Further insight into the dual COX-2 and 15-LOX anti-inflammatory activity of 1,3,4-thiadiazole-thiazolidinone hybrids: The contribution of the substituents at 5th positions is size dependent

Article abstract of DOI:10.1016/j.bioorg.2020.103657

Herin we report the design, synthesis, full characterization and biological investigation of new 15-LOX/COX dual inhibitors based on 1,3-thiazolidin-4-one (15-lipoxygenase pharmacophore) and 1,3,4-thiadiazole (COX pharmacophore) scaffolds. This series of molecular modifications is an extension of a previously reported series to further explore the structural activity relationship. Compounds 3a, 4e, 4n, 4q, 7 and 8 capable of inhibiting 15-LOX at (2.74, 4.2, 3.41, 10.21, 3.71 and 3.36 μM, respectively) and COX-2 at (0.32, 0.28, 0.28, 0.1, 0.28 and 0.27 μM, respectively). The results revealed that binding to 15-LOX and COX is sensitive to the bulkiness of the substituents at the 5 positions. 15-LOX bind better with small substituents, while COXs bind better with bulky substituents. Compounds 3a, 4r and 4q showed comparable in vivo anti-inflammatory activity to the reference drug (celecoxib). The ulcer liability test showed no sign of ulceration which ensures the safe gastric profile. Docking study was performed to explore the possible mode of interaction of the new compounds with the active site of human 15-LOX and COX-2. This study discloses some structural features for binding to 15-LOX and COX, thus pave the way to design anti-inflammatory agents with balanced dual inhibition of these enzymes.

Full text of DOI:10.1016/j.bioorg.2020.103657

Journal Pre-proofs
Further insight into the dual COX-2 and 15-LOX anti-inflammatory activity
of 1,3,4-thiadiazole-thiazolidinone hybrids: The contribution of the substitu-
ents at 5th positions is size dependent
Yasser M. Omar, Samia G. Abdel-Moty, Hajjaj H.M. Abdu-Allah
PII:
S0045-2068(19)32121-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103657
YBIOO 103657
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
11 December 2019
28 January 2020
9 February 2020
Please cite this article as: Y.M. Omar, S.G. Abdel-Moty, H.H.M. Abdu-Allah, Further insight into the dual
COX-2 and 15-LOX anti-inflammatory activity of 1,3,4-thiadiazole-thiazolidinone hybrids: The contribution of
the substituents at 5th positions is size dependent, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/
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Further insight into the dual COX-2 and 15-LOX anti-inflammatory
activity of 1,3,4-thiadiazole-thiazolidinone hybrids: The contribution
of the substituents at 5th positions is size dependent
Yasser M. Omara*, Samia G. Abdel-Motya, Hajjaj H. M. Abdu-Allaha*,
a Department Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut
University, Assiut 71526, Egypt. E-mail: yasser_omar@aun.edu.eg,
hajjaj@aun.edu.eg
1

Abstract
The current study reports the design, synthesis, full characterization and biological
investigation of new 15-LOX/COX dual inhibitors based on 1,3-thiazolidin-4-one (15-
lipoxygenase pharmacophore) and 1,3,4-thiadiazole (COX pharmacophore) scaffolds.
This series of molecular modifications is an extension of a previously reported series to
further explore the structural activity relationship. Compounds 3a, 4e, 4n, 4q, 7 and 8
capable of inhibiting 15-LOX at (2.74, 4.2, 3.41, 10.21, 3.71 and 3.36 µM, respectively)
and COX-2 at (0.32, 0.28, 0.28, 0.1, 0.28 and 0.27 µM, respectively). The results
revealed that binding to 15-LOX and COX is sensitive to the bulkiness of the
substituents at the 5 positions. 15-LOX bind better with small substituents, while COXs
bind better with bulky substituents. Compounds 3a, 4r and 4q showed comparable in
vivo anti-inflammatory activity to the reference drug (celecoxib). The ulcer liability test
showed no sign of ulceration which ensures the safe gastric profile. Docking study was
performed to explore the possible mode of interaction of the new compounds with the
active site of human 15-LOX and COX-2. This study discloses some structural features
for binding to 15-LOX and COX, thus pave the way to design anti-inflammatory agents
with balanced dual inhibition of these enzymes.
Keywords: Anti-inflammatory; 15-LOX; COX; inhibitory potency; 1,3,4-thiadiazole;
1,3-thiazolidin-4-one; structure-activity; safety
2

1. Introduction
Lipoxygenases (LOX) belongs to the family of non-heme iron-containing enzymes
which catalyze lipid peroxidation [1]. They classified according to the peroxidation site
of arachidonic acid (AA) into 5-LOX, 12-LOX and 15-LOX [1]. They have been
implicated in a number of physiological processes and in the pathogenesis of
inflammatory, hyperproliferative and neurodegenerative diseases [1]. LOX enzymes
which are well conserved among mammalian species [2], catalyze the production of
pro-inflammatory mediators from arachidonic acid (AA) like leukotrienes, eoxins and
lipoxins [3-5]. 15-Lipoxygenase (15-LOX) is one of the major metabolic pathways that
converts arachidonic acid into 15-Hydroxyeicosatetraenoic acid (15-HETE) and other
pro-inflammatory mediators [6, 7]. Evidence continues to reveal the role of 15-LOX
and its metabolites in a plethora of diseases [6]. Recent literature showed the direct
contribution of 15-HETE in airway inflammation [7], induced dysfunction of the retina
in diabetic retinopathy [8], various types of cancer [9-11], osteoarthritis [12] and
multiple sclerosis [13]. To date, zileuton which acts by chelating the iron metal located
in the active site of the LOX enzyme, is the only approved LOX inhibitor [14].
However, it has unfavourable pharmacokinetic properties and has been related with
liver toxicity [15, 16]. Moreover, the increased demand on anti-LOX therapies has
enhanced the interest in developing new safe and effective LOX inhibitors. Although
several lipoxygenase inhibitors have been reported [17, 18], it is proved to be
challenging to generate potent small inhibitors with favourable physicochemical
properties [19]. In addition to LOX enzymes, cyclooxygenases (COX-1 and COX-2)
is the other metabolic pathway of arachidonic acid [20, 21]. They convert arachidonic
acid into prostaglandin, thromboxane, and prostacyclin which play a major role in
inflammation [21]. However, the use of cyclooxygenase inhibitors (NSAIDs) shunts
the metabolism of arachidonic acid toward 15-LOX [22]. In fact, this can worsen the
patient's condition if the 15-LOX enzyme has a significant role in the pathogenesis of
the disease like in asthma [23-25]. Accordingly, the use of multitarget-directed ligands
(MTDLs) to inhibit both enzymes (15-LOX and COX) is a promising strategy in drug
therapy with maximum efficiency and minimal side effects [26-28]. On the other hand,
4-thiazolidinone and 1,3,4-thiadiazole are common scaffolds in anti-inflammatory
agents. For example, a series of compounds containing 4-thiazolidinone moiety (I and
II Fig.1) has been reported to inhibit 15-LOX enzyme with IC50 (17.7 and 52 µM,
respectively) [29, 30]. However, 1,3,4-thiadiazole ring containing compounds (III and
3

