Synthesis of new N-phenylpyrazole derivatives with potent antimicrobial activity

Article abstract of DOI:10.1016/j.bmc.2008.02.043

The versatile synthons 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H -pyrazole (2) were used as precursors for the synthesis of a series of phenylpyrazoles with different aromatic ring systems at position 4. The antimicrobiological evaluation of the newly synthesized compounds was carried out in vitro assays for antifungal and antibacterial activities. Amongst the tested compounds, 4-acetyl-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (1), 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H -pyrazole (2), 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-(2-aminothiazol-4-yl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (17) showed interesting antimicrobial properties. In particular, all tested compounds produced inhibitory effects against pathogenic yeast (Candida albicans) similar or superior to those of reference drug. In addition, compound 3 showed excellent activity against pathogenic mould (Aspergillus). From structure-activity relationship (SAR) point of view, the attachment of bromoacetyl moiety to pyrazole ring can be considered as a breakthrough in developing a new therapeutic antifungal agent related to phenylpyrazole system.

Full text of DOI:10.1016/j.bmc.2008.02.043

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4569–4578
Synthesis of new N-phenylpyrazole derivatives
with potent antimicrobial activity
Ahmad M. Farag,a,* Abdelrahman S. Mayhoub,b
Saber E. Barakatb and Ashraf H. Bayomic
aDepartment of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Naser City, Cairo 11884, Egypt
cDepartment of Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Naser City, Cairo 11884, Egypt
Received 31 October 2007; revised 6 February 2008; accepted 12 February 2008
Available online 15 February 2008
Abstract—The versatile synthons 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-[(E)-3-(dimethyl-
amino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (2) were used as precursors for the synthesis of a series of
phenylpyrazoles with different aromatic ring systems at position 4. The antimicrobiological evaluation of the newly synthesized
compounds was carried out in vitro assays for antifungal and antibacterial activities. Amongst the tested compounds, 4-acetyl-5-
methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (1), 4-[(E)-3-(dimethylamino)acryloyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-
1H-pyrazole (2), 4-(2-bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (3) and 4-(2-aminothiazol-4-yl)-5-methyl-
1-phenyl-3-phenylcarbamoyl-1H-pyrazole (17) showed interesting antimicrobial properties. In particular, all tested compounds
produced inhibitory effects against pathogenic yeast (Candida albicans) similar or superior to those of reference drug. In addition,
compound 3 showed excellent activity against pathogenic mould (Aspergillus). From structure–activity relationship (SAR) point of
view, the attachment of bromoacetyl moiety to pyrazole ring can be considered as a breakthrough in developing a new therapeutic
antifungal agent related to phenylpyrazole system.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tion with the hydrophobic residues in the largely non-
polar active site of the enzyme.2
Fungi are causing cutaneous, sub-cutaneous or systemic
infections, such as oral thrush, Tenia pedis, Tenia corpo-
ris or Tenia capidis. The azoles (imidazoles and triazoles)
represent a class of versatile antifungal agents which are
used to treat generalized systemic fungal infections. The
mechanism of their antifungal action includes the inhibi-
tion of cytochrome P450 51 (CYP51), which is essential
for ergosterol biosynthesis at the step of lanosterol-14-
demethylation.1
In recent years, the widespread use of imidazoles and tri-
azoles antifungal agents has resulted in the development
of resistance to these drugs by pathogenic microorgan-
isms, causing an increase in morbidity and mortality.
Therefore, new trend of antifungal related to phenylpyr-
azole is developed in order to get an effective antifungal
agent without known resistance.3–17
Although many reported phenylpyrazoles showed sig-
nificant activity against pathogenic yeast (Candida),
unfortunately, no significant effect was obtained against
pathogenic moulds such as Aspergillus.3–17
The high affinity of the azole antifungals for CYP51 iso-
zymes appears to be determined primarily by their
bulky, polycyclic structure, giving favourable interac-
As part of our ongoing research programme aiming at
the synthesis of variety of heterocyclic systems for bio-
logical and pharmacological evaluation,18–27 we report
here the synthesis of several phenylpyrazoles having dif-
ferent aromatic bulky structures at position 4 in order to
increase the selectivity of this promising new group to-
wards true pathogenic fungi; for example, Aspergillus
Keywords: Phenylcarbamoylpyrazole; 1,2,4-Triazolo[4,3-a]pyrimidine;
Pyrazolo[1,5-a]pyrimidine; Imidazo[2,1-b]benzothiazole; Enaminones;
Antibacterial; Antifungal; Anticandidal; Structure–activity relation-
ship (SAR); 2-Aminothiazole.
*
Corresponding author. Tel.: +20 12377 3794; fax: +20 235727556;
e-mail: afarag49@yahoo.com
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.043

4570
A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
sp. The present study includes also antibacterial evalua-
tion of the newly synthesized compounds.
The spectral data of the product recommended the cyclic
structure 4 as its 1H NMR spectrum displayed two
broad singlets (D2O-exchangeable) at d 10.6 and at d
13.0 indicating two highly deshielded NH protons. The
two protons of the formed pyrazole ring displayed on
1H NMR spectrum as two doublets at d 7.985 and d
7.769 with the same coupling constant (J = 7.5 Hz).
The two NH groups displayed on IR spectrum of 4 as
2. Results and discussion
2.1. Chemistry
Treatment of 4-acetyl-5-methyl-1-phenyl-3-phenylcar-
bamoyl-1H-pyrazole (1) with bromine in glacial acetic
acid afforded the 4-(2-bromoacetyl)-5-methyl-1-phenyl-
3-phenylcarbamoyl-1H-pyrazole (3) in a good yield
(Scheme 1).
two broad bands at 3240 and 3417 cmÀ1
.
The 13C NMR spectrum showed one carbonyl carbon
signal at d 160.93, pyrazole-attached methyl group ap-
peared at d 12.03, other aromatic carbons arranged be-
tween d 105.59 and 138.85. Mass spectrum of compound
4 showed prominent molecular ion peak at m/z 343 as
the base peak.
