Design, synthesis and biological screening of new 4-thiazolidinone derivatives with promising COX-2 selectivity, anti-inflammatory activity and gastric safety profile

Article abstract of DOI:10.1016/j.bioorg.2015.11.001

Two series of new thiazolidin-4-one derivatives 4a-c and 8a-e were designed and prepared. All the synthesized compounds were evaluated for their in vitro COX-2 selectivity and anti-inflammatory activity in vivo. Compounds 8c and 8d showed the best overall

Full text of DOI:10.1016/j.bioorg.2015.11.001

Bioorganic Chemistry 64 (2016) 1–12
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological screening of new 4-thiazolidinone
derivatives with promising COX-2 selectivity, anti-inflammatory activity
and gastric safety profile
Khaled R.A. Abdellatif a, , Mohamed A. Abdelgawad a,b, Heba A.H. Elshemy a, Shahinda S.R. Alsayed a
⇑
a Department of Pharmaceutical Organic Chemistry, Beni Suef University, Beni Suef 62514, Egypt
b Pharmaceutical Chemistry Department, College of Pharmacy, Al Jouf University, Sakaka, Al Jouf 2014, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of new thiazolidin-4-one derivatives 4a–c and 8a–e were designed and prepared. All the syn-
thesized compounds were evaluated for their in vitro COX-2 selectivity and anti-inflammatory activity
in vivo. Compounds 8c and 8d showed the best overall in vitro COX-2 selectivity (selectivity indexes of
4.56 and 5.68 respectively) and in vivo activities (edema inhibition % = 61.8 and 67 after 3 h, respectively)
in comparison with the reference drug celecoxib (S.I. = 7.29, edema inhibition % = 60 after 3 h). In addi-
tion, 8c and 8d were evaluated for their mean effective anti-inflammatory doses (ED50 = 27.7 and
Received 10 September 2015
Revised 31 October 2015
Accepted 3 November 2015
Available online 6 November 2015
Keywords:
Thiazolidinone
Cyclooxygenase inhibition
Anti-inflammatory
Molecular modeling
18.1 lmol/kg respectively, celecoxib ED50 = 28.2 lmol/kg) and ulcerogenic liability (reduction in ulcero-
genic potential versus celecoxib = 85%, 92% respectively. Molecular docking studies were performed and
the results were in agreement with that obtained from the in vitro COX inhibition assays.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
prophylactic doses resulting in gastro intestinal side effects
ranging from ulcers to perforation and bleeding [7]. For these rea-
Non steroidal anti-inflammatory drugs (NSAIDs) are considered
one of the most widely used therapeutics to alleviate pain and
inflammation, especially arthritis [1]. The anti-inflammatory
activity of NSAIDs arises from their ability to inhibit cyclooxyge-
nase (COX) enzyme which catalyzes the production of pro-
inflammatory prostaglandins (PGs) and thromboxanes (TXs) [2,3].
Currently, It is well known that cyclooxygenase enzyme exists in
at least two distinct isoforms, a constitutive form (COX-1) and an
inducible form (COX-2) [4]. The constitutively expressed COX-1
isoform plays a critical role as a housekeeping enzyme which is
responsible for the maintenance of physiological functions such
as protection of gastric mucosa, vascular homeostasis and platelet
aggregation. The inducible COX-2 isoform is significantly upregu-
lated during acute and chronic inflammation, pain and oncogenesis
[5,6]. Traditional nonselective NSAIDs such as aspirin, ibuprofen
and indomethacin interact with both forms (COX-1 and COX-2);
this broad inhibitory profile accounts for their anti-inflammatory
activity in addition to their pronounced side effects resulting from
the inhibition of gastro protective PGs synthesized through COX-1
pathway. Therefore, their long term administration even at low
sons, the synthesis of selective COX-2 inhibitory drugs (coxibs)
takes much consideration in recent years that achieves the same
anti-inflammatory efficacy as traditional NSAIDs, but with minimal
risk of the concomitant gastric and renal toxicity mediated through
the inhibition of COX-1 enzyme [4,6]. Hence, a number of selective
COX-2 inhibitors such as celecoxib I, rofecoxib II and valdecoxib III
(coxibs) has been developed and approved for marketing by virtue
of their fewer gastrointestinal side effects compared to traditional
NSAIDs (Fig. 1). However, when the coxibs were marketed, evi-
dence for increased the risk of cardiovascular adverse effects
appeared that led to rofecoxib ban in 2004 followed by the volun-
tary withdrawal of some other coxibs from the market [8]. In this
respect, celecoxib has advantages of that it is not associated with
an increased incidence of cardiovascular events compared with
placebo and with nonselective NSAIDs [9]. However, there are
some characteristics of celecoxib that need to be improved. For
example, celecoxib has some gastrointestinal side effects and is
not effective in all patients [10,11]. In earlier studies, we reported
some derivatives of celecoxib IV–VIII [12–17] (Fig. 1) with compa-
rable activities to celecoxib as COX-2 selective compounds.
In continuation with our work related to synthesis of safe
anti-inflammatory agents, we now describe the synthesis, in vitro
evaluation as COX-1/COX-2 inhibitors, in vivo anti-inflammatory
(AI) activity, and ulcerogenic liability for two new groups of
⇑
Corresponding author at: Department of Pharmaceutical Organic Chemistry,
Faculty of Pharmacy, Beni Suef University, Beni Suef 62514, Egypt.
E-mail address: khaled.ahmed@pharm.bsu.edu.eg (K.R.A. Abdellatif).
http://dx.doi.org/10.1016/j.bioorg.2015.11.001
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.

2
K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
Me
SO2NH2
SO2Me
SO2NH2
N
O
O
N
O
N
Me
Rofecoxib
Celecoxib
I
F3C
Valdecoxib
III
II
R
NHR1
H
N
SO2Me
SO2R2
SO2R
N
N
N
N
N
HOOC
N
R = H, F, Me
IV
F3C
R
1 = Me, Et; R2 = Me, NH2
F3C
R = Me, NH2
VI
V
R1
OH
N
O
SO2R2
SO2R
N
N
N
N
F3C
F3C
R1 = OH, COOH; R2 = Me, NH2
VII
R = Me, NH2
VIII
Fig. 1. Chemical structures of the selective cyclooxygenase-2 (COX-2) inhibitors celecoxib (I), rofecoxib (II), valdecoxib (III), and some reported celecoxib derivatives IV–VIII.
4-thiazolidinone derivatives 4a–c and 8a–e in which, (i) the
pyrazole ring of celecoxib was replaced with thiazole moiety in
compounds 4a–c or 4-thiazolidinone nucleus in compounds
8a–e, (ii) tolyl group was replaced with iminothiazolidin-4-one
in compounds 4a–c and maintained or replaced with different aryl
moieties in compounds 8a–e, (iii) the aminosulfonylphenyl group
which is the major determinant for COX-2 selectivity and in vivo
efficacy [18–20] was replaced with aryl moiety substituted at para
position with bulky group in compounds 4a–c or replaced with
more bulky aminosulfonylphenylamino group in compounds
8a–e. This bulky substitution could maximize the interaction with
the hydrophobic residues within COX-2 active site and enhance
COX-2 selectivity [21], and (iv) the trifluoromethyl group of cele-
coxib was replaced with methyl group (8a–e) since it was reported
that the substituent at pyrazole C-3 has very few steric restrictions
with respect to COX-2 suggesting that COX-2 inhibition should be
retained [22] (Fig. 2).
dioxane under reflux conditions. Finally, cyclization of acetamide
derivatives 3a–c with ammonium thiocyanate in ethanol afforded
thiazolidinone derivatives 4a–c in moderate yield (40–50%).
All the newly synthesized compounds have been characterized
by IR, 1H NMR, 13C NMR, mass spectra and elemental analyses. The
suggested mechanism of heterocyclization of 3a–c and the theoret-
ically tautomeric forms of target compounds 4a–c are shown in
Scheme 2. The
c lactam structure of 4a–c was confirmed based
on the 1H NMR spectra which exhibited an NH proton appeared
at d = 12.12–12.21 ppm which was in the same region as the previ-
ously reported NH appeared at d ꢀ 12.35 ppm. These findings
proved that this proton accounts for a lactam proton but not for
an imine proton which is expected to appear at a much higher field
at d = 9.0 ppm. [23–25].
The second series of the target 4-thiazolidinones 8a–e were
synthesized according to the method described by Neuenfeldt
et al. [26]. This new method allows the synthesis of the
4-thiazolidinones derived from phanylhydrazines even if they have
electron withdrawing groups on the phenyl ring. Accordingly, the
hydrazones 7a–e were prepared in high yields (80–90%) by heating
2. Results and discussion
4-hydrazinobenzenesulfonamide
hydrochloride
(5)
with
2.1. Chemistry
4-substituted-aldehydes (6a–e) in presence of sodium acetate in
ethanol. Heating the formed hydrazones 7a–e with excess thiolac-
tic acid at 60 °C under solvent free conditions afforded the 4-
thiazolidinone derivatives 8a–e in moderate yields (35–60%)
(Scheme 3). The 1H NMR spectra of 8a–e displayed a peak appeared
as two doublets at d 1.52–1.53 and 1.54–1.55 ppm corresponding
to thiazolidinone CH3. In addition, another peak appears as two
The target compounds 4-thiazolidinones 4a–c were synthesized
via a reaction sequence illustrated in Scheme 1 starting from the
reaction of 4-substituted-acetophenone (1a–c) with iodine and
thiourea at 60 °C in water bath to provide the corresponding 2-a
mino-4-(4-substituted-phenyl)thiazole (2a–c) in high yields
(70–85%). Then, compounds 3a–c were obtained in good yield
(65–70%) via the reaction of 2a–c with chloroacetyl chloride in
singlets at
d 5.82–6.02 and 5.87–6.06 ppm corresponding to

