Design, synthesis and biological evaluation of some pyrazole derivatives as anti-inflammatory-antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2004.01.037

The synthesis of novel series of structurally related 1H-pyrazolyl derivatives is described. All the newly synthesized compounds were tested for their in vivo anti-inflammatory activity by two different bioassays namely; cotton pellet-induced granuloma and sponge implantation model of inflammation in rats. In addition, COX-1 and COX-2 inhibitory activities, ulcerogenic effects and acute toxicity were determined. The same compounds were evaluated for their in vitro antimicrobial activity against Escherichia coli, as an example of Gram negative bacteria, Staphylococcus aureus as an example of Gram positive bacteria, and Candida albicans as a representative of fungi. The combined anti-inflammatory data from local and systemic in vivo animal models showed that compounds 4, 5, 8, 9, 11 and 12a exhibited anti-inflammatory activity comparable to that of indomethacin with no or minimal ulcerogenic effects and high safety margin (LD₅₀>500 mg/Kg). In addition, compounds 4, 7, 10, 12a and 12b displayed appreciable antibacterial activities when compared with ampicillin, especially against S. aureus. Compounds 4 and 12a are the most distinctive derivatives identified in the present study because of their remarkable in vivo and in vitro anti-inflammatory potency and their pronounced antibacterial activities comparable to ampicillin against Gram positive. On the other hand, compound 12a exhibited good selective inhibitory activity against COX-2 enzyme. Therefore, such compound would represent a fruitful matrix for the development of anti-inflammatory-antimicrobial candidates.

Full text of DOI:10.1016/j.bmc.2004.01.037

Bioorganic & Medicinal Chemistry 12 (2004) 1935–1945
Design, synthesis and biological evaluation of some pyrazole
derivatives as anti-inflammatory-antimicrobial agents
Adnan A. Bekhita,* and Tarek Abdel-Aziemb
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria 21521,
Egypt
bDepartment of Pharmacology, Faculty of Medicine, University of Alexandria, Alexandria 21521,
Egypt
Received 17 June 2003; accepted 23 January 2004
Abstract—The synthesis of novel series of structurally related 1H-pyrazolyl derivatives is described. All the newly synthesized
compounds were tested for their in vivo anti-inflammatory activity by two different bioassays namely; cotton pellet-induced gran-
uloma and sponge implantation model of inflammation in rats. In addition, COX-1 and COX-2 inhibitory activities, ulcerogenic
effects and acute toxicity were determined. The same compounds were evaluated for their in vitro antimicrobial activity against
Escherichia coli, as an example of Gram negative bacteria, Staphylococcus aureus as an example of Gram positive bacteria, and
Candida albicans as a representative of fungi. The combined anti-inflammatory data from local and systemic in vivo animal models
showed that compounds 4, 5, 8, 9, 11 and 12a exhibited anti-inflammatory activity comparable to that of indomethacin with no or
minimal ulcerogenic effects and high safety margin (LD50>500 mg/Kg). In addition, compounds 4, 7, 10, 12a and 12b displayed
appreciable antibacterial activities when compared with ampicillin, especially against S. aureus. Compounds 4 and 12a are the most
distinctive derivatives identified in the present study because of their remarkable in vivo and in vitro anti-inflammatory potency and
their pronounced antibacterial activities comparable to ampicillin against Gram positive. On the other hand, compound 12a
exhibited good selective inhibitory activity against COX-2 enzyme. Therefore, such compound would represent a fruitful matrix for
the development of anti-inflammatory-antimicrobial candidates.
# 2004 Elsevier Ltd. All rights reserved.
1. Introduction
zenesulfonamide (Fig. 1); occupies a unique position as
a potent and GI safe anti-inflammatory and analgesic
agent. It is considered as a typical model of the diaryl
heterocycle template that is known to selectively inhibit
the COX-2 enzyme.1
The prescription of co-administration of multiple drugs
for treatment of inflammatory conditions associated
with some microbial infections may inflect added health
problems especially in patients with impaired liver or
kidney functions. A mono therapy of an anti-inflamma-
tory drug with antimicrobial properties will be better from
the pharmaco-economic point, this will enhance patient
compliance and in case that this anti-inflammatory- anti-
microbial agent shows minimum adverse effects and high
safety margin, this drug will be highly desirable.
On the other hand, much attention has been focused
towards pyrazoles as antimicrobial,2,3 antiviral4,5 and
anticancer6,7 agents after the discovery of the natural
pyrazole C-glycoside, pyrazofurin; 4-hydroxy-3-b-d-
ribofuranosyl-1H-pyrazole-5-carboxamide (Fig. 1). This
antibiotic was reported to possess a broad spectrum of
antimicrobial and antiviral activities in addition to
being active against several tumor cell lines.8
Among the already marketed COX-2 inhibitors that
comprise the pyrazole nucleus, celecoxib; 4-[5-(4-
methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]ben-
Motivated by the afore-mentioned findings, and as a
continuation of our on going program in the field of
pyrazoles as anti-inflammatory- antimicrobial agent,9ꢀ15
it was designed to synthesise novel series of pyrazole
derivatives (A & B) that would act as dual anti-
microbial- non-acidic anti-inflammatory agents with
Keywords: 1H-pyrazoles; Anti-inflammatory activity; COX inhibitory
activities; Ulcerogenic effect; Acute toxicity; Antimicrobial activity.
* Corresponding author. Tel.: +20-3-4871317; fax: +20-3-4873273;
e-mail: adnbekhit@hotmail.com
0968-0896/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.01.037

1936
A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
pyrazolin-5-yl]-1-phenyl-1H-pyrazole 8, 3-(5-bromo-2-
thienyl)- 4-[1 - methanesulfonyl- 3-(4 - methylphenyl)-2-
pyrazolin-5-yl]-1-phenyl-1H-pyrazole 9 or 3-(5-bromo-
2-thienyl)-4-[1-(4-methylphenylsulfonyl)-3-(4-methyl-
phenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole 10. It is
worth-mentioning that heating the intermediate 2 with
hydrazine hydrate in boiling ethanol for 6 h afforded the
pyrazole derivative 11, namely; 3-(5-bromo-2-thienyl)-4-
[3-(4-methylphenyl) - 1H - pyrazol - 5 - yl] - 1 - phenyl-1H-
pyrazole, which is the oxidation product of 5.
Compound 4 was also obtained by acetylation of com-
pound 5. Moreover, compound 11 could also obtained
by oxidation of compound 5 using bromine.
On the other hand, compounds 12a,b, namely; 3-(5-
bromo-2-thienyl)-4-[1-aryl-3-(4-methylphenyl)-1H-pyra-
zol-5-yl]-1-phenyl-1H-pyrazole; were obtained in excel-
lent yields by cyclizing the a,b-unsaturated ketone 2
with phenylhydrazine or p-tolylhydrazine respectively.
Figure 1. Structures of celecoxib, pyrazofurin and the novel series of
pyrazole A and B.
3. Results and discussion
minimal GI side effects and high safety margin. The
substitution pattern of the pyrazole ring was rational-
ised so as to be correlated to the diaryl heterocycles
template of compounds that are known to act selectively
as COX-2 inhibitors such as celecoxib. It should be
pointed out that, in addition to the targeted anti-
inflammatory and antimicrobial activities, the ulcero-
genic and acute toxicity profiles of the newly synthesised
compounds were determined.
3.1. Anti-inflammatory activity
3.1.1. Cotton pellet-induced granuloma bioassay. The
anti-inflammatory activity of the synthesised compounds
2–11 and 12a,b was evaluated by applying the cotton-
pellet granuloma bioassay in rats16 using indomethacin
as a reference standard. The ED50 values were deter-
mined for each compound (Table 1).