The IR spectrum of compound 3 showed two strong
absorption bands at 1651 and 1705 cmÀ1 assignable to
amide and ketonic carbonyl groups, respectively. Other
important band revealed at 3244 cmÀ1 characterized for
amide NH. The 1H NMR spectrum showed a singlet at d
2.44 corresponding to pyrazole-attached methyl group
and a singlet at d 4.82 due to active methylene of bromo-
acetyl moiety. Other important signal appeared at d 8.8
(D2O-exchangeable) due to NH protons. Furthermore,
the 13C NMR spectrum of compound 3 displayed three
important signals at d 37.17, 161.22 and 189.76 corre-
sponding to the active methylene of bromoacetyl, amide
and ketonic carbonyl carbons, respectively. The mass
spectrum of compound 3 revealed molecular ion peaks
at 397 and 399 reflecting the isotopes of bromine.
When enaminone 2 was treated with hydroxylamine in
refluxing ethanol, it afforded a single product identified
as 4-(isoxazol-3-yl)-5-methyl-1-phenyl-3-phenylcarba-
moyl-1H-pyrazole (5) (Scheme 2).
1
The H NMR spectrum of compound 5 exhibited two
doublets at d 6.88 and 8.65 with (J = 2.1 Hz) due to isox-
azole protons. The mass spectrum of 5 is characterized
by the presence of molecular ion peak at m/z 344, and
a fragment ion peak at m/z 68 corresponding to isoxaz-
olyl radical cation.
On the other hand, when enaminone 2 was treated with
thiosemicarbazide in refluxing ethanol, it afforded the
open structure 4-[(E)-3-(hydrazinothiocarbamido)acry-
loyl]-5-methyl-1-phenyl-4-phenylcarbamoyl-1H-pyra-
zole (6). All attempts to cyclize the 4-side chain of 6 to
give 5-methyl-1-phenyl-4-phenylcarbamoyl-4-(1-thioc-
arbamoyl-1H-pyrazol-5-yl)-1H-pyrazole (7) using differ-
ent solvents and catalysis were unsuccessful (Scheme 2).
The reactivity of 4-[(E)-3-(dimethylamino)acryloyl]-5-
methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (2)18
towards some nitrogen nucleophiles was investigated.
Thus, the treatment of compound 2 with hydrazine
hydrate, in refluxing ethanol, afforded white crystals of
5-methyl-1-phenyl-3-phenylcarbamoyl-4-(3-pyrazolyl)-
1H-pyrazole (4) (Scheme 2).
O
O
N
H
N
N
1
Me
Me
MeO
MeO
N
Br2 /glacial acetic acid
80-90
º
C/ 30 min.
dry xylene
O
O
O
O
Br
N
H
N
N
H
N
N
N
N
2
3
Scheme 1.

A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
4571
Ar
N
N
N
O
O
N
N
N
N
Ph
N
H
N
H
N
N
N
N
Ph
Ph
9
11a,b
Ar
H
EtOH/Piperidine/
reflux, 24 h
Pyridine/ reflux,
48 h
N
NH2
-H2O
N
N
-H2O
H2N
N
8
-Me2NH
N
H
-Me2NH
10a,b
a, Ar = C6H5
b, Ar = C6H4-
pCl
2
NH2NH2 / glacial acetic acid
NH2CSNHNH2
EtOH/reflux, 7days
EtOH/reflux, 3h
- Me2NH
- H2O
NH OH
RT/ 12 h
- Me2NH
- H2O
2
- Me2NH
O
O
H
N
O
O
N
NH
N
O
Ph
NH2
N
H
N
H
Ph
Ph
N
H
N
N
S
H
N
N
N
N
N
Ph
6
Ph
4
Ph
-H2O
S
5
H2N
N
O
N
N
Ph
N
H
N
Ph
7
Scheme 2.
1
The H NMR spectrum of compound 6 revealed four
(D2O-exchangeable) singlets at d 5.85, 8.21, 8.77 and
10.38 due to amino and three amide protons,
respectively.
NMR spectrum revealed also a singlet, of one proton, at
d 8.57 corresponding to triazole H-3 proton. Its 13C
NMR spectrum revealed eighteen carbon types; the most
important signals include triazole-3,5-carbons, which ap-
peared at d 154.68 and 154.95. Finally, the spectrum
showed fourteen aromatic carbons arranged between d
110.80 and 146.12. The mass spectrum of 9 showed a
molecular ion peak at m/z 395, which loses a phenylamine
radical to give the base peak at m/z 303.
The reactivity of enaminone 2 towards some heterocy-
clic amines was also investigated. Thus, the treatment
of compound 2 with 3-amino-1,2,4-triazole (8) in the
presence of a catalytic amount of piperidine afforded
5-methyl-1-phenyl-3-phenylcarbamoyl-4-([1,2,4]triazolo
[4,3-a]pyrimidin-7-yl)-1H-pyrazole (9).
A
possible
When enaminone 2 was treated with 5-amino-1H-pyra-
zole derivatives 10a,b in refluxing pyridine, it afforded
the corresponding 5-methyl-4-(3-methyl-4-phenylpyraz-
olo[1,5-a]pyrimidin-6-yl)-1-phenyl-3-phenylcarbamoyl-1H-
pyrazole (11a) and its chloro derivative 11b (Scheme 2).
mechanism for the formation of compound 9 may in-
volve an initial Michael-type addition of the amino
group of 3-amino-1,2,4-triazole to the activated double
bond in enaminone 2 followed by the elimination of
dimethylamine and water molecules (Scheme 2).
The formation of products 11 is assumed to take place
via the addition of the amino group in aminopyrazoles
10a,b to a,b-unsaturated moiety in enaminone 2 fol-
lowed by the elimination of water and dimethylamine
molecules to give the final products 11a,b. The struc-
tures of compounds 11a,b were established on the basis
of their elemental and spectral data (see Section 3).
The absorption band of carbonyl group at position 4 dis-
appeared in the IR spectrum of compound 9 in compari-
son with the IR spectrum of 2. The 1H NMR spectrum of 9
showed two doublets, each of one proton, in the region of
d 7.49–8.96 with (J = 4.5 Hz), which can be attributed to
1
the two adjacent protons of pyrimidine ring. The H

4572
A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
O
S
O
O
Ph
O
N
N
H
N
Ph
N
O
N
H
N
N
Ph
N
19
Ph
16
O
O
CHCl3/ ref. 1h
K+ K2CO3
N
EtOH/ ref. 48h
- H2O
NH2
15
O
O
O
S
O
17
Br
18
PhHN
N
o-Phenylendiamine
Thiourea
N
EtOH/ ref. 1h
Ph
3
EtOH/ ref. 4h
- H2O
NH2
O
HN
O
N
S
Ph
NH
Ph
N
H
N
H
N
N
N
N
Ph
13
Ph
12
Oxidation
O
N
Ph
N
N
H
N
N
Ph
14
Scheme 3.