IV Fig. 1) explored significant inhibition to COX-2 enzyme (IC50 = 0.42 and 0.09 µM,
respectively) [31, 32]. Therefore, it seems interesting to investigate the anti-
inflammatory activity of 1,3,4-thiadiazole-thiazolidinone hybrid and its structural
modifications.
OH
O
S
N
OCH3
N
N
S
N
N
S
S
HN
H
O
H3CO
II
I
15-LOX IC50= 52.0 µM
15-LOX IC50= 17.7 µM
F
H
N
N
S
N
S
N
N
F
NH
NH
O
N
N
HN
S
S
N
O
S
H
N
S
N
O
HN
S
III
COX-2 IC50= 0.42 µM
IV
COX-2 IC50= 0.09 µM
Fig. 1. Some reported compounds with 15-LOX and COX-2 inhibitory activities.
In a previous study we developed a series of 1,3,4-thiadiazole-thiazolidinone hybrids
as potent dual COX-2 and 15-LOX inhibitors with good in vivo anti-inflammatory
activity by structural modifications at the 5th position of thiazolidinone moiety [33]. In
the current study, the contribution of the substituents at the 5th positions of thiadiazole
and thiazolidinone moieties to the anti-inflammatory activity was investigated.
2. Result and discussion
2.1. Design
As mentioned above, in a recent study from our group, hybrid compounds containing
both 1,4-thiazoldin-4-one (15-LOX pharmacophore) and 1,3,4-thiadiazole (COX
pharmacophore) were capable of inhibiting both 15-LOX and COX enzymes at low
micromolar concentration [33]. Also, it was found that the 15-LOX inhibitory potency
of the test compound was inversely proportional to the volume of the substituent at 5th
4

position of 1,4-thiazolidin-4-one ring, while the activity against COX-2 enzyme has a
direct relation [33]. To get further insight into the SAR of this class of 15-LOX
inhibitors, we developed compounds 3a-i (Scheme 1). This series were synthesized to
study the contribution of the substituent at the 5th position of 1,3,4-thiadiazole moiety
to the anti-inflammatory activity. Accordingly, p-hydroxyphenyl moiety in the recently
published series [33] was replaced with methyl 3a or un/substituted phenyl 3b-i (Fig.
2). The volume of the compounds was calculated using MOE software. Also, the new
compounds were tested for their 15-LOX and COX inhibitory activity. From data
shown in Table 1, it is clear that the inhibitory activities against 15-LOX are inversely
proportional to the size of the substituents at the 5th position of the 1,3,4-thiadiazole
ring. Whereas the unsubstituted phenyl and methyl group (3a and 3b) showed the
highest potencies, the 3,4,5-trimethoxyphenyl (3i) showed the lowest one. Despite the
fact that the methyl (3a) and the unsubstituted phenyl group (3b) have almost similar
potency against 15-LOX, the methyl group (3a) has better selectivity against COX-2
enzyme. For this reason, compound 3a was chosen as lead compound for further
optimization by using the substitutions on arylidene moiety (5th position of 1,4-
thiazolidin-4-one) to modify the inhibitory potency against both 15-LOX and COX
enzymes (Scheme 2). Additionally, ulcer liability test was performed to ensure the
safety of gastric profile.
Table 1.
15-LOX, COX-1, and COX-2 IC50 of the synthesized compounds 3a-i and references
drugs.
O
HN
N
N
R1
S
N
S
Compounds
15-LOX
COX-1
COX-2
Selectivity
indexb
12.44
8.80
R1
Volumec
No.
3a
3b
3c
3d
3e
3f
3g
3h
IC50a (µM) IC50 (µM) IC50 (µM)
Methyl
Phenyl
2.74
2.54
4.13
3.11
4.86
7.64
4.33
5.22
3.98
3.87
5.21
6.48
6.78
8.74
5.41
6.33
0.32
0.44
0.45
0.41
0.42
0.34
0.36
0.29
168.1
231.3
236.3
2-hydroxyphenyl
4-hydroxyphenyl
4-methylphenyl
4-bromophenyl
4-methoxyphenyl
3,4- dimethoxyphenyl
11.58
16.86
16.14
25.71
15.03
21.83
247.1
256.4
257.3
282.1
5

3i
3,4,5- trimethoxyphenyl
7.89
-
-
9.11
15.1
5.37
-
0.19
0.049
0.32
-
47.95
308.16
16.78
-
307.9
Celecoxib
Diclofenac Sod.
Zileuton
-
-
-
-
-
-
15.6
a IC50 value represents the concentration of the compound that produce 50% inhibition
of COX-1, COX-2, or 15-LOX which is the mean value of two determinations where
the deviation from the mean is < 10% of the mean value. b Selectivity index (COX-1
IC50 /COX-2 IC50). ccalculated by vol descriptor
Fig. 2. The design of new compounds with higher 15-LOX activity.
2.2. Chemistry
The synthesis of thiazolidinones 3a-i and 5-arylidenes of 3a (4a-r and 5-8) is illustrated
in Schemes 1 and 2, respectively. The synthesis of compound 3d was recently reported
by our group [33]. Firstly, 1,3,4-thiadiazole-2-amines (1a-i) were synthesized from the
appropriate carboxylic acid by reaction with thiosemicarbazide and phosphorus
oxychloride as reported [33-35]. These were chloroacetylated by refluxing with
chloroacetyl chloride in dry benzene to give 2a-i [36, 37]. Heterocyclization of 2a-i by
refluxing with ammonium thiocyanate in ethanol afforded the key thiazolidinones 3a-
i. Cyclization was confirmed by the slight shifting of the CH2 signal to lower δ 4.05-
4.10 in 1H-NMR spectra. Their IF-IR spectra showed increase of the C=O frequency to
1712-1737 cm-1 due to formation of five-membered ring. These compounds are fully
characterized, and their spectral data and the elemental analysis correlate very well with
the proposed structures. As shown in Scheme 2, Knoevenagel condensation of the 1,3-
thiazolidin-4-one active methylene of 3a with different aldehydes using acetic acid/
sodium acetate buffer afforded compounds (4a-r and 5-8). The structures of these
compounds were confirmed by shifting of the amidic carbonyl peaks to lower
6

wavenumber due to conjugation (range from 1724 to 1683 to cm-1) in the IR spectrum
1
and the disappearance of the active methylene signal at 4.08 ppm in the H-NMR.
Moreover, the disappearance of the active methylene carbon signal (at 35.90 ppm) in
13C-NMR spectrum confirms cyclization. Verification of all synthesized compounds
purity was performed using elemental analysis and their chemical structures were fully
O
O
N
N N
N
N
HN
a
b
N
R1
Cl
R1
R1
NH2
N
S
S
S
N
S
H
(3a-i)
(1a-i)
(2a-i)
R1
CH3
Ph
2-OH-Ph
4-OH-Ph
2-amino
1a
2-Chloroacetamido
Thiazolidinone
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
1b
1c
1d
1e
4-CH3-Ph
4-Br-Ph
1f
4-CH3O-Ph
3,4-(CH3O)2-Ph
3,4,5-(CH3O)3Ph
1g
1h
1i
2g
2h
2i
3g
3h
3i
characterized by IR, NMR and mass spectrometry.
Scheme 1. Synthesis of 1,3,4-thiadiazole-thiazolidinone hybrids. Reagents and
conditions: (a) ClCH2COCl, benzene, reflux, 3-5 h, 76-96% yield; (b) NH4SCN, EtOH,
reflux, 3-5 h, 83-93% yield.
O
N
HN
N
S
S
N
4a-r
R
a
O
S
O
HN
N
N
HN
b
c
N N
S
3a
S
N
S
N
5
8
e
d
O
O
N
HN
N
N
HN
N
S
S
N
S
S
N
6
7
N
S
Scheme 2. Synthesis of target compounds 4a-r and 5–8. Reagents and conditions:
Appropriate aldehyde (a: substituted benzaldehyde, b: cinnamaldehyde, c:
7

