Synthesis and antimicrobial activity of some new 4-hetarylpyrazole and furo[2,3-c]pyrazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2011.03.045

In continuation of our efforts to find a new class of antimicrobial agents, a series of 4-hetarylpyrazoles and furo[2,3-c]pyrazoles were prepared via the reaction of 2-chloro-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)ethanone (1) with an appropriate

Full text of DOI:10.1016/j.ejmech.2011.03.045

European Journal of Medicinal Chemistry 46 (2011) 2555e2561
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activity of some new 4-hetarylpyrazole
and furo[2,3-c]pyrazole derivatives
*
Samir Bondock , Wesam Khalifa, Ahmed A. Fadda
Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
In continuation of our efforts to find a new class of antimicrobial agents, a series of 4-hetarylpyrazoles
and furo[2,3-c]pyrazoles were prepared via the reaction of 2-chloro-1-(5-hydroxy-3-methyl-1-phenyl-
1H-pyrazol-4-yl)ethanone (1) with an appropriate nucleophilic reagents. These compounds were
screened for their antibacterial activity against Gram-positive bacteria (Bacillus subtilis and Bacillus
thuringiensis), Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and antifungal
activity against Fusarium oxysporum and Botrytis fabae. Among the synthesized compounds, 1-(5-(5-
hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-yl)-2-methylfuran-3-yl)ethanone (12) showed equal activity
Received 27 November 2010
Received in revised form
20 March 2011
Accepted 22 March 2011
Available online 30 March 2011
Keywords:
Pyrazole
Furan
Furo[2,3-c]pyrazole
Antimicrobial activity
with chloramphenicol against B. subtilis (MIC 3.125 mg/mL), while its activity was 50% lower than of
chloramphenicol against B. thuringiensis. N-[(4Z)-3-Methyl-1-phenyl-1H-furo[2,3-c]pyrazol-4(5H)-yli-
dene]-1H-benzimidazol-2-amine (7) and 2-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-4H-furo
[3,2-c]chromen-4-one (13) were found to exhibit the most potent in vitro antifungal activity with MICs
(6.25 mg/mL) against B. fabae and F. oxysporum.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
agent Cicerfuran, obtained from the roots of wild species of
chickpea, Cicer bijugum, reported to be a major factor in the defense
system against Fusarium wilt [29]. In addition, Nitrofurantoin and
Furazolidone are potent nitrofuran drugs. They have been reported
to possess broad antimicrobial properties, including activity against
trypanosomes [30,31].
Prompted by the above-mentioned results, it was planned to
study structure variation by attaching some biologically active
heterocycles such as quinoxaline, benzothiazine, benzoxazine,
furan, benzofuran and furo[3,2-c]coumarin at position 4 of the
main pyrazole moiety as well as construction of some furo[2,3-c]
pyrazoles. These combinations were suggested in an attempt to
investigate the possible synergistic influence of such structure
hybridizations on the anticipated activity, hoping to discover a new
lead structure that would have a significant antimicrobial activity at
very small concentration.
In continuation of our recent work aiming at the synthesis of
heterocyclic systems with remarkable biological importance
[32e37], we report herein on the utility of 2-chloro-1-(5-hydroxy-
3-methyl-1-phenyl-1H-pyrazol-4-yl)ethanone as building blocks
for the synthesis of functionalized 4-hetarylpyrazoles and furo
[2,3-c]pyrazoles, and study their antimicrobial activity in order to
get a new compounds that could be optimized for potent antimi-
crobial agents.
The pyrazole ring is a prominent structural motif found in
numerous pharmaceutically active compounds. This is mainly due
to the ease preparation and the important pharmacological activity.
Therefore, the synthesis and selective functionalization of pyr-
azoles have been the focus of active research area over the years
[1e5]. Pyrazoles have been reported to possess antibacterial
activity and inhibitor activity against DNA gyrase and top-
oisomerase IV at their respective ATP-binding sites [6e8]. More-
over, pyrazole-containing compounds have received considerable
attention owing to their diverse chemotherapeutic potentials
including versatile antineoplastic activities. Literature survey
revealed that some pyrazoles have been implemented as antileu-
kemic [9e11], antitumor [12e16], antiproliferative agents [17,18],
GABA receptor antagonists and insecticides [19], anti-inflammatory
and antimicrobial agents [20e23].