K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
3
Me
R
O
R
SO2NH2
NH
N
N
S
SO2NH2
S
N
NH
N
S
N
F3C
O
H3C
4a-c
Celecoxib
I
8a-e
R= NO2, isobutyl and NHSO2CH3
R= H, CH3, Cl, F and CF3
Fig. 2. Representative example of selective COX-2 inhibitor (celecoxib I) and the designed thiazolidinones 4a–c and 8a–e.
COCH3
Cl
R
R
a
b
O
N
N
NH2
NH
S
S
R
2a-c
3a-c
1a-c
R
NO2
isobutyl
NHSO2CH3
c
a
b
c
O
R
HN
N
S
N
S
4a-c
Scheme 1. General synthetic procedure for title compounds 4a–c. Reagents and conditions: (a) I2, thiourea, heat at 60 °C, 3–5 h; (b) chloroacetyl chloride, dioxan, reflux, 24 h;
(c) ammonium thiocyanate, ethanol, reflux, 24 h.
thiazolidinone H-2. Another D2O exchangeable peak appears as
two singlets at d 8.67–8.81 and 8.74–8.87 ppm corresponding to
NH group and these findings are due to presence of two chiral cen-
ters at C2 and C5 as previously reported [27,28], so compounds 8a–
e are present as diastereoisomers. The separation of two isomers
was not possible to be done by chromatography because of similar
retention times between them in different solvents [29].
activity against COX-1 (IC50 = 10.5–29.1
l
M) and against COX-2
M for
(IC50 = 1.9–8.7 M) with celecoxib (IC50 = 9.7 and 1.33
l
l
COX-1 and COX-2, respectively). Within the biologically active
series (8a–e), the chloro (8c) and fluoro (8d) analogs were more
selective COX-2 inhibitors (selectivity indexes of 4.56 and 5.68
respectively) than the unsubstituted (8a), methyl (8b) and trifluro-
romethyl (8e) analogs (selectivity indexes of 3.34, 3.50 and 3.47
respectively) in comparison with celecoxib (S.I. = 7.29).
2.2. Biological evaluation
2.2.2. In vivo anti-inflammatory activity
2.2.1. In vitro cyclooxygenase (COX) inhibition assay
The anti-inflammatory (AI) activities exhibited by compounds
4a–c and 8a–e using dose 10 mg/kg according to a previously
reported procedures [30] are listed in Table 2. Compounds 4a
and 4b showed moderate COX-2 inhibitory activity (IC50 = 4.6
The target of the in vitro biological activity tests was to study
the ability of tested compounds to inhibit ovine COX-1 and human
recombinant COX-2 using an enzyme immunoassay (EIA) kit. The
obtained data (Table 1) showed that all the tested compounds
(4a–c and 8a–e) were weak inhibitors of COX-1 isozyme
and 6.1 lM, respectively) with moderate selectivity index (S.I.
= 2.91 and 2.88, respectively) were found to possess anti-
inflammatory activity (52.7% and 56.7% reduction in inflammation
after 3 h, respectively) close to celecoxib (60% reduction in inflam-
mation after 3 h). While, 4-thiazolidinone derivatives 8c and 8d
which showed COX-2 inhibitory activity in vitro comparable to
celecoxib, showed in vivo anti-inflammatory activity (edema inhi-
bition % = 61.8 and 67 after 3 h, respectively) higher than the refer-
ence drug celecoxib (edema inhibition % = 60 after 3 h). Moreover,
compounds 8b and 8e with hydrophobic methyl or trifluoromethyl
groups that had good COX-2 inhibitory activity exhibited moderate
anti-inflammatory effect in vivo (edema inhibition % = 22.3 and
23.7 after 3 h, respectively).
(IC50 = 10.5–29.1
isozyme inhibitory activities (IC50 = 1.9–8.7
l
M
range) and exhibited moderate COX-2
M). Also, the results
l
showed COX-2 selectivity indexes in the 2.84–5.68 range
(celecoxib COX-2 S.I. = 7.29). The structure activity data acquired
showed that when the two bulky moieties attached to the central
heterocyclic thiazole ring are not vicinal (4a–c), lower inhibitory
activity against both COX-1 (IC50 = 13.4–17.6
l
M) and COX-2
M for
(IC50 = 4.6–6.1 M) than celecoxib (IC50 = 9.7 and 1.33
l
l
COX-1 and COX-2, respectively) were obtained. While, when
the two bulky moieties attached to the central heterocyclic
4-thiazolidinone moiety are vicinal (8a–e) comparable inhibitory

4
K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
Cl
R
O
N
NH
S
3a-c
NH4SCN
-NH4Cl
R
R
S
S
N
O
N
O
N
HN
H
S
N
N
S
O
R
O
R
HN
N
N
S
N
S
N
S
HN
S
4a-c
O
O
HO
N
R
R
R
N
N
S
H
S
S
N
N
N
N
NH
N
S
S
S
Scheme 2. Mechanistic pathway for compounds 4a–c and their tautomers.
SO2NH2
R
H
N
N
a
R
H2NO2S
7a-e
NHNH2.HCl
CHO
5
6a-e
b
R
a
b
c
d
e
H
CH3
Cl
F
CF3
R
SO2NH2
NH
N
S
O
H3C
8a-e
Scheme 3. General synthetic procedure for compounds 8a–e. Reagents and conditions: (a) sodium acetate, ethanol, reflux, 12–18 h; (b) thiolactic acid, heat at 60 °C, 3 h.