cyclohexanecarboxaldehyde, d: pyridine-3-carboxaldehyde, e: thiophene-3-
carboxaldehyde), sodium acetate/ acetic acid buffer, reflux, 24 h, 55–78% yield.
2.3. Biology
2.3.1. In vitro Lipoxygenase inhibitory assay
The synthesized compounds were tested in vitro for their inhibitory potencies against
15-LOX using 15-LOX enzyme of soybean. The IC50s are shown in Table 2.
Table 2
15-LOX, COX-1, and COX-2 IC50 of the synthesized compounds 4a-r, 5-8 and
references drugs
O
HN
N
N
R2
S
N
S
Compounds
No.
15-LOX
COX-1
IC50 (µM)
COX-2
IC50 (µM)
Selectivity
indexb
R2
IC50a (µM)
4a
4.98
6.33
9.24
6.25
5.42
11.42
6.41
7.24
12.34
5.63
9.71
7.42
7.42
5.87
8.42
8.41
11.42
15.11
8.71
5.74
4.98
4.52
15.1
0.33
0.26
0.17
0.37
0.28
0.11
0.34
0.41
0.1
15.09
24.35
54.35
16.89
19.36
103.82
18.85
17.66
123.40
14.82
74.69
23.19
23.94
20.96
20.05
42.05
114.20
151.10
24.19
22.96
17.79
16.74
308.16
4-methylphenyl
4-propylphenyl
3.11
5.24
8.33
5.47
4.21
10.24
5.23
5.87
10.94
4.62
8.42
6.17
4.65
3.41
7.36
6.98
10.21
13.11
6.55
4.62
3.71
3.36
-
4b
4c
4-isopropylphenyl
4-hydroxyphenyl
2- hydroxyphenyl
3,4-dihydroxyphenyl
4-methoxyphenyl
4d
4e
4f
4g
4h
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
4i
4j
0.38
0.13
0.32
0.31
0.28
0.42
0.2
4-hydroxy-3-methoxyphenyl
4-dimethylaminophenyl
4-chlorophenyl
3-chlorophenyl
2-chlorophenyl
2,4-dichlorophenyl
3,4-dichlorophenyl
4-bromophenyl
pentafluorophenyl
2-phenylethenyl
3-pyridyl
4k
4l
4m
4n
4o
4p
4q
0.1
4r
0.1
5
0.36
0.25
0.28
0.27
0.049
6
7
3-thienyl
Cyclohexyl
8
Celecoxib
-
8

Diclofenac
Sod.
Zileuton
-
-
-
5.37
-
0.32
-
16.78
-
15.6
a IC50 value represents the concentration of the compound that produce 50% inhibition
of COX-1, COX-2, or 15-LOX which is the mean value of two determinations where
the deviation from the mean is < 10% of the mean value. b Selectivity index (COX-1
IC50 /COX-2 IC50). The most promising results are highlighted in bold.
All the synthesized compounds revealed higher potency than zileuton. Compound 3a
devoted from arylidene group showed the highest activity with IC50 = 2.74 µM. Within
the arylidene series, compound 4a with 4-methylphenyl exhibited the highest potency
(IC50 = 3.11 µM) which is slightly lower than 3a. That means 4-methylphenyl is
tolerated. Increasing the length of the aliphatic substituent on the phenyl ring decreased
the LOX inhibitory activity (4b and 4c). Compound 4e with 2-hydroxyphenyl showed
higher potency than 4d with 4-hydroxyphenyl group. Increasing the number of
hydroxyl groups led to decrease in the inhibitory activity (4f). Similar observation
obtained by increasing the number of methoxy groups (4h, 4i, and 4j). These results
may indicate that the interaction at this position is mainly lipophilic in nature. It is worth
mentioning that compound 4j containing 3-methoxy-4-hydroxyphenyl group showed
higher potency then both 4d (with 4-hydroxyphenyl group) and 4h (with mono
methoxyphenyl group). Moreover, Compound 4n containing 2-chlorophenyl group
explored the highest potency among the halogenated phenyl series. Any attempt to
change the position (4l and 4m), increase the number of chlorine atom (4o and 4p) or
replace chlorine by bromine atom (4q) led to decrease in the 15-LOX inhibitory
potency. Additionally, Compounds (7 and 8) containing (3-thienyl and cyclohexyl
group, respectively) exhibited similar activity to 4a. Finally, the insertion of an ethenyl
spacer (5) or the replacement of phenyl group with 3-pyridyl group (6) decreased the
inhibitory activity. The results clearly show that small hydrophobic group, such as 4-
methylphenyl, 3-thienyl or cyclohexyl, at the 5th position of thiazolidinone is required
for good activity.
2.3.2. In vitro cyclooxygenase inhibitory assay
All the tested compounds showed inhibitory potencies against COX-2 ranging from 0.1
to 0.42 µM and selectivity index ranging from 12.44 to 151.10 (Table 2). The most
selective compounds were 4r, 4i, 4q, and 4f with selectivity index of 151.10, 123.40,
114.20 and 103.82, respectively. Also, these compounds showed higher potency than
9

Diclofenac sodium. Moreover, compound 3a that lack of arylidene moiety showed the
lowest potency and selectivity among all synthesized compounds. The structure-
activity relationship of compound 4i showed that, decreasing the number of methoxy
group resulted in marked decrease in the potency and selectivity (4g and 4h). Similar
observation obtained when decreasing the number of hydroxy groups as for compound
4f. Additionally, replacement of the bromine atom in compound 4q by chlorine atom
results in marked decrease in potency and selectivity (4l). Therefore, bulky substituent
(4-bromo or trimethoxyphenyl) is required at the 5th position of thiazolidinone moiety
for good activity and selectivity. Compounds with phenyl (4a), 3-thienyl (7) or
cyclohexyl (8) are potent dual inhibitors of 15-LOX and COX-2 due to presence of
moderately bulky substituents. These compounds were also less selective than
celecoxib. This property, dual balanced inhibition of COX-2 and 15-LOX, is the basis
for developing safe anti-inflammatory agents [27, 28].
2.3.3. In vivo anti-inflammatory activity
Carrageenan-induced paw edema in rats was chosen as an animal model to study the in
vivo anti-inflammatory activity of the selected compounds (3a, 4f, 4i, 4q, and 4r)
against celecoxib as a reference drug (Table 3). The synthesized compounds and
celecoxib were administered i.p at a dose of 28 µg/Kg. All the tested compounds
reached its maximal activity after 3 h and showed comparable activity (non-significant
statistical difference) to the reference drug (Table 3). Compounds (3a, 4q and 4r)
exhibited the highest percent of inhibition among the tested compounds.
Table 3
In vivo anti-inflammatory in rats using celecoxib as a reference drug.
% Inhibition of Edema ± SE
Compounds
No.
1h
2 h
3 h
4 h
Celecoxib
24.1 ± 2.5 c
17.2 ± 4.2 a
21.8 ± 5.6
8.0 ± 3.6
17.2 ± 5.6
21.4 ± 8.4 a
44.0 ± 3.4d
36.0 ± 2.5 d
52.0 ± 5.8 d
38.0 ± 3.7 d
42.0 ± 6.6 d
48.0 ± 7.3 d
56.1 ± 7.1 d
49.5 ± 4.8 d
45.8 ± 3.5 d
42.1 ± 1.9 d
60.7 ± 3.5 d
57.9 ± 4.8 d
40.0 ± 3.1 d
44.8 ± 7.0 d
27.6 ± 2.3 d
27.6 ± 2.3 d
27.6 ± 2.3 d
25.7 ± 1.9 d
3a
4f
4i
4q
4r
a P < 0.05, b P < 0.01, c P < 0.001, d P < 0.0001.
The calculated LogP (Table 4) reflects the high lipophilicity of compounds 4q and 4r
which may contribute to their high in vivo anti-inflammatory activity.
10

2.3.4. Ulcerogenic liability
Ulcerogenic liability test was performed to ensure the safety of gastric profile for the
most active compounds (3a, 4i and 4r) in the in vivo anti-inflammatory test. Double the
dose (20 mg/kg) of the in vivo test was chosen to measure the tolerability of the stomach
at a higher dose. Also, indomethacin at 10 mg/kg was selected as a positive control.
After oral administration of the tested compounds and indomethacin, gastric mucosa
was examined using light microscope. In compare to the negative control, the gastric
mucosa following the administration of the selected compounds showed no sign of
ulceration edema or desquamation of the epithelial cells in contrast to indomethacin
which showed edema, dilatation of the blood vessel and detached of the epithelial cells
(Fig. 3 and 4).
Fig. 3. Gastric mucosa at 4x magnification and 200µm scale bare of the negative control (C),
tested compounds (3a, 4i and 4r) and indomethacin (I). The arrows in the indomethacin Fig
clarify the edema and detached epithelial cells.
11

