Novel 1-[4-(Aminosulfonyl)phenyl]-1H-1,2,4-triazole derivatives with remarkable selective COX-2 inhibition: Design, synthesis, molecular docking, anti-inflammatory and ulcerogenicity studies

Article abstract of DOI:10.1016/j.ejmech.2014.06.049

A novel series of 1,2,4-triazole derivatives were synthesized and confirmed with different spectroscopic techniques. The prepared compounds exhibited remarkable anti-inflammatory activity comparable to that of indomethacin and celecoxib after 3 h. The tested compounds exhibited very low incidence of gastric ulceration compared to indomethacin. Most of the newly developed compounds showed excellent selectivity towards human COX-2 with selectivity indices (COX-1 IC₅₀/COX-2 IC₅₀) ranged from 62.5 to 2127. Docking studies results revealed that the highly selective tested compounds 6h and 6j showed lower CDOCKER energies, which means that they require less energy for proper interaction with the enzyme. The additional H-bonds with the oxygen of the amide and/or H of NH of the amide with the amino acid residues may be responsible for the higher binding affinity of this group of compounds towards COX-2.

Full text of DOI:10.1016/j.ejmech.2014.06.049

European Journal of Medicinal Chemistry 83 (2014) 398e408
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 1-[4-(Aminosulfonyl)phenyl]-1H-1,2,4-triazole derivatives with
remarkable selective COX-2 inhibition: Design, synthesis, molecular
docking, anti-inflammatory and ulcerogenicity studies
,
Gamal El-Din A.A. Abuo-Rahma a, Mohamed Abdel-Aziz a *, Nahla A. Farag b,
,
c
Tamer S. Kaoud a
a Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Abbassia, 11566 Cairo, Egypt
c Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 1,2,4-triazole derivatives were synthesized and confirmed with different spectroscopic
techniques. The prepared compounds exhibited remarkable anti-inflammatory activity comparable to
that of indomethacin and celecoxib after 3 h. The tested compounds exhibited very low incidence of
gastric ulceration compared to indomethacin. Most of the newly developed compounds showed excel-
lent selectivity towards human COX-2 with selectivity indices (COX-1 IC50/COX-2 IC50) ranged from 62.5
to 2127. Docking studies results revealed that the highly selective tested compounds 6h and 6j showed
lower CDOCKER energies, which means that they require less energy for proper interaction with the
enzyme. The additional H-bonds with the oxygen of the amide and/or H of NH of the amide with the
amino acid residues may be responsible for the higher binding affinity of this group of compounds to-
wards COX-2.
Received 15 February 2014
Received in revised form
8 June 2014
Accepted 24 June 2014
Available online 24 June 2014
Keywords:
1,2,4-Triazole
Anti-inflammatory
Ulcerogenicity
© 2014 Elsevier Masson SAS. All rights reserved.
Cyclooxygenase selectivity
Docking study
1. Introduction
the traditional NSAIDs [15e17]. In view of the gastric side-effects of
conventional NSAIDs and the recent withdrawal of selective COX-2
Non-steroidal anti-inflammatory drugs (NSAIDs) are considered
one of the most useful clinical therapies for the treatment of pain,
fever, and inflammation [1]. They exert their anti-inflammatory
activity through inhibition of cyclooxygenase (COX) derived pros-
taglandin synthesis. Two isoforms of COX are found; COX-1 which
is a housekeeping enzyme responsible for providing the physio-
logical maintenance actions (vascular and renal homeostasis, gas-
troprotection) [2]. On the other hand, COX-2 isozyme which is
induced by proinflammatory stimuli resulting in inflammatory
actions [3]. Thus, selective inhibition of COX-2 is useful for the
treatment of inflammations with reduced gastrointestinal toxicities
[4,5]. The chronic use of NSAIDs, is associated with gastrointestinal
[6e10], renal [11e13], and hepatic side effects [14] that are mainly
due to inhibition of the beneficial COX-1 isozyme. Diaryl hetero-
cycles have become the major class of selective COX-2 inhibitors,
such as celecoxib, rofecoxib, parecoxib and valdecoxib (Fig. 1),
which display improved gastrointestinal safety profile compared to
inhibitors from the market due to their adverse cardiovascular side-
effects, there is considerable impetus to develop alternative anti-
inflammatory agents with reduced gastric and cardiovascular side
effects [18]. Various 1,4- and 1,5-diaryl substituted 1,2,3-triazoles
[19] (Fig. 2A) were evaluated as COX-2 inhibitors. From this study,
it was concluded that compounds containing a vicinal diaryl sub-
stitution pattern display higher COX-2 inhibition potency compared
to their corresponding 1,4-diaryl-substituted counterparts. 1,2,4-
Triazol derivative [20] (Fig. 2B) exhibited a high in vitro COX-2
selectivity (COX-1 IC50
¼
20.5 nM; COX-2 IC50
¼
1.8 nM;
SI ¼ 11.39) relative to the reference drug celecoxib (COX-1
IC50 ¼ 3.7 nM; COX-2 IC50 ¼ 2.2 nM; SI ¼ 1.68) and also showed
good anti-inflammatory activity compared to celecoxib in
a
carrageenan-induced rat paw edema assay. A recent research study
revealed that the presence of an additional ester group in the ortho-
carbaboranyl analogues [21] with the small methyl ester group
could further fine-tune the COX selectivity profile. Similarly, it was
reported that the carboxyl moiety of acidic NSAIDs such as flurbi-
profen interacts with Arg120 in both COX isoforms via hydrogen
bonding or electrostatic interactions [22].
* Corresponding author.
E-mail addresses: abulnil@hotmail.com, abulnil2002@yahoo.com (M. Abdel-
Aziz).
http://dx.doi.org/10.1016/j.ejmech.2014.06.049
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
399
Fig. 1. Selective COX-2 inhibitors.
Promoted with the above-mentioned studies and as a continu-
2. Results and discussion
ation of our research interest [23e27] in the synthesis and bio-
logical evaluation of novel and safe NSAIDs; the present study is
concerned with synthesis of novel 1-[4-(aminosulfonyl)phenyl]-
1H-1,2,4-triazole derivatives containing triazole moiety as a bio-
isostere of the heterocyclic ring of the well known selective COX-2
inhibitors, keeping in mind the vicinal diaryl substituents with one
having the 4-sulfamoyl group that fulfills the best pharmacophore
for selective COX-2 inhibitors. In addition, the designed compounds
contain an amide group, as a bioisostere for ester moiety, as an
extra-binding site towards COX-2 that may enhance the selectivity.
The prepared compounds are evaluated for their anti-inflammatory
activity using carrageenan-induced rat paw edema and compared
to the well-known NSAIDs, celecoxib and indomethacin. The ability
of the prepared compounds to induce gastric toxicity and the
selectivity to COX-1 and COX-2 isozymes has been also evaluated.