Out of the compounds tested, compound 12a displayed
anti-inflammatory activity (ED50 8.97 mmol) compar-
able to that of indomethacin (ED50 9.17 mmol); (Table
1). Compounds 8, 9, 11 (ED50 9.56, 9.57 and 9.23 mmol);
were nearly equipotent with indomethacin at the same
dose level and test conditions. Furthermore, compounds
4 and 5 exhibited moderate anti-inflammatory activity
(Table 1). The rest of the compounds were found to be
weakly active or inactive as anti-inflammatory agents
(ED50 values range 13.00–17.46 mmol).
2. Chemistry
Synthesis of the intermediate and target compounds
were performed by the reactions illustrated in Figure 2.
Compound 2, namely; 3-(5-bromo-2-thienyl)-4-[3-(4-
methylphenyl)-3-oxopropenyl]-1-phenyl-1H-pyrazole;
was synthesised in an excellent yield by condensing 3-(5-
bromo-2-thienyl)-1-phenyl-1H-pyrazole-4-carboxalde-
hyde10 1 with 4-methylacetophenone in the presence of
potassium hydroxide. This a,b-unsaturated ketone 2
was converted to the 3-(5-bromo-2-thienyl)-4-[1-formyl-
3-(4-methylphenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyra-
zole 3 by the reaction with hydrazine hydrate in boiling
formic acid. Analogously, heating 2 with hydrazine
hydrate in boiling acetic acid yielded the corresponding
3-(5-bromo-2-thienyl)-4-[1-acetyl-3-(4-methylphenyl)-2-
pyrazolin-5-yl]-1-phenyl-1H-pyrazole 4. Furthermore,
the target compound 5; 3-(5-bromo-2-thienyl)-4-[3-(4-
methylphenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole;
was obtained by cyclizing the intermediates 2 with
hydrazine hydrate in boiling ethanol for 30 min. The
pyrazoline derivative 5 was allowed to react with phenyl
isothiocyanate, NaNO2, benzoyl chloride, methane-
sulfonyl chloride or 4-toluenesulfonyl chloride to give
rise to 3-(5-bromo-2-thienyl)-4-[1-phenylthiocarbamoyl-
3-(4-methylphenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyra-
zole 6, 3-(5-bromo-2-thienyl)-4-[1-nitroso-3-(4-methyl-
phenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole 7, 3-(5-
bromo-2-thienyl)-4-[1-benzoyl-3-(4-methylphenyl)-2-
Table 1. The anti-inflammatory activity (ED50, mmol), ulcerogenic
effects and LD50 values of the test compounds
Test compound
ED50 (mmol)
% Ulceration
LD50 (mg/kg)
—
Control
Phenylbutazone
—
—
0.0
60
Indomethacin
2
3
4
5
6
7
8
9
10
11
12a
12b
P
9.17ꢁ1.19
17.46ꢁ1.25
12.64ꢁ1.55
11.75ꢁ1.27
10.10ꢁ1.08
13.68ꢁ1.10
14.82ꢁ1.40
9.56ꢁ0.94
9.57ꢁ1.18
15.76ꢁ1.49
9.23ꢁ1.01
8.97ꢁ0.87
14.82ꢁ1.14
<0.001
100
NTa
10
—
NT
>500
>500
>500
NT
10
0.0
NT
NT
10
0.0
NT
10
NT
>500
>500
NT
>500
>500
NT
0.0
NT
a NT=not tested.

A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
1937
3.2. Sponge implantation model of inflammation in rats
will change its light absorption properties, as detected
spectrophotometrically.
The anti-inflammatory activity of the newly synthesised
compounds 2–11 and 12a,b was further evaluated in
vivo using the sponge implantation model of inflamma-
tion in rats with indomethacin as a standard. Polyester
sponge implantation, the animal model chosen in this
study, has the merit of triggering a non-immune acute
type of inflammatory response. This model was used to
assess the possibility of some of the synthesised
compounds altering the course of inflammation and
they have been claimed to potentially modulate inflam-
mation. This was assessed by determining their effects
on the inflammatory exudate parameters: exudate
volume, Total Leucocyte Cell Count (TLC), Differential
Leucocyte Cell Count (DLC), reduced cytochrome C
and interleukin-1 beta (IL-1b) levels. Indomethacin was
used as a standard anti-inflammatory drug (Tables 2
and 3).
The superoxide radical (O2.ꢀ) is a highly reactive inter-
mediate produced in biological system by one-electron
reduction of oxygen. It is generated by phagocytes dur-
ing the respiratory burst. It is an intermediate in the
formation of H2O2 and HOCl which are considered
powerfull antimicrobial agents. The detection of O2.ꢀ is
based on its ability to reduce cytochrome C.
3.3. Effect on exudate volume
Results presented in Table 2 revealed that pre-treatment
with indomethacin significantly reduced the exudate
volume in comparison to the inflammatory control
group( p<0.001). The mean values were 0.236ꢁ0.012
and 0.362ꢁ0.014 mL respectively.
Pre-treatment with the investigated compounds 3, 4, 5, 8,
9, 11 and 12a significantly reduced the exudate volume as
compared to the inflammatory control group (p<0.001).
The mean values were 0.238ꢁ0.013, 0.282ꢁ0.014,
0.256ꢁ0.012, 0.274ꢁ0.015, 0.251ꢁ0.014, 0.251ꢁ0.015
and 0.244ꢁ0.013 mL respectively, compared to a control
mean value of 0.362ꢁ0.014 mL. On the other hand, pre-
treatment with the investigated compounds 2, 6, 7, 10
and 12b did not significantly alter the exudate volume
compared to the inflammatory control group (p>0.05).
The neutrophil phagocytic function was determined
as one of the most important indices used to assess
the different neutrophilic functions. This is because
it is the ultimate and major stepin neutrohpilic
response to acute inflammation. The main step in
this function is the respiratory burst, that reflects an
important face of immune system integrity. This was
evaluated by the cytochrome C reduction test, where
the reduction of cytochrome C by superoxide anion
Figure 2. Synthesis of the intermediate and target compounds.

1938
A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
3.4. Effect on total leucocyte cell count (TLC)
3.5. Effect on differential leucocyte cell count (DLC)
Results presented in Table 2 showed that pre-treatment
with indomethacin significantly reduced the exudate
TLC, as compared to the inflammatory control group
(p<0.001). The mean value was 126.60ꢁ14.28 compared
to a control mean value of 274.40ꢁ15.22 cell/cm3. Pre-
treatment with the investigated compounds 3, 4, 5, 8, 9,
11 and 12a significantly reduced the exudate TLC as
compared to the inflammatory control group
(P<0.001). The mean values were 138.40ꢁ12.56,
192.80ꢁ15.26, 180.80ꢁ14.16, 192.50ꢁ13.68, 146.90
ꢁ13.82, 136.80ꢁ13.24 and 148.40ꢁ12.62 cell/cm3
respectively, compared to a control mean value of
276.40ꢁ15.22 cell/cm3. However, pre-treatment with
the investigated compounds 2, 6, 7, 10 and 12b did not
significantly reduce the exudate TLC, in comparison to
the control group( p>0.05).
Results presented in Table 2 showed that pre-treatment
with indomethacin significantly reduced the exudate
neutrophil% as compared to the inflammatory control
group( p<0.001). The mean value was 56.60ꢁ2.26%
compared to a control mean value of 83.40ꢁ3.16%.
Pre-treatment with the investigated compounds 3, 4,
5, 8, 9, 11 and 12a significantly reduced the exudate
neutrophil% as compared to the inflammatory control
group( p<0.001). The mean value were 56.40ꢁ1.98,
63.60ꢁ2.16, 60.20ꢁ2.52, 62.40ꢁ2.48, 59.80ꢁ2.64,
59.20ꢁ2.24 and 63.40ꢁ2.36%, compared to a control
mean value of 83.40ꢁ3.16%. However, pre-treatment
with the investigated compounds 2, 6, 7, 10 and 12b did
not significantly alter the exudate neutrophil%, as
compared to the control group (p>0.05).
