Discovery of novel pyridazine-based cyclooxygenase-2 inhibitors with a promising gastric safety profile

Article abstract of DOI:10.3390/molecules25092002

Cyclooxygenase-2 (COX-2) is implicated in the development of chronic inflammatory diseases. Recently, pyridazine derivatives have emerged as a novel prototype to develop COX-2 inhibitors. Accordingly, some pyridazine-based COX-2 inhibitors are reported herein. The reaction of aldehyde 3 and different hydrazines yielded the corresponding hydrazones. The hydrazones were further derivatized to the title compounds, which were assessed for COX-1 and COX-2 inhibitory action, gastric ulcerogenic effects, and lipid peroxidation properties. Molecular docking studies and determination of the physicochemical parameters were also carried out. The allocated structures of the reported compounds were coherent with their spectroscopic data. The compounds 9a (IC₅₀ = 15.50 nM, 114.77%), 9b (IC₅₀ = 17.50 nM, 101.65%), 12 (IC₅₀ = 17.10 nM, 104.03%), 16b (IC₅₀ = 16.90 nM, 105.26%), and 17 (IC₅₀ = 17.70 nM, 100.5%) displayed better COX-2 inhibition than celecoxib (IC₅₀ = 17.79 nM, 100%). These outcomes were harmonious with the molecular docking studies of 9a, 9b, 12, 16b, and 17. These compounds also displayed comparable onset and the duration of action concerning celecoxib and indomethacin in the in vivo studies. No ulcerogenic effects were observed for 9a and 12, whereas 9b, 16b, and 17 showed an insignificant ulcerogenic effect compared to celecoxib. The compounds 9a, 9b, 12, 16b, and 17 displayed a better lipid peroxidation profile than celecoxib and indomethacin. The compounds 9a (%ABS = 84.09), 9b (%ABS = 84.09), 12 (%ABS = 66.87), 16b (%ABS = 75.02), and 17 (%ABS = 81.42) also displayed appreciable calculated absorption compared to celecoxib (%ABS = 82.09). The compounds 9a, 9b, 11, 16b, and 17 have been recognized and postulated as non-ulcerogenic COX-2 inhibitors with promising physicochemical parameters and gastric safety profile. These compounds may be useful candidates to combat diseases caused by higher levels of COX-2.

Full text of DOI:10.3390/molecules25092002

molecules
Article
Discovery of Novel Pyridazine-Based
Cyclooxygenase-2 Inhibitors with a
Promising Gastric Safety Profile
1
,
1
2
3
Abida Khan * , Anupama Diwan , Hamdy Kh. Thabet , Mohd Imran
and
Md. Afroz Bakht 4
1
School of Pharmaceutical Sciences, Apeejay Stya University, Sohna - Palwal Road, Sohna 122103, India;
anupama.diwan@asu.apeejay.edu
2
3
4
Department of Chemistry, Faculty of Arts and Science, Northern Border University, Rafha 91911,
Saudi Arabia; hamdykhamees@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Northern Border University,
Rafha 91911, Saudi Arabia; imran.pchem@gmail.com
Department of Chemistry, College of Science and Humanities, Prince Sattam Bin Abdulaziz University,
Al-Kharj 11942, Saudi Arabia; m.bakht@psau.edu.sa
*
Correspondence: aqua_abkhan@yahoo.com; Tel.: +91-8053027613
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 25 March 2020; Accepted: 21 April 2020; Published: 25 April 2020
Abstract: Cyclooxygenase-2 (COX-2) is implicated in the development of chronic inflammatory
diseases. Recently, pyridazine derivatives have emerged as a novel prototype to develop COX-2
inhibitors. Accordingly, some pyridazine-based COX-2 inhibitors are reported herein. The reaction of
aldehyde 3 and different hydrazines yielded the corresponding hydrazones. The hydrazones were
further derivatized to the title compounds, which were assessed for COX-1 and COX-2 inhibitory
action, gastric ulcerogenic effects, and lipid peroxidation properties. Molecular docking studies and
determination of the physicochemical parameters were also carried out. The allocated structures of the
reported compounds were coherent with their spectroscopic data. The compounds 9a (IC50 = 15.50 nM,
1
14.77%), 9b (IC50 = 17.50 nM, 101.65%), 12 (IC50 = 17.10 nM, 104.03%), 16b (IC50 = 16.90 nM, 105.26%),
and 17 (IC50 = 17.70 nM, 100.5%) displayed better COX-2 inhibition than celecoxib (IC50 = 17.79 nM,
00%). These outcomes were harmonious with the molecular docking studies of 9a 9b 12 16b
1
,
,
,
,
and 17. These compounds also displayed comparable onset and the duration of action concerning
celecoxib and indomethacin in the in vivo studies. No ulcerogenic effects were observed for 9a and
1
2
, whereas 9b
compounds 9a
indomethacin. The compounds 9a (%ABS = 84.09), 9b (%ABS = 84.09), 12 (%ABS = 66.87), 16b
%ABS = 75.02), and 17 (%ABS = 81.42) also displayed appreciable calculated absorption compared
to celecoxib (%ABS = 82.09). The compounds 9a 9b 11 16b, and 17 have been recognized and
,
,
16b, and 17 showed an insignificant ulcerogenic effect compared to celecoxib. The
9b 12 16b, and 17 displayed a better lipid peroxidation profile than celecoxib and
,
,
(
,
,
,
postulated as non-ulcerogenic COX-2 inhibitors with promising physicochemical parameters and
gastric safety profile. These compounds may be useful candidates to combat diseases caused by
higher levels of COX-2.
Keywords: pyridazine; thiazole; 4-thiazolidinone; cyclooxygenase-2; non-ulcerogenic; lipid
peroxidation; Lipinski’s rule
Molecules 2020, 25, 2002; doi:10.3390/molecules25092002
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2002
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1
. Introduction
Inflammation is a self-protective reaction of human body tissues towards injurious stimuli like
infection, irritants, poisonous substances, irradiation, and tissue injury [ ]. The inflammation can be
divided into acute inflammation and chronic inflammation. The symptoms of acute inflammation
comprise of swelling, heat, immobility, redness, and pain, which may last for several days [ ]. However,
1
2
persistent inflammation for a longer time or chronic inflammation may lead to the development of
diseases like gout, ankylosing spondylitis, osteoarthritis, rheumatoid arthritis, Alzheimer’s disease,
ulcerative colitis, depression, epilepsy, irritable bowel diseases, kidney injury, cancer, asthma, hepatitis,
pancreatitis, and atherosclerosis [
of the age > 60 years in the UK and Saudi Arabia, respectively, are suffering from arthritis [
is estimated that about 1/4th of the adults in the USA will be affected by osteoarthritis by 2030 [
1
,
3
]. According to the published data, 40% and 61% of the population
]. It
].
4
5
Non-steroidal anti-inflammatory drugs (NSAIDs) are regularly prescribed for chronic inflammatory
diseases. NSAIDs inhibit the cyclooxygenase enzyme (COX). COX is accountable for transforming
arachidonic acid into prostaglandins, which initiate the inflammatory events in a cell [
constitutive COX-1 is responsible for the maintenance functions of the cell, including the safety of the
gastric mucosa, aggregation of platelets, and control of the renal blood flow [ ]. COX-2 cannot be
1,2]. The
6,7
detected in healthy cells. However, this inducible enzyme is produced intracellularly after harmful
stimuli. It is responsible for the development of inflammatory events in a cell, which ultimately lead
to the development of inflammatory diseases [
effects after prolonged use because they inhibit both COX-2 and COX-1 [
of NSAIDs is credited to the inhibition of COX-1, and the anti-inflammatory action is credited to the
inhibition of COX-2 [ ]. Based on this understanding, celecoxib, rofecoxib, and etoricoxib were
developed as specific COX-2 inhibitors [10 12]. However, cerebrovascular risk and cardiac toxicity
have been reported as adverse effects of some particular COX-2 inhibitors at the standard dose, for
example, rofecoxib [12 13]. Accordingly, medicinal chemists are looking forward to developing new
1,3
]. The commonly used NSAIDs cause ulcerogenic
6,7]. The ulcerogenic effect
8,9
–
,
anti-inflammatory agents, which lack the aforementioned adverse effects and have a promising gastric
safety profile [14].
Pyridazine, a famous diazine ring, is part of many pharmacodynamic agents like
indolidan (antihypertensive), bemoradan (antihypertensive), levosimendan (congestive heart failure),
pimobendan (congestive heart failure), milrinone (cardiotonic), minaprine (antidepressant), imazodan
(
(
PDE3 inhibitor), zardaverine (PDE3 inhibitor), and olaparib (anticancer) [15
Pentoil), an analgesic and anti-inflammatory pyridazine derivative, is in clinical use in Japan and
–19]. Emorfazone
is claimed to lack gastric side effects [19]. Similarly, Zomipirac has been derivatized to a pyridazine
derivative, which had a COX-1/COX-2 selectivity index > 1500 [20 21]. Novel targets for developing
,
anti-inflammatory agents bearing the pyridazine scaffold along with the general chemistry, mechanism
of action, and the structure–activity relationship (SAR) have also been reported [19]. Ulcerogenic effects
or gastrointestinal bleeding are the most frequently encountered adverse effects of the clinically used
NSAIDs [13]. As per the recent publications, the pyridazine moiety is a new template for developing
COX-2 inhibitors [15
–
19]. The possibility of the various structural modifications in the pyridazine
21
ring, and the literature revealing the COX-2 inhibitory potential of the pyridazine nucleus [15
–
]
makes it a promising scaffold to develop non-ulcerogenic COX-2 inhibitors. As per the published
reports, incorporation of the thiazole moiety and the 4-thiazolidinone moiety in a compound imparts
selectivity towards COX-2 inhibition and also improves its gastric safety profile [22
–28]. Accordingly,
as an extension of our work to develop superior anti-inflammatory compounds [29
–
32], we report
herein novel pyridazine-based thiazole and 4-thiazolidinone derivatives as cyclooxygenase-2 inhibitors
with an improved gastric safety profile.