Next, when bromoacetylpyrazole 3 was treated with
thiourea in refluxing ethanol, it afforded a product iden-
tified as 4-(2-aminothiazol-4-yl)-5-methyl-1-phenyl-3-
phenylcarbamoyl-1H-pyrazole (12) (Scheme 3). The IR
spectrum of product 12 showed three characteristic
absorption bands, two bi-forked bands at 3355 and
3290 cmÀ1 assignable to amino group and a band at
A plausible mechanism may involve the condensation of
one of phenylenediamine amino groups with the car-
bonyl group of bromoacetyl moiety, while the second
amino group replaced bromine atom via nucleophilic
substitution. The expected product is the dihydroqui-
noxalinyl derivative 13; however, the spectral data of
the isolated product established that the dihydroquinox-
alinyl derivative 13 was oxidized under the reaction con-
ditions to give 5-methyl-1-phenyl-3-phenylcarbamoyl-4-
(quinoxalin-2-yl)-1H-pyrazole (15).
1
3190 cmÀ1 due to the amide NH group. Its H NMR
spectrum revealed a singlet, of one proton, appeared
in aromatic region at 6.77 corresponding to thiazole-5-
CH, and D2O-exchangeable singlets at d 7.553 and d
10.86 corresponding to amino and amide protons,
respectively. The 13C NMR spectrum of the same com-
pound revealed sixteen carbon types; the most impor-
tant signals appeared at d 104.98 corresponding to C5
of thiazole ring, d 160.6 and 168.03 characterized for
amide carbonyl and thiazole-C2. The mass spectrum
of 12 showed a molecular ion peak at m/z 374.
The IR spectrum of product 15 showed one absorption
band at 3274 cmÀ1 assignable to one NH group. The 1H
NMR spectrum showed a singlet, of one proton, at d
9.12 due to quinoxaline-3-CH. The presence of 19 aro-
matic carbon types on 13C NMR spectrum of the iso-
lated product between d 118.29 and 148.29, in addition
to one aliphatic carbon at d 11.34 and one carbonyl car-
bon at d 160.42, confirmed structure 15.
Treatment of compound 3 with o-phenylenediamine in
refluxing ethanol afforded a crystalline product identi-
fied as 5-methyl-1-phenyl-3-phenylcarbamoyl-4-(qui-
noxalin-2-yl)-1H-pyrazole (14) in an excellent yield
(Scheme 3).
Cyclocondensation reaction of bromoacetylpyrazole 3
with some heterocyclic amine was also examined. Thus,
when compound 3 was allowed to react with 2-amino-
benzothiazole (15) in ethanol, at reflux temperature, it

A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
4573
a
NH2
O
HN
O
HO
HN
O
NH
HN
Ph
O
NH O
NH
O
NH2
O
O
NH
NH2
b
O
HN
HN
NH2
OHNH
O
O
O
O
Ph
HN
Br
HN
N
N
Ph
NH2
Figure 1. Chelation parts of polymyxin B1 and compound 3.
afforded 4-(imidazo[2,1-b]benzothiazole-3-yl)-5-methyl-1-
phenyl-3-phenylcarbamoyl-1H-pyrazole (16) (Scheme 3).
vious assumption can be excluded. The second
assumption is that this compound exerts its action on
bacteria and fungi via two different mechanisms; to con-
firm this suggestion, further study is in progress based
on molecular modeling.
The preliminary antimicrobial test showed unexpected
result that the phenylpyrazole derivative that carries
the least non-polar side chain, that is, bromoacetylpy-
razole 3, has good activity against both fungi and bacte-
ria, which is assumed that it exerts its action via
chelation of some vital cellular divalent cations as
observed in other anti-infective agents such as poly-
2.2. Biological testing
2.2.1. Antimicrobial screening. The results of the antimi-
crobial evaluation of the tested compounds are presented
in Table 1. They showed that most of the tested com-
pounds have a significant antibacterial effect against
Staphylococcus aureus (S. A.), and Pseudomonas aerugin-
osa (P. A.). Acetylpyrazole 1 showed the highest potency
against Gram-positive bacteria S. A., while pyrazolopyr-
imidines 11a,b exhibited the lowest activity. Antipseudo-
monal activity was observed with compounds 1, 3 and 12
(Table 1 and Fig. 2). These compounds also possess a po-
tent antifungal activity against Candida albicans (C. A.),
mostly higher than that of reference drug (terbinafine).
However, they have no effect against Aspergillus fumiga-
tus (A. F.) except compound 3. The latter compound
was found to be more effective than terbinafine against
A. F. (Table 1 and Fig. 3). Moreover, compound 3 showed
an antifungal activity against C. A., 1.6 higher than that of
the reference drug.
28
myxin B1 (Fig. 1a). In other words, the bromoacetyl
moiety acts as a chelating part (Fig. 1b). Therefore, we
decided to synthesize a phenylpyrazole derivative with
a strong chelating moiety at position 4 to prove this
assumption. Thus bromoacetylpyrazole 3 was allowed
to react with 5,5-dimethylcyclohexane-1,3-dione (dime-
done) (17) in refluxing chloroform in the presence of
potassium carbonate, which reaction afforded a product
identified as 4-[2-(4,4-dimethyl-2,6-dioxocyclohexyl)-1-
oxoethyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyra-
zole (19).
The suggested reaction mechanism involves the conver-
sion of dimedone (17) into its enolate potassium salt 18
in the presence of potassium carbonate. The latter inter-
mediate is considered as a strong carbon nucleophile
that can replace bromine atom from bromoacetylpyraz-
ole 3 to produce the final product 19 as shown in Scheme
Based on the antimicrobial evaluation, compound 3 was
selected for further assessment. The minimum inhibitory
concentration (MIC) of compound 3 against A. F. was
25 lg/ml, which is less than that of miconazole and high-
er than amphoteracin B as reference drugs as depicted in
Table 2.