3.6. Effect on neutrophil phagocyte function
Table 2. The effect of inflammation induced by sponge implantation,
in drug pre-treated rats (meanꢁS.E., n=10) on non-immunological
parameters
The neutrophil phagocyte function was measured using
the cytochrome C reduction test. In this test, both the
basal level of reduced cytochrome C (unstimulated), as
well as its level after stimulation of granulocytes by
means of Zymosan particles (stimulated) were esti-
Test compd Exudate volumea
TLCb
DLCc(neutrophil%)
X(ꢁS.E.)
X(ꢁS.E.)
X(ꢁS.E.)
mated. The results were expressed as nmol O.ꢀ2
/
Control
0.362(ꢁ0.014) 276.40(ꢁ15.22)
83.40(ꢁ3.16)
56.60(ꢁ2.26)
72.40(ꢁ2.64)
56.40(ꢁ1.98)
63.60(ꢁ2.16)
60.20(ꢁ2.52)
76.40(ꢁ3.12)
81.20(ꢁ2.58)
62.40(ꢁ2.48)
59.80(ꢁ2.64)
68.40(ꢁ2.82)
59.20(ꢁ2.24)
63.40(ꢁ2.36)
71.20(ꢁ2.14)
<0.001
2ꢂ106 PMN/h. Results presented in Table 3 showed
that pre-treatment with indomethacin significantly
reduced the unstimulated reduced cytochrome C level
as compared to the inflammatory control group
(p<0.001). The mean value was 0.74ꢁ0.02 compared to
a control mean value of 1.42ꢁ0.02. Similarly, the
results showed that pre-treatment with the investigated
compounds 3, 4, 5, 6, 8, 9, 10, 11, 12a and 12b sig-
nificantly reduced the cytochrome C level as compared
to the inflammatory control group( p<0.001). The
mean values were 0.82ꢁ0.04, 0.87ꢁ0.04, 0.79ꢁ0.04,
Indomethacin 0.236(ꢁ0.012) 126.60(ꢁ14.28)
2
3
4
5
6
7
8
9
10
11
12a
12b
P
0.312(ꢁ0.016) 252.20(ꢁ13.24)
0.238(ꢁ0.013) 138.40(ꢁ12.56)
0.282(ꢁ0.014) 192.80(ꢁ15.26)
0.256(ꢁ0.012) 180.80(ꢁ14.16)
0.336(ꢁ0.013) 240.40(ꢁ13.62)
0.321(ꢁ0.016) 262.30(ꢁ16.24)
0.274(ꢁ0.015) 192.50(ꢁ13.68)
0.251(ꢁ0.014) 146.90(ꢁ13.82)
0.336(ꢁ0.013) 202.60(ꢁ14.26)
0.251(ꢁ0.015) 136.80(ꢁ13.24)
0.244(ꢁ0.013) 148.40(ꢁ12.62)
0.347(ꢁ0.014) 248.20(ꢁ12.20)
<0.001
<0.001
0.92ꢁ0.02,
0.72ꢁ0.02,
0.86ꢁ0.03,
0.94ꢁ0.01,
0.81ꢁ0.03, 0.84ꢁ0.03 and 0.98ꢁ0.01 respectively,
compared to a control value of 1.42ꢁ0.02. However,
pre-treatment with the investigated compounds 2 and 7
a Exudate volume (expressed in mL).
b Total leucocytic count (TLC) (expressed as cell/cm3).
c Differential leucocytic count (DLC) (expressed as exudate neutrophil%).
Table 3. The effect of inflammation induced by sponge implantation, in drug pre-treated rats (meanꢁS.E., n=10) on the immunological
parametersa,b
Test compd
Unstimulated reduced
cytochrome C level X(ꢁS.E.)
Stimulated reduced
cytochrome C level X(ꢁS.E.)
Unstimulated IL-1b level
X(ꢁS.E.)
LPS stimulated IL-1b level
X(ꢁS.E.)
Control
Indomethacin
2
3
4
5
6
7
8
1.422(ꢁ0.018)
0.742(ꢁ0.016)
1.17(ꢁ0.02)
0.82(ꢁ0.04)
0.87(ꢁ0.04)
0.79(ꢁ0.04)
0.92(ꢁ0.02)
1.36(ꢁ0.04)
0.72(ꢁ0.02)
0.86(ꢁ0.03)
0.94(ꢁ0.01)
0.81(ꢁ0.03)
0.84(ꢁ0.03)
0.98(ꢁ0.01)
<0.001
1.522(ꢁ0.036)
0.868(ꢁ0.022)
1.29(ꢁ0.02)
0.96(ꢁ0.05)
1.11(ꢁ0.03)
0.89(ꢁ0.04))
1.24(ꢁ0.05)
1.42(ꢁ0.03)
1.04(ꢁ0.07)
0.98(ꢁ0.02)
1.21(ꢁ0.03)
0.89(ꢁ0.03)
0.97(ꢁ0.05)
0.99(ꢁ0.03)
<0.001
196.40(ꢁ15.42)
102.20(ꢁ13.26)
172.60(ꢁ12.34)
116.40(ꢁ14.24)
122.80(ꢁ13.28)
134.40(ꢁ14.32)
182.40(ꢁ14.62)
172.20(ꢁ16.28)
145.40(ꢁ14.28)
116.30(ꢁ14.42)
160.60(ꢁ15.24)
114.40(ꢁ12.38)
138.40(ꢁ13.42)
185.30(ꢁ14.20)
<0.001
281.60(ꢁ16.24)
126.20(ꢁ13.22)
260.40(ꢁ12.40)
146.40(ꢁ12.82)
178.40(ꢁ14.28)
179.40(ꢁ14.42)
268.20(ꢁ13.24)
262.40(ꢁ15.34)
161.80(ꢁ13.68)
138.40(ꢁ14.66)
206.80(ꢁ13.98)
138.40(ꢁ14.12)
176.30(ꢁ13.62)
265.20(ꢁ15.30)
<0.001
9
10
11
12a
12b
P
a Neutrophil Phagocyte Function (expressed in nmol O2/2ꢂ106 PMN/h) on both unstimulated and stimulated reduced cytochrome C level.
b The assay of Interleukin-1b (IL-1b) (expressed in pg/mL) on both unstimulated IL-1b level and LPS stimulated IL-1b level.

A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
1939
did not significantly reduce the unstimulated reduced
cytochrome C level as compared to the control group
(p>0.05).
The combined anti-inflammatoty results showed that it
is not easy to draw structure–activity relationship.
However, The types of the newly synthesised ring sys-
tem whether pyrazoline (compounds 3, 4, 5, 8, 9) or
pyrazole (compounds 11, 12a) did not much affect the
anti-inflammatory activity. In addition, substitution of
N1of the pyrazoline or pyrazole ring has only a minor
influence on the activity. When N1 is substituted by
formyl (3), acetyl (4), phenylthiocarbamoyl (6), nitroso
(7), 4-methylbenzenesulfonyl (10) or 4-methylphenyl
(12b), the recorded anti-inflammatoty activity was
found to be less than that of indomethacin. Whereas
compounds unsubstituted on N1 (5, 11), N1-benzoyl (8),
N1-methanesulfonyl (9) or N1-phenyl (12a), showed
anti-inflammatoty activity comparable to that of indo-
methacin.
