Design, synthesis, biological evaluation, and computational studies of novel thiazolo-pyrazole hybrids as promising selective COX-2 inhibitors: Implementation of apoptotic genes expression for ulcerogenic liability assessment

Article abstract of DOI:10.1016/j.bioorg.2021.104883

A novel series of thiazolo-pyrazole hybrids has been prepared and assessed for their in vitro COX-1/COX-2 inhibitory activity. Compound 6c exhibited the most selective COX-2 inhibition profile (SI of 264) not far of Celecoxib (2 9 4). In-vivo anti-inflamm

Full text of DOI:10.1016/j.bioorg.2021.104883

Bioorganic Chemistry 111 (2021) 104883
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, biological evaluation, and computational studies of novel
thiazolo-pyrazole hybrids as promising selective COX-2 inhibitors:
Implementation of apoptotic genes expression for ulcerogenic
liability assessment
,
Adel A. Marzouka, Ehab S. Taherb *, Montaser Sh. A. Shaykoona, Ping Lanc,
,
f
Walaa Hamada Abd-Allahd, Adel M. Aboregelae , Mohammed Farrag El-Behairyg
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, 71524 Assiut, Egypt
b Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, 71524 Assiut, Egypt
c Institute for Advanced and Applied Chemical Synthesis, Jinan University, Guangzhou 510632, China
d Pharmaceutical Chemistry Department, Collage of Pharmaceutical Science and Drug Manufacturing, Misr University for Science and Technology, P.O. 77, 6th of
October City, Giza, Egypt
e Basic Medical Science, College of Medicine, University of Bisha, Saudi Arabia
f Human Anatomy and Embryology, faculty of medicine, Zagazig University, Zagazig, Egypt
g Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Menoufiya 32897 Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
A novel series of thiazolo-pyrazole hybrids has been prepared and assessed for their in vitro COX-1/COX-2
inhibitory activity. Compound 6c exhibited the most selective COX-2 inhibition profile (SI of 264) not far of
Celecoxib (294). In-vivo anti-inflammatory activity revealed that compound 6d exhibited the highest activity
(97.30% inhibition of edema) exceeding reference standard Indomethacin (84.62% inhibition of edema). The
ulcerogenic liability tested, using gross, microscopic, biochemical analysis and apoptotic genes expression,
showed that compound 6b matched the optimal candidate activity (ulcer index = 120, selectivity index of ~ 162
and 77% in-vivo inhibition of edema). Meanwhile, compound 6 m (ulcer index = 0) showcased the highest safety
profile. Molecular modeling analysis and drug likeness studies presented appreciated agreement with the bio-
logical evaluation.
COX-2
Thiazolo-pyrazole hybrids
Molecular modeling
Apoptosis
Gene expression
Ulcerogenic liability
1. Introduction
such as antibacterial, anti-tuberculosis, anticancer, antidepressant,
antioxidant, antifungal, as well as antiviral agents [3,4]. Certainly, their
Dual insult of traditional non-steroidal anti-inflammatory drugs
(NSAIDs) includes direct erosion of gastric mucosa by acidic carboxylate
moiety and the harmful outcomes of constitutive COX-1 inhibition.
Unfortunately, it represents a major challenge in NSAIDs clinical use [1].
Several approaches have been applied to circumvent the unwanted ef-
fects of NSAIDs such as direct gastro-protection, enhancement of
mucosal defense mechanisms, and the development of safer NSAIDs.
Indeed, the most promising strategy was to innovate new safer agents
which exhibit not only substantial clinical usefulness, but also little to no
gastrointestinal adverse effects [2].
therapeutic potential for the treatment of inflammation with COX-2
selective inhibitory effect have been delivered to the clinic as safer
anti-inflammatory agents (e.g. Celecoxib) [5].
On the same note, thiazole moiety represents a crucial pharmaco-
phore that has been found in numerous biologically active molecules of
natural origin (e.g., thiamine, cystothiazole C). Moreover, it contains
synthetic compounds possessing various pharmacological activities such
as antimicrobial, antiviral, anti-tubercular, anti-inflammatory, anxio-
lytic, anesthetic, and anticonvulsant [6]. Furthermore, it is portrayed in
a large number of drugs that has gained application on clinical level and
was delivered to the market as anthelmintic (tiabendazole), antibacte-
rial (sulfathiazole), anticonvulsant (riluzole), anti-ulcer (alizatidine),
Literately, pyrazole scaffolds have been demonstrated as a spectac-
ular class of heterocycles that display diverse pharmacological activities,
* Corresponding author at: Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Assiut 71524, Egypt.
E-mail address: ehabtaher@azhar.edu.eg (E.S. Taher).
https://doi.org/10.1016/j.bioorg.2021.104883
Received 26 January 2021; Received in revised form 26 March 2021; Accepted 27 March 2021
Available online 31 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
antiparkinsonian (talipexole), antiviral (ritonavir), and anti-
inflammatory (meloxicam & fentiazac) (Fig. 1). [6–9].
were synthesized and their anti-inflammatory activity was evaluated in-
vivo utilizing a standard formalin-induced paw edema in rats and in-vitro
by COX-1/COX-2 enzyme inhibition assay. The most active candidates
were evaluated for ulcerogenic liability via gross, microscopic,
biochemical analyses and apoptotic genes expression and then
compared to Indomethacin and Celecoxib as reference standards. Mo-
lecular docking simulations were executed to investigate the binding
outline of compounds 6a-m in the active sites of COX-1, and COX-2
enzymes to study the binding modes and key interactions with the re-
ceptor in comparable to lead compound I.
Celecoxib, which represents the coxibs family, has the ability to
inhibit COX-2 isozyme selectively, thus, minifying the gastric ulcers
risks. Notably, higher specificity to COX-2 inhibition leads to higher
risks of cardiovascular (CV) side effects, possibly due to the prevention
of the biosynthesis of PGI2 that possess vasodilatory and anti-
aggregatory effects [10]. This remark is fostered by the fact that Cele-
coxib is the only coxib member that still approved by the Food and Drug
Administration (FDA) compared to other relevant ones (Rofecoxib,
Valdecoxib). The minimal CV toxicity of Celecoxib has been linked to its
higher percentage of COX-1 inhibition than other coxibs [11]. Subse-
quently, to insure optimal activity and safety, the pharmacological
profile dogma recommended that new anti-inflammatory drug should be
selective but not specific to COX-2 i.e. COX-1/COX-2 ratio to be more
than that of Meloxicam and less than that of Celecoxib. Noteworthy, the
advantages of combining thiazole with pyrazole in one compact struc-
ture, incorporating cyclic amide linker i.e. pyrazolone, produced non-
carboxylate candidates that showed potent and selective COX-2 inhibi-
tion [8]. In this regard, computationally predicted pharmacodynamic
profile recognized compound I as a lead candidate (Fig. 2).
2. Rational design of target compounds
To the best of our knowledge, the new functionality associated with
tethering open chain amide linker between the useful chirons thiazole
and pyrazole has not been explored yet. In continuation with the pre-
vious works [8], our design is principally based on a molecular hybrid
tactic of thiazole moiety with pyrazole while incorporating hydrazide
primes in one compact skeleton as shown in Fig. 2. Primarily, the pyr-
azolone nucleus in the lead compound I was bioisoterically replaced
with hydrazide moiety as a relevant open chain. Secondly, one of the
diaryl systems carried on the pyrazole scaffold was replaced by a small
fragment to study the electronic effect. The second aryl was modified
with variant substituents (where R = Me, Et & p-Cl-Ph) to investigate the
proper bioactive fragment core to the structure. Furthermore, the p-
Motivated by the above mentioned promising activity and selectivity
of thiazole-pyrazole hybrids [8], compound I was considered a lead
compound for rational design of a new set of 3,5-substituted-1,3-thiazo-
lopyrazole hybrids with novel linkers (Fig. 2). Thus compounds (6a-m)
Fig. 1. Pharmaceutical drugs containing pyrazole and thiazole units.
Fig. 2. Design of open chain analogues 6a-m (Phase II).
2

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Scheme 1. Synthesis of pyrazoles 4a-c.