1
3. The H NMR spectrum of the reaction product dis-
played five signals at d 0.95, 2.13, 2.36, 2.48 and 3.64
corresponding to methyl and methylene groups of dime-
done, methylene protons of acetyl moiety, pyrazole-at-
tached methyl group and dimedone-CH, respectively.
The mass spectrum of compound 19 is characterized
by the presence of molecular ion peak at m/z 457.
From the structure–activity relationship (SAR), we can
conclude that the bromoacetyl moiety is essential for
the activity against true fungi (moulds). Amongst the
dozens of synthesized phenylpyrazoles found in recent
The antimicrobial tests showed that the activity of com-
pound 19 is lower than compound 3; therefore, the pre-

4574
A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
Table 1. Antimicrobial activity of new compounds and reference drugs
Sample
Inhibition zone diameter (mm/mg sample)
(G –ve) Mould
(G +ve)
Yeast
S. A.
Pot.a
P. A.
17
Pot.a
A. F.
Pot.a
C. A.
Pot.a
1
21 1.3
14 0.7
1
2
0.85
0.7
1
0
0
0
15 1.2
15 1.2
16 1.2
1.5
1.5
1.6
1.2
1.3
1.2
1.2
1
2
0.66
0.85
0.61
0.61
0.47
0.61
0.38
0.47
0.61
0.52
0.57
0.52
0
14 0.9
20 1.3
0
3
18
3
12 0.5
0
1.2
0
4
13 1.1
13 1.2
10 0.5
13 1.2
13
13
8
1
2
0.65
0.65
0.4
0.65
0
12
13 1.2
1
5
0
0
6
1
0
0
12
12
10
10
13
10
12
10
0
1
1
1
1
1
1
1
1
9
11a
13
0
1
0
0
8
10
0.5
1
0
0
11b
12
0
0
0
0
1
13 1.0
11 1.0
12 1.0
11 0.8
0
18 1.3
10 1.0
10 1.1
0
0.9
0.5
0.5
0
0
0
1.3
1
14
16
0
0
0
0
1.2
1
19
Control
0
0
0
0
0
0
0
Chloramphenicol
Terbinafine
21 2.2
Ntb
1
—
20 1.4
Ntb
1
—
Ntb
10 0.5
—
1
Ntb
—
1
10 0.5
a Potency.
b Not tested.
S.A. P.A
1.2
1
0.8
0.6
0.4
0.2
0
Tested Compounds
Figure 2. Antibacterial potency of the tested compounds in comparison with reference drug.
few years,3–17 compound 3 showed one of the highest
observed activities against Aspergillus; so that, it can
be considered as a lead compound in this field. Further
studies are in progress on the same compound to in-
crease its efficacy and understand its QSAR. Also,
aminothiazolyl moiety is important to increase anti-
pseudomonal activity as observed in compound 12.
the alarmingly high antibiotics resistance rates.29,30 Even
with the most effective antibiotics against this pathogen,
namely carbapenems (imipenem and meropenem), the
resistance rates were detected as 15–20.4% amongst
152 P. A. strains.29,30 This pathogen was found to be
sensitive to compounds 1, 3 and 12 (Table 1 and
Fig. 2). Candida albicans and other Candida species
causing candidiasis are increasingly important diseases
that are distributed worldwide due to the fact that they
are frequent opportunistic pathogens in AIDS pa-
tients.31 This fungus was found to be sensitive to most
of the synthesized compounds especially bro-
moacetylpyrazole 3 (Table 1 and Fig. 3). Aspergillus
The overall results of the present study can be consid-
ered very promising in the perspective of new drugs dis-
covery, with respect to the medical importance of the
tested microorganisms. P. A. has emerged as one of
the most problematic Gram-negative pathogens, with

A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
4575
A.F. C.A.
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Tested Compound
Figure 3. Antimycotic effect of the tested compounds in comparison with reference drug.
Table 2. MIC (lg/ml) of compound 3 and fluconazole against A. F.
3.1.2. 4-(2-Bromoacetyl)-5-methyl-1-phenyl-3-phenylcar-
MICa (lg/ml)
A. F.
bamoyl-1H-pyrazole (3).
methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole
A
solution of 4-acetyl-5-
(1)
(31.9 g, 100 mmol) in glacial acetic acid (100 ml) was
heated to 80–90 ꢁC with vigorous stirring. To this hot
solution, bromine (16 g, 100 mmol) in glacial acetic acid
(20 ml) was added dropwise over a period of 30 min with
stirring and maintaining the temperature at 80–90 ꢁC.
After complete addition of bromine, the reaction mix-
ture was stirred vigorously at room temperature for fur-
ther 1 h till the evolution of hydrogen bromide gas was
ceased, then poured onto crushed ice with stirring. The
solid that formed was collected, washed with water
and dried. Crystallization ethanol from 70% afforded
Compound 3
Miconazole
Amphoteracin B
25 0.2
27.5 0.1
11 0.1
a Minimum inhibitory concentration.
causes chronic necrotizing pulmonary aspergillosis
(CNPA),32 which is a subacute infection seen in patients
with an underlying lung disease. About 50% of reported
cases have shown no response to amphotericin B.31 Such
a pathogen was found to be highly sensitive to com-
pound 3 (Table 1 and Fig. 3).
compound
3
in 78% yields, mp 140–141 ꢁC.
C19H16BrN3O2 (398.25), Analysis % Calcd (Found): C:
57.30 (57.39), H: 4.05 (4.02), N: 10.55 (10.52). IR
(KBr) tmax/cmÀ1: 3244 (NH), 1705 (C@O), 1651
1
(C@O), 1600 (C@N). H NMR (DMSO-d6): d 2.44 (s,
3. Experimental
3.1. Chemistry
3H, CH3), 4.82 (s, 2H, CH2), 7.14–7.66 (m, 10H, ArH’s),
8.8 (D2O-exchangeable) (br s, 1H, NH). 13C NMR
(DMSO-d6): d 12.59 (CH3, aliphatic), 37.17 (CH2),
118.32, 120.82, 121.23, 124.66, 126.42, 129.79, 130.12,
138.4, 139.02, 145.57, 146.7 (11 CH, ArC’s), 161.22
(C@O, amide), 189.76 (C@O, ketonic). MS (m/z, aband.
