Novel benzenesulfonamide and 1,2-benzisothiazol-3(2H)-one-1,1-dioxide derivatives as potential selective COX-2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.03.042

Two new series of 1,2-benzisothiazol-3(2H)-one-1,1-dioxide derivatives containing either five membered heterocyclic rings or aryl hydrazones were synthesized and evaluated for their in vitro COX-1/COX-2 inhibitory activity. In vivo anti-inflammatory evalu

Full text of DOI:10.1016/j.ejmech.2019.03.042

European Journal of Medicinal Chemistry 171 (2019) 372e382
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel benzenesulfonamide and 1,2-benzisothiazol-3(2H)-one-1,1-
dioxide derivatives as potential selective COX-2 inhibitors
,
,
,
Ehab S. Taher a *, Tarek S. Ibrahim b c, Mohamed Fares d e, Amany M.M. AL-Mahmoudy c,
Abdullah F. Radwan f, Khaled Y. Orabi g, Osama I. El-Sabbagh h
,
i
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, 71524, Assiut, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, King Abdulaziz University, 21589, Saudi Arabia
c Pharmaceutical Organic Chemistry Department Faculty of Pharmacy, Zagazig University, Zagazig, 44519, Egypt
d School of Chemistry, University of Wollongong, Wollongong, 2522, NSW, Australia
e School of Chemistry, The University of Sydney, 2006, NSW, Australia
f Biochemistry Department, Faculty of Pharmacy, Egyptian Russian University, 11829, Cairo, Egypt
g Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Health Sciences Center, Kuwait University, 13110, Safat, Kuwait
h Pharmaceutical Chemistry Department, Faculty of Pharmacy, Taif University, 11099, Taif, Saudi Arabia
i Medicinal Chemistry Department, Faculty of Pharmacy, Zagazig University, 44519, Zagazig, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new series of 1,2-benzisothiazol-3(2H)-one-1,1-dioxide derivatives containing either five membered
heterocyclic rings or aryl hydrazones were synthesized and evaluated for their in vitro COX-1/COX-2
inhibitory activity. In vivo anti-inflammatory evaluation revealed that benzenesulfonamides bearing
pyrazole moiety 19, 20 and its cyclized form 23 exhibited the highest anti-inflammatory activity with
comparable potency to celecoxib. Furthermore, the ulcerogenic activity evaluation showed that com-
pounds 19, 20 and 23 exerted the minimal ulcer index in comparison to indomethacin as a reference
drug. Docking studies of the most selective COX-2 derivatives were also carried out against COX-2 active
site. Benzenesulfonamide derivatives 19 and 20 displayed higher predicted binding affinities inside the
COX-2 active site. Molecular modelling simulation and drug likeness studies showed good agreement
with the obtained biological evaluation.
Received 15 December 2018
Received in revised form
1 March 2019
Accepted 16 March 2019
Available online 20 March 2019
Keywords:
Anti-inflammatory
COX-2
Benzenesulfonamide
Benzisothiazol
Saccharin
Crown Copyright © 2019 Published by Elsevier Masson SAS. All rights reserved.
Celecoxib
1. Introduction
succeeded in the synthesis of new NSAIDs as selective COX-2 in-
hibitors, namely “coxibs” to minimise the danger of gastric ulcers
Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the
most widely prescribed and cost-effective drugs for pain relief.
They represent approximately 5e10% of all medications prescribed
each year [1]. These medications inhibit the cyclooxygenase en-
zymes (COX-1 and COX-2), which are responsible for arachidonic
acid metabolism to prostaglandin H2 (PGH2), the precursor for
prostacyclin (PGI2), prostaglandin-E2 (PGE2) and thromboxane-A2
(TXA2) synthesis [2]. Chronic use of NSAIDs has been implicated in
sever gastric ulcers and renal disorders [3] due to inhibition of COX-
1. These side effects of the traditional NSAIDs are the main reason
for drug intolerability and discontinuation [4]. Previous studies
[5], however the cardiovascular adverse effects were significant
[6e8]. Within the coxibs class of drugs, Celecoxib has the advantage
of lowering cardiovascular toxicity compared to other coxibs [9].
Various attempts for safer NSAIDs have been made in the last
few years, through optimization of benzenesulfonamide-based
derivatives, considering celecoxib 1, bearing
a pyrazole ring
linked to benzenesulfonamide ring at N-1 and p-tolyl group at C-5,
as a lead compound (Fig. 1) [10].
Saccharin 2 has played a crucial role in the development of
several biologically active agents such as repinotan, supidimide and
ipsapirone [11]. Meanwhile, piroxicam 3, the lead compound of
oxicams NSAIDs family (Fig. 1), was discovered as a saccharin de-
rivative through utilizing the GabrieleColman rearrangement un-
der basic conditions [12].
* Corresponding author. Lecturer of Pharmaceutical Organic chemistry at the
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar
University, 71524, Assiut, Egypt.
The varied functionality associated with sodium saccharin, as a
useful chiron, has promoted many attempts for synthesis of a wide
E-mail address: ehabtaher@azhar.edu.eg (E.S. Taher).
https://doi.org/10.1016/j.ejmech.2019.03.042
0223-5234/Crown Copyright © 2019 Published by Elsevier Masson SAS. All rights reserved.

E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
373
Fig. 1. Structure of celecoxib 1, sodium saccharin 2 and piroxicam 3.
range of benzenesulfonamides. Several studies have investigated
the analgesic and anti-inflammatory activities of the benzene-
sulfonamides scaffold attached to a pyrazole moiety [13]. For
instance, compounds 4 and 5 possessed higher analgesic and anti-
inflammatory activities than celecoxib [13,14]. In addition, it was
reported that 1,5-diphenyl pyrazoles exhibited anti-inflammatory
activities less than diclofenac against COX-1 but they showed a
reasonable in vitro COX-2 inhibitory activity with IC50 value of
dipyrazole derivative 23, to adopt a preferred binding conformation
to COX-2 and reduce the entropic loss, which might enhance the
potency and efficacy as well.
2. Discussion
2.1. Chemistry
0.45 mM [15]. Likewise, compounds 6 displayed high COX-2 selec-
In this work, novel series of acyclic benzenesulfonamide de-
rivatives bearing 1,3,4-oxadiazole or triazole moieties were pre-
pared as shown in Schemes 1 and 2. N-alkyl saccharins 10,17 and 21
were prepared via the reaction of benzyl bromide, isopropyl iodide
and diethyl bromomalonate respectively, with sodium saccharin by
heating the reactants in dimethylformamide (DMF) for 2 h [19]. The
key hydrazide intermediates 11,18 and 22 were prepared by stirring
N-benzyl, N-isopropyl saccharins or its diester derivative 10, 17 and
21, respectively with excess hydrazine hydrate at room tempera-
ture for 15e20 min following the reported procedure [13]. The
hydrazide 11 was cyclized through its reaction with carbon disul-
fide in basic condition to afford the targeted 1,3,4-oxadiazole bio-
isoster 12 in high yield (70%). Subsequent alkylation of oxadiazole
12 with benzyl bromide and ethyl iodide in ethanol containing
potassium hydroxide at room temperature gave the desired novel
thioalkylated derivatives 13a and 13b, respectively.
tivity and high anti-inflammatory activity compared to celecoxib
[16]. Celecoxib derivative 7 was found to have higher anti-
inflammatory activity and COX-2 selectivity with reasonable ulcer
index [17]. The pyrazolyl benzenesulfonamides linked to thiazoli-
dinone 8 and pyrazole 9 displayed higher COX-2 selectivity with
minimal potential for gastric injury (Fig. 2) [18].
