Synthesis and biological evaluation of some thiazolylpyrazole derivatives as dual anti-inflammatory antimicrobial agents

Article abstract of DOI:10.1016/j.ejmech.2010.10.001

The synthesis of a novel series of 4-thiazolylpyrazolyl derivatives is described in the present report. All the newly synthesized compounds were examined for their anti-inflammatory activity using cotton pellet-induced granuloma and carrageenan-induced rat paw edema bioassays. Their inhibitory activities of cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), ulcerogenic effect and acute toxicity were also determined. Furthermore, all compounds were evaluated for their in vitro antimicrobial activity against Escherichia coli, Staphylococcus aureus and Candida albicans. A docking pose for compounds 8b, 10a and 10b separately in the active site of the human COX-2 enzyme and DNA-gyrase B was also obtained. The results revealed that compounds 8b, 10a and 10b exhibited good anti-inflammatory activity with no or minimal ulcerogenic effect and good safety margin. Compounds 10a and 10b were found to be the most potent anti-inflammatory agents in the present study. Meanwhile, 10a and 10b displayed higher selective inhibitory activity towards COX-2 compared to indomethacin. Moreover, compounds 10a and 10b exhibited promising antibacterial against both E. coli and S. aureus. Docking studies for 8b, 10a and 10b with COX-2 (PDB ID: 1CX2) and DNA-gyrase B (PDB ID: 1EI1) showed good binding profile.

Full text of DOI:10.1016/j.ejmech.2010.10.001

European Journal of Medicinal Chemistry 45 (2010) 6027e6038
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of some thiazolylpyrazole derivatives as dual
anti-inflammatory antimicrobial agents
,
b
a,
Adnan A. Bekhit a , Hesham T.Y. Fahmy , Sherif A.F. Rostom c, Alaa El-Din A. Bekhit d
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt
b Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Box 2202C, Brookings, SD 57007, USA
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, P.O. 80260, Jeddah 21589, Saudi Arabia
d Food Science, Otago University, Dunedin, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2010
Received in revised form
14 September 2010
Accepted 1 October 2010
Available online 20 October 2010
The synthesis of a novel series of 4-thiazolylpyrazolyl derivatives is described in the present report. All
the newly synthesized compounds were examined for their anti-inflammatory activity using cotton
pellet-induced granuloma and carrageenan-induced rat paw edema bioassays. Their inhibitory activities
of cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2), ulcerogenic effect and acute toxicity were
also determined. Furthermore, all compounds were evaluated for their in vitro antimicrobial activity
against Escherichia coli, Staphylococcus aureus and Candida albicans. A docking pose for compounds 8b,
10a and 10b separately in the active site of the human COX-2 enzyme and DNA-gyrase B was also
obtained. The results revealed that compounds 8b, 10a and 10b exhibited good anti-inflammatory
activity with no or minimal ulcerogenic effect and good safety margin. Compounds 10a and 10b were
found to be the most potent anti-inflammatory agents in the present study. Meanwhile, 10a and 10b
displayed higher selective inhibitory activity towards COX-2 compared to indomethacin. Moreover,
compounds 10a and 10b exhibited promising antibacterial against both E. coli and S. aureus. Docking
studies for 8b, 10a and 10b with COX-2 (PDB ID: 1CX2) and DNA-gyrase B (PDB ID: 1EI1) showed good
binding profile.
Keywords:
Thiazolylpyrazoles
anti-Inflammatory activity
COX inhibitory activity
Acute toxicity
Ulcerogenic effect
Antimicrobial activity
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
shown to be a potent and a gastrointestinal safe anti-inflammatory
and analgesic agent. Celecoxib is considered a typical model of pyr-
Non-steroidal anti-inflammatory drugs (NSAIDs) have been
recognized as important therapeutic agents for the treatment of
rheumatoid arthritis and its variants. However, large doses of NSAIDs
or long term use usually results in gastrointestinal mucosal damage,
intolerance and renal toxicity [1e4]. Despite the efforts that had been
made to improve the pharmacological profile of NSAIDs, ulcer-
ogenicity remains the most limiting problem in their clinical use. A
major breakthrough in anti-inflammatory research occurred when it
was discovered that COX exists in three isoforms COX-1, COX-2 and
COX-3 which are regulated differently [5,6]. The discovery of the
inducible isoform COX-2 spurred the search for novel anti-inflam-
matory agents devoid of the undesirable effects associated with
classical non selective NSAIDs. Consequently, a new generation of
COX-2 selective inhibitors has been clinically used with the hope that
they would exhibit a reduced risk in gastrointestinal events. Among
this class of compounds, celecoxib; 4-[5-(4-methylphenyl)-3-(tri-
fluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide (Fig. 1) was
azole containing diaryl heterocyclic template that is known to inhibit
COX-2 selectively [7]. Moreover, several other compounds containing
pyrazole functionality were also reported to exhibit anti-inflamma-
tory activity [8e12]. Furthermore, much attention has been focused
on pyrazoles as antimicrobial agents after the discovery of the natural
pyrazole C-glycoside pyrazofurin; 4-hydroxy-3-b-D-ribofuranosyl-
1H-pyrazole-5-carboxamide (Fig. 1) which demonstrated a broad
spectrum of antimicrobial activities [13,14]. This led to the synthesis
of several pyrazole derivatives that exhibited antimicrobial activity
by Tanitame and coworkers [15e17].
Co-administration of multiple drugs for treatment of inflamma-
tory conditions associated with microbial infection is a major risk
especially in the case of patients with impaired liver or kidney
functions. A mono therapy of a drug with dual anti-inflammatory
antimicrobial activity would be preferred from the pharmacoeco-
nomic and patient compliance point of view. This premise was one of
the goals of our research program aimed at the discovery of new
pyrazoles that would possess dual anti-inflammatory antimicrobial
activities [18e29]. Some of our reported pyrazolyl compounds
[18e28] showed pronounced dual activities. Four of these lead
* Corresponding author. Tel.: þ20 3 4871317; fax: þ20 3 4873273.
E-mail address: adnbekhit@hotmail.com (A.A. Bekhit).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.001

6028
A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
NH
2
HO
OH
HO
O
CF
3
N
N
N
HO
N
O
H
NH
2
S
O
O
Pyrazofurin
Celecoxib
HN
S
H
N
S
N
H3C
O
CN
CN
N
N
N
N
N
N
N
N
N
N
N
Cl
CH3
N
C
D
B
A
N
S
CH3
SO2NH2
SO2NH2
O
O
H
SO2NH2
Fig. 1. Structures of celecoxib and reported active pyrazole derivatives A, B, C and D.
compounds A [26], B [26] C [27] and D [28] displayed appreciable
anti-inflammatory and selective COX-2 inhibitory activity (Fig.1). On
the other hand, thiazole derivatives were reported to possess anti-
microbial [20,30] or anti-inflammatory [20,31] activities. Encour-
aged by these results it was decided to synthesize novel compounds
having essentially in their structure the pyrazole counterpart linked
to different thiazole derivatives in order to investigate the effects of
such molecular variation on the anti-inflammatory antimicrobial
activity. This type of molecular variation could regulate other bio-
logical functions; therefore the new compounds were investigated
for their inhibitoryactivities of COX-1 and COX-2 enzymes. A docking
pose of the most active compounds separately in the active site of the
human COX-2 enzyme and DNA-gyrase B was also obtained. Addi-
tionally, the ulcerogenic effect and acute toxicity profiles of the active
compounds were determined.
