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

Article abstract of DOI:10.1002/ardp.200500197

Some hydroxypyrazole derivatives 2-7 were synthesized by cyclocondensation of the keto-ester 1 with hydrazines hydrate or substituted hydrazines followed by reduction and acylation with acetic anhydride or trifluoroacetic anhydride. The newly synthesized

Full text of DOI:10.1002/ardp.200500197

Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
A. A. Bekhit et al.
81
Full Paper
Synthesis and Biological Evaluation of Some Hydroxypyrazole
Derivatives as Anti-inflammatory-Antimicrobial Agents
Adnan A. Bekhit1, Hamdy M. Abdel-Rahman2, Aida A. Guemei3
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2 Pharmaceutical Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt
3 Department of Pharmacology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt
Some hydroxypyrazole derivatives 2–7 were synthesized by cyclocondensation of the keto-ester
1 with hydrazines hydrate or substituted hydrazines followed by reduction and acylation with
acetic anhydride or trifluoroacetic anhydride. The newly synthesized compounds were evalua-
ted for their anti-inflammatory, antimicrobial activities. In addition, the ulcerogenic and acute
toxicity profiles were determined. Compounds N-(4-(5-hydroxy-1-trifluoroacetyl-1H-pyrazol-3-
yl)phenyl) trifluoroacetamide 4b, 3-(4-nitrophenyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-ol 5b, and
N-(4-(5-hydroxy-1-methyl-1H-pyrazol-3-yl)phenyl)trifluoroacetamide 7b were proved to be the
most active anti-inflammatory, antimicrobial agents in the present study with a good safety
margin and minimal or no ulcerogenic effect.
Keywords: Hydroxypyrazole / Anti-inflammatory / Antimicrobial / Ulcerogenic effect / Acute Toxicity /
Received: August 28, 2005; accepted: October 9, 2005
DOI 10.1002/ardp.200500197
Introduction
Identification of novel compounds which treat both
infectious and inflammatory states more effectively and
which lack side effects associated with current therapies
remains a major challenge in biomedical research. The
use of several drugs to treat inflammatory conditions
associated with infection is a problem, especially in case
of patients with impaired liver or kidney functions, or to
avoid drug-drug interaction. In addition, from the phar-
macoeconomic cost-effective stand-point and seeking for
a better patient compliance, a dual anti-inflammatory,
antimicrobial agent with minimum adverse effects and a
high safety margin is highly desirable. This promoted us
searching for agents that have a dual effect as anti-
Figure 1. Structures of reported active pyrazole derivatives A,
B, C, and D.
inflammatory, antimicrobial agents. Compounds con-
taining pyrazole functionality have been reported to
exhibit anti-inflammatory activity [1–5]. Moreover, Tani-
tame et al. reported the antimicrobial activities of several
pyrazole derivatives [6–8]. We have already reported the
anti-inflammatory and the antimicrobial activities of
some lead compounds containing the pyrazole moiety
attached to different heterocyclic rings [9-17]. Some of
our reported compounds A [11], B [12], C [13, 14], and D
[15] showed pronounced dual anti-inflammatory, antimi-
Correspondence: Adnan A. Bekhit, Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria
21521, Egypt.
E-mail: adnbekhit@hotmail.com
Fax: +20 3 487-3273
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82
A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
Scheme 1. Synthesis route of target compounds.
crobial activity (Figure 1). The present investigation deals was allowed to react with substituted hydrazines to
with the synthesis of some hydroxypyrazole derivatives afford the pyrazole derivatives 5a, 5b. The amino deriv-
in order to investigate their anti-inflammatory and anti- atives 6a, 6b were obtained by treating 5a, 5b with hydro-
microbial activities. It should be pointed out that, in gen in the presence of palladium and carbon. Acylation
addition to the targeted anti-inflammatory and antimi- of compound 6a, 6b with acetic anhydride or trifluoroa-
crobial activities, the ulcerogenic and acute toxicity pro- cetic anhydride gave the acylated compounds 7a–7d. It is
files of the newly synthesized compounds were deter- worth to mention that the newly synthesized pyrazole
mined. The results revealed that some derivatives showed compounds are present in the hydroxypyrazole form
promising activities.
rather than the pyrazolone tautomers as revealed from
IR and1H-NMR spectra.