In our ongoing investigation of the structure activity relation-
ship of benzenesulfonamide anti-inflammatory activity, we
designed and synthesized a novel set of benzenesulfonamide de-
rivatives starting from commercially available sodium saccharin.
The pivotal features of our approach aimed to do some structural
variations on celecoxib skeleton to explore the shifting effect of
pyrazole ring located at para-position of the benzensulfonamide
moiety as seen in celecoxib to ortho-position, as in our targets 19
and 20 on not only COX-2 selectivity but also to explore the new
possible receptor interactions. Moreover, the effect of replacement
of the ortho pyrazole ring with its bioisosteres, such as, 1,3,4-
oxadiazole i.e. 12 and 13 and triazole, i.e. 15 and 16 was also
considered. Another focus of our investigation was rigidification of
the benzenesulfonamide moiety, by ring closure towards the
In addition, the aminotriazole bioisostere 15 was synthesized
via heating the intermediate 14 at reflux with hydrazine hydrate.
The set of hydrazones 16a-d were then prepared through
condensation of aminotriazole 15 with appropriate aromatic alde-
hydes through heating at reflux in acetic acid for 20 min.
Fig. 2. Literature survey for benzensulfonamide derivatives with anti-inflammatory and analgesic activities.

374
E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
Scheme 1. Reagents and conditions: (i) benzyl bromide, DMF, reflux, 2 h; (ii) NH2NH2, r.t, 15 min; (iii) CS2, KOH, EtOH, reflux, 6 h; (iv) benzyl bromide and or ethyl iodide, EtOH,
KOH, r.t, 2 h.
Scheme 2. Reagents and conditions: (i) EtOH, CS2, KOH, r.t, 14 h; (ii) NH2NH2, reflux, 1 h; (iii) Ar-CHO, AcOH, reflux, 20 min.
New pyrazoles 19, 20, 23 and dihydrazones derivatives 24a-c
were synthesized as shown in Schemes 3 and 4. The hydrazides 18
and 22 were cyclized with b-diketones; namely benzoylacetoace-
appropriate aromatic aldehydes via heating the reactants in ethanol
containing catalytic amounts of glacial acetic acid to give the
anticipated novel dihydrazone derivatives 24a-c.
tate and benzoylacetone; via heating equimolar amounts of the
reactants under reflux in ethanol containing catalytic amount of
acetic acid for 12 h to afford the novel pyrazole derivatives 19, 20
and 23 in higher yields, 80%, 85% and 75%, respectively.
The novel benzenesulfonamide derivatives were characterized
by using thin layer chromatography and melting points tech-
niques. Structures of novel compounds were determined using
elemental analyses, IR, 1H NMR, 13C NMR and mass spectroscopic
methods.
In a related vein, the dihydrazide 22 was condensed with
Scheme 3. Reagents and conditions: (i) Isopropyl iodide, DMF, reflux, 2 h; (ii) NH2NH2, r.t, 2 h; (iii) benzoylacetoacetate and/or benzoylacetone, EtOH, AcOH, reflux, 12 h.

E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
375
Scheme 4. Reagents and conditions: (i) diethyl 2-bromomalonate, DMF, reflux, 2 h; (ii) NH2NH2, r.t, 20min; (iii) benzoyl acetone, EtOH, AcOH, reflux, 12 h; (iv) Ar-CHO, EtOH, AcOH
reflux, 2 h.
2.2. Biology
0.39
mM) showed slight improvement in activity but its ethyl de-
rivative 13b (IC50: 0.71
mM) was less effective. This improvement in
2.2.1. In vitro COX-1/COX-2 enzyme inhibition assay
activity may be due to increasing the bulkiness of oxadiazole ring.
In general, oxadiazole ring and its alkylated derivatives showed
All the compounds were tested for their in vitro COX-1 and COX-
2 inhibitory effect as depicted in Table 1 [20]. The efficacies of tested
compounds were measured as the concentration causing 50%
enzyme inhibition (IC50) as shown in Table 1, while selectivity
indices (SI values) defined as IC50 (COX-1)/IC50 (COX-2) were
calculated and then compared to the selective COX-2 inhibitor,
celcoxib. The results indicated the tested compounds have strong to
weak inhibition of COX-2 (IC50: 0.39e0.71
mM) in comparison with
celecoxib as a reference drug (IC50: 0.05
mM) and therefore, the
oxadiazole ring is not preferable for COX-2 inhibitory effect. It was
noted however, that replacement of oxadiazole ring with a triazole
as seen in compound 15 (Table 1) enhanced COX-2 inhibition ac-
tivity (IC50 ¼ 0.23
mM).
fair COX-1 inhibition (IC50: 1.98e12.23
ecoxib (IC50: 14.8 M).
mM), in comparison to cel-
The introduction of bulky groups on the free amino group of the
m
triazole moiety (15) through hydrazone formation (16a-d) caused a
The structure activity data acquired suggested that installing the
oxadiazole pharmacophore into the benzenesulfonamide as seen in
decrease for both COX-2 activity (IC50: 0.38e0.65
selectivity (SI ¼ 6.9e12.2) in comparison with celecoxib (IC50
0.05
assist binding on the COX-2 active site. Hence, it is proposed ben-
zenesulfonamides bearing the ortho-triazole ring (15) is preferen-
tial over the oxadiazole (12) for COX-2 inhibitory activity.
mM, Table 1) and
:
compound 12 caused a weak inhibition of COX-2 (IC50: 0.52
mM)
m
M, SI ¼ 296). It was noted that the free amino group may
while its alkylated derivative 13a bearing benzyl group (IC50
:
Conversely, a remarkable improvement in COX-2 inhibitory ac-
tivity as well as selectivity was observed upon replacement of the
five membered triazole ring with pyrazole as seen in compound 19,
Table 1
Data of the in vitro COX-1/COX-2 enzyme inhibition assay of the designed
compounds.
(IC50 0.09
m
M, SI ¼ 135.9) and compound 20 (IC50 ¼ 0.06
mM,
Compounds
IC50
(
m
M)a
SIb
SI ¼ 154.0). Introduction of an extra pyrazole ring to the cyclized
COX-1 COX-2
benzenesulfonamide skeleton (compound 23) caused marked in-
12
4.52
3.68
8.21
2.98
6.45
5.71
4.21
3.25
12.23
9.24
11.81
1.98
2.78
7.65
14.8
0.52
0.39
0.71
0.23
0.53
0.65
0.38
0.47
0.09
0.06
0.05
0.29
0.12
0.61
0.05
8.70
9.40
crease of both COX-2 potency and selectivity (IC50 ¼ 0.05
SI ¼ 236) which is comparable to celecoxib (IC50 0.05
mM,
M,
13a
13b
15
16a
16b
16c
16d
19
20
23
24a
24b
24c
Celecoxib
:
m
11.60
13.00
12.20
8.80
11.00
6.90
135.90
154.00
236.20
6.80
23.20
12.50
296.00
SI ¼ 296). Considering structural modification of the later com-
pound through replacement of the di-pyrazole rings with dihy-
drazone moieties (24a-c) led to a deleterious effect on both COX-2
potency and selectivity (Table 1). It was noted that benzenesulfo-
namide derivatives bearing pyrazole or dipyrazole 5-membered
rings showed higher COX-2 inhibitory activity and selectivity than
their corresponding dihydrazone ones. This may contribute to the
molecule's ability to be engaged in intermolecular interactions with
COX-2 active site. The order of activity of 5-membered rings to-
wards COX-2 inhibitory activity is cyclized benzenesulfonamide
bearing dipyrazole > pyrazole > triazole > oxadiazole.
a
IC50 value is the compound concentration required to produce 50% inhibition of
COX-1 or COX-2 for means of two determinations and deviation from the mean is
<10% of the mean value.