In addition to, furans and their derivatives with substituent(s) at
C-2 and/or C-3 have attracted strong interest due to their wide-
spread in a large number of natural products and for their useful
biological and pharmacological properties [24e28]. The antifungal
* Corresponding author. Tel.: þ20 502369267; fax: þ20 502246781.
E-mail address: Bondock@mans.edu.eg (S. Bondock).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.045

2556
S. Bondock et al. / European Journal of Medicinal Chemistry 46 (2011) 2555e2561
2. Results and discussion
the chloride by the hydroxyl group to form compound 5 that
underwent in situ condensation with heterocyclic amines to afford
the target molecules.
2.1. Chemistry
In contrast to the behavior of compound 1 toward heterocyclic
primary amines, it has been found that refluxing of compound 1
with heterocyclic secondary amines such as piperidine and/or
morpholine in absolute ethanol furnished the substitution products
9aeb rather than the enamines 8aeb. The structures of compounds
9aeb were determined by elemental analyses, which give the
molecular formula C17H21N3O2 for 9a and C16H19N3O3 for 9b, as
well as spectral data (see experimental).
Moreover, we investigated the reactivity of 1 toward sali-
cyaldehyde, 2-hydroxyacetophenone, acetylacetone and 4-hydrox-
ycoumarin as a possible synthetic route to attain furan, benzofuran
and furo[3,2-c]chromenone derivatives. Thus, treatment of 1 with
salicylaldehyde and/or 2-hydroxyacetophenone in dimethylsulf-
oxide in the presence of anhydrous potassium carbonate furnished
benzofuran derivatives 10 and 11, respectively. Structure of the
isolated products 10 and 11 was confirmed on the basis of their
elemental analyses and spectral data. For example, the IR spectrum
of compound 11 revealed an absorption bands at 3390 and
1635 cmꢀ1 characteristic to OH and C]O groups. The lower
frequency of a ketonic carbonyl group is attributed to the hydrogen
bonding formation with hydroxyl group. Its 1H NMR spectrum
The synthetic strategies adopted to obtain the target
compounds are depicted in Schemes 1e3. The starting material,
2-chloro-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)etha-
none (1) was prepared by refluxing 5-pyrazolone with chloroacetyl
chloride in
a basic dioxane solution following a procedure
described earlier by Jensen et al. [38]. Treatment of 1 with o-phe-
nylenediamine in boiling ethanol gave a sole product that was
identified as 4-(3,4-dihydroquinoxalin-2-yl)-3-methyl-1-phenyl-
1H-pyrazol-5-ol (2a). In the same manner, compound 1 reacts with
2-aminothiophenol and 2-aminophenol to afford product that may
be formulated as 1,4-benzothiazine 2b and 1,4-benzoxazine 2c
derivatives based on both elemental analysis and spectral data
(Scheme 1).
Next, we studied the reactivity of 1 toward some heterocyclic
amines namely, 2-aminopyridine, 2-aminopyrazine, 3-amino-4H-
1,2,4-triazole and 2-aminobenzimidazole as a possible synthetic
route to get bridghead heterocyclic nitrogen compounds of
potential pharmacological interest. Surprisingly, the reaction of 1
with either 2-aminopyridine or 2-aminopyrazine in refluxing
ethanolic triethylamine afforded in each case a single product
identified as furo[2,3-c]pyrazoles 3aeb rather than the expected
imidazo[1,2-a]pyridine 4a and imidiazo[1,2-a]pyrazine 4b. The
chemical structures of compounds 3aeb were elucidated on the
basis of elemental analyses, spectral data and an alternative
synthetic route. Thus, treatment of 3-methyl-1-phenyl-1H-furo
[2,3-c]pyrazol-4(5H)-one (5), which was synthesized from 1 by
boiling in ethanol containing a catalytic amount of triethylamine,
with 2-aminopyridine or 2-aminopyrazine in boiling ethanol gave
product identical in all aspects (mp., mixed mp., and spectra) with
3aeb (Scheme 2).
