Synthesis of new thiazolo-celecoxib analogues as dual cyclooxygenase-2/15-lipoxygenase inhibitors: Determination of regio-specific different pyrazole cyclization by 2D NMR

Article abstract of DOI:10.1016/j.ejmech.2016.04.049

Two new series of 1,5-diaryl pyrazoles (5a, 5b, 7a, 7b and 10) and 1,5-diaryl pyrazoline (12a and 12b) were prepared as both Cyclooxygenase-2 and 15-lipoxygenase inhibitors. Carrageenan-induced rat paw edema, ulcer index and anti-COX-1/COX-2 and 15-LOX in

Full text of DOI:10.1016/j.ejmech.2016.04.049

Accepted Manuscript
Synthesis of new thiazolo-celecoxib analogues as dual cyclooxygenase-2 / 15-
lipoxygenase inhibitors: Determination of regio-specific different pyrazole cyclization
by 2D NMR
Eman K.A. Abdelall, Gehan M. Kamel
PII:
S0223-5234(16)30342-7
10.1016/j.ejmech.2016.04.049
EJMECH 8564
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 February 2016
Revised Date: 17 April 2016
Accepted Date: 19 April 2016
Please cite this article as: E.K.A. Abdelall, G.M. Kamel, Synthesis of new thiazolo-celecoxib analogues
as dual cyclooxygenase-2 / 15-lipoxygenase inhibitors: Determination of regio-specific different
pyrazole cyclization by 2D NMR, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.04.049.
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ACCEPTED MANUSCRIPT
Synthesis of new thiazole-celecoxib analogues as dual cyclooxygenase-2 / 15-lipoxygenase
inhibitors: Determination of regiospecific different Pyrazole Cyclization by 2D NMR
Eman K. A. Abdelalla* and Gehan M. Kamelb
*Corresponding author; eman.ahmed@pharm.bsu.edu.eg-
Graphical abstract
SO2R
RO2S
N
N
N
N
N
N
S
X
OCH3
S
5, 7, R = CH3 or SO2NH2
X= H orCF3
12, R = CH3 or SO2NH2
effective
anti-inflammatory
agents
more safer
non-ulcerogenic
agents
COX-2 / 15-LOX
inhibitors

ACCEPTED MANUSCRIPT
Synthesis of new thiazolo-celecoxib analogues as dual cyclooxygenase-2 / 15-
lipoxygenase inhibitors: Determination of regio-specific different Pyrazole
Cyclization by 2D NMR
Eman K. A. Abdelalla* and Gehan M. Kamelb
aDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef
University, Beni-Suef, 62514, Egypt.
bPharmacology Department, Faculty of Veterinary, Cairo University, Cairo, Egypt.
*Corresponding author; eman.ahmed@pharm.bsu.edu.eg-
Abstract: Two new series of 1,5-diaryl pyrazoles (5a, 5b, 7a, 7b and 10) and
1,5-diaryl pyrazoline (12a and 12 b) were prepared as both Cyclooxygenase-2 and
15-lipoxygenase inhibitors. Carrageenan-induced rat paw edema, ulcer index and
anti-COX-1/ COX-2 and 15-LOX inhibition assays were also included. Cyclization
of different pyrazoles was discussed using 2D NMR such as HSQC, HMBC and
NOSEY determinations. Compound 5a is more effective with ED50 = 0.98 and 3.98
µM against COX-2 and 15-lipoxygenase respectively, than the references celecoxib
(1.54 µM) and meclofenamate sodium (5.64 µM).
________________________________
Keywords:
Cyclooxygenase
inhibitors,
celecoxib
analogues,
SO2NH2
pharmacophores, 15-Lipoxygenase inhibitors, anti-inflammatory, DMFDMA, Ethyl
trifloroacetate.
*Corresponding author: Tel: +002 01141021524 fax: +002 2319397;
e-mail:; eman.ahmed@pharm.bsu.edu.eg-
1

ACCEPTED MANUSCRIPT
1. Introduction
Arachidonic acid (AA) is biologically transformed into a variety of inflammatory
mediators through two metabolic pathways, cyclooxygenase and lipoxygenase.
Cyclooxygenases are responsible for the production of Prostaglandins (PGs),
thromboxanes (TXA2) and prostacyclin (PGI2) while lipoxygenases produce
leukotrienes (LTs) and also catalyse the oxidation of lipoproteins (LDL, HDL) to
atherogenic forms [1,2]. Cyclooxygenase isozymes are classed into a constitutive
COX-1, induced COX-2 and COX-3 that still remain under investigation [3,4]. All
the above-mentioned mediators are highly expressed in many inflammatory diseases,
allergic reaction [5-7] and neo-angiogenesis [8,9]. The traditional non-steroidal anti-
inflammatory drugs (NAISDs) such as aspirin or even the potent indomethacin exert
their anti-inflammatory effect through rough inhibition of both COX-1 and 2.
Reasonably, their action is associated with gastric bleeding due inhibition of
constitutive COX-1. Moreover, selective COX-2 [10,11] inhibitors pronounced as
Coxibs including celecoxib I (celebrexTM) [12] (Fig.1), rofecoxib (vioxxTM) or
valdecoxib (bextraTM) greatly inhibited the induced COX-2, but their administration
is associated with myocardial thrombotic event and this is the reason of rofecoxib and
valdecoxib withdrawal from the pharmaceutical market. Coxibs block the
cyclooxygenase pathway, therefore metabolism of AA shunted to LOX pathway
resulted in increasing the incidence of the unfavourable cardiovascular thrombotic
event. According to the above findings, the development of new anti-inflammatory
agent with a dual COX-2\LOX inhibition [13] activity, will introduce an effective
cardio-safe drug with no ulcerogenic property. Celebrex is being a lead, due its high
anti-inflammatory activity with a minimum gastrointestinal side effect. It has been
belonged to a vicinal diaryl stelbene like structure with a pyrazole central ring and a
sulfonamide (SO2NH2) at p- position of one of aryl groups. Also exploring many
selective COX\LOX inhibitors such as darbufelone II and CI-987 (III) [14-16]
(Fig.1), stated that they all have a thiazole moiety. So the aim of this research was
directed to the synthesis of new celecoxib analogues on two designs (IV and V). The
first design IV has three criteria that one of its diaryl was replaced with a thiazolyl
moiety in order to maintain their COX-2\15-LOX inhibitory activities. Additional
modification is adding or removing of CF3 in position -3 of pyrazoles to
investigate their essentiality for activity beside to keep both COX-2 inhibitory
2

