Synthesis, antimicrobial, antioxidant, anti-inflammatory, and analgesic activities of some new 3-(2′-thienyl)pyrazole-based heterocycles

Article abstract of DOI:10.1007/s00044-011-9661-x

1-Phenyl-3-(thiophen-2-yl)-1H-pyrazole-4-carbaldehyde was used as a synthon for the synthesis of new thiazole and pyrazoline heterocycles having 2-thienylpyrazole moiety. The antimicrobial, anti-inflammatory, and analgesic activities of the synthesized compounds were evaluated using agar diffusion method, carrageenan-induced paw edema, and writhing assays, respectively. It was found that the majority of the tested compounds exhibited both analgesic and anti-inflammatory activities. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9661-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1418–1426
DOI 10.1007/s00044-011-9661-x
ORIGINAL RESEARCH
Synthesis, antimicrobial, antioxidant, anti-inflammatory,
and analgesic activities of some new 3-(20-thienyl)pyrazole-based
heterocycles
•
Bakr F. Abdel-Wahab Hassan Abdel-Gawad
•
•
Ghada E. A. Awad Farid A. Badria
Received: 15 February 2011 / Accepted: 5 May 2011 / Published online: 18 May 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract 1-Phenyl-3-(thiophen-2-yl)-1H-pyrazole-4-carb-
aldehyde was used as a synthon for the synthesis of new
thiazole and pyrazoline heterocycles having 2-thienylpy-
razole moiety. The antimicrobial, anti-inflammatory, and
analgesic activities of the synthesized compounds were
evaluated using agar diffusion method, carrageenan-
induced paw edema, and writhing assays, respectively. It
was found that the majority of the tested compounds
exhibited both analgesic and anti-inflammatory activities.
biological activities including antibacterial (Sarma et al.,
2010), antifungal (Zhang et al., 2010), herbicidal (Yuhan
et al., 2010), and insecticidal (Finkelstein and Strock,
1997). They are also found in some commercial pesticides,
including fenpyroximate, pyrazolate, and pyrazoxyfen (Li-
Rong et al., 2004). In addition, pyrazole derivatives are
important pharmaceuticals, for instance, they have been
found to possess analgesic and anti-inflammatory activities
because of their cyclooxygenase and 5-lipoxygenase
inhibiting properties (Ochi et al., 1999; Chauhan et al.,
2011). Also, they are also useful as biodegradable agro-
chemicals (Crosscurt et al., 1979) and as intermediates in
the dye industry (Lubs, 1970). Furthermore, 5-thienyl
pyrazole derivatives have CB1 receptor antagonistic
activity (Srivastava et al., 2009) and antimicrobial activity
by inhibition of cell wall, nucleic acid, and protein in
bacteria (Kulikova and Cherkesova, 1974). In view of such
applications and in connection with our previous study on
synthesis of biologically active heterocycles (Abdel-Aziz
et al., 2010; Abdel-Wahab et al., 2008, 2009a,b), we syn-
thesized various thienylpyrazoles that have antimicrobial,
antioxidant, anti-inflammatory, and analgesic activities.
Keywords 2-Thienylpyrazoles ꢀ Thiazoles ꢀ
Heterocycles ꢀ Antimicrobial ꢀ Antioxidant ꢀ Analgesic ꢀ
Anti-inflammatory activities
Introduction
Pyrazole derivatives represent one of the most active
classes of compounds possessing a wide spectrum of
B. F. Abdel-Wahab (&)
Chemistry Department, Faculty of Science and Arts, King
Abdulaziz University, Khulais Branch, Saudi Arabia
e-mail: balshoaibi@kau.edu.sa
Results and discussion
URL: http://sak.njb.kau.edu.sa/Default.aspx
Chemistry
H. Abdel-Gawad
Applied Organic Chemistry Department, National Research
Centre, Giza 12622, Egypt
Heating of an equimolar amounts of 1-phenyl-3-(thiophen-
2-yl)-1H-pyrazole-4-carbaldehyde 1 (Bratenko et al., 2005)
with thiosemicarbazide in ethanol in the presence of glacial
acetic acid under reflux condition afforded thiosemicarba-
zone (2) in 70% yield. The target compounds 7–10 were
obtained by reaction of equimolar quantities of thiosemi-
carbazide 2 with phenacyl bromides 3a,b, 2-chloro-N-
G. E. A. Awad
Chemistry of Natural and Microbial Products, National Research
Center, Dokki, Giza 12622, Egypt
F. A. Badria
Pharmacognosy Departments, Faculty of Pharmacy, Mansoura
University, Mansoura 35516, Egypt
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Med Chem Res (2012) 21:1418–1426
1419
(thiophen-2-yl)acetamide 4, N0-(4-fluorophenyl)-2-oxo-
propanehydrazonoyl chloride 5, or 2,3-dichloroquinoxaline
6 (Scheme 1).
