Cyclooxygenase-2 and 15-lipoxygenase inhibition, synthesis, anti-inflammatory activity and ulcer liability of new celecoxib analogues: Determination of region-specific pyrazole ring formation by NOESY

Article abstract of DOI:10.1016/j.bmcl.2016.04.046

Two new series of 1,5-diaryl pyrazoline (3a-f) and 1,5-diaryl pyrazole (5a and 5b) were designed as both COX-2 and 15-LOX inhibitors. All the prepared compounds were fully characterized by all spectral and element analysis. Their anti-inflammatory activity and ulcer index were included. Pyrazoline 3f is the most effective with IC₅₀ = 1.14 and 4.7 μM against COX-2 and 15-LOX respectively, and more potent than celecoxib and meclofenamate references. In addition 3a, 3b, 5a, and 5b were safer with low ulcer index than celecoxib. Docking study was performed for the most active compounds such as 2b, 3a, and 3f on COX-2 and 15-LOX enzymes.

Full text of DOI:10.1016/j.bmcl.2016.04.046

Accepted Manuscript
Cyclooxygenase-2 and 15-lipoxygenase inhibition, synthesis, anti-inflammato-
ry activity and ulcer liability of new celecoxib analogues: Determination of
region-specific pyrazole ring formation by NOESY
Eman K.A. Abdelall, Phoebe F. Lamie, Waleed A.M. Ali
PII:
S0960-894X(16)30419-X
http://dx.doi.org/10.1016/j.bmcl.2016.04.046
BMCL 23808
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 March 2016
10 April 2016
16 April 2016
Please cite this article as: Abdelall, E.K.A., Lamie, P.F., Ali, W.A.M., Cyclooxygenase-2 and 15-lipoxygenase
inhibition, synthesis, anti-inflammatory activity and ulcer liability of new celecoxib analogues: Determination of
region-specific pyrazole ring formation by NOESY, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.04.046
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Cyclooxygenase-2 and 15-lipoxygenase inhibition, synthesis, anti-inflammatory activity and
ulcer liability of new celecoxib analogues: Determination of region-specific pyrazole ring
formation by NOESY.
Eman K. A. Abdelalla*, Phoebe F. Lamiea, and Waleed A.M. Alib
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514,
Egypt
bDepartment of Biochemistry, Cairo General hospital, Cairo, Egypt
______________________________________________________________________________
Abstract ― Two new series of 1,5-diaryl pyrazoline (3a-f) and 1,5-diaryl pyrazole (5a and 5b)
were designed as both COX -2 and 15-LOX inhibitors. All the prepared compounds were fully
characterized by all spectral and element analysis. Their anti-inflammatory activity and ulcer
index were included. Pyrazoline 3f is the most effective with IC50 = 1.14 & 4.7 µM against COX-
2 and 15-LOX respectively, and more potent than celecoxib and meclofenamate references. In
addition 3a, 3b, 5a, and 5b were safer with low ulcer index than celecoxib. Docking study was
performed for the most active compounds such as 2b, 3a, and 3f on COX-2 and 15-LOX
enzymes.
_____________________________________________________________________________
Prostaglandins (PGs), prostacyclin (PGI2), thromboxanes (TXA2), and leukotrienes (LTs) are
some metabolites of arachidonic acid (AA). Arachidonic acid is the most abundant
polyunsaturated fatty acid bound to cell membranes. PGs and PGI2 are produced through
cyclooxgenase (COX) pathway type 1-3. Cyclooxygenase isozymes are classed into a constitutive
COX-1, an induced COX-2, and COX-3 that remains under investigation. In contrast,
4
leukotrienes (LTs) are the final metabolites of AA via LOX pathway . There are four different

2
isoforms of LOX enzymes such as 5, 8, 12 and 15-LOX. Two of them (5 & 15-LOX) were
commonly observed in humans and are responsible for the undesirable physiological effects. For
example, 5-LOX is expressed in many pathological conditions as inflammatory diseases, allergic
reactions and bronco-spasm 5, while 15-LOX is involved in atherosclerosis 6.
Many classical non-steroidal anti-inflammatory drugs such as aspirin exert its anti-inflammatory
effect through rough inhibition of both COX-1 and COX-2 and its action is accompanied by
gastric bleeding due inhibition of constitutive COX-17. Development of more selective COX-2
inhibitors like celecoxib I (celebrexTM)8 (Fig.1), rofecoxib (vioxxTM) and valdecoxib (bextraTM)
9, was the great target. However, it was noticed that selective COX-2 resulted in many disorders
which were associated with myocardial thrombotic infarctions. This was the reason for
withdrawal of rofecoxib and valdecoxib from the pharmaceutical market. The development of
new anti-inflammatory agents with a dual COX-2\ LOX inhibitory activity would be effective as
a non-ulcerogenic-cardio-safe drug. Exploring Celebrex I, found that it is as an effective anti-
inflammatory with a minimum gastrointestinal side effect. It is belonged to be a vicinal di aryl
stilbene like structure with a sulfonamide (SO2NH2) group at p- position of one of the aryls. Also
10-12
looking to selective COX-2\ LOX inhibitors as darbufelone II and CI-987
III (Fig. 1), they
all contain a thiazolyl moiety and an electron rich aryl. So, it was of interest to design and
synthesize two classes hybrids (3a-f) and (5a&b) with the required pharmacophores of both
COX-2 selective inhibitors: i- vicinal diaryl structure, ii- SO2NH2 and SO2CH3 groups; and
LOX criteria: i- thiazolyl moiety, and ii- aryl bearing electron-rich substituent either di-or tri-
methoxyphenyl. Accordingly and to a continuation of previous work 13-16, we synthesized and
evaluate the anti-inflammatory activity of the new targeted compounds. Moreover, COX-1/2 and
15-LOX assays were done. Finally, docking studies were performed on COX- 2 and 15-LOX

3
enzymes to explore the possible binding mode of the most active compounds inside the two
enzymes.
H3C
O
O
t-bu
HO
t-bu
HO
CF3
N
N
S
S
N
N
t-bu
darbufelone II
t-bu
NH2
OH
1 (Celecoxib)
CI-987 III
H2NO2S
Selective COX\LOX-inhibitors
selective COX-2 inhibitores
SO2R
SO2R
CH3
S
N
N
N
R1
R1
H3C
N
N
N
COOCH3
R1
H3CO
S
3a-f
5a & b
R= CH3, NH2
R1= H, OCH3
keeping sulphoneyl group
replacement of one phenyl
with a phenylthiazolyl
moiety
inseration of electron richaryl
Fig.1: Representative examples of selective COX-2 inhibitors such as Celecoxib I, COX-
2/LOX inhibitors like Drabufelone II, and CI-987 III and the design for the newly
synthesized compounds 3a-f and 5a &b.
The reaction of 1-(4-methyl-2-phenyl-thiazol-5-yl)-ethanone (1) with the different
methoxylated aldehyde under Claisen –Schmidt condition, gave a series of new intermediate

