129.81, 130.63, 130.89, 133.15, 133.66, 158.50, 160.68, 165.69, 167.39, 168.95
(C=O); Elemental analysis for C18H12N4O2S2 (380.44): Calculated/Found:
56.83/57.07 (%C), 3.18/3.31 (%H), 14.73/15.02 (%N) and 16.86/16.79 (%S).
3.1.5.2.(5Z)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-[(4-
methylphenyl)methylidene]-1,3-thiazolidin-4-one(6b).
Yellow solid; Yield 83.7%; mp >300 °C; IR (cm-1, KBr): broad band at 3115 (NH,
1
OH), 3021 (sp2C-H), 2909 (sp3C-H), 1708 (C=O), 1594 (C=N); H-NMR (400 MHz,
δ ppm DMSO- d6): 2.38 (s, 3H, CH3), 6.91,7.77 (dd, J =8.31-8.68, 4H, Ar-H), 7.40,
7.57 (dd, J =7.46-7.58, 4H, Ar-H), 7.75 (s, 1H, =C-H), 10.13 (br. s, 1H, OH), 12.85
(br. s, 1H, NH); Elemental analysis for C19H14N4O2S2 (394.47): Calculated/Found:
57.85/58.02 (%C), 3.58/3.80 (%H), 14.20/14.37 (%N) and 16.26/16.39 (%S).
22

3.1.5.3.(5Z)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-[(4-
propylphenyl)methylidene]-1,3-thiazolidin-4-one (6c).
Yellow solid; Yield 71%; mp 263-265 °C; IR (cm-1, KBr): broad band at 3421 (NH,
1
OH), 3022 (sp2C-H), 2927 (sp3C-H), 1719 (C=O), 1597 (C=N); H-NMR (400 MHz,
δ ppm DMSO- d6): 0.90 (t, J =7.34, 3H, CH3), 1.61 (sxt, J =7.41, 2H, CH2), 2.61 (t, J
=7.52, 2H, CH2), 6.91, 7.77 (dd, J =8.56-8.68, 4H, Ar-H), 7.40, 7.58 (dd, J = 7.95,
4H, Ar-H), 7.75 (s, 1H, =C-H) 10.19 (br. s, 1H, OH), 12.89 (br. s, 1H, NH);
Elemental analysis for C21H18N4O2S2 (422.52): Calculated/Found: 59.69/60.02 (%C),
4.29/4.37 (%H), 13.26/13.43 (%N) and 15.18/15.29 (%S).
3.1.5.4.(5Z)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-{[4-(propan-2-
yl)phenyl]methylidene}-1,3-thiazolidin-4-one (6d).
Yellow solid; Yield 73.4%; mp 182-184 °C; IR (cm-1, KBr): broad band at 3372 (NH,
1
OH), 3022 (sp2C-H), 2958 (sp3C-H), 1696 (C=O), 1595 (C=N). H-NMR (400 MHz,
δ ppm DMSO- d6): 1.23 (d, J =6.85, 6H, (CH3)2), 2.69 (m, 1H, CH), 6.92 (d, J =8.56,
2H, Ar-H), 7.47, 7.61 (dd, J = 7.70, 4H, Ar-H), 7.74-7.80 (m, 3H, Ar-H, 1H, =C-H)
10.19 (br. s, 1H, OH), 12.90 (br. s, 1H, NH); Elemental analysis for C21H18N4O2S2
(422.52): Calculated/Found: 59.69/59.98 (%C), 4.29/4.41 (%H), 13.26/13.19 (%N)
and 15.18/15.31 (%S).
3.1.5.5.(5Z)-5-[(4-hydroxyphenyl)methylidene]-2-{[5-(4-hydroxyphenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6e).
Yellow solid; Yield 75.7%; mp >300 °C; IR (cm-1, KBr): broad band at 3404 (NH,
1
OH), 3012 (sp2C-H), 1694 (C=O), 1580 (C=N); H-NMR (400 MHz, δ ppm DMSO-
d6): 6.92 (br. s, 2H, Ar-H), 6.96 (br. s, 2H, Ar-H), 7.53, 7.69, 7.75 (m, 5H, Ar-H, =C-
H), 10.04 (br. s, 1H, OH), 10.22 (br. s, 1H, OH), 12.66 (br. s, 1H, NH); Elemental
analysis for C18H12N4O3S2 (396.44): Calculated/Found: 54.53/54.81 (%C), 3.05/3.22
(%H), 14.13/14.5 (%N) and 16.18/16.35 (%S).