revealed three singlet signals at
d 2.31, 2.50 and 12.80 ppm assign-
able to CH3 of pyrazole ring, CH3 of furan ring and OH protons,
respectively, besides a multiplet centered around 6.93e8.01 ppm
due to aromatic protons. The 13C NMR spectrum of compound 11
revealed eighteen carbon types. The carbonyl carbon is displayed at
d
165.5 ppm. Other important signals are displayed at
d 12.3 and
16.4 ppm characteristics for two methyl carbons, while C-5 pyrazole
was displayed downfield at
d 154.2 ppm. Moreover, the mass
spectrum of 11 showed a molecular ion peak at m/z ¼ 333 (Mþ)
corresponding to a molecular formula C20H16N2O3.
It is worthwhile to mention that compounds 3aeb can exist in
two diastereomeric E- and Z-isomers. The preferential formation of
Z-isomer was supported from molecular orbital calculation using
Hyperchem 7, semi-empirical method (PM3), which revealed that
the heat of formation of Z-isomer is lower than E-isomer by about
0.5 kcal/mol.
Analogously, the appropriate 3-amino-4H-1,2,4-triazole or
2-aminobenzimidazole was reacted with compound 1 under the
same experimental conditions gave N-(3-methyl-1-phenyl-1H-furo
In a similar manner, 1-(5-(5-hydroxy-3-methyl-1-phenyl-1H-pyr-
azole-4-yl)-2-methylfuran-3-yl)ethanone (12) and 2-(5-hydroxy-3-
methyl-1-phenyl-1H-pyrazole-4-yl)-4-H-furo[3,2-c]chromen-4-one
(13) were obtained in a good yield upon treatment of compound 1
with acetylacetone and 4-hydroxycoumarin in ethanolic sodium
ethoxide solution. Structures 12 and13 wereestablished on thebasis
of their elemental analyses and spectral data. For example, the IR
spectrum of compound 12 revealed an absorption bands at 1695
(acetyl C]O) and 3440 (OH). The 1H NMR spectrum of compound 12
[2,3-c]pyrazol-4(5H)-ylidene-4H-1,2,4-triazol-3-amine
(6)
and
displayed three singlet signals at d 2.33, 2.43 and 2.61 ppm charac-
N-(3-methyl-1-phenyl-1H-furo[2,3-c]pyrazol-4(5H)-ylidene)-1H-
benzimidazol-2-amine (7), respectively. The assigned structures 6
and 7 were confirmed on the basis of elemental analyses and spectral
data (see experimental).
The plausible mechanism for the formation of compounds 3aeb,
6, and 7 may be attributed to the first nucleophilic displacement of
teristic for three methyl groups and broad D2O-exhangeable signal
at 12.82 characteristic for OH, in addition to multiplets centered
around 7.15e7.84 ppm due to aromatic protons and furan H-4. Its
mass spectrum revealed a molecular ion peak at m/z ¼ 296 (Mþ) and
the base peak at 173 (100%) due to the fragment [1-phenyl-3-
methyl-5-hydroxypyrazole]þ.
The formation of compounds 10e13 may be rationalized by two
step reactions, which involve the base promote alkylation of the
hydroxyl group by the
a-chloroketone followed by in situ intra-
molecular CeC bond formation of the initial O-alkyl adduct. It is
worthwhile to mention that all trials to isolate the initially formed
O-alkyl adduct was failed. The preference formation of angular furo
[3,2-c]coumarin 13 is also in the line with similar report by Risitano
and coworkers [39].
N
O
X
Cl
H3C
H3C
NH2
EtOH / TEA
+
N
N
OH
OH
N
N
XH
Ph
Ph
1
X = NH, S, O
2
X
3. Pharmacology
2a-c
a
b
c
NH
S
3.1. Antimicrobial evaluation
O
The tested microorganisms were obtained from the culture
collection at the Microbiology laboratory, National Organization for
Scheme 1. Synthesis of pyrazolyl-quinoxaline, 1,4-benzothiazine, and 1,4-benzoxazine
2aec.