The results presented in Table 3 also showed that pre-
treatment with indomethacin significantly reduced the
stimulated cytochrome C level as compared to the
inflammatory control group( p<0.001). The mean value
was 0.87ꢁ0.02 compared to control value of
1.52ꢁ0.04. Pre-treatment with the investigated com-
pounds 3, 4, 5, 8, 9, 11, 12a and 12b significantly
decreased the stimulated reduced cytochrome C level in
comparison to the inflammatory control group
(p<0.001). Their mean value were 0.96ꢁ0.05,
1.11ꢁ0.03,
0.89ꢁ0.04,
1.04ꢁ0.07,
0.98ꢁ0.02,
0.89ꢁ0.03, 0.97ꢁ0.05 and 0.99ꢁ0.03 respectively,
compared to a control mean value of 1.52ꢁ0.04. On the
other hand, pre-treatment with the investigated com-
pounds 2, 6, 7 and 10 did not significantly alter the sti-
mulated reduced cytochrome C level as compared to the
control group( p>0.05).
3.8. Human COX-1and COX-2 enzymatic assay
Compounds 3, 4, 5, 8, 9, 11 and 12a that exhibited
moderate to potent anti-inflammatory profiles in the
pre-mentioned animal models were further tested for
their ability to inhibit human COX-2 and COX-1
enzymes in vitro appling the methodology of Wakitani
et al.17 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 Hammarstrom and Falardeau.18
COX-2 assay was performed utilising human recombi-
nant COX-2 (hrCOX-2) purchased from Sigma-Aldrich.
The concentration of the compound causing 50%
enzyme inhibition (IC50 mmol) was estimated. The
results are recorded in Table 4. The results showed that
the test compounds exhibited weak inhibitory activity
against COX-1 enzyme (IC50 values between 76.8 and
>100 mmol) when compared with indomethacin
(IC50=0.26 mmol), but several were more potent than
celecoxib (IC50=>100 mmol). In addition, the test
compounds showed higher inhibitory profile against
COX-2 when compared with indomethacin. All test
compounds showed approximate selectivity ratio
(COX-1/COX-2) lower than that of celecoxib. In gen-
eral, pyrazoline derivatives (compounds 3, 4, 5, 8, 9)
showed lower selectivity toward COX-2 enzyme than
pyrazole derivative (compound 12a), although, pyrazole
compound 11 showed low selectivity. Substitution of
3.7. Effect on interleukin-1ꢀ (IL-1ꢀ) level
The assay of Interleukin-1b using an IL-1b ELISA Kit
was done by estimating both the unstimulated level of
IL-1b [spontaneously released as well as the lipo-poly-
saccharide (LPS) stimulated release of IL-1b]. The
results were expressed in pg/mL. The results presented
in Table 3 showed that pre-treatment with indometha-
cin significantly decreased the unstimulated IL-1b level
as compared to the inflammatory control group
(p<0.001). The mean value was 102.20ꢁ13.26 com-
pared to the control value of 196.40ꢁ15.42. Pre-treat-
ment with the investigated compounds 3, 4, 5, 8, 9, 11
and 12a significantly decreased the unstimulated IL-1b
levels in comparison to the inflammatory control group
(p<0.001). The mean values were 116.40ꢁ14.24,
122.80ꢁ13.28, 134.40ꢁ14.32, 145.40ꢁ14.28, 116.30
ꢁ14.42, 114.40ꢁ12.38 and 138.60ꢁ13.42 respectively,
compared to a control mean value of 196.40ꢁ15.42. On
the other hand, pre-treatment with the investigated
compounds 2, 6, 7, 10 and 12b did not significantly alter
the unstimulated IL-1b levels as compared to the con-
trol group( p>0.05).
Results presented in Table 3 also showed that pre-
treatment with indomethacin significantly decreased the
LPS-stimulated IL-1b levels as compared to the inflam-
matory control group( p<0.001). The mean value was
126.20ꢁ13.22 compared to a control mean value of
281.60ꢁ16.24. Pre-treatment with the investigated
compounds 3, 4, 5, 8, 9, 11 and 12a significantly
decreased the LPS-stimulated IL-1b levels in compar-
ison to the inflammatory control group( p<0.001). The
mean values were 146.40ꢁ12.82, 178.40ꢁ14.28,
179.40ꢁ14.24, 161.80ꢁ13.68, 138.40ꢁ14.66, 138.40
ꢁ14.12 and 176.30ꢁ13.62 respectively, compared to
a control mean value of 281.60ꢁ16.24. However,
pre-treatment with the investigated compounds 2, 6,
7, 10 and 12b did not significantly alter the LPS-stimu-
lated IL-1b levels as compared to the control group
(p>0.05).
Table 4. In vitro human COX-2a and COX-1b enzymes inhibitory
activities of compounds 3, 4, 5, 8, 9, 11 and 12a
Test compd COX-2 IC50 COX-1 IC50 Approximate Selectivity Ratio
(mM)c
(mM)c
COX-1/COX-2
Indomethacin
Celecoxib
3
4
5
8
9
11
12a
2.63
<0.3
1.48
1.32
1.72
0.82
0.98
1.52
0.37
0.26
>100
>100
0.098
333
67.
64
44
117
102
51
84.71
76.83
96.35
>100
78.46
>100
270
a Human recombinant COX-2 enzyme.
b Human COX-1 enzyme from human platelets.
c Values are means of at least four experiments.

1940
A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
N1of the pyrazoline or pyrazole ring affects the selectiv-
ity. When N1 is substituted by benzoyl (8), methane-
sulfonyl (9) or phenyl (12a), the recorded selectivity
ratio was found higher than other test compounds, but
lower than that of celecoxib. Compound 12a is the most
selective COX-2 inhibitor (approximate selectivity
ratio=220) in the present study as compared with cel-
ecoxib (approximate selectivity ratio >333).
Table 5. Minimal inhibitory concentrations (MIC mg/mL) of test
compounds
Test compd
E. coli
S. aureus
C. albicans
2
3
4
5
6
7
8
9
10
>200
200
200
>200
100
100
>200
200
100
200
50
>200
100
25
>200
50
12.5
>200
100
50
>200
25
25
>200
>200
>200
100
>200
200
>200
>200
>200
100
>200
50
—
3.9. Ulcerogenic effects
Compounds 3, 4, 5, 8, 9, 11 and 12a that exhibited
moderate to potent anti-inflammatory profiles in the
pre-mentioned animal models were evaluated for their
ulcerogenic potential in rats.19 Phenylbutazone and
indomethacin were tested as reference drugs. All the
active compounds revealed a superior GI safety profiles
(0–10% ulceration) when compared with phenylbuta-
zone (60% ulceration) or indomethacin (100% ulcera-
tion) under the same experimental conditions (Table 1).
11
12a
12b
Ampicillin
Clotrimazole
200
25
—
12.5
—
12.5
activity as ampicillin against S. aureus. The rest of the
tested compounds were weakly active against both
organisms with MIC values ranging between 100–>200
mg/mL (Table 5). All the tested compounds showed
weak antifungal activity against C. albicans (MIC values
of 50–>200 mg/mL) when compared with the reference
antifungal agent clotrimazole (Cansten1, Bayer) (MIC
12.5 mg/mL).
Gross observation of the isolated rat stomach showed a
normal stomach texture for compounds 5, 9 and 12a
(0% ulceration), whereas the others showed slight
hyperemia (10% ulceration).
3.10. Acute toxicity
The biologically significant compounds were further
evaluated for their approximate LD50 in male mice
using a literature method.20,21 The results (Table 1)
indicated that most of the tested compounds proved to
be non-toxic and well tolerated by the experimental
animals as evidenced by their LD50 values (>500 mg/
Kg).