Results and discussion
Biological screening
Chemistry
The target compounds were synthesized according to the Anti-inflammatory activity
presented Scheme 1. Cyclocondensation of the key inter- Cotton pellet-induced granuloma bioassay
mediate keto-ester 1 with hydrazine hydrate resulted in The anti-inflammatory activity of the synthesized com-
pyrazole compounds 2. The nitro group of the latter com- pounds 2–7 was evaluated applying the cotton-pellet
pound was reduced with hydrogen in the presence of pal- granuloma bioassay in rats [18] using indomethacin as a
ladium and carbon to give rise to amino compound 3. reference standard. The ED50 values were determined for
Acylation of compound 3 with acetic anhydride or tri- each compound. Data were expressed as the mean € SEM.
fluoroacetic anhydride gave the diacylated compounds Significant difference between the control and the trea-
4a, 4b. On the other hand, the key compound keto-ester 1 ted groups was evaluated using Student's t-test (Table 1).
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Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
Anti-inflammatory-Antimicrobial Hydroxypyrazoles
83
Table 1. The anti-inflammatory (ED50 [lmol])* and ulcerogenic
activities* of test compounds.
Table 2. Effect of compounds 3, 4b, 5b, and 6b on carragee-
nan-induced rat paw edema [mL], % protection, and activity rela-
tive to indomethacin.
Test compound
ED50 [lmol]
% Ulceration
Test
compound
Increase in paw % Protection Activity relative
Indomethacin
2
3
9.68
11.44
9.84
10.82
9.96
11.76
9.94
11.36
11.34
14.48
8.34
100
0.0
0.0
10
0.0
10
edema
to Indometha-
cin
[mL] l SEMa, b)
4a
4b
5a
5b
6a
6b
7a
7b
7c
7d
Control
Indomethacin 0.25 l 0.024
3
4b
5b
7b
0.98 l 0.027
0.0
74.4
68.3
73.4
71.4
77.5
0.0
100
91.80
98.55
95.96
104.16
0.31 l 0.026
0.26 l 0.015
0.28 l 0.012
0.22 l 0.036
10
0.0
0.0
10
0.0
0.0
10
a)
b)
SEM denotes the standard error of the mean.
All data are significantly different from control (P a 0.001).
12.46
13.28
*
All data are significantly different from control (P a 0.001).
under the same experimental conditions (Table 1). Gross
observation of the isolated rat stomachs showed a nor-
The difference in results was considered significant mal stomach texture for compounds 2, 3, 4b, 6a, 6b, 7b,
when P a0.001 (Table 1). All test compounds showed sig-
nificant anti-inflammatory activity. Compounds 3, 4b,
and 7c (0% ulceration).
5b, and 7b (ED50 = 9.84, 9.96, 9.94, and 8.34 lmol, respec- Acute toxicity
tively) possessed anti-inflammatory activity comparable Compounds 3, 4b, 5b, and 7b were further evaluated for
to that of indomethacin (ED50 = 9.68 lmol). The amino
derivative 3 showed better anti-inflammatory activity
their oral acute toxicity in male mice using the method
described in the literature [21, 22]. The results indicated
than its nitro-compound precursor 2. On other hand, acy- that most of the tested compounds proved to be non-
lation of the amino group of 3 did not affect the activity.
Moreover, reduction of nitro compound 5b greatly
toxic and well tolerated by the experimental animals up
to 120 mg/kg. Moreover, these compounds were tested
reduced the activity, whereas the activity of amino com- for their toxicity through parenteral route [12]. The
pound 6a was greatly enhanced after introduction of tri- results revealed that all the test compounds were non-
flouroacetyl group to form compound 7b. In general, the
trifluoroacetyl derivatives have higher activity than the
acetyl derivatives.
toxic up to 55 mg/kg.