S. Bondock et al. / European Journal of Medicinal Chemistry 46 (2011) 2555e2561
2557
X
O
H3C
N
NH2
X
N
N
O
N
N
Ph
3
X
5
N
N
N
H
a
b
CH
N
N
H3C
H3C
H2N
EtOH / TEA
N
H
N
O
N
O
N
Ph
N
X
N
O
3
EtOH / TEA
Cl
Ph
7
H3C
X
NH2
N
N
N
OH
N
Ph
1
N
N
H
N
N
EtOH / TEA
N
N
H3C
H3C
H2N
N
H
N
O
N
N
Ph
X
OH
EtOH / TEA
N
6
NH
4
Ph
X
X
N
O
N
H3C
9
X
H3C
a
b
CH
O
N
N
Ph
N
OH
N
O
8
Ph
9
Scheme 2. Synthesis of furo[2,3-c]pyrazole derivatives.
Drug Control and Research (NODCAR). Thirteen of the newly
synthesized target compounds were evaluated for their in vitro
antibacterial activity against Bacillus subtilis (ATCC 6633) and
Bacillus thuringiensis (ATCC 6051) as examples of Gram-positive
bacteria and Escherichia coli (ATCC 14169) and Pseudomonas aeru-
ginosa (ATCC 27853) as examples of Gram-negative bacteria. They
were also evaluated for their in vitro antifungal potential against
Fusarium oxysporum (ATCC 16417) and Botrytis fabae (ATCC 14862)
fungal strains.
Agar-diffusion method was used for the determination of the
preliminary antibacterial and antifungal activity. Chloramphenicol,
cephalothin and cycloheximide were used as reference drugs. The
results were recorded for each tested compound as the average
diameter of inhibition zones (IZ) of bacterial or fungal growth
around the disks in mm. The minimum inhibitory concentration
(MIC) measurement was determined for compounds showed
significant growth inhibition zones (>14 mm) using twofold serial
The inhibition zone diameters values cited in Table 1 between
brackets are attributed to the tested original concentration (1 mg/
mL) as a preliminary test. The results depicted in Table 1 revealed
that most of tested compounds displayed variable inhibitory effects
on the growth of the tested Gram-positive and Gram-negative
bacterial strains, and also against fungal strains.
In general, most of the tested compounds revealed better
activity against the Gram-positive rather than the Gram-negative
bacteria. It would be also noticed that compounds belonging to the
furo[2,3-c]pyrazoles and furylpyrazole series (Schemes 2 and 3)
exhibited better antibacterial potentials than members of the pyr-
azolyl-quinoxaline, pyrazolyl-1,4-benzothiazine and pyrazolyl-1,4-
benzoxazine (Scheme 1).
Regarding the activity of the furylpyrazoles against Gram-posi-
tive bacteria, the results revealed that compounds 10, 11, and 12
exhibited divergent antibacterial activity against the tested organ-
isms. In this view, compound 12 was equipotent to chloramphenicol
dilution method [40]. The MIC (
m
g/mL) and inhibition zone diam-
in inhibiting the growth of B. subtilis (MIC 3.125
mg/mL), while its
eters values are recorded in Table 1.
activity was 50% lower than of chloramphenicol against
H
O
O
O
H
O
H
C
O
H
3C
H
C
3
H
C
H
3C
Ph
3
N
N
O
H
O
O
O
H
C
N
3
O
N
Ph
K
2CO3
DM
/
SO
Et Na
O
H
C
3
12
10
H
O
1
H
O
N
Ph
H
3C
H
3C
N
H
COC
3
O
H
3C
H
O
O
O
O
O
N
Et Na
O
K
DM
/ SO
2CO3
H
O
N
13
Ph
O
O
11
Scheme 3. Synthesis of furan, benzofuran and furo[3,2-c]coumarin derivatives.

2558
S. Bondock et al. / European Journal of Medicinal Chemistry 46 (2011) 2555e2561
Table 1
Minimal inhibitory concentrations (MIC,
m
g/mL) and inhibition zone (mm) of some new synthesized compounds.
Compound no.