Additionally, quantitative structure activity relationship (QSAR)
studies were carried out for validation of the observed biological
properties of the target compounds and for determination of the
most important parameters controlling these properties. Moreover,
docking studies were carried out using “Discovery studio software
program”. The scoring functions and hydrogen bonds formed with
the surrounding amino acids are used to predict their binding
modes, their binding affinities and orientation of these compounds
at the active site of COX-2.
2.1. Chemistry
The synthetic route used to prepare the target compounds 4, 5
and 6aej is outlined in Scheme 1. Heating of hippuric acid 1 with
acetic anhydride afforded the intermediate 2. The key intermediate
3 was prepared using Kuskov like reaction through coupling of the
diazonium salt of sulfanilamide with the active methylene of 2 in
presence of sodium acetate. Compounds 5 and 6aej were prepared
via Sawdey rearrangement [28] of compound 3. Heating at reflux of
3 in methanolic potassium hydroxide afforded the methyl ester 4.
Hydrazinolysis of the ester 4 using hydrazine monohydrate (98%) in
ethanol afforded the corresponding hydrazide 5 in a good yield.
Moreover, reaction of the intermediate 3 with ammonia, or
appropriate aliphatic amine including; isopropylamine, cyclohex-
ylamine or benzylamine in methanol afforded the corresponding
amides 6aed. Also, treatment of the intermediate 3 with the
appropriate primary aromatic amines in acetic acid in presence of
sodium acetate afforded the amides 6eej. The 1H NMR of the am-
ides 6aej showed the characteristic eNH signal at
d
10.74e9.29 ppm and the eSO2NH2 protons at d 7.45e8.00 ppm.
All the newly prepared compounds were identified using 1H NMR,
13C NMR and high resolution mass spectroscopy.
Fig. 2. 1,2,3-and 1,2,4-triazole derivatives as COX-2 inhibitors.

400
G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
Scheme 1. Synthesis of the target compounds 4, 5 and 6aej.
2.2. Biological investigations
1, 2, 3, 4 and 5 h after carrageenan injection. The anti-inflammatory
activity of the tested compounds, celecoxib and indomethacin was
calculated as the percentage decrease in edema thickness induced
by carrageenan and was determined using the following formula:
2.2.1. Screening of anti-inflammatory activity
The synthesized compounds 4, 5 and 6aej were evaluated for
their anti-inflammatory activity using carrageenan-induced paw
edema method in rats described by Winter et al. [29] The test
compounds and the reference drugs indomethacin and celecoxib
were administered orally at a dose level of 0.28 mmol/kg; 30 min
before carrageenan injection at the right hind paw of Albino male
rats. The thickness of both paws was measured at different times of
ðVR ꢀ VLÞcontrol ꢀ ðVR ꢀ VLÞtreated
% of edema inhibition ¼
ðVR ꢀ VLÞcontrol
Where VR represents the mean right paw thickness and VL
represents the mean left paw thickness.
Table 1
The anti-inflammatory activity at different times and ulcer indices of compounds 4, 5 and 6aej compared to celecoxib and indomethacin.
Compound
% Of edema inhibition (% mean S.E.M)
1 h
2 h
3 h
4 h
5 h
Control
4
5
6a
6b
6c
6d
6e
6f
6g
6h
6i
0
0
0
0
0
38.00 1.80***
32.00 1.60***
43.00 1.00***
21.00 1.46***
20.00 2.38***
42.00 1.30***
40.00 1.55***
53.00 1.18***
22.00 1.21***
36.00 1.31***
53.00 1.12***
20.00 1.16***
44.00 1.00***
54.00 0.81***
48.00 1.32***
53.00 1.27***
48.00 1.00***
43.00 2.00***
43.00 1.20***
47.00 2.00***
47.00 2.00***
57.00 2.42***
52.00 1.26***
62.00 2.00***
68.00 1.33***
32.00 1.40***
50.00 1.25***
66.00 2.00***
69.00 1.44***
60.00 1.10***
52.00 1.40***
55.00 1.70***
65.00 1.31***
58.00 2.00***
76.00 1.00***
78.00 1.31***
72.00 1.00***
67.00 2.00***
73.00 2.47***
54.00 1.50***
69.00 1.30***
79.00 1.17***
61.00 1.20***
45.00 1.70***
49.00 0.94***
44.00 0.70***
53.00 1.28***
36.00 1.60***
71.00 2.70***
73.00 1.30***
63.00 1.93***
53.00 2.20***
64.00 1.30***
44.00 1.77***
59.00 2.00***
81.00 2.39***
46.00 1.00***
32.00 2.00***
42.00 1.32***
43.00 1.80***
42.00 2.61***
29.00 0.90***
65.00 2.00***
55.00 1.40***
60.00 1.16***
44.00 1.09***
42.00 0.78***
38.00 1.20***
52.00 2.09***
84.00 1.87***
6j
Celecoxib
Indomethacin
***
Significantly different from control group at P < 0.001.
Note. one way ANOVA test was applied to determine the significance of the difference between the control group and rats treated with the tested compounds. (n ¼ 4),
***p < 0.001, significant difference from control group.

G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
401
(VR ꢀ VL)control represents the mean increase in paw thickness in
the control group of rats.
inflammatory activity compared to the presence of 3,4-dimethoxy
moieties (6g).
(VR ꢀ VL)treated represents the mean increase in paw thickness in
rats treated with the test compounds.
2.2.2. Screening of ulcerogenicity
The results listed in Table 1 show the percentage of edema in-
hibition induced by carrageenan for the tested compounds, cele-
coxibs and indomethacin versus time in h. The obtained results
indicated that most of the test compounds revealed significant
(p < 0.001) inhibition against carrageenan-induced paw edema in
rats and the maximum anti-inflammatory activity was obtained
after 3 h. Then, the activity decreased gradually for the next 2 h.
Indomethacin showed an inhibitory activity of 79% while celecoxib
exhibited an inhibitory activity of 69% against carrageenan-induced
paw edema after 3 h. Compounds 6f, 6e, 6i and 6g exhibited good
anti-inflammatory activity of 78%, 76%, 73% and 72%, respectively,
which represents 99%, 96%, 92% and 91% of indomethacin activity
and 113%, 110%, 106% and 104% of celecoxib activity, respectively,
The in vivo ulcerogenic liability was evaluated for the synthe-
sized compounds 4, 5 and 6aej relative to both celecoxib and
indomethacin according to a reported procedure [30]. Ulcers were
classified into levels, level I, ulcer areas less than 1 mm2, level II,
ulcer areas from 1 to 3 mm2 and level III, ulcer areas more than
3 mm2, and the ulcer index (UI) was calculated as follows:
UI ¼ 1 ꢁ ðnumber of ulcers level IÞ
þ 2 ꢁ ðnumber of ulcers level IIÞ
þ 3 ꢁ ðnumber of ulcers level IIIÞ; etc……
The UIs of compounds 4, 5 and 6aej were calculated and listed
in Table 2 as (mean
S.E.M). The results of ulcerogenic liability
after
3 h (Table 2). The ester 4 exhibited moderate anti-
revealed that indomethacin caused significant ulcerogenic toxicity
with UI of 39, while celecoxib exhibited very low UI of 0.5. All of the
synthesized compounds exhibited very lower UIs compared to
indomethacin. Compounds 4 and 5 exhibited lower ulcerogenic
liability relative to indomethacin with UI of 3 (Fig. 3A and B).