K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
5
Table 1
NSAID ibuprofen (50 mg/kg). From the obtained data in Table 3, it
had been observed that compounds 8c and 8d caused much less
gastric ulceration effect with nearly negligible ulcerogenic liability
(ulcer index of compound 8c: 1.2 and 8d: 0.6) in the experimental
animals, compared to that of the standards, ibuprofen (ulcer index:
14.6) and celecoxib (ulcer index: 7.8). Therefore, the potential
value of these compounds as anti-inflammatory agents is that they
have highly better safety margin on gastric mucosa than celecoxib
and ibuprofen. These promising ulcer protective properties of the
designed 4-thiazolidinone derivatives 8c and 8d greatly supported
our main objective to avoid gastric injuries caused by COX-1 inhi-
bition. Moreover, compound 8c showed about 85% reduction in
ulcerogenic potential versus celecoxib. While, compound 8d with
best overall profile in animal efficacy model showed about 92%
reduction in ulcerogenic potential versus celecoxib. Hence, this
successful result supported the aim of the present work to develop
novel series of celecoxib analogs bearing thiazolidinone moiety as
COX-2 inhibitors with diminished gastrointestinal side effects.
In vitro COX-1 and COX-2 inhibitory activity and COX-2 selectivity index of
thiazoldinones 4a–c, 8a–e as well as celecoxib.
Compounds
IC50
(
l
M)a
S.I.b
COX-1
COX-2
4a
4b
4c
8a
8b
8c
8d
8e
13.4
17.6
15.1
29.1
16.8
10.5
10.8
11.8
9.7
4.6
6.1
5.3
8.7
4.8
2.3
1.9
3.4
1.33
2.91
2.88
2.84
3.34
3.5
4.56
5.68
3.47
7.29
Celecoxib
a
IC50 value represents the concentration of the compound required to produce
50% inhibition of COX-1 or COX-2 which is the mean value of two determinations
where the deviation from the mean is <10% of the mean value.
b
Selectivity index (COX-1 IC50/COX-2 IC50).
Table 2
Anti-inflammatory activity at different time intervals of the tested compounds 4a–c,
8a–e and celecoxib using 10 mg/kg dose employing carrageenan-induced paw edema
method in mice.
2.3. Molecular modeling
With the aim to understand the protein-inhibitor interaction
and the structure features of COX-2 active site taking into account
that COX-2 is the most relevant enzymatic system for inflamma-
tion and the target of our compounds, a molecular modeling study
was performed using the crystal structure of COX-2 (3LN1) [33].
Docking of compounds 4a, 4b, 8c, 8d and celecoxib as the reference
ligand into the crystal structure of COX-2 enzyme was performed
using the MOE 2008.10 modeling software (Molecular Operating
Environment) [34]. The docking results of compounds 4a, 4b, 8c,
8d and celecoxib are presented in Table 4. The docking score of
celecoxib = ꢁ26.44 kcal/mol, while the tolyl moiety is bound in
the primary hydrophobic pocket, the trifluoromethyl group is
bound in an adjacent pocket formed by Val335, Tyr341, Leu345
and Leu517. The phenyl sulfonamide moiety is inserted in the sec-
ondary pocket where the sulfonamide group exhibited three
hydrogen bonding interaction with Gln178, Leu338 and Ser339
(distance = 3.11, 2.76 and 2.97 Å, respectively), such interactions
are almost essential for COX-2 inhibitory activity [33,35]. Also,
the selectivity of celecoxib seems to result from the binding of
the sulfonamide group to this secondary pocket which is more
restricted in COX-1 [35]. The docking models of 4-thiazolidione
compounds 4a and 4b that exhibited the highest anti-
inflammatory activities in vivo in Scheme 1 showed that they are
oriented in COX-2 active site in a close manner to celecoxib (Figs. 3
and 4). These compounds had moderate binding affinities (docking
scores: ꢁ13.30 and ꢁ 6.05 kcal/mol, respectively), Table 4. The
iminothiazolidin-4-one is positioned in the primary hydrophobic
pocket occupied by the tolyl moiety of celecoxib. Also, the
4-nitro/isobutylphenyl moiety is oriented toward the secondary
pocket, in which it doesn’t exhibit any hydrogen bond interactions
with the amino acids present in COX-2 secondary pocket. Accord-
ingly, the lack of the H-bond interactions with COX-2 side pocket
for the analogs 4a and 4b might be responsible for their reduced
aforementioned in vitro COX-2 inhibitory activities.
Compounds
Edema inhibition %
1 h
3 h
5 h
4a
4b
4c
8a
8b
8c
8d
8e
42.2
43.1
2.9
52.7
56.7
22.36
17
22.3
61.8
67
57
60
29.1
22.8
30.4
64.5
69.6
30.4
62.5
8.7
11.6
48.3
50.7
4.3
23.7
60
Celecoxib
50.1
On the basis of the aforementioned in vitro and in vivo results,
compounds 8c and 8d which showed COX-2 inhibitory potency
and selectivity indexes very close to celecoxib and in vivo anti-
inflammatory activity higher than celecoxib, their corresponding
mean effective anti-inflammatory doses (ED50) were calculated
according to the method of Litchfield and Wilcoxon [31], Table 3.
The oral anti-inflammatory activity (ED50 value) exhibited by the
chloro derivative 8c (ED50 = 27.7 mg/kg) was found to be nearly
equipotent to celecoxib (ED50 = 28.2
lmol/kg) and < the fluoro
derivative 8d (ED50 = 18.1 mol/kg). The ED50 data obtained con-
l
firmed the high in vitro and in vivo results of 8c and 8d suggesting
the promising activity of those two derivatives as selective COX-2
inhibitors.
2.2.3. Ulcerogenic liability
Compounds 8c and 8d which showed the most potent COX-2
inhibitory activity and showed higher anti-inflammatory activity
than celecoxib were evaluated for their ulcerogenic potential
according to a previously reported method [32]. The ulcerogenic
effect was compared to both celecoxib (50 mg/kg) and the classical
Table 3
The geometries of the top ranked poses of 4-thiazolidinone
compounds with the best overall in vivo and in vitro profiles 8c
and 8d indicate that celecoxib and those two compounds adopt
nearly superimposed orientations in the binding site and interact
with many of the same amino acid residues in the crystal structure
Mean effective anti-inflammatory doses (ED50
celecoxib and ibuprofen.
) and ulcerogenic liability 8c, 8d,
Compounds
ED50 (mg/kg)a
ED50
(l
mol/kg)a
Ulcer indexb
8c
8d
Celecoxib
Ibuprofen
11.0
6.9
27.7
18.1
28.2
NDc
1.2
0.6
7.8
3LN1 (Figs.
5
and 6). It is interesting to note that
10.8
NDc
14.6
4-sulfamoylphenylamino moiety of 8c and 8d is positioned in the
COX-2 secondary pocket, surrounded by His75, Ser339, Arg499
and Gln178. As aforementioned, this secondary pocket is
considered to be responsible for the selectivity and occupied by
the P-SO2NH2 pharmacophore of selective COX-2 inhibitors [35].
a
Anti-inflammatory activity represented by ED50 which was the effective dose
calculated after 3 h.
b
Ulcerogenic liability using 50 mg/kg dose.
Not determined.
c

6
K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
Table 4
Docking score and bond interactions of celecoxib and synthesized compounds 4a, 4b, 8c and 8d with amino acids of COX-2.
Compounds
Docking score (kcal/mol)
No. of H-bonds
Distance
Amino acids involved
Molecular structure
c logPa
4a
4b
8c
ꢁ13.30
ꢁ6.05
–
–
3
–
–
–
–
–
–
2.76
4.05
3.07
ꢁ16.40
3.16
2.02
2.69
2.14
2.78
2.97
2.76
3.11
His75
Ser339
Arg499
Ser339
Arg499
Ser339
Leu338
Gln78
H of SO2NH2
H of SO2NH2
O of SO2NH2
H of SO2NH2
O of SO2NH2
N of SO2NH2
N of SO2NH2
N of SO2NH2
8d
ꢁ17.15
ꢁ26.44
2
3
2.55
3.82
Celecoxib
a
Calculated using Ligand Properties tool in MOE (The Molecular Operating Environment, Version 2008.10).
Fig. 3. The orientation of 4a (pink) in COX-2 active site and celecoxib (turquoise) top ranked docking pose in the COX-2 active site. (For interpretation of the references to
color in this figure legend, the reader is to the web version of this article).
Fig. 4. The orientation of 4b (pink) in COX-2 active site and celecoxib (turquoise) top ranked docking pose in the COX-2 active site. (For interpretation of the references to
color in this figure legend, the reader is to the web version of this article).

K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
7
Fig. 5. Overlay of 8c (pink) and celecoxib (turquoise) top ranked docking pose in the COX-2 active site and hydrogen bonds are shown in green. (For interpretation of the
references to color in this figure legend, the reader is to the web version of this article).
Fig. 6. Overlay of 8d (pink) and celecoxib (turquoise) top ranked docking pose in the COX-2 active site and hydrogen bonds are shown in green. (For interpretation of the
references to color in this figure legend, the reader is to the web version of this article).
Furthermore, the CH3 group of compounds 8c and 8d is placed
within the same region of the CF3 moiety of celecoxib. Compound
8c forms three hydrogen bonds with the amino acids present in the
COX-2 secondary pocket in which one of the sulfonamide hydrogen
atoms forms two hydrogen bonds with the nitrogen atom of
His75 (distance = 3.16 Å) and the oxygen atom of Ser339
(distance = 2.02 Å) and one of the sulfonamide oxygen atoms forms
a hydrogen bond with the NH of Arg499 (distance = 2.69 Å) (Fig. 5).
While, compound 8d forms two hydrogen bonds with the amino
acids present in the COX-2 secondary pocket in which one of the
sulfonamide hydrogen atoms forms a hydrogen bond with the oxy-
gen atom of Ser339 (distance = 2.14 Å) and one of the sulfonamide
oxygen atoms forms a hydrogen bond with the NH of Arg499
(distance = 2.78 Å) (Fig. 6).
The profound enhancement in COX-2 inhibitory effect of the
chlorophenyl and fluorophenyl derivatives 8c and 8d suggested
that they bound to the enzyme in such way to maximize their
interaction with the binding site. As seen in Table 4, these com-
pounds exhibited high binding affinities (docking scores: ꢁ16.40
and ꢁ17.15 kcal/mol, respectively) with docking scores close to
that of celecoxib, (docking score: ꢁ26.44 kcal/mol). This may be
attributed to the highly electronegative nature of chlorine or fluo-
rine atom which led to a more efficient p–p stack interaction of the
phenyl ring with Tyr371 and Trp373 [36,37]. Moreover, the
difference in electronegativity between chlorine or fluorine and
carbon creates a large dipole moment in this bond which may con-
tribute to the molecule’s ability to be engaged in intermolecular
interactions with the COX-2 active site [38,39].