Fig. 4. Gastric mucosa at 10x magnification and 100 µm scale bare of the negative control (C),
tested compounds (3a, 4i and 4r) and indomethacin (I1 and I2). The arrows in the indomethacin
figures clarify the edema and detached epithelial cells.
2.4. Docking and molecular modelling studies
Descriptors and docking scores were calculated using MOE 2019 software to explain
the activity of the synthesized compounds (Table 3). Also, Human 15-LOX enzyme
with PDB code (4NRE) and human COX-2 enzyme with PDB code (5KIR) were
chosen to perform the docking study.
Table 4. Descriptors and docking scores of the synthesized compounds.
Compound No. Volume a
LogP b
1.08
3.93
4.84
4.77
3.32
3.32
3.05
3.58
3.33
M.Wt
214.27
316.40
344.45
344.45
318.37
318.37
334.37
332.40
362.43
ΔG COX-2 c ΔG 15-LOX c
3a
4a
4b
4c
4d
4e
4f
168.3
278.4
313.0
312.4
267.4
265.0
272.8
284.5
311.5
-5.89
-7.58
-8.46
-8.4
-5.87
-7.37
-6.72
-7.52
-7.11
-7.15
-7.23
-7.63
-6.18
-7.32
-7.46
-7.58
-7.81
-8.38
4g
4h
12

4i
340.4
292.8
310.4
275.4
276.9
276.1
289.6
291.1
285.4
275.4
290.4
253.9
249.0
278.1
-
3.07
3.31
3.54
4.22
4.26
4.22
4.85
4.85
4.43
4.38
4.27
2.39
3.28
3.64
-
392.45
348.40
345.44
336.82
336.82
336.82
371.26
371.26
381.27
392.33
328.41
303.36
308.40
308.42
-
-8.78
-7.52
-8.28
-7.56
-7.06
-7.05
-7.37
-7.78
-7.78
-7.32
-7.6
-7.46
-7.87
-7.67
-6.98
-7.28
-7.25
-7.14
-7.54
-7.67
-7.15
-7.50
-6.98
-6.48
-7.19
-8-49
4j
4k
4l
4m
4n
4o
4p
4q
4r
5
6
-7.23
-7.1
7
8
-7.49
-
Rofecoxib
a: calculated by vol descriptor b: calculated by LogP (o/w) descriptor c: ΔG (Kcal/mol)
2.4.1. Docking study on human 15-LOX
The calculated volume and molecular weight of the synthesized compounds showed a
rough inverse correlation with their IC50. This may explain the high potency of
compound 3a among the synthesized compound and match with the proposed design.
Docking study was performed to explore the possible interaction between the
synthesized compounds and the active site of the 15-LOX enzyme which consists of
iron metal complexed with four amino acids (His 373, His 378, His 553 and Ile 676)
(Fig. 5).
13

Fig. 5. The active site of 15-LOX active site.
Docking score ranging from -5.89 to -8.78. All the compounds bearing arylidene moiety
(4-8) showed a pi-pi interaction between the 1,3thiazolidin-4-one ring (the 15-LOX
pharmacophore) and His 378. Moreover, the sulfur atom of the 1,3-thiazolidin-4-one
ring forms a hydrogen bond with the C-terminal amino acid Ile 676. For example,
compounds (4a and 8) explore a pi-pi interaction with His 373 at a distance of (3.80,
3.79 Å) and formation of a hydrogen bond with Ile 676 at a distance of (3.17, 3.39 Å),
respectively (Fig. 6 and 7). In addition to the previous interaction, compound 4a showed
a pi- hydrogen interaction with Glu 369 (at 4.60 Å) which may explain its high potency
(IC50 = 3.11 µM) (Fig. 6).
14

Fig. 6. Interaction between 4a and 15-LOX active site.
Fig. 7. Interaction between 8 and 15-LOX active site.
15

Compound 3a which lacks arylidene moiety showed different types of interaction with
the 15-LOX active site (Fig. 8). Besides the formation of hydrogen bonding between
the amidic NH of the 1,3-thiazolidin-4-one ring (at 2.93 Å), The carbonyl group
interacts with the iron metal to form a metal complex (at 2.48 Å). In fact, these types
of interactions give a reasonable explanation to the high activity of compound 3a (IC50
= 2.74 µM) which consider the highest among the newly synthesized compounds.
Fig. 8. Interaction between 3a and 15-LOX active site.
2.4.2. Docking study on human COX-2
Despite the reduction of the new synthesized compounds volume and molecular weight,
they still maintain relative selectivity towards COX-2 enzyme as predicted from the
design. The docking scores (ranging from -5.87 to -7.87) reflect the good binding of
the newly synthesized compounds with the COX-2 enzyme. All the new compounds
explore a hydrogen bonding between the 1,3,4-thiadiazole ring (COX-2
pharmacophore) and the amino acid Arg 513 (except compounds 4c, 4l, and 4q which
bind with Arg 120). For example, compounds 4i and 4r bind with Arg 513 at a distance
of (2.88 and 3.14 Å) respectively (Fig. 9 and 10). Also, compound 4i shows additional
binding with Gln192.
16

Fig. 9. Interaction between 4i and COX-2 enzyme.
Fig. 10. Interaction between 4r and COX-2 enzyme.
17

Compound 4q showed a hydrogen bonding with Arg 120 instead of Arg 513 and a pi
hydrogen interaction with Tyr 355 (Fig. 11).
Fig. 11. Interaction between 4q and COX-2 enzyme.
3. Conclusion
In this study, we gained further insight into the SAR of thiadiazole-thiazolidinone
hybrids as dual inhibitors of 15-LOX and COX-2. For good activity against 15-LOX,
small hydrophobic group (methyl) should be introduced at the 5th positions of
thiadiazole moiety, while the 5th position of thiazolidinone moiety should be
unsubstituted (3a) or substituted by small hydrophobic groups, such as 4-methylphenyl
(4a), 3-thienyl (7) or cyclohexyl (8). In contrary introducing a bulky hydrophobic
substituent at that position improved the binding for COX-2 as shown by 3,4,5-
trimethoxyphenyl (4i), 4-bromophenyl (4q) and pentafluorophenyl (4r). The balance
between 15-LOX binding and COX-2 binding was achieved by introducing methyl
group at the 5th position of thiadizole moiety and phenyl, 3-thienyl or cyclohexyl at the
5th position of thiazolidinone moiety. Therefore, compounds 4a, 7 and 8 are balanced
and potent dual inhibitors of 15-LOX and COX-2. The in vivo anti-inflammatory
evaluation revealed that compounds 3a, 4q and 4r are equipotent to the reference drug
18

celecoxib and exhibited good gastric safety profile. Molecular docking studies
disclosed important binding modes of these compounds with COX-2 and 15-LOX.
4. Experimental
4.1. Chemistry
Commercial grade solvents and reagents were used to synthesize the specified
compounds without further treatment except for benzene which dried using sodium
metal. Cole-Parmer -Electrothermal [model IA9100, UK] melting point apparatus was
used to determined melting points and they were uncorrected. Pre-coated TLC sheets
(kieselgel 0.25 mm, 60G F254, Merck, Germany) were used to monitor the progress of
reactions. estimate the reaction time. Spots were visualized by an ultraviolet lamp at
254 nm wavelength (Spectroline, model CM-10, USA). Thermo Scientific Nicolet IS10
FT IR spectrometer was used to generate IR spectrum (Thermo Fischer Scientific,
USA) at Faculty of Science, Assiut University, Assiut, Egypt. 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 (1a, 3b, 3e, 3f and 3g) were performed on a Varian EM-390 NMR
spectrometer (60 MHz, Varian, CA, USA) at Faculty of Pharmacy, Assiut University,
Assiut, Egypt and compounds (2a, 3c, 3h and 3i) 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 performed on Direct Probe Controller
Inlet Part TO Single Quadrupole 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, Cairo, Egypt.
Elemental microanalyses were carried out on elemental analyzer model flash 2000
Thermo-fisher at the Regional Center for Mycology and Biotechnology (RCMB),
Faculty of Science, Al-Azhar University, Cairo, Egypt.
4.1.1. Synthesis of 2-amino-5-methyl-1,3,4-thiadiazole (1a)
A mixture of acetic acid (1 mL, 16.6 mmol,), thiosemicarbazide (1.5 g, 16.6 mmol, 1
equv.) and phosphorus oxychloride (7.5 mL, 80.2 mmol, 8.2 equv.) were refluxed for
2 h. The reaction mixture was cooled to room temperature, ice water (25 mL) was
added, and the reaction was refluxed for further 4 h. After cooling the reaction mixture
was alkalinized by conc ammonium hydroxide solution (20%), the formed precipitate
19