MICa in
Bacteria
mg/mL, and zone of inhibition (mm)
Fungi
Gram-positive bacteria
Gram-negative bacteria
B. subtilis
B. thuringiensis
E. coli
P. aeruginosa
F. oxysporum
B. fabae
2a
2b
2c
3a
3b
6
7
9a
9b
10
11
12
50 (19)
100 (15)
50 (20)
100 (15)
100 (15)
50 (19)
100 (15)
100 (15)
50 (19)
6.25 (38)
50 (20)
100 (15)
50 (19)
50 (19)
100 (16)
100 (15)
25 (27)
100 (15)
100 (15)
50 (19)
100 (15)
100 (15)
100 (15)
100 (16)
12.5 (31)
100 (15)
100 (14)
100 (14)
50 (19)
50 (20)
50 (19)
100 (15)
50 (20)
100 (16)
50 (19)
6.25 (37)
12.5 (31)
50 (19)
50 (19)
100 (15)
25 (26)
100 (15)
100 (16)
100 (15)
100 (15)
100 (16)
100 (16)
50 (20)
100 (16)
50 (20)
12.5 (33)
50 (19)
25 (26)
25 (25)
12.5 (32)
6.25 (38)
3.125 (44)
3.125 (43)
12.5 (32)
6.25 (38)
6.25(37)
3.125 (42)
12.5 (33)
100 (15)
6.25 (38)
6.25 (37)
6.25 (38)
6.25 (38)
6.25 (37)
6.25 (37)
6.25 (37)
100 (15)
25 (26)
100 (15)
50 (20)
50 (18)
6.25 (38)
13
6.25 (37)
Reference drugs
Chloramphenicol
Cephalothin
Cycloheximide
3.125 (42)
6.25 (36)
NT
3.125 (44)
6.25 (37)
NT
6.25 (37)
6.25 (38)
NT
6.25 (38)
6.25 (37)
NT
NTb
NT
3.125 (43)
NT
NT
3.125 (42)
a
MIC: Minimal inhibitory concentration values.
NT: Not tested.
b
1,4-benzoxazine to pyrazole nucleus at position 4 in compounds
2a, 2b, and 2c unfortunately produced weak antimicrobial
activity.
B. thuringiensis. Compounds 10 and 11 showed 50% of the activity of
chloramphenicol (MIC 6.25 g/mL) but they were equipotent to
cephalothin in inhibiting the growth of B. subtilis and B. thuringiensis
(MIC 6.25 g/mL).
m
ꢁ High biological activity can be correlated with low electron
m
density of ring systems.
On the other hand, compounds 2a, 2b, 2c, 3a, 3b, 9b and 13
exhibited weak to moderate growth inhibitory activity against Gram-
In conclusion, the objective of the present study was to synthe-
size and investigate the antimicrobial activities of some new func-
tionalized 4-hetarylpyrazoles and furo[2,3-c]pyrazoles with the
hope of discovering new structure leads serving as potent antimi-
crobial agents. Our aim has been verified by the synthesis of three
different groups of structure hybrids comprising basically the pyr-
azole moiety attached to either quinoxaline, 1,4-benzothiazine, 1,4-
benzoxazine, polysubstituted furan, benzofuran, furo[3,2-c]chro-
menone, or fused to furan counter parts through various linkages of
synthergistic purpose. The obtained results clearly revealed that
compounds derived from furan, benzofuran and furo[2,3-c]pyr-
azoles exhibited better antimicrobial activity than their quinoxaline,
1,4-benzothiazine and 1,4-benzoxazine structure variants.
positive bacteria as revealed from their MIC values (25e100
Among these compounds 3b and 13 showed relatively good growth
inhibitory profiles against B. subtilis (MIC 12.5 g/mL) which were
about 25% of the activity of chloramphenicol and 50% of cephalothin
against the same organism. Moreover, distinctive anti-Gram-positive
profile was displayed by compound 7 where it proved to be equi-
mg/mL).
m
potent as chloramphenicol against B. subtilis (MIC 3.125
together with a significant activity against B. thuringiensis (MIC
6.25 g/mL).