Meanwhile, the anilides 6aej exhibited very low gastric toxicity
compared to indomethacin with UI ranging from zero to 3 (Fig. 3A
and B). The obtained results revealed that all of the tested com-
pounds exhibit safer ulcerogenic liability relative to indomethacin
and comparable to that of celecoxib.
inflammatory activity of 69% against carrageenan-induced paw
edema after 3 h; that represents 87% and 100% of indomethacin and
celecoxib activities, respectively. The anilides 6h and 6c showed
remarkable anti-inflammatory activities of 67% and 65% against
carrageenan-induced paw edema after 3 h; that represents 85% and
82% of indomethacin and 97% and 94% of celecoxib activities,
respectively. The remaining anilides showed a variable anti-
inflammatory activity ranging from 52% to 62% that represent
from 66% to 76% of indomethacin and 75%e87% of celecoxib ac-
tivities, respectively (Tables 1 and 2). The aforementioned results
indicated that substitution on the amide nitrogen by an aromatic
moiety e.g. 4-methoxyphenyl (6f), 4-methylphenyl (6e), 4-
acetylphenyl (6i) 3,4-dimethoxyphenyl (6g), and 4-chlorophenyl
(6h) is associated with increasing of the anti-inflammatory activ-
ity compared to substitution on the amide nitrogen by an aliphatic
moiety e.g. cyclohexyl (6c), isopropyl (6b), the unsubstituted amide
(6a) and the hydrazide (5). The results in Table 1 also indicated that
the substitution on the amide nitrogen by a p-substituted phenyl
with electron donating groups such as CH3 or OCH3 (6f and 6e) is
associated with increasing the anti-inflammatory activities of these
compounds compared with similar derivatives with electron
withdrawing moiety such as eCOCH3, eCl and -benzothiazol 6i, 6h
and 6j, respectively. Additionally, the presence of only one 4-
methoxy (6f) is associated with a little increase of the anti-
2.3. Selectivity to COX-1 and COX-2
The effects of compounds 6beh and 6j on the activity of ovine
COX-1 (using SC-560 as a reference [31,32]) and on human re-
combinant COX-2 using (DuP-697 as a reference [33]) were
A
5 0
4
5
4 0
3 0
2 0
1 0
0
6 a
6 b
6 c
6 d
c e le c o x ib
in d o m e th a c in
Table 2
% Activity and ulcer indices of the tested compounds 4, 5 and 6aej relative to cel-
ecoxib and indomethacin.
4
5
c
6
d
e
b
n
i
6 a
6 b
6
c
x
c i
a
o
h
t
e c
in
l
m e
Compound
% Activity relative to
% Activity relative to UI
d o
indomethacin after 3 h celecoxib after 3 h
mean S.E.M
Control
4
5
6a
6b
6c
6d
6e
6f
6g
6h
6i
0
87
76
66
70
82
73
96
99
91
85
92
68
0
100
87
75
80
0.0 0.00
3.0 0.40***
3.0 0.70***
2.0 0.33**
0.0 0.0***
0.0 0.0***
3.0 0.60**
1.0 0.33**
2.3 0.18**
0.0 0.0***
0.5 0.11***
0.5 0.23**
1.0 0.25***
0.5 0.40***
39.0 0.60***
5 0
4 0
3 0
2 0
1 0
0
B
6 e
6 f
6 g
6 h
94
84
6 i
110
113
104
97
106
78
6j
c e le c o x ib
in d o m e th a c in
6j
i
j
b
i
n
6
6
6 f
i
6 e
6 g
6 h
x
o
c
Celecoxib
Indomethacin 100
100
c
e
h a
t
e
e l
m
c
o
d
**
Significantly different from control group at P < 0.01.
Significantly different from control group at P < 0.001.
in
***
Note. one way ANOVA test was applied to determine the significance of the differ-
ence between the control group and rats treated with the tested compounds. (n ¼ 4),
**p < 0.01, ***p < 0.001, significant difference from control group.
Fig. 3. A. UI of compounds 4, 5 and 6aed compared to indomethacin and celecoxib
expressed as mean S.E.M. B. UI of compounds 6eej compared to indomethacin and
celecoxib expressed as mean S.E.M.

402
G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
Table 3
estimated using fluorescent inhibitor screening assay kit (Cayman
Chemical, Ann Arbor, MI, USA). The dose response curves
(Fig. 4AeH) and IC50 values (Table 3) were determined for each
compound. Unlike indomethacin (COX-1 selective inhibitor), the
newly developed compounds exhibited remarkable selectivity to-
wards human COX-2 if compared to ovine COX-1 (Fig. 4). The
in vitro COX-2 selectivity indices are ranging from 62.5 to 2127
(Table 3) for compounds 6b, 6c, 6d, 6e, 6f, 6g, 6h and 6j while it was
reported to be 0.004 for indomethacin [34,35] and 600 for cele-
coxib (COX-2 selective inhibitor) [36]. It is obvious that there is no
remarkable effect on the selectivity index upon changing the sub-
stitution on the amide nitrogen by an aliphatic or aromatic moiety.
The N-cyclohexyl 6c and N-benzyl 6d derivatives exhibited lower
selectivity index compared to N-isopropyl derivative 6b. Addi-
tionally, substitution on the amide nitrogen by a p-chlorophenyl or
2-benzothiazolyl moieties revealed the best selectivity indices
compared to other derivatives.
In vitro COX-1 and COX-2 enzyme inhibition data for compounds 6beh and6j
compared to indomethacin, celecoxib, SC-560 and DuP-697.
Compound
COX-1 IC50
(
m
M)a
COX-2 IC50
16 2.00
(
m
M)a
SIb
6b
6c
6d
6e
6f
6g
6h
6j
>1000
>1000
>1000
516 74.0
>1000
346 15.0
>1000
798 100
62.50
1515
588
271
1123
266
0.66 0.120
1.7 0.180
1.9 0.140
0.89 0.200
1.3 0.150
0.47 0.021
0.38 0.050
2127
2100
a
The in vitro test compound concentration required to produce 50% inhibition of
either COX-1 or COX-2 activity, calculated from the dose response curve of inhibi-
tion. The result (IC50, mM) is the mean of three determinations acquired using the
COX Inhibitor Screening Assay Kit (Catalog No. 560131, Cayman Chemicals Inc., Ann
Arbor, MI, USA).