Scheme 2. Synthesis of target compounds 6a-m.
chlorophenyl group located at position 4 was changed by another reli-
able hydrophobic methyl group at the same position within the thiazole
ring. Ultimately, the impact of the installment of the aliphatic and/or
aromatic fragments’ substituents at N-position within the later moiety
has been investigated. This new approach could be a good lead to
declare the role of the amidic NH group and highlight the impact of
converting an H-bond acceptor (in cyclic compounds like compound I)
to an H-bond donor (in open chain analogues 6a-m) which will help
better understand the detail of the interactions of 6a-m compounds with
COX-2 structural model and its consequences on the anti-inflammatory
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A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Table 1
Table 3
in-vitro COX-1/COX-2 enzyme inhibition assay of our compounds 6a-m, Cele-
Gross evaluation of ulcerogenic effects of celecoxib, indomethacin and the nine
chosen compounds with the highest anti-inflammatory effects in rats. (n = 5/
group).
coxib and Indomethacin.
Compounds
IC 50 (µM)a
SIb
COX-1
COX-2
Compound
Percentage
Ulcer score
Ulcer index
of ulceration
Celecoxib
14.70
14.30
0.10
0.05
0.03
0.08
0.07
0.07
0.05
0.08
0.17
0.07
0.14
0.27
0.23
0.07
0.06
0.31
0.29
294.00
476.67
1.25
Rofecoxib
Control
0
0.0 ± 0.00
0
Indomethacin
Celecoxib
0
0.0 ± 0.00
0
Indomethacin
100
100
20
3.8 ± 0.905 a,b
5.7 ± 0.185 a,b
1.2 ± 0.048 a,b,c
3.9 ± 0.332 a,b
4.6 ± 0.155 a,b
4.3 ± 0.644 a,b
1.1 ± 0.025 a,b,c
0.9 ± 0.014 a,b,c
3.2 ± 0.482 a,b
0.0 ± 0.00
380
570
120
390
460
430
110
90
6a
6b
6c
12.30
11.30
13.20
10.40
8.40
175.72
161.43
264.00
130
6a
6b
6c
6d
6e
6f
100
100
100
20
49.41
158.57
60.00
36.30
23.04
145.72
220.00
22.26
25.86
6d
6f
11.10
8.40
6 g
6 h
6i
6 h
6i
9.80
20
5.30
6 l
100
0
320
0
6j
10.20
13.20
6.90
6 m
6 k
6 l
6 m
a
significant to control.
b
c
significant to indomethacin.
significant to celecoxib.
7.50
a
IC50 value is the compound concentration required to produce 50% Inhibi-
tion of COX-1 or COX-2 for means of two determinations and Deviation from the
mean is <10% of the mean value.
thiosemicarbazides 5a-h [12], which was then subjected to successful
cyclization through reaction with appropriate phenacyl bromide de-
rivatives [13] as per method reported for the Hantzsch-thiazole syn-
thesis [14] gave the target compounds 6a-m (scheme 2).
b
SI: Selectivity index IC50 (COX-1)/IC50 (COX-2).
potential. This could help to further explore their mechanism of action
as well.
All novel compounds (6a-m) were confirmed by variant IR, NMR
spectroscopic and HRMS techniques where characteristic features were
–
–
–
–
identified. For instance, IR showed amidic NH, C O, and imine C N at
3. Results and discussion
3150–3200, 1650–1690, and 1550–1590 cmꢀ 1, respectively. 1H NMR
displayed characteristic thiazole and pyrazole protons at δH 6.0–6.2 and
5.5–5.8 ppm, respectively, while methylene bridge protons were
observed at δH 4.6–4.9 ppm and amidic NH at δH 8.0–10.0 ppm.
Regarding 13C NMR, a typical carbonyl carbon was the most de-shielded
carbon at δc 165–170 ppm followed by the imine carbon at δc 160–165
3.1. Chemistry
The synthesis of our rationalized thiazolo-pyrazole targets 6a-m in-
volves two key structural elements: (i) 2-(5-substituted-3-methyl-1H-
pyrazol-1-yl) acetohydrazide moiety (scheme 1), and (ii) instalment of
thiazole moiety (scheme 2). The two concise steps leading to 2-(5-
substituted-3-methyl-1H-pyrazol-1-yl)acetohydrazide (4a-c) are shown
in Scheme 1. Cyclocondensation of the appropriate 1,3-dicarbonyl
compounds 1a-c, which represent a three-carbon moiety including
two electrophilic carbons in a 1,3-relationship, with ethyl amino-
glycinate hydrochloride 2, which acts as a double nucleophile, led to the
formation of pyrazole ester derivatives 3a-c which then underwent
hydrazinolysis in alcoholic solution to afford the corresponding hydra-
zides 4a-c.
–
–
ppm. Furthermore, the C N of pyrazole were detected at δ 150 ppm.
c
HRMS spectra of all compounds were in accordance with their antici-
pated molecular mass (see experimental and supporting information
file).
3.2. Biology
3.2.1. In vitro COX-1/COX-2 enzyme inhibition Assay:
Compounds (6a, 6b, 6c, 6d, 6f, 6j, and 6 k) showed powerful COX-2
inhibitory activity with an IC50 of 0.05–0.08 µM similar to the reference
standards which showed IC50 of 0.05 and 0.08 µM, respectively. Besides,
Hydrazides 4a-c were reacted with appropriate isothiocyanate de-
rivatives to afford the corresponding key intermediates
Table 2
Results of in-vivo anti-inflammatory activity of compounds 6a-m, Celecoxib and Indomethacin.
1 h
SD
2 h
SD
3 h
SD
4 h
SD
Compound
Edema±
%Protection
Edema±
%Protection
Edema±
%Protection
Edema±
%Protection
SE (mm)
SE (mm)
SE (mm)
SE (mm)
6a
1.48 ± 0.13
0.97 ± 0.06
1.03 ± 0.10
0.87 ± 0.01
1.67 ± 0.03
1.87 ± 0.03
1.22 ± 0.03
1.4 ± 0.03
0.18
0.09
0.14
0.01
0.04
0.04
0.04
0.04
0.03
0.03
0.04
0.04
0.06
0.04
0.04
/
14.24
18.10
28.19
35.78
13.23
15.18
22.49
1.15
1.00 ± 0.10
0.86 ± 0.03
0.70 ± 0.03
0.74 ± 0.01
0.97 ± 0.02
1.29 ± 0.07
1.17 ± 0.03
1.31 ± 0.02
0.59 ± 0.01
1.14 ± 0.01
1.48 ± 0.01
1.36 ± 0.03
0.86 ± 0.01
1.58 ± 0.04
0.91 ± 0.04
2.08 ± 0.03
0.14
0.04
0.04
0.01
0.04
0.11
0.05
0.04
0.01
0.02
0.01
0.05
0.01
0.06
0.06
/
42.05
26.12
50.77
45.09
49.58
41.39
25.49
5.33
0.71 ± 0.06
0.49 ± 0.06
0.34 ± 0.06
0.28 ± 0.01
0.78 ± 0.05
1.15 ± 0.05
0.95 ± 0.05
1.09 ± 0.05
0.48 ± 0.02
1.1 ± 0.01
0.09
0.09
0.09
0.01
0.07
0.07
0.07
0.08
0.03
0.01
0.01
0.04
0.07
0.01
0.01
/
58.67
58.38
76.22
79.17
59.44
47.82
39.62
20.75
55.15
46.20
26.32
58.40
55.15
0
0.21 ± 0.09
0.26 ± 0.02
0.11 ± 0.05
0.03 ± 0.01
0.43 ± 0.02
0.59 ± 0.03
0.79 ± 0.05
0.22 ± 0.05
0.13 ± 0.03
0.72 ± 0.01
0.88 ± 0.03
0.23 ± 0.01
0.14 ± 0.03
2.06 ± 0.03
0.27 ± 0.03
2.52 ± 0.13
0.13
0.04
0.07
0.01
0.03
0.04
0.07
0.07
0.04
0.05
0.04
0.01
0.04
0.04
0.04
/
87.84
77.14
92.20
97.30
77.53
72.99
49.98
84.06
87.51
64.81
51.99
86.45
89.54
0
6b
6c
6d
6e
6f
6 g
6 h
6i
1.06 ± 0.02
1.33 ± 0.02
1.59 ± 0.03
1.65 ± 0.03
1.28 ± 0.04
1.2 ± 0.03
0.85
44.69
43.91
19.83
21.76
38.75
0
6j
34.61
13.89
5.16
6 k
1.36 ± 0.01
0.72 ± 0.03
0.63 ± 0.04
1.87 ± 0.01
0.75 ± 0.01
2.34 ± 0.16
6 l
6 m
9.07
Control
Indomethacin
Celecoxib*
0
1.19 ± 0.03
1.52 ± 0.15
34.04
26.90
49.22
36.50
58.09
40.60
84.62
36.60
*Adopted from reference [17].