derivatives 6a,b. 1H NMR spectra of these compounds revealed
double doublet and singlet at 3.68e3.71 ppm and at
d
¼
d
¼ 5.62e5.68 ppm which are characteristics for methylene and
methine protons of thiazolidinone ring, respectively. Heating,
under reflux, a solution of pyrazole carboxaldehyde 1a,b and
L-cysteine in aqueous ethanol [35], followed by N-protection [36],
using (Boc)2O resulted in compounds 7a,b. These compounds 7a,b
either subjected to N-deprotection using 4 N HCl/dioxane leading
to thiazolidine carboxylic acid derivatives 8a,b or allowed to react
with NH4OH in the presence of DCC/HOBt to afford the corre-
sponding carboxamide derivatives 9a,b. The latter when subjected
to N-deprotection resulted in carboxamide derivatives 10a,b. It is
worth mentioning that all trials to separate diastereomers of
compounds 7, 8, 9 and 10 went in vain. In 2,4-disubstituted thia-
zolidines, the cis and trans diastereomers freely interconverted in
solution [37]. The ratio of diastereomers observed for the
compounds in the present study was 1:1 based on 1H NMR spec-
troscopy. Diastereomerization was at the C-2 center via an iminium
intermediate [35]. This property prevents the independent exam-
ination of the biological activities of the two diastereomers; hence
the structure was drawn and discussed throughout this paper as
diastereomeric mixture.
2. Chemistry
Synthesis of the intermediate and target compounds was per-
formed according to the reactions illustrated in Scheme 1. The
intermediates 3-aryl-1-phenyl-1H-pyrazole-4-aldoxime 2a,b were
obtained in good yields by the condensation of 3-aryl-1-phenyl-
1H-pyrazole-4-carboxaldehyde [32] 1a,b with hydroxylamine
hydrochloride in ethanol containing anhydrous sodium acetate. 1H
NMR spectra of these compounds revealed a singlet characteristic
3. Results and discussion
for hydroxyl group proton at
d
¼ 11.87e11.91 ppm and iminomethyl
proton at
d
¼ 7.42e7.46 ppm confirming existence of these
3.1. Anti-inflammatory activity
compounds as E-isomers [33]. Dehydration of the oximes 2a,b with
acetic anhydride [26] afforded the cyano derivatives 3a,b. The IR
spectra for 3a,b showed the specific band for the cyano group at
2228e2232 cmꢀ1.Cyclization of the cyano derivatives 3a,b with
cysteamine hydrochloride in the presence of sodium hydroxide
resulted in thiazolidine derivatives 4a,b [34]. 1H NMR spectra of
thiazolidine 4a,b showed a two triplets assigned for SCH2 and NCH2
3.1.1. Cotton pellet-induced granuloma bioassay
The anti-inflammatory activities of the target compounds 4a,b,
6a,b, 8a,b and 10a,b were evaluated by applying the cotton pellet-
induced granuloma bioassay in rats as described earlier [38] using
indomethacin and celecoxib as reference standards. The ED50 value
for each of the test compounds was expressed as the mean ꢁ SEM.
Significant difference between the control and treated groups was
estimated using one way analysis of variance (ANOVA) and the
significance of the difference between means was determined by
Tukey’s test at P < 0.001 (Table 1). Three of the test compounds 8b,
10a and 10b possessed anti-inflammatory activity (ED50 ¼ 9.74, 8.11
protons at
d
¼ 3.34e3.37 ppm and ¼ 4.46e4.48 ppm, respectively.
d
Condensation of the aldehydes 1a,b with 4-fluorobenzylamine gave
rise to Schiff’s bases 5a,b. 1H NMR spectra of these compounds
revealed
a singlet characteristic for iminomethyl proton at
d
¼ 7.42e7.46 ppm confirming the existence of these compounds as
E-isomers [33]. Reaction of 5a,b with thioglycolic acid in the pres-
and 7.86
mmol, respectively) surpassing that of indomethacin and
ence of anhydrous zinc chloride resulted in the thiazolidinone
celecoxib (ED50 ¼ 9.64 and 16.74
mmol, respectively). The results

A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
6029
R
R
COOH
S
COOH
S
NH
N
i) 4N HCl/dioxane
O
N
R = H, CH3
N
O
N
N
ii) Et3N
8a,b
R
7a,b
R
CONH2
S
CONH2
NH
S
i) Stirring
ii) (Boc)2O
i) 4N HCl/dioxane
ii) Et3N
N
i) DCC, HOBt
ii) NH4OH
N
N
O
O
N
N
HS
O
H2N
OH
10a,b
9a,b
+
R
R
R
O
S
H
H
H2N
O
SH
F
N
O
N
F
HO
N
N
N
+
N
N
N
F
5a,b
6a,b
1a,b
NH2OH
R
R
R
S
H
N
CN
NOH
SH
H2N
Ac2O
N
N
N
N
N
N
NaOH
4a,b
2a,b
3a,b
Scheme 1.
revealed that thiazolidine carboxamide derivatives 10a,b and
thiazolidine carboxylic acid derivative 8a,b appeared to possess
higher anti-inflammatory activity than thiazolidine derivatives 4a,b
or thiazolidinone 6a,b. Within thiazolidine derivatives 8a,b and
10a,b, compounds 8b, 10b (ED50 ¼ 9.74 and 7.86 mol, respectively)
containing p-tolyl substitution were more active than compounds
mol, respectively) which contained
phenyl substitution. This may affect the structure conformation to
have hydrogen bonding with the backbone of COX-2 enzyme as will
be discussed in the docking section. Compound 10b was proved to
m
8a, 10a (ED50 ¼ 11.86 and 8.11
m
Table 1
The anti-inflammatory activity (ED50, mmol/cotton pellet) and ulcerogenic activity.
Test Compound
ED50
(
m
mol)
% Ulceration
be the most potent anti-inflammatory agent (ED50 ¼ 7.86
mmol)
Control
Indomethacin
Celecoxib
4a
4b
6a
6b
8a
8b
e
100z
100z
0.0x
NDa
ND
whereas compound 6b (ED50 ¼ 28.42
m
mol) was found to be the
9.64 ꢁ 0.28t
16.74 ꢁ 0.22v
22.37 ꢁ 0.18w
26.62 ꢁ 0.24y
25.08 ꢁ 0.44x
28.42 ꢁ 0.32z
11.86 ꢁ 0.34u
9.74 ꢁ 0.38t
8.11 ꢁ 0.15s
7.86 ꢁ 0.14r
least active as anti-inflammatory agent (Table 1).
3.1.2. Carrageenan-induced rat paw edema bioassay
ND
ND
Compounds showing promising anti-inflammatory activity in the
cotton pellet-induced granuloma bioassay (8b, 10a and 10b) were
further evaluated for their in vivo systemic effect using carrageenan-
induced paw edema bioassay in rats [39]. The results showed that
compounds 8b, 10a and 10b displayed systemic anti-inflammatory
activity (% protection ¼ 78.4 and 79.4, 82.5, respectively) comparable
or slightly higher than celecoxib (% protection ¼ 77.3) but higher
than indomethacin (% protection ¼ 73.2). Thiazolidine derivative 10b
ND
70y
0.0x
0.0x
10a
10b
rezMeans within each activity with different superscripts are significantly different
from control (P < 0.001).
a
ND ¼ Not determined.