In conclusion, among tested compounds, benzenesulfonamides
bearing pyrazole ring 19 and 20 showed high activity against COX-2
with IC50: 0.09 (SI ¼ 135.9), 0.06 M (SI ¼ 154), respectively, while
b
SI: Selectivity index IC50 (COX-1)/IC50 (COX-2).
m

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E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
cyclized benzenesulfonamide bearing dipyrazole rings 23 (IC50
0.05
M, SI ¼ 236) was the most potent, with a comparable activity
to celecoxib (IC50: 0.05
:
correlation between in vitro selective COX-2 inhibition and in vivo
anti-inflammatory effect was established.
m
m
M, SI ¼ 296.00), thus, these compounds
(19, 20 and 23) can be considered as improved drug leads as
2.2.2.2. Ulcerogenic liability. The ulcerogenic activity for the
selected derivatives was determined at 18 mg/kg body weight dose
level using celecoxib and indomethacin as reference drugs [22].
The data obtained from Table 3 revealed that benzenesulfona-
mide with triazole 15 and dihydrazone 24b can cause stomach
ulceration (ulcer index: 15: 360; and 24b: 120) in comparison with
both the selective COX-2 inhibitor celecoxib (ulcer index: 3) and
indomethacin as ulcerogenic drug (ulcer index: 480) and these
results agree with in vitro COX-2 inhibitory activity. A remarkable
and nearly equipotent effect to celecoxib (ulcer index: 3) was
observed for benzenesulfonamide carrying pyrazole ring 19, 20 and
its cyclized derivative bearing dipyrazole rings 23 (ulcer index: 4).
Therefore, the pivotal anti-inflammatory value of these com-
pounds 19, 20 and 23 is that they possess significantly improved
safety margin on gastric mucosa than indomethacin. These positive
results supported the aim of our protocol to develop a novel series
of benzenesulfonamide derivatives as COX-2 inhibitors with mini-
mal gastrointestinal side effects.
opposed to celecoxib.
2.2.2. In vivo anti-inflammatory evaluation and ulcerogenic
liability
The novel benzenesulfonamide derivatives 15, 19, 20, 23 and
24b, which showed the highest in vitro COX-2 inhibitory activity,
were selected to evaluate their in vivo anti-inflammatory and ul-
cerogenic activities.
2.2.2.1. Anti-inflammatory effect evaluation. Anti-inflammatory ac-
tivity was evaluated by employing the carrageenan rat hind paw
edema method using celecoxib as a reference drug [21]. Mean
changes in paw edema thickness after 1, 2, 3 and 4 h from induction
of inflammation and percentages of edema inhibition given by the
tested compounds and celecoxib at 18 mg/kg body weight dose
level are displayed in Table 2.
It was noted that all the tested compounds displayed a very
similar figure regarding anti-inflammatory activity which ranges
from 49.19 to 65.32% reduction in inflammation after 4 h in com-
parison to celecoxib (62.90% reduction in inflammation after 4 h).
The benzenesulfonamide derivative bearing triazole ring 15
which showed a very weak in vitro COX-2 inhibitory activity, had
the lowest anti-inflammatory activity with edema inhibition
% ¼ 39.73 after 1 h to 49.19 after 4 h.
2.2.3. Molecular docking
The inhibitory profiles of celecoxib and the compounds 19, 20
and 23 (Table 1) were further studied by docking into the COX-2
active site (PDB code: 3ln1) to gain insight into the potential
binding modes [23]. Celecoxib exploited the side pocket near
Val523 in COX-2 (which is not present in COX-1) and inserted its
phenyl sulfonamide part into it. The sulfonamide NH2 forms three
hydrogen bonds with the hydrophilic side chains of Ser339 and
Gln178 in the side pocket and the carbonyl group at residue Leu338.
The p-tolyl and the trifluoromethyl methyl moieties of the cele-
coxib occupied hydrophobic regions in the active site (Fig. 3).
By a careful analysis of the docked poses of compounds 19, 20
and 23, it was clear that both compound 19 and 20 can occupy the
same pocket as celecoxib, but compound 23 could not, likely due to
its bulkiness. While the predominant binding site nature of COX
enzymes is hydrophobic, hydrophilic amino acids residues were
found in the side pocket of COX-2, including Ser339, Tyr341, Tyr371,
Arg499 and Ser516. The sulfone in compound 19 was observed to
be capable of forming a hydrogen bond with the Tyr341 of the COX-
2 active site (Fig. 3). The sulfonamide group was surrounded by
In contrast, benzenesulfonamide derivatives bearing pyrazole
ring 19, 20 and the cyclized dipyrazole form 23 which exhibited
potent in vitro COX-2 inhibitory activity and selectivity (Table 1) had
high anti-inflammatory activity (edema inhibition % ¼ 56.45, 65.32,
61.29, respectively after 4 h).
Unexpectedly, the benzenesulfonamide derivative bearing
dihydrazone 24b, which exhibited a weak in vitro COX-2 inhibitory
activity, showed a high anti-inflammatory effect (edema inhibition
% ¼ 54.26 after 4 h).
The higher in vivo potency of pyrazole motifs 19, 20 and 23
might reflect their better pharmacokinetic properties and higher
systemic bioavailability. This might be partly due to their enhanced
aqueous solubility and thereby, increased rate of dissolution
through different biological membranes. In conclusion,
a
Table 2
Anti-inflammatory activity of the tested compounds and Celecoxib (18 mg/kg p.o.) against formalin-induced paw edema in rats. (Mean S.D; n ¼ 6).
Treatment
Initial thickness
Zero time
Rat paw thickness (mm)
1 h
2 h
3 h
4 h
Control
Celecoxib
15
19
20
0.24 .024
0.24 .024
0.24 .024
0.24 .024
0.24 .024
0.24 .024
0.24 .024
0.73 0.03
0.41 0.02a
0.44 0.02a
0.42 0.01a
0.32 0.02a
0.36 0.02a
0.36 0.01a
0.84 0.02
0.44 0.01a
0.48 0.01a
0.50 0.02a
0.36 0.01a
0.41 0.01a
0.40 0.02a
0.94 0.02
0.44 0.03a
0.56 0.02a,b
0.52 0.02a
0.38 0.03a
0.41 .029a
0.43 0.01a
1.24 0.02
0.46 0.03a
0.63 0.02a,b
0.54 0.01a
0.43 0.03a
0.48 0.01a
0.55 0.01a
23
24b
Treatment
Edema inhibition %
1 h
2 h
3 h
4 h
Control
Celecoxib
15
19
20
————
————
————
————
————
————
————
0
0
0
0
43.84
39.73
42.47
56.17
50.69
50.69
47.62
42.86
43.48
57.14
51.19
52.38
53.19
45.43
44.68
59.58
56.38
54.25
62.90
49.19
56.45
65.32
61.29
54.26
23
24b
a
Significantly different from normal control at p < 0.05.
Significantly different from Celecoxib at p < 0.05 within the same column.
b

E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
377
Table 3
Ulcerogenic effect of Celecoxib, 15, 19, 20, 23 and 24b (18 mg/kg) in rats and Indomethacin (18 mg/kg). (Mean S.D, n ¼ 6).