4
chalcones 2a-c. The structure of 2a-c was confirmed by all element and spectral data. IR of 2a
revealed the appearance of C-H aromatic at 3061cm-1 and C-H aliphatic 2989 cm-1 and
tautomeric C=O at 1651 cm-1; Also, its 1HNMR characterized by a doublet peak at δ 7.39 due to
COCH=CH with a high coupling J = 15.6 Hz and another doublet at δ 8.11 due to COCH=CH
with the same J-coupling in a manner corresponding to chalcones and also was fully
characterized by 13C NMR that revealed the presence of CH=CH carbons at δ 124.96 and 148.87
respectively. Further cyclization of chalcones with p-methylsulfonylphenylhydrazine
7,17
18
hydrochloride
or p-sulfamoylphenyl-hydrazine hydrochloride resulted in a new series of
pyrazoline derivatives 3a-f (Scheme1). The structure of 3a-f was characterized and confirmed.
According to IR of 3a showed an absorption peak at 3045 cm-1 due to (C-H aromatic) and at
2929 cm-1 related (C-H aliphatic) and at 1592 cm-1 to (C=N) and also a two peaks at 1344,
1138 cm-1 due to (SO2). The 1H NMR of 3a characterized by the presence of a singlet peak at δ
3.08 due to (SO2CH3) another doublet of doublet peak at δ 3.22 related to Ha with a Jab = 17.2
Hz and, Jax= 5.2 Hz, another doublet of doublet at δ 4.15 due to Hb with a Jab= 17.2 Hz and Jbx
= 12.4 Hz and a third doublet of doublet at δ 5.76 due Hx, with a Jbx= 12.4 Hz and Jax = 5.2
Hz.

5
R1
O
O
O
N
N
a
S
R2 2a, R1 = OCH3, R2 = H, R3 = H;
2b, R1 = H, R2 = OCH3, R3 = H;
2c, R1 = H, R2 = OCH3, R3 = OCH3
S
R3
2a-c
1
b
R1
Hx
O
Hb
Ha
N
R2
3a, R1 = OCH3, R2 = H, R3 = H, R4 = SO2CH3;
3b, R1 = H, R2 = OCH3, R3 = H, R4 = SO2CH3;
3c, R1 = H, R2 = OCH3,R3=OCH3,R4=SO2CH3;
3d, R1 = OCH3, R2 = H, R3 = H, R4 = SO2NH2;
3e, R1 = H, R2 = OCH3, R3 = H, R4 = SO2NH2;
3f, R1 = H, R2 = OCH3, R =OCH3,R4=SO2NH2
S
N
N
R3
R4
3a-f
Scheme 1. Synthesis of compounds 2a-c and 3a-f.
Reagents and conditions: (a) aromatic aldehyde derivatives, KOH, RT, 3-6 h; (b) p-
methanesulfonylphenylhydrazine hydrochloride or p-benzenesulfonamidephe-
nylhydrazine hydrochloride, EtOH, reflux 8-12 h.
Additionally, compound 1 reacted with diethyl oxalate under mixed Claisen condition resulting
in the ester derivatives 4 that further cyclized with the same substituted phenyl hydrazine
derivatives furnishing 5a and 5b through Knorr reaction (Scheme 2) and their mechanism of
formation is illustrated in (Fig. 2).

6
O
OH
O
N
a
O
N
S
O
S
1
4
b
/
/
N
O
O
S
O
N
N
N
N
N
S
O
5a, R4 = SO2CH3,
5b, R4 = SO2NH2
B
R4
R4
5a & b
Scheme 2. Synthesis of compounds 4 and 5a & 5b.
Reagents and conditions: (a) Diethyl oxalate, Me-ONa,
2
h; (b) p-
methanesulfonylphenylhydrazine hydrochloride or p-benzenesulfonamidephe-
nylhydrazine hydrochloride, EtOH, reflux 5-8 h.

7
O
O
-O
S
O
S
O
S
O
CH3ONa
- H+
O
O
O
N
N
N
O
-OCH3
trans anion
1
R4
HO
O
O
S
R4
O
N
O
O
HO
N
N
N
S
O
O
HN
4
OH
N
- 2 H2O
N
S
5a & b
NHNH2
5a, R4 = SO2CH3,
5b, R4 = SO2NH2
R4
A
R4
R4
H
- 2 H2O
OH
N
//
N
HO
O
N
N
S
S
S
O
O
N
N
O
B
Fig. 2. A suggested mechanism for formation of compounds 5a & b.
This pyrazole cyclization could be occurred either through the formation of intermediate A or B.
Herein, a trial was made to postulate which pathway is favourable. Compound 5a is illustrated
1
through IR, H NMR, 13C NMR spectral data and element analysis, but all not excluded, which
form is formed either A or B. Additional spectral NMR experiment [NOESY] is operated to
the full elucidation of structure 5a. Upon studying both forms of 5a (A and B) in 3D mode,
form A showed a proton-proton correlation between methyl ester protons and H-2 & H-6 of
methansulphonylphenyl hence, they are close in space in a distance of 2.52, 2.87 and 3.92 Ao

8
while form B do not show this NOE correlations. As illustrated on the spectrum, the NOE
correlations is existed and 5a is confirmed to be in form A (Fig.3). All data of all synthesized
compounds were explained in experimental parts.
Fig. 3: 3D models of 5a in form A and B and NOESY scan of compound 5a.

9
The target of the in vitro biological activity assay was to explore the ability of synthesized
compounds to inhibit both bovine COX-1 and COX-2 using a colorimetric enzyme immunoassay
(EIA) kit to screen the isozyme-specific inhibition. This COX assay is a time- saving tool for
screening vast numbers of inhibitors. The potency of testing compounds was determined as the
concentration causing 50% enzyme inhibition (IC50)19. Also, the COX-2 selectivity indexes (SI
values) which are defined as IC50 (COX-1) /IC50 (COX-2) were calculated and compared with
that of the standard drug celecoxib. All compounds were tested and the data obtained are listed
in Table1. The results showed that all the chalcones (2a-c), tri-aryl pyrazolines (3a-f) and the di-
aryl pyrazoles (5a and 5b) are weak inhibitors of the COX-1 enzyme that showed (IC50 = 3.9 –
7.2 μM range and IC50 = 5.8 - 10.2 μM range and 6.5, 6.8 μM respectively). Compound 3d
inhibit COX-1 at higher dose (IC50 = 10.20 μM), is a pyrazoline structure with (SO2NH2)
pharmacophore that anticipate its good safety profile that practically measured with an (UI =
2.93) as celecoxib. While, the three series exhibited good COX-2 enzyme inhibitory activities
(IC50 = 0.74 – 1.89 μM range and IC50 = 1.14 – 3.21 μM range and 1.40, 1.76 respectively). Two
common features between two active compounds 3c (IC50 = 1.98 μM) and 3f (IC50 = 1.14 μM
against COX-2) are pyrazoline and the tri-methoxy phenyl part as being the highest electron-
rich aryl that evolved a hyposis of their essentiality for reactivity.
Also, the results showed COX-2 selectivity index ranges for intermediate chalcones 2a-c (3.80 -
5.27), 3a-f (2.89- 5.20) and for 5a and 5b (3.86 , 4.64). Within the synthesized compounds, five
compounds 2b, 2c, 3a, 3f and 5b showed a better COX-2 selectivity index (S.I. = 4.58 - 5.27)
compared to celecoxib (S.I. = 4.93) . Compounds 2b, 3a, and 3f exhibited the highest selectivity
index while 2c and 5b each showed a COX-2 selectivity index close to celecoxib.