ACCEPTED MANUSCRIPT
pharmacophores (SO2NH2 or SO2CH3) as illustrated in Fig.1. Another design of
compounds V which has three modifications. 1-one of aryl is replaced with the
thiazole one. 2- keeping sulphonyl groups and 3- an electron rich group (OCH3) was
inserted as noticed in drabufelone II in order to maintain synergistic dual COX-
2\15-LOX inhibitory activities. Herein, two new designs of highly effective drug
hybrid (celecoxib/darbufelone) that might encourage the higher effectiveness of
resulted compounds as anti-inflammatory agents with high safety profiles. Also, The
dual inhibition of COX-2 / 15- LOX would decrease the cardiovascular adverse side
effect. Accordingly and to a continuation of previous work [17-20], we synthesized
and evaluate anti-inflammatory activity of the new targeted compounds. Moreover,
in vitro COX-1/ 2 and 15-LOX inhibitory assays were done. Finally, the way of
different pyrazole cyclization was discussed and proved by 2D NMR. These 2 D
results has been converted the research to be a unique one, hence, many works of
literature [21] prove their structures using theoretical computational studies without
practical findings.
H3C
O
O
t-bu
HO
t-bu
HO
CF3
N
N
S
S
N
N
NH2
t-bu
t-bu
OH
darbufelone II
Selective COX\LOX-inhibitors
1 (Celecoxib)
selective COX-2 inhibitores
CI-987 III
H2NO2S
RO2S
new more selective
COX-2/LOX inhibitores
SO2R
CH3
S
N
N
CH3
N
N
N
N
X
S
OCH3
IV, R = CH3 or SO2NH2
X= H, CF3
V, R = CH3 or SO2NH2
1-replacement of one phenyl
1-replacement of one phenyl
with a phenylthiazolyl moiety
with a phenylthiazolyl moiety
2--inseration of electron rich
2-with or without a CF3 moiety
3-keeping sulphoneyl groups
aryl as in drabufelone
3-keeping sulphoneyl groups
Fig 1: selective COX-2 inhibitors and selective COX \ LOX and Design of targeted
compounds
3

ACCEPTED MANUSCRIPT
2. Result and discussion
2.1. Chemistry
The Reaction of 1-(4-methyl-2-phenyl-thiazol-5-yl)-ethanone (1) with ethyl
trifloroacetate under mixed Claisen condition resulted in the formation of β-diketo
intermediate 3 which further cyclized with the use of un-symmetric hydrazines 4a
and/or 4b [9,22,23] giving one and sole products 5a or 5b (scheme 1). Many reports
stated about such reaction which constitutes the main synthetic approach for 1,5-di-
substituted pyrazoles considered that either NH or NH2 could start the attack to one
carbonyl but which one of them?. In a way to answer such question, two possible
forms of 5a could be obtained either A or B (Fig.2) and definitely, the reaction is a
region-specific one that one form A is predominantly formed. Many reports tried to
explain their mechanism of formation and using quantum mechanics and computer
programs to estimate which di-hydroxpyrazolidine intermediate A or B was
energetically favoured and it is clearly obvious from this research findings that the
favoured is intermediate A. Herein, we elucidate the structure of 5a by the use of 2D
HNMR (HMBC, HSQC, and NOESY) in order to discriminate between two forms
A or B. NOESY spectrum of 5a (R = CH3) was achieved. Upon studying 3D of two
possible forms of 5a (A and B), the methyl protons of thiazole CH3 showed two
correlations on space. First, with C2-and C-6 H protons of methansulfonylphenyl
hence, they are close in space in distance = 3.33, 3.88, 4.01 Ao and the other with
the pyrazole H in distance = 3.88 Ao. Such interactions could be measured in NOSY
experiment. From NOESY scan (on supplementary), we can identify a diagonal and
a series of associated-off diagonal cross peaks. From these cross peaks, two peaks
which are due to positive NOE signals for the concerned atoms confirming a
vicinal diaryl structure A as the only formed compound while the other form B do
not show these correlations.
4

ACCEPTED MANUSCRIPT
OH
O
O
O
S
CH3
CH3
N
CH3
N
CF3
CH3
+
O
CF3 OEt
2
O
a
N
S
CF3
S
1
3
R
NH-NH2
O
S
O
b
CH3
S
SO2R
4a.b
N
N
N
CF3
a, R = CH3
b, R =NH2
5a,b
Scheme1: synthesis of triflourodiketo-intermediate 3 and pyrazole derivatives 5a
and 5b. Reagent and condition: (a) CH3ONa, ethyl triflouroacetate, diethyl ether, (b)
phenylhydrazine derivative, ethanol, 12h.
O
OH
CF3
O
O
O
-OEt
OEt CF3
CH3ONa
CH2
CH3
Ar
Ar
Ar
SO2R
SO2R
SO2R
-2H2O
NH2
NH
OH N
N
N
H
N
Ar
CF3
Ar
OH
CF3
SO2R
SO2R
A
intermediate A
H
N
N
N
-2H2O
N
OH
Ar
intermediate B
OH
CF3
Ar
CF3
B
Ar = 5-dimethyl-2-phenylthiazolyl
Fig.2: mechanism and possible forms of structures of 5a and 5b
5

ACCEPTED MANUSCRIPT
Additionally, another pathway was adapted to get another pyrazoles 7a and/or 7b.
Herein, these pyrazoles were prepared by two different methods. The first method, a
green method involved the reaction of 1 with ethyl formate affording sodium salt of
3-hydroxy-1-(4-methyl-2-phenylthiazolyl-5-yl)prop-2-en-1-one (6) which in-situ
reacted with respective phenylhydrazine in water as an eco-friendly solvent in the
presence of a catalytic amount of acetic acid. The second method, it operated via
formation of enaminone 8 which further reacted with phenylhydrazine 4a or 4b in
ethanol to get the pyrazoles 7a or 7b (Scheme 2). Pyrazole 7a structure was
elucidated by all spectral data and using NOESY scan to prove 5a. From the 3D of
both forms of 7a (A and B), the form A showed that the protons of thiazole CH3 are
in space with C2-and C-6 H protons of methanesulfonylphenyl in a distance = 3.3,
4.05 and 4.8 Ao and with pyrazole H-3 in distance = 3.2 Ao, such correlations could
be measured in NOSY experiment while the form B do not show these correlations
in space. Data of NOESY scan prove that the two aryls are vicinal to each other.
O
O
H3C
N
ONa
H3C
N
CH3
a
S
S
HCOOC2H5
+
b
c
6
1
RO2S
RO2S
NH
N
S
N(CH3)2
H3C
N
CH3
N
-H2O
N
OH
O
N
-NH(CH3)2
N(CH3)2
H3C
S
7a,R= CH3
b,R= NH2
N
S
d
SO2R
A
SO2R
N
HN
N(CH3)2
H3C
N
CH3
N
8
N
OH
N
S
S
B
Scheme 2: synthesis of pyrazole derivatives 7a and 7b. Reagent and condition:(a)
CH3ONa, ethyl formate diethyl ether, (b) phenylhydrazine derivative, H2O, AcOH,
1h. (C) DMFDMA, toluene, 12h., (d) phenylhydrazine derivative, AcOH, ethanol,
12h.
6