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
compounds 6beh and 6j. The 2D molecular descriptors such as
AlogP, molecular properties, molecular property counts, and sur-
face area volume were used as input molecular properties that
could describe the molecules [37]. MLR technique was used in the
present study for selecting a significant set of descriptors in order to
2.4. Docking studies
2.4.1. QSAR modeling
The 2D structures of the training set analogs were imported into
the Discovery Studio to calculate various molecular descriptors for
Fig. 4. AeH. The effects of compounds 6b (A), 6c (B), 6d (C), 6e (D), 6f (E), 6g (F), 6h (G) and 6j (H) on the activity of ovine COX-1 (-) and human recombinant COX-2 (C). Inhibition
of ovine COX-1 by 330 nM SC-560 and human recombinant COX-2 by 300 nM DuP-697 (I). The results are expressed as percent of control in the absence of inhibitor (100% maximum
activity).

G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
403
Table 4
build the significant models. Equation 1 represents the best per-
forming QSAR models (Fig. 5) show the corresponding scatter plots
of the experimental versus estimated bioactivity values for the
training set compounds as COX-2 inhibitor. The MLRTempModel
equation bearing the relevant descriptors is given as follow:
CDocker energy interaction, amino acid residues which form hydrogen bonds, IC50
COX-2 in M and length of hydrogen bond (A) with ligand for compounds 6aej
compared to SC-558.
m
Compound -(CDocker
energy
Amino acid residues
which form hydrogen
interaction) bonds with ligand
IC50 COX-2
in
Length of
hydrogen
bond (A)
m
M
ꢀLogIC50¼ꢀ0:622161ALogP ꢀ0:0135553MolecularWeight
ꢀ0:436435NumAromaticRings
SC-558
6a
49.0774
Arg120 HeF of CF3
2.12
2.02
2.07
2.02
2.24
2.49
1.82
2.13
2.36
2.43
2.47
2.38
1.93
2.38
2.00
2.09
2.06
Arg 513 HeO of SO2NH2
Ser 353 OeNH of SO2NH2
Arg 120 HeO of SO2NH2
Arg 315 HeO of CONH2
Arg 120 HeO of SO2NH2
Arg 513 HeO of SO2NH2
Ser 353 OeH of SO2NH2
Leu 352 OeH of SO2NH2
Leu 352 OeH of CONH
Arg 120 HeN of SO2NH2
Leu 352 OeH of CONH
Arg 120 HeN of SO2NH2
Arg 120 HeO of SO2NH2
Arg 513 HeO of SO2NH2
Glu 524 OeH of SO2NH2
Glu 524 OeH of SO2NH2
His 90 NeH of CONH
Ser 353 OeH of CONH
Leu 352 H of NHeO of
SO2NH2
þ0:269189NumHAcceptorsþ1:45282NumRings
ꢀ0:0364895NumRotatableBonds
ꢀ0:0493985MolecularFractionalPolarSurfaceArea
ꢀ0:0440254:
40.6659
NA
16
6b
6c
53.1608
44.2243
2
0.66 0.12
6d
6e
6f
36.9765
38.0162
41.1984
23.9268
1.7 0.18
1.9 0.14
0.89 0.2
1.3 0.15
Where ALogP is a measure of the hydrophobicity of the mole-
cule; it is calculated in Discovery Studio as the Log of the octa-
nolewater partition coefficient.
Molecular Fractional Polar Surface Area is the ratio of the polar
surface area divided by the total surface area of the molecule.
6g
6h
6i
30.7184
41.0442
0.47 0.021 2.35
2.25
NA
ꢀlogIC50 : ðthe negative logarithmic value of the concentration
required to produce 50% inhibition of COX
1.98
ꢀ2 enzyme compared to the control experimentÞ:
Gln 192 HeO of SO2NH2
Arg 120 HeO of SO2NH2
Arg 513 HeO of CONH
Gln 192 HeN of
benzothiazole
Leu 352 OeH of CONH
2.33
2.42
2.46
2.42
6j
47.0161
0.38 0.05
The method used to build the model was Least-Squares.
(N ¼ 7, r2 ¼ 1.000, r2 adjusted ¼ 1.000, r2 prediction ¼ 1.000),
where r2 (adj) is r2 adjusted for the number of terms in the model;
r2 (pred) is the prediction r2, equivalent to q2 from a leave-1-out
cross-validation. Number of hydrogen donor descriptor was
excluded from the model for being constant. Where, it shows the
direct relationship of hydrogen acceptor count, and number of rings
descriptors reveals their importance for selective potent molecule
design.
1.87
2.4.3. Docking analysis
To predict the anti-inflammatory data on a structural basis,
automated docking studies were carried out using Discovery studio
software program [37]. The scoring functions and hydrogen bonds
formed with the surrounding amino acids are used to predict their
binding modes, their binding affinities and orientation of these
compounds at the active site of the COX-2 enzyme. The protein-
ligand complex was constructed based on the X ray structure of
COX-2 (prostaglandin-endoperoxide synthase 2) complex with a
selective inhibitor SC-558 available through the RCSB Protein Data
Bank (PDB entry 1CX2) [38]. The scoring functions of the com-
pounds were calculated from minimized ligand protein complexes.
In order to compare the binding affinity of the newly synthesized
derivatives, compounds 6aej were docked into the empty binding
site of COX-2 (1CX2) and the results are listed in Table 4. SC-558
2.4.2. Validation of QSAR
External validation of the determined QSAR models was per-
formed utilizing the synthesized analogs 6beh and 6j.
It should be noted that the predicted anti-inflammatory activ-
ities by QSAR models were very close to those experimentally
observed, where predicted IC50 of 6h (use identified as statistical
outlier) is 0.31 mM and its experimental IC50 is 0.47 mM, indicating
that these models can be safely applied for predication of more
effective hits having the same skeletal framework as that of the
potent selective COX-2 inhibitor compounds.
Fig. 5. Predicted versus experimental-log IC50 of the tested compounds as COX-2 inhibitors.