peak at m/z 466 and 453 because of their molecular ions,
respectively.
4-Thiazolidinone compound 11 was readily obtained
from the reaction of thiazosemicarbazone 2 with chloro-
acetic acid in glacial acetic acid and in the presence of
anhydrous sodium acetate (Scheme 1). The 1H NMR
spectrum of 11 is consistent with the assigned structure.
For example, the thiazolidinone CH2 protons resonated as a
singlet at 3.91 ppm.
The structures of thiazoles 7, 9, and 10 were established
1
on the basis of their spectral data. For example, the H
NMR spectra of 7a showed the thiazole CH proton as a
broad singlet that resonated at 5.12 ppm, while the methyl
protons of compound 9 resonated as a singlet at 2.67 ppm.
Also, the mass spectra of compounds 8 and 10 showed a
Scheme 1 Reaction of
thiosemicarbazone 2 with
halogenated compounds
Ar
S
N
3 & 7 Ar
Ar
N
NH
Br
3
HC
a
b
O
O
O
EtOH, reflux
2h, 70-75%
N
N
Ph
S
O
7
OHC
O
S
Cl
N
N
H
H
C
S
4
N
Ph
S
S
N
N
HN
N
N
Ph
1
EtOH, reflux
5h, 70%
S
O
S
NH2
H2N
8
N
H
NH2
EtOH/AcOH
reflux, 2h
70%
F
F
S
O
NH2
N
N
N
NH
N
N
H3C
N
H
S
N
HN
HC
5
HC
S
Cl
N
CH3
EtOH-Et3N, reflux
5h, 65%
N
N
N
Ph
S
Ph
9
2
N
N
Cl
Cl
N
N
S
N
6
N
NH
HC
EtOH, reflux
5h, 70%
N
N
Ph
S
10
S
O
N
N
N
HC
H
Cl
CO2H
NaOAc-AcOH,
reflux, 6h
85%
S
N
N
Ph
11
123

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Med Chem Res (2012) 21:1418–1426
Treatment of 1 with 4-chloroaceteophenone in ethanol
Table 1 Characteristic data of the synthesized compounds
and 10% sodium hydroxide at room temperature for 1 h
afforded the aldol-type product 12. Reaction of 12 with
hydrazine hydrate produced pyrazoline derivative 13.
Finally, 4-tolylisothiocyanate reacted with 13 to afford
thioamide 14 in 45% yield (Scheme 2).
Compd. Mol. formula
no.
Calcd. found
C% H% N%
Mp. ꢁC Yield
(M. Wt.)
%
2
C15H13N5S2
(327.43)
55.02 4.00 21.39 226–227 70
55.13 3.92 21.43
The structures of compounds 12–14 were confirmed by
their spectroscopic data (Table 1).
7a
7b
8
C
25H17N5OS2 64.22 3.66 14.98 178–179 70
(467.57)
64.13 3.72 15.03
C26H17N5O2S2 63.01 3.46 14.13 242–243 75
Antimicrobial activity
(495.58)
62.89 3.51 13.99
C21H18N6OS3 54.06 3.89 18.01 275–276 70
Table 2 shows the antimicrobial activity of the synthesized
compounds against target pathogens using the agar diffusion
assay (Perez et al., 1990). Compound 2 showed excellent
antibacterial activities for all the tested pathogen including
gram-positive, gram-negative bacteria, and yeast with min-
imal inhibitory concentration (MIC) that ranged between
83.3 and 20.8 lg/ml (Table 2),whereas compound 12
showed antibacterial activities with MIC ranging between
333.3 and 41.6 lg/ml against the tested pathological strains.