10
Table 1: In vitro COX-1, COX-2 inhibition, 15-LOX- and anti-inflammatory activity of
chalcones 2a-c, pyrazoline 3a-f, pyrazoles 5a & 5b and the reference drug celecoxib for COX
and meclofenamte sodium for LOX.
Compound no
aCOX-1
(IC50) µM
aCOX-2
(IC50)
µM
COX-2
S.I.b
a15-
Lipoxygenase
(IC50) µM
6.22
ED50 (μmol/Kg)c
7.20
3.90
6.70
6.50
8.40
7.50
10.20
8.60
5.80
6.80
6.50
7.60
0.30
dND
1.89
3.80
5.27
4.58
5.20
2.89
3.78
3.18
3.63
5.09
3.86
4.64
4.93
0.13
dND
59
80
45
70
52
87
47
55
45
2a
2b
2c
3a
3b
3c
3d
3e
3f
0.74
1.46
1.23
2.90
1.98
3.21
2.37
1.14
1.76
1.40
1.54
2.40
dND
2.80
5.80
5.60
7.40
6.40
8.90
7.40
4.70
5.90
6.02
dND
dND
5.64
90
74
47
5a
5b
Celebrex
Aspirin
714
dND
Meclofenamate
sodium
aThe in vitro test compound concentration required to produce 50% inhibition of COX-1 or
COX-2, soya bean 15-LOX assay kit, The result (IC50, μM) is the mean of two determinations
acquired using an ovine COX-1/ COX-2 assay Kit (Cayman Chemicals Inc., Ann Arbor, MI,
b
USA) and the deviation from the mean is < 10% of the mean value. In Vitro COX-2 selectivity
c
index (COX-1 IC50/COX-2 IC50). Inhibitory activity in a Carrageenan induced rat paw edema
assay. The results are expressed as the ED50 value (μmol/kg) after
3 hours after oral
d
administration of the test compound. Nd = not determined.
For in vitro Lipoxygenase (LOX) inhibition assay, such Lipoxygenases are non-heme iron-
containing dioxygenase to catalyze the addition of molecular oxygen to fatty acids containing
cis, cis 1.4-pentadiene. The initial product of this reaction is 4-hydroxy-cis-trans-1,3-conjugated
pentadiene moiety. 15-LOX enzyme is designated from other LOXs that introduce

11
20
hydroperoxidate to lineolate and arachidonate substrates. Enzyme assay kit
was used to
measure the concentration of hydroxy peroxidase. The data obtained is listed in Table 1.
Regarding 15-LOX inhibitory activity, compound 2b was the highest 15-LOX inhibitor with
(IC50 = 2.8 μM) compared that of meclofenamate sodium (IC50 = 5.64 μM). Four compounds 2c,
3a, 3f and 5a showed inhibitory activity against 15-LOX enzyme in the range of (IC50 = 4.7-5.9
μM), nearly similar to meclofenamate sodium. One of the most active compounds is 3f which
structure of a pyrazoline and a tri-methoxy phenyl part, such combination is the preferable
toward lipoxygenase inhibitory activity.
The anti-inflammatory (AI) activity exhibited by the synthesized compounds using a Carrageen-
induced rat foot paw edema model21,22 and the concentration causing 50% edema inhibition
(ED50) was determined in comparison to the reference drugs celecoxib (Table 1). All synthesized
compounds showed AI activity with the range of (ED50 = 45-90 μmol/kg). The most active
compounds were the tri-methoxy chalcone 2c, tri-aryl pyrazoline sulfonamide derivitive 3f and
the pyrazoline methane sulfonyl derivative 3d with (ED50 = 45, 45 and 47 μmol/kg) respectively
in comparison with the reference drugs celecoxib (ED50 = 47 μmol/kg). Close sight at chalcones
The tri-methoxy derivative was more potent 2c > 2,3-di-methoxy (2a) > 3,4-di-methoxy (2b).
While the tri aryl pyrazolines (3a-f) showed anti-inflammatory activity in order of 3f > 3d > 3b >
3e > 3a > 3c. On the other hand pyrazoles 5a and 5b showed moderate activity when compared
with reference in spite that 5a was the least active among all synthesized compounds.
Surprisingly, 3b, 3d and 3f are the most active as anti-inflammatory and belong to a pyrazoline
with either dimethoxy phenyl part for 3b or the trimethoxyphenyl part structure for 3d and 3f
that encourage the previously assumed hyposis. Ulcerogenic liability of the tested compounds
was determined following oral dosing in rats (50 mg/kg) as once daily for three consecutive

12
days 23. Most of the compounds showed excellent activity when compared to reference where
compounds 3a, 3c, 5a and 5b were safer than reference celecoxib (UI = 2.9) itself that they
showed ulcer index = 2.65, 2.78, 2.48, 2.67 respectively. While, the rest were close to celecoxib.
Chalcone 2a is the least safe one that showed the highest ulcer index = 6.63 when compared to
reference celecoxib (UI = 2.9).
Table 2: In vivo anti-inflammatory activity of the tested compounds (orally administered) against
Carrageenan-induced rat paw edema.
Ulcer index
Compound Average severity
Average no of ulcers
0.6 ± 0.012***
0.3 ± 0.004***
0.4 ± 0.005
% incidence/10
1.03 ± 0.025***
0.57 ± 0.013**
0.52 ± 0.017
0.25± 0.008***
0 .85 ± 0.035***
0.18 ± 0.004***
0.73 ± 0.017***
0.68 ± 0.02***
0.53 ± 0.013
5
3
3
2
3
2
2
3
3
3
2
2
2
0
6.63
3.87
3.92
2.65
4.35
2.78
2.93
4.08
3.83
3.79
2.48
2.67
2.9
2a
2b
2c
3a
3b
3c
3d
3e
3f
0.4 ± 0.006
0.5 ± 0.011***
0.6 ± 0.013***
0.2 ± 0.004***
0.4 ± 0.008
0.3± 0.005***
0.4 ± 0.003
0.39±0.004***
0.28 ± 0.015***
0.17 ±0.003***
0.5 ± 0.013
4
0.2 ± 0.007***
0.5 ± 0.012***
0.4 ± 0.006
5a
5b
Celecoxib
DMSO
0
0
0
Values represent means ± SEM for ten animals for each group.
Significance levels **p<0.01, ***p<0.001as compared with the respective celecoxib
A molecular docking simulation was performed on the most active compounds 2b, 3a, and 3f to
investigate the binding interaction of these active compounds within the binding site of COX-2