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K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
Table 5
ADME of celecoxib and synthesized compounds 4a, 4b, 8c and 8d using mipc-Molinspiration property calculator.
Compounds
MW
No. of H-bond donors
No. of H-bond acceptors
milogP
No. of rotatable bonds
TPSA
No. of violations
4a
4b
8c
8d
320.355
331.466
397.909
381.454
381.379
1
1
3
3
2
7
4
6
6
5
2.1
4
5
4
4
4
100.178
54.354
92.501
92.501
77.991
0
0
0
0
0
3.442
2.811
2.297
3.611
Celecoxib
MW: molecular weight.
milogP: octanol–water partition coefficient (logP predicted at Molinspiration).
TPSA: topological polar surface area.
2.4. ADME profiling
synthesized compounds were evaluated for their COX-1/COX-2
inhibitory activity in vitro and anti-inflammatory activity in vivo.
Compounds 8c and 8d showed COX-2 inhibitory potency
The bioavailability of the reference drug celecoxib and the most
biologically active compounds 4a, 4b, 8c and 8d was assessed
using mipc-Molinspiration Property Calculator [40]. In particular,
we calculated the compliance of compounds to Lipinski’s ‘‘rule of
five” to evaluate the drug-likeness [41]. The rule describes molec-
ular properties which are important for drug’s pharmacokinetics in
the human body, including their absorption, distribution, metabo-
lism and elimination (ADME) and is used to make sure that drug-
like physicochemical properties are maintained during drug
design. This simple rule states that orally active drug has no more
than one violation of the following criteria: molecular weight less
than 500 Da; no more than five hydrogen bond donors; no more
than 10 hydrogen bond acceptors; and calculated octanol–water
partition coefficient (c logP) not greater than 5 [42]. Moreover,
topological polar surface area (TPSA) together with the number
of rotatable bonds have been considered to be very good descrip-
tors of oral bioavailability of drugs. Compounds which meet the
following two criteria: ten or fewer rotatable bonds and polar sur-
face area equal to or less than 140 Å2 are predicted to exhibit good
oral bioavailability [43]. The calculated parameters presented in
Table 5 showed good bioavailability of studied compounds. The
most active compounds 4a, 4b, 8c and 8d fulfilled all rules, similar
to the clinically used drug celecoxib. Theoretically, these four com-
pounds should exhibit good passive oral absorption and differ-
ences in their bioactivity cannot be attributed to these
properties. Compounds 4a and 4b exhibited anti-inflammatory
activity with edema inhibition % (42.2 and 43.1, respectively) after
1 h indicating their high anti-inflammatory activity and rapid
onset of action which might be attributed to their enhanced aque-
ous solubility (miLogP: 2.1 and 3.442, respectively). Also, the deter-
mined logP values were 2.76 and 4.05, respectively, which
affirmed the hydrophilic characteristics of these compounds. In
the same context, compounds 8c and 8d which showed the highest
inhibitory activity and selectivity for COX-2 enzyme in the in vitro
(IC50 = 1.9 and 2.3
(S.I. = 4.56 and 5.68, respectively) very close to celecoxib
(IC50 = 1.33 M and S.I. = 7.29). Moreover, compounds 8c and 8d
were found to be potent and much more selective toward COX-2
isozyme (IC50 = 1.9 and 2.3 M, respectively) than COX-1 isozyme
(IC50 = 10.5 and 10.8 M, respectively). In addition, the synthesized
compounds were evaluated for their in vivo anti-inflammatory
activity. Compounds 4a, 4b, 8c and 8d were found to have close
to higher anti-inflammatory activity (edema inhibition % = 52.7,
56.7, 61.8 and 67 after 3 h, respectively) than celecoxib (edema
inhibition % = 60 after 3 h). ED50 was calculated for the most potent
compounds 8c and 8d that showed the highest in vitro and in vivo
results. It is further interestingly confirmed their promising anti-
inflammatory activities with ED50 values indicating that compound
8c is equipotent to celecoxib and compound 8d is much more
lM, respectively) and selectivity indexes
l
l
l
potent than celecoxib (ED50 = 27.7, 18.1 and 28.2 lmol/kg for 8c,
8d and celecoxib, respectively). The ulcerogenic liability of com-
pounds 8c and 8d was also determined which was another proof
of their promising activity as safe anti-inflammatory compounds
with nearly negligible ulcerogenic liabilities compared to celecoxib
and ibuprofen (Ulcer index = 1.2, 0.6, 7.8 and 14.6 for 8c, 8d, cele-
coxib and ibuprofen, respectively).
Molecular docking simulations and analysis of the binding
modes of the new inhibitors within COX-2 active site were also
performed to rationalize the highly obtained anti-inflammatory
activity results. The H-bonding capability of sulfonamide group
in the COX-2 active site and the electronic effect (electronegativity)
of halogen atom on the phenyl ring seems to be the most crucial
factors affecting the anti-inflammatory activity and exerted the
major influence on the COX-2 inhibitory potency of 3-(4-aminosul
fonylphenylamino)-2-(4-chloro/fluorophenyl)-5-methyl-4-thiazoli
dinone (8c and 8d). The promising anti-inflammatory activity of 8c
and 8d which was higher than celecoxib as well as their reduced
ulcerogenic potential by 85% and 92%, respectively compared to
celecoxib, make them good lead candidates for further optimiza-
tion and development of potent and safe anti-inflammatory agents.
assay (IC50 = 1.9 and 2.3 lM, respectively), exhibited anti-
inflammatory activity with edema inhibition % (48.3 and 50.7,
respectively) after 1 h. The high potency and the rapid onset of
action of 3-(4-aminosulfonylphenylamino)-2-(4-chloro/fluorophe
nyl)-5-methyl-4-thiazolidinone derivatives (8c and 8d) might be
attributed to their enhanced aqueous solubility (miLogP: 2.81
and 2.29, respectively). In this context, aqueous solubility controls
the rate of dissolution of the compound together with the maxi-
mum concentration reached in the gastrointestinal fluid and may
be the main factor ensuring a good level of distribution of these
compounds in vivo [44]. The determined logP values were 3.07
and 2.55, respectively, which affirmed the hydrophilic characteris-
tics of compounds.
4. Experimental
4.1. Chemistry
Melting points were determined on a Griffin apparatus and are
uncorrected. Infrared (IR) spectra were recorded on a Shimadzu
435 spectrometer using KBr discs. 1H NMR was measured on a Bru-
ker 400 MHz spectrometer and 13C NMR spectra were measured on
a Bruker 100 MHz spectrometer (Faculty of Pharmacy, Beni Suef
University, Beni Suef, Egypt) in D2O or DMSO-d6 with TMS as the
internal standard, where J (coupling constant) values were esti-
mated in hertz (Hz). Mass spectra were run on Hewlett Packard
5988 spectrometer. Microanalyses were performed for C, H, N at
the (Micro Analytical Center, Cairo University, Egypt) and were
3. Conclusion
The present study reported the design and synthesis of novel
thiazoldinone derivatives as selective COX-2 inhibitors. The