%): 399 (M+2, 4.1), 397 (M+, 3.6), 318 (100), 307(13.0),
305 (13.9), 304 (12.7), 279 (0.7), 277 (0.9), 276 (0.7),
227 (22.0), 199 (1.1), 157 (3.4), 123 (0.4), 121(0.4), 118
(27.7), 95 (5.7), 93 (5.7), 81 (0.5), 79 (0.7), 77 (40.4).
3.1.1. General. All melting points were measured on a
Gallenkamp melting point apparatus. The infrared spec-
tra were recorded in potassium bromide discs on a Pye
Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared
spectrophotometers. The NMR spectra were recorded
1
on a Varian Mercury VX-300 NMR spectrometer. H
(300 MHz) and 13C NMR (75.46 MHz) were run in deu-
terated chloroform (CDCl3) or dimethylsulphoxide
(DMSO-d6). Chemical shifts were related to that of the
solvent. Mass spectra were recorded on a Shimadzu
GCMS-QP 1000 EX mass spectrometer at 70 eV. Ele-
mental analyses were carried out at the Microanalytical
Center of Cairo University, Giza, Egypt. Acety-
lphenylpyrazole 1,18 and enaminone 218 were prepared
following the procedures reported in the literature.
3.1.3. 5-Methyl-1-phenyl-3-phenylcarbamoyl-4-(3-pyraz-
olyl)-1H-pyrazole (4). Hydrazine hydrate (2 ml) was
added to a stirred solution of enaminone 2 (0.748 g,
2 mmol) dissolved in acetic acid (10 ml). Stirring was
continued for 12 h at room temperature and the solid
product obtained was filtered off, washed with cold
water, dried and finally recrystallized from ethanol/

4576
A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
DMF to afford 5-methyl-1-phenyl-3-phenylcarbamoyl-
4-(3-pyrazolyl)-1H-pyrazole (4) in 85% yield, mp 197–
198 ꢁC. C20H17N5O (343.4), Analysis % Calcd (Found):
C: 69.96 (70.04), H: 4.99 (5.00), N: 20.40 (20.42). IR
(KBr) tmax/cmÀ1: 3417 (NH), 3240 (NH), 1654 (C@O),
1596 (C@N). 1H NMR (DMSO-d6): d 2.44 (s, 3H,
CH3), 6.56–7.78 (m, 12H, ArH’s), 7.595 (d, 1H, pyra-
zole-4-CH, J = 7.5 Hz), 7.769 (d, 1H, pyrazole-3-CH,
J = 7.5 Hz), 10.6 (D2O-exchangeable) (s, 1H, NH),
13.0 (D2O-exchangeable) (br s, 1H, NH). 13C NMR
(DMSO-d6): d 12.03 (CH3, aliphatic), 105.59, 119.84,
123.69, 125.41, 128.7, 129.39, 138.85 (14 ArC’s),
160.93 (C@O, amide). MS (m/z, aband. %): 343 (M+,
100), 251 (97.8), 93 (21), 118 (14.5%), 77 (54.3).
was refluxed for 24 h in the presence of catalytic amount
of piperidine, then allowed to cool. The solid that
formed was filtered off, washed with cold water and
dried. Crystallization from DMF afforded compound 9
in 65% yield, mp 278–279 ꢁC. C22H17N7O (396.4), Anal-
ysis % Calcd (Found): C: 66.82 (66.89), H: 4.33 (4.34),
N: 24.80 (24.84). IR (KBr) tmax/cmÀ1: 3417 (NH),
1670 (C@O), 1596 (C@N). 1H NMR (DMSO-d6): d
2.37 (s, 3H, CH3), 7.05–7.77 (m, 11H, ArH’s), 7.49 (d,
1H, pyrimidine-5-CH, J = 4.5 Hz), 8.961 (d, 1H, pyrim-
idine-6-CH, J = 4.5 Hz), 8.57 (s, 1H, (1,2,4-triazole-5-
CH)), 10.36 (D2O-exchangeable) (br s, 1H, NH). 13C
NMR (DMSO-d6): d 11.22 (CH3, aliphatic), 110.80,
111.48, 120.30, 123.78, 125.57, 128.59, 129.23, 129.58,
138.26, 138.49, 141.61, 142.22, 146.01, 146.12 (14
ArC’s), 154.68, 154.95 (triazole-3,5-Carbons), 159.57
(C@O, amide). MS (m/z, aband. %): 396 (M+, 4.7),
303 (100), 184 (0.5), 118 (3.3), 93 (3), 77 (17.7), 65 (5.5).
3.1.4. 4-(Isoxazol-3-yl)-5-methyl-1-phenyl-3-phenylcarba-
moyl-1H-pyrazole (5). To a solution of 2 (0.748 g,
2 mmol), in ethanol (10 ml), were added hydroxylamine
hydrochloride (0.14 g, 2 mmol) and ammonium acetate
(0.3 g). The reaction mixture was heated under reflux
for 3 h, then poured onto ice cold water. The resulting
solid was filtered off, washed with cold water, dried
and recrystallized from 70% ethanol to afford 4-(3-isox-
azolyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyra-
zole (5) in 88% yield, mp 183–185 ꢁC. C20H16N4O2
(344.4). Analysis % Calcd (Found): C: 69.76 (69.81),
3.1.7. 5-Methyl-4-(3-methyl-4-(aryl)pyrazolo[1,5-a]pyr-
imidin-6-yl)-1-phenyl-3-phenylcarbamoyl-1H-pyrazole
(11a,b): General procedure. A mixture of the enaminone
2 (0.374 g, 1 mmol) and the appropriate aminopyrazole
derivatives 10a,b (1 mmol), in dry pyridine (20 ml),
was refluxed for 48 h. The formed solid product was fil-
tered off, washed with ethanol and crystallized from eth-
anol/DMF to afford the pyrazolo[1,5-a]pyrimidine
derivatives 11a,b in 65–70% yield.
