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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
a
Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Naser City, Cairo 11884, Egypt
c
Department 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- In recent years, the widespread use of imidazoles and tri-
ris or Tenia capidis. The azoles (imidazoles and triazoles) azoles antifungal agents has resulted in the development
represent a class of versatile antifungal agents which are of resistance to these drugs by pathogenic microorgan-
used to treat generalized systemic fungal infections. The isms, causing an increase in morbidity and mortality.
mechanism of their antifungal action includes the inhibi- Therefore, new trend of antifungal related to phenylpyr-
tion of cytochrome P450 51 (CYP51), which is essential azole is developed in order to get an effective antifungal
for ergosterol biosynthesis at the step of lanosterol-14- agent without known resistance.3–17
demethylation.1
Although many reported phenylpyrazoles showed sig-
The high affinity of the azole antifungals for CYP51 iso- nificant activity against pathogenic yeast (Candida),
zymes appears to be determined primarily by their unfortunately, no significant effect was obtained against
bulky, polycyclic structure, giving favourable interac- pathogenic moulds such as Aspergillus.3–17

As part of our ongoing research programme aiming at

the synthesis of variety of heterocyclic systems for bio-
Keywords: Phenylcarbamoylpyrazole; 1,2,4-Triazolo[4,3-a]pyrimidine;
logical and pharmacological evaluation,18–27 we report
Pyrazolo[1,5-a]pyrimidine; Imidazo[2,1-b]benzothiazole; Enaminones;
Antibacterial; Antifungal; Anticandidal; Structure–activity relation-
here the synthesis of several phenylpyrazoles having dif-
ship (SAR); 2-Aminothiazole. ferent aromatic bulky structures at position 4 in order to
* Corresponding author. Tel.: +20 12377 3794; fax: +20 235727556; increase the selectivity of this promising new group to-
e-mail: afarag49@yahoo.com wards true pathogenic fungi; for example, Aspergillus

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

O O

N
H
N
N

1
MeO Me
Br 2 /glacial acetic acid N
MeO Me
80-90 ºC/ 30 min. dry xylene

O O O O
Br N
N N
H H
N N
N N

3 2

Scheme 1.
 A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578 4571

Ar
N N
N
O N N O N N
Ph
N N
H H
N N
N N
Ph Ph
11a,b 9

Pyridine/ reflux, Ar H EtOH/Piperidine/

N reflux, 24 h
48 h
NH2
-H 2O N N -H 2O
-Me2 NH H 2N N N
-Me2 NH
H 8
10a,b
a, Ar = C 6H 5
b, Ar = C6 H 4- pCl
2

NH2 NH2 / glacial acetic acid NH2 CSNHNH 2

- Me2 NH RT/ 12 h - Me2 NH NH2 OH EtOH/ref lux, 7days - Me NH
2
- H2 O - H2 O EtOH/ref lux, 3h
O O
O O H
N NH N O Ph N NH 2
N N
Ph Ph H H
N N N S
H H N
N N
N N Ph
6
Ph Ph
-H2 O
4 5
S
H2 N
O N N
Ph
N
H
N
N
Ph
7

Scheme 2.

The 1H NMR spectrum of compound 6 revealed four NMR spectrum revealed also a singlet, of one proton, at
(D2O-exchangeable) singlets at d 5.85, 8.21, 8.77 and d 8.57 corresponding to triazole H-3 proton. Its 13C
10.38 due to amino and three amide protons, NMR spectrum revealed eighteen carbon types; the most
respectively. important signals include triazole-3,5-carbons, which ap-
peared at d 154.68 and 154.95. Finally, the spectrum
The reactivity of enaminone 2 towards some heterocy- showed fourteen aromatic carbons arranged between d
clic amines was also investigated. Thus, the treatment 110.80 and 146.12. The mass spectrum of 9 showed a
of compound 2 with 3-amino-1,2,4-triazole (8) in the molecular ion peak at m/z 395, which loses a phenylamine
presence of a catalytic amount of piperidine afforded radical to give the base peak at m/z 303.
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-
mechanism for the formation of compound 9 may in- zole derivatives 10a,b in refluxing pyridine, it afforded
volve an initial Michael-type addition of the amino the corresponding 5-methyl-4-(3-methyl-4-phenylpyraz-
group of 3-amino-1,2,4-triazole to the activated double olo[1,5-a]pyrimidin-6-yl)-1-phenyl-3-phenylcarbamoyl-1H-
bond in enaminone 2 followed by the elimination of pyrazole (11a) and its chloro derivative 11b (Scheme 2).
dimethylamine and water molecules (Scheme 2).
The formation of products 11 is assumed to take place
The absorption band of carbonyl group at position 4 dis- via the addition of the amino group in aminopyrazoles
appeared in the IR spectrum of compound 9 in compari- 10a,b to a,b-unsaturated moiety in enaminone 2 fol-
son with the IR spectrum of 2. The 1H NMR spectrum of 9 lowed by the elimination of water and dimethylamine
showed two doublets, each of one proton, in the region of molecules to give the final products 11a,b. The struc-
d 7.49–8.96 with (J = 4.5 Hz), which can be attributed to tures of compounds 11a,b were established on the basis
the two adjacent protons of pyrimidine ring. The 1H of their elemental and spectral data (see Section 3).
4572 A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578