K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
9
within 0.4% of theoretical values. All other reagents, purchased
from the Acros Chemical Company, were used without further
purification; celecoxib was prepared while ibuprofen was pur-
chased from the Aldrich Chemical Company (Milwaukee, WI),
Compounds 2a [45], 2b [46], 2c [47], 3a [48], 5 [49] and 7a–e
[50] were prepared according to reported procedures.
nitrophenyl H-3, H-5), 12.21(s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6) d 35.5 (thiazolidinone C-5), 115.5 (thiazole C-5),
124.7 (nitrophenyl C-3, C-5), 127.0 (nitrophenyl C-2, C-6), 140.3
(nitrophenyl C-1), 147.0 (nitrophenyl C-4), 149.1 (thiazole C-4),
164.7 (thiazolidinone C-2), 170.1 (thiazole C-2), 174.6 (C@O); MS
(m/z): 320 (M+., 100%); Anal. Calcd for C12H8N4O3S2: C, 44.99; H,
2.52; N, 17.49. Found: C, 44.96; H, 2.33; N, 17.20.
4.1.1. General method for preparation of 2-chloroacetamido-4-(4-
substituted-phenyl)thiazoles (3b–c)
4.1.2.2. 2-(4-(4-Isobutylphenyl)thiazol-2-ylimino)thiazolidin-4-one
(4b). Yield: 40%; brown solid; mp 250–252 °C; IR (KBr): 3136
(NH), 3030 (CH aromatic), 2935, 2821 (CH aliphatic), 1725 (C@O)
To a solution of 2-amino-4-(4-substituted-phenyl)thiazole (2b
or 2c) (2 mmol) in dioxane (20 mL), chloroacetyl chloride (4 mmol,
0.44 gm) was added dropwise then the reaction mixture was
heated under reflux for 24 h. The solid precipitated on hot was fil-
tered, washed with aqueous sodium carbonate followed by cold
water, dried and crystallized from ethanol to give the respective
cmꢁ1 1H NMR (DMSO-d6) d 0.87 (d, J = 6 Hz, 6H, CH2CH(CH3)2),
;
1.83–1.86 (m, 1H, CH2CH(CH3)2), 2.47 (d, J = 6.4 Hz, 2H, CH2CH
(CH3)2), 4.03 (s, 2H, CH2), 7.22 (d, J = 8 Hz, 2H, isobutylphenyl
H-3, H-5), 7.74 (s, 1H, thiazole H-5), 7.85 (d, J = 8 Hz, 2H, isobutyl-
phenyl H-2, H-6), 12.12 (s, 1H, NH, D2O exchangeable); 13C NMR
2-chloroacetamido-4-(4-substituted-phenyl)thiazoles
(3b–c).
Physical and spectral data for 3b–c are listed below.
(DMSO-d6) d 22.5 (CH2CH(CH3)2), 30.0 (CH2CH(CH3)2), 35.4
(thiazolidinone C-5), 44.7 (CH2CH(CH3)2), 110.1 (thiazole C-5),
125.9 (isobutylphenyl C-2, C-6), 129.8 (isobutylphenyl C-3, C-5),
132.0 (isobutylphenyl C-1), 141.7 (isobutylphenyl C-4), 151.5
(thiazole C-4), 164.0 (thiazolidinone C-2), 169.3 (thiazole C-2),
174.9 (C@O); MS (m/z): 331 (M+., 100%); Anal. Calcd for
C16H17N3OS2: C, 57.98; H, 5.17; N, 12.68. Found: C, 57.66; H,
5.36; N, 12.58.
4.1.1.1. 2-Chloroacetamido-4-(4-isobutylphenyl)thiazole (3b). Yield:
65%; buff solid; mp 143–145 °C; IR (KBr): 3425 (NH), 3047 (CH aro-
matic), 2923, 2867 (CH aliphatic), 1698 (C@O) cmꢁ1 1H NMR
;
(DMSO-d6) d 0.87 (d, J = 6.8 Hz, 6H, CH2CH(CH3)2), 1.83–1.89 (m,
1H, CH2CH(CH3)2), 2.47 (d, J = 7.2 Hz, 2H, CH2CH(CH3)2), 4.41 (s,
2H, CH2Cl), 7.22 (d, J = 8 Hz, 2H, isobutylphenyl H-3, H-5), 7.62
(s, 1H, thiazole H-5), 7.80 (d, J = 8 Hz, 2H, isobutylphenyl H-2,
H-6), 12.63 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6)
d 22.6 (CH2CH(CH3)2), 30.0 (CH2CH(CH3)2), 42.7 (CH2Cl), 44.7
(CH2CH(CH3)2), 108.2 (thiazole C-5), 125.9 (isobutylphenyl C-2,
C-6), 129.7 (isobutylphenyl C-3, C-5), 132.2 (isobutylphenyl C-1),
141.4 (isobutylphenyl C-4), 149.6 (thiazole C-4), 157.7 (thiazole
C-2), 165.5 (C@O); MS (m/z): 308 (M+., 47%), 309 (M+1, 9%), 310
(M+2, 18%), 265 (100%); Anal. Calcd for C15H17ClN2OS: C, 58.34;
H, 5.55; N, 9.07. Found: C, 58.57; H, 5.62; N, 9.31.
4.1.2.3. 2-(4-(4-Methanesulfonamidophenyl)thiazol-2-ylimino)thia-
zolidin-4-one (4c). Yield: 45%; buff solid; mp 260–262 °C; IR
(KBr): 3155, 3113 (2 NH), 3063 (CH aromatic), 2923, 2867 (CH ali-
phatic), 1730(C@O) cmꢁ1 1H NMR (DMSO-d6) d 3.35 (s, 3H, CH3),
;
4.05 (s, 2H, CH2), 7.15 (s, 1H, methanesulfonamido NH, D2O
exchangeable), 7.59 (d, J = 8.4 Hz, 2H, 4-methanesul-
fonamidophenyl H-3, H-5), 7.98 (s, 1H, thiazole H-5), 8.04 (d,
J = 8.4 Hz, 2H, 4-methanesulfonamidophenyl H-2, H-6), 12.12 (s,
1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) d 35.4 (thiazolidi-
none C-5), 43.5 (CH3), 112.8 (thiazole C-5), 127.1 (4-methanesul-
fonamidophenyl C-3, C-5), 131.9 (4-methanesulfonamidophenyl
C-2, C-6), 133.7 (4-methanesulfonamidophenyl C-1), 135.9
(4-methanesulfonamidophenyl C-4), 150.1 (thiazole C-4), 164.3
(thiazolidinone C-2), 169.7 (thiazole C-2), 174.7 (C@O); MS (m/z):
367 (Mꢁ1, 100%), 368 (M+., 21%); Anal. Calcd for C13H12N4O3S3: C,
42.38; H, 3.28; N, 15.21. Found: C, 42.49; H, 3.25; N, 15.45.
4.1.1.2. 2-Chloroacetamido-4-(4-methanesulfonamidophenyl)thiazole
(3c). Yield: 70%; buff solid; mp 230–232 °C; IR (KBr): 3441, 3394 (2
NH), 3041 (CH aromatic), 2937 (CH aliphatic), 1690 (C@O) cmꢁ1
;
1H NMR (DMSO-d6) d 3.33 (s, 3H, CH3), 4.10 (s, 1H, methanesulfon-
amido NH, D2O exchangeable), 4.43 (s, 2H, CH2Cl), 7.58
(d, J = 8.4 Hz, 2H, 4-methanesulfonamidophenyl H-3, H-5), 7.86
(s, 1H, thiazole H-5), 8.00 (d, J = 8.4 Hz, 2H, 4-methanesulfona-
midophenyl H-2, H-6), 12.68 (s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6) d 42.7 (CH3), 43.5 (CH2Cl), 110.8 (thiazole C-5),
127.0 (4-methanesulfonamidophenyl C-3, C-5), 131.8 (4-methane-
sulfonamidophenyl C-2, C-6), 133.6 (4-methanesulfonamido-
phenyl C-1), 136.0 (4-methanesulfonamidophenyl C-4), 148.2
(thiazole C-4), 158.1 (thiazole C-2), 165.7 (C@O); MS (m/z): 344
(Mꢁ1, 100%), 345 (M+., 20%), 346 (M+1, 44%), 347 (M+2, 10%); Anal.
Calcd for C12H12ClN3O3S2: C, 41.68; H, 3.50; N, 12.15. Found: C,
41.85; H, 3.55; N, 12.52.
4.1.3. General method for preparation of 3-(4-aminosulfonyl-
phenylamino)-2-aryl-5-methyl-4-thiazolidinones (8a–e)
A mixture of the appropriate phenyl hydrazones (7a–e, 2 mmol)
and excess of thiolactic acid (2 mL) was heated at 60 °C for 3 h. The
reaction was cooled then saturated solution of sodium carbonate
(3 ꢂ 20 mL) was added and the precipitate was filtered. The
residue was washed with water (1 ꢂ 10 mL), dried and crystallized
from aqueous ethanol to afford 4-thiazolidinone derivatives 8a–e.
Physical and spectral data for 8a–e are listed below.
4.1.2. General method for preparation of 2-(4-(4-substituted-phenyl)
thiazol-2-ylimino)thiazolidin-4-ones (4a–c)
4.1.3.1. 3-(4-Aminosulfonylphenylamino)-5-methyl-2-phenyl-4-thia-
zolidinone (8a). Yield: 50%; white solid; mp 138–140 °C; IR (KBr):
3267 (NH), 3091 (CH aromatic), 2928 (CH aliphatic), 1689 (C@O),
To a solution of 2-chloroacetamido-4-(4-substituted-phenyl)th
iazole (3a–c) (2 mmol) in ethanol (20 mL), ammonium thiocyanate
(4 mmol, 0.3 gm) is added then the reaction mixture was heated
under reflux for 24 h. The separated solid was filtered, washed with
water, dried then crystallized from ethanol to afford corresponding
compounds 4a–c. Physical and spectral data for 4a–c are listed
below.
1328, 1154 (SO2) cmꢁ1 1H NMR (DMSO-d6) d 1.52, 1.54 (2d,
;
J = 7.2 Hz, 3H, thiazolidinone CH3), 4.14–4.28 (m, 1H, thiazolidi-
none H-5), 5.87, 5.92 (2s, 1H, thiazolidinone H-2), 6.70–6.74 (m,
2H, aminosulfonylphenyl H-2, H-6), 7.07 (s, 2H, NH2, D2O
exchangeable), 7.33–7.43 (m, 5H, phenyl H-2, H-3, H-4, H-5, H-
6), 7.56–7.61 (m, 2H, aminosulfonylphenyl H-3, H-5), 8.73, 8.80
(2s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) d 14.6, 19.6
(thiazolidinone CH3), 17.7, 20.3 (thiazolidinone C-5), 37.7, 38.6
(thiazolidinone C-2), 111.6, 111.8 (aminosulfonylphenyl C-2, C-6),
126.5 (phenyl C-4), 127.6, 127.7 (phenyl C-2, C-6), 127.9
(phenyl C-3, C-5), 129.1, 129.3 (aminosulfonylphenyl C-3, C-5),
4.1.2.1. 2-(4-(4-Nitrophenyl)thiazol-2-ylimino)thiazolidin-4-one (4a).
Yield: 50%; yellow solid; mp 300–302 °C; IR (KBr): 3428 (NH),
3100, 3052 (CH aromatic), 2920 (CH aliphatic), 1716 (C@O)
cmꢁ1 1H NMR (DMSO-d6) d 4.07 (s, 2H, CH2), 8.18–8.19 (m, 3H,
;
nitrophenyl H-2, H-6, thiazole H-5), 8.31 (d, J = 8.4 Hz, 2H,