Treatment
Average severity (ulcer score)
% Incidence of gastric ulceration
Ulcer index
Control
0.00 0.00
0
0
Celecoxib
Indomethacin
15
19
20
23
24b
0.00 0.00
0
3
4.80 0.20a,b
3.60 0.24a,b,c
0.20 0.20c
0.20 0.20c
0.20 0.20c
1.20 0.20a,b,c
100
100
20
20
20
100
480
360
4
4
4
120
a
Significantly different from normal control at p < 0.05.
Significantly different from Celecoxib at p < 0.05.
Significantly different from Indomethacin at p < 0.05.
b
c
basic residues Arg499 and His75 in case of compound 19 and His75
in the case of compound 20. Van der Waals interactions with
Val335, Leu338, Ala513 and Val509 dominate the remainder of the
compound 20 and 23 interactions. Overlaying of celecoxib and
compound 20 illustrated they possess a similar active conformation
of the trifluoropyrazole and the p-tolyl of celecoxib with the 5-
methylpyrazole and the phenyl in compound 20, respectively
(Fig. 3). However, celecoxib phenyl sulfonamide was perpendicular
on its congener in compound 20, possibly due to the presence of the
carbonyl group between the phenyl sulphonamide and the pyr-
azole moiety in compound 20.
Compound 23 binds to the exposed part of the receptor, pre-
sumably due to its bulky size (Fig. 4). Clusters of hydrophilic amino
acids, including three basic residues His80, His75 and His337 were
found in the pocket. The phenyl group in 3-oxobenzo[d]isothiazole
and the methyl group of the pyrazole moiety formed a pi-pi hy-
drophobic interaction with Pro500 and Pro177, respectively.
In general, data obtained from docking studies showed that
compounds 19 and 20 can occupy the binding site of the enzyme
with binding interaction energy ranging from ꢀ10.6 kcal/mol
to ꢀ11.3 kcal/mol relative to celecoxib ꢀ13.8 kcal/mol. Compound
23 binds to an exposed pocket of COX-2 with
energy ꢀ6.3 kcal/mol.
a binding
2.2.4. Pharmacokinetic properties and drug likeness
The drug likeness, HBD and HBA scores of the most active
compounds 15, 19, 20, 23, 24b were calculated using the MolSoft
online calculation kits [24]. Prediction of the lipophilicity was
performed using ALOGPS 2.1 program 5, while calculation of TPSA,
the number of rotatable bonds, used the Molinspiration property
calculation kit 6. The compounds have 5 rotatable bonds except 24b
has 7 rotatable bonds (Table 4). Reduced molecular flexibility is
measured by the number of rotatable bonds and is greatly linked to
oral bioavailability [25]. The number of rotatable bonds less or
equal to ten potentially increases the oral bioavailability [26]. To-
pological polar surface area (TPSA), for these five compounds, with
range from 81.07 to 137.38, is a very valuable parameter for the
prediction of drug transport properties and oral bioavailability. The
Fig. 3. 2D representations of the proposed binding patterns of celecoxib (A), 19 (B), 20 (C) and 23 (E) into the cyclooxygenase 2 (PDB code 3ln1) and overlaying of celecoxib and
compound 20 (D).

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E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
Fig. 4. Representations of the docked compound 23 into the cyclooxygenase 2 (PDB code 3ln1) (A) and representation of the two binding pockets identified in the COX-2, where
pocket 1 (active site) is occupied by celecoxib, compounds 19 and 20 and the exposed pocket 2 occupied by compound 23 (B). Pink: polar; green: hydrophobic; red: exposed. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Table 4
Drug likeness calculations and Lipinski parameters of the compounds 15, 19, 20, 23 and 24b.
M. Wta
log Pb
HBAc
HBDd
n violationse
nrot
TPSAg
Volumeh
DLs
f
15
19
20
23
361.45
385.44
383.47
565.61
525.49
2.15
2.10
3.26
4.49
2.72
6
6
5
7
7
4
1
1
0
2
0
0
0
1
1
5
5
5
5
7
102.91
95.91
81.07
124.25
137.38
293.50
329.21
337.40
472.37
415.20
ꢀ0.58
ꢀ0.34
ꢀ0.64
0.26
24b
1.07
a
Molecular weight.
Lipophilicity.
Number of hydrogen bond acceptors.
Number of hydrogen bond donors.
Number of violations.
Number of rotatable bonds.
Topological polar surface area.
Molecular volume. DLs, Drug likeness score.
b
c
d
e
f
g
h
drug likeness score (DLS) of all these compounds was from ꢀ0.64 to
1.07 (Table 4). The dipyrazolyl 23 and dihydrazone 24b benzene-
sulfonamide counterparts possessed maximum drug-likeness score
of 0.26 and 1.07, respectively (Table 4).
4. Experimental
4.1. Chemistry
Our physicochemical calculation indicated that these com-
pounds fulfilled the Lipinski guidelines and have drug-likeness
scores, which enables them to be potential oral bioavailable drugs.
Melting points were determined with a Gallenkamp (London,
U.K.) melting point apparatus and are uncorrected. IR spectra (KBr,
cmꢀ1) 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-200 (200 MHz, Foster
City, Calif., USA), 400 and 600 spectrometers using dime-
thylsulphoxide DMSO/D2O as a solvent and tetramethylsilane (TMS)
3. Conclusion
Two new series, namely, benzenesulfonamide and 1,2-
benzisothiazol-3(2H)-one-1,1-dioxide derivatives were synthe-
sized using sodium saccharin as cheapest starting synthon. In vitro
COX-2 inhibitory activity study revealed that benzenesulfonamides
bearing pyrazole ring 19 and 20 showed high activity against COX-2
as an internal standard (chemical shift in d, ppm). Mass spectra
were determined using Mass spectrometer GC/MS Shimadzu QP
1000 EX (Shimadzu Corporation, Tokyo, Japan) with ionization
energy 70 eV. Elemental analyses were determined using Manual
Elemental Analyzer Heraeus (Germany) and Automatic Elemental
Analyzer CHN Model 2400 Perkin Elmer (USA) at Microanalytical
Center, Faculty of Science, Cairo University, Egypt. All the results of
elemental analyses corresponded to the calculated values within
experimental error. Progress of the reaction was monitored by thin-
layer chromatography (TLC) using precoated TLC sheets with Ul-
traviolet (UV) fluorescent silica gel (Merck 60F254) and spots were
visualized by iodine vapours or irradiation with UV light (254 nm).
All the chemicals were purchased from Sigma-Aldrich or Lancaster
Synthesis Corporation (U.K.). Intermediates 10e12, 17 and 18 were
prepared according to the reported procedure [19,27,28].
with IC50: 0.09 (SI ¼ 135.9) and 0.06
meanwhile the cyclized benzenesulfonamide bearing dipyrazole
rings 23 (IC50: 0.05
M, SI ¼ 236) was the most potent, with a
comparable activity to celecoxib (IC50: 0.05
M, SI ¼ 296.0).
m
M (SI ¼ 154), respectively,
m
m
Moreover, anti-inflammatory activity and ulcer liability evalua-
tion demonstrated compounds 19, 20 and 23 possessed significant
anti-inflammatory activity with low gastric ulceration which was
comparable to celecoxib. Docking study showed that compounds
19 and 20 can occupy the binding site of the enzyme with binding
interaction energy ranging from ꢀ10.6 kcal/mol to ꢀ11.3 kcal/mol
compared to celecoxib ꢀ13.8 kcal/mol. Compound 23 binds to an
exposed pocket of COX-2 with binding energy ꢀ6.3 kcal/mol. The
obtained results from the in silico pharmacokinetic properties
calculation, for compounds 19, 20 and 23, suggested that these
compounds could be promising leads for further investigation to
explore more about the structure inhibitor relationship and
develop novel selective COX-2 inhibitors agent.