was filtered off and washed with cold water. The product used for the next step without
-1
o
further purification. Brownish solid; Yield (52%); mp 224-225 C; IR (cm , KBr):
1
3259, 3069 (NH2), 1640 (C=N); H-NMR (60 MHz, δ ppm DMSO-d6): 2.6 (s, 3H,
CH3), 7.1 (br. s, 2H, NH2), EI-MS [m/z (%)]: 115.29 (M+, 48.11%), 45.98 (100% base
peak); Elemental analysis for C3H5N3S (115.16): Calculated/Found: 31.29/31.53 (%C),
4.38/4.61(%H), 36.49/36.70 (%N) and 27.84/27.69 (%S).
4.1.2. Synthesis of 2-chloro-N-(5-methyl-1,3,4-thiadiazol-2-yl)acetamide (2a)
Compound 1a (1.15 g, 10 mmol) was stirred in dry benzene (15 mL). Chloroacetyl
chloride (1.3 mL, 16 mmol, 1.6 equv.) in dry benzene (10 mL) was added in a dropwise
manner. The reaction mixture was refluxed for 3-5 h. The reaction was monitored by
TLC using ethyl acetate: hexane (1:1 v/v) system. The products were filtered while hot
and washed with a small amount of cold methanol. The The reaction mixture was
filtered while hot, washed with a small amount of cold methanol and recrystallized from
dioxane-water to give white crystalline solid. Yield (89.5%); mp decompose at 236 oC;
1
IR (cm-1, KBr): 3434(NH), 1698 (C=O), 1580 (C=N); H-NMR (90 MHz, δ ppm
DMSO-d6): 2.6 (s, 3H, CH3), 4.4 (s, 2H, CH2), 12.9 (br. s, 1H, NH), Elemental analysis
for C5H6ClN3OS (191.64): Calculated/Found: 31.34/31.49 (%C), 3.16/3.28 (%H),
21.93/21.87 (%N) and 16.73/16.94 (%S).
4.1.3. General procedure for heterocyclization
Compound (2a-i) (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 reaction mixture
was filtered while hot, the collected product was washed with cooled water and
recrystallized from dioxane-water.
4.1.3.1. Synthesis of 2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one
(3a)
Brown crystals. Yield (91%); mp 269-271 oC; IR (cm-1, KBr): 3429 (NH), 1734 (C=O),
1
1556 (C=N); H-NMR (400 MHz, δ ppm DMSO-d6): 2.64 (s, 3H, CH3), 4.08 (s, 2H,
13
CH2), 12.24 (br. s, 1H, NH); C-NMR (100 MHz, δ ppm DMSO-d6): 16.14 (CH3),
35.90 (CH2), 162.49, 165.41, 170.55, 174.33(C=O); EI-MS [m/z (%)] 215.96 (M++2,
2.42%), 213.99 (M+,21.93%), 140.01 (80.25%), 141.04 (93.40%), 58.95 (100.00);
Elemental analysis for C6H6N4OS2 (214.27): Calculated/Found: 33.63/33.39 (%C),
2.82/2.48 (%H), 26.15/26.12 (%N) and 29.93/29.79 (%S).
20

4.1.3.2. 2-[(5-Phenyl -1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one (3b)
White crystalline solid; Yield 92%; mp 280-282 °C (reported mp 128 – 130 °C [32]).
1
IR (cm-1, KBr): 3100 (NH), 1726 (C=O), 1566 (C=N); H-NMR (60 MHz, δ ppm
DMSO-d6): 4.1 (s, 2H, CH2), 7.3-8.2 (m, 5H, Ar-H); Elemental analysis for
C11H8N4OS2 (276.34): Calculated/Found: 47.81/48.12 (%C), 2.92/2.96 (%H),
20.27/20.54 (%N) and 23.21/23.37 (%S).
4.1.3.3. 2-[(5-(2-Hydroxyphenyl) -1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one
(3c)
White crystalline solid; Yield 85%; mp decompose at 263 °C (reported mp 160 – 161
1
°C [32]); IR (cm-1, KBr): 3438 (NH, OH), 1729 (C=O), 1564 (C=N); H-NMR (90
MHz, δ ppm DMSO-d6): 4.1 (s, 2H, CH2), 6.8-7.15 (m, 2H, Ar-H),7.2-7.5 (m, 1H, Ar-
H), 8.1 (d, J = 9, 1H, Ar-H) off set (br. s, 1H, OH), off set (br. s, 1H, NH); Elemental
analysis for C11H8N4O2S2 (292.34): Calculated/Found: 45.19/45.00 (%C), 2.76/2.73
(%H), 19.17/19.35 (%N) and 21.94/21.85 (%S).
4.1.3.4 2-[(5-(4-Methylphenyl) -1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one
(3e)
White crystalline solid; Yield 92%; mp decompose at 260 °C (reported mp 122 – 123
°C [32]); IR (cm-1, KBr): 3100 (NH), 1740 (C=O), 1589 (C=N); 1H-NMR (60 MHz, δ
ppm DMSO-d6): 2.3 (s, 3H, CH3), 4.05 (s, 2H, CH2), 7.33 (d, J = 8.4, 2H, Ar-H); ,7.80
(d, J = 8.4, 2H, Ar-H); elemental analysis for C12H10N4OS2 (290.36):
Calculated/Found: 49.64/49.51 (%C), 3.47/3.75 (%H), 19.30/19.53 (%N) and
22.09/21.78 (%S).
4.1.3.5. 2-[(5-(4-Bromophenyl) -1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one
(3f)
White crystalline solid; Yield 93%; mp 295-296 °C (reported mp 135 – 136 °C [32]);
1
IR (cm-1, KBr): 3437 (NH), 1722 (C=O), 1548 (C=N); H-NMR (60 MHz, δ ppm
DMSO-d6): 4.4 (s, 2H, CH2), 7.55-8.1 (m, 4H, Ar-H); Elemental analysis for
C11H7BrN4OS2 (355.23): Calculated/Found: 37.19/37.54 (%C), 1.99/1.82 (%H),
15.77/15.73 (%N) and 18.05/17.84 (%S).
4.1.3.6 2-[(5-(4-Methoxyphenyl) -1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one
(3g)
White crystalline solid; Yield 92.5%; mp decompose at 268 °C (reported mp 108 – 110
°C [32]); IR (cm-1, KBr): 3100 (NH), 1719 (C=O), 1589 (C=N); 1H-NMR (60 MHz, δ
21