10
K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
133.9, 134.6 (aminosulfonylphenyl C-4), 134.7, 135.7 (phenyl C-1),
148.2, 149.9 (aminosulfonylphenyl C-1), 172.1 (C@O); MS (m/z):
363 (M+., 39%), 77 (100%); Anal. Calcd for C16H17N3O3S2: C, 52.87;
H, 4.71; N, 11.56. Found: C, 52.99; H, 4.79; N, 11.71.
4.1.3.5.
3-(4-Aminosulfonylphenylamino)-5-methyl-2-(4-trifluo-
romethylphenyl)-4-thiazol-idinone (8e). Yield: 45%; white solid;
mp 143–145 °C; IR (KBr): 3269 (NH), 3095 (CH aromatic), 2931
(CH aliphatic), 1696 (C@O), 1326, 1157 (SO2) cmꢁ1 1H NMR
;
(DMSO-d6) d 1.53, 1.55 (2d, J = 7.6 Hz, 3H, thiazolidinone CH3),
4.19–4.32 (m, 1H, thiazolidinone H-5), 6.02, 6.06 (2s, 1H, thiazo-
lidinone H-2), 6.72–6.76 (m, J = 8.8 Hz, 2H, aminosulfonylphenyl
H-2, H-6), 7.09 (s, 2H, NH2, D2O exchangeable), 7.58–7.71 (m, 4H,
trifluoromethylphenyl H-2, H-3, H-5, H-6), 7.73–7.78 (m, 2H,
aminosulfonylphenyl H-3, H-5), 8.81, 8.87 (2s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) d 15.5, 19.4 (thiazolidinone
CH3), 18.0, 20.4 (thiazolidinone C-5), 37.6, 38.7 (thiazolidinone
C-2), 111.7, 111.9 (aminosulfonylphenyl C-2, C-6), 123.1, 125.8
(trifluoromethyl), 126.1, 126.9 (trifluoromethylphenyl C-3, C-5),
127.7, 127.8 (trifluoromethylphenyl C-2, C-6), 127.9, 128.9
4.1.3.2. 3-(4-Aminosulfonylphenylamino)-5-methyl-2-(4-methylphe-
nyl)-4-thiazolidinone (8b). Yield: 40%; white solid; mp
145–147 °C; IR (KBr): 3285 (NH), 3096 (CH aromatic), 2925 (CH
aliphatic), 1690 (C@O), 1330, 1154 (SO2) cmꢁ1 1H NMR (DMSO-
;
d6) d 1.52, 1.54 (2d, J = 7.2 Hz, 3H, thiazolidinone CH3), 2.29 (s,
3H, methylphenyl CH3), 4.12–4.26 (m, 1H, thiazolidinone H-5),
5.82, 5.87 (2s, 1H, thiazolidinone H-2), 6.69–6.73 (m, 2H,
aminosulfonylphenyl H-2, H-6), 7.06 (s, 2H, NH2, D2O exchange-
able), 7.17–7.30 (m, 4H, methylphenyl H-2, H-3, H-5, H-6), 7.56–
7.60 (m, 2H, aminosulfonylphenyl H-3, H-5), 8.67, 8.74 (2s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO-d6) d 19.7, 20.2 (thiazo-
lidinone CH3), 21.23 (methylphenyl CH3), 21.21, 21.4 (thiazolidi-
none C-5), 37.7, 38.6 (thiazolidinone C-2), 111.6, 111.7
(aminosulfonylphenyl C-2, C-6), 126.5 (methylphenyl C-3, C-5),
127.6, 127.9 (methylphenyl C-2, C-6), 129.6, 129.7 (aminosul-
fonylphenyl C-3, C-5), 134.5, 134.6 (aminosulfonylphenyl C-4),
138.5, 138.6 (methylphenyl C-1), 138.8 (methylphenyl C-4),
149.8, 149.9 (aminosulfonylphenyl C-1), 171.7 (C@O); MS (m/z):
377 (M+., 62%), 137 (100%); Anal. Calcd for C17H19N3O3S2: C,
54.09; H, 5.07; N, 11.13. Found: C, 54.23; H, 5.18; N, 11.30.
(aminosulfonylphenyl C-3, C-5), 129.4,
129.7
(trifluo-
romethylphenyl C-4), 134.6, 134.9 (aminosulfonylphenyl C-4),
145.2, 147.8 (trifluoromethylphenyl C-1), 149.5, 149.7 (aminosul-
fonylphenyl C-1), 172.0 (C@O); MS (m/z): 431 (M+., 1%), 64
(100%); Anal. Calcd for C17H16F3N3O3S2: C, 47.32; H, 3.74; N, 9.74.
Found: C, 47.45; H, 3.78; N, 9.88.
4.2. Biological evaluation
4.2.1. In vitro cyclooxygenase inhibition assay
The ability of the tested compounds to inhibit both COX-1 and
COX-2 isozymes was measured using colorimetric COX (ovine)
Inhibitor Screening Assay Kit (Kit catalog number 760111, Cayman
Chemical, Ann Arbor, MI, USA) following the manufacturer’s
instructions and as mentioned before [51]. Different concentrations
of celecoxib or tested compounds were incubated with the enzymes
for a period of 5 min at 25 °C. After the incubation period an addi-
tion of the colorimetric substrate and arachidonic acid was done
then the absorbance was measured at 590 nm using plate reader.
4.1.3.3.
3-(4-Aminosulfonylphenylamino)-2-(4-chlorophenyl)-5-
methyl-4-thiazolidinone (8c). Yield: 60%; white solid; mp
150–152 °C; IR (KBr): 3276 (NH), 3095 (CH aromatic), 2929 (CH
aliphatic), 1692 (C@O), 1328, 1154 (SO2) cmꢁ1 1H NMR (DMSO-
;
d6) d 1.52, 1.54 (2d, J = 7.2 Hz, 3H, thiazolidinone CH3), 4.15–4.28
(m, 1H, thiazolidinone H-5), 5.89, 5.93 (2s, 1H, thiazolidinone H-
2), 6.69–6.73 (m, 2H, aminosulfonylphenyl H-2, H-6), 7.08 (s, 2H,
NH2, D2O exchangeable), 7.42–7.47 (m, 4H, 4-chlorophenyl H-2,
H-3, H-5, H-6), 7.56–7.61 (m, 2H, aminosulfonylphenyl H-3, H-5),
8.72, 8.79 (2s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6) d
17.7, 19.6 (thiazolidinone CH3), 17.8, 20.3 (thiazolidinone C-5),
37.6, 38.6 (thiazolidinone C-2), 111.6, 111.8 (aminosulfonylphenyl
C-2, C-6), 127.6 (chlorophenyl C-3, C-5), 127.73, 127.75 (chloro-
phenyl C-2, C-6), 129.1, 129.2 (aminosulfonylphenyl C-3, C-5),
133.6 (aminosulfonylphenyl C-4), 133.8 (chlorophenyl C-4),
134.7, 134.8 (chlorophenyl C-1), 149.6, 149.7 (aminosul-
fonylphenyl C-1), 171.7 (C@O); MS (m/z): 397 (M+., 48%), 398
4.2.2. In vivo anti-inflammatory activity
White albino adult female mice, weighing approximately
18–22 g were used throughout the study of anti-inflammatory
activity. While, white adult male albino rats weighing approxi-
mately 100–150 g were used throughout the study of ulcerogenic
liability. The animals were housed in micro isolator cages (five
per cage) at laboratory temperature of 24 1 °C with 40–80% rela-
tive humidity with access to food and water. The animals were
allowed to adapt to the experimental environment for 7 days
before experimentation. All procedures relating to animal care
and treatments were performed following the protocols approved
by the Research Ethical Committee, Beni-Suef University (2014-
Beni-Suef, Egypt). The white albino mice were divided into ten
groups of ten animals each. The first group was acting as a negative
control given 5% aqueous solution of DMSO (v/v). The second
groups were orally administered celecoxib as a reference standard
(10 mg/kg). The eight tested compounds 4a–c and 8a–e in the form
of 5% aqueous solution of DMSO were given orally to the rest
groups, one group for each compound, in which each compound
was administered in a dose 100 mg/kg body weight and treatment
began 1 h before induction of inflammation. Induction of Paw
edema was performed by single sub-plantar injection of 0.02 ml
of freshly prepared 1% carrageenan (Sigma) in normal saline [30].
The paw thickness was measured using vernier calipers and the
difference between paw volumes was calculated after 1, 3 and
5 h of carrageenan injection. The anti-inflammatory activity was
calculated as percentage inhibition of edema thickness in treated
animals in comparison to the control group, Table 2 according to
the following formula
(M+1
16H16ClN3O3S2: C, 48.30; H, 4.05; N, 10.56. Found: C, 48.47; H,
4.13; N, 10.67.
, , 21%) 213 (100%); Anal. Calcd for
10%), 399 (M+2
C
4.1.3.4.
3-(4-Aminosulfonylphenylamino)-2-(4-fluorophenyl)-5-
methyl-4-thiazolidinone (8d). Yield: 35%; white solid; mp
140–142 °C; IR (KBr): 3281 (NH), 3089 (CH aromatic), 2929 (CH
aliphatic), 1693 (C@O), 1329, 1154 (SO2) cmꢁ1 1H NMR (DMSO-
;
d6) d 1.53, 1.55 (2d, J = 7.2 Hz, 3H, thiazolidinone CH3), 4.15–4.30
(m, 1H, thiazolidinone H-5), 5.89, 5.93 (2s, 1H, thiazolidinone H-
2), 6.68–6.72 (m, 2H, aminosulfonylphenyl H-2, H-6), 7.07 (s, 2H,
NH2, D2O exchangeable), 7.19–7.23 (m, 2H, fluorophenyl H-3, H-
5), 7.49–7.61 (m, 4H, flourophenyl H-2, H-6, aminosulfonylphenyl
H-3, H-5), 8.70, 8.78 (2s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6) d 17.5, 19.6 (thiazolidinone CH3), 17.7, 20.2 (thiazolidi-
none C-5), 37.7, 38.6 (thiazolidinone C-2), 111.6, 111.8 (aminosul-
fonylphenyl C-2, C-6), 115.8, 116.0 (flourophenyl C-3, C-5), 127.6,
127.7 (flourophenyl C-2, C-6), 130.0, 130.1 (aminosulfonylphenyl
C-3, C-5), 134.6, 134.7 (aminosulfonylphenyl C-4), 149.6, 149.8
(flourophenyl C-1), 161.3, 161.4 (aminosulfonylphenyl C-1),
163.8, 163.9 (flourophenyl C-4), 171.9 (C@O); MS (m/z): 381 (M+.,
33%), 213 (100%); Anal. Calcd for C16H16FN3O3S2: C, 50.38; H,
4.23; N, 11.02. Found: C, 50.49; H, 4.31; N, 11.17.
Edema inhibition ð%Þ ¼ ððNa ꢁ NbÞ=NaÞ ꢂ 100