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A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
Table 2
derivatives 10a,b proved to have superior gastrointestinal safety
profiles (0.0% ulceration) in the population of test animals at oral
Effect of compounds 8b, 10a and 10b on carrageenan-induced rat paw edema (ml), %
protection and activity relative to indomethacin.
doses of 30
mmol/kg per day, compared to indomethacin, the
Test compound
Increase in paw
% Protection
Activity relative
to indomethacin
reference drug, which was found to cause 100% ulceration under
the same experimental conditions (Table 1). Thiazolidine carboxylic
acid derivative 8b showed a significant ulcerogenic effect (70%),
however this was lower than that of indomethacin. Gross obser-
vation of the isolated rat stomachs showed a normal stomach
texture for compounds 10a,b.
edema (ml) ꢁ SEMa,b
Control
Indomethacin
Celecoxib
8b
10a
10b
0.97 ꢁ 0.028
0.26 ꢁ 0.024
0.22 ꢁ 0.014
0.21 ꢁ 0.016
0.20 ꢁ 0.026
0.17 ꢁ 0.018
0.0
73.2
77.3
78.4
79.4
82.5
0.0
100
105.60
107.10
108.46
112.70
a
3.1.5. Acute toxicity
SEM denotes the standard error of the mean.
All data are significantly different from control (P < 0.001).
b
Compounds 8b, 10a and 10b were further evaluated for their oral
acute toxicity in male mice using a previously reported method [43].
The results indicated that the test compounds proved to be non-
toxic and well tolerated by experimental animals up to 150 mg/kg.
Moreover, these compounds were tested for their toxicity through
the parenteral route [20]. The results revealed that all test
compounds were non-toxic up to 80 mg/kg.
containing p-tolyl substitution also showed higher activity than
compound 10b containing phenyl substitution in this assay (Table 2).
3.1.3. Human COX-1 and COX-2 enzymatic activity
Compounds 8b, 10a and 10b that showed potent anti-inflam-
matory profiles in animal models were further tested for their
ability to inhibit human COX-1 and COX-2 enzymes in vitro as
described by Wakitani et al. [40]. COX-1 assay was carried out using
platelets microsome fraction. Human platelets were prepared from
NSAID-free normal human volunteers according to the method of
Hammarström and Falardeau [41]. COX-2 assay was performed
using human recombinant COX-2 purchased from SigmaeAldrich.
The inhibitory activity was expressed as the concentration of the
3.1.6. Docking studies
Molecular docking studies of compound 8b, 10a and 10b were
performed using Molecular Operating Environment (MOE-Dock
2006) module [44] in order to rationalize the obtained biological
results. Molecular docking studies further helps in understanding
the various interactions between the ligand and enzyme active site
in detail.
The determination of the three-dimensional co-crystal structure
of COX-2 complex with a selective inhibitor, SC-558 (Fig. 2; PDB ID:
1CX2) has led to the development of a model for the topography of
the NSAIDs binding site in human COX-2 [45]. Therefore, herein we
performed the docking studies using this COX-2 co-crystal struc-
ture with SC-558 as a template.
Figs. 3e5 show the binding interactions of compounds 8b, 10b
and 10b to the active site of COX-2, respectively, where they
exhibited some similar interactions as with SC-558. Compound 8b
displayed hydrogen bond interaction with Tyr 385, in addition to
hydrophobic interactions with His 90, Val 349, Leu 352, Ser 353, Leu
359, Arg 513, Phe 518 and Val 523. On the other hand, compound
10a showed hydrogen bond interactions with Leu 352 and Ser 353,
in addition to hydrophobic interactions with His 90, Arg 120, Val
349, Leu 352, Ser 353, Tyr 355, Tyr 385, Val 523 and Ala 527.
Compound 10b displayed hydrogen bond interactions with Tyr 355
and Arg 513, in addition to hydrophobic interactions with His 90,
Val 116, Arg 120, Val 349, Leu 352, Ser 353, Tyr 355, Arg 513, Val
523, Gly 526, Ala 527 and Leu 531.
compound causing 50% enzyme inhibition (IC50 mmol) (Table 3).
The results revealed that the test compounds exhibited weak
inhibitory activity against COX-1 enzyme (IC50 values ranged
between 96.22 and >100
(IC50 ¼ 0.26 mol), however compound 8b (IC50 ¼ 96.22
more potent than celecoxib (IC50 ¼ > 100 mol). The test
compounds showed higher inhibitory profile against COX-2 (IC50
values between 0.38 and 0.94 mol) compared with indomethacin
(IC50 ¼ 2.63 mol). All test compounds showed approximate
mmol) compared with indomethacin
m
mmol) was
m
m
m
selectivity ratio (COX-1/COX-2) lower than that of celecoxib. In
general, the results revealed that compounds having thiazolidine
carboxamide moiety (compounds 10a and 10b) possessed higher
selectivity towards COX-2 enzyme than compound containing
thiazolidine carboxylic acid moiety (compound 8b). However,
within thiazolidine carboxamide class of compounds, the selective
COX-2 inhibition of the tolyl derivative 10b was greater than the
phenyl derivative 10a. Compounds 10a and 10b, the most selective
COX-2 inhibitors in the present study (approximate selectivity
ratio ¼ 212.77 and 263.16, respectively), had lower selectivity
compared to celecoxib (approximate selectivity ratio > 333).
3.1.4. Ulcerogenic effect
The most active compounds 8b, 10a and 10b were evaluated for
their ulcerogenic potential in rats [42]. Thiazolidine carboxamide
H2N
O
S
O
Table 3
In vitro human COX-2a and COX-1b enzymes inhibitory activities of compounds 8b,
10a and 10b.
Test compound
COX-2 IC50
M)c
COX-1 IC50
(m
M)c
Approximate selectivity ratio
COX-1/COX-2
(
m
Indomethacin
Celecoxib
8b
10a
10b
2.63 ꢁ 0.02
<0.3
0.94 ꢁ 0.02
0.47 ꢁ 0.05
0.38 ꢁ 0.06
0.26 ꢁ 0.32
>100
0.098
>333
102.13
>212.77
>263.16
N
N
96.22 ꢁ 0.24
Br
>100
>100
a
CF3
Human recombinant COX-2 enzyme.
Human COX-1 enzyme from human platelets.
Values are means of at least four independent experiments.
b
c
Fig. 2. Structure of SC-558.

A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
6031
Fig. 3. 3D View from a molecular modeling study, of the minimum-energy structure of the complex of 8b docked in COX-2 (PDB ID: 1CX2). Viewed using Molecular Operating
Environment (MOE) module.
3.1.7. Antimicrobial activity
DNA-gyrase. Compound 10a showed a hydrogen bond donor
interaction with Asn 46, a hydrogen bond acceptor interaction with
Val 118, and hydrophobic interactions with Asp 73, Ile 78, Pro 79, Ile
94, Gly 117, Val 118, Glt 119, Val 120 and thr 1165. Compound 10b
displayed 2 hydrogen bonds donor and one hydrogen bond
acceptor interaction with Tyr 109, in addition to hydrophobic
interactions with Glu 50, Asp 73, Arg 76, Gly 77, Ile 78, Gly 102, Lys
103, Ser 108, Tyr 109, Gly 117, Val 118, Gly 119, Val 120 and Thr 165.