Antimicrobial activity
The designed compounds 2–7 have been evaluated for
their antimicrobial activity. The microdilution suscept-
ibility test in Mꢀller-Hinton Broth (Oxoid) and Sabouraud
Carrageenan-induced rat paw edema
Compounds 3, 4b, 5b, and 7b that showed anti-inflamma-
tory activity comparable to that of indomethacin in the Liquid Medium (Oxoid) were used for the determination
cotton pellet-induced granuloma bioassay, were further of antibacterial and antifungal activity [23]. The minimal
evaluated for their in vivo systemic effect using the carra-
geenan-induced paw edema bioassay in rats [19]. The
inhibitory concentration (MIC) listed in Table 3 showed
that although all test compounds have antifungal activ-
results (Table 2) revealed that test compounds 3, 4b, 5b, ity lower than that of clotrimazole (Canestenm, Bayer). All
and 7b exhibited systemic anti-inflammatory activity (%
protection 68.3, 73.4, 71.4, and 77.5, respectively) com-
parable to that of indomethacin (% protection 74.4).
test compounds possessed lower activity against B. subtilis
than that of ampicillin but active against E. coli and S.
typhimurium. Compounds 3, 4a, 4b, 5a, 6a, 6b, and 7b pos-
sessed half-fold the activity of ampicillin against E. coli,
while 5b showed comparable activity to that of ampicil-
lin against E. coli. On the other hand, compounds 4a, 4b,
Ulcerogenic effects
All synthesized compounds were evaluated for their
ulcerogenic potential in rats [20]. All the test compounds 5b, and 6b showed half-fold the activity of ampicillin
proved to have a superior GI safety profile (0–10% ulcera- against S. typhimurium. The amino derivative 3 and its
tion) in the population of the test animals at oral doses of
30 mmol/kg per day when compared with indomethacin.
acylated derivatives 4a and 4b showed better antibacter-
ial activity than their nitro-compound precursor 2
This reference drug was found to cause 100% ulceration against E. coli, while reduction of nitro compound 5b
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A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
plet; m: multiplet. Elemental analyses were performed on Per-
kin-Elmer 2400 elemental analyzer (Perkin-Elmer, Hitachi,
Tokyo, Japan) and were found within l0.4% of the theoretical
values. Follow up of the reactions and checking the purity of the
compounds were made by TLC on silica gel-precoated alumi-
nium sheets (Type 60 GF254, Merck) and the spots were detected
by exposure to UV lamp at k 254 nm for few seconds.
Table 3. Minimal inhibitory concentration (MIC) of test com-
pounds [lg/mL].
Test
compound
E. coli
S. typhimu- S. aureus B. subtilis C. albicans
rium
Ampicillin
25
12.5
12.5
12.5
Clotrimazole
2
3
12.5
A200
100
50
50
50
50
25
50
50
100
50
100
50
25
25
100
25
50
25
A200
50
100
100
A200
A200
100
100
50
100
100
A200
100
100
100
100
A200
100
A200
A200
A200
A200
A200
A200
A200
A200
A200
A200
A200
3-(4-Nitrophenyl)-1H-pyrazol-5-ol (2), 1-methyl-3-(4-
nitrophenyl)-1H-pyrazol-5-ol 5a and 1-(4-methoxy-
phenyl)- 3-(4-nitrophenyl)-1H-pyrazol-5-ol 5b
A mixture of ethyl 3-(4-nitrophenyl)-3-oxopropanoate 1 (4.74 g,
20 mmol) and hydrazine hydrate, methyl hydrazine, or 4-meth-
oxyphenylhydrazine (24 mmol) in ethanol (40 mL) was heated
under reflux for 4 h. The reaction mixture was allowed to attain
room temperature; the precipitated solid product was filtered,
washed with water and crystallized from ethanol (Table 4).