ppm DMSO-d6): 3.9 (s, 3H, O-CH3), 4.1 (s, 2H, CH2), 7.10 (d, J = 9, 2H, Ar-H) ,7.88
(d, J = 9, 2H, Ar-H);; Elemental analysis for C12H10N4O2S2 (306.36):
Calculated/Found: 47.04/47.19 (%C), 3.29/3.35 (%H), 18.29/18.48 (%N) and
20.93/21.04 (%S).
4.1.3.7. 2-[(5-(3,4-Dimethoxyphenyl) -1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-4-
one (3h)
White crystalline solid; Yield 91%; mp decompose at 258 °C; IR (cm-1, KBr): 3435
(NH), 1712 (C=O), 1571 (C=N); 1H-NMR (90 MHz, δ ppm DMSO-d6): 3.8 (s, 6H, 2
O-CH3), 4.05 (s, 2H, CH2), 6.9-7.5 (m, 3H, Ar-H), 12.4 (br. s, 1H, NH); Elemental
analysis for C13H12N4O3S2 (336.39): Calculated/Found: 46.42/46.54 (%C), 3.60/3.78
(%H), 16.66/16.86 (%N) and 19.06/19.11 (%S).
4.1.3.8. 2-[(5-(3,4,5-Trimethoxyphenyl)-1,3,4-thiadiazol-2-yl)imino]-1,3-thiazolidin-
4-one (3i)
White crystalline solid; Yield 92.6%; mp decompose at 280 °C; IR (cm-1, KBr): 3446
(NH), 1737 (C=O), 1583 (C=N); 1H-NMR (90 MHz, δ ppm DMSO-d6): 3.7 (s, 3H, O-
CH3), 3.8 (s, 6H, 2 O-CH3), 4.05 (s, 2H, CH2), 7.05 (s, 2H, Ar-H), 12.5 (br. s, 1H, NH);
Elemental analysis for C14H14N4O4S2 (366.42): Calculated/Found: 45.89/45.61 (%C),
3.85/4.04 (%H), 15.29/14.99 (%N) and 17.5/17.46 (%S).
4.1.4. General procedure for Knoevenagel condensation of an appropriate aldehyde
with compound (3a) to give compounds (4a-r and 6-8)
Compound (3a) (0.21 g, 1 mmol) and an aldehyde derivative (1.5 mmol, 1.5 equv.)
were stirred in glacial acetic acid (8 mL). Sodium acetate (0.16 g, 2 mmol, 2 equv.) was
added and the reaction mixture was heated under reflux for 24 h. The precipitate was
filtered off while hot from the reaction mixture (except compounds 4r, 6 and 7 were
collected after adding few drops of distilled water and cooling).
4.1.4.1. (5Z)-5-[(4-methylphenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4a)
Yellow solid; Yield (69.5%); mp > 300 oC; IR (cm-1, KBr): 3412 (NH), 2821 (sp3C-
H), 1712 (C=O), 1568 (C=N). 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.37 (s, 3H,
CH3), 2.67 (s, 3H, CH3), ), 7.38 (d, J = 7.7 Hz, 2H, Ar-H), 7.54 (d, J = 7.7 Hz, 2H,
Ar-H), 7.72 (s, 1H, C=CH), 12.78 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm
DMSO-d6): 16.30 (CH3), 21.61, 123.23, 130.5, 130.73, 130.88, 133.18, 141.27,
22

158.37, 163.28, 167.53, 170.38 (C=O); Elemental analysis for C14H12N4OS2 (316.40):
Calculated/Found: 53.14/53.45 (%C), 3.82/3.97 (%H), 17.71/17.82 (%N) and
20.27/20.35 (%S).
4.1.4.2. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-[(4-propylphenyl)-methyli-
dene]-1,3-thiazolidin-4-one (4b)
Yellow solid; Yield (77%); mp decompose at 292 oC; IR (cm-1, KBr): 3415 (NH), 2951
(sp3C-H), 1716 (C=O), 1565 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 0.92 (t, J
= 7.09 Hz, 3H, CH3), 1.63 (m, 2H, CH2), 2.63 (t, J = 7.46 Hz, 2H, CH2), 2.67 (s, 3H,
CH3), 7.40 (d, J = 7.2 Hz, 2H, Ar-H), 7.57 (d, J = 7.2 Hz, 2H, Ar-H), , 75 (br. s, 1H,
NH); Elemental analysis for C16H16N4OS2 (344.45): Calculated/ Found: 55.79/55.96
(%C), 4.68/4.80 (%H), 16.27/16.34 (%N) and 18.62/18.84 (%S).
4.1.4.3. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-{[4-(propan-2-yl)phenyl]-
methylidene}-1,3-thiazolidin-4-one (4c)
Yellow solid; Yield (78%); mp 286-288 oC; IR (cm-1, KBr): 3435 (NH), 2958 (sp3C-H),
1
1715 (C=O), 1573 (C=N); H-NMR (400 MHz, δ ppm DMSO-d6): 1.24 (d, J = 6.72
Hz, 6H, 2CH3), 2.68 (s, 3H, CH3), 2.92-2.99 (m, 1H, CH), 7.46 (d, J = 7.2 Hz, 2H, Ar-
H), 7.59 (d, J = 7.2 Hz, 2H, Ar-H), 7.7 (s, 1H, C=CH), 12.75 (br. s, 1H, NH); Elemental
analysis for C16H16N4OS2 (344.45): Calculated/Found: 55.79/56.04 (%C), 4.68/4.89
(%H), 16.27/16.38 (%N) and 18.62/18.91 (%S).
4.1.4.4. (5Z)-5-[(4-hydroxyphenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4d)
Yellow solid; Yield 67%; mp > 300 oC; IR (cm-1, KBr): broad band at 3385 (NH, OH),
2825 (sp3C-H), 1702 (C=O), 1583 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67
(s, 3H, CH3), 6.97 (d, J = 8.4 Hz, 2H, Ar-H), 7.53 (d, J = 8.5 Hz, 2H, Ar-H), 7.69 (s,
1H, C=CH), 10.36 (s, 1H, OH), 12.74 (br. s, 1H, NH); EI-MS [m/z (%)]: 317.91 (M+,
57.85%), 149.92 (100% base peak); Elemental analysis for C13H10N4O2S2 (318.37):
Calculated/Found: 49.04/49.38 (%C), 3.17/3.19 (%H), 17.60/17.68 (%N) and
20.14/20.41 (%S).
4.1.4.5. (5Z)-5-[(2-hydroxyphenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4e)
-1
o
Yellow solid; Yield 56.5%; mp Decompose at 258 C; IR (cm , KBr): broad band at
3418 (NH, OH), 2825 (sp3C-H), 1707 (C=O), 1588 (C=N); 1H-NMR (400 MHz, δ ppm
23

DMSO-d6): 2.66 (s, 3H, CH3), 6.92-7.07 (m, 2H, Ar-H), 7.27-7.38 (m, 1H, Ar-H), 7.45
(d, J = 7.4 Hz, 1H, Ar-H), 8.01 (s, 1H, C=CH), 10.51 (s, 1H, OH), 12.72 (br. s, 1H,
NH); Elemental analysis for C13H10N4O2S2 (318.37): Calculated/Found: 49.04/49.31
(%C), 3.17/3.44 (%H), 17.60/17.89 (%N) and 20.14/20.33 (%S).
4.1.4.6. (5Z)-5-[(3,4-dihydroxyphenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4f)
Brown solid; Yield 63%; mp >300 oC; IR (cm-1, KBr): broad band at 3492 (NH, OH),
2964 (sp3C-H), 1702 (C=O), 1586 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67
(s, 3H, CH3), 6.91(d, J = 8.07 Hz, 1H, Ar-H), 7.03 (d, J = 7.82 Hz, 1H, Ar-H), 7.09 (s,
1H, Ar-H) 7.60 (s, 1H, C=CH), 9.57 (s, 1H, OH), 9.78 (s, 1H, OH), 12.66 (br. s, 1H,
NH); Elemental analysis for C13H10N4O3S2 (334.37): Calculated/Found: 46.70/46.52
(%C), 3.01/3.07 (%H), 16.76/16.54 (%N) and 19.18/19.42 (%S).
4.1.4.7. (5Z)-5-[(4-methoxyphenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4g)
Yellow solid; Yield 60%.; mp 290-292 oC; IR (cm-1, KBr): 3400 (NH), 2817 (sp3C-H),
1710 (C=O), 1564, 1592 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.71 (s, 3H,
CH3), 3.89 (s, 3H, OCH3), 7.18 (d, J = 8.4 Hz, 2H, Ar-H), 7.66 (d, J = 8.5 Hz, 2H, Ar-
13
H), 7.76 (s, 1H, C=CH), 12.70 (br. s, 1H, NH); C-NMR (100 MHz, δ ppm DMSO-
d6): 16.29 (CH3), 55.97 (OCH3), 115.49, 121.24, 126.11, 132.75, 133.17, 158.46,
161.53, 163.14, 167.59, 170.41(C=O); EI-MS [m/z (%)]: 332.48 (M+, 18.91%), 123.89
(100% base peak); Elemental analysis for C14H12N4O2S2 (332.40): Calculated/Found:
50.59/50.82 (%C), 3.64/3.78 (%H), 16.86/17.01 (%N) and 19.29/19.38 (%S).
4.1.4.8. (5Z)-5-[(3,4-dimethoxyphenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4h)
Yellow solid; Yield 55%; mp 274-276 oC; IR (cm-1, KBr): 3432 (NH), 2813 (sp3C-H),
1697 (C=O), 1568, 1580,1593 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.71 (s,
3H, CH3), 3.88 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 7.24 (d, J = 8 Hz, 1H, Ar-H),7.29,
7.32 (br. s, 2H, Ar-H), 7.78 (s, 1H, C=CH), 12.71 (br. s, 1H, NH); Elemental analysis
for C15H14N4O3S2 (362.43): Calculated/Found: 49.71/49.97 (%C), 3.89/3.94 (%H),
15.46/15.75 (%N) and 17.69/17.52 (%S).
24