S O
O O
O N Ph
N N
Ph H
N N O
H N
N
N Ph
19
Ph O O
16
CHCl3 / ref. 1h K+ K2 CO3
N
EtOH/ ref. 48h NH2
S 15 O O O O
- H2O
Br 18 17
PhHN
N
o-Phenylendiamine N Thiourea
EtOH/ ref. 1h Ph 3 EtOH/ ref. 4h
- H 2O

NH 2
O HN O N
Ph NH S
N Ph
H N
N H
N
N
N
Ph
Ph
13
Oxidation 12

O N
Ph N
N
H
N
N
Ph
14

Scheme 3.

Next, when bromoacetylpyrazole 3 was treated with A plausible mechanism may involve the condensation of
thiourea in refluxing ethanol, it afforded a product iden- one of phenylenediamine amino groups with the car-
tified as 4-(2-aminothiazol-4-yl)-5-methyl-1-phenyl-3- bonyl group of bromoacetyl moiety, while the second
phenylcarbamoyl-1H-pyrazole (12) (Scheme 3). The IR amino group replaced bromine atom via nucleophilic
spectrum of product 12 showed three characteristic substitution. The expected product is the dihydroqui-
absorption bands, two bi-forked bands at 3355 and noxalinyl derivative 13; however, the spectral data of
3290 cm 1 assignable to amino group and a band at the isolated product established that the dihydroquinox-
3190 cm 1 due to the amide NH group. Its 1H NMR alinyl derivative 13 was oxidized under the reaction con-
spectrum revealed a singlet, of one proton, appeared ditions to give 5-methyl-1-phenyl-3-phenylcarbamoyl-4-
in aromatic region at 6.77 corresponding to thiazole-5- (quinoxalin-2-yl)-1H-pyrazole (15).
CH, and D2O-exchangeable singlets at d 7.553 and d
10.86 corresponding to amino and amide protons, The IR spectrum of product 15 showed one absorption
respectively. The 13C NMR spectrum of the same com- band at 3274 cm 1 assignable to one NH group. The 1H
pound revealed sixteen carbon types; the most impor- NMR spectrum showed a singlet, of one proton, at d
tant signals appeared at d 104.98 corresponding to C5 9.12 due to quinoxaline-3-CH. The presence of 19 aro-
of thiazole ring, d 160.6 and 168.03 characterized for matic carbon types on 13C NMR spectrum of the iso-
amide carbonyl and thiazole-C2. The mass spectrum lated product between d 118.29 and 148.29, in addition
of 12 showed a molecular ion peak at m/z 374. 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- Cyclocondensation reaction of bromoacetylpyrazole 3
fied as 5-methyl-1-phenyl-3-phenylcarbamoyl-4-(qui- with some heterocyclic amine was also examined. Thus,
noxalin-2-yl)-1H-pyrazole (14) in an excellent yield when compound 3 was allowed to react with 2-amino-
(Scheme 3). benzothiazole (15) in ethanol, at reflux temperature, it
 A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578 4573

a
NH2
4
O HN
O HO O
NH
HN Ph NH O HN
O O NH2
O NH
NH
NH2
O HN b
HN NH2 O
Ph O O
Br
O OH NH HN
HN
O 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- vious assumption can be excluded. The second