The docking results showed that compound 10b had better
interactions with the backbone of DNA-gyrase enzyme than
compound 10a.
The designed compounds 4a,b; 6a,b; 8a,b and 10a,b were
evaluated for their in vitro antimicrobial activity against E. coli ATCC
25922, as an example of Gram-negative bacteria, S. aureus ATCC
19433 as an example of Gram-positive bacteria, and C. albicans as
a representative of fungi. The micro-dilution susceptibility test in
Müller-Hinton Broth (Oxoid) and Sabouraud liquid Medium (Oxoid)
were used for the determination of antibacterial and antifungal
activities [46]. The minimal inhibitory concentration (MIC; mg/ml)
of the test compounds are shown in Table 4. The results revealed
that some of the newly synthesized compounds exhibited appre-
ciable antibacterial activity. Thiazoline derivatives 4a,b and thia-
zolidinone derivatives 6a,b did not show appreciable antimicrobial
activity. Compounds 8a, 8b and 10a exhibited antibacterial activity
4. Conclusion
against E. coli (MIC ¼ 25
compound 10b (MIC ¼ 12.5
terial activity of ampicillin. Both compounds 10a and 10b
g/ml) exhibited antibacterial activity against S. aureus
mg/ml) comparable to ampicillin, whereas
mg/ml) showed two fold the antibac-
Compounds 8b, 10a and 10b exhibited good anti-inflammatory
activity with no or minimal ulcerogenic effect and good safety
margin. Compounds 10a and 10b were found to be the most potent
anti-inflammatory agents in the present study. Meanwhile 10a and
10b displayed higher selective inhibitory activity towards COX-2
compared to indomethacin. Moreover, compounds 10a and 10b
exhibited promising antibacterial against both E. coli and S. aureus.
Docking studies for both 10a and 10b with COX-2 (PDB ID: 1CX2)
and DNA-gyrase B (PDB ID: 1EI1) showed good binding profile.
Therefore, compounds 10a and 10b would represent a fruitful
matrix for the development of a new class of dual anti-inflamma-
tory antimicrobial agents that would deserve further investigation
and derivatization.
(MIC ¼ 12.5
m
comparable to ampicillin. However, none of the screened
compounds showed significant activity against C. albicans (MIC
values >200) compared with the reference antifungal agent clo-
trimazole (MIC 12.5 mg/ml).
In a trial to study the antimicrobial mechanism of the most
active thiazolindine carboxamide 10a,b; the compounds were
tested for their inhibition of DNA-gyrase [47] (full data will be
published elsewhere). Compounds 10a and 10b showed promising
DNA gyrase inhibitory activity, with IC50 of 5 mg/ml and 2.5 mg/ml
respectively. Furthermore, we performed the docking studies of 10a
and 10b with DNA-gyrase B (PDB ID: 1EI1). Figs. 6 and 7 show the
binding interactions of compounds 10a and 10b to the active site of
It is worth mentioning that compounds 10a and 10b displayed
anti-inflammatory profile comparable to our previously reported

6032
A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
Fig. 4. 3D View from a molecular modeling study, of the minimum-energy structure of the complex of 10a docked in COX-2 (PDB ID: 1CX2). Viewed using Molecular Operating
Environment (MOE) module.
pyrazolyl benzenesulfonamide compounds A [26], B [26] and C
[27]. However, the selective COX-2 inhibitory activity of
compounds 10a and 10b was higher than those found with
compounds A, B and C.
acetate (0.5 g, 6.1 mmol) in ethanol (15 ml) was heated under reflux
for 4 h. The reaction mixture was concentrated and left to attain
room temperature. The separated solid product was filtered,
washed with water, dried and crystallized from an appropriate
solvent (Table 5).
5. Experimental
IR (cmꢀ1), 2a: 3378 (OH), 1647, 1596 (C]N). IR (cmꢀ1). 1H NMR
(CDCl3), 2a:
8.91 (s, 1H, pyrazole-C5 H), 11.87 (s, 1H, OH, D2O exchangeable). 13
NMR (CDCl3), 2a: 33.1, 56.4, 106.5, 120.4, 126.7, 127.8, 128.7, 129.2,
129.6, 130.4, 133.2, 139.8, 151.1, 164.1.
IR (cmꢀ1), 2b: 3369 (OH), 1643, 1595 (C]N). 1H NMR (CDCl3),
2b: 2.36 (s, 3H, CH3), 7.45 (s, 1H, N]CH), 7.49e7.58 (m, 5H,
d 7.42 (s, 1H, N]CH), 7.48e7.69 (m, 10H, phenyl-H),
5.1. Chemistry
C
d
Melting points were determined in open glass capillaries using
Thomas Capillary melting point apparatus and are uncorrected.
Infrared (IR) spectra were recorded using KBr discs on Per-
kineElmer 1430 infrared spectrophotometer. 1H NMR spectra were
scanned on Jeol-400 MHz Spectrometer, and chemical shifts are
d
phenyl-H), 7.73 (d, 2H, J ¼ 7.78 Hz, tolyl-C3,5H), 7.82 (d, 2H,
J ¼ 7.78 Hz, tolyl-C2,6H) 8.90 (s, 1H, pyrazole-C5 H), 11.86 (s, 1H, OH,
given in
d
(ppm) downfield from tetramethylsilane (TMS) as
D2O exchangeable). 13C NMR (CDCl3), 2b:
d 24.6, 33.2, 56.2, 106.4,
internal standard. Splitting patterns were designated as follows; s:
singlet; d: doublet; t: triplet; m: multiplet. Elemental analyses were
performed on Vario El Fab.-Nr. elemental analyzer, Faculty of
Pharmacy, Assiut University, Assiut, Egypt and were found to be
within ꢁ0.4% of the theoretical values. Follow up of the reactions
and checking the purity of the compounds was made by thin layer
chromatography (TLC) on silica gel-precoated aluminium sheets
(Type 60 GF254 Merck) and the spots were detected by exposure to
UV lamp at l254 nm for few seconds.
120.6, 126.5, 127.5, 129.1, 129.7,130.1,130.5, 138.5, 139.7, 151.3, 164.4.
5.1.2. 3-Aryl-1-phenyl-1H-pyrazole-4-carbonitrile (3a,b)
A solution of the selected oxime 2a,b (5.85 mmol) in acetic
anhydride (10 ml) was heated under reflux for 3 h and the reaction
mixture was then left over night at room temperature. The separated
solid product was filtered, washed with water, dried and crystallized
from an appropriate solvent (Table 5).
IR (cmꢀ1), 3a: 2232 (CN),1638 (C]N). IR (cmꢀ1).1H NMR (CDCl3),
3a:
d 7.33e7.73 (m, 10H, phenyl-H), 8.87 (s, 1H, pyrazole-C5 H).