13
and 15-LOX enzymes. Using Molecular Operating Environment (MOE, 2010, Version 8,
Chemical Computing Group Inc., Montreal, Quebec, Canada) was a software of choice used in
the docking experiments.
The X-ray coordinates were deposited in the protein data bank with codes (PDB: ID 1CX2) 24 for
COX-2 and (PDB: ID 1IK3) 25 for 15-LOX. The crystal structures of SC-558 bound at COX-2
and 13-(S)-hydroperoxy-9-(Z),11-(E)-octadecadienoic acid bound at the 15-LOX active sites
were used to investigate the selectivity of our compounds as dual COX-2/15-LOX inhibitors.
SC-558 – A selective COX-2 inhibitor- was used as a ligand for COX-2 isoform, His90 and
Tyr355 amino acids were responsible for two hydrogen bonding interactions both with -SO2
group and N-2 of pyrazole ring, in a distance equal to 2.44 and 2.58 Ao, respectively. The
energy score was found to be -8.3732Kcal/mol. On the other hand, docking 13-(S)-hydroperoxy-
9-(Z),11-(E)-octadecadienoic acid into 15-LOX enzyme resulted in one hydrogen bonding
interaction with Asp766 amino acid and OH of the ligand in a distance of 2.23 Ao and the energy
associated with the intermolecular interaction was -11.9176 Kcal/mol26.
The docking study of the most active compounds 2b, 3a, and 3f toward COX-2 showed that
Arg120, Tyr355, Tyr385 and Ser630 amino acids established the hydrogen bonding interactions
with –NH2, -SO2, and -OCH3 groups of the active compounds with a distance ranged from 2.40
to 3.04 Ao. The energy scores were equal to -12.6171, -12.6515 and -13.0955 Kcal/mol for 2b,
3a, 3f, sequentially.
Concerning to 15-LOX, compounds 2b, 3a, and 3f showed appreciable binding interactions with
binding energy ranged from -11.1472 to -19.5893 Kcal/mol with one or two hydrogen bonding
interactions between Ser510, Gln716, and Asp766 amino acids and –OCH3, -SO2, and –NH2
groups of the docked compounds in a distance between 2.64 to 2.92 Ao.

14
From all the above, we noticed that the selected compounds 2b, 3a, and 3f were in response to
both COX-2 and 15-LOX and that are consistent with their experimentally observed data
(Figures 4 and 5 and Table 3).
(A)
(B)
Fig. 4: Binding of the most active compound 3f inside COX-2. (A) 2D interaction of the
proposed binding mode of 3f inside the active site of COX-2 resulting from docking, the
most important amino acids are shown together with their respective numbers. It forms
five H-bonds with Arg120 and Tyr355, Tyr385 and Ser530; (B) 3D interaction of 3f.

15
(A)
(B)
Fig.5: Binding of the most active compound 3f inside 15-LOX. (A) 2D of the proposed
binding mode of 3f inside the active site of 15-LOX resulting from docking, the most
important amino acids are shown together with their respective numbers. It forms two H-
bonds with Gln716 and Asp766; (B) 3D interaction of 3f.
Table 3: Molecular modeling data for compounds 2b, 3a, 3f, 13-(S)-hydroperoxy-9-(Z),11-(E)-
octadecadienoic acid, and SC-558 during docking in the COX-2 and 15-LOX.
compound
COX-2
15-LOX
Distance (in Ao)
Distance (in Ao)
from main
residu
Kcal/mol
Affinity
Kcal/mol
Affinity
Functional
group
Functional
group
from
main
residu
2.44 Arg120
2.76 Tyr355
2.52 Arg120
-OCH3
-OCH3
-OCH3
-OCH3
- SO2
-OCH3
-OCH3
-SO2
-
2b
12.6171-
2.69 Ser510
2.92 Gln716
-OCH3
-SO2
16.8257
-
-12.6515 2.83 Arg120
2.60 Tyr385
2.40 Arg120
2.58 Tyr355
-13.0955 2.88 Tyr385
3.04 Ser630
3a
3f
11.1472
-
2.91 Asp766
-NH2
-SO2
19.5893 2.64 Gln716
-SO2
2.60 Tyr385
-NH2
13(S)Hydroperoxy-
9-
(Z),11(E)octadeca-
dienoic acid
-
-------
------- -------
-------
2.23 Asp766
11.9176
-OH
-SO2
His90 Pyrazole
-------
-8.3732
2.44
------- -------
-------
SC-558
2.58 Tyr355 N-2

16
In conclusion, Two series were prepared such as pyrazolines 3a-f and pyrazoles 5a&b as
(darbufelone – celecoxib) drug hybrid resulting in several safer compounds than celecoxib such
as 3a, 3c, 5a and 5b. The chalcone intermediate 2b with a 3,4-dimethoxy phenyl part was the
most effective among other chalcones as anti-inflammatory and showed a dual anti-COX-2\15-
LOX inhibitory activity. Among of pyrazolines 3a-f, which is the most effective series, 3a, 3d,
and 3f were the most active of all synthesized compounds showing higher anti-inflammatory and
anti-COX-2\15-LOX inhibitory activities. The pyrazoline–trimethoxy phenyl part structure
combination that evolved the most active compounds (3d and 3f) is appreciated and could be a
point of investigation in a further research. The in vivo data of pyrazoles 5a and 5b showed that
this direction is not a successful one, hence, 5a and 5b showed the least anti-inflammatory
activity but they are active in vitro as anti-COX-2\15-LOX inhibitory agents.
Acknowledgements
The authors are grateful to all members of the Pharmaceutical Organic Chemistry, Faculty of
pharmacy, Beni-suef university for all supports during proceeding of research.