phenyl-3-phenylcarbamoyl-1H-pyrazole (16) (Scheme 3). assumption is that this compound exerts its action on
bacteria and fungi via two different mechanisms; to con-
The preliminary antimicrobial test showed unexpected firm this suggestion, further study is in progress based
result that the phenylpyrazole derivative that carries on molecular modeling.
the least non-polar side chain, that is, bromoacetylpy-
razole 3, has good activity against both fungi and bacte- 2.2. Biological testing
ria, which is assumed that it exerts its action via
chelation of some vital cellular divalent cations as 2.2.1. Antimicrobial screening. The results of the antimi-
observed in other anti-infective agents such as poly- crobial evaluation of the tested compounds are presented
myxin B128 (Fig. 1a). In other words, the bromoacetyl in Table 1. They showed that most of the tested com-
moiety acts as a chelating part (Fig. 1b). Therefore, we pounds have a significant antibacterial effect against
decided to synthesize a phenylpyrazole derivative with Staphylococcus aureus (S. A.), and Pseudomonas aerugin-
a strong chelating moiety at position 4 to prove this osa (P. A.). Acetylpyrazole 1 showed the highest potency
assumption. Thus bromoacetylpyrazole 3 was allowed against Gram-positive bacteria S. A., while pyrazolopyr-
to react with 5,5-dimethylcyclohexane-1,3-dione (dime- imidines 11a,b exhibited the lowest activity. Antipseudo-
done) (17) in refluxing chloroform in the presence of monal activity was observed with compounds 1, 3 and 12
potassium carbonate, which reaction afforded a product (Table 1 and Fig. 2). These compounds also possess a po-
identified as 4-[2-(4,4-dimethyl-2,6-dioxocyclohexyl)-1- tent antifungal activity against Candida albicans (C. A.),
oxoethyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyra- mostly higher than that of reference drug (terbinafine).
zole (19). However, they have no effect against Aspergillus fumiga-
tus (A. F.) except compound 3. The latter compound
The suggested reaction mechanism involves the conver- was found to be more effective than terbinafine against
sion of dimedone (17) into its enolate potassium salt 18 A. F. (Table 1 and Fig. 3). Moreover, compound 3 showed
in the presence of potassium carbonate. The latter inter- an antifungal activity against C. A., 1.6 higher than that of
mediate is considered as a strong carbon nucleophile the reference drug.
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
3. The 1H NMR spectrum of the reaction product dis- selected for further assessment. The minimum inhibitory
played five signals at d 0.95, 2.13, 2.36, 2.48 and 3.64 concentration (MIC) of compound 3 against A. F. was
corresponding to methyl and methylene groups of dime- 25 lg/ml, which is less than that of miconazole and high-
done, methylene protons of acetyl moiety, pyrazole-at- er than amphoteracin B as reference drugs as depicted in
tached methyl group and dimedone-CH, respectively. Table 2.
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 antimicrobial tests showed that the activity of com- the activity against true fungi (moulds). Amongst the
pound 19 is lower than compound 3; therefore, the pre- dozens of synthesized phenylpyrazoles found in recent
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) (G –ve) Mould Yeast
a a a
S. A. Pot. P. A. Pot. A. F. Pot. C. A. Pot.a
1 21 ± 1.3 1 17 ± 2 0.85 0 0 15 ± 1.2 1.5
2 14 ± 0.7 0.66 14 ± 0.9 0.7 0 0 15 ± 1.2 1.5
3 18 ± 3 0.85 20 ± 1.3 1 12 ± 0.5 1.2 16 ± 1.2 1.6
4 13 ± 1.1 0.61 13 ± 1 0.65 0 0 12 ± 1 1.2
5 13 ± 1.2 0.61 13 ± 2 0.65 0 0 13 ± 1.2 1.3
6 10 ± 0.5 0.47 8±1 0.4 0 0 12 ± 1 1.2
9 13 ± 1.2 0.61 13 ± 1 0.65 0 0 12 ± 1 1.2
11a 8 ± 0.5 0.38 0 0 0 0 10 ± 1 1
11b 10 ± 1 0.47 0 0 0 0 10 ± 1 1
12 13 ± 1.0 0.61 18 ± 1.3 0.9 0 0 13 ± 1 1.3
14 11 ± 1.0 0.52 10 ± 1.0 0.5 0 0 10 ± 1 1
16 12 ± 1.0 0.57 10 ± 1.1 0.5 0 0 12 ± 1 1.2
19 11 ± 0.8 0.52 0 0 0 0 10 ± 1 1
Control 0 0 0 0 0 0 0 0
Chloramphenicol 21 ± 2.2 1 20 ± 1.4 1 Ntb — Ntb —
Terbinafine Ntb — Ntb — 10 ± 0.5 1 10 ± 0.5 1
a
Potency.
b
Not tested.