Molecules 2020, 25, 2002
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2
. Results and Discussion
2
.1. Chemistry
The intermediates
2,
3,
4b, and 4c were reported in our previous publication [30]. The intermediates
1, 5a, 5b, and 13 are available commercially. The condensation of aldehyde 3 with hydrazine hydrate,
thiosemicarbazide, and phenylthiosemicarbazide afforded hydrazone 4a and the thiosemicarbazone
derivatives 4b,c, respectively (Scheme 1).
Scheme 1. Preparation of 4a
–
c
(Reagents and conditions: (a) NH NH
·
H O, EtOH, reflux; (b) Br /AcOH,
2
2
2
2
(c) 4-fluorobenzaldehyde, DMSO/K CO , reflux; (d) RNH , EtOH, few drops AcOH, reflux).
2
3
2
The cycloalkylation of the thiocarbamoyl group of 4b with the ethyl
ethyl
acetate at the reflux temperature afforded the corresponding 4-thiazolidinone derivatives, 6a and
, respectively (Scheme 2). The formation of 6a and 6b is expected to proceed through the initial
S-alkylation via the loss of sodium chloride followed by the intramolecular cyclization with the
elimination of ethanol. The Hantzsch reaction of 4b with the ethyl -chloro acetoacetate in acetic acid
in the presence of sodium acetate led to the formation of 4-methyl-thiazole derivative (Scheme 2).
The possibility of compound was excluded based on the spectral analyses. The treatment of 4b with
-substituted phenacyl bromides in refluxing ethanol in the presence of anhydrous sodium acetate
produced the corresponding thiazole derivatives, 9a and 9b (Scheme 2). The cyclization reaction of
with dimethyl acetylenedicarboxylate in methanol provided the 4-thiazolidinone derivative 10
Scheme 2).
α
-chloroacetate (5a) and
α
-chloropropionate (5b) in glacial acetic acid comprising a catalytic amount of the fused sodium
6
b
α
8
7
4
4
b
(

Molecules 2020, 25, 2002
4 of 19
Scheme 2. Preparation of 6a,b, 8, 9a,b, and 10.
The condensation of the active methylene group of 6a with electrophilic dimethylformamide-
dimethylacetal (DMF-DMA) in dry dioxane afforded 5-dimethylaminomethylidine derivative 11
(
Scheme 3). Similarly, the treatment of 6a with isatin in dioxane comprising a catalytic amount of
piperidine provided compound 12 (Scheme 3). The treatment of 6a with -cinnamonitriles in dioxane
containing a catalytic amount of piperidine furnished the benzylidene derivatives 16a , wherein
other possible structures 15a were ruled out based on the spectral data. Another synthetic route
of compounds 16a was achieved via the Knoevenagel condensation of compounds 6a with the
corresponding aromatic aldehydes (Scheme 3).
α
,b
,
b
,
b

Molecules 2020, 25, 2002
5 of 19
Scheme 3. Preparation of 11, 12, and 16a,b.
The cyclocondensation of the thiocarbamoyl group of 4c with the ethyl
α
-chloroacetate in acetic acid
containing a catalytic amount of the anhydrous sodium acetate provided 4-thiazolidinone derivative
7 (Scheme 4).
1

Molecules 2020, 25, 2002
6 of 19
Scheme 4. Preparation of 17.
Finally, the reaction of 4c with dimethyl acetylenedicarboxylate in methanol at the reflux
temperature afforded the compound 18 (Scheme 5). The formation of 18 is expected to proceed
via the nucleophilic addition of S-atom to the sp-carbon of the dimethyl acetylenedicarboxylate to
generate the intermediate A. The intermediate A after the intramolecular cyclization through the
nucleophilic amino group of the thiosemicarbazone yielded 18.
Scheme 5. Preparation of 18.
There is a possibility of at least four or more geometrical isomers for some of the compounds,
for example, 10
Therefore, we have not mentioned the E or Z configuration in the nomenclature of our compounds.
The structures of 4a 6a 6b 9a 9b 10 11 12 16a 16b 17, and 18 were proven on the basis of their
spectroscopical data. The detailed spectroscopical data are provided in the experimental part.
, 11, 12, 16a, 16b, 17, and 18. However, these isomers may be able to interconvert.
,
,
,
8,
,
,
,
,
,
,
,
2
.2. Biological Activity
2
.2.1. In Vitro COX Inhibitory Action
The compounds 4a, 6a, 6b, 8, 9a, 9b, 10, 11, 12, 16a, 16b, 17, and 18 were examined as COX-1 and
COX-2 inhibitors along with indomethacin and celecoxib. It was performed by the 10-fold dilution
technique utilizing test packs of the human COX-1/COX-2 (Cayman Chemicals, 560131, Ann Arbor, MI,
USA) [30]. Indomethacin and celecoxib were used as standard drugs. Indomethacin is an ulcerogenic