mg/mL)
m
Concerning the antibacterial activity of the compounds 2c, 3b, 7,
and 9a revealed weak growth inhibitory against the tested Gram-
negative bacteria (MIC 50
3a and 13 showed equipotent activity as chloramphenicol and
cephalothin (MIC 6.25 g/mL) against E. coli and P. aeruginosa.
mg/mL). On the other hand, compounds
m
Regarding the activity 4-hetarylpyrazoles and furo[2,3-c]pyr-
azoles, against fungal strains, the results revealed that compounds
7 and 13 were 50% lower than cycloheximide in inhibitory the
4. Experimental
All melting points were measured on a Gallenkamp electro-
thermal melting point apparatus. IR spectra were recorded for KBr
disc on a Mattson 5000 FTIR spectrophotometer. 1H NMR spectra
were measured on a Bruker AC 300 (300 MHz) in CDCl3 or DMSO-d6
as solvent, using TMS as an internal standard, and chemical shifts
are expressed as dppm. Mass spectra were determined on Finnigan
Incos 500 (70 ev). Elemental analyses were carried out in the
Microanalytical Unit of the Faculty of Science, Cairo University.
2-Chloro-1-(5-hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)etha-
none (1) [38] was prepared according to literature procedure.
growth of B. fabae and F. oxysporum (MIC 6.25
reactivity of compound 3b was 25% lower than cycloheximide
against F. oxysporum (MIC 12. 5 g/mL).
The results of the antimicrobial screening demonstrated the
following assumptions about the structural activity relationship
(SAR):
mg/mL), while the
m
ꢁ It is interesting to point out that furylpyrazoles 10e12 having
electron withdrawing groups such as COCH3 or ArCO
(Ar ¼ benzofuryl) recorded higher antibacterial activity.
ꢁ The incorporation of furan or benzofuran to pyrazole nucleus
at position 4 directly (in case of furan) or via a carbonyl
linker (in case of benzofuran) produced a high antimicrobial
activity.
4.1. General procedure for reaction of compound 1 with aromatic
and heterocyclic amines
To a solution of compound 1 (2.50 g, 10 mmol) in absolute
ethanol (30 mL), an equimolar amount of the appropriate aromatic
or heterocyclic amines and two drops of triethylamine was added
and the mixture was heated under reflux for 8 h, then left to cool.
ꢁ Conversion of compound
1 to furo[2,3-c]pyrazoles 3aeb
enhanced also the antimicrobial activity. On the other hand,
incorporation of quinoxaline, 1,4-benzothiazine and/or

S. Bondock et al. / European Journal of Medicinal Chemistry 46 (2011) 2555e2561
2559
The solid products that obtained was collected by filtration, washed
with ethanol, dried well and recrystallized from the appropriate
solvent.
(Mþ, 8.4), 251 (31.5), 198 (100), 105 (8.24), 91 (18.1), 77 (15). Anal.;
For C17H14N4O (290.32) Calcd.: C 70.33; H 4.86; N 19.30%, Found: C
70.23; H 4.90; N 19.36%.
4.1.5. N-[(4Z)-3-Methyl-1-phenyl-1H-furo [2,3-c]pyrazol-4(5H)-
ylidene]pyrazin-2-amine (3b)
4.1.1. 4-(3,4-Dihydroquinoxalin-2-yl)-3-methyl-1-phenyl-1H-
pyrazol-5-ol (2a)
This compound was prepared from 2-aminopyrazine (0.95 g,
10 mmol) by heating for 12 h under reflux and recrystallized from
DMF.
This compound was prepared from o-phenylenediamine (1.08 g,
10 mmol) by heating for 8 h under reflux and recrystallized from
DMF.