4

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
(97.30% inhibition of edema) among tested compounds and reference
standards.
Table 4
Docking binding scores (-CDOCKER ENERGY) for the compounds with
higher affinity and best docking scores in the active site of COX-2 and the
reference compound celecoxib.
3.2.3. Ulcerogenic liability
Compound
Energy score(Kcal/mol)Cox-2 (1CVU)
3.2.3.1. Gross evaluation. Ulcerative percentages and indices were
assessed to all tested compounds and shown in Table 3. The control
stomachs were completely free of ulcers, either macroscopic or micro-
scopic, similar to that of celecoxib treated stomachs. Indomethacin
treated rats showed aggressive several ulcers in all stomachs with ulcers
score mean ± SD 3.8 ± 0.905. Compounds 6a, 6c, 6d, 6f and 6 l showed
high ulcer indices with significant difference towards control and Cel-
ecoxib. While compounds 6b, 6 h and 6i showed mild to moderate
mucosal injuries in some stomachs with an ulcer index significantly
different from indomethacin, control and celecoxib. Compound 6 m
compound showed no gross apparent lesions in the stomachs with 0.0
ulcer index.
6a
ꢀ 8.8
ꢀ 5.7
ꢀ 9.4
ꢀ 7.5
ꢀ 6.2
ꢀ 8.9
6b
6c
6j
6 k
Celecoxib
they displayed higher preference of COX-2 over COX-1 inhibition.
Compound 6c and 6 k were the most selective COX-2 ones with selec-
tivity indices (SIs) of 264 and 220, respectively, which are better than
that of indomethacin (1.25) and close to that of celecoxib (294). SIs of
compounds 6a, 6b, 6f, and 6j were in the range of 145–175 (Table 1).
3.2.2. In vivo anti-inflammatory evaluation
3.2.3.2. Light microscopic evaluation. Light microscopic examinations of
haematoxylin and eosin-stained specimens were carried out to confirm
the gross findings. It was interesting to describe different microscopic
grades of gastric ulcers mediated by different compounds. The gastric
wall in celecoxib treated group showed no difference when compared to
the control stomachs with clearly identifiable layers (mucosa, muscu-
laris mucosa, submucosa, musculosa and adventitia) and full thickness
The anti-inflammatory potential of 6a-m was estimated in-vivo by the
acute formalin induced paw edema standard method in rats [15,16] as
depicted in Table 2. Candidates 6a, 6b, 6c, 6d, 6i, 6 l, and 6 m revealed
greater anti-inflammatory properties (77.14 – 97.30% inhibition of
edema) compared to that of indomethacin (84.62% inhibition of edema)
after 4 hrs. Indeed, compound 6d demonstrated the highest activity
Fig. 3. Histopathological changes in the gastric mucosa after treatment with different compounds. (): the whole thickness of gastric mucosa. (): fissuring of the
mucosa. (): erosion and exfoliation of mucosa. (*): focal mucosal degeneration.
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A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Fig. 4. Levels of oxidative stress markers (MDA, CAT, SOD and GSH) in the stomach tissue of different tested groups. Values are presented as means ± SD. High
significance (***) when P value < 0.001, Moderate significant (**) at 0.01 > P value > 0.001, low significance (*) when 0.05 > P value > 0.01and non-significant
(ns) when p value > 0.05.
Fig. 5. Quantification of mRNA expression of apoptotic genes (Caspase3, BAX and BCL2) in gastric mucosa of different tested groups. Values are normalized to
β-actin and presented as means ± SD. High significance (***) when P value < 0.001, Moderate significant (**) at 0.01 > P value > 0.001, and low significance (*)
when 0.05 > P value > 0.01.
intact mucosa. Indomethacin affected extensively the gastric mucosa
that appeared highly eroded with congestion and fissuring. A similar
pathological picture was observed in the stomachs of compounds 6a and
6d treated groups with wide exfoliation of mucosa. The changes were
less in the stomachs treated with compounds 6c, 6f and 6 l which
showed little fissuring, congestion and localized mucosal injuries and
exfoliations in certain areas with maintenance of mucosal thickness and
integrity elsewhere. Indeed, the stomachs treated with 6b, 6 h and 6i
had better mucosa than the previous ones showing only localized lesions
in the form of tissue loss or exfoliation. It was amazing to see the
stomachs of the 6 m compound treated group with completely intact and
full thickness mucosa with uniform integrity and no injuries (Fig. 3).
and CAT activities were evaluated and the results were presented in
Fig. 4 and table S1. Indomethacin, 6a, 6c, 6d, 6f, and 6 l compounds
exerted strong oxidative stress on the gastric tissue that appeared in the
highly significant difference between the levels of antioxidants
compared to control. This might be the preliminary cause of the ulcers
found in the gross and microscopic examination. Compounds 6b, 6 h
and 6i expressed highly significant difference to control in all parame-
ters except in MDA activities in which they showed moderate to non-
significant difference with control. However, Celecoxib and 6 m com-
pounds displayed variable differences in the antioxidant contents and
activities ranging from moderately significant to non-significant in
comparable with control.
3.2.3.3. Biochemical analysis. The antioxidant activities were assessed
in order to explore the potential implication of oxidative stress in
induced gastric mucosal injuries. MDA and GSH content as well as SOD
3.2.3.4. Quantitative real time (PCR) RT-qPCR. To evaluate the degree
of mRNA expression of different apoptotic genes in the stomachs of the
four compounds that showed the least injury by gross, microscopic and
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A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Fig. 6. 2D-interaction for compound 6i in the active site of COX-1 (pdb : 3kk6), 2D-interaction for compounds 6c, compound I and celecoxib in the active site of
COX-2 (pdb: 1CVU).
biochemical analysis (6b, 6 h, 6i and 6 m), RT-qPCR was conducted
(Fig. 5 and table S2). Caspase3, BAX and BCL2 mRNA expressions were
quantified and normalized to β-actin. Celecoxib treated group showed a
very mild difference compared to the control group. However, the
stomachs of indomethacin treated group expressed the caspase3 and
BAX in a highly significant manner and showed very minute expression
of BCL2 which was also significantly different from control. All the
tested compounds showed variable levels of mRNA expression of the
nominated genes with very high significant difference to the control
except 6 m. It was interesting to see that 6 m treated stomachs with very
mild significantly different expression of caspase3 and BCL2 mRNA
although BAX showed moderate significant difference with control.
Compound I exhibited good interaction with the essential amino acids, it
showed H-bond interaction with Ser 530, Van der Waal interaction with
Arg 120, Pi-sulfur interaction with Tyr 385 and pi-alkyl interaction with
Val 523 which indicate its ability to interact with COX-2. Studying the
docking results of the target compounds on the active site of COX-1,
certain compounds showed good interaction. Compounds 6i and 6 l
showed good binding interaction on the active site of COX-1. Concerning
compound 6i, it showed hydrogen bond interaction with Ile 523 residue
which is crucial for binding in cox-1 active cite. Additionally, it
exhibited van der Waal interaction with essential amino acids such as
Arg 120, Ser 530 and Tyr 385. Furthermore, it showed pi-pi interaction
with Tyr 355 and pi-alkyl interaction with Ala 527, Leu 531, Val 349,
Tyr 385 and His 90 (Fig. 6).
From the molecular docking point of view, all the synthesized
compounds had higher affinity and docking score into the active site of
COX-2 comparing with Celecoxib and compound I. Compounds 6a-c, 6
k, and 6j exhibited the highest docking scores and best binding in-
teractions on the COX-2 active site. A comparative binding study for
compound 6c showed an H-bond interaction with Arg 120as one of the
joint shared conserved residues in binding pockets of COX-1 and COX-2.