S.A. P.A

1.2

0.8
Potency

0.6

0.4

0.2

0
l
12

19
1

14

16

.
tro

or
11

11

hl
on

C
C

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 the alarmingly high antibiotics resistance rates.29,30 Even
observed activities against Aspergillus; so that, it can with the most effective antibiotics against this pathogen,
be considered as a lead compound in this field. Further namely carbapenems (imipenem and meropenem), the
studies are in progress on the same compound to in- resistance rates were detected as 15–20.4% amongst
crease its efficacy and understand its QSAR. Also, 152 P. A. strains.29,30 This pathogen was found to be
aminothiazolyl moiety is important to increase anti- sensitive to compounds 1, 3 and 12 (Table 1 and
pseudomonal activity as observed in compound 12. Fig. 2). Candida albicans and other Candida species
causing candidiasis are increasingly important diseases
The overall results of the present study can be consid- that are distributed worldwide due to the fact that they
ered very promising in the perspective of new drugs dis- are frequent opportunistic pathogens in AIDS pa-
covery, with respect to the medical importance of the tients.31 This fungus was found to be sensitive to most
tested microorganisms. P. A. has emerged as one of of the synthesized compounds especially bro-
the most problematic Gram-negative pathogens, with moacetylpyrazole 3 (Table 1 and Fig. 3). Aspergillus
 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
Potency