Among all the compounds tested, compound 8 showed
an excellent (MIC) 20.8 lg/ml against Staphylococcus
aureus ATCC 29213 and Enterobacter Cloaca ATCC3047.
Compounds 10, 11, and 13 showed moderate antimicrobial
activities. However, no compound showed inhibition
against fungi Aspergillus niger as observed by the absence
of inhibition zone (data not shown).
(466.60)
53.98 3.98 18.20
9
C
24H18FN7S2
(487.58)
23H15N7S2
(453.54)
59.12 3.72 20.11 140–142 65
59.33 3.79 20.26
10
11
12
13
14
C
60.91 3.33 21.62 244–245 70
61.10 3.46 21.73
C
17H13N5OS2 55.57 3.57 19.06 273–274 85
(367.45) 55.49 3.63 18.97
22H15ClN2OS 67.60 3.87 7.17 130–131 85
C
(390.89)
67.51 3.91 7.26
C22H17ClN4S
65.26 4.23 13.84 184–185 70
65.39 4.49 14.01
(404.92)
C30H24ClN5S2 65.02 4.37 12.64 245–246 45
(554.13) 65.18 3.46 12.50
scavenging activity was determined by the decrease in
absorbance at 517 nm, due to the reduction by the anti-
oxidant (AH) or the reaction with a radical species, as
shown in the Eq. 1 (Gordon, 2001).
DPPH free radical scavenging activity
DPPH test, which is based on the ability of DPPH, a stable
free radical, to decolorize in the presence of antioxidants, is
a direct and reliable method for determining radical scav-
enging action (Niki, 1987). Therefore, the DPPH radical
DPPHꢁ þ Rꢁ ! DPPH ꢂ R
According to the results of DPPH free radical
ð1Þ
scavenging activity showed in Table 3, compounds 7a,
Scheme 2 Route to bipyrazole
derivative 14
Cl
Cl
S
CHO
S
NH2NH2.H2O
4-ClC6H4COCH3
N
H
C
S
10% NaOH, stirring
1h, rt, 85%
N
H
EtOH, reflux, 3h
70%
N
N
N
N
N
C
H
N
Ph
O
1
Ph
Ph
13
12
Cl
S
4-MeC6H4NCS
N
Me
EtOH, reflux, 6h
45%
N
N
N
N
S
Ph
H
14
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Med Chem Res (2012) 21:1418–1426
1421
Table 3 CI50 for DPPH inhibition of chemical compounds
Chemical compounds
DPPH IC50 (lg/ml)
2
500
25
7a
7b
8
25
1000
25
9
10
11
12
13
14
500
500
1000
750
500
Fig. 1 Percent anti-inflammatory activity of the tested compounds
(carrageenan-induced paw edema test in rats)
7b, and 9 were the strongest radical scavenger among the
tested compounds with CI50 25 lg/ml, followed by 2, 10,
11, and 14 with CI50 500 lg/ml. However, the other
compounds were moderate radical scavengers with CI50
that ranged between 750 and 1000 lg/ml.
The anti-inflammatory activity
The anti-inflammatory activity of the synthesized
pyrazoles was evaluated using carrageenan-induced
paw edema test in rats. The anti-inflammatory activity
data (Fig. 1) indicated that the most compounds pro-
tected rats from carrageenan-induced inflammation.
Compounds 8, 9, and 11 were equipotent as diclofenac
sodium in terms of anti-inflammatory activity. However,
other compounds showed moderate activity. Compound
7a was proved to be the least active among tested
compounds.
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Med Chem Res (2012) 21:1418–1426
reported in this article may be a helpful guide for the
medicinal chemists who are working in this area.