4.1.4.9. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-[(3,4,5-trimethoxyphenyl)
methylidene]-1,3-thiazolidin-4-one (4i)
-1
3
o
Yellow solid; Yield 68.8%; mp 233-236 C; IR (cm , KBr): 3435 (NH), 2836 (sp C-
1
H), 1694 (C=O), 1578 (C=N); H-NMR (400 MHz, δ ppm DMSO-d6): 2.70 (s, 3H,
CH3), 3.81 (s, 3H, OCH3), 3.90 (s, 6H, 2 (OCH3)), 7.01 (s, 2H, Ar-H), 7.76 (s, 1H,
C=CH), 12.73 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm DMSO-d6): 16.23 (CH3),
56.57, 60.71, 108.43, 123.54, 129.18, 133.4, 140.09, 153.66, 158.27, 163.21, 167.35,
170.31 (C=O); Elemental analysis for C16H16N4O4S2 (392.45): Calculated/Found:
48.97/49.13 (%C), 4.11/4.15 (%H), 14.28/14.54 (%N) and 16.34/16.49 (%S).
4.1.4.10. (5Z)-5-[(4-hydroxy-3-methoxyphenyl)methylidene]-2-[(5-methyl-1,3,4-
thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one (4j)
Yellow solid; Yield 73%; mp Decompose at 281 oC; IR (cm-1, KBr): 3512, 3400 (NH,
1
OH), 2825 (sp3C-H), 1706 (C=O), 1575 (C=N); H-NMR (400 MHz, δ ppm DMSO-
d6): 2.67 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 7.00 (d, J = 8.31, 1H, Ar-H), 7.14 (d, J =
8.68, 1H, Ar-H), 7.28 (br. s, 1H, Ar-H), 7.71 (s, 1H, C=CH), 10.02 (s, 1H, OH), 12.75
(br. s, 1H, NH); Elemental analysis for C14H12N4O3S2 (348.40): Calculated/Found:
48.26/48.42 (%C), 3.47/3.43 (%H), 16.08/16.28 (%N) and 18.41/18.63 (%S).
4.1.4.11. (5Z)-5-{[4-(dimethylamino)phenyl]methylidene}-2-[(5-methyl-1,3,4-
thiadiazol-2-yl)imino]-1,3-thiazolidin-4-one (4k)
-1
3
o
Reddish solid; Yield 70%; mp > 300 C; IR (cm , KBr): 3100 (NH), 2822 (sp C-H),
1683 (C=O), 1577 (C=N);1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67 (s, 3H, CH3),
3.03 (s, 6H, N(CH3)2), 6.88 (d, J = 8.7 Hz, 2H, Ar-H), 7.51 (d, J = 8.7 Hz, 2H, Ar-H),
7.66 (s, 1H, C=CH), 12.63 (br. s, 1H, NH); EI-MS [m/z (%)]: 344.89 (M+, 17.79%),
177.19 (100% base peak); Elemental analysis for C15H15N5OS2 (345.44):
Calculated/Found: 52.15/52.38 (%C), 4.38/4.44 (%H), 20.27/20.48 (%N) and
18.56/18.70 (%S).
4.1.4.12. (5Z)-5-[(4-chlorophenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4l)
Yellow solid; Yield 72.5%; mp > 300 oC; IR (cm-1, KBr): 3417 (NH), 2901 (sp3C-H),
1711 (C=O), 1568 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67 (s, 3H, CH3),
7.60 – 7.75 (m, , 4H, Ar-H), 7.78 (s, 1H, C=CH), 12.93 (br. s, 1H, NH); Elemental
25

analysis for C13H9ClN4OS2 (336.82): Calculated/Found: 46.36/46.09 (%C), 2.69/2.88
(%H), 16.63/16.97 (%N) and 19.04/19.42 (%S).
4.1.4.13. (5Z)-5-[(3-chlorophenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4m)
Yellow solid. Yield 75%. mp 263-265 oC; IR (cm-1, KBr): 3435 (NH), 2899 (sp3C-H),
1711 (C=O), 1587 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67 (s, 3H, CH3),
7.60 – 7.75 (m, 5H, C=CH, Ar-H), 12.87 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm
DMSO-d6) 16.24 (CH3), 126.37, 128.42, 130.37 (br. peak), 131.35, 131.61, 134.44,
135.88, 157.82, 163.43, 167.18, 170.24(C=O). Elemental analysis for C13H9ClN4OS2
(336.82): Calculated/Found: 46.36/46.50 (%C), 2.69/2.77 (%H), 16.63/16.89 (%N) and
19.04/19.27 (%S).
4.1.4.14. (5Z)-5-[(2-(chlorophenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4n)
-1
3
o
Yellow solid; Yield 74%; mp > 300 C; IR (cm , KBr): 3435 (NH), 2989 (sp C-H),
1710 (C=O), 1574 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67 (s, 3H, CH3),
7.45 – 7.75 (m, 4H, Ar-H), 7.92 (s, 1H, C=CH) 12.96 (br. s, 1H, NH); Elemental
analysis for C13H9ClN4OS2 (336.82): Calculated/Found: 46.36/46.54 (%C), 2.69/2.62
(%H), 16.63/16.75 (%N) and 19.04/19.38 (%S).
4.1.4.15. (5Z)-5-[(2,4-(dichlorophenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4o)
Yellow solid; Yield 68%; mp 262-264 oC; IR (cm-1, KBr): 3412 (NH, OH), 2918 (sp3C-
1
H), 1717 (C=O), 1584 (C=N); H-NMR (400 MHz, δ ppm DMSO-d6): 2.66 (s, 3H,
CH3), 7.62-7.93 (M, 4H, Ar-H, C=CH), 13.03 (br. s, 1H, NH); Elemental analysis for
C13H8Cl2N4OS2 (371.26): Calculated/Found: 42.06/42.19 (%C), 2.17/2.2 (%H),
15.09/15.34 (%N) and 17.27/17.38 (%S).
4.1.4.16. (5Z)-5-[(3,4-dichlorophenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4p)
Yellow solid. Yield 77.5%; mp > 300 oC; IR (cm-1, KBr): 3412 (NH), 2895 (sp3C-H),
1713 (C=O), 1586 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.67 (s, 3H, CH3),
7.61 (d, J = 8.3 Hz, 1H, Ar-H), 7.75 (s, 1H, C=CH), 7.84 (d, J = 8.07 Hz, 1H, Ar-H),
7.92 (s, 1H, Ar-H), 12.88 (br. s, 1H, NH); Elemental analysis for C13H8Cl2N4OS2
26