17
References and notes
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http: //www.rcsb.org/pdb/explore/explore.do?structureId=1CX2.
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Abdellatif, K. R. A.; Lamie, P. F. ; Omar, H. M. J. Enz. Inhib .Med. Chem 2015,1.
General procedure for the synthesis of chalcones 2a-c and 3a-f. for 2a-c: A mixture of 1-(4-
methyl-2-phenylthiazol-5-yl)-ethanone (1) (2.17 g, 0.01mol), appropriate aromatic aldehyde
(0.01mol) and potassium hydroxide (0.56 g, 0.01 mol) in a minimum volume of methanol
(15 mL) was stirred at room temperature for about 3-6 h., (monitored by TLC). The resulting
solid was filtered off, rinsed with water, dried, and crystallized from methanol:chloroform
(9:1V/V).
[E]3-(2,3-Dimethoxyphenyl)-1-(4-methyl-2-phenylthiazol-5-yl)-prop-2-en-one

19
(2a). Yellow powder; Yield: 98%; mp: 96-98oC; IR (KBr, cm-1): 3061(C-H aromatic), 2989
1
(C-H aliphatic), 1651 (C=O), 1583 (C=N); H NMR (DMSO-d6); δ 2.89 (s, 3H, CH3), 3.91
(s, 3H, OCH3), 3.99 (s, 3H, OCH3), 6.96 (d, 1H, J = 8 Hz, dimethoxyphenyl H-4), 7.11 (t, J =
8 Hz, dimethoxyphenyl H-5), 7.26 (d, 1H, J = 8 Hz, dimethoxyphenyl H-6), 7.39 (d, 1H, J =
15.6 Hz, COCH=CH), 7.46 (m, 3H, phenyl H-3, H-4, H-5), 8.01 (m, 2H, phenyl H-2, H-6),
13
8.11 (d, 1H, J = 15.6 Hz, COCH=CH); C NMR (DMSO-d6); 18.90, 56.35, 61.40, 115.87,
120.07, 124.96, 126.14, 127.02, 127.10, 128.08, 129.93, 132.04, 132.63, 138.64, 148.87,
153.30, 159.67, 169.13, 182.79; MS (m/z,%): 365 [(M)+., 56.84%], 324 [100%]; Anal.
Calcd.for C21H19NO3S: C, 69.02; H, 5.24; N, 3.83. Found: C, 68.99; H, 5.17; N, 3.65%.
[E]3-(3,4-Dimethoxyphenyl)-1-(4-methyl-2-phenylthiazol-5-yl)-prop-2-en-one (2b). Brown
powder; Yield: 92%; mp: 132-134oC; IR (KBr, cm-1): 3052 (C-H aromatic), 2991 (C-H
aliphatic), 1645 (C=O), 1579 (C=N); 1H NMR (DMSO-d6); δ 2.88 (s, 3H, CH3), 3.95 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 6.92 (d, 1H, J = 8.4 Hz, dimethoxyphenyl H-2), 7.13 (d, 1 H, J =
15.2 Hz, COCH=CH), 7.14 (d, 1H, J = 10 Hz, dimethoxyphenyl H-5), 7.25 (dd, 1H, J = 8.4,
2 Hz, dimethoxyphenyl H-6), 7.47 (m, 3H, phenyl H-3, H-4, H-5), 7.78 (d, 1H, J = 15.2 Hz,
COCH=CH), 8.02 (m, 2H,phenyl H-2, H-6); 13C NMR (DMSO-d6); 18.80, 56.11, 56.20,
111.46, 112.12, 122.83, 124.35, 127.03, 127.41, 129.91, 131.92, 132.11, 132.71, 145.03,
149.49, 152.03, 159.25, 168.72, 182.71; MS (m/z,%): 365 [(M)+., 29%], 151[100%]; Anal.
Calcd.for C21H19NO3S: C, 69.02; H, 5.24; N, 3.83. Found: C, 69.13; H, 5.25; N, 4.01%.
[E]1-(4-Methyl-2-phenylthiazol-5-yl)-3-(3,4,5-trimethoxyphenyl)-prop-2en-one (2c). Grey
powder; Yield: 96%; mp: 118-120oC; IR (KBr, cm-1): 3059 (C-H aromatic), 2932 (C-H
aliphatic), 1657 (C=O), 1594 (C=N); 1H NMR (DMSO-d6); δ 2.78 (s, 3H, CH3), 3.72 (s, 3H,
p-OCH3), 3.86 (s, 6H, 2m-OCH3), 7.19 (s, 2H, trimethoxyphenyl H-2, H-6), 7.46 (d, 1 H, J =

20
15.2 Hz, COCH=CH), 7.57 (m, 3H, phenyl H-3, H-4, H-5), 8.01 (d, 1H, J = 15.2 Hz,
COCH=CH), 8.04 (m, 2H, phenyl H-2, H-6); 13C NMR (DMSO-d6); 18.82, 56.60, 60.63,
107.04, 124.58, 127.05, 129.93, 130.18, 131.05, 131.98, 132.65, 140.45, 144.98, 153.60,
159.57, 168.92, 182.86; MS (m/z,%): 395 [(M)+., 97.53%], 229 [100%]; Anal. Calcd.for
C22H21NO4S: C, 66.82; H, 5.35; N, 3.54. Found: C, 66.65; H, 5.17; N, 3.71%
General Procedure for the synthesis of 5-aryl-3-(4-methyl-2-phenylthiazole)-4,5-dihydro-1-
(4-substituted phenyl)-1H-pyrazoles (3a–f). A solution of the appropriate chalcone 2a, 2b or
2c (0.01mol) and the corresponding phenyl hydrazine derivative (0.01mol) in absolute
ethanol (30 mL) was refluxed for 8-12 h, (monitored by TLC). The solid separated on hot
was filtered, dried, then crystallized from 95% ethanol.
(S)5-[5-(2,3-Dimethoxyphenyl)-1-(4-(methanesulfonyl)-phenyl)-4,5-dihydro-1H-pyrazol-3-
yl]-4-methyl-2-phenylthiazole (3a). Yellow powder; Yield: 47%; mp: 210-212oC; IR (KBr,
cm-1): 3045 (C-H aromatic), 2929 (C-H aliphatic), 1592 (C=N), 1344, 1138 (SO2); 1H NMR
(DMSO-d6); δ 2.65 (s, 3H, CH3), 3.08 (s, 3H, SO2CH3), 3.22 (dd, 1H, Ha, Jab = 17.2 Hz, Jax=
5.2 Hz), 3.82 (s, 6H, 2OCH3), 4.15 (dd, 1H, Hb, Jab= 17.2 Hz, Jbx = 12.4 Hz), 5.76 (dd, 1H,
Hx, Jbx= 12.4 Hz, Jax = 5.2 Hz), 6.67 (m, 1H, dimethoxyphenyl H-4), 7.01 (m, 2H,
dimethoxyphenyl H-5, H-6), 7.04 (d, 2H, J = 8.8 Hz, methanesulfonylphenyl H-2, H-6), 7.51
(m, 3H, phenyl H-3, H-4, H-5), 7.69 (d, 2H, J = 8.8 Hz, methanesulfonylphenyl H-3, H-5),
7.96 (m, 2H, phenyl H-2, H-6); 13C NMR (DMSO-d6); 17.86, 44.59, 44.70, 56.15, 58.79,
60.71, 112.45, 113.12, 125.07, 125.24, 126.62, 129.16, 127.01, 129.76, 129.80, 131.13,
133.02, 134.42, 145.60, 146.13, 146.98, 153.10, 153.23, 165.40; MS (m/z,%): 533 [(M)+.,
72.22%], 520[100%]; Anal. Calcd.for C28H27N3O4S2: C, 63.02; H, 5.10; N, 7.87. Found: C,
63.15; H, 5.24; N, 7.69%.