4. Conclusion
As the results obtained from cotton pellet-induced
granuloma bioassay are comparable to that revealed
from sponge implantation model of inflammation, one
can conclude from this study that the sponge implanta-
tion model of inflammation seemed to be a reliable
method for collection of an adequate amount of exudate
necessary for the evaluation of different inflammatory
markers. Moreover, it can be concluded from the pre-
sent study that pre-treatment with the investigated
compounds 3, 4, 5, 8, 9, 11 and 12a significantly altered
the inflammatory markers, not only, the non-immuno-
logical parameters (exudate volume, TLC and DLC),
but also the immunological parameters (reduced cyto-
chrome C and IL-1b levels). Therefore, the investigated
compounds 3, 4, 5, 8, 9, 11 and 12a were capable of
modulating the inflammatory response and were
assumed to have in vivo anti-inflammatory activity
similar to that of indomethacin. Moreover, they showed
minimum ulcerogenic activity as compared to phe-
nylbutazone and indomethacin. Meanwhile, they dis-
played distinct in vitro inhibitory activity against COX-
2 than COX-1 when compared with indomethacin and
celecoxib. Moreover, they are well tolerated by experi-
mental animals and showed high safety margin as
revealed from their LD50 values (>500 mg kgꢀ1). These
speculations remain to be further expanded in other
models of experimental inflammation and confirmed by
clinical trials before a final drug design is to be made.
On the other hand, compounds 4 and 12a showed
moderate antimicrobial activity against S. aureus. The
latter compounds would represent a fruitful matrix for
the development of a new class of dual non-acidic anti-
inflammatory-antimicrobial agents that would deserve
3.11. In vitro antimicrobial activity
Compounds 2–11 and 12a,b have been evaluated for
their in vitro antimicrobial activity against Escherichia
coli (E. coli ATCC 25922), as an example of Gram
negative bacteria, Staphylococcus aureus (S. aureus
ATCC 19433) as an example of Gram positive bacteria,
and Candida albicans (C. albicans) as a representative of
fungi. The microdilution susceptibility test in Muller-
Hinton Broth (Oxoid) and Sabouraud Liquid Medium
(Oxoid) were used for the determination of antibacterial
and antifungal activity.22 The minimal inhibitory
concentrations (MICs, mg/mL) of the tested compounds
are recorded in Table 5.
The results revealed that most of the newly synthesised
compounds exhibited promising antibacterial activities
but they showed poor antifungal activity. Generally, the
test compounds showed better activity against the Gram
positive bacteria (Table 5). Out of the compounds tes-
ted, compound 12a (MIC 50 mg/mL) exhibited moder-
ate antibacterial activity against the Gram negative E.
coli as compared with the broad spectrum antibiotic
ampicillin (MIC 25 mg/mL). In addition, compounds 4,
12a and 12b (MIC 25 mg/mL) was 50% as active as
ampicillin (MIC 25 mg/mL) against S. aureus (Table 4).
It is worth-mentioning that compound 7 showed equal

A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
1941
further investigation and derivatization. It is worth-
mentioning that compound 12a showed anti-inflamma-
tory activity profile similar to our priviously published
compound (3-phenyl-2-[3-(4-methylphenyl)-1-phenyl-
1H-pyrazol-4-methylidene-hydrazono]-5-thioxo-2,3-di-
hydrothiazolo[4,5-d]pyrimidin-7(6H)-ones),14 but with
less antimicrobial activity. In addition, compound 12a
exhibited good selectivity against COX-2 enzyme.
(cmꢀ1): 1646 (C¼O), 1622 (C=N). 1H NMR (CDCl3): d
2.40 (s, 3H, CH3), 2.94-3.03 (dd, J1=10 Hz, J2=9 Hz,
1H, pyrazoline-C-4-H), 3.34–3.44 (dd, J1=10 Hz, J2=9
Hz, 1H, pyrazoline-C-4-H), 5.08–5.16 (dd, J1=10 Hz,
J2=10 Hz, 1H, pyrazoline-C-5-H), 7.11 (d, J=3.96 Hz,
1H, thioph C-3-H), 7.21–7.77 (m, 9H, Phenyl-H ), 7.80
(d, J=3.96 Hz, 1H, thioph C-4-H), 8.43 (s, 1H, pyrazol
C-5-H)) 8.43 (s, 1H, CHO). Analysis calculated for
C24H19BrN4OS (491.404). Physical and analytical data
ꢃ
of compound 4; yield: 83%; mp208–209 C. IR (cmꢀ1):
1652 (C¼O), 1628 (C=N). 1H NMR (CDCl3): d 2.38 (s,
3H, CH3), 2.43 (s, 3H, CH3), 2.98–3.07 (dd, J1=10 Hz,
J2=9 Hz, 1H, pyrazoline-C-4-H), 3.32–3.46 (dd, J1=10
Hz, J2=9 Hz, 1H, pyrazoline-C-4-H), 5.11–5.17 (dd,
J1=10 Hz, J2=10 Hz, 1H, pyrazoline-C-5-H), 7.12 (d,
J=3.96 Hz, 1H, thioph C-3-H), 7.18-7.82 (m, 9H,
Phenyl-H), 7.83 (d, J=3.96 Hz, 1H, thioph C-4-H), 8.38
(s, 1H, pyrazol C-5-H)). Analysis calculated for
C25H21BrN4OS (505.430).
5. Experimental
5.1. Chemistry
All chemicals used in this study were purchased from E.
Merck, Fluka AG and Aldrich companies. Melting
points were determined in open-glass capillaries using
Thomas capillary melting point apparatus and are
uncorrected. The infrared (IR) spectra were recorded on
470-Shimadzu infrared spectrophotometer using the
1
KBr disc technique. The H NMR spectra were recor-
5.4. 3-(5-Bromo-2-thienyl)-4-[3-(4-methylphenyl)-2-pyra-
zolin-5-yl]-1-phenyl-1H-pyrazole (5)
ded on a Varian XL-200 MHz Spectrometer, and the
chemical shifts are given in d (ppm) down field from
tetramethylsilane (TMS) as an internal standard. Split-
ting patterns were designated as follows: s: singlet; d:
doublet; m: multiplet. Elemental analyses were per-
formed at the Microanalytical Unit, Faculty of Science,
Cairo University, Cairo, Egypt, and the found values
were within ꢁ0.4% of the theoretical values. Follow up
of the reactions and checking the homogeneity of the
compounds were made by TLC on silica gel-protected
aluminum sheets (Type 60 F254, Merck) and the spots
were detected by exposure to UV-lamp at l 254 nm for
few seconds.
A mixture of 2 (2.2g, 5 mmol) and hydrazine hydrate
(1.6 mL, 5 mmol) in ethanol (15 mL) was heated under
reflux for 30 min. The white solid product obtained on
cooling was filtered, washed with ethanol and recrys-
tallized from ethanol. Yield: 87%; mp134–136 ꢃC. IR
1
(cmꢀ1): 3380 (NH), 1630 (C=N). H NMR (CDCl3): d
2.37 (s, 3H, CH3), 2.96–3.08 (dd, J1=10 Hz, J2=9 Hz,
1H, pyrazoline-C-4-H), 3.33–3.46 (dd, J1=10 Hz, J2=9
Hz, 1H, pyrazoline-C-4-H), 5.07–5.15 (dd, J1=10 Hz,
J2=10 Hz, 1H, pyrazoline-C-5-H), 7.13 (d, J=3.96 Hz,
1H, thioph C-3-H), 7.17–7.81 (m, 10H, phenyl-H, NH ),
7.82 (d, J=3.96 Hz, 1H, thioph C-4-H), 8.44 (s, 1H,
pyrazol C-5-H)). Analysis calculated for C23H19BrN4S
(463.393).