Molecules 2020, 25, 2002
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non-specific COX-1 and COX-2 inhibitor, whereas celecoxib is considered as a non-ulcerogenic-specific
COX-2 inhibitor [6,10].
It is a well known fact that the inhibition of COX-1 is mainly responsible for the ulcerogenic effect
of NSAIDs like indomethacin [6,7]. It is also well documented that specific COX-2 inhibitors like
celecoxib are potent anti-inflammatory agents and possess a better gastric safety profile because they
do not inhibit COX-1 [10]. Therefore, for a better comparison, the %COX-1 inhibition of indomethacin
was normalized to 100% for COX-1, and the %COX-2 inhibition of celecoxib was normalized to 100%
for COX-2 (Table 1). The selectivity index of celecoxib was also normalized to 100%. All the compounds
comprising celecoxib (IC50 = 320 nM) displayed greater IC50 against COX-1, when compared to
indomethacin (IC50 = 220 nM) (Table 1, Figure 1). This result points out that our compounds should
have a better gastric safety profile than indomethacin [
fact that our compounds showed better inhibition of COX-2 in contrast to COX-1. The compounds
(IC50 = 15.50 nM, 114.77%), 9b (IC50 = 17.50 nM, 101.65%), 12 (IC50 = 17.10 nM, 104.03%), 16b
IC50 = 16.90 nM, 105.26%), and 17 (IC50 = 17.70 nM, 100.5%) demonstrated better COX-2 inhibition than
celecoxib (IC50 = 17.79, 100%). The selectivity index (SI) of 9a (SI = 21.29, 118.40%) and 16b (SI = 18.63,
03.61%) was superior to celecoxib (SI = 17.98, 100%). The SI of 9b (SI = 15.71, 87.37%), 12 (SI = 17.25,
5.93%), and 17 (SI = 16.10, 89.54%) was also comparable to celecoxib (SI = 17.98, 100%). Based on the
data mentioned above, 9a, 9b, 12, 16b, and 17 were chosen for the in vivo anti-inflammatory activity.
8,9]. Our belief is further strengthened by the
9
a
(
1
9
Table 1. In vitro cyclooxygenase inhibitory effect (N = 3, Mean
6a, 16b, 17, and 18.
±
SD) of 4a, 6a, 6b, 8, 9a, 9b, 10, 11, 12,
1
COX-1
IC , nM *)
%COX-1
Inhibition
COX-2
%COX-2
Inhibition
Compound
SI
%SI
(
(IC , nM *)
5
0
50
4
6
a
a
235 ± 0.11
260 ± 0.22
280 ± 0.50
250 ± 0.12
330 ± 0.28
275 ± 0.42
285 ± 0.15
270 ± 0.24
295 ± 0.28
275 ± 0.10
315 ± 0.33
285 ± 0.16
255 ± 0.20
320 ± 0.01
220 ± 0.01
93.61
84.61
78.57
88.0
66.66
80.0
77.19
81.48
74.57
80.0
69.84
77.19
86.27
68.75
100.0
26.19 ± 0.50
25.10 ± 0.15
22.75 ± 0.10
24.18 ± 0.14
15.50 ± 0.55
17.50 ± 0.60
22.11 ± 0.55
25.11 ± 0.11
17.10 ± 0.19
22.16 ± 0.44
16.90 ± 0.16
17.70 ± 0.18
23.15 ± 0.50
17.79 ± 0.69
67.72 ± 0.62
67.92
70.87
78.19
73.57
114.77
101.65
80.46
70.84
104.03
80.27
105.26
100.5
76.84
100.0
26.26
8.97
49.88
57.56
68.40
57.45
10.35
12.30
10.33
21.29
15.71
12.89
10.75
17.25
12.40
18.63
16.10
11.01
17.98
3.24
6
b
8
9
a
118.40
87.37
71.69
59.78
95.93
68.96
103.61
89.54
61.23
100.0
18.02
9
b
1
1
1
0
1
2
1
6a
1
6b
1
1
7
8
Celecoxib
Indomethacin
*
p < 0.5 (SPSS); SI (Selectivity index): IC50 for COX-1/IC50 for COX-2.
The novel pyridazine derivatives can be categorized as thiazole derivatives (8, 9a, and 9b) and
-thiazolidinone derivatives (6a 6b 10 11 12, 16a 16b 17, and 18). It is apparent from Table 1 that
the thiazole derivative 9a (4-phenyl thiazole group) was more potent than thiazole derivative 9b
4-bromophenyl thiazole group). This reflects that the incorporation of bromine in the structure of
decreases its COX-2 inhibitory potential. This result is in concurrence with the earlier report [22].
However, the COX-2 inhibitory potential of the corresponding chrolo, fluoro, iodo, and nitro derivatives
of 9b should also be assessed for a better understanding of this observation. The thiazole derivative
4-methyl-thiazole-5-carboxylate group) displayed a further decrease in the COX-2 inhibitory potential.
4
,
,
,
,
,
,
(
9
a
8
(
This also indicates that the incorporation of methyl and carboxylate groups in the structure of 9a
decreases its COX-2 inhibitory activity.

Molecules 2020, 25, 2002
8 of 19
Figure 1. The IC50 (nM) and the % selectivity index (SI) of compounds 4a
,
6a,
6b,
8,
9a,
9b
,
10
,
11
,
12
,
1
6a, 16b, 17, 18, celecoxib, and indomethacin.
In case of 4-thiazolidinone derivatives, the COX-2 inhibitory activity increases as 11
<
6a
<
18 < 6b
<
16a < 10 < celecoxib < 17 < 12 < 16b. The presence of the 5-dimethylaminomethylidene group (11),
unsubstituted 4-thiazolidinone ring (6a), 5-methoxycarbonylmethylidene-3-phenyl group (18), methyl
group (6b), 4-chlorophenylmethylidene group (16a), and 5-methoxycarbonylmethylidene group (10
at position 5 of the 4-thiazolidinone ring provides compounds with lesser or average COX-2 inhibitory
action. However, when the chlorine of the 16a is replaced with a methoxy group, a potent COX-2
inhibitor 16b is obtained. This observation indicates that the presence of the electron donor group in
these types of compounds increases the COX-2 inhibitory action. This reflection is also in concurrence
with the earlier reports that the presence of the electron donor group may increase the COX-2 inhibitory
)
action of thiazolidinone ring-bearing compounds [22
derivative (17) has been recognized as a potent inhibitor of COX-2. Some earlier reports also support
this fact [26 28]. We also believe that this kind of other 2,3-disubstituted derivatives may provide
,24]. The 2,3-disubstituted-4-thiazolidinone
,
potent COX-2 inhibitors. The isatin-bearing compound 12 also provided a potent inhibitor of COX-2.
The incorporation of the isatin moiety is reported to potentiate the COX-2 inhibitory activity of a
compound [33]. Recently, we reported the isomeric 4-thiazolidinone-bearing pyridazine derivatives,
which had a methylidine linker joining the phenyl ring and the 4-thiazolidinone ring [30]. The presently
reported 4-thiazolidinone-bearing pyridazine derivatives contain a methylidene hydrazinyl linker
between the phenyl ring and the 4-thiazolidinone ring. A comparison of the COX-2 inhibitory activity
among these isomeric compounds reveals that the incorporation of a methylidene hydrazinyl linker
provides potent COX-2 inhibitors. This observation is in concurrence with our earlier report that states
that compounds bearing a hydrazine moiety display higher COX-2 inhibition [30].
2
.2.2. In Vivo Anti-Inflammatory Activity
The compounds 9a 9b 12 16b, and 17 were chosen for the in vivo anti-inflammatory action
because they displayed better COX-2 inhibition and lesser COX-1 inhibition. It was performed by the
carrageenan-induced rat paw edema method [30 34]. A total of eight groups of rats were utilized,
wherein each group comprised of six rats. The compounds were administered orally (10 mg/kg)
Table 2, Figure 2).
,
,
,
,
(