21
(S)5-[5-(3,4-Dimethoxyphenyl)-1-(4-(methanesulfonyl)-phenyl)-4,5-dihydro-1H-pyrazol-3-
yl]-4-methyl-2-phenylthiazole (3b). Yellow powder; Yield: 45%; mp: 199-201oC; IR (KBr,
cm-1): IR (KBr, cm-1): 3013 (C-H aromatic), 2932 (C-H aliphatic), 1592 (C=N), 1325, 1130
1
(SO2); H NMR (DMSO-d6); δ 2.65 (s, 3H, CH3), 3.08 (s, 3H, SO2CH3), 3.33 (m, 1H, Ha),
3.71 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 4.11 (dd, 1H, Hb, Jab= 17.2 Hz, Jbx = 12 Hz), 5.59
(dd, 1H, Hx, Jbx= 12 Hz, Jax = 5.2 Hz), 6.71 (dd, 1H, J = 8.4, 1.6 Hz, dimethoxyphenyl H-2),
6.90 (d, 1H, J = 8.4 Hz, dimethoxyphenyl H-5), 6.98 (d, 1H, J = 1.6 Hz, dimethoxyphenyl
H-6),6.98 (s, 1H, dimethoxyphenyl H-2), 7.00 (d, 2H, J = 8.8 Hz, methanesulfonylphenyl H-
3, H-5), 7.51 (m, 3H, phenyl H-3, H-4, H-5), 7.69 (d, 2H, J = 8.8 Hz,
methanesulfonylphenylH-2, H-6), 7.96 (d, 2H, J = 3.6 Hz, phenyl H-2, H-6); 13C NMR
(DMSO-d6); 17.85, 44.60, 45.59, 55.92, 55.96, 63.16, 110.25, 112.67, 112.82, 117.96,
125.30, 126.61, 129.03, 129.80, 129.90, 131.13, 133.00, 133.81, 145.30, 147.28, 148.76,
149.60, 153.13, 165.42; MS (m/z,%): 533 [(M)+., 54.10%], 104 [100%]; Anal. Calcd.for
C28H27N3O4S2: C, 63.02; H, 5.10; N, 7.87. Found: C, 63.13; H, 4.98; N, 7.73%.
(S)5-[1-(4-(Methanesulfonyl)-phenyl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-
yl]-4-methyl-2-phenylthiazole (3c). Yellow powder; Yield: 54%; mp: 243-245oC; IR (KBr,
cm-1): 3055 (C-H aromatic), 2933 (C-H aliphatic), 1590 (C=N), 1344, 1134 (SO2); 1H NMR
(DMSO-d6); δ 2.64 (s, 3H, CH3), 3.09 (s, 3H, SO2CH3), 3.39 (m, 1H, Ha), 3.63 (s, 3H, p-
OCH3), 3.72 (s, 6H, 2OCH3), 4.12 (dd, 1H, Hb, Jab= 18 Hz, Jbx = 12 Hz), 5.54 (dd, 1H, Hx,
Jbx= 12 Hz, Jax = 6.8 Hz), 6.62 (s, 2H, trimethoxyphenyl H-2, H-6), 7.12 (d, 2H, J = 7.6 Hz,
methanesulfonylphenyl H-3, H-5), 7.51 (m, 3H, phenyl H-3, H-4, H-5), 7.71 (d, 2H, J = 7.6
Hz, methanesulfonylphenyl H-2, H-6), 7.96 (d, 2H, J = 4.4 Hz, phenyl H-2, H-6); 13C NMR
(DMSO-d6); 17.83, 44.58, 45.56, 56.36, 56.63, 60.40, 103.44, 107.08, 112.90, 126.62,

22
127.06, 129.08, 129.81, 129.93, 132.13, 134.02, 137.37, 145.53, 147.59, 153.87, 161.90,
164.30; MS (m/z, %): 563 [(M)+., 100%], Anal. Calcd.for C29H29N3O5S2: C, 61.79; H, 5.19;
N, 7.45. Found: C, 61.55; H, 4.97; N, 7.50%.
(S)4-[5-(2,3-Dimethoxyphenyl)-3-(4-methyl-2-phenylthiazol-5-yl)-4,5-dihydro-pyrazol-1-yl]-
benzenesulfonamide (3d). Yellow powder; Yield: 43%; mp: 214-216oC; IR (KBr, cm-1):
3383, 3330 (NH2, forked peak), 3045 (C-H aromatic), 2936 (C-H aliphatic), 1593 (C=N),
1342, 1156 (SO2); 1H NMR (DMSO-d6); δ 2.63 (s, 3H, CH3), 3.19 (dd, 1H, Ha, Jab = 18 Hz,
Jax= 5.2 Hz), 4.15 (m, 9H, 2OCH3, Hb, NH2, D2O exchangeable), 5.72 (dd, 1H, Hx, Jbx= 12.4
Hz, Jax = 5.2 Hz), 6.64 (m, 1H, dimethoxyphenyl H-4), 6.98 (m, 4H, dimethoxyphenyl H-5,
H-6, benzensulfonamide H-2, H-6), 7.60 (m, 3H, phenyl H-3, H-4, H-5), 7.69 (d, 2H, J = 4.4
13
Hz, benzensulfonamide H-3, H-5), 7.94 (m, 2H, phenyl H-2, H-6); C NMR (DMSO-d6);
17.82, 44.57, 56.14, 60.69, 112.27, 113.03, 124.97, 125.44, 126.57, 127.71, 129.80, 131.07,
133.04, 133.73, 134.53, 144.71, 145.75, 145.80, 152.70, 153.21, 165.13; MS (m/z,%): 534
[(M)+., 62.26%], 369 [100%]; Anal. Calcd.for C27H26N4O4S2: C, 60.65; H, 4.90; N, 10.48.
Found: C, 60.43; H, 5.11; N, 10.63%.
(S)4-[5-(3,4-Dimethoxyphenyl)-3-(4-methyl-2-phenylthiazol-5-yl)-4,5-dihydro-pyrazol-1-yl]-
benzenesulfonamide (3e). Orange powder; Yield: 42%; mp: 261-263oC; IR (KBr, cm-1): 3466
(NH2, broad band), 3045 (C-H aromatic), 2931 (C-H aliphatic), 1594 (C=N), 1333, 1127
1
(SO2); H NMR (DMSO-d6); δ 2.64 (s, 3H, CH3), 3.25 (dd, 1H, Ha, Jab = 17.6 Hz, Jax= 5.6
Hz), 3.70 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 4.10 (dd, 1H, Hb, Jab= 17.6 Hz, Jbx = 11.6 Hz),
5.57 (dd, 1H, Hx, Jbx= 11.6 Hz, Jax = 5.6 Hz), 6.71 (dd, 1H, J = 8.4, 1.6 Hz, dimethoxyphenyl
H-6), 6.90 (d, 1H, J = 8.4 Hz, dimethoxyphenyl H-5), 6.96 (d, 1H, J = 1.6 Hz,
dimethoxyphenyl H-6), 7.051 (s, 2H, NH2, D2O exchangeable), 7.052 (d, 2H, J = 8.8 Hz,