ACCEPTED MANUSCRIPT
In a way to prepare compound 7 via another synthetic pathway, a trial was made
through the formation of the hydrazone derivatives 9b that reacted with DMFDMA
in toluene that due to high acidic properties of SO2NH2 it reacted first then
cyclization was completed giving the pyrazole derivative 10 (Scheme 3).
HN
N
SO2R
O
S
H3C
CH3
H3C
SO2
N
N
N
a
S
b
N
H3C
N
N
N
R=NH2
10
S
9a,b
Scheme 3: synthesis of hydrazones 9a and 9b and pyrazole 10; Reagent and
conditions (a) phenylhydrazine derivative HCl 4a and 4b, ethanol, (b) DMFDMA,
toluene 24hr
Adapting Claisen –Schemidt condensation of the starting acetyl with 4-methoxy
benzaldehyde, chalcone 11 was obtained that further cyclized with the substituted
hydrazines 4a or 4b in boiling ethanol to the pyrazolines 12a or 12b (scheme 4).
R4
O
O
H3C
CH3
H3C
OCH3
b
N
OCH3
S
N
S
N
N
Hx
Ha
S
1
Hb
CH3
11
N
12a&b
12a, R = SO2CH3,
12b,R= SO2NH2
Scheme 4: Synthesis of chalcone 11 and pyrazolines 12a and 12b. Reagents and
conditions: (a) 4-methoxybenzaldhyde, KOH, RT, 3-6 h; (b) p-substituted
phenylhydrazine hydrochloride derivatives, EtOH, reflux and 8-12 h.
2.2. Anti-inflammatory activity
7

ACCEPTED MANUSCRIPT
2.2.1. In vitro cyclooxygenase (COX) inhibition assay
The in vitro biological activity assay was operated to investigate the ability of
synthesized compounds to inhibit both bovine COX-1 and COX-2. A colorimetric
enzyme immunoassay (EIA) kit was used to screen the isozyme-specific inhibition
[24]. Such COX assay is a time-saving tool for screening a vast no of inhibitors. The
potency of testing compounds was determined as the concentration causing 50%
enzyme inhibition (IC50). Also, the COX-2 selectivity indexes (SI values) were
calculated , is defined as IC50 (COX-1) / IC50 (COX-2) and compared with that of the
standard drug celecoxib. All compounds were tested and the data obtained are listed
in Table 1. For the COX-1 inhibition assay, pyrazoles 7a and 7b inhibit COX-1 in
higher doses (10.30 and 9.80 µM, respectively) while pyrazoles 5a and 5b with a
(CF3) moiety inhibit COX-1 at doses (IC50 = 4.80 and 6.30 µM in sequent). Also, SI,
for 7a and 7b (3.46, 4.39) don’t differ so much from that of 5a and 5b and this may
postulate that (CF3) is not essential for reactivity. When compared 9a to 9b,
compound 9a inhibit COX-2 with (IC50 = 1.11 µM) that is more potent than 9b
(2.23 µM). This previous comparison concludes that SO2CH3 is more preferable
than the other COX-2 pharmacophore (SO2NH2). Pyrazoline 12a is the most active
in vitro with SI = 5.08 which is higher than celecoxib its self with SI = 4.93.
pyrazoline 12a with a COX-2 inhibitors pharmacophore (SO2CH3) which maintain a
higher lipid affinity with the highest partition coefficient (4.72) of all synthesized
compounds. The other Most of the compounds showed moderate SI relative to the
standard celecoxib that 5a, 9a, 9b, 7b showed SI = 4.89, 4.86, 4.39, 4.39
respectively. Pyrazole 5b with (SO2NH2) pharmacophore, 10 which formed
unexpectedly without a COX-2 pharmacophores and chalcone 11 which also without
COX-2 pharmacophores were the least active compounds with a lower SI ~3.6.
2.2.2. In vitro lipoxygenase (LOX) inhibition assay
Different lipoxygenases such as (5, 8, 12, 15) are non-heme iron-containing
dioxygenase that catalyze the addition of molecular oxygen to fatty acids containing
cis, sis 1,4-pentadiene. The initial product of this reaction is 4-hydroxy-cis, trans-1,3-
conjugated pentadienyl moiety. 15-LOX enzyme is designated from other LOXs that
introduce hydroperoxide to lineolate and arachidonate substrates. Using the LOX
enzyme assay kit [24] to measure the concentration of hydroxy peroxidase. The data
obtained is listed in Table 1. (Clog P) values were calculated using (Chem. Draw
8

ACCEPTED MANUSCRIPT
Ultra 3D
,
thremodynmic octanol /Water) in a way to anticipate the lipoxygenase
(
activity as whole most potent compounds showed reasonable higher liopxygenase
inhibitory activity. Regarding 15-LOX inhibitory activity, compounds 5a , 5b, 12a
were the most active as 15-LOX inhibitor with (IC50 = 3.98, 5.41 and 4.71 ꢀM)
compared that of meclofenamate sodium (IC50 = 5.64 ꢀM) and also they nearly
showed a higher partition coefficient (= 4.84, 4.72 and 4.72) from all synthesized
compounds. From previous results (C-LogP) could be used as an indicator to
anticipate liopoxygenase inhibitor potency. 12b, 9a, 7a, 10 and 11 showed moderate
inhibitory activity against 15-LOX enzyme in the range of (IC50= 5.90-7.68 ꢀM)
while 9b and 7b have the lowest of the partition coefficient results (3.37, 3.79) and
were the lowest as lipoxygenase inhibitors with (IC50 = 8.60 , 8.68 ꢀM for both).
Table 1: In vitro COX-1, COX-2 inhibition, 15-Lox- and C-LogP of pyrazoles (5a,
5b, 7a, 7b and 10), hydrazones (9a , 9b), Chalcone 11 and pyrazolines (12a , 12b)
and the reference drug celecoxib for COX and meclofenamte sodium for LOX.
Compound no
aCOX-1
aCOX-2
COX-1/
COX-2
S.I.b
a15-Lipoxygenase
IC50(µM)
cC-LogP
IC50 (µM)
IC50 (µM)
5a
5b
4.80
6.30
10.30
9.80
5.40
9.80
8.70
8.60
5.80
6.80
7.60
0.98
1.71
2.97
2.23
1.11
2.23
2.41
2.37
1.14
1.76
1.54
4.89
3.68
3.46
4.39
4.86
4.39
3.60
3.62
5.08
3.86
4.93
3.98
5.41
7.49
8.68
6.35
8.60
7.68
7.40
4.71
5.90
dND
4.84
4.72
3.92
3.79
2.98
3.37
4.07
4.28
4.72
4.52
-
7a
7b
9a
9b
10
11
12a
12b
Celebrex
9