5.1.1. 3-Aryl-1-phenyl-1H-pyrazole-4-aldoxime (2a,b)
IR (cmꢀ1), 3b: 2228 (CN), 1642 (C]N). 1H NMR (CDCl3), 3b:
d 2.36
A solution of the selected aldehyde 1a,b (6.1 mmol), hydroxyl-
amine hydrochloride (0.42g, 6.1 mmol) and anhydrous sodium
(s, 3H, CH3), 7.31e7.58 (m, 5H, phenyl-H), 7.73 (d, 2H, J ¼ 7.78 Hz, tolyl-
C3,5H), 7.82 (d, 2H, J ¼ 7.78 Hz, tolyl-C2,6H), 8.9 (s, 1H, pyrazole-C5 H).

A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
6033
Fig. 5. 3D View from a molecular modeling study, of the minimum-energy structure of the complex of 10b docked in COX-2 (PDB ID: 1CX2). Viewed using Molecular Operating
Environment (MOE) module.
5.1.3. 2-(3-Aryl-1-phenyl-1H-pyrazole-4-yl)-4,5-dihydrothiazole
(4a,b)
IR (cmꢀ1), 4b: 1636 (C]N).1H NMR (CDCl3), 4b:
d 2.36 (s, 3H, CH3),
3.37 (t, 2H, J ¼ 12 Hz, SCH2), 4.46(t, 2H, J ¼ 12 Hz, NCH2), 7.32e7.64 (m,
5H, phenyl-H), 7.71 (d, 2H, J ¼ 7.78 Hz, tolyl-C3,5H), 7.84 (d, 2H,
J ¼ 7.78 Hz, tolyl-C2,6H), 8.87 (s, 1H, pyrazole-C5 H).
A 10 ml of 1 N NaOH was added to a solution of the selected
nitrile 3a,b (10 mmol) and cysteamine hydrochloride (2.25 g,
10 mmol) in ethanol (20 ml). The reaction mixture was heated
under reflux with stirring for 6 h, then concentrated under vacuum
and left to attain room temperature. The separated solid product
was filtered, washed with water, dried and crystallized from an
appropriate solvent (Table 5).
5.1.4. 3-Aryl-1-phenyl-4-(4-fluorobenzyliminomethyl)-1H-pyrazole
(5a,b)
A solution of the appropriate aldehyde 1a,b (10 mmol) in
ethanol was added to an equivalent amount of 4-fluorobenzyl-
amine (1.25 g, 10 mmol). The reaction mixture was heated under
reflux for 4 h, concentrated and left to attain room temperature.
The separated solid product was filtered, washed with ethanol,
dried and crystallized from proper solvent (Table 5).
IR (cmꢀ1), 4a: 1632 (C]N). IR (cmꢀ1). 1H NMR (CDCl3), 4a:
d 3.34
(t, 2H, J ¼ 12 Hz, SCH2), 4.48 (t, 2H, J ¼ 12 Hz, NCH2), 7.32e7.67 (m,
10H, phenyl-H), 8.89 (s, 1H, pyrazole-C5 H).
IR (cmꢀ1), 5a: 1638,1632 (C]N). IR (cmꢀ1).1H NMR (CDCl3), 5a:
d
4.37 (s, 2H, NCH2), 7.14 (d, 2H, J ¼ 7.64 Hz, fluorophenyl-C2,6H),
7.33e7.68 (m, 12H, fluorophenyl-C3,5H and phenyl-H), 8.18 (s, 1H,
CHN), 8.91 (s,1H, pyrazole-C5 H). IR (cmꢀ1), 5b: 1637,1630 (C]N).1H
Table 4
Minimal inhibitory concentrations (MIC
m
g/ml) of test compounds.
Test compound
E. coli
S. aureus
C. albicans
NMR (CDCl3), 5b:
7.64 Hz, fluorophenyl-C2,6H), 7.27-7.69 (m, 7H, fluorophenyl-C3,5
d
2.36 (s, 3H, CH3), 4.38 (s, 2H, NCH2), 7.16 (d, 2H, J ¼
H
4a
4b
6a
6b
8a
8b
10a
10b
100
50
50
>200
25
25
25
12.5
25
50
>200
25
100
50
25
12.5
12.5
12.5
e
>200
>200
>200
>200
>200
>200
>200
>200
e
and phenyl-H), 7.72 (d, 2H, J ¼ 7.78 Hz, tolyl-C3,5H), 7.85 (d, 2H, J ¼
7.78 Hz, tolyl-C2,6H), 8.21 (s, 1H, CHN), 8.88 (s, 1H, pyrazole C5 H).
5.1.5. 2-(3-Aryl-1-phenyl-1H-pyrazole-4-yl)-3-(4-fluorobenzyl)-4-
oxo-thiazolidine (6a,b)
Few crystals of anhydrous zinc chloride and thioglycolic acid
(0.11 g,12 mmol) were added to a solution of the selected Schiff’s base
5a,b (10 mmol) in anhydrous dimethylformamide (10 ml). The
Ampicillin
Clotrimazole
e
12.5

6034
A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
Fig. 6. 3D View from a molecular modeling study, of the minimum-energy structure of the complex of 10a docked in DNA-gyrase B (PDB ID: 1EI1). Viewed using Molecular
Operating Environment (MOE) module.
reaction mixture was heated under reflux for 8 h, cooled, and then
poured into ice-cold water (50 ml). The separated solid was filtered,
washed with water, dried and crystallized from aqueous ethanol
(Table 5).
washed with ether (2 ꢂ 15 ml). The aqueous phase was acidified
with crystalline citric acid and extracted by ethyl acetate (3 ꢂ15 ml).
The combined organic extract was evaporated under vacuum and
the residue was crystallized from an appropriate solvent (Table 5).
IR (cmꢀ1), 7a: 3378 (br., OH),1734 (C]O),1708 (C]O),1632 (C]
IR (cmꢀ1), 6a:1682(C]O),1634(C]N).1HNMR(CDCl3), 6a:
d3.72
(2d, 2H, J ¼ 10 Hz, thiazol-C5H) 4.48 (s, 2H, NCH2), 5.69 (s,1H, thiazol-
C2H), 7.13 (d, 2H, J ¼ 7.64 Hz, fluorophenyl-C2,6H), 7.31e7.69 (m, 12H,
fluorophenyl-C3,5H and phenyl-H), 8.91 (s, 1H, pyrazole-C5 H). 13C
NMR (CDCl3), 6a: 34.2, 46.7, 48.3,115.8,117.6,120.7,123.4,126.8,127.9,
128.9, 129.2, 129.4, 129.8, 132.5, 133.4, 139.9, 150.2, 161.5, 171.8.
IR (cmꢀ1), 6b: 1684 (C]O), 1637 (C]N). 1H NMR (CDCl3), 6b:
N), 1210, 1135 (CeOeC). IR (cmꢀ1). 1H NMR (CDCl3), 7a:
d 1.39 (s, 9H,
3 (CH3)), 3.21e3.78 (m, 2H, thiazol-C5H), 4.50 (t, J ¼ 7.80 Hz, 1H,
thiazol-C4H), 6.07 (s, 0.5H, thiazol-C2H), 6.17 (s, 0.5H, thiazol-C2H),
7.32e7.71 (m, 10H, phenyl-H), 8.87 (s, 1H, pyrazole-C5 H).