3.1.5.6.(5Z)-5-[(2-hydroxyphenyl)methylidene]-2-{[5-(4-hydroxyphenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6f).
Yellow solid; Yield 65.6%; mp 264-266 °C; IR (cm-1, KBr): broad band at 3377 (NH,
OH), 3032 (sp2C-H), 1690 (C=O), 1592 (C=N).1H-NMR (400 MHz, δ ppm DMSO-
d6) 6.92,7.76 (dd, J = 6.97-7.21, 4H, Ar-H), 7.00 (br. s, 2H, Ar-H), 7.33 (br. s, 1H,
Ar-H), 7.47 (d, 2H, J = 5.99, Ar-H), 8.03 (s, 1H, =C-H), 10.04 (br. s, 1H, OH), 10.44
23

(br. s, 1H, OH), 12.71 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm DMSO- d6):
116.36, 121.62, 129.32,160.55,116.76, 120.24, 120.73, 122.91, 128.79,129.32 (br.
signal), 132.75, 157.75, 158.99, 165.55, 167.58, 169.05(C=O); Elemental analysis for
C18H12N4O3S2 (396.44): Calculated/Found: 54.53/54.79 (%C), 3.05/3.11 (%H),
14.13/14.37 (%N) and 16.18/16.32 (%S).
3.1.5.7. (5Z)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-[(4-
methoxyphenyl)methylidene]-1,3-thiazolidin-4-one (6g).
Yellow solid; Yield 70.7%; mp > 300 °C; IR (cm-1, KBr): 3348 (NH, OH), 3013
1
(sp2C-H), 2814 (sp3C-H), 1712 (C=O), 1588 (C=N); H-NMR (400 MHz, δ ppm
DMSO- d6): 3.83 (s, 3H, OCH3), 6.91, 7.75 (dd, J =8.44-8.56, 4H, Ar-H), 7.15, 7.63
(dd, J = 8.19, 4H, Ar-H), 7.73 (s, 1H, =C-H), 10.12 (br. s, 1H, OH), 12.78 (br. s, 1H,
NH); Elemental analysis for C19H14N4O3S2 (410.47): Calculated/Found: 55.60/55.94
(%C), 3.44/3.70 (%H), 13.65/13.87 (%N) and 15.62/15.89 (%S).
3.1.5.8. (5Z)-5-[(3,4-dimethoxyphenyl)methylidene]-2-{[5-(4-hydroxyphenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6h).
Yield 61.3%; mp 286-288 °C; IR (cm-1, KBr): 3423 (NH, OH), 3010 (sp2C-H), 2939
1
(sp3C-H), 1692 (C=O), 1588 (C=N); H-NMR (400 MHz, δ ppm DMSO- d6): 3.825
(s, 3H, OCH3), 3.834 (s, 3H, OCH3), 6.91, 7.75 (dd, J = 8.56, 4H, Ar-H), 7.19 (d, J =
8.22, 1H, Ar-H), 7.22-7.32 (m, 3H, Ar-H), 7.73 (s, 1H, =C-H), 10.12 (br. s, 1H, OH),
12.77 (br. s, 1H, NH); Elemental analysis for C20H16N4O4S2 (440.50):
Calculated/Found: 54.53/54.8 (%C), 3.66/3.67 (%H), 12.72/12.59 (%N) and
14.56/14.32 (%S).
3.1.5.9.(5Z)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-[(3,4,5-
trimethoxyphenyl)methylidene]-1,3-thiazolidin-4-one (6i).