0.8

0.6

0.4

0.2

.
1

12

14

16

19

tro

rb
11

11

Te
on
C
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) bamoyl-1H-pyrazole (3). A solution of 4-acetyl-5-
A. F. methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole (1)
Compound 3 25 ± 0.2
(31.9 g, 100 mmol) in glacial acetic acid (100 ml) was
Miconazole 27.5 ± 0.1 heated to 80–90 C with vigorous stirring. To this hot
Amphoteracin B 11 ± 0.1 solution, bromine (16 g, 100 mmol) in glacial acetic acid
a (20 ml) was added dropwise over a period of 30 min with
Minimum inhibitory concentration.
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-
causes chronic necrotizing pulmonary aspergillosis ther 1 h till the evolution of hydrogen bromide gas was
(CNPA),32 which is a subacute infection seen in patients ceased, then poured onto crushed ice with stirring. The
with an underlying lung disease. About 50% of reported solid that formed was collected, washed with water
cases have shown no response to amphotericin B.31 Such and dried. Crystallization ethanol from 70% afforded
a pathogen was found to be highly sensitive to com- compound 3 in 78% yields, mp 140–141 C.
pound 3 (Table 1 and Fig. 3). C19H16BrN3O2 (398.25), Analysis % Calcd (Found): C:
57.30 (57.39), H: 4.05 (4.02), N: 10.55 (10.52). IR
(KBr) tmax/cm 1: 3244 (NH), 1705 (C@O), 1651
3. Experimental (C@O), 1600 (C@N). 1H NMR (DMSO-d6): d 2.44 (s,
3H, CH3), 4.82 (s, 2H, CH2), 7.14–7.66 (m, 10H, ArH’s),
3.1. Chemistry 8.8 (D2O-exchangeable) (br s, 1H, NH). 13C NMR
(DMSO-d6): d 12.59 (CH3, aliphatic), 37.17 (CH2),
3.1.1. General. All melting points were measured on a 118.32, 120.82, 121.23, 124.66, 126.42, 129.79, 130.12,
Gallenkamp melting point apparatus. The infrared spec- 138.4, 139.02, 145.57, 146.7 (11 CH, ArC’s), 161.22
tra were recorded in potassium bromide discs on a Pye (C@O, amide), 189.76 (C@O, ketonic). MS (m/z, aband.
Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared %): 399 (M+2, 4.1), 397 (M+, 3.6), 318 (100), 307(13.0),
spectrophotometers. The NMR spectra were recorded 305 (13.9), 304 (12.7), 279 (0.7), 277 (0.9), 276 (0.7),
on a Varian Mercury VX-300 NMR spectrometer. 1H 227 (22.0), 199 (1.1), 157 (3.4), 123 (0.4), 121(0.4), 118
(300 MHz) and 13C NMR (75.46 MHz) were run in deu- (27.7), 95 (5.7), 93 (5.7), 81 (0.5), 79 (0.7), 77 (40.4).
terated chloroform (CDCl3) or dimethylsulphoxide
(DMSO-d6). Chemical shifts were related to that of the 3.1.3. 5-Methyl-1-phenyl-3-phenylcarbamoyl-4-(3-pyraz-
solvent. Mass spectra were recorded on a Shimadzu olyl)-1H-pyrazole (4). Hydrazine hydrate (2 ml) was
GCMS-QP 1000 EX mass spectrometer at 70 eV. Ele- added to a stirred solution of enaminone 2 (0.748 g,
mental analyses were carried out at the Microanalytical 2 mmol) dissolved in acetic acid (10 ml). Stirring was
Center of Cairo University, Giza, Egypt. Acety- continued for 12 h at room temperature and the solid
lphenylpyrazole 1,18 and enaminone 218 were prepared product obtained was filtered off, washed with cold
following the procedures reported in the literature. 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- was refluxed for 24 h in the presence of catalytic amount
4-(3-pyrazolyl)-1H-pyrazole (4) in 85% yield, mp 197– of piperidine, then allowed to cool. The solid that
198 C. C20H17N5O (343.4), Analysis % Calcd (Found): formed was filtered off, washed with cold water and
C: 69.96 (70.04), H: 4.99 (5.00), N: 20.40 (20.42). IR dried. Crystallization from DMF afforded compound 9
(KBr) tmax/cm 1: 3417 (NH), 3240 (NH), 1654 (C@O), in 65% yield, mp 278–279 C. C22H17N7O (396.4), Anal-
1596 (C@N). 1H NMR (DMSO-d6): d 2.44 (s, 3H, ysis % Calcd (Found): C: 66.82 (66.89), H: 4.33 (4.34),