IR (cmꢀ1), 7b: 3372 (br., OH), 1732 (C]O), 1712 (C]O), 1635 (C]
N), 1205, 1138 (CeOeC). 1H NMR (CDCl3), 7b:
d 1.40 (s, 9H, 3 (CH3)),
d
2.34 (s, 3H, CH3), 3.68 (2d, 2H, J ¼ 10 Hz, thiazol-C5H) 4.51 (s, 2H,
2.33 (s, 3H, CH3), 3.22e3.76 (m, 2H, thiazol-C5H), 4.53 (t, J ¼ 7.80 Hz,
1H, thiazol-C4H), 6.05 (s, 0.5H, thiazol-C2H), 6.15 (s, 0.5H, thiazol-
C2H), 7.32e7.68 (m, 5H, phenyl-H), 7.71 (d, 2H, J ¼ 7.78 Hz, tolyl-
C3,5H), 7.80 (d, 2H, J ¼ 7.78 Hz, tolyl-C2,6H), 8.90 (s,1H, pyrazole-C5 H).
NCH2), 5.71 (s,1H, thiazol-C2H), 7.16 (d, 2H, J ¼ 7.64 Hz, fluorophenyl-
C2,6H), 7.28e7.66 (m, 7H, fluorophenyl-C3,5H and phenyl-H), 7.73 (d,
2H, J ¼ 7.78 Hz, tolyl-C3,5H), 7.82 (d, 2H, J ¼ 7.78 Hz, tolyl-C2,6H), 8.89
(s,1H, pyrazole-C5 H).13C NMR (CDCl3), 6b:24.5, 34.3, 46.5, 48.6,115.7,
117.6, 121.1, 123.6, 126.7, 127.6, 129.3, 129.7, 130.1, 130.3, 132.6, 138.6,
139.8, 150.4, 161.8, 172.1.
5.1.7. (2RS,4R)-2-(3-Aryl-1-phenyl-1H-pyrazole-4-yl)thiazolidine-
4-carboxylic acid (8a,b)
Anisole (1.1 ml, 10.4 mmol) was added at 0 ꢃC to a solution of
7a,b (5.2 mmol) in 4 N HCl/dioxane (10 ml). The reaction mixture
was stirred for 1 h at room temperature. The solvent was removed
in vacuo at room temperature, ether was added, and then the
mixture was centrifuged. The obtained solid product was crystal-
lized from a proper solvent (Table 5).
5.1.6. (2RS,4R)-2-(3-Aryl-1-phenyl-1H-pyrazole-4-yl)-3-(ter-
butyloxycarbonyl)-thiazolidine-4-carboxylic acid (7a,b)
L-Cysteine (1.21 g,10 mmol) and selected pyrazole aldehyde 1a,b
were suspended in 60% aqueous ethanol (20 ml) and the mixture
was heated under reflux for 5 h, then cooled and concentrated under
vacuum. The resulted slurry was treated with 2 N NaOH (6 ml) and
(Boc)2O (2.34 g, 11 mmol) in 5 ml dioxane. After stirring for 5 h at
room temperature, the reaction mixture was diluted with water and
IR (cmꢀ1), 8a: 3363 (br., OH), 3158 (NH), 1716 (C]O), 1637 (C]
N). IR (cmꢀ1). 1H NMR (CDCl3), 7a:
d
3.24e3.76 (m, 2H, thiazol-C5H),
4.48 (t, J ¼ 7.80 Hz, 1H, thiazol-C4H), 6.05 (s, 0.5H, thiazol-C2H), 6.16

A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
6035
Fig. 7. 3D View from a molecular modeling study, of the minimum-energy structure of the complex of 10b docked in DNA-gyrase B (PDB ID: 1EI1). Viewed using Molecular
Operating Environment (MOE) module.
(s, 0.5H, thiazol-C2H), 7.35e7.74 (m, 10H, phenyl-H), 8.84 (s, 1H,
pyrazole-C5 H). 13C NMR (CDCl3), 8a: 35.6, 60.4, 65.2, 117.5, 120.1,
123.3, 126.2, 127.6, 128.7, 129.2, 129.6, 133.4, 140.1, 150.3, 175.2.
IR (cmꢀ1), 8b: 3368 (br., OH), 3154 (NH), 1714 (C]O), 1631 (C]
IR (cmꢀ1), 9b: 3349, 3187 (NH2), 1735 (C]O), 1684 (C]O), 1634
(C]N), 1208, 1136 (CeOeC). 1H NMR (CDCl3), 9b:
1.40 (s, 9H, 3
(CH3)), 2.35 (s, 3H, CH3), 3.22e3.76 (m, 2H, thiazol-C5H), 4.52 (t,
J ¼ 7.80 Hz, 1H, thiazol-C4H), 6.06 (s, 0.5H, thiazol-C2H), 6.16 (s,
0.5H, thiazol-C2H), 7.33e7.71 (m, 5H, phenyl-H), 7.72 (d, 2H,
J ¼ 7.78 Hz, tolyl-C3,5H), 7.81 (d, 2H, J ¼ 7.78 Hz, tolyl-C2,6H), 8.29
(br. s, 2H, NH2, D2O exchangeable), 8.91 (s, 1H, pyrazole-C5 H).
d
N). 1H NMR (CDCl3), 7b:
d 2.31 (s, 3H, CH3), 3.25e3.73 (m, 2H,
thiazol-C5H), 4.52 (t, J ¼ 7.80 Hz, 1H, thiazol-C4H), 6.07 (s, 0.5H,
thiazol-C2H), 6.16 (s, 0.5H, thiazol-C2H), 7.33e7.69 (m, 5H, phenyl-
H), 7.31 (d, 2H, J ¼ 7.78 Hz, tolyl-C3,5H), 7.82 (d, 2H, J ¼ 7.78 Hz, tolyl-
C2,6H), 8.89 (s, 1H, pyrazole-C5 H). 13C NMR (CDCl3), 8b: 24.7, 35.4,
60.3, 65.4, 117.3, 120.4, 123.3, 126.0, 127.5, 129.4, 129.6, 130.3, 138.2,
139.7, 150.5, 174.8.
5.1.9. (2RS,4R)-2-(3-Aryl-1-phenyl-1H-pyrazole-4-yl)thiazolidine-
4-carboxamide (10a,b)
Anisole (1.1 ml, 10.4 mmol) was added at 0 ꢃC to a solution of
9a,b (5.2 mmol), in 4 N HCl/dioxane (10 ml) and the reaction
mixture was stirred for 1 h at room temperature. The solvent was
removed in vacuo at room temperature, ether was added, and the
mixture was centrifuged. The obtained solid product was crystal-
lized from a proper solvent (Table 5).
5.1.8. (2RS,4R)-2-(3-Aryl-1-phenyl-1H-pyrazole-4-yl)-3-(ter-
butyloxycarbonyl)-thiazolidine-4-carboxamide (9a,b)
To a solution of the selected acid 7a,b (10 mmol) in DMF (15 ml);
HOBT$H2O (1.84 g, 12 mmol) and DCC$HCl (2.48 g, 12 mmol) were
added while stirring at 0 ꢃC, and the mixture was further stirred at
0 ꢃC for 1 h. Ammonium hydroxide solution (28%, 1.56 ml,40 mmol)
was added and the mixture was stirred over night at room
temperature. The mixture was filtered, the filtrate was evaporated
under vacuum, and the residue was crystallized from an appro-
priate solvent (Table 5).