23
benzensulfonamide H-2, H-6), 7.51 (t, 3H, J = 2 Hz, phenyl H-3, H-4, H-5), 7.60 (d, 2H, J =
13
8.8 Hz, benzensulfonamide H-2, H-6), 7.95 (dd, 2H, J = 5.6, 2 Hz, phenyl H-2, H-6); C
NMR (DMSO-d6); 17.82, 45.47, 55.91, 55.95, 63.25, 110.27, 112.60, 112.66, 117.98,
125.49, 126.57, 127.58, 129.79, 131.06, 133.05, 133.88, 133.91, 144.43, 146.08, 148.69,
149.56, 152.75, 165.14; MS (m/z,%): 534 [(M)+., 84.54%], 518 [100%]; Anal. Calcd.for
C27H26N4O4S2: C, 60.65; H, 4.90; N, 10.48. Found: C, 60.34; H, 4.99; N, 10.51%.
(S)4-[3-(4-Methyl-2-phenylthiazol-5-yl)-5-(3,4,5-trimethoxyphenyl)-4,5-dihydro-pyrazol-1-
yl]-benzenesulfonamide (3f). Orange powder; Yield: 52%; mp: 244-246oC; IR (KBr, cm-1):
3371, 3268 (NH2, forked peak), 3045 (C-H aromatic), 2936 (C-H aliphatic), 1593 (C=N),
1335, 1153 (SO2);1H NMR (CDCl3); δ 2.76 (s, 3H, CH3), 3.29 (dd, 1H, Ha, Jab = 17.20 Hz,
Jab = 6 Hz), 3.84 (s, 3H, p-OCH3), 3.85 (s, 6H, 2OCH3), 3.97 (dd, 1H, Hb, Jab = 17.20 Hz, Jbx
= 12.4 Hz), 4.66 (s, 2H, NH2, D2O exchangeable), 5.27 (dd, 1H, Hx, Jbx = 12.4 Hz, Jax = 6
Hz), 6.48 (s, 2H, trimethoxyphenyl H-2, H-6), 7.09 (d, 2H, J = 8.8 Hz, benzensulfonamide
H-3, H-5), 7.46 (t, 3H, J = 1.6 Hz, phenyl H-3, H-4, H-5), 7.76 (d, 2H, J = 8.8 Hz,
benzensulfonamide H-2, H-6), 7.96 (d, 2H, J = 2.8 Hz, phenyl H-2, H-6); 13C NMR (CDCl3);
15.77, 28.11, 39.20, 56.62, 62.67, 102.18, 112.81, 127.28, 129.07, 130.19, 130.75, 133.00,
135.26, 137.70, 143.52, 144.83, 150.37, 154.87, 155.25, 161.07, 164.64; MS (m/z,%): 564
[(M)+., 82%], 64 [100%]; Anal. Calcd.for C28H28N4O5S2: C, 59.56; H, 5.00; N, 9.92. Found:
C, 59.51; H, 4.89; N, 9.79%. Synthesis of 2-hydroxy-4-(4-methyl-2-phenylthiazol-5-yl)-4-
oxo-but-2-enoic acid methyl ester (4). A mixture of 1-(4-methyl-2-phenyl-thiazol-5-yl)-
ethanone (1) (2.17 g, 0.01mol) and sodium methoxide (0.23 g / 5 mL methanol) in a
minimum volume of methanol (15 mL) was stirred at room temperature for about 2 h.
Diethyl oxalate (1.46 g, 0.01 mol) was added and the reaction mixture was stirred at room

24
temperature for 5 h. The resulting solid was filtered off, rinsed with water, dried, and
crystallized from 95%ethanol.Yellow powder; Yield: 59%; mp: 197-199oC; IR (KBr, cm-1):
3043 (C-H aromatic), 2924 (C-H aliphatic), 1726 (C=O), 1627 (C=N); 1H NMR (DMSO-d6);
δ 2.60 (s, 3H, CH3), 2.72 (s, 2H, COCH2CO), 3.50 (s, 3H, OCH3), 3.70 (s, 2H, CH2CO), 7.48
(m, 2H, phenyl H-3, H-5), 7.55 (t, 1H, J = 5.6 Hz, phenyl H-4), 7.93 (m, 1H, phenyl H-2),
8.00 (dd, 1H, J = 2.4, 5.6 Hz, phenyl H-6); 13C NMR (DMSO-d6); 17.86, 44.59, 44.70, 56.15,
58.79, 60.71, 112.45, 113.12, 125.07, 125.24, 126.62, 129.16, 127.01, 129.76, 129.80,
131.13, 133.02, 134.42, 145.60, 146.13, 146.98, 153.10, 153.23, 165.40; MS (m/z,%): 303
[(M)+., 16.76%], 69 [100%]; Anal. Calcd.for C15H13NO4S: C, 59.39; H, 4.32; N, 4.62. Found:
C, 59.51; H, 4.57; N, 4.59%. General Procedure for the synthesis of 1-(4-
substitutedfonylphenyl)-5-(4-methyl-2-phenylthiazol-5-yl)-1H-pyrazole-3-carboxylic
acid
methyl ester (5a&b). A solution of enoic acid ethyl ester 4 (3.17 g, 0.01mol) and the
corresponding phenyl hydrazine derivative (0.01 mol) in ethanol (30 mL) was refluxed for 5-
8 h, (monitored by TLC). The solid separated on hot was filtered, dried then crystallized from
95%ethanol.1-(4-(Methanesulfonyl)phenyl)-5-(4-methyl-2-phenylthiazol-5-yl)-1H-pyrazole-
3-carboxylic acid methyl ester (5a). Yellow powder; Yield: 44%; mp: 318-320oC; IR (KBr,
cm-1): 3019 (C-H aromatic), 2954 (C-H aliphatic), 1731 (C=O), 1638 (C=N), 1372, 1183
(SO2); 1H NMR (DMSO-d6); δ 2.19 (s, 3H, CH3), 3.28 (s, 3H, SO2CH3), 3.90 (s, 3H, OCH3),
7.34 (s, 1H, pyrazole H-4), 7.51 (m, 3H, phenyl H-3, H4, H-5), 7.72 (d, 2H, J = 8.4 Hz,
methanesulfonylphenylH-3, H-5), 7.89 (m, 2H, phenyl H-2, H-6), 8.05 (d, 2H, J = 8.4 Hz,
methanesulfonylphenyl H-2, H-6); 13C NMR (DMSO-d6); 16.34, 43.71, 52.59, 113.56,
126.02, 126.59, 128.91, 129.85, 131.38, 141.19, 142.66, 144.75, 153.94, 162.01, 168.07; MS
(m/z,%): 453 [(M)+., 100%]. Anal.Calcd.for C22H19N3O4S2: C, 58.26; H, 4.22; N, 9.27.