Molecules 2020, 25, 2002
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Table 2. In vivo anti-inflammatory activity of 9a, 9b, 12, 16b, and 17.
0
h
1 h
2 h
3 h
4 h
Compound
%
Edema
%
Edema
%
Edema
%
Edema
PD *
PD
PD
PD
PD
Control
3.40 ± 0.20
3.45 ± 0.20
3.44 ± 0.18
3.51 ± 0.16
3.50 ± 0.32
3.49 ± 0.13
3.45 ± 0.22
4.70 ± 0.42
3.89 ± 0.14
3.89 ± 0.10
3.98 ± 0.62
3.89 ± 0.21
3.88 ± 0.10
3.95 ± 0.22
3.88 ± 0.26
38.23
12.75
13.08
13.39
11.14
11.17
14.49
11.49
4.95 ± 0.21
3.81 ± 0.12
3.80 ± 0.28
3.91 ± 0.10
3.85 ± 0.30
3.85 ± 0.13
3.84 ± 0.22
3.81 ± 0.40
45.58
10.43
10.46
11.39
10.0
10.31
11.30
9.48
5.15 ± 0.10
3.72 ± 0.14
3.74 ± 0.16
3.78 ± 0.22
3.77 ± 0.50
3.82 ± 0.12
3.77 ± 0.33
3.77 ± 0.14
51.47
7.82
8.72
7.69
7.71
9.45
9.27
8.33
5.25 ± 0.32
3.58 ± 0.36
3.66 ± 0.40
3.64 ± 0.15
3.67 ± 0.42
3.81 ± 0.14
3.62 ± 0.14
3.71 ± 0.33
54.41
3.76
6.39
3.70
4.85
9.16
4.92
6.60
9a
9b
12
16b
17
Celecoxib
Indomethacin 3.48 ± 0.44
*
p < 0.5 (SPSS); PD: Paw diameter (mm).
Figure 2. In vivo action of 9a, 9b, 12, 16b, 17, celecoxib, and indomethacin.
The compounds 9a (3.76%), 12 (3.70%), and 16b (4.85%) reduced the % edema more than celecoxib
4.92%) and indomethacin (6.60%) after 4 h of drug administration. The compound 9b (6.39%) reduced
(
the % edema more than indomethacin (6.60%) but less than celecoxib (4.92%) after 4 h of drug
administration. However, compound 17 (9.16%) reduced the % edema less than indomethacin (6.60%)
and celecoxib (4.92%). The compounds 9a, 9b, 12, 16b, and 17 also have a comparable onset of action
plus duration of action concerning celecoxib and indomethacin (Table 2, Figure 2).
Accordingly, it was determined that the novel pyridazine-based thiazole and 4-thiazolidinone
derivatives are active lead compounds to develop future COX-2 inhibitors [22,24,26,28].
2
.2.3. Ulcerogenic Activity
The compromising gastric safety profile of the existing NSAIDs is a concern [6,7]. The ulcerogenic
effects are more pronounced in non-specific COX inhibitors like indomethacin, whereas specific
COX-2 inhibitors like celecoxib lack this side effect [ 10]. Accordingly, 9a 9b 12 16b, and 17 were
6,
,
,
,
evaluated for their gastric safety profile. It was performed by the indomethacin-induced gastric erosion
method [30]. A total of eight groups of rats were utilized, wherein each group comprised of six rats.
The compounds were administered orally (10 mg/kg) (Table 3, Figure 3).

Molecules 2020, 25, 2002
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Table 3. The gastric safety profile of 9a, 9b, 12, 16b, and 17.
Score
MDA Content in nmol
Group
(100 mg Tissue)
No of Gastric Ulcers *
Severity Lesions *
Control
0
0
0
0
3.24 ± 0.32
3.05 ± 0.12
3.89 ± 0.50
2.95 ± 0.44
3.45 ± 0.28
4.11 ± 0.18
6.11 ± 0.50
8.90 ± 0.18
9
a
9
b
0.33 ± 0.38
0.26 ± 0.16
1
2
0
0
1
6b
0.12 ± 0.30
0.42 ± 0.10
2.51 ± 0.32
8.40 ± 0.60
0.18 ± 0.22
0.20 ± 0.50
5.82 ± 0.44
12.15 ± 0.10
1
7
Celecoxib
Indomethacin
*
p < 0.5, Mean ± SE, (SPSS).
Figure 3. The ulcerogenic and lipid peroxidation activity effect of 9a, 9b, 11, 15b, 17, celecoxib,
and indomethacin.
The compounds 9a and 12 did not produce any ulcerogenic impact. The compounds 9b
also exhibited insignificant ulcerogenic effects. The negligible ulcerogenic effect produced by 9a
16b, and 17 is attributed to their higher COX-2 inhibitory potential plus their lesser potential to
]. It is well established that the presence of the -COOH group in NSAIDs
aids in their ulcerogenic effect [ ]. Another prospect of the insignificant ulcerogenic effect produced by
a, 9b, 12, 16b, and 17 may be the absence of the -COOH moiety in their structure.
,
16b, and
1
1
7
2
,
9b,
,
inhibit COX-1 (Table 1) [8,9
6
9
2
.2.4. Lipid Peroxidation Studies
The lipid peroxidation inhibitory activity of a compound makes it less ulcerogenic [35
,
36]. The
lipid peroxidation in tissue increases the malondialdehyde (MDA) content in the tissue [35]. A decrease
in the MDA content is a measure to assess the non-ulcerogenic effect of a compound. Accordingly, the
compounds 9a
Figure 3). The compounds 9a
celecoxib and indomethacin, which provides concurrence to the ulcerogenic activity data (Table 3).
,
9b
,
12
,
16b, and 17 were evaluated for their lipid peroxidation effects [35
,36] (Table 3,
,
9b 12 16b, and 17 displayed a better lipid peroxidation profile than
,
,

Molecules 2020, 25, 2002
11 of 19
2
.3. Molecular Modelling
Molecular docking studies of 9a
,
9b
,
12
,
16b, and 17 were carried out to validate the results of
their biological activities [30,37–39] (Table 4).
Table 4. Molecular docking data of 9a, 9b, 12, 16b, and 17.
Binding Affinity
Hydrogen Bonding Interacting
Residues *
Compound
Celecoxib
Other Interacting Residues
(kcal/mol)
MET 522 (-NH of sulphonamide
group), ARG 120 (Fluorine of
TRP 387, LEU 352, ALA 527,
PHE 518, VAL 349, VAL 523,
LEU 359
−9.4
-
CF group)
3
CYS 47 (imine group of
-CH=N-), TYR 30 (C=O of the
pyridazinone)
CYS 47 (imine group of
-CH=N-), TYR 30 and PRO 153
TYR 136, GLY 135, PRO 156,
PRO 154, PRO 153, LYS 468
9
a
−10.8
−10.2
PRO 514, PRO 156, SER 353,
LYS 468
9
b
(
C=O of the pyridazinone)
o
ARG 44 (2 -amino group of
=
N-NH- moiety), HIS 122
ASP 125, LYS 137, VAL 46,
PRO 153, CYS 47, CYS 36
1
2
−10.5
o
(
2 -amino group of isatin), TYR
30 (phenyl ring)
TYR 130 (C=O of the
1
TYR 136, ARG 469, LEU 152,
PRO 153, CYS 47, CYS 36
1
6b
−10.6
−9.5
pyridazinone), ASN 34 (C=O of
the thiazolidinoe)
VAL 89, PRO 84, TYR 115,
GLU 524, PRO 86
1
7
LYS 473 (phenyl ring)
*
Dark green bonds in the Supplementary figures of the molecular docking.
The molecular docking data of celecoxib was described in our earlier publication [30]. The docking
score (DS in kcal/mol) of compounds 9a (DS = 10.8), 9b (DS = 10.2), 12 (DS = 10.5), 16b (DS = 10.6),
and 17 (DS = 9.5) was superior to celecoxib (DS = 9.4) (Table 4). The superior docking scores of
9b 12 16b, and 17 support the in vitro COX-2 inhibitory analysis results of 9a (IC50 = 15.50 nM,
14.77%), 9b (IC50 = 17.50 nM, 101.65%), 12 (IC50 = 17.10 nM, 104.03%), 16b (IC50 = 16.90 nM, 105.26%),
and 17 (IC50 = 17.70 nM, 100.5%), and celecoxib (IC50 = 17.79 nM, 100%). Table 4 also mentions the
hydrogen bonding pattern of celecoxib, 9a 9b 12 16b, and 17 with the COX-2 protein. We also
−
−
−
−
−
−
9
a
,
,
,
1
,
,
,
consider that the superior COX-2 inhibitory action of celecoxib, 9a, 9b, 12, 16b, and 17 is attributed to
their hydrogen bonding interactions [30].
2
.4. Physicochemical Parameters
The molecular properties of 9a, 9b, 12, 16b, and 17 were determined by the available online
software, Molinspiration [40]. The calculated absorption (%ABS), number of H-bond acceptors (nON),
number of H-bond donors (nOHNH), number of rotatable bonds (nrotb), and the topological polar
surface area (tPSA) are provided in Table 5.
Table 5. The physicochemical parameters of 9a, 9b, 12, 16b, 17, celecoxib, and indomethacin.
nrotb ≤
nON ≤
nOHNH
miLogP
≤ 5
MW ≤
n Violations
Compound
%ABS
tPSA
10
10
≤ 5
500
≤ 1
9a
84.09
84.09
66.87
75.02
81.42
82.09
85.35
72.18
72.18
122.11
98.48
79.93
77.99
68.54
6
6
6
9
8
7
5
5
1
1
2
1
0
2
1
5.10
5.91
4.37
4.66
4.24
3.61
3.99
449.54
528.43
518.56
507.57
465.54
381.38
357.79
1
2
1
1
0
0
0
9b
6
5
7
5
4
4
12
16b
17
Celecoxib
Indomethacin