5.2. 3 - (5 - bromo - 2 - thienyl) - 4 - [3 - (4 - methylphenyl)-3-
oxopropenyl]-1-phenyl-1H-pyrazole (2)
An equimolar mixture of 3-(5-bromo-2-thienyl)-1-
phenyl-1H-pyrazole-4-carboxaldehyde10 1 (3.33 g, 10
mmol) and p-methylacetphenone (1.34 g, 10 mmol) in
30 mL ethanolic KOH (3%) was stirred at room temp-
erature for 4 h. The precipitate formed was filtered,
washed with ethanol, dried and crystallized from
chloroform/ethanol (3:1). Yield: 94%; mp159–161 ꢃC.
IR (cmꢀ1): 1660 (C¼O), 1625 (C=N). 1H NMR
(CDCl3): d 2.41 (s, 3H, CH3), 7.11 (d, J=3.96 Hz, 1H,
5.5. 3-(5-Bromo - 2 - thienyl)-4-[1-phenylthiocarbamoyl-3-
(4-methylphenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole
(6)
To a solution of 5 (2.31 g, 5 mmol) in dry ether (30 mL)
was added an equal amount of phenyl isothiocyanate.
The reaction mixture was stirred for 5 h. The solid pro-
duct separated was filtered, washed with ethanol, dried
and crystallized from ethanol. Yield: 78%; mp215–
216 ꢃC. IR (cmꢀ1): 1630 (C=N), 1524, 1315, 1140 and
945 (NCS). 1H NMR (CDCl3): d 2.38 (s, 3H, CH3),
2.94–3.09 (dd, J1=10 Hz, J2=9 Hz, 1H, pyrazoline-C-
4-H), 3.34–3.46 (dd, J1=10 Hz, J2=9 Hz, 1H, pyrazo-
line-C-4-H), 5.05–5.16 (dd, J1=10 Hz, J2=10 Hz, 1H,
pyrazoline-C-5-H), 7.15 (d, J=3.96 Hz, 1H, thioph C-3-
H), 7.18–7.80 (m, 15H, phenyl-H, NH), 7.82 (d, J=3.96
Hz, 1H, thioph C-4-H), 8.43 (s, 1H, pyrazol C-5-H)).
Analysis calculated for C30H24BrN5S2 (598.581).
thioph C-3-H), 7.21-7.77 (m, 11H, Phenyl-H
&
CH=CH), 7.80 (d, J=3.96 Hz, 1H, thioph C-4-H), 8.47
(s, 1H, pyrazol C-5-H)). Analysis calculated for
C23H17BrN2OS (449.364).
5.3. 3-(5-Bromo-2-thienyl)-4-[1-formyl-3-(4-methylphe-
nyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole 3 and 3-(5-
bromo-2-thienyl)-4-[1-acetyl-3-(4-methylphenyl)-2-pyra-
zolin-5-yl]-1-phenyl-1H-pyrazole (4)
A mixture of 2 (2.2 g, 5 mmol) and hydrazine hydrate
(1.6 mL, 5 mmol) in formic acid or acetic acid (15 mL)
was heated under reflux for 6 h. The solid product
obtained on cooling was filtered, washed with ethanol
and crystallized from ethanol. Physical and analytical
data of compound 3; yield: 76%; mp216–218 ꢃC. IR
5.6. 3-(5-Bromo-2-thienyl)-4-[1-nitroso-3-(4-methyl-
phenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole (7)
To an ice cold solution of 5 (2.31 g, 5 mmol) in hydro-
chloric acid (50%, 20 mL) was added sodium nitrite
(0.35 g, 5 mmol). The reaction mixture was stirred for

1942
A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
2 h. The solid product separated was filtered, washed
with ethanol, dried and crystallized from ethanol. Yield:
5.8. 3-(5-Bromo-2-thienyl)-4-[3-(4-methylphenyl)-1H-
pyrazol-5-yl]-1-phenyl-1H-pyrazole (11)
ꢃ
54%; mp163–164 C. IR (cmꢀ1): absence of 3380 (NH),
1
1630 (C=N). H NMR (CDCl3): d 2.37 (s, 3H, CH3),
A mixture of 2 (2.2g, 5 mmol) and hydrazine hydrate
(1.6 mL, 5 mmol) in ethanol (15 mL) was heated under
reflux for 6 h. The reddish white solid product obtained
on cooling was filtered, washed with ethanol and crys-
tallized from ethanol. Physical and analytical data of
compound 11; yield: 64%; mp172–174 ꢃC. IR (cmꢀ1):
2.97–3.10 (dd, J1=10 Hz, J2=9 Hz, 1H, pyrazoline-C-
4-H), 3.33–3.46 (dd, J1=10 Hz, J2=9 Hz, 1H, pyrazo-
line-C-4-H), 5.08–5.15 (dd, J1=10 Hz, J2=10 Hz, 1H,
pyrazoline-C-5-H), 7.12 (d, J=3.96 Hz, 1H, thioph C-3-
H), 7.18–7.81 (m, 9H, phenyl-H), 7.81 (d, J=3.96 Hz,
1H, thioph C-4-H), 8.42 (s, 1H, pyrazol C-5-H)). Ana-
lysis calculated for C23H18BrN5OS (492.392).
1
3385 (NH), 1628 (C=N). H NMR (CDCl3): d 2.38 (s,
3H, CH3), 6.97 (s,1H, pyrazol-C-4-H), 7.12 (d, J=3.96
Hz, 1H, thioph C-3-H), 7.17–7.80 (m, 10H, phenyl-H,
NH ), 7.83 (d, J=3.96 Hz, 1H, thioph C-4-H), 8.42 (s,
1H, pyrazol C-5-H)). Analysis calculated for
C23H17BrN4S (461.378).
5.7. 3-(5-bromo-2-thienyl)-4-[1-acetyl-3-(4-methyl-
phenyl)-2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole 4, 3-(5-
bromo-2-thienyl)-4-[1-benzoyl-3-(4-methylphenyl)-2-
pyrazolin-5-yl]-1-phenyl-1H-pyrazole 8, 3-(5-bromo-2-
thienyl)-4-[1-methanesulfonyl-3-(4-methylphenyl)-2-pyra-
zolin-5-yl]-1-phenyl-1H-pyrazole 9 or 3-(5-bromo-2-thie-
nyl)-4-[1-(4-methylbenzenesulfonyl)-3-(4-methylphenyl)-
2-pyrazolin-5-yl]-1-phenyl-1H-pyrazole (10)
5.9. 3-(5-Bromo-2-thienyl)-4-[1-aryl-3-(4-methylphenyl)-
1H-pyrazol-5-yl]-1-phenyl-1H-pyrazole (12a&b)
To a solution of 2 (2.2 g, 5 mmol) in absolute ethanol
(20 mL) was added the proper arylhydrazine hydro-
chloride (5 mmol) and anhydrous sodium acetate (0.49
g, 5 mmol). The reaction mixture was heated under
reflux for 6 h, then cooled and poured into cold water
(50 mL). The precipitated solid was filtered, washed
with water, dried and crystallized from ethanol. Physical
and analytical data of compound 12a; yield: 82%; mp
196–198 ꢃC. IR (cmꢀ1): 1625 (C=N). 1H NMR
(CDCl3): d 2.39 (s, 3H, CH3), 6.98 (s,1H, pyrazol-C-4-
H), 7.13 (d, J=3.96 Hz, 1H, thioph C-3-H), 7.15–7.80
(m, 14H, phenyl-H), 7.82 (d, J=3.96 Hz, 1H, thioph C-
4-H), 8.42 (s, 1H, pyrazol C-5-H)). Analysis calculated
for C29H21BrN4S (537.474). Physical and analytical
data of compound 12b; yield: 78%; mp184–186 ꢃC. IR
To a solution of 5 (0.46 g, 1 mmol) in dry pyridine (5
mL) was added an equivalent amount of acetyl chloride,
benzoyl chloride, 4-methanesulphonyl chloride or 4-
toluenesulphonyl chloride. The reaction mixture was
heated on a boiling water bath for 20 min, cooled
and then poured onto crushed ice (30 g). The solid
product separated was filtered, washed with water,
dried and crystallised from ethanol. Physical and
analytical data for compound 4; yield: 93%, mp,
1
IR and H NMR were identical to the compound pre-
pared by the afore-mentioned method. Physical and
analytical data of compound 8: yield 89%; mp233–
235 ꢃC. IR (cmꢀ1): 1665 (C¼O), 1632 (C=N). 1H
NMR (CDCl3): d 2.41 (s, 3H, CH3), 2.95–3.09 (dd,
J1=10 Hz, J2=9 Hz, 1H, pyrazoline-C-4-H), 3.33–
3.45 (dd, J1=10 Hz, J2=9 Hz, 1H, pyrazoline-C-4-
H), 5.06–5.15 (dd, J1=10 Hz, J2=10 Hz, 1H, pyr-
azoline-C-5-H), 7.12 (d, J=3.96 Hz, 1H, thioph C-3-H),
7.14–7.81 (m, 14H, phenyl-H), 7.83 (d, J=3.96 Hz,
1H, thioph C-4-H), 8.39 (s, 1H, pyrazol C-5-H)).