ACCEPTED MANUSCRIPT
Aspirin
0.30
dND
2.40
dND
0.13
dND
dND
5.64
-
-
Meclofenamate
sodium
a
The 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, ꢀM) is the
mean of two determinations acquired using an ovine COX-1/ COX-2 assay Kits
(Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean
is <10% of the mean value. b In Vitro COX-2 selectivity index (COX-1 IC50/COX-
D
2 IC50).
cClog, p partition coefficient calculated (octanol /Water) ND = not
determined.
2.2.3. In vivo anti-inflammatory activity [25,26]
The anti-inflammatory (AI) activities exhibited by the synthesized compounds were
determined using a Carrageen-induced rat foot paw edema model and the percent in
% edema inhibition was determined after 2and 4h and their relative potency of
synthesized compounds to standard celecoxib was determined (Table 2). All
synthesized compounds showed AI activity represented as a ratio relative to
celecoxib with the range of 0.88-0.43. The most active compound was 5b which is a
trifluoropyrazole with a sulphonamide pharmacophore. Most of prepared pyrazoles
showed good activity in order of 5b > 5a> 7b > 10 > 7a. On contrary to in vitro
assays, AI activity stated that (SO2NH2) as a COX-2 pharmacophore was more
effective than (SO2CH3). The methoxy chalcon 11 was active with a relative potency
equal to 0.73 higher than the pyrazoline derivatives 12a and 12b those showing
lower relative potencies (0.43 and 0.64 respectively). Pyrazolines 12a and hydrazone
9a were with the lowest AI as they showed the least relative potencies that conclude
pyrazole is the preferred pharmacophores as they all showed good in vivo activities.
10

ACCEPTED MANUSCRIPT
Table 2: In vivo anti-inflammatory activity of the tested compounds (orally
administered) against Carrageenan-induced rat paw edema.
Increase in paw thickness
(mm)
aRelative
potency
EIP %
4h
Compound
no
Dose
(mg/kg)
2h
4h
2h
2h
4h
0.29 ±0.013*
0.29 ±0.031*
0.33 ±0.016*
0.28 ±0.012*
0.31±0.015* 53.23 51.56 0.89 0.79
0.27 ± 0.033* 53.22 57.81 0.89 0.88
0.33 ±0.017* 46.77 48.44 0.78 0.73
0.29 ±0.014* 54.84 54.69 0.92 0.83
0.46 ±0.028* 27.42 28.13 0.46 0.43
0.39 ±0.034* 33.87 37.09 0.57 0.57
0.31±0.015* 53.23 51.56 0.89 0.79
0.34 ±0.023* 43.75 46.88 0.73 0.71
0.46 ±0.028* 27.48 28.14 0.47 0.45
0.37 ± 0.025* 37.09 42.19 0.62 0.64
5a
5b
7a
7b
0.45 ± 0.026*
0.41±0.032*
0.29 ±0.013*
0.36 ±0.022*
0.45 ± 0.026*
0.39 ±0.024*
0.62 ±0.017
0.25 ±0.008*
9a
9b
10
50
15
11
12a
12b
Control
Celecoxib
0.64 ±0.019
0
0
-
-
0.22 ±0.01* 59.68 65.63
1
1
The results are expressed as means ± SEM (n = 5)Significance levels *p<0.05 as
a
compared with the respective celecoxib. Inhibitory activity in a carrageenan-induced
rat paw edema assay. The results are expressed as the relative potency at 2 and 4
hours after oral administration of 50 mg/kg the test compound.
2.2.4. In vivo ulcerliability activity [27,28]
The ability of compounds to Ulcer formation was determined following oral dosing
in rats (50mg/kg) as once daily for three consecutive days. Most of the tested
compounds showed excellent activity (Table 3) when compared to reference where
compounds 5a, 5b, 9b and 7b showed ulcer index = 2.78, 2.67, 2.65, 2.48
respectively and were safer than reference celecoxib (UI = 2.9) itself. Close to 7b
which is the safest compound of all synthesized compounds it belong to pyrazole
series with a sulphonamide moiety and it would introduce a good safety profile
drug. While the rest of the compound was close to celecoxib. Chalcone 11 which
deviated from any sulphonamide pharmacophore, is the least safe one that showed the
highest ulcer index (4.08).
11

ACCEPTED MANUSCRIPT
Table 3: Ulcerogenic liability of the tested compounds following oral dosing in rats
(50mg/kg) once daily for three consecutive days.
Comp.no
Average severity
Average no of ulcers
%
Ulcer
incidence/10 index
5a
5b
0.18 ± 0.004***
0.17 ±0.003***
0 .85 ± 0.035***
0.28 ± 0.015***
0.57 ± 0.013**
0.25± 0.008***
0.73 ± 0.017 ***
0.68 ± 0.02***
0.53 ± 0.013**
0.39±0.004***
0.5 ± 0.013
0.6 ± 0.013***
0.5 ± 0.012***
0.5 ± 0.011***
0.2 ± 0.007 ***
0.3 ± 0.004***
0.4 ± 0.006***
0.2 ± 0.004***
0.4 ± 0.008**
0.3± 0.005 ***
0.4 ± 0.003***
0.4 ± 0.006
2
2
3
2
3
2
2
3
3
3
2
0
2.78
2.67
4.35
2.48
3.87
2.65
2.93
4.08
3.83
3.79
2.9
7a
7b
9a
9b
10
11
12a
12b
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.
3. Conclusion
In this study, several thiazolo-celecoxib analogs (5a, 5b, 7a, 7b, 9a, 9b, 10, 12a, 12b)
were designed and prepared as dual cyclooxygenase and lipoxygenase inhibitors. All
designed compounds are good inhibitors for such enzymes specially pyrazoline 12a
((COX-1/COX-2) SI = 5.08 and IC50 = 4.70ꢀM against Lipoxygenase) which is more
selective than celecoxib and meclofenamate sodium references. Carrageenan-
induced rat paw edema assay results showed that pyrazole 5a is with the highest
relative potency in comparison with celecoxib. UI liability showed a promising safety
profile to most of the synthesized compounds.
4. Experimental protocols
4.1. Chemistry
All melting points were determined on a Griffin apparatus and were uncorrected. IR
spectra were recorded on (Schimadzu IR 435) using KBr discs and values were
12