CH3), 6.56–7.78 (m, 12H, ArH’s), 7.595 (d, 1H, pyra- N: 24.80 (24.84). IR (KBr) tmax/cm 1: 3417 (NH),
zole-4-CH, J = 7.5 Hz), 7.769 (d, 1H, pyrazole-3-CH, 1670 (C@O), 1596 (C@N). 1H NMR (DMSO-d6): d
J = 7.5 Hz), 10.6 (D2O-exchangeable) (s, 1H, NH), 2.37 (s, 3H, CH3), 7.05–7.77 (m, 11H, ArH’s), 7.49 (d,
13.0 (D2O-exchangeable) (br s, 1H, NH). 13C NMR 1H, pyrimidine-5-CH, J = 4.5 Hz), 8.961 (d, 1H, pyrim-
(DMSO-d6): d 12.03 (CH3, aliphatic), 105.59, 119.84, idine-6-CH, J = 4.5 Hz), 8.57 (s, 1H, (1,2,4-triazole-5-
123.69, 125.41, 128.7, 129.39, 138.85 (14 ArC’s), CH)), 10.36 (D2O-exchangeable) (br s, 1H, NH). 13C
160.93 (C@O, amide). MS (m/z, aband. %): 343 (M+, NMR (DMSO-d6): d 11.22 (CH3, aliphatic), 110.80,
100), 251 (97.8), 93 (21), 118 (14.5%), 77 (54.3). 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
3.1.4. 4-(Isoxazol-3-yl)-5-methyl-1-phenyl-3-phenylcarba- ArC’s), 154.68, 154.95 (triazole-3,5-Carbons), 159.57
moyl-1H-pyrazole (5). To a solution of 2 (0.748 g, (C@O, amide). MS (m/z, aband. %): 396 (M+, 4.7),
2 mmol), in ethanol (10 ml), were added hydroxylamine 303 (100), 184 (0.5), 118 (3.3), 93 (3), 77 (17.7), 65 (5.5).
hydrochloride (0.14 g, 2 mmol) and ammonium acetate
(0.3 g). The reaction mixture was heated under reflux 3.1.7. 5-Methyl-4-(3-methyl-4-(aryl)pyrazolo[1,5-a]pyr-
for 3 h, then poured onto ice cold water. The resulting imidin-6-yl)-1-phenyl-3-phenylcarbamoyl-1H-pyrazole
solid was filtered off, washed with cold water, dried (11a,b): General procedure. A mixture of the enaminone
and recrystallized from 70% ethanol to afford 4-(3-isox- 2 (0.374 g, 1 mmol) and the appropriate aminopyrazole
azolyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyra- derivatives 10a,b (1 mmol), in dry pyridine (20 ml),
zole (5) in 88% yield, mp 183–185 C. C20H16N4O2 was refluxed for 48 h. The formed solid product was fil-
(344.4). Analysis % Calcd (Found): C: 69.76 (69.81), tered off, washed with ethanol and crystallized from eth-
H: 4.68 (4.74), N: 16.27 (16.23). IR (KBr) tmax/cm 1: anol/DMF to afford the pyrazolo[1,5-a]pyrimidine
3359 (NH), 1670 (C@O), 1600 (C@N). 1H NMR derivatives 11a,b in 65–70% yield.
(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), 3.1.7.1. 5-Methyl-4-(2-methyl-3-phenylpyrazolo[1,5-
8.625 (d, 1H, isoxazole-5-CH, J = 2.1 Hz), 10.34 (D2O- a]pyrimidin-7-yl)-1-phen-yl-3-phenylcarbamoyl-1H-pyra-
exchangeable) (br s, 1H, NH). 13C NMR (DMSO-d6): zole (11a). Yield (65%), mp. 222–223 C (ethanol/DMF).
d 12.02 (CH3, aliphatic), 120.31, 123.89, 125.61, C30H24N6O (484.2). Analysis % Calcd (Found): C: 74.36
125.72, 128.67, 129.01, 129.17, 129.46, 138.49, 138.59, (74.41), H: 4.99 (4.98), N: 17.34 (17.33). IR (KBr) tmax/
140.9, 144.12, 150.98 (13 ArC’s), 154.97 (isoxazole-5- cm 1: 3259 (NH), 1666 (C@O), 1596 (C@N). 1H NMR
CH), 160.31 (C@O, amide). MS (m/z, aband. %): 344 (DMSO-d6): d 2.45 (s, 3H, CH3), 2.48 (s, 3H, CH3),
(M+, 29.2), 252 (76), 118 (58.4), 93 (19.8), 77 (100), 68 6.92–7.78 (m, 17H, ArH’s), 10.56 (D2O-exchangeable)
(15.9). (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 3.1.7.2. 5-Methyl-4-(2-methyl-3-(4-chlorophenyl)pyr-
a solution of enaminone 2 (0.748 g, 2 mmol), in ethanol azolo[1,5-a]pyrimidin-7-yl)-1-phenyl-3-phenylcarbamoyl-
(10 ml), was added thiosemicarbazide (0.182 g, 1H-pyrazole (11b). Yield (70%), mp. 230–231 C (ethanol/
10 mmol). The reaction mixture was heated under reflux DMF). C30H23ClN6O (518.16). Analysis % Calcd
for 7 days, and then poured onto ice cold water. The (Found): C: 69.43 (69.39), H: 4.47 (4.37), N: 16.19
resulting solid product was filtered off, washed with cold (16.15). IR (KBr) tmax/cm 1: 3132 (NH), 1666 (C@O),
water, dried and crystallized from ethanol 70% to afford 1600 (C@N). 1H NMR (DMSO-d6): d 2.45 (s, 3H, CH3),