Experimental
Chemistry
All melting points were taken on Electrothermal IA 9000
series digital melting point apparatus. Elemental analytic
data were carried from the microanalytic unit, National
Research Centre, Dokki, Giza, Egypt. The IR spectra were
recorded in potassium bromide disks on a Shimadzu CVT-
04 spectrophotometer. The 1H NMR spectra were recorded
at 270 MHz on a Varian EM-360 spectrometer using TMS
as an internal standard. Chemical shifts values (d) are given
in parts per million (ppm). The electron impact mass
spectra were performed using mass Varian MAT CH-5
spectrometer at 70 eV. 1-Phenyl-3-(thiophen-2-yl)-1H-
pyrazole-4-carbaldehyde 1 (Bratenko et al., 2005), 1-
(benzofuran-2-yl)-2-bromoethanone 3a (Shriner and
Anderson, 1939), 3-(2-bromoacetyl)-2H-chromen-2-one
3b (Koelsch, 1950), 2-chloro-N-(thiazol-2-yl)acetamide 4
(Abdel-Rahman et al., 1981), N0-(4-fluorophenyl)-2-ox-
opropanehydrazonoyl chloride 5 (Biere et al., 1982), and
2,3-dichloroquinoxaline 6 (Krishnan et al., 1999) were
prepared according to the reported procedures.
Fig. 2 Percent analgesic activity (peripheral, writhing test)
Analgesic activity
The analgesic activity was determined by the hot plate test
(central analgesic activity) and acetic acid induced writhing
assay. The obtained data (Fig. 2) revealed that all tested
compounds exhibited good analgesic activity. Most of the
tested compounds have comparable activity to diclofenac
sodium. Compounds 8 and 9 exhibit activities higher than
that of the reference drug in peripheral analgesic activity
testing. The remaining compounds have strong to moderate
activity.
2-((1-phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-
yl)methylene)hydrazinecarbothioamide (2)
Structure–activity Relationship (SAR) correlations
Compound 1 (2.54 g, 10 mmol) was dissolved in absolute
ethanol (20 ml) and thiosemicarbazide (0.91 g, 10 mmol)
was added. Three drops of glacial acetic acid was also
added. The reaction mixture was refluxed for 5 h. The solid
obtained was isolated by filtration, washed with ethanol,
dried, and recrystallized from (EtOH-DMF; 2:1 by v/v) to
give 2.
•
•
Pyrazole nucleus is essential for all of the biological
activities
The antimicrobial and antioxidant activities of the
synthesized thien-2-ylpyrazole derivatives are due to
the presence of thiosemicarbazide fragment (compound
2), chalcone (12), and thiazole substitute (8).
The anti-inflammatory and analgesic activities are due
to the presence of thiosemicarbazone moiety (com-
pound 8); fluorine substituent (compounds 9), 4-thia-
zolidinone nucleus (compounds 11), and coumarin
residue (compounds 7b).
1
IR (KBr) mmax/cm-1 3330–3172 (NH2, NH); H NMR
•
(DMSO-d6) d 7.48–7.69 (m, 8H, Ar–H), 7.80 (s, 1H, pyr-
azole-H), 8.32 (s, 1H, CH = N), 9.16 (s, 2H, NH2, D2O-
exchangeable), 11.51 (s, H, NH, D2O-exchangeable); MS
m/z (%): 327 (M?, 33), 63 (100%).
Synthesis of thiazoles 7–10
Conclusion
Equimolar amounts of 2 (0.33 g, 1 mmol) and appropriate
phenacyl bromides 3; 2-chloro-N-(thiophen-2-yl)acetamide
4, N0-(4-fluorophenyl)-2-oxopropanehydrazonoyl chloride
5, or 2,3-dichloroquinoxaline 6 (1 mmol) in absolute eth-
anol (30 cm3) {in the presence of few drops of triethyl-
amine as a catalyst in the case of 5} was heated under
reflux for 3 h, then left to cool. The solid formed was
The results of this investigation revealed that the observed
increase in analgesic, anti-inflammatory, and antimicrobial
activities are attributed to the presence of 3-thien-2-ylpy-
razole nucleus, thiosemicarbazone, and 4-F in phenyl of
synthesized compounds containing pyrazole. The data
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Med Chem Res (2012) 21:1418–1426
1423
2-{[(1-Phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-
yl)methylene]hydrazono}thiazolidin-4-one (11)
isolated by filtration, washed with ethanol, dried, and re-
crystallized from EtOH-DMF.