As well as, van der Waal interaction with Ser 530 which represents the
crucial feature for most of COX-2 inhibitory complexes and Hist 90
which is an vital side pocket of the COX active site that formulate the
main connecting point which contributes to the substrate inhibitory
binding and Tyr-348 holds a very key therapeutic position since it is
located in the active site of COX-2. Additionally, it showed alkyl inter-
action with the key amino acid Val 523, Leu 352 whereasVal 349 is
characterized to play vital role in COX-2 inhibition [8] (Fig. 6). The
3.3. In silico studies
3.3.1. Molecular docking
Docking simulation study using CDOCKER protocol was demon-
strated via the selection of the X-ray crystallography of COX-1 and COX-
2 taken from PDB database www.rcsb.org (PDB code 3KK6 and 1CVU,
respectively) [18]. To indorse the suitability of the docking procedure,
docking of co-crystallized ligands was performed into the active sites of
COX-1 and COX-2 complexes [19]. Before investigating the results of
docking simulation, the sequence alignment of human COX-1 and COX-2
showed that, the major residues included in catalysis or heme binding
are, Arg-120, Tyr-385, His-386, His-206 and His-388 all conserved,
along with the residue which is acetylated by aspirin (Ser-530)
[8,20–22]. One difference was observed at cyclooxygenase binding site,
where the isoleucine at position 523 in COX-1 is a valine in COX-2 [20].
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A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Table 5
Pharmacokinetic effects of compounds 6a-m on the enzymes and transporter involved in the metabolism of the four FDA-approved PIs.
Compound
Pgp substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
6a
6b
6c
No
No
No
No
No
No
Yes
No
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
6d
6e
6f
6 g
6 h
6i
6j
6 k
6 l
6 m
Table 6
Predicted physicochemical properties of compounds 6a-m using SWISSADME server.
Compound
Lipinski’s rule of five (RO5)
#H-bond acceptors
#H-bond donors
MR
Consensus Log P
MW
Lipinski #violations
6a
6b
6c
3
3
3
3
3
3
3
3
3
3
3
4
4
1
1
1
1
1
1
1
1
1
1
1
1
1
104.52
104.56
109.32
109.37
124.99
125.03
142.14
130.04
147.15
147.11
150.27
151.8
3.33
3.45
3.67
3.85
4.3
369.48
389.9
0
0
0
0
0
0
2
0
2
2
2
2
2
383.51
403.93
431.55
451.97
506.06
486.42
540.51
520.09
548.49
564.49
544.07
6d
6e
6f
4.5
6 g
6 h
6i
5.45
5.04
6.05
5.73
6.17
5.81
5.54
6j
6 k
6 l
6 m
151.76
Table 7
Predicted ADME parameters, drug-likeness, and pharmacokinetic of compounds 6a-m using SWISSADME server.
Compound
Pharmacokinetics
GI absorption
Medicinal Chemistry
Lead
BBB permeant
Bioava-
ilability
Score
ESOL Class
solubility
Synthetic Accessibility
Drug likeness
Veber #violations
likeness #violations
(Lipinski
#violation)
6a
6b
6c
High
High
High
High
High
High
Low
High
Low
Low
Low
Low
Low
No
No
No
No
No
No
No
No
No
No
No
No
No
0.55
0.55
0.55
0.55
0.55
0.55
0.17
0.55
0.17
0.17
0.17
0.17
0.17
Moderate
Moderate
Moderate
Moderate
Moderate
Moderate
Poor
2
2
2
2
2
2
2
2
2
2
2
3
3
3.68
3.55
3.79
3.66
3.97
3.85
4.21
3.85
4.21
4.33
4.09
4.1
Yes
0
0
0
0
0
0
0
0
0
0
0
0
0
Yes
Yes
6d
6e
6f
Yes
Yes
Yes
6 g
6 h
6i
No (2)
Yes
Poor
Poor
No (2)
No (2)
No (2)
No (2)
No (2)
6j
Poor
6 k
6 l
6 m
Poor
Poor
Poor
4.21
presence of the amide moiety in our synthesized targets helped in for-
mation of H-bond with Arg120 residue of COX-2 which is highly
conserved among mammalian COX enzymes in comparable with lead
compound I.
expected to be inhibited by all compounds, while CYP1A2, and CYP2D6
are predicted to have no possibility of inhibition. Compounds 6 g, 6i,
and 6j were the only predicted substrates (inducers) of P-gp.
3.3.3. Measurement of pharmacokinetics properties and drug likeliness
All compounds were predicted not to cross the BBB while compounds
6a-f and 6 h were expected to be highly absorbed passively through the
GIT (Table 7). This could be retrieved to its moderate solubility,
meanwhile 6 g-m were poorly soluble. The pharmacokinetic properties
(ADME) of compounds 6a-m were also estimated based on Lipinski’s
rule of five from the scores got from SwissADME online server (Table 6).
Compounds 6a-f and 6 h did not violate Lipinski’s RO5 indicating good
oral bioavailability of these molecules (Table 6). Concerning descriptor
by Veber et al. [23], All our compounds fulfilled the descriptors of
3.3.2. Prediction of enzymes and transporters targets
SWISSADME server was used to predict the enzymes and transporters
involved in the metabolism of compounds 6a-m (Table 5). The SWIS-
SADME provided a prediction about the possible pharmacokinetic im-
pacts on some cytochrome P450 enzymes (CYP450) such as CYP1A2,
CYP2C19, CYP2C9, CYP2D6 and CYP3A4 and the probability to be
substrates (inducers) of Permeability glycoprotein (P-gp). The pharma-
cokinetic effects of compounds 6a-m on the Cytochrome P450 enzymes
and P-glycoprotein showed that CYP2C19, CYP2C9, and CYP3A4 are
8

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Fig. 7. Structural activity relationship (SAR) of the target thiazolopyrazole hybrids.
Veber’s rule, which designates these compounds as transportable
edema reduction of 92% and 97% with good SIs of 264 and 130,
respectively. However, their safety (expressed in high ulcer indices with
significant difference against control and celecoxib) was very low
compared to safer candidates such as 6b and 6 m. In a related vein, 6 m
demonstrated potent reduction in edema volume 89% with supreme
safety profile but its SI was very low to be considered as selective COX-2
candidate. The presence of electron donating substituents on both the
pyrazole (di-methyl) and the thiazole (tolyl) rings i.e. compound 6c
(where SI = 264) has confirmed the observations of Kamble et al that
electron donating substituent as possible pharmacophore for selective
inhibition of COX-2 [8]. While, the replacement of alky substituent with
electronegative one i.e. compound 6d led to a higher potency (97%
reduction of edema). The dropping SI of the later compound to 130
could be retrieved to the electron withdrawing moiety (p-Cl-phenyl
group) on thiazole ring which was also in-line with Kamble et. al ob-
servations [8]. The highest safety of 6 m probably due to the profitable
of an extra lipophilic and more electron donating group (anisyl) at the N-
position of thiazole moiety.Results declared that the structure–activity
relationships of our synthesized targeted hybrids are as following
(Fig. 7):
through membranes and can be considered latent drug molecules.
3.4. SAR study
All hybrids displayed high docking scores and good binding in-
teractions on the COX-2 active site. This could be possibly due to the
presence of the carbonyl and NH (i.e. open chain linkers) which helped
in the formation of further H-bond with the highly conserved Arg120
residue of mammalian COX-II enzymes when compared to lead com-
pound I(Fig. 2 and tableS3). Accordingly, selectivity of compounds (6a-
d, 6f, 6j, and 6 k) was confirmed by the in-vitro COX-1/COX-2 assay
through displaying good selectivity indices in the range 130–264.
Similarly, the in-vivo anti-inflammatory evaluation revealed that com-
pounds (6a-c and 6f) possess potent COX-2 selectivity. However, the
minimal in-vivo profile of compounds 6j and 6 k could be attributed to
their predicted ADME properties (table7) that demonstrated low GI
absorption, low bioavailability scores, poor solubility, and no drug
likeness via Lipinski’s violation. Surprisingly, compounds 6d, 6 h, 6i, 6 l
and 6 m displayed an experienced in-vivo activity albeit their low in-vitro
efficacy. The ulcerogenic liability study revealed that compound 6b was
one of the safest congeners (ulcer index = 120) with the beneficial of
high selectivity index of ~ 162 and 77% in-vivo inhibition of edema (in
contrast to 6 h and 6i where SI<50) while supreme safety profile of 6 m
which was found equal to that of Celecoxib (ulcer index = 0). Indeed,
both 6c and 6d offered potent in-vivo anti-inflammatory activity with
The substitutions on thiazole moiety (electron donating or with-
drawing) play a pivotal role in the selectivity and safety i.e. com-
pound 6 m.