Yellow solid; Yield 72%; mp decompose at 280 °C; IR (cm-1, KBr): 3435 (NH, OH),
3010 (sp2C-H), 2944 (sp3C-H), 1699 (C=O), 1600 (C=N); 1H-NMR (400 MHz, δ ppm
DMSO- d6): 3.75 (s, 3H, OCH3), 3.85 (s, 6H, O(CH3)2), 6.89, 7.74 (dd, J = 8.56, 4H,
Ar-H), 6.96 (s, 2H, Ar-H), 7.70 (s, 1H, =C-H), 10.11 (br. s, 1H, OH), 12.82 (br. s, 1H,
NH); Elemental analysis for C21H18N4O5S2 (470.52): Calculated/Found: 53.61/53.89
(%C), 3.86/3.94 (%H), 11.91/12.23 (%N) and 13.63/13.78 (%S).
24

3.1.5.10.(5Z)-5-{[4-(dimethylamino)phenyl]methylidene}-2-{[5-(4-hydroxyphenyl)-
1,3,4-thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6j).
Yellow solid; Yield 79.2%; mp 294-297 °C; IR (cm-1, KBr): broad bande at 3323
(NH, OH), 3000 (sp2C-H), 2902 (sp3C-H), 1659 (C=O), 1566 (C=N).1H-NMR (400
MHz, δ ppm DMSO- d6): 3.00 ( s, 6H, N(CH3)2), 6.83,7.47 (dd, J =8.56-8.44, 4H, Ar-
H), 6.90, 7.74 (dd, J =8.44, 4H, Ar-H), 7.61 (s, 1H, =C-H), 10.15 (br. s, 1H, OH),
12.31 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm DMSO- d6): (2 CH3 signals
overlapped with DMSO signal), 116.56, 121.62, 129.29,160.55, 112.53, 132.89,
116.61, 120.41,134.43, 151.94, 159.04, 165.06, 167.65, 169.16 (C=O); Elemental
analysis for C20H17N5O2S2 (423.51): Calculated/Found: 56.72/56.89 (%C), 4.05/4.12
(%H), 16.54/16.80 (%N) and 15.14/15.23 (%S).
3.1.5.11. (5Z)-5-[(4-chlorophenyl)methylidene]-2-{[5-(4-hydroxyphenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6k).
Yellow solid; Yield 74.7%; mp >300 °C; IR (cm-1, KBr): broad band at 3376 (NH,
1
OH), 3019 (sp2C-H), 1715 (C=O), 1606 (C=N); H-NMR (400 MHz, δ ppm DMSO-
d6): 6.91 (d, J = 6.36, 2H, Ar-H), 7.55-7.85 (m, 7H, Ar-H, =C-H), 10.13 (br. s, 1H,
OH), 12.92 (br. s, 1H, NH); Elemental analysis for C18H11ClN4O2S2 (414.89):
Calculated/Found: 52.11/52.40 (%C), 2.67/2.75 (%H), 13.50/13.78 (%N) and
15.46/15.57 (%S).
3.1.5.12. (5Z)-5-[(3,4-dichlorophenyl)methylidene]-2-{[5-(4-hydroxyphenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6l).
Orange solid; Yield 64.3%; mp >300 °C; IR (cm-1, KBr): broad band at 3371 (NH,
1
OH), 3011 (sp2C-H), 1729 (C=O), 1604 (C=N). H-NMR (400 MHz, δ ppm DMSO-
d6): 6.91 (br. s, 2H, Ar-H), 7.45-8.05 (m, 6H, Ar-H, =C-H), 10.19 (br. s, 1H, OH),
13
12.00 (br. s, 1H, NH); C-NMR (100 MHz, δ ppm DMSO- d6): 116.60, 121.44,
126.88, 129.35, 129.50, 130.38, 131.94, 132.53, 132.69, 133.2, 134.37, 157.73,
160.74, 165.85, 167.09, 168.79 (C=O); Elemental analysis for C18H10Cl2N4O2S2
(449.33): Calculated/Found: 48.11/48.39 (%C), 2.24/2.31 (%H), 12.47/12.8 (%N)
and 14.27/14.56 (%S).
25

3.1.5.13. (5Z)-5-[(4-bromophenyl)methylidene]-2-{[5-(4-hydroxyphenyl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (6m).