Molecules 2020, 25, 2002
12 of 19
The compounds 9a
,
12
,
16b, and 17 passed Lipinski’s rule [40
,
41]. This rule defines the molecular
properties of a drug, which governs its pharmacokinetic performance, for example, the absorption
of a drug. It also helps to evaluate a compound’s ability to be orally active based on its molecular
properties [40,41], which are mentioned in Table 5. To pass Lipinski’s rule, a compound should not
have more than one violation of the calculated properties. As per a recent report, Lipinski’s rule
of five does not have any significant deficiency in defining the druggability of a compound and is
still useful today [42]. It can be observed that compound 9a displayed a better calculated absorption
(%ABS = 84.09) than celecoxib (%ABS = 82.09) (Figure 4). The compound 9b (%ABS = 84.09) also
displayed a better calculated absorption than celecoxib. The compound 9b did not pass Lipinski’s rule.
However, there are many clinically used natural compounds and drugs that violate Lipinski’s rule [42].
The compound 12 (%ABS = 66.87), compound 16b (%ABS = 75.02), and compound 17 (%ABS = 81.42)
also exhibited appreciable calculated absorption. These observations revealed that the compounds 9a
,
9
b, 12 16b, and 17 possess not only better COX-2 inhibitory action than indomethacin and celecoxib
,
but also possess a promising pharmacokinetic/physicochemical profile.
Figure 4. Calculated degree of absorption (%ABS) of 9a, 9b, 12, 16b, 17, celecoxib, and indomethacin.
3
. Materials and Methods
The uncorrected melting points, FTIR spectra (KBr), NMR spectra ( H-NMR & 13C-NMR), mass
1
spectra, and elemental analyses were obtained by Gallenkamp apparatus (MFB-595- 010M; Weiss
Gallenkamp, London, UK), Shimadzu 440 spectrometer (Shimadzu, Tokyo, Japan), Varian Gemini
5
00 MHz spectrometer (Palo Alto, CA, USA), GCMS/QP 1000 Ex mass spectrometer (70 eV) (Shimadzu,
Tokyo, Japan), and the VARIO El Elementer apparatus (Langenselbold, Germany), respectively. The
◦
−1
1
melting points are mentioned in C, IR signals are mentioned as (KBr,
mentioned as (500 MHz, DMSO-d6;
in ppm), 13C-NMR signals are mentioned as (125 MHz, DMSO-d6,
in ppm), and the mass peaks (MS) are represented as (m/z). The intermediates 4b, and 4c were
reported in our previous publications [30 43]. SciFinder was used to ascertain the novelty of 4a
9a, 9b 10 11 12 16a 16b, 17, and 18. The preparation methods of 4a 6a 6b 9a 9b 10 11
6b, 17, and 18 are depicted in Schemes 1–5.
ν in cm ), H-NMR signals are
δ
δ
2, 3,
,
,
6a, 6b
,
,
,
8
1
,
,
,
,
,
,
,
,
,
8
,
,
,
,
,
12 16a

Molecules 2020, 25, 2002
13 of 19
3
.1. Chemistry
General preparation of 4a–c
A mixture comprising
3
(3.62 mmoles), hydrazine hydrate or thiosemicarbazide or
◦
phenylthiosemicarbazide (3.62 mmoles), AcOH (5 mL), and EtOH (50 mL) was refluxed at 80 C for
3–5 h. The separated precipitate was filtered and recrystallized by MeOH. The compounds 4b and 4c
were reported in our previous publications [30].
◦
-(4-(Hydrazineylidenemethyl)phenyl)-6-phenylpyridazin-3(2H)-one (4a): Yield (75%); M.P.: 235–236 C;
2
1
IR: 3371 & 3216 (NH ), 3063, 1662 (C=O); H-NMR: 6.98 (s, 2H, NH ), 7.14 (d, 1H, Ar-H), 7.42 (q, 3H,
2
2
13
Ar-H), 7.61 (dd, 4H, Ar-H), 7.79 (s, 1H, azomethine-H), 7.88 (d, 2H, Ar-H), 8.05 (d, 1H, Ar-H); C-NMR:
1
1
7
59.06 (C=O), 144.59, 140.95, 137.56, 136.59, 134.64, 131.45, 131.27, 129.99, 129.37 (2C), 128.45 (2C),
+
26.46 (2C), 126.08 (2C), 125.52 (2C); Mass: 290 (M , 100% base peak); Anal. Calcd. for C H N O: C,
17
14
4
0.33; H, 4.86; N, 19.30. Found: C, 70.22; H, 4.70; N, 19.24.
General synthesis of 6a, 6b, 7, 9a, and 9b
A mixture comprising 4b (10 mmoles), ethyl 2-chloroacetate (10 mmoles) or ethyl
2-chloropropanoate (10 mmoles) or ethyl 2-chloro-3-oxobutanoate (10 mmoles) or 2-bromo-1-
phenylethan-1-one (10 mmoles) or 2-bromo-1-(4-bromophenyl)ethan-1-one (10 mmoles) and
◦
CH COONa (20 mmoles) in 40 mL of AcOH was heated to 125 C for 4–6 h. The precipitate
3
was collected and recrystallized by dioxane.
2
-(2-(4-(6-Oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazineyl)thiazol-4(5H)-one (6a): Yield (65%);
◦
1
M.P.: 250–252 C; IR: 3150 (NH), 2942, 2850, 1720 & 1665 (C=O), 1584; H-NMR: 3.92 (s, 2H, -S-CH -),
2
7.20 (d, 2H, Ar-H), 7.48 (q, 3H, Ar-H), 7.80 (d, 2H, Ar-H), 7.89 (d, 2H, Ar-H), 8.14 (d, 2H, Ar-H),
8
.48 (s, 1H, azomethine-H), 12.12 (s, 1H, NH); 13C-NMR: 170.43 (C=O), 159.06 (C=O), 155.87, 154.58,
1
44.88, 143.59, 134.57, 134.15, 131.64, 130.14 (2C), 129.43 (2C), 128.25 (2C), 126.56 (2C), 126.34 (2C), 33.54
+
(
S-CH -); Mass: 389 (M ), 274 (100% base peak); Anal. Calcd. for C H N O S: C, 61.68; H, 3.88; N,
2
20 15
5
2
1
7.98. Found: C, 61.47; H, 3.76; N, 17.89.
5
(
-Methyl-2-(2-(4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazineyl)thiazol-4(5H)-one (6b): Yield
◦
67%): M.P.: 285–286 C; IR: 3115 (NH), 3059, 1714 & 1668 (C=O), 1595; H-NMR: 1.57 (d, 3H, CH ),
3
1
4
.22 (q, 1H, CH), 7.22 (d, 1H, Ar-H), 7.46 (q, 3H, Ar-H), 7.80 (d, 2H, Ar-H), 7.94 (dd, 4H, Ar-H), 8.14
13
(d, 1H, Ar-H), 8.48 (s, 1H, azomethine-H), 12.02 (s, 1H, NH); C-NMR: 174.21 (C=O), 159.06 (C=O),
1
1
55.91, 153.47, 144.89, 143.61, 134.58, 134.15, 131.65, 130.14 (2C), 129.43 (2C), 128.26 (2C), 126.57 (2C),
+
26.35 (2C), 42.71 (-S-CH -), 19.20 (-CH ); Mass: 403 (M ), 274 (100% base peak); Anal. Calcd. for
2
3
C H N O S: C, 62.52; H, 4.25; N, 17.36. Found: C, 62.50; H, 4.22; N, 17.35.
21
17
5
2
Ethyl 4-methyl-2-(2-(4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazineyl) thiazole-5-carboxylate (
8
):
Yield (74%): M.P.: 245–246 C; IR: 3115 (NH), 3059, 1715 & 1665 (C=O), 1595; H-NMR: 1.43 (s, 3H,
CH ), 2.26 (s, 3H, CH ), 4.20 (q, 2H, CH ), 7.24 (d, 2H, Ar-H), 7.49–7.60 (m, 4H, Ar-H), 8.05 (m, 4H,
◦
1
3
3
2
13
Ar-H), 8.20 (d, 1H, Ar-H), 8.49 (s, 1H, azomethine-H), 9.77 (s, 1H, NH); C-NMR: 170.51 (C=O), 167.15
C=O), 160.95, 160.02, 159.13, 157.72, 145.10, 143.47, 135.19, 134.58, 134.45, 133.39, 131.78, 131.14, 130.98,
(
1
4
6
30.19, 129.47, 128.93, 126.65, 126.19, 116.91, 60.89 (-O-CH -), 17.27 (-CH ), 14.75 (Ester -CH ); Mass:
2 3 3
+
59 (M ), 274 (100% base peak); Anal. Calcd. for C H N O S: C, 62.73; H, 4.61; N, 15.24. Found: C,
24
21
5
3
2.70; H, 4.55; N, 15.24.
6
-Phenyl-2-(4-((2-(4-phenylthiazol-2-yl)hydrazinylidene)methyl)phenyl)pyridazin-3(2H)-one (9a): Yield (72%):
◦
1
M.P.: 280–281 C; IR: 3115 (NH), 3059, 1666 (C=O), 1595; H-NMR: 7.04–7.50 (m, 9H, Ar-H + thiazole-H),