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1
represented in cm-1. HNMR and 13C were carried out on a spectrophotometer
(Bruker 400 MHz) at the faculty of pharmacy, Beni-suef Univeristy, Beni-suef,
Egypt. In DMSO-d6 or CDCl3 and D2O using TMS as an internal standard, chemical
shifts were recorded in ppm on δ scale, J (coupling constant) were estimated in hertz.
The electron impact (EI) mass spectra were recorded on Hewlett 5988 spectrometer
(Palo Alto, CA). Microanalysis was performed for C, H, N at Micro-analytical
center, Cairo university, Cairo, Egypt and was + 0.4% of theoretical values. Thin
layer chromatography (TLC) silica gel plates were employed using a UV lamp to
monitor the time of reaction and check the purity of products. Reagent and solvent
used were of Aldrich (Milwaukee, WI) and used without further purification.
Compounds 9 [18] and 11 [29] were prepared as previously reported procedures.
4.1.1. Synthesis of (Z)-1,1,1-Trifluoro-4-hydroxy-4-(4-methyl-2-phenylthiazol-5-
yl)but-3-en-2-one (3)
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 ether (15 mL) was stirred at room
temperature for about 2 h. Ethyl trifloroacetate was added (1.42 g, 0.01 mol) was
added and the reaction mixture was stirred at room temperature for 5 h. The resulting
mixture was evaporated, then suspended in water and neutralized with acetic acid
using PH meter to PH= 5.5 and, then extracted with ether. The ether extract was
evaporated, then dried, and crystallized from ethyl acetate –methanol.
Yellow powder; Yield: 59%; mp: 260-262oC; IR (KBr, cm-1): 3423 (OH, broad
1
band), 3060 (C-H aromatic), 2961 (C-H aliphatic), 1684 (C=O), 1623 (C=N); H
NMR(DMSO-d6); δ 2.66 (s, 3H, CH3 of thiazole), 5.68 (s, 1H, CHCO-CF3), 7.46-
4.52 (m, 3H, phenyl H-3, H-5, and H-4), 7.99 (t, 2H, phenyl H-2 and H-6 J = 2.4, 6.4
Hz,), OH (not appeared);
13C NMR (DEPTQ); 18.17 (CH3of thiazole), 90.82 (CH-COCF3), 115.12, 118.02,
120.92, 123.83 [q] (CF3); 126.99 (C3 and C5-phenyl); 129.67 (C2 and C6 phenyl);
130.99 (C4-phenyl); 133.21 (C-1 of phenyl); 136.06 (C5-of thiazole); 153.28 (C-4 of
thiazole); 165.24 (C-2 of thiazole); 169.13, 169.41, 169.70, 169.98 [q] CF3-C=O);
179.25 (HO-C=CH) ; MS (m/z,%): 313 [(M)+., 1.3%], 43 [100%] ; Anal. Calcd. for
C14H10F3NO2S: C, 53.67; H, 3.22; N, 4.47, Found: C, 53.21; H, 3.47; N, 4.59 %.
4.1.2. General method for pyrazoles 5a and 5b:
13

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A solution of 3 (3.13 g, 0.01mol) and the corresponding phenyl hydrazine derivative
(0.01 mol) in ethanol (30 mL) was refluxed for 10-12 h., (monitored by TLC). The
solid separated on hot was filtered, dried, then crystallized from ethyl acetate-
methanol
5-(Trifluoromethyl)-3-(4-methyl-2-phenylthiazol-5-yl)-1-(4-methyl-sulfonyl) phenyl)-
1H-pyrazole (5a)
Yellow powder; Yield: 45%; mp: 190-192 oC; IR (KBr, cm-1): 3013 (C-H aromatic),
1
2932 (C-H aliphatic), 1656 (C=N), 1389, 1130 (SO2); H NMR (CDCl3); δ 2.26 (s,
3H, CH3), 3.09 (s, 3H, SO2CH3), 6.89 (s, 1H, pyrazole H-4), 7.46-7.48 (m, 3H,
phenyl H-3, H4, H-5), 7.65 (d, 2H, J = 8.4 Hz, methanesulfonyl phenyl H-2, H-6),
7.90-7.92 (m, 2H, phenyl H-2, H-6), 8.00 (d, 2H, J = 8.4 Hz, methanesulfonyl phenyl
H-5, H-3); 13C NMR (DEPTQ); 16.18 (thiazole CH3), 44.49 (methansulfonyl CH3)
109.25( C-4 pyrazole), (116.71,119.38, 122.06,124.74)[q] (CF3), 117.04 (C-1 of
methansulfonyl phenyl), 125,09 (C3,C5 of phenyl), 126.59 (C2, C6 of phenyl),
128.83 (C-2, C-6 methanesulfonyl phenyl), 129.15 (C-3,C-5 methanesulfonyl
phenyl), 130.97 (C-5 of phenyl), 132.58 (C-1 of phenyl), 135.50 (C-4 of
methanesulfonylphenyl phenyl), 140.35 (C-3 of pyrazole), 142.88(C-5 of
thiazole),(143.90,144.28,144.67, 145.06) [q] (C-5 of pyrazole), 153.94 (C-4 of
thiazole), 169.34 (C-2 of thiazole). (HMBC, HSQC and NOESY in supplementary
materials, MS (m/z, %): 463 [(M) +., 100 %], 465 [(M) +. +2, 12.44 %]. Anal.Calcd.for
C21H16F3N3O2S2 : C, 54.42; H, 3.48; N, 9.07; Found: C, 54.17; H, 3.33; N, 9.05%.
5-(Trifluoromethyl)-3-(4-methyl-2-phenylthiazol-5-yl)-1-(4-sulfamoyl-phenyl)-1H-
pyrazole (5b).
o
Yellow powder; Yield: 60%; mp: 300-302 C; IR (KBr, cm-1): 3381 (NH2, broad
band),3030 (C-H aromatic), 2926 (C-H aliphatic), 1668 (C=N), 1376, 1163 (SO2);1H
NMR (DMSO); δ 2.22 (s, 3H, CH3), 7.44 (s, 1H, pyrazole H-4), 7.44-7.51 (m, 3H,
phenyl H-3, H4, H-5), 7.53 (s, 2H, NH2 (D2O, exchangeable),7.66 (d, 2H, J = 8.4 Hz,
methanesulfonylphenyl H-2, H-6), 7.87-7.89 (m, 2H, phenyl H-2, H-6), 7.91 (d, 2H, J
13
= 8.4 Hz, methanesulfonylphenyl H-5, H-3); C NMR (DEPTQ); 16. 49 ( thiazole
CH3) , 109.28( C-4 pyrazole), (117.34,120.01,122.69,125.36) [q] (CF3), 117.51 (C-
1of sulfamoyl phenyl), 126.15 (C-3, C-5 of phenyl ), 126.55 (C-2, C-6 of phenyl),
127.46 (C-2, C-6 of sulfamoyl phenyl), 129.88 (C-3, C-5 of sulfamoyl phenyl),
131.57 (C4- of phenyl ), 132.30 (C-1 of phenyl), 135.5 (C-4 of sulfamoyl ), 140.90
14