Pharmacokinetics has a crucial role in the activity of tested com-
pounds i.e. compounds 6j and 6 k.
Fig. 8. Emerging lead compounds based on SAR study.
9

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Presence of methyl lipophilic group rather than chlorine atom at the
phenyl ring has dramatically increased selectivity towards COX-2
where R = R” = methyl and R’ = propyl i.e. 6c > 6b > 6a.
Docking study was very beneficial since all compounds that exhibi-
ted high docking score and good binding interaction on the COX-2
active site (6a-c, 6 k, and 6j), showed high selectivity indices.
commercially available and purchased from Sigma-Aldrich or Lancaster
Synthesis Corporation (U.K.).
5.1.1. General procedures for the preparation of 3a-c
Ethyl hydrazine acetate hydrochloride 2 (3.85 g, 25 mmol) was
dissolved in 8 mL distilled water neutralized with sodium bicarbonate,
then a solution of ß-diketones 1a-c (25 mmol) in glacial acetic acid (25
mL) was added. The reaction mixture was refluxed for 2 h and the
resulting solution was poured onto crushed ice, extracted with ethyl
acetate (3 × 25 mL) and dried over anhydrous sodium sulfate. The
resulting solution was evaporated to dryness under vacuum and purified
by column chromatography using ethyl acetate-petroleum-ether (b.p.
60–70 ◦C) mixture as 1:3 v/v, yields are 81% for 3a [24] [m.p 58–60 ◦C,
Lit., 60 ◦C], 78% for 3b [25] [m.p 62–64 ◦C], and 85% for 3c [26] [m.p
65 ◦C, Lit., 64–65 ◦C].
These findings postulate that compounds 6b (optimal candidate,
emerging lead), 6 m (most safe) 6c (most selective), and 6d (most
potent) could open the door for further development of potent, selective
and safe NSAIAs (Fig. 8).
4. Conclusion
A novel series of thiazolo-pyrazole hybrids 6a-m has been rational-
ized and synthesized. All the novel structures have been confirmed by
variant NMR spectroscopic, elemental analyses and HRMS techniques.
This new hybrid approach had the advantage of combining pyrazole and
thiazole rings via an open chain linker which hasn’t been explored so far
as selective COX-2 inhibitors. The ability of compounds 6a-m for se-
lective COX-2 inhibition was evaluated using in-vitro COX-1/COX-2
enzyme inhibition and in-vivo anti-inflammatory assays. Compound 6c
exhibited the most selective COX-2 inhibitory profile (SI of 264) not far
of Celecoxib (294). In-vivo anti-inflammatory activity revealed that
compound 6d possessed the highest activity (97.30% inhibition of
edema) surpassing reference standard indomethacin (84.62% inhibition
of edema). Furthermore, compounds that showed potential activity were
selected for ulcerogenicity testing using gross, microscopic, biochemical
analysis and apoptotic genes expression. The ethylthiazole- derivative
6b introduced as the optimal candidate (ulcer index = 120, selectivity
index of ~ 162 and 77% in-vivo inhibition of edema). The existence of an
extra lipophilic and more electron donating group (anisyl) at the N-
position of thiazole moiety i.e. 6 m has displayed the highest safety
profile. Molecular modeling study demonstrated high docking scores
and good binding interactions on the COX-2 active site for all the
rationalized compounds where the open chain linker fashioned addi-
tional H-bond with the highly conserved Arg120 residue of mammalian
COX-II enzymes. Overall, our novel hybrids have established the impact
of the open chain linker for such hybrids in order to higher COX-2
inhibitory activity. Indeed, compounds 6b-d and 6 m are likely to be
excitable leads for additional consideration to discover more about the
structure activity relationship and evolve novel potent, selective and
safe COX-2 inhibitors agent.
5.1.2. General procedures for the preparation of 4a-c
To a mixture of the appropriate 3a-c (5 mmol) in ethanol, hydrazine
hydrate (5 mmol, 100%) was added dropwise with continuous stirring.
The reaction mixture was then refluxed for 3 h and allowed to cool to
room temperature. The separated product was filtered, dried and crys-
tallized from ethanol. Yields for compounds 4a, b, and c were 87, 85,
and 85% and m.p. were 189–190, 164–167, 196–197 ◦C [12]
respectively.
5.1.3. Compounds 5a-h
Compounds 5a-h were prepared according to the reported method
[12].
5.1.4. General procedures for the preparation of 6a-m
To a solution of thiosemicarbazide 5a-h (3 mmol) and the appro-
priate phenacyl bromide derivatives (3 mmol) in absolute ethanol (20
mL), a catalytic amount of triethylamine (0.1 mL) was added. Then, the
reaction mixture was heated under reflux for 3 h, and was allowed to
cool. The precipitated product was filtered off, washed with ethanol,
dried, and finally recrystallized from ethanol.
5.1.4.1. 2-(3,5-diMethyl-1H-pyrazol-1-yl)-N’-(3-ethyl-4-(p-tolyl)thiazol-2
(3H)-ylidene)acetohydrazide (6a). Yellowish white solid, Yield: 70%; m.
p.: 110–112 ◦C; IR:
υ
= 3165 (NH), 3071 (CH, aromatic), 2992 (CH,
ꢀ 1
aliphatic), 1645 (C O), 1572 (C N), 1506 (C C) cm
.
1H NMR
–
–
–
–
–
–
[(CD3)2SO, 400 MHz] δ: 1.05 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.22 (s, 3H,
CH3), 2.37 (s, 3H, CH3), 3.67 (d, J = 5.5 Hz, 2H, CH2CH3), 4.67 (s, 2H,
CH2), 5.81 (s, 1H, pyrazole H-4), 6.17 (s, 1H, thiazole, H-5), 7.26–7.40
(m, 4H, Ar-H) 10.18 (s, 1H, NH) ppm. 13C NMR [(CD3)2SO, 100 MHz] δ:
10.72, 12.61, 13.28, 20.82, 50.06, 59.66, 96.54, 104.90, 127.80,
127.95, 128.65, 129.38, 138.61, 138.95, 139.91, 145.80, 153.78,
161.26, 168.02 ppm. HRMS (M + Na)+ calcd for C19H23N5OSNa
392.1521, found 392.1521.
5. Experimental
5.1. Chemistry
Melting points were determined with a Gallenkamp (London, U.K.)
melting point apparatus and are uncorrected. Infrared spectra (νmax)
were recorded on Bruker Vector, 22FT-IR [Fourier Transform Infrared
(FTIR), Germany] spectrometer. 1H NMR and 13C NMR spectra were
recorded on a Varian Gemini-400 (400 MHz, Foster City, Calif., USA)
spectrometer using chloroform (CDCl3),) dimethylsulphoxide and/or
(DMSO /D2O) as solvents and tetramethylsilane (TMS) as an internal
standard (chemical shift in δ, ppm). 1H NMR data are recorded as fol-
lows: chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), rela-
tive integral] where multiplicity is defined as s = singlet; d = doublet; t
= triplet; q = quartet; and m = multiplet or combinations of the above.
High-resolution measurements were conducted on a time-of-flight in-
strument. All the results of elemental analyses corresponded to the
calculated values within experimental error. reaction progress was
observed by thin-layer chromatography (TLC) using precoated TLC
sheets with Ultraviolet (UV) fluorescent silica gel (Merck 60F254) and
spots were visualized by irradiation with UV light (254 nm) or iodine
vapors. All the starting materials and reagents were generally
5.1.4.2. N’-(4-(4-Chlorophenyl)-3-ethylthiazol-2(3H)-ylidene)-2-(3,5-
dimethyl-1H-pyrazol-1-yl) Acetohydrazide (6b). Yellowish white solid,
Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3168 (NH), 3064 (CH, aromatic),
2913 (CH, aliphatic), 1646 (C O), 1588 (C N), 1486 (C C) cmꢀ 1. 1H
–
–
–
–
–
–
NMR (CDCl3, 400 MHz) δ: 1.15 (t, 3H, J = 6.8 Hz, CH3CH2), 2.29 (s, 3H,
CH3), 2.31 (s, 3H, CH3), 3.80 (dd, J = 27.7, 7.1 Hz, 2H, CH2), 4.80 (s, 2H,
CH2), 5.84 (s, 1H, pyrazole H-4), 5.93 (s, 1H, thiazole, H-5), 7.27–7.45
(m, 4H, Ar-H) 8.29 (s, 1H, NH) ppm. 13C NMR (CDCl3, 100 MHz) δ:
11.21, 13.37, 13.64, 41.02, 51.49, 97.41, 106.41, 128.21, 129.27,
130.35, 135.79, 140.22, 141.07, 149.76, 163.37, 165.90 ppm. HRMS
(M + H)+ calcd for C18H21Cl35N5OS 390.1155, found 390.1158 and
C
18H21Cl37N5OS 392.1126, found 392.1133.