Molecules 2020, 25, 2002
14 of 19
7
.79–8.11 (m, 9H, Ar-H + azomethine-H), 12.28 (s, 1H, NH); 13C-NMR: 168.60 (C=O), 159.07, 144.82,
1
1
42.46, 140.67, 135.13, 134.64, 134.44, 132.37, 131.60, 131.52, 130.10, 129.43, 129.08 (2C), 128.02 (2C),
26.79 (2C), 126.55 (2C), 126.34 (2C), 126.0 (2C), 104.33; Mass: 449 (M ), 77 (100% base peak); Anal.
+
Calcd. for C H N OS: C, 69.47; H, 4.26; N, 15.58. Found: C, 69.45; H, 4.25; N, 15.55.
26
19
5
2
-(4-((2-(4-(4-Bromophenyl)thiazol-2-yl)hydrazineylidene)methyl)phenyl)-6-phenylpyridazin-3(2H)-one (9b):
◦
1
Yield (70%): M.P.: 261–262 C; IR: 3115 (NH), 3059, 1670 (C=O), 1595; H-NMR: 7.20–8.13 (m, 17H,
Ar-H + thiazole-H5 + azomethine-H), 12.31 (s, 1H, NH); 13C-NMR: 168.78 (C=O), 159.08, 149.92, 144.83,
1
42.53, 141.64, 140.87, 134.66, 134.36, 132.02, 131.62, 131.51, 130.11, 129.44, 128.59 (2C), 128.04, 127.91
+
+
(
2C), 126.84, 126.57 (2C), 126.34, 126.15, 121.03, 105.24; Mass: 527 (M ), 529 (M + 2), 274 (100% base
peak); Anal. Calcd. for C H BrN OS: C, 59.10; H, 3.43; N, 13.25. Found: C, 59.11; H, 3.42; N, 13.21.
26
18
5
Synthesis
of
Methyl2-(4-oxo-2-((4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazineylidene)
-
thiazolidin-5-ylidene)acetate (10). A mixture comprising 4b (5 mmoles), dimethyl acetylene
◦
dicarboxylate (5 mmol), and MeOH (30 mL) was reacted at 80 C for 1 h. The resultant residue was
cooled at 25 C. The solid was filtered, and recrystallized by dioxane. Yield (75%): M.P.: 290–291 C;
IR: 3183 (NH), 2963, 2776, 1724 (C=O), 1710 & 1678 (C=O), 1614; H-NMR: 3.77 (s, 3H, OMe), 6.69 (s,
◦
◦
1
1
1
1
H, CH=C), 7.24–7.39 (m, 8H, Ar-H), 7.75–7.92 (m, 3H, Ar-H), 8.34 (s, 1H, azomethine-H), 12.29 (hump,
H, NH); 13C-NMR: 175.93 (C=O), 167.57 (C=O), 161.05 (C=O), 159.43, 159.13, 157.51, 155.24, 145.11,
43.50, 134.58, 134.32, 132.42, 132.30, 131.78, 130.19, 129.48, 129.04, 128.71, 126.66, 126.12, 122.12, 121.93,
+
5
6.37 (-OCH ); Mass: 459 (M ), 274 (100% base peak); Anal. Calcd. for C H N O S: C, 60.12; H, 3.73;
3
23 17
5
4
N, 15.24. Found: C, 60.10; H, 3.69; N, 15.22.
Synthesis of 5-((dimethylamino)methylene)-2-(2-(4-(6-oxo-3-phenylpyridazin-1(6H)-yl) benzylidene)
hydrazineyl) thiazol-4(5H)-one (11). A mixture comprising 6a (0.01 mole), dimethylformamide-
dimethylacetal (DMF-DMA) (0.01 moles) and dioxane (30 mL) was refluxed for 4 h. The resultant
◦
residue was cooled at 25 C. The solid was isolated, and recrystallized with EtOH-dioxane mixture
(
◦
1
1:1). Yield (70%): M.P.: 284–285 C; IR: 3183 (NH), 2963, 2776, 1724 & 1678 (C=O), 1614; H-NMR: 2.94
3.13 (2s, 6H, N(CH ) ), 7.18 (d, 1H, Ar-H), 7.49 (brs, 3H, Ar-H + methine-H), 7.74 (d, 3H, Ar-H), 7.94
q, 3H, Ar-H), 8.12 (d, 2H, Ar-H), 8.25 (s, 1H, azomethine-H), 12.34 (s, 1H, NH); C-NMR: 172.72
C=O), 167.35 (C=O), 159.07, 157.10, 144.91, 144.85, 144.59, 143.74, 143.33, 134.57, 134.47, 134.04, 131.63,
30.14, 129.43, 128.38, 128.06, 127.69, 126.55, 126.35, 126.27, 42.37 (2C, 2CH ); Mass: 444 (M ), 274
&
(
(
3
2
13
+
1
3
(
100% base peak); Anal. Calcd. for C H N O S: C, 62.15; H, 4.54; N, 18.91. Found: C, 62.13; H, 4.52;
23 20 6 2
N, 18.91.
Synthesis of 2-(2-(4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazineyl)-5- (2-oxoindolin-3-ylidene)
thiazol-4(5H)-one (12). A mixture comprising 6a (0.01 mole), indoline-2,3-dione (0.01 mole), piperidine
(
0.5 mL), and dioxane (40 mL) was refluxed for 2 h. The solid was filtered hot, and recrystallized with
◦
dioxane. Yield (77%); M.P.: 255–256 C; IR: 3169 & 3149 (NH), 3054, 2973, 1723 (C=O), 1705 & 1672
(
1
C=O), 1613; H-NMR: 6.76 (d, 1H, Ar-H), 6.93 (d, 1H, Ar-H), 7.05 (d, 2H, Ar-H), 7.22 (d, 2H, Ar-H),
7
.50 (brs, 3H, Ar-H), 7.87, 7.92 (2brs, 5H, Ar-H), 8.18 (d, 1H, Ar-H), 8.36 (s, 1H, azomethine-H), 11.20,
1
1
2.51 (2s, 2H, 2NH); 13C-NMR: 175.43 (C=O), 168.22 (C=O), 161.15 (C=O), 159.53, 159.23, 157.51, 155.34,
45.12, 143.60, 134.58, 134.38, 132.33, 132.24, 131.68, 130.29, 129.48 (2C), 129.14 (2C), 128.71 (2C), 126.67
+
(3C), 126.22 (2C), 122.14, 121.92; Mass: 518 (M ), 274 (100% base peak); Anal. Calcd. for C H N O S:
28
18
6
3
C, 64.86; H, 3.50; N, 16.21. Found: C, 64.70; H, 3.37; N, 16.10.
General Synthesis of 16a and 16b
A mixture comprising 6a (10 mmoles), the requisite cinnamonitrile (10 mmoles), piperidine
(0.5 mL), and dioxane (40 mL) was refluxed for 4–6 h. The solid produced was collected and