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(C-3 of pyrazole), 143.9, 144.28, 144.67, 145.06 [q] (C-5 of pyrazole), 144.48 (C-5
of thiazole), 154.17 (C-4-of thiazole), 168.62 (C-2 of thiazole), MS (m/z,%): 464
[(M)+., 97.86 %],
466 [M+.+2,12.25%], 105.08 [100%]. Anal. Calcd. for
C20H15F3N4O2S2: C, 51.72; H, 3.26; N, 12.06 Found: C, 52.01; H, 3.33; N, 11.99%.)
4.1.3 .General procedure for preparation of pyrazoles 7a and 7b.
Method A: A mixture of 1 (2.17 g, 0.01mol) and sodium methoxide (0.54 g,
0.01mol) in ether 20mL and ethyl formate (0.74 g, 0.01mol) was stirred at room
temperature for 3hr., the salt formed was filtered , dried and further reacted with
hydrazines 4a or 4b ( 0.01mol) in water (30mL) and acetic acid (1.2 g, 0.02mol) and
heated for 1hr and a solid formed, filtered and crystallized form ethanol / ethylacetate
mixture (1:1).
Method B: a solution of enaminone 8 (2.72, 0.01mol) and substituted hydrazines (
0.01mol) in ethanol was heated for 12hr., the reaction mixture was evaporated, dried
and crystallized.
5-(4-Methyl-2-phenylthiazol-5-yl)-1-(4-(methylsulfonyl)phenyl)-1H-pyrazole
7a:
Yellow cubes ; Yield: 44%; mp: 177-179 oC; IR (KBr, cm-1): 3058 (C-H aromatic),
2932 (C-H aliphatic), 1656 (C=N), 1360, 1199 (SO2);1H NMR (CDCl3); δ 2.25 (s,
3H, CH3), 3.08 (s, 3H, SO2CH3), 6.65 (d, 1H, J = 2.0 Hz, pyrazole H-4), 7.45-7.49
(m, 3H, phenyl H-3, H4, H-5), 7.62 (d, 2H, J = 8.8 Hz, methanesulfonylphenyl H-2,
H-6), 7.85 (d, 1H, J = 2.0 Hz, pyrazole H-5), 7.92-7.95 (m, 2H, phenyl H-2, H-6),
7.96 (d, 2H, J = 8.8 Hz, methanesulfonylphenyl H-5, H-3); 13C NMR (DEPTQ) ;
16.17 (thiazole CH3), 44.54 (methansulfonyl CH3) 112.01 ( C-4 pyrazole), 118.91 (C-
1of methansulfonylphenyl), 124.37 (C3,C5 phenyl), 126.62 (C2, C6 of phenyl),
128.67 (C-2, C-6 methanesulfonylphenyl), 128.79 (C-3, C-5 methanesulfonyl
phenyl), 130.88(C-5 of phenyl), 132.51(C-1 of phenyl), 139.12 (C-4 of
methanesulfonyl phenyl ), 140.27(C-3 of pyrazole), 141.65(C-5 of pyrazole), 143.78
(C-5 of thiazole), 152.73 (C-4 of thiazole), 168.74(C-2 of thiazole). ( NOESY in
supplementary materials); MS (m/z,%): 395 [(M)+., 100%], 397[M+.+2,18.73%];
Anal.Calcd.for C20H17N3O2S2 : C, 60.74; H, 4.33; N, 10.62; Found: C, 60.50; H,
4.30; N, 10.25%.
5-(4-Methyl-2-phenylthiazol-5-yl)-1-(4-(sulfamoyl)phenyl)-1H-pyrazole 7b:
15

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o
Yellow cubes; Yield: 60%; mp: 202-204 C; IR (KBr, cm-1): 3431,3307 (NH2, ),
1
3184 (C-H aromatic), 3030 (C-H aliphatic), 1594 (C=N), 1376, 1148 (SO2); H
NMR (CDCl3); δ 2.26 (s, 3H, CH3), 5.02 (s, 2H, NH2 (D2O, exchangeable), 6.65 (d,
1H, J = 1.6 Hz, pyrazole H-4), 7.45-752 (m, 3H, phenyl H-3, H4, H-5), 7.53 (d, 2H, J
= 8.4 Hz, sulfamoylphenylH-2, H-6), 7.85 (d, 1H, J = 1.6 Hz, pyrazole H-5), 7.92-
7.97 (m, 4H, phenyl H-2, H-6 and sulfamoyl phenyl H-5, H-3); 13C NMR (DEPTQ) ;
15.88 (thiazole CH3), 111.80 ( C-4 pyrazole), 119.08 (C-1of sulfamoyl phenyl),
124.26 (C3, C5 phenyl), 126.67 (C2, C6 of phenyl), 127.76 (C-2, C-6
sulfamoylphenyl), 129.16 (C-3, C-5 sulfamoyl phenyl), 130.95 (C-5 of phenyl),
132.33(C-1 of phenyl), 133.01 (C-4 of sulfamoyl phenyl), 140.05 (C-3 of pyrazole),
140.91 (C-5 of pyrazole), 142.75 (C-5 of thiazole), 152.50 (C-4 of thiazole),168.72
(C-2 of thiazole). MS (m/z,%): 396 [(M)+., 100%], 398 [M+.+2,13.53%]. Anal.
Calcd. For C19H16N4O2S2: C, 57.56; H, 4.07; N, 14.13; Found: C, 57.50; H, 4.03; N,
14.05%.
4.1.4. General procedure for synthesis of 9a and 9b
A solution of 1 (2.17 g, 0.01mol) and the corresponding phenyl hydrazine derivative
(0.01 mol) in ethanol (30 mL) containing a catalytic amount of acetic acid and heated
under reflux for 3 h., (monitored by TLC). The solid separated on hot was filtered,
dried,then crystallized from ethanol \ dimethyformamide mixture (5:1).
(E)-1-(1-(4-methyl-2-phenylthiazol-5-yl)ethylidene)-2-(4-methylsulfonyl)phenyl)
hydrazine (9a)
Yellow Needles; Yield: 80%; mp: 204-206 oC; IR (KBr, cm-1): 3597 (NH hydrazono),
3164, 3082 (C-H aromatic), 2938 (C-H aliphatic), 1593 (C=N), 1381, 1137 (SO2);
1H NMR (DMSO-d6); δ 2.40 (s, 3H, CH3), 2.66 (s, 3H, CH3-C=N) 3.16 (s, 3H,
SO2CH3), 7.34 (d, 2H, J = 8.4 Hz , H-2, H-6 of methanesulfonylphenyl ),7.49-7.42
(m, 3H, phenyl H-3, H-4, H-5), 7.75 (d, 2H, J = 8.4 Hz, methanesulfonylphenyl H-
3, H-5), 7.92-7.93 (m, 2H, phenyl H-2, H-6), 10.04 (s, 1H, NH (D2O exchangeable);
13C NMR (DEPTQ); 17.10 (CH3, thiazole), 18.82 (CH3-C=N), 49.06 (CH3,
methanesulfonyl) 112.78 (C-2, C-6 methanesulfonylphenyl), 126,34 (C3, C5 of
methansulfonylphenyl), 129.20 (C3, C5 of phenyl), 129.71 (C2, C6- of phenyl),
130.47 (C-5 of phenyl) ,130.70 (C-1 of methanesulfonylphenyl), 133.34 (C=NH-),
133.37 (C-1 of phenyl), 149.82 (C-4 of methanesulfonylphenyl ), 149.88 (C-5 of
thiazole), 150.15 (C-4 of thiazole), 163.57(C-2 of thiazole). MS (m/z,%): 385 [(M)+.,
16