A mixture of 2 (0.33 g, 1 mmol) and chloroacetic acid
(0.1 g, 1 mmol) in glacial acetic acid (30 ml) containing
anhydrous sodium acetate (0.33 g, 4 mmol) was heated
under reflux for 6 h. The reaction mixture was cooled, and
the solid formed was filtered and recrystallized from eth-
anol to give 11. IR (KBr) mmax/cm-1 3193 (NNH), 1689
(C=O); 1H NMR (DMSO-d6) d 3.91 (s, 2H, thiazolidinone-
CH2), 7.14–7.90 (m, 8H, Ar–H), 8.51 (s, 1H, pyrazole-H),
8.95 (s, 1H, CH = N), 11.82 (s, 1H, NH, D2O-exchange-
able); MS m/z (%): 367 (M?, 30), 62 (100%).
4-(Benzofuran-2-yl)-2-{[(1-phenyl-3-(thiophen-2-yl)-
1H-pyrazol-4-yl)methylene]hydrazono}-2,
5-dihydrothiazole (7a)
1
IR (KBr) mmax/cm-1 3185 (NH); H NMR (DMSO-d6) d
5.12 (s, 1H, thiazole-CH), 7.02 (s, 1H, benzofuryl-H),
7.21–7.81 (m, 12H, Ar–H), 7.91 (s, 1H, pyrazole-H), 8.27
(s, 1H, CH = N), 11.33 (s, H, NH, D2O-exchangeable);
MS m/z (%): 467 (M?, 2.5), 76 (100%).
3-(2-{[(1-Phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-
yl)methylene]hydrazono}-2,5-dihydrothiazol-4-yl)-2H-
chromen-2-one (7b)
1-(4-Chlorophenyl)-3-(1-phenyl-3-(thiophen-2-yl)-1H-
pyrazol-4-yl)prop-2-en-1-one (12)
1
IR (KBr) mmax/cm-1 1679 (C=O), 3181 (NH); H NMR
A suspension of 1 (2.5 g, 10 mmol) and 4-chloroacete-
opheneone (1.54 g, 10 mmol) in ethanol (20 ml) contain-
ing sodium hydroxide (10%; 5 ml) was stirred for 1 h at
room temperature and then left overnight to stand. The
solid obtained was filtered, dried, and recrystallized from
(DMSO-d6) d 6.32 (s, 1H, thiazole-CH), 7.19–7.76 (m,
12H, Ar–H), 7.90 (s, 1H, pyrazole-H), 8.10 (s, 1H,
coumarinyl-H), 8.62 (s, 1H, CH = N), 11.99 (s, H, NH,
D2O-exchangeable); MS m/z (%): 495 (M?, 4.5), 76
(100%).
EtOH-DMF (5:1 by volume) to give 12. IR (KBr) mmax
/
1
cm-1 1732 (C=O); H NMR (DMSO-d6) d 6.54 (d, 1H,
CH, J = 8.5 Hz), 6.62 (d, 1H, CH, J = 8.5 Hz), 7.15–7.64
(m, 12H, Ar–H), 8.31 (s, H, pyrazole-H); MS m/z (%): 392
(M?, 52), 63 (100).
2-Oxo-2-(thiophen-2-ylamino)ethyl N0-(1-phenyl-3-
(thiophen-2-yl)-1H-pyrazol-4-
yl)methylenecarbamohydrazonothioate (8)
4-(3-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-5-yl)-1-
phenyl-3-(thiophen-2-yl)-1H-pyrazole (13)
IR (KBr) mmax/cm-1 1668 (C=O), 3181–3390 (NH2,NH);
1H NMR (DMSO-d6) d 3.91 (s, 2H, CH2), 7.15 (d, 1H,
thiazole-CH), 7.16 (d, 1H, thiazole-H), 7.36–7.61 (m, 8H,
Ar–H), 7.88 (s, 1H, pyrazole-H), 8.52 (s, 1H, CH = N),
8.95 (s, 2H, NH2, D2O-exchangeable), 11.76 (s, H, NH,
D2O-exchangeable); MS m/z (%): 466 (M?, 0.5), 76
(100%).
A mixture of 12 (0.39 g, 1 mmol), hydrazine hydrate
(0.5 g, 10 mmol) in absolute ethanol (20 ml) was heated
under reflux for 3 h. The reaction mixture was left to cool
and the solid formed was filtered, washed with ethanol,
dried and recrystallized from ethanol to give 13.1H NMR
(DMSO-d6) d 3.38 (dd, 1H, CH, J = 16.8; 7.02 Hz), 4.05
(dd, 1H, CH, J = 16.7; 6.98 Hz), 6.21 (t, 1H, CH),
7.15–7.98 (m, 12H, Ar–H), 8.15 (s, H, pyrazole-H), 10.01
(s, H, NH, D2O-exchangeable); MS m/z (%): 404 (M?, 18),
63 (100).