5.1.4.3. 2-(3,5-diMethyl-1H-pyrazol-1-yl)-N’-(3-propyl-4-(p-tolyl)
thiazol-2(3H)-ylidene)aceto hydrazide (6c). Yellowish white solid,
10

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Yield: 70%; m.p.: 110–112 ◦C; IR:
υ
= 3173 (NH), 3071 (CH, aromatic),
508.1752, found 508.1756.
2920 (CH, aliphatic), 1652 (C O), 1595 (C N), 1460 (C C) cmꢀ 1. 1H
–
–
–
–
–
–
NMR [(CD3)2SO, 400 MHz] δ: 0.65 (t, J = 6.0 Hz, 3H CH2CH2CH3),
1.42–1.65 (m, 2H, CH2CH2CH3), 2.08 (s, 3H, CH3), 2.22 (s, 3H, CH3),
2.37 (s, 3H, CH3), 3.65–3.71 (complex m, 2H, –CH2CH2CH3), 4.67 (s,
2H, CH2), 5.81 (s, 1H, pyrazole H-4), 6.17 (s, 1H, thiazole, H-5),
7.25–7.40 (m, 4H, Ar-H) 10.18 (s, 1H, NH) ppm. 13C NMR [(CD3)2SO,
100 MHz] δ: 10.72, 10.79, 13.28, 20.11, 20.85, 46.44, 50.21, 96.61,
104.75, 128.03, 128.62, 129.36, 138.80, 139.83, 140.41, 145.91,
162.61, 166.82 ppm. HRMS (M + Na)+ calcd for C20H25N5OSNa
406.1678, found 406.1675.
5.1.4.8. N’-(4-(4-Chlorophenyl)-3-ethylthiazol-2(3H)-ylidene)-2-(5-(4-
chlorophenyl)
ꢀ 3-methyl-1H-pyrazol-1-yl)acetohydrazide
(6
h).
Yellowish white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3144 (NH),
–
–
–
3077 (CH, aromatic), 2940 (CH, aliphatic), 1649 (C O), 1562 (C N),
–
1488 (C C) cmꢀ 1.1H NMR (CDCl3, 400 MHz) δ: 1.15 (t, J = 6.8 Hz, 3H,
–
–
–CH3CH2), 2.36 (s, 3H, CH3), 3.80 (dd, J = 13.6, 6.7 Hz, 2H, –CH2CH3),
4.81 (s, 2H, CH2), 5.83 (s, 1H, pyrazole H-4), 6.19 (s, 1H, thiazole, H-5),
7.28–7.43 (m, 8H, Ar-H), 8.57 (s, 1H, NH) ppm. 13C NMR (CDCl3, 100
MHz) δ: 13.26, 13.61, 40.95, 51.95, 97.29, 106.77, 129.17, 129.22,
130.10, 130.24, 135.09, 139.73, 143.44, 144.70, 149.99, 151.17,
156.16, 169.50, 170.41 ppm. HRMS (M + H)+ calcd for C23H22Cl325N5OS
486.0922, found 486.0920 and C23H22Cl327N5OS 488.0893, found
488.0893.
5.1.4.4. N’-(4-(4-Chlorophenyl)-3-propylthiazol-2(3H)-ylidene)-2-(3,5-
dimethyl-1H-pyrazol-1-yl) Acetohydrazide (6d). Yellowish white solid,
Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3167 (NH), 3061 (CH, aromatic),
2973 (CH, aliphatic), 1648 (C O), 1587 (C N), 1485 (C C) cmꢀ 1. 1H
–
–
–
–
–
–
NMR (CDCl3, 400 MHz) δ: 0.47 (t, 3H, CH2CH2CH3), 1.32–1.37 (com-
plex m, 2H, CH2CH2CH3), 2.03 (s, 3H, CH3), 2.05 (s, 3H, CH3),
3.43–3.47 (m, 2H, CH2CH2CH3), 4.53 (s, 2H, CH2), 5.56 (s, 1H, pyrazole
H-4), 5.66 (s, 1H, thiazole, H-5), 7.00–7.18 (m, 4H, Ar-H) 7.98 (s, 1H,
NH) ppm. 13C NMR (CDCl3, 100 MHz) δ: 11.04, 11.15, 13.63, 20.92,
47.28, 51.47, 97.19, 106.33, 129.17, 129.27, 129.56, 130.29, 135.65,
140.45, 140.86, 149.66, 163.84, 166.86 ppm. HRMS (M + H)+ calcd for
5.1.4.9. N’-(4-(4-Chlorophenyl)-3-cyclohexylthiazol-2(3H)-ylidene)-
2-(5-(4-chloro phenyl)-3-methyl-1H -pyrazol-1-yl)acetohydrazide
(6i). Yellowish white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3192
–
(NH), 3070 (CH, aromatic), 2929 (CH, aliphatic), 1676 (C O), 1557
–
(C N), 1490 (C C) cmꢀ 1. 1H NMR [(CD3)2SO, 400 MHz)] δ: 1.02–1.70
–
–
–
–
(m, 10H, cyclohexyl), 2.20 (s, 3H, CH3), 2.43–2.59 (complex m, 1H,
cyclohexyl), 4.69 (s, 2H, CH2), 6.22 (s, 1H, thiazole, H-5), 7.42–7.64 (m,
9H, 8Ar-H + 1H, thiazole, H-4), 10.37 (s, 1H, NH) ppm. 13C NMR
(CDCl3, 100 MHz) δ: 13.26, 13.65, 13.74, 21.34, 40.91, 51.91, 96.54,
106.56, 127.96, 128.64, 128.83, 128.89, 128.93, 129.47, 129.66,
132.20, 139.24, 139.60, 141.56, 141.62, 146.09, 149.97, 163.25,
166.34 ppm. HRMS (M + H)+ calcd for C27H28Cl235N5OS 540.1392,
found 540.1392 and C27H28Cl327N5OS 544.1333, found 544.1348.
C
19H23Cl35N5OS 404.1312, found 404.1309 and C19H23Cl37N5OS
406.1282, found 406.1281.
5.1.4.5. N’-(3-Ethyl-4-(p-tolyl)thiazol-2(3H)-ylidene)-2-(3-methyl-5-
phenyl-1H-pyrazol-1-yl)aceto Hydrazide (6e). Yellowish white solid,
Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3162 (NH), 3072 (CH, aromatic),
2932 (CH, aliphatic), 1651 (C O), 1558 (C N), 1495 (C C) cmꢀ 1. 1H
–
–
–
–
–
–
NMR [(CD3)2SO, 400 MHz)] δ: 1.06 (t, J = 6.7 Hz, 3H, –CH3CH2), 2.21
(s, 3H, CH3), 2.37 (s, 3H, CH3), 3.68 (dd, J = 12.8, 5.97 Hz, 2H,
–CH2CH3), 4.69 (s, 2H, CH2), 6.19 (s, 1H, pyrazole H-4), 6.20 (s, 1H,
thiazole, H-5), 7.32–7.59 (m, 9H, Ar-H), 10.26 (s, 1H, NH) ppm. 13C
NMR [(CD3)2SO, 100 MHz)] δ: 13.16, 13.77, 21.32, 36.98, 51.08, 97.23,
105.88, 128.38, 128.58, 128.94, 129.18, 129.86, 130.81, 139.36,
140.61, 141.06, 144.94, 145.18, 147.38, 163.28, 166.97, 175.84 ppm.
HRMS (M + Na)+ calcd for C24H25N5OSNa 454.1678, found 454.1685.