IR (cmꢀ1), 10a: 3344, 3186, 3158 (NH2, NH), 1686 (C]O), 1632
(C]N). 1H NMR (CDCl3), 10a:
d 3.21e3.77 (m, 2H, thiazol-C5H), 4.48
(t, J ¼ 7.80 Hz, 1H, thiazol-C4H), 6.09 (s, 0.5H, thiazol-C2H), 6.15 (s,
0.5H, thiazol-C2H), 7.33e7.73 (m,11H, NH, phenyl-H), 8.29 (br. s, 2H,
NH2, D2O exchangeable), 8.86 (s, 1H, pyrazole-C5 H). 13C NMR
(CDCl3), 10a: 36.1, 60.2, 71.6, 117.4, 120.1, 123.3, 126.6, 127.4, 128.7,
129.2, 129.6, 133.2, 140.1, 149.7, 177.8.
IR (cmꢀ1), 9a: 3352, 3194 (NH2), 1736 (C]O), 1682 (C]O), 1633
(C]N), 1211, 1138 (CeOeC). 1H NMR (CDCl3), 9a:
d
1.41 (s, 9H, 3
IR (cmꢀ1), 10b: 3339, 3192, 3166 (NH2, NH), 1683 (C]O), 1635
(CH3)), 3.21e3.75 (m, 2H, thiazol-C5H), 4.55 (t, J ¼ 7.80 Hz, 1H,
thiazol-C4H), 6.07 (s, 0.5H, thiazol-C2H), 6.16 (s, 0.5H, thiazol-C2H),
7.28e7.74 (m, 10H, phenyl-H), 8.31 (br. s, 2H, NH2, D2O exchange-
able), 8.89 (s, 1H, pyrazole-C5 H).
(C]N). 1H NMR (CDCl3), 10b:
d 2.35 (s, 3H, CH3), 3.23e3.74 (m, 2H,
thiazol-C5H), 4.55 (t, J ¼ 7.80 Hz, 1H, thiazol-C4H), 6.07 (s, 0.5H,
thiazol-C2H), 6.16 (s, 0.5H, thiazol-C2H), 7.31e7.68 (m, 6H, NH,
phenyl-H), 7.73 (d, 2H, J ¼ 7.78 Hz, tolyl-C3,5H), 7.82 (d, 2H,

6036
A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
Table 5
Physical and analytical data of compounds 2e10.
Comp. No.
R
Yield %
MP (ꢃC)
Mol. Formula
(Mol. wt)
Elemental analysis %, Calcd/Found
Cryst. Solvent
C%
H%
N%
S%
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
H
83
84
85
82
72
76
72
77
84
89
87
85
73
76
81
77
75
76
243e241
(EtOH)
258e259
(EtOH)
252e254
(EtOH)
262e264
(EtOH)
266e268
(EtOH/H2O)(6:1)
273e275
(EtOH/H2O)(6:1)
259e260
(EtOH/H2O)(6:1)
268e269
(EtOH/H2O)(6:1)
279e281
(EtOH/H2O)(6:1)
291e293
(EtOH/H2O)(6:1)
257e258
(ethyl acetate/n-hexane) (1:1)
284e185
ethyl acetate/n-hexane) (1:1)
250e252
ethyl acetate/n-hexane) (1:1)
268e269
ethyl acetate/n-hexane) (1:1)
285e286
(EtOH/H2O)(6:1)
291e292
(EtOH/H2O)(5:1)
257e258
(EtOH/H2O)(6:1)
276e278
C16H13N3O
(263.29)
C17H15N3O
(277.32)
C16H11N3
(245.27)
C17H13N3
(259.30)
72.99
72.64
73.67
73.42
78.35
78.51
78.74
8.92
4.94
5.11
5.45
5.61
4.52
4.29
5.05
4.83
4.95
5.16
5.36
5.53
5.10
4.92
5.46
5.28
4.69
4.38
5.00
4.78
5.58
5.21
5.85
5.59
4.88
4.61
5.24
5.52
5.82
5.68
6.07
6.32
5.18
4.92
5.53
5.78
15.96
16.21
15.15
14.92
17.13
16.92
16.20
15.89
13.76
13.85
13.15
12.87
11.82
12.01
11.37
11.57
9.78
9.92
9.47
9.28
9.31
9.53
9.03
8.87
11.96
12.18
11.50
11.28
12.44
12.65
12.06
11.84
15.99
16.21
15.37
15.12
CH3
H
CH3
H
C
18H15N3S
(305.34)
19H17N3S
(319.42)
23H18FN3
70.79
70.92
71.44
71.62
77.73
77.52
78.03
77.86
69.91
70.15
70.41
70.64
63.84
63.62
64.50
64.80
64.94
65.22
65.73
65.50
63.98
64.24
64.63
64.37
65.12
64.86
65.91
66.23
10.50
10.21
10.04
9.84
CH3
H
C
C
(355.40)
C24H20FN3
(369.41)
CH3
H
C
25H20FN3OS
(429.51)
26H22FN3OS
7.47
7.61
7.23
6.92
7.10
6.86
6.89
7.01
9.12
8.81
8.77
8.98
7.12
6.87
6.90
7.20
9.15
9.42
8.80
8.51
CH3
H
C
(443.53)
C24H25N3O4S
(451.54)
C25H27N3O4S
(465.56)
CH3
H
C
19H17N3O2S
(351.42)
C20H19N3O2S
(365.45)
CH3
H
C
24H26N4O3S
(450.55)
C25H28N4O3S
(464.58)
CH3
H
C
19H18N4OS
(350.44)
20H20N4OS
(364.14)
CH3
C
(EtOH/H2O)(6:1)
J ¼ 7.78 Hz, tolyl-C2,6H), 8.30 (br. s, 2H, NH2, D2O exchangeable),
8.89 (s, 1H, pyrazole-C5 H). 13C NMR (CDCl3), 10b: 24.6, 36.3, 59.8,
71.3, 117.5, 120.3, 123.1, 126.4, 127.5, 129.4, 129.7, 130.3, 138.2, 139.8,
149.5, 177.5.
granuloma from control value was also calculated. The ED50 values
were determined through dose response curves using doses of 4, 7,
10 and 15 mmol for each compound.