4a
4b
5a
5b
6a
6b
7a
7b
7c
7d
100
A200
A200
100
A200
A200
A200
A200
A200
100
A200
50
3-(4-Aminophenyl)-1H-pyrazol-5-ol (3), 3-(4-
aminophenyl)-1-methyl-1H-pyrazol-5-ol 6a and 3-(4-
reduced its activity against E. coli. Although reduction of
nitro compound 2 resulted in active amino compound 3
against S. typhimurium, the activity was increased after
acylation of 3. Furthermore, reduction of nitro com-
pound 5b to amino compound 6b did not affect the activ-
ity against S. typhimurium.
It can be safely concluded that compound 3, 4b, 5b,
and 7b were proved to be the most active anti-inflamma-
tory, antimicrobial agents in the present study with
minimal or no ulcerogenic effect and a good safety mar-
gin. Compound 5b showed anti-inflammatory activity
comparable to that of indomethacin with minimal
ulcerogenic effect and no toxicity up to 120 mg/kg orally.
In addition, its antibacterial activity against E. coli is com-
parable to that of ampicillin, while its activity against S.
typhimurium is about 50% that of ampicillin.
aminophenyl)-1-(4-methoxyphenyl)-1H-pyrazol-5-ol 6b
To a solution of the appropriate nitro compound 2, 5a, or 5b
(10 mmol) in methanol (100 mL), was added 0.5 g of 10% Pd/C.
The mixture was stirred overnight under a balloon of hydrogen
gas. The reaction mixture was filtered over celite, followed by
evaporation of the filtrate under reduced pressure. The obtained
solid product was crystallized from ethanol (Table 4).
N-(4-(5-Hydroxy-1-substituted-1H-pyrazol-3-
yl)phenyl)acetamides 4a, 7a, 7c and N-(4-(5-hydroxy-1-
substituted-1H-pyrazol-3-yl)phenyl)trifluoroacetamides
4b, 7b, 7d
Acetic anhydride or trifluoroacetic anhydride (25 mmol) was
added slowly at room temperature to the selected amino com-
pound 3, 6a, or 6b (10 mmol), followed by warming the mixture
till dissolution of all solids. The reaction mixture was stirred at
room temperature for 15 min, then water was added and
allowed to stand for overnight. The separated white solid prod-
uct was filtered, washed with water, and crystallized from etha-
nol (Table 4).
The authors wish to thank Dr. Elsayed Aboulmagd, Depart-
ment of Microbiology, Faculty of Pharmacy, University of Alex-
andria, Egypt for his assistance during antimicrobial testing.
In case of compounds 4a, 4b the mixture was stirred at room
temperature with 2N NaOH (5 mL) for 5 h followed by neutrali-
zation with diluted HCl before the crystallization step.
Biological screening
Experimental
Chemistry
Anti-inflammatory testing
All chemicals were purchased from E. Merck (Whitehouse Sta-
tion, NJ, USA), Fluka AG (Buchs SG, Switzerland), TCI (Tokyo,
Japan), and Aldrich (St. Louis, MO, USA). Melting points were
determined in open glass capillaries using a Thomas capillary
melting point apparatus (Philadelphia, PA, USA) and are uncor-
rected. Infrared (IR) spectra were recorded on 470-Shimadzu
infrared spectrophotometer using the KBr disc technique (Shi-
Adult male Sprague-Dawley rats (120–140 g) were used (Medi-
cal Research Institute, Alexandria University). They were accli-
mated 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 (6 rats each).
Cotton pellet (35 l 1 mg) cut from dental rolls were impreg-
nated with 0.2 mL (containing 0.01 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
1
madzu Corp., Kyoto, Japan). H-NMR spectra were recorded on
JEOL JNM-AL 300 FT NMR spectrometer (DMSO-d6) (JEOL, Tokyo,
Japan), and the chemical shifts are given in d (ppm) downfield
from tetramethylsilane (TMS) as an internal standard. Splitting
patterns were designated as follows: s: singlet; d: doublet; t: tri-
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Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
Anti-inflammatory-Antimicrobial Hydroxypyrazoles
85
Table 4. Physical and analytical data of compounds 2–7.