5.1.4.10. 2-(5-(4-Chlorophenyl)-3-methyl-1H-pyrazol-1-yl)-N’-(3-cyclo-
hexyl-4-(p-tolyl)thiazol-2(3H)-ylidene)acetohydrazide
(6j). Yellowish
white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3185 (NH), 3026 (CH,
–
–
–
aromatic), 2924 (CH, aliphatic), 1674 (C O), 1592 (C N), 1493
–
ꢀ 1
–
(C C) cm
–
.
1H NMR (CDCl3, 400 MHz) δ: 1.04–1.77 (m, 10H, cyclo-
hexyl), 2.37 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.47–2.62 (complex m, 1H,
cyclohexyl), 4.81 (s, 2H, CH2), 5.70 (s, 1H, pyrazole H-4), 6.20–7.46 (m,
9H, 8Ar-H + 1H, thiazole, H-5), 8.43 (s, 1H, NH) ppm. (CDCl3, 100 MHz)
δ: 13.63, 21.36, 24.79, 25.89, 26.00, 28.19, 28.42, 52.05, 59.41, 96.18,
106.68, 128.73, 128.82, 128.89, 129.19, 129.37, 129.50, 130.13,
135.10, 139.34, 142.46, 144.74, 150.01, 163.24, 168.94 ppm. HRMS
(M + H)+ calcd for C28H30Cl35N5OS 520.1938, found 520.1929 and
5.1.4.6. N’-(4-(4-Chlorophenyl)-3-ethylthiazol-2(3H)-ylidene)-2-(3-
methyl-5-phenyl-1H-pyrazol-1-yl)acetohydrazide
(6f). Yellowish
white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3150 (NH), 3072 (CH,
–
–
–
aromatic), 2936 (CH, aliphatic), 1651 (C O), 1562 (C N), 1490
C28H31Cl37N5OS 522.1908, found 522.1909.
–
ꢀ 1
–
(C C) cm
–
.
1H NMR (CDCl3, 400 MHz) δ: 1.17 (t, J = 7.0 Hz, 3H,
–CH3CH2), 2.39 (s, 3H, CH3), 3.82 (q, J = 6.9, Hz, 2H, –CH2CH3), 4.87
(s, 2H, CH2), 5.84 (s, 1H, pyrazole H-4), 6.22 (s, 1H, thiazole, H-5),
7.28–7.45 (m, 9H, Ar-H), 8.63 (s, 1H, NH) ppm. 13C NMR (CDCl3, 100
MHz) δ: 13.28, 13.64, 40.92, 51.89, 97.30, 106.57, 128.82, 128.94,
129.15, 129.32, 129.63, 130.23, 135.70, 140.15, 146.12, 150.01,
163.71, 166.26 ppm. HRMS (M + H)+ calcd for C23H23Cl35N5OS
452.1312, found 452.1313 and C23H23Cl37N5OS 454.1282, found
454.1289.
5.1.4.11. N’-(4-(4-Chlorophenyl)-3-(m-tolyl)thiazol-2(3H)-ylidene)-2-(5-
(4-chloro phenyl)-3-methyl-1H-pyrazol-1-yl)acetohydrazide (6 k).
Yellowish white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ
= 3195 (NH),
–
–
–
3112 (CH, aromatic), 2952 (CH, aliphatic), 1676 (C O), 1591 (C N),
–
1504 (C C) cmꢀ 1. 1H NMR (CDCl3, 400 MHz) δ: 2.39 (s, 3H, CH3), 3.83
–
–
(s, 3H, OCH3), 4.84 (s, 2H, CH2), 6.12 (s, 1H, pyrazole H-4), 6.22 (s, 1H,
thiazole, H-5), 7.09–7.49 (m, 12H, Ar-H) 8.69 (s, 1H, NH) ppm. 13C NMR
(CDCl3, 100 MHz) δ: 13.56, 51.97, 55.39, 98.31, 106.71, 114.59,
114.82, 127.59, 128.08, 128.65, 128.87, 129.03, 129.19, 129.28,
129.38, 129.45, 129.57, 130.10, 134.75, 135.14, 139.88, 144.72,
150.03, 159.10, 163.19, 168.87, 169.16 ppm. HRMS (M + H)+ calcd for
5.1.4.7. N’-(4-(4-Chlorophenyl)-3-cyclohexylthiazol-2(3H)-ylidene)-
2-(3-methyl-5-phenyl-1H-pyrazol-1-yl)acetohydrazide
(6
g).
Yellowish white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ
= 3204 (NH),
C
28H24Cl235N5O2S 564.1028, found 528.1036 and C28H24Cl237N5O2S
–
–
–
3059 (CH, aromatic), 2929 (CH, aliphatic), 1676 (C O), 1585 (C N),
566.0998, found 566.1007.
–
1489 (C C) cmꢀ 1.1H NMR [(CD3)2SO, 400 MHz)] δ: 1.02–1.71 (m, 10H,
–
–
cyclohexyl), 2.20 (s, 3H, CH3), 2.47–2.62 (complex m, 1H, cyclohexyl),
4.69 (s, 2H, CH2), 6.20 (s, 1H, pyrazole H-4), 6.22 (s, 1H, thiazole, H-5),
7.35–7.58 (m, 9H, 9Ar-H), 10.33 (s, 1H, NH) ppm. 13C NMR [(CD3)2SO,
100 MHz)] δ: 13.77, 24.78, 25.03, 25.93, 27.95, 32.43, 51.00, 59.06,
98.55, 105.85, 128.69, 128.98, 129.19, 129.42, 130.82, 131.01, 134.45,
140.27, 145.24, 147.41, 163.28, 167.69 ppm. HRMS (M + H)+ calcd for
5.1.4.12. N’-(4-(4-Chlorophenyl)-3-(4-methoxyphenyl)thiazol-2(3H)-yli-
dene)-2-(5-(4-chlorophenyl)-3-methyl-1H-pyrazol-1-yl)acetohydrazide (6
l). Yellowish white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3185
–
(NH), 3071 (CH, aromatic), 2927 (CH, aliphatic), 1676 (C O), 1598
–
ꢀ 1
(C N), 1504 (C C) cm
.
1H NMR (CDCl3, 400 MHz) δ: 2.29 (s, 3H,
–
–
–
–
CH3), 2.33 (s, 3H, CH3), 3.77 (s, 3H, OCH3), 4.78 (s, 2H, CH2), 6.01 (s,
C
27H29Cl35N5OS 506.1781, found 506.1781and C27H29Cl37N5OS
1H, pyrazole H-4), 6.17 (s, 1H, thiazole, H-5), 6.98–7.43 (m, 12H, Ar-H)
11

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
8.57 (s, 1H, NH) ppm. 13C NMR (CDCl3, 100 MHz) δ: 13.66, 21.33,
52.12, 55.47, 97.24, 106.80, 114.52, 114.84, 123.14, 128.13, 128.22,
128.36, 129.03, 129.12, 129.28, 129.34, 129.61, 129.90, 130.21,
evaluated statistically. Quantitative variables from normal distribution
were represented as means ± standard error. The anti-inflammatory
activity was calculated as percentage inhibition of edema volume in
treated animals compared to the control group following the equation:
159.01, 163.26, 169.54 ppm. HRMS (M
+
H)+ calcd for
C29H27Cl35N5O2S 544.1574, found 544.1572 and C29H27Cl37N5O2S
546.1544, found 546.1544.
˜
%inhibition = ((Vc ꢀ Vt)A⋅Vc)X100
Vc: the mean of edema volume of the rat paw in the control group
after injection of formalin,
5.1.4.13. 2-(5-(4-Chlorophenyl)-3-methyl-1H-pyrazol-1-yl)-N’-(3-(4-
methoxyphenyl)-4-(p-tolyl)thiazol-2(3H)-ylidene)acetohydrazide (6 m).
Vt : the mean of edema volume after formalin injection of rat paw in
the tested and reference groups.
Yellowish white solid, Yield: 70%; m.p.: 110–112 ◦C; IR:
υ = 3195 (NH),
–
–
–
3030 (CH, aromatic), 2932 (CH, aliphatic), 1692 (C O), 1573 (C N),
–
1494 (C C) cmꢀ 1.1H NMR (CDCl3, 400 MHz) δ: 2.32 (s, 3H, CH3), 4.73
–
–
5.2.4. Statistical analysis
(s, 2H, CH2), 6.06 (s, 1H, pyrazole H-4), 6.16 (s, 1H, thiazole, H-
5),7.11–7.34 (m, 12H, Ar-H), 8.21 (s, 1H, NH) ppm. HRMS (M + H)+
calcd for C27H21Cl325N6O3S 579.0773, found 579.0776 and
C27H21Cl327N6O3S 581.0743, found 581.0751.