Yield 50.1%; sublimate at 290; IR (cm-1, KBr): 3392 (NH, OH), 3109 (sp2C-H), 1705
1
(C=O), 1603 (C=N); H-NMR (400 MHz, δ ppm DMSO- d6): 6.90 (d, J =8.19, 2H,
Ar-H), 7.58 (d, J =7.46, 2H, Ar-H), 7.65-7.87 (m, 5H, Ar-H, =C-H), 10.06 (br. s, 1H,
OH), 12.88 (br. s, 1H, NH); Elemental analysis for C18H11BrN4O2S2 (459.34):
Calculated/Found: 47.07/47.26 (%C), 2.41/ 2.44 (%H), 12.2/12.46 (%N) and
13.96/14.05 (%S).
3.1.5.14. (5Z)-2-{[5-(4-Hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-[(pyridin-3-
yl)-methylidene]-1,3-thiazolidin-4-one (7).
Orange solid; Yield 78.4%; mp >300 °C; IR (cm-1, KBr): broad band at 3409 (NH,
1
OH), 3012 (sp2C-H), 1721 (C=O), 1605 (C=N); H-NMR (400 MHz, δ ppm DMSO-
d6): 6.91,7.77 (dd, J = 8.31-8.19 Hz, 4H, Ar-H), 7.36 (br. s, 1H, Ar-H), 7.82 (s, 1H,
=C-H), 8.04 (d, J = 6.97 Hz, 1H, Ar-H), 8.65 (br. s, 1H, Ar-H), 8.89 (br. s, 1H, Ar-H),
10.21 (br. s, 1H, OH), 13.05 (br. s, 1H, NH); Elemental analysis for C17H11N5O2S2
(381.43): Calculated/Found: 53.53/53.71 (%C), 2.91/2.98 (%H), 18.36/18.52 (%N)
and 16.81/16.93 (%S).
3.1.5.15.(5Z)-5-(cyclohexylmethylidene)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-
yl]imino}-1,3-thiazolidin-4-one (8).
White solid; Yield 77.6%; mp Decompose at 281 °C; IR (cm-1, KBr): 3114 (NH, OH),
3025 (sp2C-H), 2926 (sp3C-H), 1703 (C=O), 1580 (C=N); 1H-NMR (400 MHz, δ ppm
DMSO- d6): 1.15-1.42 (m, 5H, cyclohexyl), 1.57-182 (m, 5H, cyclohexyl), 2.25 (m,
1H, CH), 6.78 (d, J = 9.41 Hz, 1H, C=CH), 6.91, 7.75 (dd, J =8.68, 4H, Ar-H), 10.18
(br. s, 1H, OH), 12.58 (br. s, 1H, NH); Elemental analysis for C18H18N4O2S2 (386.49):
Calculated/Found: 55.94/56.21 (%C), 4.69/4.78 (%H), 14.50/14.63 (%N) and
16.59/16.71 (%S).
3.1.5.16.(5Z)-2-{[5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl]imino}-5-[(2E)-3-
phenylprop-2-en-1-ylidene]-1,3-thiazolidin-4-one (9).
Orange solid; Yield 65%; mp 277-280 °C; IR (cm-1, KBr): broad band at 3393 (NH,
1
OH), 3023 (sp2C-H), 2958 (sp3C-H), 1712 (C=O), 1582 (C=N); H-NMR (400 MHz,
δ ppm DMSO- d6): 6.92 (d, J =5.62, 2H, Ar-H), 7.12- 8.00 (m, 10H, Ar-H, CH=CH,
=C-H), 10.06 (br. s, 1H, OH), 12.63 (br. s, 1H, NH); Elemental analysis for
26

C20H14N4O2S2 (406.48): Calculated/Found: 59.10/59.42 (%C), 3.47/3.60 (%H),
13.78/13.96 (%N) and 15.78/16.01 (%S).
3.2. Biological screening.
3.2.1 In vitro cyclooxygenase inhibition assay.
Cyclooxygenase inhibition studies were carried out at the department of
Biochemistry, Faculty of Medicine, Cairo University, Cairo, Egypt. Synthesized
compounds were tested for their ability to inhibit ovine COX-1 and human
recombinant COX-2 using a COX inhibitor screening assay kit (Catalog No. 560131,
Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s
instructions and the deviation from the mean is < 10% of the mean value.