404
G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
(the original ligand) revealed CDOCKER interaction energy
of ꢀ49.08 and forms three hydrogen bonds with Arg-120, Arg-513
and Ser-353 (Table 4). Compound 6j exhibited the highest inhibi-
tory activity with IC50 of 0.38 revealed scoring value of ꢀ47.02 and
forms four hydrogen bonds with Arg-120, Arg-513, Gln-192, and
Leu-352 (Table 4, Fig. 6). Compound 6h of IC50 of 0.47 exhibited
scoring value of ꢀ30.72 and form two hydrogen bonds with His-90,
and Ser-353 (Table 4, Fig. 7). From the obtained results in Table 4,
Figs. 6 and 7 it is obvious that the highly selective COX-2 (com-
pounds 6j and 6h) showed lower CDOCKER energies, which means
that they require less energy for proper interaction with the re-
ceptor. Also it was found that compounds 6a, 6d, 6f, 6h and 6j form
additional H-bonds with the oxygen of the amide and/or H of NH of
the amide with the amino acid residues which may lead to higher
binding affinity with the enzyme and accordingly higher inhibition
ability. Additionally, all of the docked compounds exhibit Pi cation
interaction between aromatic phenyl of benzene sulfonamido and/
or triazole moieties with the cation of Arg-120 resulting in high
binding affinity and hence high inhibitory activity for COX-2
enzyme.
Fig. 7. Binding mode of compound 6j in the binding site of COX-2 (for interpretation of
the references to color in the text, the reader is referred to the web version of this
article).
of this novel 1,2,4-triazole derivatives with additional binding
moieties to COX-2 looks a promising approach to improve the
safety of NSAIDs and may be a challenge in the field of COX-2
inhibitory therapy.
3. Conclusion
A series of novel 1,2,4-triazole derivatives was prepared and
confirmed with different spectroscopic techniques. Most of the
synthesized compounds showed significant anti-inflammatory ac-
tivity with low incidence of gastric ulceration comparable to that of
indomethacin and celecoxib. Most of the newly developed com-
pounds showed excellent selectivity towards human COX-2
compared to COX-1. The remarkable binding affinity to COX-2
enzyme may be attributed to the formation of additional H-bonds
with the oxygen of the amide and/or H of NH of the amide with the
amino acid residues of the enzyme. Additionally, all of the docked
compounds exhibit Pi cation interaction between aromatic phenyl
of benzene sulfonamido and/or triazole moieties with the cation of
Arg-120 resulting in high binding affinity and consequently high
inhibitory activity for COX-2 enzyme. In summary, the introduction
4. Experimental section
Reactions were monitored by TLC: Pre-coated plastic sheets,
0.2 mm silica gel with fluorescent indicator (MachereyeNagel).
Melting points were determined on Stuart electrothermal melting
point apparatus and were uncorrected. IR spectra were recorded on
Nicolet iS5 (ATR) FT-IR spectrometer. 1H NMR spectra were recor-
ded using a 400 MHz Bruker AM 400 spectrometer and Bruker
Advance 400 and 300 MHz NMR spectrometer. 13C NMR spectra
were recorded using 100 MHz Bruker AM 400 spectrometer and
Bruker Advance (75 MHR) spectrometer. Chemical shifts
d values
are given in parts per million (ppm) using CDCl3 (7.29 for proton
and 76.98 for carbon), DMSO-d6 (2.5) as solvents and coupling
constants (J) in Hertz. Splitting patterns are designated as follows:
s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublet; m,
multiplet; bs, broad singlet instrument. High resolution mass
spectra (HRESIMS) were obtained on Thermo Scientific Q Exactive
Orbitrap LC-MS/MS System and were reported as mass/charge (m/
z) with percent relative abundance.
4.1. Chemistry
4.1.1. 4-[2-(5-Oxo-2-phenyl-4,5-dihydro-1,3-oxazol-4-yliden)
hydrazino]-1-benzenesulfonamide 3
Hippuric acid 1 (11.5 g, 0.065 mol) in acetic anhydride (40 mL)
was heated to 80 ꢂC until a clear solution of the intermediate 2 was
obtained. The resulting solution was cooled to room temperature
(Solution A). To a cold solution of sulphanilamide (8.6 g, 0.05 mol)
in 5 M HCl (17.5 mL) in an ice bath (0e5 ꢂC), a solution of sodium
nitrite (4.48 g, 0.065 mol) in water (10 ml) was added in a dropwise
manner. The reaction mixture was left for 15 min (Solution B).
Solution A was added to solution B in presence of anhydrous so-
dium acetate (7.5 g, 0.09 mol). The reaction mixture was stirred at
(0e5 ꢂC) for 4 h, the formed precipitate was filtered off and dried.
The product was obtained as orange powder; mp: 236e237 ꢂC;
Yield ¼ 16.57 g, 75.00%; 1H NMR (400 MHz, DMSO-d6)
d 10.70 (s,
1H, NH), 8.80e9.00 (m, 2H, eSO2NH2), 7.20e7.60 (m, 3H, AreH),
7.70 (d, 2H, J ¼ 7.80 Hz, AreH), 7.85 (d, 2H, J ¼ 7.80 Hz, AreH),
7.86e7.90 (m, 1H, AreH), 8.18 (d, 1H, J ¼ 7.80 Hz, AreH); 13C NMR
(100 MHz, DMSO-d6) 171.29, 166.45, 162.56, 161.16, 145.30, 138.23,
133.96, 133.76, 131.40, 129.38, 126.67, 114.49.
Fig. 6. Binding mode of compound 6h in the binding site of COX-2 (for interpretation
of the references to color in the text, the reader is referred to the web version of this
article).

G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
405
4.1.2. Methyl 1-[4-(aminosulfonyl)phenyl]-5-phenyl-1H-1,2,4-
4.1.7. N-cyclohexyl-1-[4-(aminosulfonyl)phenyl]-5-phenyl-1H-
triazole-3-carboxylate 4
1,2,4-triazole-3-carboxamide 6c
To a stirred suspension of 3 (3.44 g, 0.01 mol) in methanol
(30 mL), potassium hydroxide (5%, 5 mL) was added, with contin-
uous stirring for 1 h at room temperature. The solvent was evap-
orated under reduced pressure; the formed precipitate was filtered,
dried and recrystallized from methanol affording compound 4 as
orange powder; mp: 218e220 ꢂC Yield ¼ 2.33 g, 65%; FT-IR (cmꢀ1):
Brown powder; mp: 129e130 ꢂC; Yield ¼ 2.72 g, 64%; 1H NMR
300 MHz, DMSO-d6,
d ppm: 10.50 (bs, 1H, eCONHe), 8.41 (s, 1H,
eSO2NH2); 8.38 (s, 1H, eSO2NH2); 7.93 (d, 2H, J ¼ 8.40 Hz, AreH),
7.64 (d, 2H, J ¼ 8.40 Hz, AreH), 7.49e7.45 (m, 5H, AreH), 3.82e3.80
(m, 1H, Cyclohexyl-H), 1.70e1.22 (m, 10H, Cyclohexyl-H). 13C NMR,
75 MHz, DMSO-d6,
d ppm: 157.60, 156.89, 154.73, 144.68, 139.77,
3352, 3212, 1751, 1596, 1563; 1H NMR (400 MHz, DMSO-d6)
d
7.90
130.66, 129.02, 128.79, 127.34, 126.99, 126.91, 126.23, 48.7, 32.09,
25.11, 24.92, 24.73, 23.92; HRESIMS (LC-MS/MS) Calcd. for
(d, 2H, J ¼ 8.00 Hz, AreH) 7.58e7.48 (m, 4H, AreH þ eSO2NH2),
7.44e7.38 (m, 3H, AreH); 7.34e7.27 (m, 2H, AreH), 3.90 (s, 3H,
eCH3); 13C NMR (100 MHz, DMSO-d6) 159.77, 155.38, 153.70,
144.87, 139.67, 133.93, 130.79, 129.05, 128.81, 128.79, 127.03, 126.76,
C
21H23N5O3S [MꢀH]þ 426.15944, Found: 426.15884.