All mentioned computations were performed by using Microsoft
Excel. One-way ANOVA followed by Tukey’s posttest was used to
compare applied doses of each compound with the control group. P <
0.05 was considered significant in all tests.
5.2.5. Ulcerogenic liability
5.2. Biology
5.2.5.1. Experimental animals and dosing. The rats were divided
randomly into twelve equal groups (5/ group). The first group (control)
was administered 1% gum acacia (suspending vehicle), the second
group was dosed with celecoxib at a dose of 18 mg/kg/day, while the
third group was received indomethacin at a dose of 18 mg/kg/day. The
remaining nine groups were treated with the nine chosen compounds
with the highest anti-inflammatory effects (6a, 6b, 6c, 6d, 6f, 6 h, 6i, 6l
and 6 m) at a dose of 18 mg/kg/day. All the drugs were administered via
oral gavage once a day for three successive days after fasting for 18 h
every day before dosing. Six hours after the last dose, the animals were
anesthetized by over dosage of ether and the stomachs were rapidly
removed and opened along the greater curvature. The stomachs were
washed with cold saline to remove the contents so as to evaluate the
extent of gastric damage.
5.2.1. Materials
Healthy adult male albino rats (aged 2:3 months and weighted from
120 to 130 gm) of Wister strain were obtained from the animal house of
Zagazig Scientific and Medical Research Center (ZSMRC), Egypt. All rats
were kept ad libitum for two weeks in groups of 5 per standard special
cages made from plastic to accommodate at [Temperature: 24–26 ◦C;
and in a 12 h light/12 h dark cycle]. Human COX-1 and COX-2 inhibitor
screening kit supplied by Cayman chemicals (catalog number 701,070
and 701080, respectively, Ann Arbor, MI, USA). Buffer solution for COX-
1/COX-2 inhibition test (160 mL, 0.1 M TriseHCl, pH 8 containing 5 mM
ethylenediamine tetra acetate (EDTA) and 2 mM phenol). Ellman’s re-
agent (200 mL), a reagent that contains substrate to acetyl cholines-
terase. Animals’ treatment protocol was approved by the ethical
committee of Misr University of Science and Technology (approval ID
MC4).
5.2.5.2. Gross evaluation. The ulcerogenic activity was checked using
celecoxib and indomethacin as reference drugs [27,28]. The percentage
of ulcerated stomachs, the ulcer scores and the ulcer index were calcu-
lated (Table 2) according to the scoring system previously described
[29]. In brief, the ulcers were scored 0 when normal colored stomach,
0.5 if hyperemic, 1 with spot ulcer, 1.5 if had hemorrhagic streak, 2
when deep ulcers are present and 3 if perforated. Ulcer score = the mean
of scores in each group, and the ulcer index (UI) = ulcer score * 100. The
prevention percentage = [(UI control-UI test) /UI control] × 100 [30].
5.2.2. In vitro COX-1 and COX-2 inhibitory [17]
Ability of compounds 6a-m, celecoxib and indomethacin to inhibit
human COX-1 and COX-2 (IC50 value, µM) was estimated using human
COX-1 and COX-2 inhibitor screening kit. Solution of compounds 6a-m,
celecoxib and indomethacin in dimethylsulfoxide (DMSO), enzyme
(COX-1 or COX-2, 10 mL), heme (10 mL) and buffer solution were kept
at 37 ◦C. After 10 min, arachidonic acid (10 mL, final concentration in
reaction mixture 100 mM) was added. Two mins later, COX reactions
were stopped by stannous chloride (30 mL) then incubated for 5 min at
room temperature. Enzyme-linked immunosorbent assay (ELISA) was
5.2.5.3. Light microscopic evaluation. Random stomach samples were
selected from each group and placed in 10% formalin at room temper-
ature to be processed and inserted in paraffin. Serial sections from the
used to quantitatively estimate PGF2α formed in the samples. Sampled
were then transfered to a 96-well plate and stored at room temperature.
After 18 hrs, unbound reagent were washed and then Ellman’s reagent
(200 mL) was added and incubated at rt for 1.0–1.5 hrs till the absor-
bance of B0 well is in the range 0.3–0.8 A U at 410 nm. The IC50 values
were estimated by an ELISA plate reader via the comparison of the
sample treated incubations to control incubations.
stomachs of 4 –5 μm thickness were set, mounted on separate slides and
stained with hematoxylin and eosin [31]. All sections were examined
and photographed using Optika B-510–5 microscope with FAST3 cam-
era in Bisha University.
5.2.5.4. Biochemical analysis. Some stomach tissue from different
groups was immediately homogenized in Tris buffer (20 mM, pH 7.5) on
ice. Homogenates were filtered and centrifuged at 11,940 g at 4 ◦C for
10 min. Malonaldehyde level (MDA), Superoxide dismutase activity
(SOD), glutathione content (GSH), and Catalase activity (CAT) were
determined in the supernatants. MDA and GSH were measured accord-
ing to [32] while, SOD and CAT activities were determined following
what was described by [33].
5.2.3. Formalin-induced paw edema test [15,16]
The anti-inflammatory activity of compounds (6a-m), celecoxib and
indomethacin were evaluated using the rat paw edema test using
formalin as a phlogistic agent. Untreated rats hind paw volume was
calculated using plethysmometer (7150 UGO Basile, Varese, Italy). Af-
terwards, inflammation and edema were induced by injection of 40 μl
formalin 5% sub-plantarly in the paw. Compounds 6a-m, and reference
standards were administered orally as suspension in 1% gum acacia at a
dose of 18 mg/kg/day and only gum acacia was administered for control
group. plethysmometer used to estimate volumes of paw edema in time
intervals of 1, 2, 3 and 4 hrs. For each time interval, the difference of
average values for treated and control groups was calculated and
5.2.5.5. Quantitative real time (PCR) RT-qPCR. Total RNA was
extracted from gastric tissue homogenate using RNA Mini kit according
to the manufacturer’s protocol (Qiagen RNeasy, Germany). The ho-
mogenates were derived from seven groups, the control, celecoxib,
12

A.A. Marzouk et al.
Bioorganic Chemistry 111 (2021) 104883
Declaration of Competing Interest
Table 8
Primer sequences of Caspase3, BAX, BCL2 and β-Actin.
The authors declared that there is no conflict of interest.
Gene
Sequence
Caspase3
Forward primer 5′ ATGGACAACAACGAAACCTC3′
Reverse primer 5′ TTAGTGATAAAAGTACAGTTCTT3′
Forward: 5′- TTG CTA CAG GGTTTCATC CA ꢀ 3′
Reverse: 5′- GAG TAC CTG AACCGG CAT CT ꢀ 3′
Forward: 5′-ATGTGTGTGGAGAGCGTCAA-3′
Reverse: 5′-AGAGACAGCCAGGAGAAATCA-3′
Forward: 5′-CAGGGCGTGATGGTGGGCATG-3′
Reverse: 5′-GCCACACGCAGCTCATTGTA-3′
Acknowledgment
Bax
We are sincerely thankful to Heba Omar Marzouk, School of
Pharmacy, Mississippi University, for manuscript proofreading.
BCL2
β-Actin
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104883.
indomethacin and the four compounds that had the lower most ul-
cerogenic effects histologically and biochemically (6b, 6 h, 6i and 6 m).
cDNA was synthesized and expression levels of mRNA were determined
by Stratagene, MX3000P quantitative (PCR) System (Agilent Technol-
ogies) and analyzed using MxPro QPCR Software (Agilent Technolo-
gies). The PCR was performed as described by [34] by adding 2.5 µl 2x
QuantiFast (PCR) Master Mix, 1 µM of each primer and 5 µl cDNA in the
following conditions: 95˚C for 5 min, then 40 cycles at 94˚C for 30 sec, and
combined annealing and extension (60˚C) for 60 sec. Melting curve
analysis was performed from 60 ◦C to 95 ◦C. Duplicates were performed
to confirm the accuracy and reproducibility of the results. Primers used
for Caspase3, BAX, BCL2 and β-Actin as housekeeping gene were
designed according to [35–37] (table 8).
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