Analysis calculated for C30H23BrN4OS (567.500).
Physical and analytical data of compound 9; yield:
91%; mp208–210 ꢃC. IR (cmꢀ1): 1632 (C=N),
1364, 1165 (SO2). 1H NMR (CDCl3): d 2.40 (s, 3H,
1
(cmꢀ1): 1628 (C=N). H NMR (CDCl3): d 2.39 (s, 3H,
CH3), 2.41 (s, 3H, CH3), 6.97 (s,1H, pyrazol-C-4-H),
7.13 (d, J=3.96 Hz, 1H, thioph C-3-H), 7.14–7.81 (m,
13H, phenyl-H), 7.82 (d, J=3.96 Hz, 1H, thioph C-4-
H), 8.41 (s, 1H, pyrazol C-5-H)). Analysis calculated for
C30H23BrN4S (551.500).
5.10. Anti-inflammatory activity
5.10.1. Cotton pellet-induced granuloma bioassay. Adult
male Sprague–Dawley rats (120–140 g) were used.16
They were acclimated 1 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 (6 rats each). Cotton pellets (35ꢁ1
mg) cut from dental rolls were impregnated with 0.2 mL
(containing 10 mmol) of a solution of the test compound
in chloroform or acetone 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
groupof animals received the standard reference indo-
methacin 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
granulomas, were removed, dried for 48 h at 60 ꢃC and
CH3), 2.95–3.10 (m, 4H, pyrazoline-C-4-H
&
CH3SO2), 3.32–3.48 (dd, J1=10 Hz, J2=9 Hz, 1H,
pyrazoline-C-4-H), 5.06–5.14 (dd, J1=10 Hz, J2=10
Hz, 1H, pyrazoline-C-5-H), 7.11 (d, J=3.96 Hz, 1H,
thioph C-3-H), 7.14–7.80 (m, 9H, phenyl-H), 7.82 (d,
J=3.96 Hz, 1H, thioph C-4-H), 8.40 (s, 1H, pyrazol C-
5-H)). Analysis calculated for C24H21BrN4O2S2
(541.485). Physical and analytical data of compound 10;
yield: 87%; mp214–215 ꢃC. IR (cmꢀ1): 1630 (C=N),
1368,1165 (SO2). 1H NMR (CDCl3): d 2.39 (s, 3H,
CH3), 2.42 (s, 3H, CH3), 2.97–3.12 dd, J1=10 Hz, J2=9
Hz, 1H, pyrazoline-C-4-H), 3.33–3.44 (dd, J1=10 Hz,
J2=9 Hz, 1H, pyrazoline-C-4-H), 5.05–5.17 (dd, J1=10
Hz, J2=10 Hz, 1H, pyrazoline-C-5-H), 7.11 (d, J=3.96
Hz, 1H, thioph C-3-H), 7.13-7.81 (m, 13H, phenyl-H),
7.82 (d, J=3.96 Hz, 1H, thioph C-4-H), 8.41 (s, 1H,
pyrazol
C30H25BrN4O2S2 (617.581).
C-5-H)).
Analysis
calculated
for

A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
1943
weighed. The increment in dry weight (difference
between the initial and final weights) was taken as a
measure of granulomaꢁSE. This was calculated for
each groupand the percentage reduction in dry weight
of 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 (Table 1).
310-sterile silk suture, Ethicon, UK)24 and the rats were
kept in their cages for 6 h.
5.13. Collection of exudates
After 6 h, the animals were reanaesthetized with ether.
The ventral incisions were quickly opened and the
sponges were carefully dissected out to avoid local
bleeding. Both sponge were gently squeezed with a 5 mL
syringe plunger and the total exudates was collected in
polyethylene centrifuge tubes containing 0.1 mL of
1.5% EDTA.24 This was used to determine non-immu-
nological markers, namely: exudates volume, total and
differential leucocyte cell counts, in addition to immu-
nological markers, namely: cytochrome C reduction test
and interleukin-1 beta levels.
5.11. Sponge implantation model of inflammation in rats
5.11.1. Material and methods. One hundred and thirty
male albino rats weighing 150–200 g were used
throughout the study. They were kept in the animal
house under standard conditions of light and temper-
ature with free access to food and water. The animals
were randomly divided into thirteen groups each of ten
rats as follows:
5.14. Cell isolation and preparations
GroupI (control group): received 2% gum acacia orally
(suspending vehicle) and served as inflammatory control
group, to estimate reference values of the parameters
studied.
Granulocyte cell suspension was prepared using a
modification of the method of Lehrer and Cline.27
5.15. Neutrophil phagocyte function estimation27ꢀ31
GroupII (indomethacin rpe-treated inflammatory
group): received indomethacin (Indomethacin-Pharco
Pharmaceuticals, Egypt) suspended in 2% gum acacia,
in a dose of 10 mg/kg body weight/day orally, divided
into two equal doses, for three successive days.23 On the
third day, the first dose was administered 30 min before
sponge implantation and the second dose 1 h before its
removal.
The isolated cells were resuspended in 1 mL Hank’s
balanced salt solution (HBSS, free from phenol
red-Gibco, England) and were adjusted to a concen-
tration of 2ꢂ105 cells/mL. This was stored at ꢀ20 ꢃC
until tested for neutrophilic function. This was eval-
uated by the cytochrome C reduction test, where the
reduction of cytochrome C by superoxide anion will
change its light absorbance properties, as detected
spectrophotometrically.
Group III–XIII (pre-treated inflammatory groups):
each being treated with the appropriate newly synthe-
sised investigational compounds 1, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12a&b respectively. The compounds were suspended
in 2% gum acacia, and were given in a dose of 10 mg/kg
body weight/day, orally, divided into two equal doses,
for three successive days, as indomethacin.
5.16. Interleukin-1 beta level estimation (IL-1ꢀ)32
The granulocyte cell layer was washed 3 times in RPMI-
1640 complete medium (Grand Island Biological Com-
pany, NY) containing 2 mmol glutamine, 100 units/mL
penicillin, 100 mg/mL streptomycin and 20 mmol N-1-
hydroxyethylpiperazine-N-2-ethane sulphonic acid
(HEPES buffer) (Nalge, Sybron Corporation-Roche-
ster, NY). The supernatant was aspirated and the cell
pellet was vigorously tapped and resuspended in a
known volume of complete RPMI medium supple-
mented with 10% inactivated fetal bovine serum (FBS),
heat inactivated at 56 ꢃC for 30 min. (Biofluid-Inc.