compound 6 in 60% yield, mp 191–192 C. C21H20N6O3 2.48 (s, 3H, CH3), 7.09–7.78 (m, 16H, ArH’s), 10.56
(404.42). Analysis % Calcd (Found): C: 59.98 (60.12), H: (D2O-exchangeable) (br s, 1H, NH). MS (m/z, aband. %):
4.79 (4.73), N: 19.99 (19.89). IR (KBr) tmax/cm 1: 3151, 520 (M+2, 2.6), 518 (M+, 6.8), 426 (100), 398 (55.0), 118
3232, 3409 (3NH, NH2 overlapped), 1681 (conjugated (13.4), 77 (40.1).
C@O), 1655 (amide C@O), 1596 (C@N). 1H NMR
(DMSO-d6): d 2.46 (s, 3H, CH3), 5.60 (d, 1H, J 3.1.8. 4-(2-Aminothiazol-4-yl)-5-methyl-1-phenyl-3-phe-
value = 12.6 Hz), 5.85 (D2O-exchangeable) (br s, 2H, nylcarbamoyl-1H-pyrazole (12). To a solution of 4-(2-
NH2), 7.1–7.77 (m, 10H, ArH’s), 7.8 (d, 1H, J bromoacetyl)-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-
value = 12.5 Hz), 8.21 (D2O-exchangeable) (br s, 1H, pyrazole (3) (3.98 g, 10 mmol) in absolute ethanol
NH), 8.77 (D2O-exchangeable) (br s, 1H, NH), 10.38 (20 ml) was added thiourea (0.76 g, 10 mmol). The mix-
(D2O-exchangeable) (br s, 1H, NH). ture was refluxed for 4 h, then afforded to cool and trea-
ted with ammonium hydroxide solution till it became
3.1.6. 5-Methyl-1-phenyl-3-phenylcarbamoyl-4-([1,2,4] alkaline (pH 9). The solid that formed was filtered off,
triazolo[4,3-a]pyrimidin-5-yl)-1H-pyrazole (9). A mixture washed with water, dried and finally recrystallized from
of enaminone 2 (0.748 g, 2 mmol) and the 3-amino-1H- dioxane to afford aminothiazole derivative 12 in a 75%
1,2,4-triazole (8) (0.184 g, 2.2 mmol), in ethanol (10 ml), yield; mp 251–252 C. C20H17N5OS, (375.45). Analysis
 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: ate (0.345 g, 2.5 mmol). The reaction mixture was re-
18.65 (18.66). IR (KBr) tmax/cm 1: 3355, 3290 (NH2), fluxed for 1 h, then allowed to cool and the
3190 (NH), 1666 (C@O), 1624, 1600 (2 C@N). 1H precipitated solid was filtered off, washed with water
NMR (DMSO-d6): d 2.44 (s, 3H, CH3), 6.77 (s, 1H, thi- and dried. Crystallization from 70% ethanol afforded
azole-5-CH), 7.08–7.77 (m, 10H, ArH’s), 7.553 (D2O- 19 in 58% yield; mp 180–181 C. C27H27N3O4,
exchangeable) (d, 2H, NH2), 10.86 (D2O-exchangeable) (457.52). Analysis % Calcd (Found): C: 70.88 (71.00),
(br s, 1H, NH). 13C NMR (DMSO-d6): d 12.09 (CH3, H: 5.95 (5.93), N: 9.18 (9.20). IR (KBr) tmax/cm 1:
aliphatic), 104.98 (thiazole-5-CH), 115.50, 119.81, 3201 (NH), 1722 (C@O), 1670 (C@O), 1596 (C@N).
1
123.55, 125.32, 128.53, 128.76, 129.35, 138.95, 138.97, H NMR (DMSO-d6): d 0.95 (s, 6H, 2 CH3), 2.13 (s,
139.01, 141.51, 144.62 (12 ArC’s), 168.03 (thiazole-2- 4H, 2CH2), 2.36 (d, 2H, CH2), 2.48 (s, 3H, CH3), 3.64
CH), 160.60 (C@O, amide). MS (m/z, aband. %): 375 (t, 1H, CH), 7.08–7.79 (m, 10H, ArH’s), 10.71 (D2O-
(48), 283 (100), 77 (11.2). exchangeable) (br s, 1H, NH). 13C NMR (DMSO-d6):
d 11.89, 28.25 (CH3, aliphatic), 31.68, 37.32, 38.73,
3.1.9. 5-Methyl-1-phenyl-3-phenylcarbamoyl-4-(quinoxa- 107.08 (aliphatic carbons), 119.90, 121.04, 123.57,
lin-2-yl)-1H-pyrazole (14). To a solution of bro- 125.63, 128.64, 128.93, 129.43, 128.23, 139.16, 141.16,
moacetylpyrazole derivative 3 (0.79 g, 2 mmol), in 147.09 (11 aromatic carbons), 161.46 (C@O, amide),
absolute ethanol (10 ml), o-phenylenediamine (0.21 g, 183.75 (C@O, ketonic), 197.57 (2C@O, ketonic). MS
2 mmol) was added. The mixture was refluxed for 1 h, (m/z, aband. %): 457 (24.4), 439 (21.5), 364 (12.9), 276
then allowed to cool and treated with ammonium ace- (6.1), 185 (26.9), 157 (2.9), 131 (3.9), 120 (5.9), 118
tate solution. The solid that formed was filtered off, (73.9), 117 (6.0), 92 (9.9), 91 (9.7), 77 (100).
washed with water, dried and finally recrystallized from