H: 4.68 (4.74), N: 16.27 (16.23). IR (KBr) tmax/cmÀ1
:
3359 (NH), 1670 (C@O), 1600 (C@N). 1H NMR
(DMSO-d6): d 2.45 (s, 3H, CH3), 7.1–7.78 (m, 10H,
ArH’s), 6.879 (d, 1H, isoxazole-4-CH, J = 2.1 Hz),
8.625 (d, 1H, isoxazole-5-CH, J = 2.1 Hz), 10.34 (D2O-
exchangeable) (br s, 1H, NH). 13C NMR (DMSO-d6):
3.1.7.1. 5-Methyl-4-(2-methyl-3-phenylpyrazolo[1,5-
a]pyrimidin-7-yl)-1-phen-yl-3-phenylcarbamoyl-1H-pyra-
zole (11a). Yield (65%), mp. 222–223 ꢁC (ethanol/DMF).
C30H24N6O (484.2). Analysis % Calcd (Found): C: 74.36
d
12.02 (CH3, aliphatic), 120.31, 123.89, 125.61,
125.72, 128.67, 129.01, 129.17, 129.46, 138.49, 138.59,
140.9, 144.12, 150.98 (13 ArC’s), 154.97 (isoxazole-5-
CH), 160.31 (C@O, amide). MS (m/z, aband. %): 344
(M+, 29.2), 252 (76), 118 (58.4), 93 (19.8), 77 (100), 68
(15.9).
(74.41), H: 4.99 (4.98), N: 17.34 (17.33). IR (KBr) tmax/
1
cmÀ1: 3259 (NH), 1666 (C@O), 1596 (C@N). H NMR
(DMSO-d6): d 2.45 (s, 3H, CH3), 2.48 (s, 3H, CH3),
6.92–7.78 (m, 17H, ArH’s), 10.56 (D2O-exchangeable)
(br s, 1H, NH). MS (m/z, aband. %): 484 (M+, 2.9),
392 (100), 364 (66.2), 118 (3.7), 77 (32.5).
3.1.5. 5-Methyl-1-phenyl-4-phenylcarbamoyl-4-[(E)-3-
(thiocarbamoylhydrazino)acryloyl]-1H-pyrazole (6). To
a solution of enaminone 2 (0.748 g, 2 mmol), in ethanol
(10 ml), was added thiosemicarbazide (0.182 g,
10 mmol). The reaction mixture was heated under reflux
for 7 days, and then poured onto ice cold water. The
resulting solid product was filtered off, washed with cold
water, dried and crystallized from ethanol 70% to afford
compound 6 in 60% yield, mp 191–192 ꢁC. C21H20N6O3
(404.42). Analysis % Calcd (Found): C: 59.98 (60.12), H:
4.79 (4.73), N: 19.99 (19.89). IR (KBr) tmax/cmÀ1: 3151,
3232, 3409 (3NH, NH2 overlapped), 1681 (conjugated
C@O), 1655 (amide C@O), 1596 (C@N). 1H NMR
(DMSO-d6): d 2.46 (s, 3H, CH3), 5.60 (d, 1H, J
value = 12.6 Hz), 5.85 (D2O-exchangeable) (br s, 2H,
NH2), 7.1–7.77 (m, 10H, ArH’s), 7.8 (d, 1H, J
value = 12.5 Hz), 8.21 (D2O-exchangeable) (br s, 1H,
NH), 8.77 (D2O-exchangeable) (br s, 1H, NH), 10.38
(D2O-exchangeable) (br s, 1H, NH).
3.1.7.2. 5-Methyl-4-(2-methyl-3-(4-chlorophenyl)pyr-
azolo[1,5-a]pyrimidin-7-yl)-1-phenyl-3-phenylcarbamoyl-
1H-pyrazole (11b). Yield (70%), mp. 230–231 ꢁC (ethanol/
DMF). C30H23ClN6O (518.16). Analysis
% Calcd
(Found): C: 69.43 (69.39), H: 4.47 (4.37), N: 16.19
(16.15). IR (KBr) tmax/cmÀ1: 3132 (NH), 1666 (C@O),
1600 (C@N). 1H NMR (DMSO-d6): d 2.45 (s, 3H, CH3),
2.48 (s, 3H, CH3), 7.09–7.78 (m, 16H, ArH’s), 10.56
(D2O-exchangeable) (br s, 1H, NH). MS (m/z, aband. %):
520 (M+2, 2.6), 518 (M+, 6.8), 426 (100), 398 (55.0), 118
(13.4), 77 (40.1).
3.1.8. 4-(2-Aminothiazol-4-yl)-5-methyl-1-phenyl-3-phe-
nylcarbamoyl-1H-pyrazole (12). To a solution of 4-(2-
bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-
pyrazole (3) (3.98 g, 10 mmol) in absolute ethanol
(20 ml) was added thiourea (0.76 g, 10 mmol). The mix-
ture was refluxed for 4 h, then afforded to cool and trea-
ted with ammonium hydroxide solution till it became
alkaline (pH 9). The solid that formed was filtered off,
washed with water, dried and finally recrystallized from
dioxane to afford aminothiazole derivative 12 in a 75%
yield; mp 251–252 ꢁC. C20H17N5OS, (375.45). Analysis
3.1.6. 5-Methyl-1-phenyl-3-phenylcarbamoyl-4-([1,2,4]
triazolo[4,3-a]pyrimidin-5-yl)-1H-pyrazole (9). A mixture
of enaminone 2 (0.748 g, 2 mmol) and the 3-amino-1H-
1,2,4-triazole (8) (0.184 g, 2.2 mmol), in ethanol (10 ml),

A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
4577
% Calcd (Found): C: 63.98 (63.87), H: 4.56 (4.60), N:
18.65 (18.66). IR (KBr) tmax/cmÀ1: 3355, 3290 (NH2),
3190 (NH), 1666 (C@O), 1624, 1600 (2 C@N). 1H
NMR (DMSO-d6): d 2.44 (s, 3H, CH3), 6.77 (s, 1H, thi-
azole-5-CH), 7.08–7.77 (m, 10H, ArH’s), 7.553 (D2O-
exchangeable) (d, 2H, NH2), 10.86 (D2O-exchangeable)
(br s, 1H, NH). 13C NMR (DMSO-d6): d 12.09 (CH3,
aliphatic), 104.98 (thiazole-5-CH), 115.50, 119.81,
123.55, 125.32, 128.53, 128.76, 129.35, 138.95, 138.97,
139.01, 141.51, 144.62 (12 ArC’s), 168.03 (thiazole-2-
CH), 160.60 (C@O, amide). MS (m/z, aband. %): 375
(48), 283 (100), 77 (11.2).
ate (0.345 g, 2.5 mmol). The reaction mixture was re-
fluxed for 1 h, then allowed to cool and the
precipitated solid was filtered off, washed with water
and dried. Crystallization from 70% ethanol afforded
19 in 58% yield; mp 180–181 ꢁC. C27H27N3O4,
(457.52). Analysis % Calcd (Found): C: 70.88 (71.00),
H: 5.95 (5.93), N: 9.18 (9.20). IR (KBr) tmax/cmÀ1
:
3201 (NH), 1722 (C@O), 1670 (C@O), 1596 (C@N).