Molecules 2020, 25, 2002
15 of 19
recrystallized using dioxane. Alternatively, 16a and 16b can also be prepared by reacting equimolar
quantities of 6a with appropriate benzaldehyde.
5
-(4-Chlorobenzylidene)-2-(2-(4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)-hydra-zinyl)thiazol-4(5H)-one
◦
1
(
16a): Yield (81%): M.P.: 297–298 C; IR: 3160 (NH), 2940, 1729 & 1670 (C=O), 1619; H-NMR: 7.24 (d,
H, Ar-H), 7.51 (brs, 3H, Ar-H), 7.84 (d, 2H, Ar-H), 7.97 (brs, 6H, Ar-H + benzylidene-H), 8.14, 8.24
2d, 4H, Ar-H), 8.40 (s, 1H, azomethine-H), 10.79 (s, 1H, NH); 13C-NMR: 180.80 (C=O), 176.51 (C=O),
1
(
1
74.15, 159.08, 156.90, 149.16, 144.77, 142.32, 140.81, 136.49, 135.88 (2C), 134.93 (2C), 134.67 (2C), 129.43
+
(4C), 126.55 (3C), 126.53 (2C), 120.69, 120.30; Mass: 511 (M ), 510 (100% base peak); Anal. Calcd. for
C H ClN O S: C, 63.34; H, 3.54; N, 13.68. Found: C, 63.25; H, 3.48; N, 13.54.
27
18
5
2
5
-(4-Methoxybenzylidene)-2-(2-(4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydra-zineyl)thiazol-4(5H)
◦
1
-one (16b): Yield (83%): M.P.: >300 C; IR: 3164 (NH), 1700 & 1665 (C=O), 1613; H-NMR: 3.91 (s, 3H,
OCH ), 6.94 (d, 2H, Ar-H), 7.24 (d, 2H, Ar-H), 7.50–7.58 (2brs, 5H, Ar-H), 7.80–8.12 (3brs, 6H, Ar-H +
3
13
CH-benzylidene), 8.18 (d, 1H, Ar-H), 8.50 (s, 1H, azomethine-H), 11.12 (s, 1H, NH); C-NMR: 168.27
(
1
C=O), 166.11 (C=O), 165.48, 159.09, 155.46, 155.17, 144.76, 134.65, 131.61 (2C), 130.98 (2C), 130.11 (2C),
+
29.44 (3C), 129.16 (3C), 127.21 (2C), 126.55 (3C), 126.16 (2C), 55.68 (-OCH ); Mass: 507 (M ), 506
3
(
100% base peak); Anal. Calcd. for C H N O S: C, 66.26; H, 4.17; N, 13.80. Found: C, 66.15; H, 4.15;
28 21 5 3
N, 13.74.
Synthesis of 2-((4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazineylidene)-3-phenyl thiazolidin-4-one
(
17). A mixture comprising 4c (10 mmoles), ethyl 2-chloroacetate (10 mmoles), CH COONa (20 mmoles),
3
◦
and AcOH (30 mL) was heated at 125 C for 4 h. The produced solid was isolated and recrystallized by
◦
1
dioxane. Yield (72%): M.P.: 283–284 C; IR: 3172 (NH), 2953, 2756, 1730 & 1699 (C=O), 1621; H-NMR:
.13 (s, 2H, CH ), 7.19 (d, 2H, Ar-H), 7.41-7.54 (m, 7H, Ar-H), 7.78 (d, 1H, Ar-H), 7.87 (d, 2H, Ar-H), 7.93
4
(
2
13
d, 2H, Ar-H), 8.13 (d, 2H, Ar-H), 8.40 (s, 1H, azomethine-H); C-NMR: 172.53 (C=O), 165.0 (C=O),
1
1
59.06, 157.36, 144.88, 135.51, 134.55, 131.63, 130.16, 129.59 (2C), 129.43 (2C), 129.19 (2C), 128.76 (2C),
+
28.59 (2C), 128.37 (2C), 126.56 (2C), 126.39 (2C), 32.85 (-S-CH -); Mass: 465 (M ), 274 (100% base peak);
2
Anal. Calcd. for C H N O S: C, 67.08; H, 4.11; N, 15.04. Found: C, 67.0; H, 4.03; N, 14.98.
26
19
5
2
Synthesis
of
Methyl2-(4-oxo-2-((4-(6-oxo-3-phenylpyridazin-1(6H)-yl)benzylidene)hydrazinylidene)
-
3-phenylthiazolidin-5-ylidene)acetate (18).
A mixture comprising 4c (5 mmoles), dimethyl
◦
acetylenedicarboxylate (5 mmol), and MeOH (30 mL) was heated to 80 C for 1 h. The
resultant mixture was cooled to 25 C. The solid separated was collected and recrystallized by
ethanol-dimethylformamide (DMF) mixture (1:1). Yield (77%): M.P.: 277–278 C; IR: 2953, 2756, 1730
(
◦
◦
1
C=O), 1699 & 1670 (C=O), 1621; H-NMR: 3.84 (s, 3H, OMe), 6.84 (s, 1H, methine-H), 7.21 (d, 1H,
Ar-H), 7.47–7.59 (m, 8H, Ar-H), 7.82 (d, 2H, Ar-H), 7.92 (d, 4H, Ar-H), 8.18 (d, 1H, Ar-H), 8.55 (s, 1H,
azomethine-H); 13C-NMR: 169.90 (C=O), 166.48 (C=O), 159.42 (C=O), 157.13, 156.46, 144.94, 142.13,
1
34.54, 133.44, 131.67, 130.18 (2C), 129.64 (3C), 129.44 (3C), 128.80 (3C), 128.70 (2C), 126.58 (3C), 126.50
+
(
2C), 53.08 (-OCH ); Mass: 535 (M ), 274 (100% base peak); Anal. Calcd. for C H N O S: C, 65.04; H,
3
29 21
5
4
3
.95; N, 13.08. Found: C, 65.0; H, 3.91; N, 13.02.
3
.2. Biological Activity
3
.2.1. In Vitro COX-1 and COX-2 Inhibition Assay
The compounds 4a
to their COX-1/COX-2 inhibitory action by a 10-fold dilution strategy (1–10
, 6a, 6b, 8, 9a, 9b, 10, 11, 12, 16a, 16b, 17, and 18 were exposed
−
4
µg/mL) utilizing
dimethylsulfoxide (DMSO) [30]. The test packs of the human COX-1/COX-2 were obtained from
Cayman Chemicals (560131, Ann Arbor, MI, USA). The supplier’s instructions were followed to
prepare the reagents as well as to perform the experiment. Briefly, samples (20
µL), the COX-1 and
COX-2 enzyme (10 L), and heme (10 L) were mixed with the buffer solution (160 µ
µ
µ
L), which was