ethanol/DMF to afford the corresponding quin- 3.2. Biological screening
oxalinylpyrazole derivative 14 in an 87% yield; mp
240–241 C. C25H19N5O (405.45). Analysis % Calcd Antimicrobial activity of eight newly synthesized com-
(Found): C: 74.06 (74.08), H: 4.72 (4.70), N: 17.27 pounds was tested by measuring the inhibitory effects
(17.30). IR (KBr) tmax/cm 1: 3274 (NH), 1654 (C@O), of such compounds against Gram-positive, Gram-nega-
1600 (C@N). 1H NMR (DMSO-d6): d 2.48 (s, 3H, tive bacteria and fungi using agar diffusion technique.
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). 3.2.1. Materials. Staphylococcus aureus, Pseudomonas
13
C NMR (DMSO-d6): d 11.34 (CH3, aliphatic), aeraginosa, C. albicans and A. fumigatus were used
118.29, 120.43, 123.84, 125.72, 128.62, 128.81, 128.89, against the tested compounds and were obtained from
129.09, 129.48, 129.81, 130.21, 138.50, 138.54, 140.07, the regional center for Mycology and Biotechnology,
141.32, 141.93, 144.47, 147.75 (18 ArC’s), 148.29 (quin- Faculty of Science, Al-Azhar University. Chloramphen-
oxaline-3-CH), 160.42 (C@O, amide). MS (m/z, aband. icol, Terbinafine, miconazole and amphoteracin B were
%): 405 (54.4), 313 (100), 129 (5), 118 (7.7), 77 (28.5). 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 3.2.2. Method
of bromoacetylpyrazole derivative3 (0.79 g, 2 mmol) 3.2.2.1. Preparation of bacterial suspensions. Suspen-
and 2-aminobenzothiazole (15) (0.3 g, 2 mmol) was re- sion of the above-mentioned micro-organisms was pre-
fluxed in ethanol for 48 h, and then allowed to cool. pared by inoculating fresh stock cultures into separate
The solid so formed was filtered off, washed with water broth tubes, each containing 7 ml of nutrient broth (pep-
and dried. Recrystallization from dioxane afforded the ton, 0.3%) beef extract (0.3%). The inoculated tubes
corresponding imidazo[2,1-b]benzothiazole derivative were incubated at 37 C for 24 h.
16 in 65% yield; mp 160–161 C. C26H19N5OS
(449.53). Analysis % Calcd (Found): C: 69.47 (69.50), 3.2.2.2. Preparation of solutions of the tested com-
H, 4.26 (4.28), N: 15.58 (15.59). IR (KBr) tmax/cm 1: pounds and reference drugs. Solutions of the tested com-
3390 (NH), 1685 (C@O), 1596 (C@N). 1H NMR pounds and reference drugs were prepared by dissolving
(DMSO-d6): d 2.49 (s, 3H, CH3), 7.08–8.10 (m, 14H, 0.5 g of the compound in 10 ml DMF.
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
d 12.91 (CH3, aliphatic), 112.84, 114.21, 114.89, were melted and poured each in an empty sterile petri-
118.36, 120.75, 121.57, 124.24, 125.51, 125.77, 126.12, dishes (100 · 15 mm) and left for 24 h. A specific culture
127.27, 129.30, 129.97, 132.48, 138.89, 139.50, 139.58, of each organism was spread with a dry sterile swab on
140.08, 144.57, 146.37, (20 ArC’s), 161.46 (C@O, the surface of the previously prepared plates. Sterile
amide). MS (m/z, aband. %): 449 (50.7), 357 (100), 118 discs of 9.6 mm diameter were impregnated with solu-
(5.1), 77 (20.2). tions of the tested compound, left to dry and were then
placed on the surface of the inoculated plate. Discs of
3.1.11. 4-[2-(4,4-Dimethyl-2,6-dioxocyclohexyl)-1-oxo- antimicrobial standard were put in the centre of the
ethyl]-5-methyl-1-phenyl-3-phenylcarbamoyl-1H-pyrazole plate agar and incubated at 37 C for 24 h. After incuba-
(19). To a solution of bromoacetylpyrazole derivative 3 tion, the plates were examined visually and the zone of
(0.79 g, 2 mmol), in chloroform (10 ml), were added inhibition was measured. The test was repeated five
dimedone (17) (0.28 g, 2 mmol) and potassium carbon- times for each compound.
4578 A. M. Farag et al. / Bioorg. Med. Chem. 16 (2008) 4569–4578

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