K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
11
In which, Na was the average difference in thickness between
the left and right hind paw of control group and Nb was that of
drug-treated group.
enzyme was 3D protonated, in which hydrogen atoms were added
to their standard geometry, the partial charges were computed and
the system was optimized. The conformers generated were docked
into the COX-2 receptor with MOE-DOCK using the triangle
matcher placement method and the London dG scoring function.
A molecular mechanics force field refinement was carried out on
the top 100 poses generated. In order to validate the docking pro-
cedure, celecoxib was docked into the active site of 3LN1. The high-
est docking score for each ligand–enzyme complex was selected.
Also, the mean effective anti-inflammatory doses (ED50) for the
prepared compounds showed the highest in vitro COX-2 inhibitory
potency and in vivo anti-inflammatory activity 8c and 8d were
determined. White albino mice were divided into ten groups of
ten animals each. In all groups, induction of inflammation was per-
formed by single sub-plantar injection of 0.02 mL of freshly pre-
pared 1% carrageenan (Sigma) in normal saline [30]. The first
group was acting as a negative control given 5% aqueous solution
of DMSO (v/v). The second, third and fourth groups were orally
administered celecoxib as a reference standard in three different
doses 5, 10 and 15 mg/kg, one dose for each group, to calculate
ED50. The two tested compounds 8c and 8d in the form of 5% aque-
ous solution of DMSO were given orally to the rest groups, three
groups for each compound, in which each compound was adminis-
tered in three different doses 5, 10 and 15 mg/kg body weight to
calculate ED50 for each compound and treatment began 1 h before
induction of inflammation. The paw thickness was measured using
vernier calipers and the difference between paw volumes was
calculated after 3 h of carrageenan injection. On the basis of
the obtained results, the corresponding mean effective
anti-inflammatory doses (ED50) were calculated according to the
method of Litchfield and Wilcoxon [31].
5. Conflict of Interest
The authors have declared no conflict of interest.
Acknowledgments
The authors wish to express their thanks to Dr. Mahmoud
Abdel-Latif, Faculty of Science, Beni Suef University for his help
in performing in vivo anti-inflammatory activity.
References
[1] G. Dannhardt, W. Kiefer, Eur. J. Med. Chem. 36 (2001) 109–126.
[2] A. Zarghi, L. Najafnia, B. Daraee, O.G. Dadrass, M. Hedayati, Bioorg. Med. Chem.
Lett. 17 (2007) 5634–5637.
[3] T. Zebardast, A. Zarghi, B. Daraie, M. Hedayati, O.G. Dadrass, Bioorg. Med.
Chem. Lett. 19 (2009) 3162–3165.
4.2.3. Ulcerogenic liability
[4] G.S. Hassan, S.M. Abou-Seri, G. Kamel, M.M. Ali, Eur. J. Med. Chem. 76 (2014)
482–493.
[5] B.J. Al-Hourani, S.K. Sharma, J.Y. Mane, J. Tuszynski, V. Baracos, T. Kniess, M.
Suresh, J. Pietzsch, F. Wuest, Bioorg. Med. Chem. Lett. 21 (2011) 1823–1826.
[6] I.G. Rathish, K. Javed, S. Ahmad, S. Bano, M.S. Alam, K.K. Pillai, S. Singh, V.
Bagchi, Bioorg. Med. Chem. Lett. 19 (2009) 255–258.
[7] A. Zarghi, T. Zebardast, B. Daraie, M. Hedayati, Bioorg. Med. Chem. 17 (2009)
5369–5373.
[8] P. Mcgettigan, D. Henry, J. Am. Med. Assoc. 296 (2006) 1633–1644.
[9] W.B. White, G. Faich, J.S. Borer, R.W. Makuch, Am. J. Cardiol. 92 (2003)
411–418.
[10] N.M. Davies, A.J. Mclachlan, R.O. Day, K.M. Williams, Clin. Pharmacokinet. 38
(2000) 225–242.
[11] J. Castellsague, N. Riera-guardia, B. Calingaert, C. Varas-lorenzo, A. Fourrier-
reglat, F. Nicotra, M. Sturkenboom, S. Perez-gutthann, Drug Saf. 35 (2012)
1127–1146.
[12] K.R.A. Abdellatif, M.A. Chowdhury, Y. Dong, E.E. Knaus, Bioorg. Med. Chem. 16
(2008) 6528–6534.
[13] M.A. Chowdhury, K.R.A. Abdellatif, Y. Dong, E.E. Knaus, Bioorg. Med. Chem. 16
(2008) 8882–8888.
[14] K.R.A. Abdellatif, M.A. Chowdhury, E.E. Knaus, J. Heterocycl. Chem. 45 (2008)
1707–1710.
[15] K.R.A. Abdellatif, M.A. Chowdhury, Y. Dong, D. Das, G. Yu, C. Velázquez, M.R.
Suresh, E.E. Knaus, Bioorg. Med. Chem. 17 (2009) 5182–5188.
[16] M.A. Chowdhury, K.R.A. Abdellatif, Y. Dong, D. Das, M.R. Suresh, E.E. Knaus,
Bioorg. Med. Chem. Lett. 18 (2008) 6138–6141.
[17] K.R.A. Abdellatif, M.A. Chowdhury, Y. Dong, C. Velázquez, D. Das, M.R. Suresh,
E.E. Knaus, Bioorg. Med. Chem. 16 (2008) 9694–9698.
[18] A.S. Kalgutkar, B.C. Crews, S.W. Rowlinson, A.B. Marnett, K.R. Kozak, R.P.
Remmel, L.J. Marnett, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 925–930.
[19] L. Di Nunno, P. Vitale, A. Scilimati, S. Tacconelli, P. Patrignani, J. Med. Chem. 47
(2004) 4881–4890.
[20] T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, S. Docter, M.J.
Graneto, L.F. Lee, J.W. Malecha, J.M. Miyashiro, R.S. Rogers, D.J. Rogier, S.S. Yu,
G.D. Anderson, E.G. Burton, J.N. Cogburn, S.A. Gregory, C.M. Koboldt, W.E.
Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, J. Med. Chem. 40
(1997) 1347–1365.
The ulcerogenic effect of 8c, 8d, celecoxib and ibuprofen was
evaluated according to
a previously reported method [32].
Twenty-five adult male albino rats were used in this study and
divided into five groups. All animals were allowed to fast for 18 h
before the administration of the drugs. The first group received
10% aqueous solution of DMSO (v/v) and kept as a control group.
The second group received celecoxib as the first reference drug in
a dose 50 mg/kg. The third group received Ibuprofen as the second
reference drug in a dose 50 mg/kg. The other groups received the
prepared compounds 8c and 8d in a dose 50 mg/kg. After 2 h of
administration of drugs, Animals were fed. Rats were given orally
the required dose for three successive days. After 2 h of the last
given dose, rats were sacrificed; the stomach of each rat was
removed and opened along the greater curvature then rinsed with
sodium chloride 0.9%. In order to examine the stomach, stretching
it by pins on a corkboard was done. With the aid of a magnifying
lens (10 xs) ulcers were searched in the stretched stomach. Calcu-
lation of the ulcer index was done as previously reported [32] in
which, ulcers were classified into levels, level I, ulcer area less than
1 mm2, level II, ulcer area is 1–3 mm2 and level III, ulcer area more
than 3 mm2, and the ulcer index was calculated according to the
following formula:
Ulcer index ðUIÞ ¼ 1 ꢂ ðnumber of ulcers level IÞ þ 2
ꢂ ðnumber of ulcers level IIÞ þ 3
ꢂ ðnumber of ulcers level IIIÞ; etc:
4.3. Molecular modeling studies
[21] O.L. Lorens, J.J. Perez, A. Palomer, D. Mauleon, J. Mol. Graph. Model. 20 (2002)
359–371.
[22] M.M. Ahlstrom, M. Ridderstrom, I. Zamora, K. Luthman, J. Med. Chem. 50
(2007) 4444–4452.
[23] H. Behbehani, H.M. Ibrahim, Molecules 17 (2012) 6362–6385.
[24] P. Vicini, A. Geronikaki, K. Anastasia, M. Incerti, F. Zani, Bioorg. Med. Chem. 14
(2006) 3859–3864.
[25] A. Bacchi, M. Carcelli, G. Pelizz, P. Vicini, Arch. Pharm. 328 (1995) 217–221.
[26] P.D. Neuenfeldt, B.B. Drawanz, G.M. Siqueira, C.R.B. Gomes, S.M.S.V. Wardell, A.
F.C. Flores, W. Cunico, Tetrahedron Lett. 51 (2010) 3106–3108.
[27] S. Ozkirimli, F. Kazan, Y. Tunali, J. Enzyme Inhib. Med. Chem. 24 (2009)
447–452.
All molecular modeling and docking studies were performed by
Molecular Operating Environment MOE version 2008.10 [34]. The
Structures of 4a, 4b, 8c and 8d were built in MOE. They were 3D
protonated and subjected to energy minimization using
MMFF94 ꢂ forcefield with 0.05 gradient. The X-ray crystal struc-
ture of celecoxib bound to the musmusculus COX-2 active site
(PDB: 3LN1) was obtained from the protein data bank [33]. Prepa-
ration of the enzyme for docking was achieved as follows: (1) The
Co-crystallized ligand and water molecules were removed. (2) The
[28] A. Kumar, C.S. Rajput, S.K. Bhati, Bioorg. Med. Chem. 15 (2007) 3089–3096.
[29] A. Zarghi, H. Arefi, O.G. Dadrass, S. Torabi, Med. Chem. Res. 19 (2009) 782–793.