4.1.1. General procedure for preparation of 13a and 13b
A mixture of compound 12 (1 g, 2.88 mmol) and benzyl bromide
(0.36 mL, 2.88 mmol) or ethyl bromide (0.21 mL, 2.78 mmol) in
ethanol (30 mL) containing potassium hydroxide (0.2 g, 3.57 mmol)
was stirred at room temperature for 2 h. The reaction mixture was

E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
379
poured onto ice water (30 mL); the separated product was filtered
4.1.6. General procedure for preparation of compounds 16a-d
and crystallized from ethanol/H2O.
A mixture of equimolar amounts of 15 (0.3 g, 0.83 mmol) and
the respective aromatic aldehyde (0.83 mmol); previously dis-
solved separately in glacial acetic acid (1.00 mL) was heated under
reflux for 20 min. The reaction mixture was cooled, diluted with
water (5.00 mL) and the precipitated azomethine was filtered and
then crystallized from ethanol.
4.1.2. N-benzyl-2-(5-(benzylthio)-1,3,4-oxadiazol-2-yl)
benzenesulfonamide (13a)
Yellowish white solid, Yield: 70%; m.p.: 110e112 ꢁC; IR:
y
¼ 3154
(NH), 3030 (CH, aromatic), 2932 (CH, aliphatic), 1548 (C¼N), 1459
(C¼C), 1331 (SO2) cmꢀ1
.
1HNMR (400 MHz, DMSO):
d
¼ 4.11 (d,
J ¼ 6.6 Hz, 2H, NCH2), 4.57 (s, 2H, S-CH2), 7.19e7.29 (m, 5H, Ar-H),
7.28 (d, J ¼ 7.2 Hz, 1H, Ar-H), 7.36 (t, J ¼ 6.6 Hz, 2H, Ar-H), 7.49 (d,
J ¼ 8.4 Hz, 2H, Ar-H), 7.76e7.85 (m, 2H, Ar-H), 7.98e8.05 (m, 1H, Ar-
H), 8.10 (t, J ¼ 6.6 Hz, 1H, Ar-H), 8.30 (s, 1H, NH) ppm. 13C NMR:
4.1.7. (E)-N-((2-(4-(benzylideneamino)-5-mercapto-4H-1,2,4-
triazol-3-yl)phenyl)sulfonyl)benzamide (16a)
Light yellow solid, Yield: 65%; m.p.: 207e209 ꢁC; IR:
y
¼ 3458
(NH), 3065 (CH, aromatic), 2977 (CH, aliphatic), 1592 (C¼N), 1497
(100 MHz, DMSO):
d
¼ 35.5 (S-CH2), 46.1 (N-CH2), 121.4, 127.2,
(C¼C), 1327 (SO2) cmꢀ1. 1H NMR (400 MHz, DMSO):
¼ 4.08 (s, 2H,
d
127.5, 127.6, 127.8, 128.2, 128.8, 129.1, 132.1, 132.5, 136.3, 137.4, 140.3
CH2), 7.23e7.30 (m, 8H, Ar-H), 7.64e7.63 (m, 2H, Ar-H), 7.72e7.75
(m, 3H, Ar-H), 7.96 (s, 1H, ArH), 7.98 (s, 1H, NH), 8.14 (s, 1H, CH¼N),
(C¼C Ar), 163.6, 164.0 (oxadiazole-C) ppm. Anal. Calcd for
C
22H19N3O3S2 (437.53): C, 60.39; H, 4.38; N, 9.60; Found: C, 60.54;
13.14 (s, 1H, SH) ppm. 13C NMR (100 MHz, DMSO):
d
¼ 46.6 (CH2),
H, 4.55; N, 9.92%.
119.3, 127.7, 128.1, 128.7, 129.2, 129.3, 131.6, 132.9, 133.1, 138.0, 139.1,
140.4, 144.0, 147.3 (phenyl-C), 149.6, (C¼N triazole), 161.7, (C¼N),
172.7 (C-SH) ppm. Anal. Calcd for C22H19N5O2S2 (449.55): C, 58.78;
H, 4.26; N, 15.58; Found: C, 59.09; H, 4.35; N, 15.16%.
4.1.3. N-benzyl-2-(5-(ethylthio)-1,3,4-oxadiazol-2-yl)
benzenesulfonamide (13b)
Yellowish white solid, Yield: 75%; m.p.: 98e100 ꢁC; IR:
y
¼ 3190
(NH), 3021 (CH, aromatic), 2970 (CH, aliphatic), 1550 (C¼N), 1460
4.1.8. (E)-N-((2-(4-((4-chlorobenzylidene)amino)-5-mercapto-4H-
1,2,4-triazol-3-yl)phenyl)sulfonyl) benzamide (16b)
(C¼C), 1334 (SO2) cmꢀ1
.
1H NMR (400 MHz, DMSO):
d
¼ 1.42 (t,
J ¼ 7.6 Hz, 3H, CH3), 3.30 (q, J ¼ 10.8 Hz, 2H, CH2), 4.10 (d, J ¼ 9 Hz,
2H, N-CH2), 7.21e7.32 (m, 3H, Ar-H), 7.75e7.80 (m, 4H, Ar-H),
8.00e8.02 (m, 1H, Ar-H), 8.32e8.35 (m, 1H, Ar-H), 8.37 (s, 1H, NH)
Light yellow solid, Yield: 60%; m.p.: 214e216 ꢁC; IR:
y
¼ 3182
(NH), 3040 (CH, aromatic), 2992 (CH, aliphatic), 1661 (C¼N), 1588
(C¼C), 1327 (SO2) cmꢀ1. 1H NMR (600 MHz, DMSO):
¼ 4.92 (s, 2H,
d
ppm. 13C NMR (100 MHz, DMSO):
d
¼ 14.9 (CH3), 26.7 (S-CH2), 46.1
CH2), 7.18e7.28 (m, 2H, Ar-H), 7.29e7.34 (m, 1H, Ar-H), 7.36 (t,
J ¼ 7.8 Hz, 2H, Ar-H), 7.40e7.54 (m, 3H, Ar-H), 7.86e7.93 (m, 1H, Ar-
H), 7.98e8.09 (m, 2H, Ar-H), 8.10 (d, J ¼ 7.8 Hz, 1H, Ar-H), 8.32 (d,
J ¼ 7.8 Hz, 1H, Ar-H), 8.72 (s, 1H, NH), 12.20 (s, 1H, CH¼N), 13.88 (s,
(N-CH2), 121.5, 127.2, 127.5, 127.7, 128.2, 128.7, 132.1, 132.4, 132.5,
137.4, 140.4 (Csp2þphenyl-C), 163.4, 164.4 (oxadiazole-C) ppm.
Anal. Calcd for C17H17N3O3S2 (375.47): C, 54.38; H, 4.56; N, 11.19;
Found: C, 54.41; H, 4.69; N, 11.42%.