5.3. Carrageenan-induced rat paw edema
5.2. Anti-inflammatory activity
Male albino rats weighing 120e150 g (Medical Research Insti-
tute, Alexandria University) were used throughout the work. They
were kept in the animal house under standard conditions of light
and temperature with free access to food and water. The animals
were randomly divided into groups of six rats each. The paw edema
was induced by subplantar injection of 50 ml of 2% carrageenan in
saline solution (0.9%). Indomethacin and the test compounds were
dissolved in DMSO and were injected subcutaneously in a dose of
5.2.1. Cotton pellet-induced granuloma bioassay
Adult male SpragueeDawley rats (120e140 g) obtained from
Medical Research Institute, Alexandria University. The rats were
acclimated one week prior to use and allowed unlimited access to
standard rat chow and water. Prior to the start of experiment, the
animals were randomly divided into groups of six rats each. Cotton
pellet (35 ꢁ 1 mg) cut from dental rolls were impregnated with
10 mmol/kg body weight, 1 h prior to carrageenan injection. Control
0.2 ml (containing 10 mmol) of a solution of the test compound in
group was injected with DMSO only. The volume of paw edema
(ml) was determined by means of plethysmometer immediately
after injection of carrageenan and 4 h later. The increase in paw
volume between time 0 and 4 h was measured as described earlier
[27]. The percentage protection against inflammation was calcu-
lated as follows:
chloroform and the solvent was allowed to evaporate. Each cotton
pellet was subsequently injected with 0.2 ml of an aqueous solution
of antibiotics (1 mg penicillin G and 1.3 mg dihydrostreptomycin/
ml). Two pellets were implanted subcutaneously, one in each axilla
of the rat, under mild general anesthesia. One group of animals
received the standard reference indomethacin and the antibiotics
at the same level. Pellets containing only the antibiotics were
similarly implanted in the control rats. Seven days later, the animals
were sacrificed and the two cotton pellets, with adhering granu-
lomas, were removed, dried for 48 h at 60 ꢃC and weighed. The
increment in dry weight (difference between the initial and final
weights) was taken as a measure of granuloma. This was calculated
for each group and the percentage reduction in dry weight of
Vc ꢀ Vd
ꢂ 100
Vc
where Vc is the increase in paw volume in the absence of test
compound (control) and Vd is the increase in paw volume after
injection of the test compound. Data were expressed as the
mean ꢁ SEM. Significant difference between the control and the

A.A. Bekhit et al. / European Journal of Medicinal Chemistry 45 (2010) 6027e6038
6037
treated groups was performed using one way ANOVA. The differ-
ence in the means was considered significant at P < 0.001 using
Tukey’s multiple comparison test. The anti-inflammatory activity of
the test compounds relative to that of indomethacin was also
calculated.
in doses of 5, 10, 20, 40, 80 mg/kg. The percentage survival was
followed up to 7 days [21].
5.7. Docking studies
Computer simulated docking experiments were carried out
under an MMFF94X force field in (PDB ID: 1CX2) using chemical
computing group’s Molecular Operating Environment (MOE-Dock
2005) software, Montréal, Canada.
5.4. Human COX-1 and COX-2 enzymatic assay
Human COX-1 and COX-2 activities were determined according
to Wakitani et al. [40]. Human COX-1 (0.3 mg protein/assay) or
COX-2 (1 mg protein/assay) was suspended in 0.2 ml of 100 mmol
Tris/HCl buffer (pH 8) containing hematin (2 mmol) and tryptophan
(5 mmol) as cofactors. The reaction mixture was pre-incubated
with each test compound individually for 5 min at 24 ꢃC. [14C]-
Arachidonic acid (100.00 dpm, 30 mmol) was added to the mixture
and then incubated for 2 min (for COX-1) or 45 min (for COX-2) at
5.8. Docking of human COX-2
The coordinate from the X-ray crystal structure of human COX-2
used in this simulation was obtained from the Protein Data Bank
(PDB ID: 1CX2), where the selective COX-2 inhibitor SC-588 is
bound to the active site. The ligand molecules were constructed
using the builder module and were energy minimized. The active
site of COX-2 was generated using the MOE-Alpha Site Finder, and
then ligands were docked within this active site using the MOE-
Dock. The lowest energy conformation was selected and the ligand
interactions (hydrogen bonding and hydrophobic interaction) with
COX-2 were determined.
24 ꢃC. The reaction was stopped by addition of 400
ml of a solution
composed of Et2O/MeOH/1 M Citric acid (30:4:1, v/v/v). After
centrifugation of the mixture at 1700 ꢂ g for 5 min at 4 ꢃC, 50
ml of
the upper phase was applied to a thin layer chromatography plate.
Thin layer chromatography was performed at 4 ꢃC with solvent
system consisting of Et2O/MeOH/AcOH (90:2:0.1, v/v/v). Enzyme
activity was calculated from the percent conversion of arachidonic
acid to PGH2 and its decomposition products, using radiometric
photographic system. The concentration of the compound causing
50% inhibition (IC50) was calculated.
5.9. Docking of DNA-gyrase B
The coordinate from the X-ray crystal structure of DNA-gyrase B
used in this simulation was obtained from the Protein Data Bank
(PDB ID: 1EI1). The ligand molecules were constructed using the
builder module and were energy minimized. The active site of
DNA-gyrase B was generated using the MOE-Alpha Site Finder, and
then ligands were docked within this active site using the MOE-
Dock. The lowest energy conformation was selected and the ligand
interactions (hydrogen bonding and hydrophobic interaction) with
DNA-gyrase B were determined.
5.5. Ulcerogenic effects
The most active compounds 8b, 10a and 10b were evaluated for
their ulcerogenic potential in rats. Indomethacin was used as
reference standard. Male albino rats (100e120 g) were fasted for
12 h prior to administration of the compounds. Water was given
ad libitum. The animals were divided into groups of six rats each.
Control group received 1% gum acacia orally. Other groups received
indomethacin or test compounds orally in two equal doses at 0 and
5.10. Antimicrobial activity
12 h for three successive days at a dose of 30 mmol/kg body weight
per day. Animals were sacrificed by diethyl ether 6 h after the last
dose and the stomach was removed. An opening at the greater
curvature was made and the stomach was cleaned by washing with
cold saline and inspected with a 3X magnifying lens for any
evidence of hyperemia, hemorrhage, definite hemorrhagic erosion
or ulcer. An arbitrary scale was used to calculate the ulcer index
which indicates the severity of stomach lesions. The percentage
ulceration for each group was calculated as follows:
The micro-dilution susceptibility test in Müller-Hinton Broth
(oxoid) and Sabouraud Liquid Medium (oxoid) were used for
determination of antibacterial and antifungal activities. Test
organisms were E. coli (E. coli) ATCC 25922 as an example of Gram-
negative bacteria, S. aureus (S. aureus) ATCC 19433 as an example of
Gram-positive bacteria and C. albicans (C. albicans) as yeast like
fungus. Ampicillin trihydrate and clotrimazole were used as stan-
dard antibacterial and antifungal agents, respectively. Solutions of
the test compounds, ampicillin trihydrate and clotrimazole were
Number of animals bearing ulcer in a group
% Ulceration ¼
prepared in DMSO at concentration of 1600
solution, two-fold dilutions of the compounds (800, 400, .
6.25 g/ml) were inoculated to the corresponding wells. Plates
mg/ml. From this stock
Total number of animals in the same group
m
ꢂ 100
were incubated at 36 ꢃC for 24e48 h, with the incubation chamber
kept sufficiently humid. At the end of the incubation period, the
minimal inhibitory concentrations (MIC) were determined.
Controls with DMSO and uninoculated media were run parallel to
the test compounds under the same conditions.
5.6. Acute toxicity
The oral acute toxicity of compounds 8b, 10a and 10b was
investigated using male mice (20 g each, Medical Research Insti-
tute, Alexandria University) according to previously reported
methods. The animals were divided into groups of six mice each.
The compounds were given orally, suspended in 1% gum acacia, in
doses of 1, 5, 10, 50, 150 mg/kg. The mortality percentage in each
group was recorded after 24 h. Additionally the test compounds
were investigated for their parenteral acute toxicity in groups of
mice of six animals each. The compounds or their vehicle,
propylene glycol (control), were given by intraperitoneal injection
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