4.1.8. N-Benzyl-1-[4-(aminosulfonyl)phenyl]-5-phenyl-1H-1,2,4-
triazole-3-carboxamide 6d
126.27, 52.63; HRESIMS (LC-MS/MS) Calcd. for
C16H14N4O4S
[MꢀH]þ 357.0663, found 357.0672.
Pale yellow powder; mp: 138e139 ꢂC; Yield ¼ 2.77 g, 64%; FT-IR
(cmꢀ1): 3379, 3330, 3200,1687, 1595, 1547; 1H NMR 300 MHz,
DMSO-d6,
d
ppm: 9.29 (t, 1H, J ¼ 6.20 Hz, eCONHe), 8.07 (s, 1H,
4.1.3. 4-[3-(Hydrazinocarbonyl)-5-phenyl-1H-1,2,4-triazol-1-yl]-1-
benzenesulfonamide 5
eSO2NH2); 8.04 (s, 1H, eSO2NH2); 7.94 (d, 2H, J ¼ 8.40 Hz, AreH),
7.67 (d, 2H, J ¼ 8.40 Hz, AreH), 7.56e7.24 (m, 10H, AreH); 4.49 (d,
2H, J ¼ 6.20 Hz, ebenzylic CH2); 10.90 (bs, 1H, eCONHe). 13C NMR,
75 MHz, DMSO-d6: 158.57, 156.68, 154.87, 144.68, 139.75, 139.32,
130.68, 129.49, 129.12, 129.02, 128.83, 128.79, 127.58, 127.40, 126.99,
126.88, 126.83, 126.55, 126.38, 126.23, 120.35, 42.30; HRESIMS (LC-
To a stirred solution of ester 4 (3.58 g, 0.01 mol) in ethanol
(50 mL), hydrazine monohydrate 98% (2.5 g, 0.05 mol) was added.
The mixture was heated at reflux for 6 h, the mixture was cooled to
room temperature. The formed precipitate was filtered, dried and
recrystallized from ethanol afforded compound 5 as a pale yellow
powder; mp: 227e228 ꢂC; Yield ¼ 2.87 g, 80.20%; FT-IR (cmꢀ1):
3362, 3337, 3243, 1655,1598, 1550; 1H NMR (300 MHz, DMSO-d6)
MS/MS) Calcd. for
434.12784.
C
22H19N5O3S [MþH]þ 434.12814, Found:
d
7.84 (d, 2H, J ¼ 8.40 Hz, AreH), 7.60 (d, 2H, J ¼ 8.40 Hz, AreH),
4.1.9. General procedure for synthesis of compounds 6eej
A solution of compound 3 (3.44 g, 0.01 mol) in acetic acid
(50 mL), the appropriate aromatic amine (0.01 mol) and anhydrous
sodium acetate (1.5 g, 0.018 mol) were added, the mixture was
heated under reflux for 3 h. The mixture was cooled to room
temperature, poured in ice water (50 mL). The formed precipitate
was filtered off, washed with water, dried and recrystallized from
ethanol to give compounds 6eej.
7.35e7.30 (m, 5H, AreH), 13C NMR (75 MHz, DMSO-d6) 170.10,
155.30, 151.75, 144.01, 142.31, 137.97, 134.10, 127.24, 126.60, 121.29,
114.07; HRESIMS (LC-MS/MS) Calcd. for C15H14N6O3S [MþH]þ
359.09209, found 359.09161.
4.1.4. General procedure for synthesis of compounds 6aed
To a solution of compound 3 (3.44 g, 0.01 mol) in methanol
(30 mL), ammonium hydroxide (25%, 50 mL) or isopropylamine
solution (1.18 g, 0.02 mol) or cyclohexylamine (1.98 g, 0.02 mol) or
benzylamine (2.14 g, 0.02 mol) was added. The mixture was heated
at reflux for 1 h, the solvent was evaporated under reduced pres-
sure, and the formed precipitate was filtered off and recrystallized
from ethanol to afford compounds 6aed.
4.1.10. N-(4-Methylphenyl)-1-[4-(aminosulfonyl)phenyl]-5-phenyl-
1H-1,2,4-triazole-3-carboxamide 6e
Brown powder; mp: 236e238 ꢂC; Yield ¼ 2.68 g, 61.90%; FT-IR
(cmꢀ1): 3379, 3330, 3201, 1687, 1595, 1540; 1H NMR 400 MHz,
DMSO-d6,
d ppm: 10.6 (bs, 1H, eCONHe), 8.06 (s, 1H, eSO2NH2);
7.95 (s, 1H, eSO2NH2); 7.82e7.70 (m, 5H, AreH); 7.60e7.40 (m, 6H,
AreH), 7.20 (d, 2H, J ¼ 8.20 Hz, AreH), 2.30 (s, 3H, CH3); 13C NMR,
100 MHz, DMSO-d6: 156.92, 156.78, 154.97, 144.73, 139.67, 135.68,
133.22, 130.74,129.07,129.04,128.79,126.99,126.77, 126.30, 120.59,
20.50; HRESIMS (LC-MS/MS) Calcd. for C22H19N5O3S [MþH]þ
434.12814, Found: 434.12766.
4.1.5. 1-[4-(Aminosulfonyl)phenyl]-5-phenyl-1H-1,2,4-triazole-3-
carboxamide 6a
Yellow powder; mp: 232e234 ꢂC; Yield ¼ 2.35 g, 68.50%; FT-IR
(cmꢀ1): 3478, 3346, 3316, 3225, 1680, 1593, 1547; 1H NMR,
300 MHz, DMSO-d6,
d ppm: 7.92 (s, 1H, eSO2NH-); 7.89 (s, 1H,
eSO2NHe); 7.78e7.74 (m, 4H, AreH), 7.57e7.46 (m, 3H, AreH), 7.25
4.1.11. N-(4-Methoxyphenyl)-1-[4-(aminosulfonyl)phenyl]-5-
phenyl-1H-1,2,4-triazole-3-carboxamide 6f
(s, 2H, AreH), 4.10 (s, 1H, eCONH-); 4.09 (s, 1H, eCONHe); 13C
NMR, 100 MHz, DMSO-d6,
d ppm: 160.24, 156.77, 154.79, 144.59,
Dark brown crystals; mp: 210e211 ꢂC; Yield ¼ 3.00 g, 66.80%;
139.75, 130.62, 128.96, 128.76, 126.96, 126.88, 126.21; HRESIMS (LC-
FT-IR (cmꢀ1):
MS/MS) Calcd. for
344.08078.