1H, SH) ppm. 13C NMR (150 MHz, DMSO):
d
¼ 46.0 (CH2), 121.6,
124.1, 125.2, 126.6, 127.6, 127.8, 128.8, 128.9, 132.0, 133.7, 135.9,
136.8, 137.6, (phenyl-C), 140.9 (C¼N of triazole), 158.6 (C¼N), 161.4
(C-SH) ppm. MS: m/z (rel. int.) ¼ 483 (M277 ,(13.00) 407 ,(10.90 ,þ
(6.50), 209 (28.30), 160 (32.50), 103 (100.00). Anal. Calcd for
4.1.4. Monopotassium(II) mono(2-(2-(N-benzylsulfamoyl)benzoyl)
hydrazinecarbodithioate) (14)
Carbon disulphide (0.2 mL, 3.38 mmol) was added dropwise to
cold solution of potassium hydroxide (0.3 g, 5.36 mmol) in ethanol
(10 mL) containing acid hydrazide 11 (0.5 g, 1.64 mmol). The
mixture was diluted with ethanol (10 mL) and stirred at room
temperature for 14 h. Dry ether (20 mL) was then added and the
separated solid was filtered and washed with ether (2 ꢂ 20 mL).
The obtained product, in nearly quantitative yield, was employed in
the next reaction without further purification.
C22H18ClN5O2S2 (483.99): C, 54.59; H, 3.75; N, 14.47. Found: C,
54.58; H, 3.41; N, 14.66%.
4.1.9. (E)-N-((2-(4-((4-bromobenzylidene)amino)-5-mercapto-4H-
1,2,4-triazol-3-yl)phenyl)sulfonyl) benzamide (16c)
Yellow solid, Yield: 70%; m.p.: 240e242 ꢁC; IR:
y
¼ 3428 (NH),
3036 (CH, aromatic), 2960 (CH, aliphatic), 1654 (C¼N), 1551 (C¼C),
1488 (C¼S), 1326 (SO2) cmꢀ1. 1H NMR (400 MHz, DMSO):
d
¼ 5.65
(s, 2H, CH2), 7.50e7.64 (m, 4H, Ar-H), 7.65e7.85 (m, 5H, Ar-H), 7.96
(d, J ¼ 12.6 Hz, 2H, Ar-H), 8.31 (s, 1H, Ar-H), 8.71 (s, 1H, Ar-H), 10.22
(s, 1H, NH), 10.87 (s, 1H, CH¼N), 13.42 (s, 1H, SH) ppm. 13C NMR
4.1.5. 2-(4-Amino-5-mercapto-4H-1,2,4-triazol-3-yl)-N- benzyl
benzenesulfonamide (15)
A suspension of compound 14 (0.40 g, 0.96 mmol) in hydrazine
hydrate 98% (0.1 mL, 2 mmol) was heated under reflux while stir-
ring for 1 h. Cold water (5 mL) was added and the mixture was then
neutralized with concentrated 2 N hydrochloric acid (1.0 mL). The
separated product was filtered, washed with cold water and crys-
tallized from ethanol/H2O. White solid, Yield: 60%; m.p.:
(100 MHz, DMSO):
128.6, 128.8, 129.4, 130.6, 132.9, 133.7, 137.2, 139.3, 139.4 (phenyl-C),
d
¼ 43.9 (CH2), 113.9, 118.5, 118.7, 122.1, 122.2,
155.1, (C¼N triazole), 155.4 (C¼N), 167.8 (C-SH) ppm. Anal. Calcd for
C22H18BrN5O2S2 (528.44): C, 50.00; H, 3.43; N, 13.25. Found: C,
49.83; H, 3.73; N, 13.24%.
210e212 ꢁC; IR:
matic), 2965 (CH, aliphatic), 1566 (C¼N), 1485 (C¼C), 1324 (SO2)
cmꢀ1 1HNMR (400 MHz, DMSO):
y
¼ 3289, 3200 (NH2), 3146 (NH), 3035 (CH, aro-
4.1.10. (E)-N-((2-(5-mercapto-4-((4-methoxybenzylidene)amino)-
4H-1,2,4-triazol-3-yl)phenyl) sulfonyl)benzamide (16d)
.
d
¼ 4.08 (d, J ¼ 9.6 Hz, 2H, CH2),
4.60 (s, 2H, NH2), 7.21e7.34 (m, 6H, Ar-H), 7.51 (d, J ¼ 10.8 Hz, 1H,
Ar-H), 7.61 (t, J ¼ 10.2 Hz, 1H, Ar-H), 7.68 (t, J ¼ 9.6 Hz, 1H, Ar-H),
7.87 (s, 1H, NH), 9.93 (s, 1H, SH) ppm. 13C NMR (100 MHz, DMSO):
Light yellow solid, Yield: 55%; m.p.: 200e202 ꢁC; IR:
y
¼ 3452
(NH), 3086 (CH, aromatic), 2969 (CH, aliphatic), 1596 (C¼N), 1501
(C¼C), 1325 (SO2) cmꢀ1. 1H NMR (600 MHz, DMSO):
¼ 3.79 (s, 3H,
d
d
¼ 46.6 (CH2), 127.8(0), 127.8(7), 128.1, 128.3, 129.5, 130.1, 132.5,
OCH3), 5.48 (s, 2H, CH2), 6.97e7.55 (m, 13H, ArH), 8.24 (s, 1H, NH),
134.1, 137.3 (Csp2þphenyl-C), 137.8 (C¼N), 167.3 (C-SH) ppm. MS:
m/z (rel. int.) ¼ 361 (M16.70) 209 ,(20.40) 241 ,(9.30) 295 ,(11.10 ,þ),
123 (11.10), 106 (18.50), 91 (100.00). Anal. Calcd for C15H15N5O2S2
(361.44): C, 49.84; H, 4.18; N, 19.38; Found: C, 49.83; H, 3.73; N,
19.59%.
10.45 (s, 1H, CH¼N), 12.90 (s, 1H, SH) ppm. 13C NMR (150 MHz,
DMSO):
d
¼ 40.0 (CH2), 55.4 (OCH3), 114.2, 114.4, 127.3, 127.9, 144.0,
(phenyl-C), 149.7 (2C ¼ N of triazole), 160.1 (C¼N), 164.3 (C-SH)
ppm. Anal. Calcd for C23H21N5O3S2 (479.57): C, 57.60; H, 4.41; N,
14.60; Found: C, 58.00; H, 3.98; N, 14.66%.

380
E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
4.1.11. General procedure for preparation of 19, 20 and 23
To a solution of the acid hydrazides 18 (0.4 g, 1.56 mmol) or 22
(0.49 g, 1.56 mmol) in ethanol (10 mL) containing glacial acetic acid
(2 mL), an equimolar amount of ethylbenzoyl acetate (0.30 g,
1.56 mol) or benzoylacetone (0.25 g, 1.56 mol) was added. The re-
action mixture was heated under reflux for 12 h, concentrated
under reduced pressure, cooled and added to ice cold water
(10 mL). The separated product was filtered, washed with water
and crystallized from ethanol.
4.1.16. 2-(1,1-Dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)-1,3-bis(3-
methyl-5-phenyl-1H-pyrazol-1-yl) propane-1,3-dione (23)
White solid, Yield: 75%; m.p.: 160e162 ꢁC; crystallized from
ethanol; IR:
y
¼ 3076 (CH, aromatic), 2965 (CH, aliphatic), 1734,
1665 (CO), 1580 (C¼N), 1505 (C¼C), 1332 (SO2) cmꢀ1
.
1H NMR
(400 MHz, DMSO):
d
¼ 2.25 (s, 6H, 2CH3), 5.48 (s, 1H, CH), 6.44 (s,
2H, 2CH of pyrazole), 7.27 (t, J ¼ 11.4 Hz, 2H, Ar-H), 7.39 (t,
J ¼ 11.4 Hz, 6H, Ar-H), 7.74 (d, J ¼ 11.4 Hz, 6H, Ar-H) ppm. 13C NMR
(100 MHz, DMSO):
d
¼ 10.6 (2CH3), 40.1 (CH), 101.1 (2CH of pyr-
azole), 121.7, 124.6, 124.9, 125.1, 126.4, 127.2, 128.6, 132.7, 135.3,
135.9, 136.9,139.4,150.4 (phenyl-Cþ pyrazole-C), 158.5,158.6,163.4
(3C ¼ O) ppm. Anal. Calcd for C30H23N5O5S (565.60): C, 63.71; H,
4.10; N, 12.38; Found: C, 64.05; H, 4.49; N, 11.94%.