12
K.R.A. Abdellatif et al. / Bioorganic Chemistry 64 (2016) 1–12
[30] D. Chattopadhyay, G. Arunachalam, A.B. Mandal, T.K. Sur, S.C. Mandal, S.K.
Bhattacharya, J. Ethnopharmacol. 82 (2002) 229–237.
[41] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv. Rev. 46
(2001) 3–26.
[31] J.T. Litchfield, F. Wilcoxon, J. Pharmacol. Exp. Ther. 96 (1949) 99–113.
[32] G.E.-D.A.A. Abuo-Rahma, M. Abdel-Aziz, M.A.E. Mourad, H.H. Farag, Bioorg.
Med. Chem. 20 (2012) 195–206.
[42] C.A. Lipinski, Drug Discov. Today Technol. 1 (2004) 337–341.
[43] D.F. Veber, S.R. Johnson, H. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, J. Med.
Chem. 45 (2002) 2615–2623.
[33] J.L. Wang, D. Limburg, M.J. Graneto, J. Springer, J.R.B. Hamper, S. Liao, J.L.
Pawlitz, R.G. Kurumbail, T. Maziasz, J.J. Talley, J.R. Kiefer, J. Carter, Bioorg. Med.
Chem. Lett. 20 (2010) 7159–7163.
[34] MOE, Chemical Computing Group, Inc., Montreal, <http://www.
chemcomp.com>.
[35] R.G. Kurumbail, A.M. Stevens, J.K. Gierse, J.J. McDonald, R.A. Stegeman, J.Y. Pak,
D. Gildehaus, J.M. Miyashiro, T.D. Penning, K. Seibert, P.C. Isakson, W.C.
Stallings, Nature 384 (1996) 644–648.
[36] A. Derhovanessian, J.B. Doyon, A. Jain, P.R. Rablen, A. Sapse, Org. Lett. 1 (1999)
1359–1362.
[44] P.R. Duchowicz, A. Talevi, C. Bellera, L.E. Bruno-Blanch, E.A. Castro, Bioorg.
Med. Chem. 15 (2007) 3711–3719.
[45] J. Banothu, K. Vaarla, R. Bavantula, P.A. Crooks, Chin. Chem. Lett. 25 (2014)
172–175.
[46] T. Giridhar, R.B. Reddy, B. Prasanna, G.V.P.C. Mouli, Indian J. Chem. 40B (2001)
1279–1281.
[47] P.M. Wehn, P.E. Harrington, T.J. Carlson, J. Davis, P. Deprez, C.H. Fotsch, M.P.
Grillo, J.Y.-L. Lu, S. Morony, K. Pattabiraman, S.F. Poon, J.D. Reagan, D.J. St Jean,
T. Temal, M. Wang, Y. Yang, C. Henley, S.E. Lively, Bioorg. Med. Chem. Lett. 23
(2013) 6625–6628.
[37] L. Schaeffer, in: Pract. Med. Chem., third ed., Elsevier Ltd., 2008, pp. 464–480.
[38] Y. Harrak, G. Casula, J. Basset, G. Rosell, S. Plescia, D. Raffa, M.G. Cusimano, R.
Pouplana, M.D. Pujol, J. Med. Chem. 53 (2010) 6560–6571.
[39] P.N.P. Rao, Q. Chen, E.E. Knaus, J. Med. Chem. 49 (2006) 1668–1683.
[40] Molinspiration Cheminformatics, <http://www.molinspiration.com/cgi-bin/
properties>.
[48] E.M. Sharshira, N.M.M. Hamada, Am. J. Org. Chem. 2 (2012) 69–73.
[49] K.R.A. Abdellatif, M.A. Chowdhury, C.A. Velázquez, Z. Huang, Y. Dong, D. Das, G.
Yu, M.R. Suresh, E.E. Knaus, Bioorg. Med. Chem. Lett. 20 (2010) 4544–4549.
[50] O. Unsal-Tan, K. Ozadali, K. Piskin, A. Balkan, Eur. J. Med. Chem. 57 (2012) 59–64.
[51] A.H. Abdelazeem, S.A. Abdelatef, M.T. El-Saadi, H.A. Omar, S.I. Khan, C.R.
McCurdy, S.M. El-Moghazy, Eur. J. Pharm. Sci. 62 (2014) 197–211.