C
15H13N5O3S [MþH]þ 344.08119, Found:
3330, 3320, 3211, 1695, 1596, 1533; 1H NMR 400 MHz, DMSO-d6,
d
ppm: 10.5 (bs, 1H, eCONHe), 8.05 (s, 1H, eSO2NH2); 7.94 (s, 1H,
eSO2NH2); 7.81e7.70 (m, 5H, AreH); 7.50e7.42 (m, 6H, AreH), 6.95
(d, 2H, J ¼ 8.00 Hz, AreH), 3.75 (s, 3H, eOCH3); 13C NMR, 100 MHz,
DMSO-d6: 168.42, 166.67, 156.82, 156.75, 155.87, 144.71, 141.83,
139.69, 138.30, 133.82, 131.25, 130.73, 129.07, 128.79, 128.33, 127.30,
126.99, 126.29, 122.20, 118.64, 113.77, 55.18; HRESIMS (LC-MS/MS)
Calcd. for C22H19N5O4S [MþH]þ 450.12305, Found: 450.12280.
4.1.6. N-isopropyl-1-[4-(aminosulfonyl)phenyl]-5-phenyl-1H-1,2,4-
triazole-3-carboxamide 6b
Pale yellow crystals; mp: 236e237 ꢂC; Yield ¼ 2.50 g, 64.90%; 1H
NMR, 300 MHz, DMSO-d6, d ppm: 8.43 (s,1H, eSO2NH2); 8.41 (s,1H,
eSO2NH2); 7.94 (d, 2H, J ¼ 8.40 Hz, AreH), 7.65 (d, 2H, J ¼ 8.40 Hz,
AreH), 7.56e7.45 (m, 5H, AreH), 4.13 (septet, 1H, J ¼ 6.80 Hz,
eCH(CH3)2), 1.18 (d, 6H, J ¼ 6.60 Hz, eCH(CH3)2); 13C NMR, 75 MHz,
4.1.12. N-(3,4-Dimethoxyphenyl)-1-[4-(aminosulfonyl)phenyl]-5-
phenyl-1H-1,2,4-triazole-3-carboxamide 6g
DMSO-d6,
d ppm: 157.65, 156.91, 154.73, 144.68, 139.78, 130.67,
129.02, 128.79, 127.01, 126.91, 126.23, 40.76, 22.09; HRESIMS (LC-
Dark brown powder; mp: 235e236 ꢂC; yield ¼ 3.16 g, 66.00%; 1H
MS/MS) Calcd. for
384.11435.
C
18H19N5O3S [MꢀH]þ 384.11358, Found:
NMR 300 MHz, DMSO-d6, d ppm: 10.39 (s, 1H, eCONHe), 7.97 (s,
1H, eSO2NH2); 7.95 (s, 1H, eSO2NH2); 7.71 (d, 2H, J ¼ 8.40 Hz,

406
G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
AreH), 7.57e7.47 (m, 9H, AreH); 6.94 (d, 1H, J ¼ 8.40 Hz, AreH),
3.75 (s, 3H, eOCH3); 3.74 (s, 3H, eOCH3); 13C NMR, 75 MHz, DMSO-
d6: 156.83, 156.71, 154.98, 148.49, 145.52, 144.76, 139.73, 131.76,
130.77, 129.09, 128.81, 127.02, 126.81, 126.30, 112.52, 111.84, 105.70,
55.72, 55.46; HRESIMS (LC-MS/MS) Calcd. for C23H21N5O5S [MþH]þ
478.11906, Found: 478.11957.
4.3.2. Screening of ulcerogenicity
After measuring the anti-inflammatory activity the rats were
sacrificed by decapitation. The stomachs were removed, collected,
opened along the greater curvature, washed with distilled water
and cleaned gently by dipping in saline. The mucosal damage for
each stomach was examined with a magnifying lens for the pres-
ence of macroscopically visible lesions. The number of lesions in
each stomach, if any, was counted and recorded. Ulcers were clas-
sified into levels, level I, in which the ulcer area is less than 1 mm2,
level II, in which ulcer area is in the range from 1 to 3 mm2 and level
III, in which the ulcer area more than 3 mm2 and this rated ac-
cording to their areas in mm2.
4.1.13. N-(4-Chlorophenyl)-1-[4-(aminosulfonyl)phenyl]-5-phenyl-
1H-1,2,4-triazole-3-carboxamide 6h
Buff powder; mp: 268e269 ꢂC; Yield ¼ 2.81 g, 62.00%; 1H NMR
300 MHz, DMSO-d6,
d ppm: 10.74 (s, 1H, eCONHe), 7.97 (s, 1H,
eSO2NH2); 7.94 (s, 1H, eSO2NH2); 7.91 (d, 2H, J ¼ 8.40 Hz, AreH),
7.71 (d, 2H, J ¼ 8.40 Hz, AreH), 7.56e7.41 (m, 9H, AreH); 13C NMR,
75 MHz, DMSO-d6: 157.19, 156.51, 155.10, 144.81, 139.65, 137.23,
130.78, 129.09, 128.81, 128.56, 127.88, 127.01, 126.73, 126.33, 122.23;
HRESIMS (LC-MS/MS) Calcd. for C21H16ClN5O3S [MꢀH]þ 452.05896,
Found: 452.05994.
The data are expressed as mean S.E.M, one way ANOVA test
was applied to determine the significance of the difference be-
tween the control group and rats treated with the tested
compounds.
4.4. Selectivity to COX-1 and COX-2
4.2. N-(4-Acetylphenyl)-1-[4-(aminosulfonyl)phenyl]-5-phenyl-
1H-1,2,4-triazole-3-carboxamide 6i
COX fluorescent inhibitor screening assay kit (catalog number
700100, Cayman Chemical, Ann Arbor, MI, USA) has been employed
to investigate the isozyme-specificity of the synthesized com-
pounds following the procedure suggested by the manufacturer.