Comp.
No
Yield
[%]
Mp.
[8C]
Mol. Formula
(Mol. wt.)
IR (KBr,
[cm-1])
1H NMR (DMSO-d6)
2
76
248-9
C9H7N3O3
(205.17)
3530 (OH), 3390
(NH), 1627(C=N),
5.63 (s, 1H, pyr-C4-H), 6.54 (d, 2H, J = 9 Hz, phenyl C2,6-H), 6.62 (s,
1H, NH, D2O exchangeable), 7.35 (d, 2H, J = 9 Hz, phenyl C3,5-H),
1562, 1343 (NO2) 10.59 (brs, 1H, OH, D2O exchangeable)
3
65
274-5
C9H9N3O
(175.18)
3525 (OH), 3385,
5.21 (brs, 2H, NH2, D2O exchangeable), 5.61 (s, 1H, pyr-C4-H),
3210 (NH2), 1625 6.55 (d, 2H, J = 9 Hz, phenyl C3,5-H), ), 6.68 (s, 1H, NH, D2O ex-
(C=N).
changeable), 7.32 (d, 2H, J = 9 Hz, phenyl C2,6-H), 10.14 (brs, 1H,
OH, D2O exchangeable)
4a
4b
84
86
268-9
262-4
C13H13N3O3
(259.26)
3515 (OH), 3395
2.42 (s, 3H, CH3), 2.48 (s, 3H, CH3), 5.78 (s, 1H, pyr-C4-H), 6.87 (s,
(NH), 1693 (C=O), 1H, NH, D2O exchangeable), 7.65 (d, 2H, J = 9 Hz, phenyl C3,5-H),
1660 (C=O), 1628 7.75 (d, 2H, J = 9 Hz, phenyl C2,6-H), 10.57 (brs, 1H, OH, D2O ex-
(C=N)
changeable)
C13H7F6N3O3
(367.20)
3515 (OH), 3390
(NH), 1720 (C=O), (brs, 1H, OH, D2O exchangeable)
5.94 (s, 1H, pyr-C4-H), 6.97–7.85 (m, 5H, phenyl –H, NH), 11.26
1698 (C=O), 1624
(C=N)
5a
5b
81
83
253-4
220-2
C10H9N3O3
(219.19)
3525 (OH), 1624
3.59 (s, 3H, pyr-CH3), 5.96 (s, 1H, pyr-C4-H), 7.94 (d, 2H, J = 9 Hz,
(C=N), 1573, 1347 phenyl C2,6-H), 8.19 (d, 2H, J = 9 Hz, phenyl C3,5-H), 10.57 (brs,
(NO2)
1H, OH, D2O exchangeable)
C16H13N3O4
(311.292)
3530 (OH), 1623
3.84 (s, 3H, CH3), 5.98 (s, 1H, pyr-C4-H), 6.97 (d, 2H, J = 9 Hz,
(C=N), 1567, 1350 methoxyphenyl C3,5-H), 7.73 (d, 2H, J = 9 Hz, methoxyphenyl
(NO2)
C2,6-H), 7.96 (d, 2H, J = 9 Hz, nitrophenyl C2,6-H), 8.22 (d, 2H, J = 9
Hz, nitrophenyl C3,5-H), 10.96 (brs, 1H, OH, D2O exchangeable)
3.59 (s, 3H, pyr-CH3), 5.12 (brs, 2H, NH2, D2O exchangeable),
6a
6b
67
61
240-2
245-6
C10H11N3O
(189.21)
3525 (OH), 3392,
3228 (NH2), 1631 5.53 (s, 1H, pyr-C4-H), 6.52 (d, 2H, J = 9 Hz, phenyl C3,5-H), 7.33
(C=N)
(d, 2H, J = 9 Hz, phenyl C2,6-H), 10.81 (brs, 1H, OH, D2O ex-
changeable)
C16H15N3O2
(281.30)
3525 (OH), 3395,
3.82 (s, 3H, CH3), 5.14 (s, 2H, NH2, D2O exchangeable), 5.94 (s,
3220 (NH2), 1624 1H, pyr-C4-H), 6.69 (d, 2H, J = 9 Hz, aminophenyl C3,5-H), 6.93 (d,
(C=N)
2H, J = 9 Hz, aminophenyl C2,6-H), 7.55 (d, 2H, J = 9 Hz, methoxy-
phenyl C3,5-H), 7.83 (d, 2H, J = 9 Hz, methoxyphenyl C2,6-H),