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100%], 387[M+.+2, 14.28 %] Anal.Calcd. for C19H19N3O2S2: C, 59.20; H, 4.97; N,
10.90, Found: C, 59.01; H, 4.63; N, 10.85%.
(E)-1-(1-(4-Methyl-2-phenylthiazol-5-yl)ethylidene)-2-(4-sulfamoyl-phenyl)
hydrazine (9b).
o
Yellow powder; Yield: 84%; mp: 275-277 C; IR (KBr, cm-1): 3382, 3300 (NH,
NH2), 3184 (C-H aromatic), 2936 (C-H aliphatic), 1592 (C=N), 1342, 1156 (SO2);
1H-NMR (DMSO-d6); δ 2.38 (s, 3H, CH3), 2.66 (s, 3H, CH3-C=N), 7.11(s, 2H, NH2)
7.28 (d, 2H, J = 8.4 Hz, H-2, H-6 of sulfamoylphenyl), 7.50 ( 3H, phenyl H-3, H-4,
H-5), 7.67 (d, 2H, J = 8.4 Hz, sulfamoylphenyl H-3, H-5), 7.94 (m, 2H, phenyl H-2,
H-6), 9.90 (s, 1H, NH (D2O exchangeable). 13C NMR (DEPTQ); 16.98 (CH3,
thiazole), 18.75 (CH3-C=N), 112.78 (C-2, C-6 sulfamoylphenyl), 126,34 (C3, C5 of
sulfamoyl phenyl), 129.20 (C3, C5 of phenyl), 129.70 (C-2, C-6 of phenyl), 130.66
(C-4 of phenyl), 133.35 (C-1of sulfamoylphenyl), 133.54 (C=NH-), 134.43 (C-1 of
phenyl), 138.95 (C-4 of sulfamoyl phenyl), 148.46 (C-5 of thiazole), 149.86 (C-4 of
thiazole), 163.40 (C-2 of thiazole); 386 [(M)+., 100%]. Anal. Calcd. for
C18H18N4O2S2 : C, 55.94; H, 4.69; N, 14.50; Found: C, 56.01; H, 4.63; N, 14.25%.
4.1.5. Synthesis 5-(4-Methyl-2-phenylthiazol-5-yl)-1-(4-(N,N-dimethylformamidine-
sulfam oyl) phenyl) - 1H-pyrazole (10).
A solution of 9b (0.38 g, 0.001mol) and DMFDMA (0.03 mol) in toluene (30 mL)
was heated under reflux for 24 h., (monitored by TLC). The reaction mixture is
evaporated solid separated , dried then separated by preparative T.L.C as 10 as Off
white powder ; Yield: 39 %; mp: 175-177 oC; IR (KBr, cm-1): 2990 (C-H aromatic),
2933 (C-H aliphatic), 1590 (C=N), 1344, 1133 (SO2); 1H NMR (DMSO-d6); δ 2.15
(s, 3H, thiazole CH3), 2.90 (s, 3H, =N-CH3), 3.1(s, 3H, =NCH3), 6.84(d, 1H, J = 2.0
Hz, pyrazole H-4), 7.49-7.53 (m, 5H, phenyl H-3, H4, H5 and H2, H-6 of
sulfomylphenyl H-2, H-6), 7.82 (d, 2H, J = 8.8 Hz, sulfamoylphenyl H-5, H-3),
7.87-7.90 (m, 2H, phenyl H-2, H-6), 7.93 (d, 1H, J = 2.0 Hz, pyrazole H-5), 8.21 (s,
1H, olfenic H); 13C NMR (DEPTQ); 16.24 (thiazole CH3), 35.59 (=NCH3), 40.48
(=NCH3 ), 111.95( C-4 pyrazole), 118.91 (C-1 of sulfamoylphenyl), 124.99 (C3, C5
phenyl), 126.51 (C2, C6 of phenyl), 127.65 (C-2, C-6 sulfamoylphenyl), 129.82 (C-3,
C-5 sulfamoylphenyl), 131.25 (C-5 of phenyl), 132.51(C-1 of phenyl), 139.12 (C-4 of
ulfamoylphenyl), 141.81(C-3 of pyrazole), 141.86(C-5 of pyrazole), 142.48(C-5 of
thiazole), 152.98(C-4 of thiazole), 160.37 (olfenic CH), 168.74 (C-2 of thiazole). (
17