5-[2-(4-Fluorophenyl)hydrazono]-4-methyl-2-{[(1-phenyl-
3-(thiophen-2-yl)-1H-pyrazol-4-yl)methylene]hydrazono}-
2,5-dihydrothiazole (9)
1
IR (KBr) mmax/cm-1 3199 (NNH); H NMR (DMSO-d6) d
2.67 (s, 3H, CH3), 7.38–7.92 (m, 12H, Ar–H), 8.08 (s, 1H,
pyrazole-H), 9.01 (s, 1H, CH = N), 10.56 (s, 1H, NH,
D2O-exchangeable); MS m/z (%): 487 (M?, 8), 62 (100%).
3-(4-Chlorophenyl)-5-(1-phenyl-3-(thiophen-2-yl)-1H-
pyrazol-4-yl)-N-p-tolyl-4,5-dihydro-1H-pyrazole-1-
carbothioamide (14)
2-{2-[(1-Phenyl-3-(thiophen-2-yl)-1H-pyrazol-4-
yl)methylene]hydrazinyl}thiazolo[4,5-b]quinoxaline (10)
A mixture of 13 (0.4 g, 1 mmol), p-tolyl isothiocyanate
(1 mmol) in absolute ethanol (20 ml) was heated under
reflux for 6 h. The solid was filtered, washed with ethanol,
dried, and recrystallized from EtOH-DMF (1:2 by volume)
to give 14. 1H NMR (DMSO-d6) d 2.46 (s, 3H, CH3), 3.36
(dd, 1H, CH, J = 16.8; 7.02 Hz), 4.02 (dd, 1H, CH,
J = 16.7; 6.98 Hz), 6.23 (t, 1H, CH), 7.16–7.97 (m, 16H,
1
IR (KBr) mmax/cm-1 3218 (NNH); H NMR (DMSO-d6) d
7.23–7.90 (m, 12H, Ar–H), 8.79 (s, 1H, pyrazole-H), 9.11
(s, 1H, CH = N), 11.15 (s, 1H, NH, D2O-exchangeable);
MS m/z (%): 453 (M?, 2), 76 (100%).
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Med Chem Res (2012) 21:1418–1426
DPPH free radical and scavenging activity
Ar–H), 8.16 (s, H, pyrazole-H), 10.11 (s, H, NH, D2O-
exchangeable); MS m/z (%): 554 (M?, 3), 63 (100).
The hydrogen atom or electron donation ability of the
corresponding compounds was measured from the
bleaching of purple colored of methanolic solution of
DPPH. This spectrophotometric assay uses stable radical
diphenylpicrylhydrazyl (DPPH) as a reagent (Cuendet
et al., 1997; Burits and Bucar, 2000). Different concen-
trations of the tested compounds were dissolved in meth-
anol to obtain a final concentration (25, 50, 50, 250, 500,
750, and 1000 lg/ml) to determine CI50 (concentrations
make 50% inhibition of DPPH color). Fifty microliters of
various sample concentrations were added to 5 ml of
0.004% methanolic solution of DPPH. After 60 min
of incubation at dark, the absorbance was measured against
a blank at 517 nm. Inhibition free radical DPPH in percent
(I %) was calculated using Eq. 2:
Antimicrobial activity
The synthetic compounds were tested for their antimicro-
bial activities against the following microorganisms:
Staphylococcus aureus ATCC 29213; B. subtilis
ATCC6633; Salmonella typhi; Enterobacter Cloaca
ATCC3047; Klebseilla peneumoniae ATCC13883; Pseu-
domonas. Aeroginosa ATCC27953; E. coli ATCC 25922;
Enterococcus faecalis ATCC29212; Mycobacterium phlei;
Saccharomyces Cervesia, and Candida Albicans NRRL
Y-477. The agar well diffusion method (Perez et al., 1990)
was applied using 100 ll of suspension containing 1 9 108
CFU/ml of pathological tested bacteria, 1 9 106 CFU/ml
of yeast, and 1 9 104 spore/ml of fungi spread on nutrient
agar (NA), Sabourand dextrose agar (SDA), and potato
dextrose agar (PDA) medium, respectively. After the
media had cooled and solidified, wells (10 mm in diameter)
were made in the solidified agar and loaded with 100 ll of
tested compound solution prepared by dissolving 50 mg of
the chemical compound in one ml of dimethyl sulfoxide
(DMSO) as solvent. The inculcated plates were then
incubated for 24 h at 37ꢁC for bacteria and 48 h at 28ꢁC for
fungi. Negative controls were prepared using DMSO
employed for dissolving the tested compound. Ciproflox-
acin (50 lg/ml) and Ketoconazole (50 lg/ml) were used as
standards for antibacterial and antifungal activity, respec-
tively. After incubation time, antimicrobial activity was
evaluated by measuring the zone of inhibition against the
test organisms and compared with that of the standard. The
observed zone of inhibition is presented in Table 2. The
experiment was carried out in triplicate and the average
zone of inhibition was calculated.