10.89 (brs, 1H, OH, D2O exchangeable)
7a
74
260-1
C12H13N3O2
(231.251)
3520 (OH), 3385
2.36 (s, 3H, CH3), 3.56 (s, 3H, pyr-CH3), 5.89 (s, 1H, pyr-C4-H),
(NH), 1663 (C=O), 7.61–7.78 (m, 5H, phenyl-H, NH), 11.32 (s, 1H, OH, D2O ex-
1630 (C=N)
changeable)
7b
7c
69
76
246-8
254-6
C12H10F3N3O2
(285.22)
3525 (OH), 3385
3.53 (s, 3H, pyr-CH3), 5.82 (s, 1H, pyr-C4-H), 7.65–7.74 (m, 5H,
(NH), 1708 (C=O), phenyl-H, NH), 11.25 (s, 1H, OH, D2O exchangeable)
1628 (C=N)
3520 (OH), 3380
C18H17N3O3
(323.34)
2.29 (s, 3H, CH3), 3.85 (s, 3H, CH3), 5.96 (s, 1H, pyr-C4-H), 6.86–
(NH), 1669 (C=O), 7.79 (m, 9H, phenyl-H, NH), 10.98 (brs, 1H, OH, D2O exchange-
1632 (C=N)
able)
7d
72
242-4
C18H14F3N3O3
(377.31)
3525 (OH), 3383
3.83 (s, 3H, CH3), 5.94 (s, 1H, pyr-C4-H), 6.78–7.93 (m, 9H, phe-
(NH), 1696 (C=O), nyl-H, NH), 11.05 (s, 1H, OH, D2O exchangeable)
1626 (C=N)
the rat, under mild general anaesthesia. One group of animals
received the standard reference indomethacin and the antibio-
tics at the same level. Pellets containing only the antibiotics
were similarly implanted in control rats. Seven days later, the
animals were sacrificed and the two cotton pellets with adher-
ing granulomas were removed, dried for 48 h at 608C, and
weighed. The increment in dry weight (difference between the
initial and final weights) was taken as a measure of granuloma
l S.E. This was calculated for each group and the percentage
reduction in dry weight of granuloma from control value was
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A. A. Bekhit et al.
Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
also calculated. The ED50 values were determined through dose-
response curves, using doses of 4, 7, 10, and 15 mmol for each
compound.
sity). Groups of mice each consisting of six animals were used.
The compounds were given orally suspended in 1% gum acacia,
in doses of 1, 10, 100, 200, 250, and 300 mg/kg, respectively. 24 h
later, the % mortality in each group and for each compound was
recorded. Moreover, these compounds were tested for their par-
enteral acute toxicity, groups of mice each consisting of six ani-
mals were used. The compounds or their vehicle, propylene gly-
col (control) were given by intraperitoneal injection in doses of
10, 25, 50, 75, and 100 mg/kg, respectively. Survival was fol-
lowed up to 7 days [12].