1H NMR (DMSO-d6): d 0.95 (s, 6H, 2 CH3), 2.13 (s,
4H, 2CH2), 2.36 (d, 2H, CH2), 2.48 (s, 3H, CH3), 3.64
(t, 1H, CH), 7.08–7.79 (m, 10H, ArH’s), 10.71 (D2O-
exchangeable) (br s, 1H, NH). 13C NMR (DMSO-d6):
d 11.89, 28.25 (CH3, aliphatic), 31.68, 37.32, 38.73,
107.08 (aliphatic carbons), 119.90, 121.04, 123.57,
125.63, 128.64, 128.93, 129.43, 128.23, 139.16, 141.16,
147.09 (11 aromatic carbons), 161.46 (C@O, amide),
183.75 (C@O, ketonic), 197.57 (2C@O, ketonic). MS
(m/z, aband. %): 457 (24.4), 439 (21.5), 364 (12.9), 276
(6.1), 185 (26.9), 157 (2.9), 131 (3.9), 120 (5.9), 118
(73.9), 117 (6.0), 92 (9.9), 91 (9.7), 77 (100).
3.1.9. 5-Methyl-1-phenyl-3-phenylcarbamoyl-4-(quinoxa-
lin-2-yl)-1H-pyrazole (14). To
a solution of bro-
moacetylpyrazole derivative 3 (0.79 g, 2 mmol), in
absolute ethanol (10 ml), o-phenylenediamine (0.21 g,
2 mmol) was added. The mixture was refluxed for 1 h,
then allowed to cool and treated with ammonium ace-
tate solution. The solid that formed was filtered off,
washed with water, dried and finally recrystallized from
ethanol/DMF to afford the corresponding quin-
oxalinylpyrazole derivative 14 in an 87% yield; mp
240–241 ꢁC. C25H19N5O (405.45). Analysis % Calcd
(Found): C: 74.06 (74.08), H: 4.72 (4.70), N: 17.27
(17.30). IR (KBr) tmax/cmÀ1: 3274 (NH), 1654 (C@O),
1600 (C@N). 1H NMR (DMSO-d6): d 2.48 (s, 3H,
CH3), 7.08–8.13 (m, 14H, ArH’s), 9.12 (s, 1H, quinoxa-
line-3-CH), 10.29 (D2O-exchangeable) (br s, 1H, NH).
13C NMR (DMSO-d6): d 11.34 (CH3, aliphatic),
118.29, 120.43, 123.84, 125.72, 128.62, 128.81, 128.89,
129.09, 129.48, 129.81, 130.21, 138.50, 138.54, 140.07,
141.32, 141.93, 144.47, 147.75 (18 ArC’s), 148.29 (quin-
oxaline-3-CH), 160.42 (C@O, amide). MS (m/z, aband.
%): 405 (54.4), 313 (100), 129 (5), 118 (7.7), 77 (28.5).
3.2. Biological screening
Antimicrobial activity of eight newly synthesized com-
pounds was tested by measuring the inhibitory effects
of such compounds against Gram-positive, Gram-nega-
tive bacteria and fungi using agar diffusion technique.
3.2.1. Materials. Staphylococcus aureus, Pseudomonas
aeraginosa, C. albicans and A. fumigatus were used
against the tested compounds and were obtained from
the regional center for Mycology and Biotechnology,
Faculty of Science, Al-Azhar University. Chloramphen-
icol, Terbinafine, miconazole and amphoteracin B were
used as reference drugs and were also obtained from the
same source.
3.1.10. 4-(Imidazo[2,1-b]benzothiazol-3-yl)-5-methyl-1-
phenyl-3-phenylcarbamoyl-1H-pyrazole (26). A mixture
of bromoacetylpyrazole derivative3 (0.79 g, 2 mmol)
and 2-aminobenzothiazole (15) (0.3 g, 2 mmol) was re-
fluxed in ethanol for 48 h, and then allowed to cool.
The solid so formed was filtered off, washed with water
and dried. Recrystallization from dioxane afforded the
corresponding imidazo[2,1-b]benzothiazole derivative
16 in 65% yield; mp 160–161 ꢁC. C26H19N5OS
(449.53). Analysis % Calcd (Found): C: 69.47 (69.50),
3.2.2. Method
3.2.2.1. Preparation of bacterial suspensions. Suspen-
sion of the above-mentioned micro-organisms was pre-
pared by inoculating fresh stock cultures into separate
broth tubes, each containing 7 ml of nutrient broth (pep-
ton, 0.3%) beef extract (0.3%). The inoculated tubes
were incubated at 37 ꢁC for 24 h.
3.2.2.2. Preparation of solutions of the tested com-
pounds and reference drugs. Solutions of the tested com-
pounds and reference drugs were prepared by dissolving
0.5 g of the compound in 10 ml DMF.