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HMBC, HSQC in supplementary materials), MS (m/z, %): 467 [(M) +., 1.76%], 71
[100%]. Anal.Calcd.for C22H21N5O2S2 : C, 58.52; H, 4.69; N, 15.51; Found: C,
58.77; H, 4.99; N, 15.75%.
4.1.6. General procedure of pyrazolines 12a and 12b
(S)-4,5-Dihydro-5-(4-Methoxyphenyl)-3-(4-methyl-2-phenylthiazol-5-yl)-1-(4-
(methylsulfonyl)phenyl)-1H-pyrazole (12a)
A solution of chalcone 11 (0.01 mol) and the corresponding phenyl hydrazine
derivative (0.01 mol) 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 ethanol/ethyl acetate mixture (1:1).
Yellow powder; Yield: 60 %; mp: 230-232; IR (KBr, cm-1): 3013 (C-H aromatic),
2932 (C-H aliphatic), 1591 (C=N), 1324, 1164 (SO2); 1H NMR (DMSO-d6); δ 2.51
(s, 3H, thiazole CH3), 3.08 (s, 3H, OCH3), 3.20 (dd, 1H, Ha, Jab = 17.2 Hz, Jax= 5.2
Hz), 3.72(s, 3H, SO2CH3), 4.10 (dd, 1H, Hb, Jab = 17.2 Hz , Jbx= 12 Hz), 5.62(dd, 1H,
Hx, Jbx= 12 Hz, Jax = 5.2 Hz), 6.92 (d, 2H, J = 8.8 Hz methoxyphenyl H3, H5), 7.07
(d, 2H, methoxyphenyl H2, H6 ), 7.21 (d, 2H, J = 8.8 Hz, H-2, H-6 of
sulfamoylphenyl), 7.50-7.51 ( m, 3H, phenyl H-3, H-4, H-5), 7.52 (d, 2H, J = 8.8 Hz,
sulfamoylphenyl H-3, H-5), 7.67-7.69 (m, 2H, phenyl H-2, H-6),). 13C
NMR(DEPTQ); 17.84 (CH3, thiazole), 44.54 (OCH3), 49.06 (CH2 of pyrazoline),
55.45 (SO2CH3), 62.65 (CH of pyrazoline), 112.77 (C-2, C-6 methoxyphenyl),
115.02 (C3, C5 of methoxy phenyl), 125.26 (C1 of methoxyphenyl), 126.61 (C3, C5
of phenyl), 127.56 (C-3, C-5 of methanesulfonyl), 129.03 (C-2,
methanesulfonylphenyl), 129.81 (C-2, C-6 of phenyl), 131.15(C-5 of phenyl), 132.98
(C-1 of methanesulfanylphenyl), 133.41(C-1 of phenyl),145.2 (C-4 of
C-6 of
methanesulfonyl phenyl and C3 of pyrazoline), 147.05 (C4-of methoxyphenyl),
153.15 (C-5 of thiazole), 159.24 (C-4 of thiazole), 165.46 (C-2 of thiazole). MS
(m/z,%): 503 [(M)+., 100%], Anal.Calcd.for C27H25N3O3S2 : C, 64.39; H, 5.00; N,
8.34, Found: C, 64.77; H, 5.30; N, 8.75%.
(S)-4,5-Dihydro-5-(4-methoxyphenyl)-3-(4-methyl-2-phenylthiazol-5-yl)-1-(4-
(sulfamoyl)phenyl)-1H-pyrazole (12b).
o
Yellow powder; Yield: 74%; MP: 273-275 C; IR (KBr, cm-1): 3335, 3318 (NH2),
3118 (C-H aromatic), 2952 (C-H aliphatic), 1592 (C=N), 1369, 1159 (SO2);
18

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1H-NMR (DMSO-d6); δ 2.64 (s, 3H, CH3), 3.18 (dd, 1H, Ha, Jab = 18 Hz, Jax= 5.2
Hz), 3.71 (s, 3H, OCH3), 4.10 (dd, 1H, Hb Jab = 18 Hz, Jbx= 12.8), 5.62 (dd, 1H, Hx,
Jbx= 12.8 Hz, Jax = 5.2 Hz) 6.91 (d, 2H, J = 8.8 Hz methoxyphenyl H3, H5), 7.02-
7.05 (m, 4H, methoxyphenyl H2, H6 and NH2, D2O exchangeable), 7.20 (d, 2H, J =
8.8 Hz, H-2, H-6 of sulfamoylphenyl), 7.51-7.52 ( m, 3H, phenyl H-3, H-4, H-5),
7.61 (d, 2H, J = 8.8 Hz, sulfamoylphenyl H-3, H-5), 7.95-7.97 (m, 2H, phenyl H-2,
H-6). 13C NMR (DEPTQ); 17.80 (CH3, thiazole), 45.44 (CH2 of pyrazoline), 55.53
(OCH3), 62.73 (CH of pyrazoline), 112.63 (C-2, C-6 methoxyphenyl), 114.95 (C3,
C5 of methoxy phenyl), 125.1 (C1 of methoxyphenyl), 126.57 (C3, C5 of phenyl),
127.59 (C-3, C-5 of sufamoylphenyl), 133.01 (C-2, C-6 of sulfamoylphenyl), 133.01
(C-2, C-6 of phenyl), 132.98 (C-1of sulfamoylphenyl), 133.76 (C-5 of phenyl),
133.88 (C-1 of phenyl), 145.2 (C-4 of sulfamoyl phenyl), 144.34 (C4-of
methoxyphenyl), 145.86 (C1 of sulfamoyl phenyl and C3 of pyrazoline), 152.80 (C-5
of thiazole), 159.27 (C-4 of thiazole), 165.24 (C-2 of thiazole). MS (m/z,%): 504
[(M)+., 59.05%], 121.11 [100%]. Anal. Calcd. for C26H24N4O3S2 : C, 61.88,; H, 4.79;
N, 11.10., Found: C, 61.64; H, 4.43; N, 11.24%.
4.2. Biological activity
4.2.1. 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, ꢀM) was determined using an enzyme immune assay (EIA) kit
(Cayman Chemical, Ann Arbor, MI, USA) according to a previously reported
methods.
4.2.2. 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, ꢀM) 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 Tris HCl, PH, 7.4).
10 µl of different compound were prepared in dissolved at the least 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 210µl. And IC50 of test compounds were determined according
to a manufacturer's instructions as reported methods.
4.2.3. In vivo anti-inflammatory activity
19

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Animals: 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. Carragenan-induced paw edema test: Anti-inflammatory
activity of ten compounds, was evaluated by employing carragenan induced rat paw
edema model according to a previously reported method after oral administration of
a dose 50mg\kg of test compounds. % Inhibition in edema thickness was determined
after 2h and 4h. % Relative potency after 2h and 4h was calculated in relation to
standard celecoxib.
4.2.4. Ulcerogenic liability
Ulcerogenic liability of 15 compounds, which showed in-vivo anti-inflammatory
activity, was evaluated according to the reported standard 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 (30.9 ꢀmol / kg), while the other groups received the tested
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 xs) for the presence of lesions in the form of haemorrhages 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.
Acknowledgements
The authors are grateful to all members of Pharmaceutical Organic Chemistry,
faculty of Pharmacy, Beni-suef university for all supports during proceeding of
research.
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•
•
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 and they showed higher
safety profiles.
Most of the compounds were effective as anti-inflammatory and more
selective towards COX-2 /15-LOX.