Tockville, USA).
5.12. Induction of inflammation
Inflammation was induced by subcutaneous implanta-
tion of dry polyester sponge (type E41-blue-kay Metze-
ler, UK).24 The polyester sponge was cut to a standard
size of 15 mm diameter, 4 mm thickness, and a weight of
17–17.5 mg. The sponges were sterilized in 70% (v/v)
ethanol, washed thoroughly twice with distilled water
and antiseptic solution (Cyteal-Pierre Fabre labora-
tories, France), and were then dried in an oven (Gal-
lenkamp-Poland) at 100 ꢃC for 1.5 h.
The granulocyte count was then adjusted to 2ꢂ106 cells/
mL by complete RPMI-1640 supplemented with 10%
heat inactivated FBS (RPMI-HIFBS). This constituted
the granulocyte cell suspension that was stored at
ꢀ20 ꢃC until tested for IL-1b levels.
For implantation, the rats were anaesthetized with die-
thyl ether and the skin of the ventral aspect of the
abdominal surface was shaved and swabbed with a 1%
solution of savlon in 70% (v/v) ethanol/water.24ꢀ26 Two
small medial incisions each of 1 cm length were made on
either side of the midline of the ventral abdominal sur-
face, and two cavities were performed by blunt dissec-
tion, separating the dermis from the muscular layers.
Lastly one dry sponge was implanted in each side, the
incisions were then closed with silk sutures (Mersilk
5.17. Stimulation of granulocytes by lipopolysaccharide
(LPS) as a mitogen (lyophilized E. coli, Sigma, USA33
100 mL of the prepared cell suspension were added to
LPS (10 mg/mL) (reconstituted in 5 mL sterile complete
RPMI-1640 medium and stored at ꢀ20 ꢃC until used) in
a flat-bottomed well of a microtitre plate. Another 100
mL of the cell suspension (without stimulant) were

1944
A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
pippetted into a flat-bottomed well to serve as a control.
Each sample was placed in triplicate well to obtain
enough supernatants for measuring IL-1b levels. Com-
plete RPMI medium was added to all wells to obtain a
final volume of 200 mL per well. The cell cultures were
incubated for 24 h at 37 ꢃC, 10% CO2 and 100%
humidity in a CO2 jar (Angelantoni Scientifica, Italy).
After 24 h, the cultures were centrifuged at 700ꢂg for 10
min and the supernatant containing putative IL-1b were
collected and stored at ꢀ20 ꢃC until tested for the cyto-
kine by ELISA kite.
Groups IV–X: received test compounds suspended in
2% gum acacia at a dose of 10 mg/kg/day orally.
The drugs were administered orally in two equal doses
at 0.0 and 12 h for three successive days. Animals were
sacrificed after 6 h of the last dose. The stomach from
each animal was removed. An opening at the greater
curvature was made and the stomach was washed with
cooled saline and inspected with 3ꢂ magnifying lens for
any evidence for hyperemia and haemorrhage, definite
haemorrhagic errosion or ulcer. The percentage ulcera-
tion for each groupwas calculated as follows:
5.18. Human COX-1 and COX-2 enzymatic assay
Number of animals bearing ulcer in a group
Human COX-1 and COX-2 activities were determined
by applying the methodology described by Wakitani et
al.17 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 com-
pound for 5 min at 24 ꢃC. [14C] Arachidonic acid
(100.00 dpm, 30 mmol) was then added to the mixture
and incubated for 2 (COX-1) or 45 min (COX-2) at
24 ꢃC. The reaction was stopped by the addition of 400
mL of a stopsolution composed of Et 2O:MeOH:1M
citric acid (30:4:1, v/v). After centrifugation of the mix-
ture at 1700 X/g for 5 min at 4 ꢃC, 50 mL of the upper
phase was applied to a thin-layer chromatography
(TLC) plate. TLC was performed at 4 ꢃC with a solvent
system consisting of Et2O:MeOH:AcOH (90:2:0.1, v/v).
Enzyme activity was calculated from the percent con-
version of arachidonic acid to PGH2 and its decom-
position products, using a radiometric photographic
system. The concentration of the compound causing
50% inhibition (IC50) was calculated.
%Ulceration ¼
Number of animals bearing ulcer in a group
ꢀ 100
Total number of animals in the same group
5.21. Acute toxicity
The biologically significant compounds were further
investigated for their approximate LD50 in male
mice.20,21 Eight groups of mice each consisting of six
animals, were used. The compounds were given orally in
doses of 1, 10, 100, 200, 250, 500 mg / kg, respectively.
Twenty four h later, the% mortality in each groupand
for each compound was recorded. The LD50 values
(Table 1) were calculated using the method described by
Litchfield and Wilcoxon.21
5.22. In vitro antimicrobial activity
The microdilution susceptibility test in Muller-Hinton
Broth (Oxoid) and Sabouraud Liquid Medium (Oxoid)
were used for the determination of antibacterial and
antifungal activity.22 The utilized test organisms were:
Escherichia coli (E. coli) ATCC 25922 as an example of
Gram-negative bacteria, Staphylococcus aureus (S. aur-
eus) ATCC 19433 as an example of Gram-Positive bac-
teria and Candida albicans (C. albicans) as yeast-like
fungi. Ampicillin trihydrate and clotrimazole were used
as standard antibacterial and antifungal agents, respec-
tively. Solutions of the test compounds, ampicillin tri-
hydrate and clotrimazole were prepared in DMSO at
concentration of 1600 mg/mL. The two-fold dilution of
the compounds were prepared (800, 400,...6.25 mg/mL).
The microorganism suspensions at 106 CFU/mL (Col-
ony Forming Unit / mL) concentration were inoculated
to the corresponding wells. Plates were incubated at
36 ꢃC for 24 h to 48 h. The incubation chamber was
kept sufficiently humid. At the end of the incubation
period, the minimal inhibitory concentrations (MIC)
were determined.
5.19. Statistical methods
Data are expressed as means with their corresponding
standard errors. Data were evaluated by the one way
analysis of variance. Then, the data were subjected to
the least significant difference ‘LSD’ test.34
5.20. Ulcerogenic effects
Compounds 3, 4, 5, 8, 9, 11 and 12a that exhibited
moderate to potent anti-inflammatory profiles in the
pre-mentioned animal models were evaluated for their
ulcerogenic potential in rats.19 Phenylbutazone and
indomethacin were tested as reference drugs. One hun-
dred male albino rats (150–200 g) were fasted for 12 h
prior to drug administration. Water was given ad libi-
tum. The animals were divided into ten groups each of
ten rats as follows:
GroupI (control group): received 2% gum acacia orally.
Group II: received phenylbutazone suspended in 2%
gum acacia at a dose of 10 mg/kg/day orally.
Acknowledgements
Authors are very grateful to Alaa Bekhit, Department
of Food Science and Technology, Faculty of Agri-
culture, University of Alexandria, and to Ji Young
GroupIII: received indomethacin suspended in 2% gum
acacia at a dose of 10 mg/kg/day orally.

A. A. Bekhit, T. Abdel-Aziem / Bioorg. Med. Chem. 12 (2004) 1935–1945
1945
Park, College of Pharmacy, Sookmyung Women’s Uni-
versity, Seoul 140-742, South Korea for their assistance
during biological testing.
Conference of Pharmaceutical Sciences, P 81, Dec. 17–19,
Cairo, Egypt, 2002.
14. Bekhit, A. A.; Fahmy, H. T. Y.; Rostom, Sh.A.F.;
Baraka, A. M. Eur. J. Med. Chem. 2003, 38, 27.
15. Bekhit, A. A.; Fahmy, H. T. Y. Archiv. Pharm. Pharm.
Med. Chem. 2003, 33, 111.
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— XXVIII International