Carrageenan-induced rat paw edema
Male albino rats weighing 120–150 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 lL of 2%
carrageenan solution in saline (0.9%). Indomethacin and the test
compounds were dissolved in DMSO and were injected subcuta-
neously in a dose of 10 mmol/kg body weight, 1 h prior to carra-
geenan injection. The controls were injected with DMSO. The
volume of paw edema (mL) was determined by means of water
plethysmometer immediately after injection of carrageenan
and 4 h later. The increase in paw volume between time 0 and +
4 h was measured [19]. The percentage protection against
inflammation was calculated as follows:
Antimicrobial activity
The microdilution susceptibility test in Mꢀller-Hinton Broth
(Oxoid) and Sabouraud Liquid Medium (Oxoid) were used for the
determination of antibacterial and antifungal activity [23]. Test
organisms: Escherichia coli (E. coli) ATCC 25922 and Salmonella
typhimurium (S. typhimurium) ATCC 3311 as Gram-negative bac-
teria, Staphylococcus aureus (S. aureus) ATCC 19433 and Bacillus
subtilis (B. subtilis) ATCC 1042 as Gram-positive bacteria and Can-
dida albicans (C. albicans) as a yeast fungus. Ampicillin trihydrate
and clotrimazole were used as standard antibacterial and anti-
fungal agents, respectively. Solutions of the test compounds,
ampicillin trihydrate and clotrimazole were prepared in DMSO
at concentration of 1600 mg/mL. From this stock different dilu-
tions of the compounds (800, 400, down to 6.25 mg/mL) were
prepared. The microorganism suspensions at 106 colony form-
ing unit/mL/1c concentration were inoculated to the corre-
sponding wells. Plates were incubated at 368C for 24 to 48 h. The
incubation chamber was kept sufficiently humid. At the end of
the incubation period, the minimal inhibitory concentrations
(MIC) were determined. Controls with DMSO and uninoculated
media were also maintained.
Vc ꢀ Vd=Vc6100
where Vc is the increase in paw volume in the absence of test
compound (control) and Vd is the increase of paw volume after
injection of the test compound. Data were expressed as the
mean l SEM. Significant difference between the control and the
treated groups was performed using Student's t-test and P values.
The differences in results were considered significant when P a
0.001. The anti-inflammatory activity of the test compounds
relative to that of indomethacin was also calculated.
Ulcerogenic effects
All synthesized compounds were evaluated for their ulcerogenic
potential in rats [20]. Indomethacin was used as reference stan-
dard. Male albino rats (100–120 g) were fasted for 12 h prior to
the administration of the compounds. Water was given ad libi-
tum. The animals were divided into groups, each consisting of
six animals. The control group received 1% gum acacia orally.
The other groups received indomethacin or the test compounds
orally in two equal doses at 0 and 12 h for three successive days
at a dose of 30 mM/kg 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
the stomach lesions [20]. The percentage ulceration for each
group was calculated as follows:
References
[1] A. K. Gadad, B. S. Kittur, S. G. Kapsi, C. S. Mahajanshetti,
S. B. Rajur, Arzneim. Forsch. 1996, 46, 1082–1085.
[2] T. D. Penning, J. J. Talley, S. R. Bertenshaw, J. S. Carter, P.
W. Collins, S. Docter, M. J. Graneto, L. F. Lee, J. W. Male-
cha, J. M. Miyashiro, R. S. Rogers, D. J. Rogier, S. S. Yu, G.
D. Anderson, E. G. Burton, J. N. Cogburn, S. A. Gregory, C.
M. Koboldt, W. E. Perkins, K. Seibert, A. W. Veenhuizen,
Y. Y. Zhang, P.C. Isakson, J. Med. Chem. 1997, 40, 1347–
1365.
[3] K. Tsuji, K. Nakamura, T. Ogino, N. Konishi, T. Tojo, T.
Ochi, N. Seki, M. Matsuo, Chem. Pharm. Bull. 1998, 46,
279–286.
% Ulceration ¼
[4] S. A. Beers, E. A. Malloy, W. Wu, M. Wachter, J. Ansell, M.
Singer, M. Steber, A. Barbone, T. Kirchner, D. Ritchie, D.
Argentieri, Bioorg. Med. Chem. Lett. 1997, 5, 779–786.