H, 4.26 (4.28), N: 15.58 (15.59). IR (KBr) tmax/cmÀ1
:
3390 (NH), 1685 (C@O), 1596 (C@N). 1H NMR
(DMSO-d6): d 2.49 (s, 3H, CH3), 7.08–8.10 (m, 14H,
ArH’s), 8.61 (s, 1H, imidazole-4-CH), 10.60 (D2O-
exchangeable) (br s, 1H, NH). 13C NMR (DMSO-d6):
3.2.2.3. Agar diffusion test.33–36 Talls of nutrient agar
were melted and poured each in an empty sterile petri-
dishes (100 · 15 mm) and left for 24 h. A specific culture
of each organism was spread with a dry sterile swab on
the surface of the previously prepared plates. Sterile
discs of 9.6 mm diameter were impregnated with solu-
tions of the tested compound, left to dry and were then
placed on the surface of the inoculated plate. Discs of
antimicrobial standard were put in the centre of the
plate agar and incubated at 37 ꢁC for 24 h. After incuba-
tion, the plates were examined visually and the zone of
inhibition was measured. The test was repeated five
times for each compound.
d
12.91 (CH3, aliphatic), 112.84, 114.21, 114.89,
118.36, 120.75, 121.57, 124.24, 125.51, 125.77, 126.12,
127.27, 129.30, 129.97, 132.48, 138.89, 139.50, 139.58,
140.08, 144.57, 146.37, (20 ArC’s), 161.46 (C@O,
amide). MS (m/z, aband. %): 449 (50.7), 357 (100), 118
(5.1), 77 (20.2).
3.1.11. 4-[2-(4,4-Dimethyl-2,6-dioxocyclohexyl)-1-oxo-
ethyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole
(19). To a solution of bromoacetylpyrazole derivative 3
(0.79 g, 2 mmol), in chloroform (10 ml), were added
dimedone (17) (0.28 g, 2 mmol) and potassium carbon-

4578
A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578
ˆ
9. Nguyet-Thanh, H.-D.; Cristina, M.-S.; Sylvie, D.; Marie-
Agnes, S.; Patrick, M. D.; Daniel, M. Arch. Biochem.
3.2.2.4. Determination of MIC. Determination of the
`
MIC of compound 3 against A. fumigatus was achieved
using 96-well microbioassay system.37 Briefly, a range of
concentrations (10–100 lg/ml) of the compound were
made using Czapeck medium containing sucrose (20 g/
l), KH2PO4 (1 g/l), NaNO2 (2 g/l), MgSO4Æ7 H2O
(0.5 g/l), FeSO4Æ7H2O (0.01 g/l) and KCl (0.5 g/l).
Biophys. 2001, 394, 189.
10. Zweers-Zeilmaker, W. M.; Wildschut, S.; Witkamp, R. F.;
Van Miert, A. A. M. Res. Vet. Sci. 1997, 63, 269.
11. Aiello, E.; Aiello, S.; Mingoia, F.; Bacchi, A.; Pelizzi, G.;
Musiu, C.; Setzu, M. G.; Pani, A.; La Colla, P.; Marongiu,
M. E. Bioorg. Med. Chem. 2000, 8, 2719.
12. Kaymakcıog˘lu, B. K.; Rollas, S. Il Farmaco 2002, 57, 595.
13. Dardari, Z.; Boudouma, M.; Sebban, A.; Bahloul, A.;
Kitane, S.; Berrada, M. Il Farmaco 2004, 59, 673.
14. Tanitame, A.; Oyamada, Y.; Ofuji, K.; Terauchi, H.;
Kawasaki, M.; Wachi, M.; Yamagishi, J. Bioorg. Med.
Chem. Lett. 2005, 15, 4299.
15. Bonacorso, H. G.; Wentz, A. P.; Lourega, R. V.; Cechinel,
C. A.; Moraes, T. S.; Coelho, H. S.; Zanatta, N.; Martins,
M. A. P.; Hoerner, M.; Alves, S. H. J. Fluorine Chem.
2006, 127, 1066.
16. Mares, D.; Romagnli, C.; Andreotti, E.; Forlani, G.;
Guccione, S.; Vicentini, C. B. Mycol. Res. 2006, 110, 686.
17. Aggarwal, R.; Kumar, V.; Tyagi, P.; Singh, S. P. Bioorg.
Med. Chem. 2006, 14, 1785.
18. Farag, A. M.; Mayhoub, A. S.; Barakat, S. E.; Bayomi, A.
H. Bioorg. Med. Chem. 2008, 16, 881.
19. Farag, A. M.; Elkholy, Y. M.; Ali, K. A. J. Heterocycl.
Chem. 2008, 45, 279.
20. Kheder, N. A.; Mabkhot, Y. N.; Farag, A. M. Hetero-
cycles 2008, 75, in press, COM-07-11286.
21. Dawood, K. M.; Farag, A. M.; Abdel-Aziz, H. A.
Heteroatom Chem. 2007, 18, 294.
22. Shaaban, R. M.; Saleh, T. S.; Osman, F. H.; Farag, A. M.
J. Heterocycl. Chem. 2007, 44, 177.
Medium (180 ll) containing appropriate concentrations
of the compound was added to the microwells. Each
concentration is repeated in triplicate. Medium controls
(medium without compound and medium with the same
range of concentration of fluconazole) were also in-
cluded. The inoculum was obtained from 48 h old fungal
cultures grown on Sabouraud agar. Each microwell was
inoculated with 20 ll of conidial suspension to obtain a
final concentration of 5 · 103 conidia of A. F. The plates
were incubated at 23 ꢁC for 72 h, and growth was ob-
served every 24 h. Growth was then evaluated by mea-
suring the absorbance of each well at 620 nm using a
microplate photometer. The experiment was repeated
three times.
3.2.3. Statistical analysis. Data are expressed as
mean SE. Differences between control and treated
tubes were tested using one-way ANOVA followed by
multiple comparisons by Duncan’s multiple rang test.
A probability value less than 0.05 was considered statis-
tically significant.
23. Shaaban, R. M.; Saleh, T. S.; Farag, A. M. Heterocycles
2007, 71, 1765.
24. Farag, A. M.; Dawood, K. M.; Khedr, N. A. J. Chem.
Res. 2007, 472.
Acknowledgements
25. Girgis, A. S.; Mishriky, N.; Farag, A. M.; El-Eraky, W. I.;
Farag, H. Eur. J. Med. Chem. 2007, in press, doi:10.1016/
j.ejmech.2007.11.025.
26. Elkholy, Y. M.; Ali, K. A.; Farag, A. M. Lett. Org. Chem.
2006, 3, 195.
The authors thank Prof. Dr. Alsaied Helal, Professor of
Microbiology and Immunology, Faculty of Pharmacy,
Al-Azhar University, for his useful discussion.
27. Dawood, K. M.; Farag, A. M.; Abdel-Aziz, H. A. .
Heteroatom Chem. 2005, 16, 621.
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