(371.26): Calculated/Found: 42.06/42.19 (%C), 2.17/2.22 (%H), 15.09/15.34 (%N) and
17.27/17.38 (%S).
4.1.4.17. (5Z)-5-[(4-bromophenyl)methylidene]-2-[(5-methyl-1,3,4-thiadiazol-2-
yl)imino]-1,3-thiazolidin-4-one (4q)
-1
3
o
Yellow solid. Yield 70%; mp >300 C; IR (cm , KBr): 3413 (NH), 2899 (sp C-H),
1712 (C=O), 1569 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.68 (s, 3H, CH3),
7.61 (d, J = 8.2 Hz, 2H, Ar-H), 7.80 (d, J = 8.2 Hz, 2H, Ar-H), 7.76 (s, 1H, C=CH),
12.92 (br. s, 1H, NH; Elemental analysis for C13H9BrN4OS2 (381.27):
Calculated/Found: 40.95/41.31 (%C), 2.38/2.52 (%H), 14.69/14.87 (%N) and
16.82/17.01 (%S).
4.1.4.18. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-[(pentafluorophenyl)-
methylidene]-1,3-thiazolidin-4-one (4r)
White solid. Yield 76.5%; mp decompose at 230 oC; IR (cm-1, KBr): 3434 (NH), 2932
(sp3C-H), 1724 (C=O), 1594 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 2.65 (s,
3H, CH3), 7.48 (s, 1H, C=CH), 12.92 (br. s, 1H, NH); Elemental analysis for
C13H5F5N4OS2 (392.33): Calculated/Found: 39.80/40.03 (%C), 1.28/1.26 (%H),
14.28/14.45 (%N) and 16.35/16.44 (%S).
4.1.4.19. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-[(2E)-3-phenylprop-2-en-
1-ylidene]-1,3-thiazolidin-4-one (5)
Yellow solid. Yield 66%; mp 258-260 oC; IR (cm-1, KBr): 3428 (NH), 3026 (sp2C-H),
1
2890 (sp3C-H), 1708 (C=O), 1610 (C=C), 1557 (C=N); H-NMR (400 MHz, δ ppm
DMSO-d6): 2.67 (s, 3H, CH3), 7.12 (t, J = 13.0 Hz, 1H, =CH-CH=CH-), 7.30 (d, J =
15.2 Hz, 1H, =CH-CH=CH-), 7.35-7.45 (m, 3H, Ar-H), 7.48 (d, J = 11.1 Hz, 1H, =CH-
CH=CH-), 7.71 (br. s, 2H, Ar-H), 12.60 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm
DMSO-d6): 16.28 (CH3), 123.82, 125.63, 128.44, 129.37, 130.19, 133.43, 136.17,
144.31, 158.27, 162.98, 167.03, 170.42 (C=O); Elemental analysis for C15H12N4OS2
(328.41): Calculated/Found: 54.86/55.13 (%C), 3.68/3.79 (%H), 17.06/17.28 (%N) and
19.53/19.67 (%S).
4.1.4.20. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-[(pyrid-3-yl)methyli-dene]-
1,3-thiazolidin-4-one (6)
Greenish solid. Yield 70.5%; mp 282-285 oC; IR (cm-1, KBr): 3416 (NH), 2900 (sp3C-
1
H), 1715 (C=O), 1587 (C=N); H-NMR (400 MHz, δ ppm DMSO-d6):2.67 (s, 3H,
27

CH3), 7.62 (dd, J = 7.78- 4.72 Hz, 1H, Ar-H), 7.81 (s, 1H, C=CH), 8.03 (d, J = 8.15
Hz, 1H, Ar-H), 8.65 (d, J = 4.08 Hz, 1H, Ar-H), 8.88 (d, J = 1.59 Hz, 1H, Ar-H), 13.01
(br. s, 1H, NH); Elemental analysis for C12H9N5OS2 (303.36): Calculated/Found:
47.51/47.69 (%C), 2.99/3.02 (%H), 23.09/23.40 (%N) and 21.14/21.29 (%S).
4.1.4.5.21. (5Z)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-5-[(thien-3-yl)methylidene]
-1,3-thiazolidin-4-one (7)
White solid. Yield 71%; mp 282-284 oC; IR (cm-1, KBr): 3413 (NH), 2825 (sp3C-H),
1712 (C=O), 1596 (C=N); 1H-NMR (400 MHz, δ ppm DMSO- d6): 2.68 (s, 3H, CH3),
7.31 (t, J = 4.22 Hz, 1H, Ar-H), 7.71 (br. s, 1H, Ar-H), 8.04 (s, 2H, Ar-H, C=CH) 12.83
(br. s, 1H, NH); 13C NMR (100 MHz δ ppm DMSO-d6): 16.27 (CH3), 137.73, 157.65,
126.19, 121.84, 167.19, 135.27, 133.76, 129.43, 170.30, 163.21 (C=O): Elemental
analysis for C11H8N4OS3 (308.40): Calculated/Found: 42.84/43.08 (%C), 2.61/2.67
(%H), 18.17/18.43 (%N) and 31.19/31.28 (%S).
4.1.4.22. (5Z)-5-(cyclohexylmethylidene)-2-[(5-methyl-1,3,4-thiadiazol-2-yl)imino]-
1,3-thiazolidin-4-one (8)
-1
3
o
White solid. Yield 75%; mp 219-222 C; IR (cm , KBr): 3425 (NH), 2935 (sp C-H),
1717 (C=O), 1594 (C=N); 1H-NMR (400 MHz, δ ppm DMSO-d6): 1.15-1.42 (m, 5H,
cyclohexyl), 1.57-182 (m, 5H, cyclohexyl), 2.24 (m, 1H, CH), 2.67 (s, 3H, CH3), 6.77
(d, J = 8.68 Hz, 1H, C=CH), 12.58 (br. s, 1H, NH); 13C-NMR (100 MHz, δ ppm DMSO-
d6): 16.25 (CH3), 25.2, 25.63, 31.11, 40.88, 125.72,142.54, 158.46, 163.05, 166.46,
170.45 (C=O); Elemental analysis for C13H16N4OS2 (308.42): Calculated/Found:
50.63/50.71 (%C), 5.23/5.4 (%H), 18.17/18.53 (%N) and 20.79/20.82 (%S).
5. Biological screening
5.1. In vitro Lipoxygenase inhibition assay
Lipoxygenase inhibitory activity was performed at the Department of Biochemistry,
Faculty of Medicine, Cairo University, Cairo, Egypt. The prepared compounds
screened against 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.
5.2. In vitro cyclooxygenase inhibition assay
Cyclooxygenase inhibitory activity was performed at the Department of Biochemistry,
Faculty of Medicine, Cairo University, Cairo, Egypt. The prepared compounds were
28

screened against 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.
5.3. In vivo anti-inflammatory activity
Compounds (3a, 4f, 4i, 4q, and 4r) were chosen to examine their in vivo anti-
inflammatory activity using the Carrageenan-induced paw edema model in rats. The
study was performed according to the reported procedures [38]. Briefly, seven groups
each of five male albino rats (120-150 g) were injected subplantar at right hind of the
paw by carrageenan suspension (0.2 mL of 1% solution in normal saline). The paw
thickness was measured by a Vernier caliper (SMIEC, China) before and 30 min after
the carrageenan injection. The selected compounds were injected i.p at a dose of 28
µM/Kg (dissolved in 1 % sodium carboxymethyl cellulose solution in normal saline).
The reference drugs (Celecoxib) were injected at the same dose while the negative
control group injected by the vehicle (1 % sodium carboxymethyl cellulose solution in
normal saline). The thickness of the right paw was measured at 1, 2, 3 and 4 h and the
percentage inhibition of edema was calculated by the following equation. Statistical
analysis was made using GraphPad Prism version 7.00 for Windows, GraphPad
Software, La Jolla California USA.
(푉푅 ― 푉푙)푐표푛푡푟표푙 ― (푉푅 ― 푉푙) 푡푟푒푎푡푒푑
% 퐸푑푒푚푎 푖푛ℎ푖푏푖푡푖표푛 =
(푉푅 ― 푉푙)푐표푛푡푟표푙
Where, VR: Average right paw thickness, VL: Average left paw thickness.
5.4. Ulcerogenic liability
Compounds (3a, 4i and 4r) were examined for their ulcerogenic liability according to
the reported method on adult male albino rats [39]. Briefly, five groups each of two rats
fasted for 12 h with free access to water. The selected compounds were administered
orally at doses of 20 mg/Kg (dissolved in 1 % sodium carboxymethyl cellulose solution
in normal saline). The positive control group received indomethacin orally at 10 mg/kg
while the negative control group received the vehicle only (dissolved in 1 % sodium
carboxymethyl cellulose solution in normal saline). The animals were sacrificed after
6 h, the stomachs were isolated and washed with saline. Stomachs were preserved in a
29