4.1.12. N-isopropyl-2-(5-oxo-3-phenyl-4,5-dihydro-1H-pyrazole-1-
carbonyl)benzenesulfonamide (19)
White solid, Yield: 80%; m.p.: 102e104 ꢁC; IR:
y
¼ 3148 (NH),
3061 (CH, aromatic), 2963 (CH, aliphatic), 1606 (CO), 1551 (C¼N),
1502 (C¼C), 1359 (SO2) cmꢀ1
.
1HNMR (200 MHz DMSO):
¼ 0.93e1.05 (m, 6H, 2CH3), 1.88e2.15 (m, 1H, CH), 2.35 (s, 2H,
4.1.17. General procedure for preparation of 24a-c
d
A mixture of equimolar amounts of the dihydrazide 22 (0.49 g,
1.56 mmol) and the appropriate aromatic aldehydes (1.56 mmol) in
ethanol (10 mL) containing glacial acetic acid (2 mL) was heated
under reflux for 2 h. The reaction mixture was cooled and the
separated product was filtered, washed with water (10 mL), dried
and crystallized from ethanol.
CH2), 6.72e7.97 (m, 10H, ArH þ NH) ppm. 13C NMR (50 MHz,
DMSO):
d
¼ 24.2 (CH3), 39.4 (CH2), 52.3 (CH), 122.8, 127.6, 127.8,
131.9, 132.5, 137.7, 135.2, 135.4, 138.5, 155.6, 163.8, 169.4. Anal. Calcd
for C19H19N3O4S (385.44): C, 59.21; H, 4.97; N, 10.90; Found: C,
59.50; H, 5.01; N, 10.62%.
4.1.13. N-isopropyl-2-(3-methyl-5-phenyl-1H-pyrazole-1carbonyl)
benzenesulfon amide (20)
1
3
4.1.18. N0 ,N0 -di((E)-benzylidene)-2-(1,1-dioxido-3-oxobenzo[d]
isothiazol-2(3H)-yl)malonohydrazide (24a)
White solid, Yield: 85%; m.p.: 150e152 ꢁC; IR:
y
¼ 3256 (NH),
White solid, Yield: 75%; m.p.: 120e122 ꢁC; IR:
y
¼ 3450, 3241
3062 (CH, aromatic), 2973 (CH, aliphatic), 1640 (CO), 1551 (C¼N),
(2NH), 3062 (CH, aromatic), 2951 (CH, aliphatic), 1749, 1672 (CO),
1475 (C¼C), 1321 (SO2) cmꢀ1. 1H NMR (400 MHz, DMSO):
d
¼ 0.88
1496 (C¼C), 1334 (SO2) cmꢀ1. 1H NMR (600 MHz, DMSO):
d
¼ 4.19
(d, J ¼ 6.4 Hz, 3H, CH3), 0.97 (d, J ¼ 6.4 Hz, 3H, CH3), 1.88 (s, 3H, CH3),
(s, 1H, CH-N), 7.29e8.00 (m, 14H, ArH), 8.29 (s, 1H, CH¼N), 8.30 (s,
3.01e3.20 (m, 1H, CH), 6.71 (s, 1H, CH of pyrazole), 6.91e7.97 (m,
1H, CH¼N), 11.39 (s, 1H, NH), 12.08 (s, 1H, NH) ppm. 13C NMR
9H, ArH),11.00 (s,1H, NH) ppm. 13C NMR (100 MHz, DMSO):
d
¼ 16.0
(150 MHz, DMSO):
d
¼ 79.3 (CH-N), 126.5, 126.9, 127.2, 128.1,
(CH3), 23.7 (2CH3), 56.1 (CH), 92.8 (CH of pyrazole), 125.2, 127.4,
127.6, 128.3, 128.9, 130.1, 131.6, 132.9, 136.6, 138.1, 139.5, 144.1, 155.5
(phenyl-Cþ pyrazole-C), 165.6 (C¼O) ppm. Anal. Calcd for
128.35, 128.7, 128.9, 129.2, 129.4, 129.7, 129.9, 130.0, 130.1, 130.3,
130.4, 132.0, 137.9, 143.8, 144.1 (phenyl C), 148.2, 148.4 (2C ¼ N),
163.9, 165.7, 169.0 (3C ¼ O) ppm. Anal. Calcd for C24H19N5O5S
(489.50): C, 58.89; H, 3.91; N, 14.31; Found: C, 58.58; H, 3.64; N,
14.10%.
C
20H21N3O3S (383.46): C, 62.64; H, 5.52; N, 10.96; Found: C, 63.01;
H, 5.98; N, 11.33%.
4.1.14. Diethyl 2-(1,1-dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
malonate (21)
1
3
4.1.19. 2-(1,1-Dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)-N0 ,N0 -
bis((E)-4-fluorobenzylidene)malono-hydrazide (24b)
To a solution of saccharin sodium 2 (0.5 g, 2.44 mmol) in dime-
thylformamide (5 mL), diethyl 2-bromomalonate (0.6 g, 2.52 mmol)
was added. The reaction was maintained at 100 ꢁC for 2 h, then
cooled and diluted with water (10 mL). The obtained product was
crystallized from ethanol. White solid, Yield: 85%; m.p.: 90e92 ꢁC; IR:
White solid, Yield: 85%; m.p.: 116e118 ꢁC; IR:
y
¼ 3460, 3181
(2NH), 3096 (CH, aromatic), 2953 (CH, aliphatic), 1746, 1680, 1628
(CO), 1460 (C¼C), 1331 (SO2) cmꢀ1
.
1H NMR (400 MHz, DMSO):
d
¼ 4.93 (s, 1H, CH-N), 7.29 (t, J ¼ 13.2 Hz, 2H, Ar-H), 7.69e7.92 (m,
3H, Ar-H), 7.97e8.29 (m, 7H, Ar-H), 8.36 (d, J ¼ 10.8 Hz, 2H,
y
¼ 3088 (CH, aromatic), 2992 (CH, aliphatic), 1737 (CO, ester), 1590
2CH ¼ N), 11.76 (s, 1H, NH), 11.83 (s, 1H, NH) ppm.13C NMR
(C¼N), 1464 (C¼C), 1332 (SO2) cmꢀ1
.
1H NMR (400 MHz, DMSO):
(100 MHz, DMSO):
d
¼ 78.7 (CH-N), 116.2, 116.3, 116.4, 122.2, 125.6,
d
¼ 1.20 (t, J ¼ 7.10 Hz, 6H, 2CH3), 4.14e4.20 (m, 4H, 2CH2), 4.62 (s,1H,
CH), 8.02e8.37 (m, 4H, ArH) ppm. 13C NMR (100 MHz, DMSO): 14.4
(CH3), 39.5 (CH2), 62.0 (CH), 122.3, 125.8, 126.4, 136.0, 136.7, 137.5
(phenyl-C), 159.0, 166.8 (-C¼O) ppm. Anal. Calcd for C14H15NO7S
(341.34): C, 49.26; H, 4.43; N, 4.10. Found: C, 49.10; H, 4.30; N, 4.45%.