Molecules 2020, 25, 2002
16 of 19
◦
supplied with the kits. The resultant combination was incubated at 37 C in a water bath for 10 min,
and arachidonic acid (10 L) was added to initiate the COX reaction. A saturated solution of stannous
chloride (30 L) was added after 2 min to halt the COX reaction. The resultant mixture was incubated
at ambient temperature for 5 min. The Prostaglandin-2 (PGF2 ) developed after the COX reaction
µ
µ
α
α
was measured through ELISA. The samples were shifted to a 96-well plate and incubated for 18 h at
◦
2
5 C. The plate was washed to get rid of the unbound components. The Ellman’s reagent (200
µL),
◦
containing the acetylcholine substrate, was mixed and further incubated at 25 C for 1–1.5 h till the
o
absorbance (410 nm) of the B well was in the range of 0.3 to 0.8 A.U. The plate was read through the
ELISA reader. The IC50 values of COX-1 and COX-2 were determined by the regression analysis.
3
.2.2. In Vivo Anti-Inflammatory Activity
The compounds 9a 9b 12 16b, and 17 were evaluated for anti-inflammatory action utilizing
Wistar rats (130–150 g) by following the rat paw edema method [30 34]. The animal approval
IAEC/KSOP/E/18/12) was obtained from the CPCSEA. A total of eight groups of rats were utilized,
,
,
,
,
(
wherein each group comprised of 6 rats. The compounds (test group), celecoxib (standard group),
and indomethacin (standard group) were administered orally (10 mg/kg) as a 10% Tween-80 solution.
Saline solution (1 mL) was given to the control group. The carrageenan solution (1%, 0.1 mL) was
administered by injection after 1 h in the right hind paw of the rats in the test, standard, and the control
groups. The volume of the paws was calculated after the carrageenan injection at 0, 1, 2, 3, and 4 h by a
plethysmometer. The % edema was calculated as follows:
paw diameter a fter carrageenan − paw diameter be fore carrageenan
%
edema =
× 100.
paw diameter be fore carrageenan
3
.2.3. Gastric Ulcerogenic Activity
The eight groups of rats were fasted for 18 h, wherein each group consisted of 6 rats. The
compounds (9a 9b 12 16b, and 17), celecoxib, and indomethacin were given orally (10 mg/kg) as
0% Tween-80 solution. Saline solution (1 mL) was given to the control group. The animals were
,
,
,
1
sacrificed after 4 h to isolate their stomachs. A longitudinal cut was made along the greater curvature,
and the presence of ulcers was evaluated. The counting of ulcers was marked as 0 (no ulcer) to 5
(
≥3 ulcers) [30].
.2.4. Lipid Peroxidation Inhibitory Activity
It was carried out on compounds 9a
3
,
9b, 12, 16b, 17, celecoxib, and indomethacin after the
ulcerogenic activity [35 36]. The gastric mucosa (100 mg) was scraped with two glass slides and
,
homogenized in ice-cold potassium chloride (1.8 mL of 1.15% KCl). Sodium dodecyl sulphate (0.2 mL
of 8.1% SDS), acetate buffer (pH = 3.5, 1.5 mL), and thiobarbituric acid (1.5 mL of 0.8% TBA) was
◦
mixed with the homogenate, and the mixture was heated to 95 C for 1 h. The obtained mixture was
extracted with a 5-mL mixture (15:1 v/v) of n-butanol and pyridine and centrifuged at 4000 rpm for
10 min. The supernatant liquid was separated, and its absorbance was measured at 532 nm using a
spectrophotometer. A standard linear curve (absorbance vs. concentration in nM) was prepared using
MDA tetrabutylammonium salt. The lipid peroxidation was calculated from the standard curve. The
−1
results are expressed as nmol MDA 100 mg tissues.
3
.3. Molecular Docking
The molecular docking of 9a
,
9b
,
12
,
16b
,
17, and celecoxib was performed by the method reported
in our earlier report [30]. This study was conducted on the HP computer system (Intel® core i5-4570
CPU, 3.20 GHz, Window 10). ChemDraw was used to design the compounds, wherein ChemBio3D
Ultra was used to generate 3-D conformations. The 3-D structure of COX-2 protein (PDB entry 5KIR)
®

Molecules 2020, 25, 2002
17 of 19
was retrieved from PDB [37
–
39]. The Auto Dock and the Auto Dock Vina were utilized to generate the
data. The detailed procedure was demonstrated in our earlier report [30].
3
.4. Physicochemical Parameters
The concept of “Lipinski’s rule” [40,41] was applied to 9a, 9b, 12, 16b, and 17. The computational
molecular properties like the molecular weight, number of H-bond acceptors and donors, log P, number
of rotatable-bonds, and molecular polar surface area were determined by the available online software,
Molinspiration [40,41]. The degree of absorption (%ABS) was computed as follows [44,45]:
%
ABS = 109 − (0.345 × tPSA)
3
.5. Statistical Analysis
It was performed by the SPSS software. The p values, N values, mean values, and standard
deviation values are mentioned at the desirable places of the manuscript.
4
. Conclusions
Five compounds, 9a
These compounds had a similar onset/duration of action to celecoxib. The compounds 9a and 12 were
devoid of any ulcerogenic effect, whereas 9b 16b, and 17 showed insignificant ulcerogenic effects. The
compounds 9a 9b 12 16b, and 17 also displayed a better lipid peroxidation profile than indomethacin
and celecoxib. They also demonstrated a considerable calculated absorption. The compounds 9a 9b,
16b, and 17 are thus recognized and postulated as non-ulcerogenic COX-2 inhibitors with promising
,
9b, 12, 16b, and 17, demonstrated superior COX-2 inhibition than celecoxib.
,
,
,
,
,
11,
physicochemical parameters and gastric safety profile. These compounds may be useful candidates to
combat diseases caused by higher levels of COX-2 like gout, ankylosing spondylitis, osteoarthritis,
rheumatoid arthritis, Alzheimer’s disease, ulcerative colitis, depression, epilepsy, irritable bowel
diseases, kidney injury, cancer, asthma, hepatitis, pancreatitis, and atherosclerosis.
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: Conceptualization, A.K., A.D., H.K.T., and M.I.; Data curation, A.K. and H.K.T.; Formal
analysis, A.K., M.I., and M.A.B.; Investigation, A.K., H.K.T., M.I., and M.A.B.; Methodology, A.K., A.D., H.K.T.,
and M.I.; Project administration, A.K.; Resources, A.D., H.K.T., M.I., and M.A.B.; Software, A.K., M.I., and M.A.B.;
Supervision, A.D., and H.K.T.; Validation, A.K., A.D., and H.K.T.; Visualization, A.K.; Writing—original draft,
A.K., H.K.T., M.I., and M.A.B.; Writing—review and editing, A.D., and M.A.B. All authors have read and agreed
to the published version of the manuscript.
Funding: This study received no external funding.
Acknowledgments: The authors thank Apeejay Stya University, Northern Border University, and Prince Sattam
Bin Abdulaziz University for providing facilities and technical support to perform this research work.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).