3.2.2. In vitro Lipoxygenase inhibition assay.
Lipoxygenase inhibition studies were carried out at the department of Biochemistry,
Faculty of Medicine, Cairo University, Cairo, Egypt. Synthesized compounds tested
for their ability to inhibit LOX enzyme using LOX inhibitor screening assay kit
(Catalog No. 760700, Cayman Chemical, Ann Arbor, MI, USA) according to the
manufacturer’s instructions and the deviation from the mean is < 10% of the mean
value.
3.2.3. In vivo Anti-inflammatory activity by carrageenan induced paw edema method.
The in vivo anti-inflammatory activity of the selected compounds (6a, 6f, 6i, 6l, 6m
and 9) were tested by carrageenan induced paw edema [26] method in rats in
comparison to Celecoxib® and Diclofenac sodium® as a reference drugs. The test is
based on the pedal inflammation in rat paws induced by subplantar injection of
carrageenan suspension (0.2 mL of 1% solution in normal saline) into the right hind
paw of the rats. Male adult albino rats (120-150 g) were divided into groups, each of
five animals. The thickness of rat paw was measured by a Vernier calliper (SMIEC,
China) before and 30 min after carrageenan injection to detect the carrageenan
induced inflammation. Each test compound at a dose of 28 µmol/Kg (dissolved in 1 %
sodium carboxymethyl cellulose solution in normal saline) was injected i.p. to
different groups of rats. Control group received a vehicle (1 % sodium carboxymethyl
cellulose solution in normal saline), while the two reference groups received
Celecoxib and Diclofenac sodium i.p. at the same dose of the tested compounds. The
difference between the thicknesses of the two paws was taken as a measure of edema.
27

The measurement was carried out at 1, 2, 3 and 4 h after injection of the test
compounds, reference drugs. The percentages of edema inhibition were calculated
according to the following equation. Two-way ANOVA followed by Dunnett’s
multiple comparisons test was performed using GraphPad Prism version 7.00 for
Windows, GraphPad Software, La Jolla California USA.
Where, VR: Average right paw thickness, VL: Average left paw thickness.
3.2.4. Ulcerogenic liability.
Compounds (6f, 6l, 6m and 9) were selected and tested for their ulcerogenic activity
according to a reported method on adult male albino rats [27]. The rats were fasted for
12 h. The test compounds were administered orally at doses of 20 mg/Kg and
indomethacin at 10 mg/kg to groups of rats each of 2 animals. After 6 h, the animals
were sacrificed, the stomachs were removed and washed with saline. Stomachs were
kept in 10 % w/v formalin solution. The surface of stomachs was examined using
light microscope.
3.2.5. Molecular modelling.
Molecular modelling study carried out using MOE 2016.09 (medicinal chemistry
department, faculty of pharmacy, Assiut university, Egypt) using human COX-2
enzyme (PDB Code 5KIR) and human 15-LOX-2 (PDB code 4NRE). The
Rofecoxib® was redocked on human COX-2 enzyme and gave root mean square
deviation (RMSD) equal to 0.843. A data base of 17 new compounds was minimized
at root mean square of 0.005 using MMFF94x force field. Compounds volume and
Log P was calculated using vsurf_D1 and Log P (o/w) descriptors, respectively.
Acknowledgment
The authors are grateful to Prof. Aml Abdel-Hafez, Faculty of Medicine, Assiut
University, Assiut, Egypt, for help in pharmacological screening. Also, authors are
greatly indebted to Medicinal Chemistry Dept., Faculty of Pharmacy, Assiut
University, in particular Dr. Ahmed Safwat for his efforts about docking studies.
28

Conflict of interest
The authors declare no conflict of interest.
Ethical conduct of research
The protocols used in the present study followed the guidelines set in “The Guide for
the Care and Use of Laboratory Animals” as found in the European Community
Guidelines (Tan 2004) and Institutional Ethical Committee Approval was obtained.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at ……………
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