125.7, 126.8, 126.9, 129.8, 129.9, 131.0, 135.8, 136.5, 137.6, 143.9
(phenyl-C), 147.0, 159.2 (2C ¼ N), 161.8 (phenyl-C-F), 162.4, 164.8,
166.2 (3C ¼ O) ppm. Anal. Calcd for C24H17F2N5O5S (525.48): C
54.86; H 3.26; N 13.20.Found: C, 54.58; H, 3.41; N, 13.20%.
4.1.15. 2-(1,1-Dioxido-3-oxobenzo[d]isothiazol-2(3H)-yl)
malonohydrazide (22)
A mixture of diesterester 21 (0.4 g, 1.17 mmol) and hydrazine
hydrate 98% (0.5 mL, 10.00 mmol) was stirred at room temperature
for 20 min. The separated product was filtered, washed with water,
dried and crystallized from ethanol. White solid, Yield: 80%; m.p.:
4.1.20. 2-Methyl-di-(4-chlorobenzylidenehydrazinocarbonyl)-1,2-
benzisothiazole-3(2H)-one-1,1-dioxide (24c)
White solid, Yield: 70%; m.p.: 130e132 ꢁC; IR:
y
¼ 3461, 3182
(2NH), 3088 (CH, aromatic), 2951 (CH, aliphatic), 1745, 1679 (CO),
1497 (C¼C), 1330 (SO2) cmꢀ1. 1H NMR (400 MHz, DMSO):
d
¼ 4.93
(s, 1H, CH-N), 7.50 (d, J ¼ 12.6 Hz, 2H, Ar-H), 7.74 (d, J ¼ 12.6 Hz,
1H, Ar-H), 7.80 (d, J ¼ 12.6 Hz, 2H, Ar-H), 7.97e8.26 (m, 6H, Ar-H),
8.36 (d, J ¼ 12.6 Hz, 1H, Ar-H), 8.72 (s, 2H, 2CH ¼ N), 11.83 (s, 1H,
100e102 ꢁC; IR:
y
¼ 3385, 3331 (2NH), 3289, 3190 (2NH2), 3097
(CH, aromatic), 1665, 1619 (CO), 1507 (C¼C), 1333 (SO2) cmꢀ1
.
1H
NMR (400 MHz, DMSO):
d
¼ 3.80 (s, 1H, CH), 4.35 (br s, 4H, 2NH2,
NH), 11.89 (s, 1H, NH) ppm. 13C NMR (100 MHz, DMSO):
d
¼ 83.2
exch.), 7.51e7.88 (m, 4H, ArH), 9.12 (s, 1H, NH, exch.), 9.89 (s, 1H,
(CH-N), 121.8, 125.2, 126.3, 128 (8), 128.9, 129.1, 130.0, 132.8, 132.9,
134.6, 135.4, 136.0, 136.9, 137.1 (phenyl-C), 143.3, 146.4 (C¼N),
158.8, 161.5, 165.8 (3C ¼ O) ppm. Anal. Calcd for C24H17Cl2N5O5S
(558.39): C, 51.62; H, 3.07; N, 12.54; Found: C, 51.32; H, 3.36; N,
12.31%.
NH, exch.) ppm. 13C NMR (100 MHz, DMSO):
d
¼ 44.07 (CH), 128.7,
129.6, 130.1, 132.8, 134.3, 137.0(phenyl-C), 166.5, 167.0, 170.9
(3C ¼ O) ppm. Anal. Calcd for C10H11N5O5S (313.29): C, 38.34; H,
3.54; N, 22.35. Found: C, 38.10; H, 3.30; N, 21.90%.

E.S. Taher et al. / European Journal of Medicinal Chemistry 171 (2019) 372e382
381
4.2. Biology
ulcer score of a group of animals similarly treated ꢂ % of ulcer-
ated animals of this group [31].
4.2.1. In vitro COX-1/COX-2 enzyme inhibition assay
Ulcer score:
The synthesized compounds 12e13a-b, 16a-d, 19, 20, 23e24a-c
were tested for their ability to inhibit COX-1 and/or COX-2 enzymes
using Cayman's colorimetric COX (ovine) assay and the results were
recorded in Table 1. The assay measures the peroxidase activity
colorimetrically by monitoring the appearance of oxidized
N,N,N0,N0-tetramethyl-p-phenylenediamine at 590 nm as reported
by Kulmacz and Lands [29].
1: 1 or 2-min sporadic punctate lesions, 2: Several small lesions,
3: One extensive lesion or multiple moderate-sized lesions, 4:
Several large lesions, 5: Several large lesions with stomach
perforation.
4.2.3. Docking
The molecular docking of the tested compounds 19, 20 and 23
was performed using Molecular Operating Environment software
utilizing the reported protein crystallographic structure of COX-2
(PDB code: 3ln1). Celecoxib was the crystallized ligand into the
COX-2 protein active site. The tested structures were created using
ChemDraw professional (version: 15.1.0.144) and were energy
minimized using the MMF94X forcefield to a constant of 0.05 kcal/
mol. The protein was prepared for docking process with removing
the water and adding the polar hydrogens. Standard Docking of 19,
20 and 23 and celecoxib into the active site of the COX-2 was per-
formed using the standard docking function in MOE. Firstly, dock-
ing was restricted at the active site, however, compound 23 did not
fit into the active site, may be due to bulky side groups, so docking
against other pockets was performed. The top 30 docking poses
finally saved and were ranked according to their interaction energy
and examined for fit and possible interactions with the active site.
4.2.2. In vivo assay
4.2.2.1. Materials. Carrageenan (carrageenan kappa-type III) and all
other reagents were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). The test compounds 15, 19, 20, 23, 24b and reference
drugs celecoxib and/or indomethacin were used in the following
assays. Housing and management of the animals and the experi-
mental protocols were made as stipulated in the Guide for Care and
Use of Laboratory Animals Guidelines of the National Institutes of
Health (NIH), and accepted by the local authorities of Zagazig
University, Zagazig, Egypt.
4.2.2.2. Animals. Mature male albino rats weighing 150e200 g
were used. All experimental animals were provided from the Fac-
ulty of Veterinary Medicine, Zagazig University, Egypt. All animals
were held under standard laboratory conditions in the animal
house (temperature 27 ꢁC) with a 12/12 light-dark cycle. Animals
were fed laboratory diet and water ad libitum. All experiments
were carried out using six animals per group. The animal experi-
ments were performed in accordance with international guidelines.
Acknowledgment
The authors are kindly appreciative to “Patrick McCosker at
School of Chemistry, University of Wollongong, for his efforts
proofreading the Manuscript."Dr Naglaa Z. Eleiwa, Zagazig Uni-
versity, Faculty of veterinary medicine is highly appreciated for her
kind co-operation in carrying out the experimental biological part.
4.2.2.3. Anti-inflammatory effect evaluation. The rat hind paw
edema method by Winter et al., 1962 [30] was applied to determine
the anti-inflammatory activity of the test compounds 15, 19, 20, 23
and 24b using celecoxib as a standard. Mature male albino rats
weighing 150e200 g was used. The animals were divided into 7
equal groups (each of six). The first group was left as a control group
while the second group was injected i.p. with celecoxib at a dose of
18 mg/kg body weight. The test compounds were injected i.p. in the
remaining groups at the same dose level. After 1 h, edema in the
right hind paw was induced by injecting 0.1 mL of 10% carrageenin.
The thickness of the paw was measured using a skin caliber 1, 2, 3,
and 4 h after the carrageenin injection to determine the anti-
inflammatory activity of the test compounds (Table 2).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.03.042.
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