Briefly, in 96-well plate, either ovine COX-1 or human recombinant
COX-2 has been incubated with different concentrations of each
tested compound in the presence of the assay buffer (100 mM
TriseHCl, pH 8.0), heme and the fluorometric substrate 10-acetyl-
3,7-dihydroxyphenoxazine (ADHP) for 20 min at room tempera-
ture. The reaction started after addition of arachidonic acid solution
for 2 min at room temperature. Fluorescence of resorufin that is
produced by the reaction between PGG2 and the fluorometric
substrate were analyzed. DuP-697 (selective COX-2 inhibitor) and
SC-560 (selective COX-1 inhibitor) were used as reference com-
pounds. The measured fluorescence intensity is proportional to the
amount of produced resorufin, which is proportional to the amount
Yellow powder; mp: 215e216 ꢂC; Yield ¼ 3.0 g, 65.10%; 1H NMR
400 MHz, DMSO-d6,
d ppm: 10.60 (bs, 1H, eCONHe), 8.15 (s, 1H,
eSO2NH2); 8.10 (s, 1H, eSO2NH2); 8.00e7.92 (m, 5H, Ar H); 7.72 (d,
2H, J ¼ 8.20 Hz, AreH), 7.65e7.42 (m, 6H, AreH), 2.60 (s, 3H,
eCOCH3); 13C NMR, 100 MHz, DMSO-d6: 195.68, 157.42, 156.38,
155.16, 144.82, 142.59, 139.62, 132.45, 130.80, 129.25, 129.10, 128.80,
127.01, 126.67, 126.36, 119.88, 26.50; HRESIMS (LC-MS/MS) Calcd.
for C23H19N5O4S [MþH]þ 462.12305, Found: 462.12268.
4.2.1. N-(Benzothiazol-2-yl)-1-[4-(aminosulfonyl)phenyl]-5-
phenyl-1H-1,2,4-triazole-3-carboxamide 6j
Yellow powder; mp: 228e229 ꢂC; Yield ¼ 3.04 g, 63.90%; 1H
NMR 300 MHz, DMSO-d6,
d ppm: 12.93 (s, 1H, eCONHe), 8.05 (s,
1H, eSO2NH2); 8.03 (s, 1H, eSO2NH2); 7.97 (d, 2H, J ¼ 8.40 Hz,
AreH), 7.80 (d, 1H, J ¼ 8.40 Hz, AreH), 7.73 (d, 2H, J ¼ 8.40 Hz,
AreH), 7.57e7.33 (m, 8H, AreH); 13C NMR, 75 MHz, DMSO-d6:
158.39, 157.88, 155.33, 155.23, 147.93, 144.88, 139.59, 131.48, 130.90,
129.07,128.86,127.04,126.60,126.35,126.20,123.99,121.88,120.51;
HRESIMS (LC-MS/MS) Calcd. for C22H16N6O3S2 [MꢀH]þ 475.06525,
Found: 475.06598.
of PGG2 present in each well during the reaction. IC50 (mM) which
corresponds to the concentration of the inhibitor that causes 50%
inhibition of COX-1 or COX-2 activity was calculated from the dose
response curve of inhibition (triplicate determinations). Dose-
response curves for data conforming to inhibition were fitted to:
ꢀ
ꢁ
i
V0 ¼ V0 ꢀ V0
þ V∞
4.3. Biology
i þ ðIC50Þ
Where: V0 is the observed rate; i is the concentration of inhibitor I;
V’ is the observed rate in the absence of inhibitor; V∞ is the
observed rate constant at saturating inhibitor, I; IC50 is the con-
centration that leads to half the maximal change in V0.
4.3.1. Screening of the anti-inflammatory activity
The experiments were performed on adult male albino rats,
weighing (120e140 g), obtained from the animal house, Minia
University. The animals were housed in stainless steel cages,
divided into groups of four animals each and deprived of food but
not water 24 h before the experiment. The anti-inflammatory ac-
tivity of the compounds under investigation was studied using
carrageenan. A suspension of the tested compounds 4, 5 and 6aej,
celecoxib and indomethacin in carboxy methyl cellulose (CMC)
solution (0.5% w/v in water) was administered orally in a dose level
of (0.28 mmol/kg). Control animals were similarly treated with
CMC solution (0.5% w/v in water). After 30 min, 0.1 mL of freshly
prepared 1% carrageenan solution in normal saline was injected
into the subplantar region of the right hind paw of rats according to
the method of Winter et al. An equal volume of saline was injected
into the left hind paw of each rat. The right paw thickness was
measured by a Vernier celiper (SMIEC) directly before and after 1, 2,
3, 4 and 5 h after carrageenan injection. The anti-inflammatory
activity of the tested compounds, celecoxib and indomethacin
was calculated as the percentage decrease in edema thickness
induced by carrageenan.
4.5. Docking studies
4.5.1. QSAR modeling
Many molecular descriptors were calculated for each compound
employing a calculated molecular properties module according to
Girgis et al. [39]. The 2D structures of the training set analogs were
imported into the Discovery Studio to calculate various molecular
descriptors for each antiinflammotory active agent. The 2D mo-
lecular descriptors such as AlogP, molecular properties, molecular
property counts, and surface area volume were used as input mo-
lecular properties that could describe the molecules. Multiple
linear regression (MLR) analysis were employed to search for
optimal QSAR model that being capable of correlating bioactivity
variation across the used training set collection. QSAR models were
validated employing leave one-out cross-validation; statistical
measures used for the evaluation of model were the number of

G.E.-D.A.A. Abuo-Rahma et al. / European Journal of Medicinal Chemistry 83 (2014) 398e408
407
compounds in regression N, the regression coefficient r2. The
regression coefficient r2 is a relative measure of fit by the regression
equation. It represents the part of the variation in the observed data
that is explained by the regression. The correlation coefficient
values closer to 1.0 represent the better fit of the regression. Vali-
dation parameter, predictive r2 (r2_pred) was calculated for evalu-
ating the predictive capacity of the model, a value of r2_pred greater
than 0.5 indicates the good predictive capacity of the QSAR model.
Statistical outliers were identified from experimental versus pre-
dicted plots. With regard to QSAR modeling, the first goal was to
establish a predictive model with a reasonable number of input
features to ensure good generalization performance. While corre-
lating various descriptors with biological activity is the most
important means to study structureeactivity relationships, the
interest lies in deciding when to stop adding a new descriptor to the
model. Thus, the optimal model should use the minimum number
of descriptors to obtain the best fit. To achieve this, a well accepted
method is to find out the saturation point, a point beyond which
there is no considerable improvement in the regression coefficient
(r2) values even if a new descriptor is added. MLR technique was
used in the present study for selecting a significant set of de-
scriptors in order to build the significant models.
and Texas Institute for Drug & Diagnostic Development (H-F-0032)
for carrying out the selectivity to Cox enzymes.
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The docking analysis was performed using Discovery Studio 2.5
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Acknowledgment
The authors thank Dr. Kevin Dalby for providing the laboratory
space and advice to T.S.K. financial support for TSK mentor “Dr.
Kevin Dalby” was from grants from the National Institute of General
Medical Sciences (R01GM059802), the Welch Foundation (F-1390),

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