25
Found: C, 58.17; H, 4.33; N, 9.05%.5-(4-Methyl-2-phenylthiazol-5-yl)-1-(4-
(sulfamoyl)phenyl)-1H-pyrazole-3-carboxylic acid methyl ester (5b). Yellow powder; Yield:
39%; mp: 194-196oC; IR (KBr, cm-1): 3335, 3227 (NH2, forked peak), 3045 (C-H aromatic),
2952 (C-H aliphatic), 1730 (C=O), 1643 (C=N), 1332, 1159 (SO2); 1H NMR (DMSO-d6); δ
2.20 (s, 3H, CH3), 3.89 (s, 3H, OCH3), 4.37 (s, 2H, NH2, D2O exchangeable), 7.31 (s, 1H,
pyrazole H-4), 7.49 (m, 4H, phenyl H-2, H-3, H5, H-6), 7.65(d, 2H, J = 2 Hz,
benzensulfonamide H-3, H-5), 7.88 (dd, 2H, J = 2.8, 4.4 Hz, phenyl H-4), 7.92 (d, 2H, J = 2
Hz, benzensulfonamide H-2, H-6); 13C NMR (DMSO-d6); 16.34, 52.55, 113.28, 118.13,
125.93, 126.56, 127.45, 129.85, 131.38, 132.66, 135.39, 141.22, 144.53, 144.59, 153.91,
162.06, 167.95; MS (m/z,%): 454 [(M)+., 100%]; Anal. Calcd.for C21H18N4O4S2: C, 55.49; H,
3.99; N, 12.33. Found: C, 55.57; H, 4.03; N, 12.18%.
28. Biological activity, In vitro cyclooxygenase (COX) inhibition assay: The ability of the test
compounds listed in Table 1 to inhibit ovine COX-1 and COX-2 (IC50 value, μM) was
determined using an enzyme immune assay (EIA) kit (Cayman Chemical, Ann Arbor, MI,
USA) according to a previously reported method.
29.In vitro 15-lipoxygenase (LOX) inhibition assay: The ability of the test compounds listed in Table
1 to inhibit soya bean 15-LOX (IC50 value, μM) and 15-LOX was determined using an enzyme
immune assay (EIA) kit (catalogue no 760709, Cayman Chemical, Ann Arbor, MI, USA). Stock
solutions were freshly prepared before use and buffer solution used (0.1 M TrisHCl, PH, 7.4). 10 µl
of different compound were prepared in dissolved at least the amount of DMSO and diluted with the
stock solution to be in concentrations of (0.001, 01, 1, 5, 10 µM) in a final volume of 210 µl and IC50
of test compounds were determined according to a manufacturer's instructions and according to
previously reported methods (Table 1).

26
30.In vivo anti-inflammatory activity: Adult male wister albino rats (100 – 150 g) were used in the
pharmacological studies. The animals (five per cage) were maintained under standard laboratory
conditions (light period of 12 h/day and temperature 27 ± 2° C), with access to food and water. The
experimental procedures were carried out in strict compliance with the Institutional Animal Ethics
Committee regulations. All experiments were performed in the morning, according to the guidelines
for the care of laboratory animals. AI was evaluated by employing carragenan induced rat paw
edema model according to a previously reported method. All of the ED50 values were determined
using at least three doses (Table 1).
31.Ulcerogenic liability: Ul of all synthesized compounds, was evaluated according to the standard
reported method. Rats were fasted for 18 hours before drug administration and classified into
separate groups (5 rats per group). All treatments were administered via the oral route. The first group
received 10% DMSO aqueous solution (v/v) and kept as control, the second group received celecoxib
in ED50 (30.9μmol/kg), while the other groups received the test compounds dissolved in DMSO in
ED50. Treatment was continued once daily for 3 successive days in all groups. One hour after the last
dose, the animals were sacrificed under general anesthesia and the stomach was removed, opened
along the greater curvature and rinsed with saline. The gastric mucosa was examined with a
magnifying lens (10 x's) for the presence of lesions in the form of hemorrhages or linear breaks and
erosions. The ulcer index was calculated and the degree of ulcerogenic effect was expressed in terms
of: i) percentage incidence of ulcer divided by 10, ii) average number of ulcers per stomach, and iii)
average severity of ulcers. The ulcer index is the value that resulted from the sum of the above three
values (Table 2).
32.
Molecular modeling Studies: In this docking study, Molecular Operating Environment
(MOE, Version 2010.08, Chemical Computing Group Inc., Montreal, Quebec, Canada) was a
software of choice used in the docking experiments. The crystal structures of SC-558 bound
at COX-2 (PDB: ID 1CX2) and 13-(S)-hydroperoxy-9-(Z),11-(E)-octadecadienoic acid

27
bound at the 15-LOX (PDB: ID 1IK3) active sites were obtained from RCSB Protein Data
Bank. Docking of the co-crystallized ligand should be carried out to study the scoring energy
(s), root mean, standard deviation (rmsd) and amino acid interactions. [Rmsd Which is the
measure of superposing was 0.50Ao for the lead compounds]. Docking was performed using
London DG force and refinement of the results was done using force field energy. The
synthesized compounds for docking was achieved via their 3D structure. Certain procedures
were taken before docking, which include 3D protonation of the structures, running
conformational analysis using systemic search, selecting the least energetic conformer and
applying the same docking protocol used with ligands. [Docking for the synthesized
compounds was applied]. Amino acid interactions and the hydrogen bond lengths were
determined(Table 3).

Graphical Abstract
Cyclooxygenase-2 and 15-lipoxygenase inhibition,
synthesis, anti-inflammatory activity and ulcer liability
Leave this area blank for abstract I nfo.
of new celecoxib analogues: Determination of region-specific pyrazole ring formation by NOESY
Eman K. A. Abdelall, Phoebe F. Lamie, and Waleed A.M. Ali.
SO2R
A r= COOCH3
= 2,3-(OCH3)2Ph
= 3,4-(OCH3)2Ph
= 3,4,5-(OCH3)3Ph
dual COX-2/LOX
inhibitores
CH3
N
N
non ulcerogenic agent
N
Ar
S
R= CH3,NH2
Ph
Keywords: Cyclooxygenase inhibitors; Sulfonamide pharmacophores; Anti-
inflammatory; 15-Lipoxygenase inhibitors; Diethyl oxalate.
*Corresponding author: Tel: +002 01141021524 fax: +002 2319397;
e-mail:; eman.ahmed@pharm.bsu.edu.eg-

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Highlights
 New thiazolo-celecoxib analogues were designed and synthesized.
 Thiazolo-celecoxib drug hybrid showed higher COX-2 / 15-LOX inhibition properties.
 Designed compounds were evaluated as anti-inflammatory activity using Carrageenan-
induced rat paw edema and proved activity.
 Ulcer liability index of compounds was determined to show higher safety profile.
 Most active 2b,3a and 3f were docked in COX-2and 15-LOX active sites