Number of animals bearing ulcer in a group
6100
Total number of animals in the same group
[5] D. H. Boschelli, D. T. Connor, D. A. Bornemeier, R. D.
Dyer, J. A. Kennedy, P. J. Kuipers, G. C. Okonkwo, D. J.
Schrier, C. D. Wright, J. Med. Chem. 1993, 36, 1802–1810.
Acute toxicity
[6] A. Tanitame, Y. Oyamada, K. Ofuji, Y. Kyoya, K. Suziki, H.
Ito, M. Kawasaki, K. Nagai, M. Wachi, J. Yamagishi, Bioorg.
Med. Chem. Lett. 2004, 14, 2857–2862.
The same biologically significant compounds were further inves-
tigated for their oral acute toxicity in male mice [21, 22] (each
20 g, supplied by Medical Research Institute, Alexandria Univer-
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2006, 339, 81–87
Anti-inflammatory-Antimicrobial Hydroxypyrazoles
87
[7] A. Tanitame, Y. Oyamada, K. Ofuji, K. Suziki, H. Ito, M.
Kawasaki, M. Wachi, J. Yamagishi, Bioorg. Med. Chem, Lett.
2004, 14, 2863–2866.
[16] A. A. Bekhit, E. Aboulmagd, A. M. Baraka, World Confer-
ence on Dosing of Antiinfectives, Nꢀrnberg, Germany,
Sept. 9–11th, 2004, Abst. P57.
[17] A. A. Bekhit, H. M. A. Ashour, A. Guemei, Arch. Pharm.
Chem. Life Sci. 2005, 338, 167–174.
[8] A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, N. Iwai,
Y. Hiyama, K. Suziki, H. Ito, H. Terauchi, M. Kawasaki, K.
Nagai, M. Wachi, J. Yamagishi, J. Med. Chem. 2004, 47,
3693–3696.
[18] R. Meier, W. Schuler, P. Desaulles, Experientia 1950, 6,
469.
[9] A. M. Farghaly, A. A. Bekhit, J. Y. Park, Arch. Pharm. Pharm.
Med. Chem. 2000, 333, 53–57.
[19] M. Di Rosa, D. A. Willoughby, J. Pharm. Pharmcol. 1971, 23,
297–298.
[10] A. A. Bekhit, Alazhar 2nd International Conference for
Pharmaceutical and Biological Sciences, 19–21 Decem-
ber 2001, Cairo, Egypt, Abst. P. 34.
[20] M. S. Abou Zeit-Har, T. Verimer, J. P. Long, Pharmazie
1982, 37, 593–595.
[21] M. Verma, M. Tripathi, A. K. Saxena, K. Shanker, Eur. J.
Med. Chem. 1994, 29, 941–946.
[11] A. A. Bekhit, H. T. Y. Fahmy, Sh. A. F. Rostom, A. M. Bar-
aka, Eur. J. Med. Chem. 2003, 38, 27–36.
[22] J. T. Litchfield, F. Wilcoxon, J. Pharmacol. Exp. Ther. 1949,
96, 99.
[12] A. A. Bekhit, H. T. Y. Fahmy, Arch. Pharm. Pharm. Med.
Chem. 2003, 336, 111–118.
[23] P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H.
Yolken in Manual of Clinical Microbiology, (Eds: G. L.
Woods, J. A. Washington), Antimicrobial Agents and Sus-
ceptibility Testing, Am. Soc. Microbiol., Washington, DC,
1995.
[13] A. A. Bekhit, T. Abdel-Azeim, Bioorg. Med. Chem. 2004, 12,
1935–1945.
[14] A. A. Bekhit, Egyptian Patent, GATT/TRIPS Treaty, Appl.
No. 1117, 12, 2003.
[15] A. A. Bekhit, T. Abdel-Azeim, Second International Con-
ference on New Biomedical Materials, Cardiff, Wales, UK,
5–8th April, 2003, Abst. P16.
i 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com