À
Á
I% ¼ Ablank ꢂ Asample=Ablank ꢃ 100
ð2Þ
where Ablank is the absorbance of the control reaction, and
Asample is the absorbance of the test sample.
Anti-inflammatory
Animals
Female Sprague-Dawley rats (150–200 g) were used in the
study of anti-inflammatory activity. Both sexes of Swiss
mice (25–30 g) were used in analgesic activity, taking into
account international principle and local regulations con-
cerning the care and use of laboratory animals. The animals
had free access to standard commercial diet and water
ad libitum, and were kept in rooms maintained at 22 1ꢁC
with 12 h light dark cycle.
Anti-inflammatory activity (carrageenan-induced rat hind
paw edema model)
Minimal Inhibitory Concentration (MIC)
The method adopted resembles essentially that described
by (Olfert et al., 1993); distilled water was selected as
vehicle to suspend the standard drugs and the synthesized
compounds. The albino rats weighing between 150 and
180 g was starved for 18 h before the experiment. The
animals were weighed, marked for identification, and
divided into 15 groups each group containing five animals.
Edema was induced in the left hind paw of all rats by
subcutaneous injection of 0.1 ml of 1% (W/V) carrageenan
in distilled water into their footpads. The first group was
kept as control and was given the prescribed volume of the
solvent (0.5 ml distilled water). The 2nd–14th groups were
orally administered aqueous suspension of the synthesized
compounds (dose; 20 mg/kg) 1 h before carrageenan
injection. The last group (standard) was administered
The bacterio-static activity of the active compounds
(having inhibition zones (IZ) C 16 mm) was then eval-
uated using the two fold serial dilution technique (Scott,
1989). Two fold serial dilutions of the tested compound
solutions were prepared using the proper nutrient broth.
The final concentrations of the solutions were 333.3;
166.6; 83.3 41.6; and 20.8 lg/ml. The tubes were
then inoculated with the test organisms, grown in their
suitable broth at 37ꢁC for 24 h, which contain
1 9 108 CFU/ml of bacteria; 1 9 106 CFU/ml of yeast;
and 1 9 104 spore/ml of fungi), each 5 ml received
0.1 ml of the above inoculum and incubated at 37ꢁC for
24 h. The lowest concentration showing no growth was
taken as the MIC.
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4-h post administration of aqueous suspension of the syn-
thesized compounds. The edema rate and inhibition rate of
each group were calculated as follows, (Edema rate
(E)% = Vt – V0/V0, Inhibition rate (I)% = Ec - Et/Ec
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(Collier et al., 1968). One hundred and two mice were
divided into 15 groups (5 in each group) starved for 16 h
pretreated as follows: the first group which served as
control positive was orally administered distilled water in
appropriate volumes. The 2nd–14th groups were adminis-
tered the aqueous suspension of synthesized compounds
orally at a specified dose (20 mg/kg). The last group was
orally administered diclofenac sodium in a dose of 20 mg/
kg. After 30 min, each mouse was administrated 0.7% of
an aqueous solution of acetic acid (10 ml/kg) and the mice
were then placed in transparent boxes for observation. The
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