Brown crystals; Yield 86%; mp 272e273 ꢂC; IR (KBr) ymax
/
Red crystals; Yield 70%; mp 235e236 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼ 2926 (CeH aliph.), 1630 (C]N). 1H NMR (DMSO-d6):
dppm ¼ 2.36 (s, 3H, CH3), 4.97 (s, 2H, OCH2), 7.18e7.95 (m, 8H,
AreH). 13C NMR (DMSO-d6): dppm ¼ 12.6 (CH3), 69.7 (CH2O), 104.9
(C-3a), 120.5, 126.6, 129.6, 136.6 (AreC), 131.0 (pyrazine C-5), 142.9
(pyrazine C-6), 145.6 (pyrazine C-3), 146.3 (C-3), 150.0 (pyrazine
C-2), 154.5 (C-4), 161.4 (C-6a). MS m/z (%): 291 (Mþ, 11.8), 198 (15.4),
185 (15.8), 172 (74.2), 105 (9.3), 91 (53.2), 77 (100). Anal.; For
C16H13N5O (291.31) Calcd.: C 65.97; H 4.50; N 24.04%, Found: C
65.37; H 4.90; N 24.16%.
cmꢀ1 ¼ 3430 (OH), 3260 (NH), 2924 (CeH aliph.), 1620 (C]N). 1H
NMR (DMSO-d6): dppm ¼ 2.44 (s, 3H, CH3), 4.54 (s, 2H, NCH2), 6.32
(s, 1H, NH), 7.0e8.10 (m, 9H, AreH), 12.75 (s, 1H, OH). 13C NMR
(DMSO-d6): dppm ¼ 12.2 (CH3), 34.4 (CH2N), 102.2 (pyrazole C-4),
116.5, 119.7, 122.5, 126.6, 130.1, 132.2, 136.6, 138.0 (AreC), 149.4
(pyrazole C-3),157.2 (quinoxaline C-2),164.4 (pyrazole C-5). MS m/z
(%): 304 (Mþ, 87.7) 210 (10.2), 119 (21.8), 169 (22), 91 (17.9), 77
(100). Anal.; For C18H16N4O (304.35) Calcd.: C 71.04; H 5.30; N
18.41%, Found: C 71.13; H 5.33; N 18.29%.
4.1.6. N-[(4Z)-3-Methyl-1-phenyl-1H-furo [2,3-c]pyrazol-4(5H)-
ylidene]-4H-1,2,4-triazol-3-amine (6)
4.1.2. 4-(2H-1,4-benzothiazin-3-yl)-3-methyl-1-phenyl-1H-
pyrazol-5-ol (2b)
This compound was prepared from 3-amino-4H-1,2,4-triazole
(0.84 g, 10 mmol) by heating for 4 h under reflux and recrystallized
from a mixture of EtOH/DMF (1:1).
This compound was prepared from o-aminothiophenol (1.25 g,
10 mmol) by heating for 8 h under reflux and recrystallized from
DMF.
Brown crystals; Yield 79%; mp 192e193 ꢂC; IR (KBr) ymax
/
Yellow crystals; Yield 75%; mp 210e211 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼3380 (NH), 2928 (CeH aliph.),1628 (C]N). 1H NMR (DMSO-
d6): dppm ¼ 2.38 (s, 3H, CH3), 5.24 (s, 2H, OCH2), 7.15e8.12 (m, 6H,
AreH). MS m/z (%): 280 (Mþ, 16.5), 252 (11.2), 212 (16.5), 198 (68.9),
174 (54.5), 105 (15,1), 91 (49.9), 77 (100). Anal.; For C14H12N6O
(280.28) Calcd.: C 59.99; H 4.32; N 29.98%, Found: C 59.89; H 4.26;
N 30.09%.
cmꢀ1 ¼ 3433 (OH), 2920 (CeH aliph.), 1610 (C]N). 1H NMR (DMSO-
d6): dppm ¼ 2.44 (s, 3H, CH3), 4.20 (s, 2H, SCH2), 7.14e8.17 (m, 9H,
AreH), 13.10 (s, 1H, OH). 13C NMR (DMSO-d6): dppm ¼ 12.4 (CH3),
23.9 (CH2S), 99.1 (pyrazole C-4), 122.5, 122.8, 123.0, 124.2, 126.4,
127.6, 128.9, 130.2, 137.7, 141.9 (AreC), 145.3 (pyrazole C-3), 156.2
(benzothiazine C-3), 165.2 (pyrazole C-5). MS m/z (%): 321 (Mþ,
29.8) 288 (24.1), 186 (9.2), 148 (3.6), 91 (14.7), 77 (100). Anal.; For
C18H15N3OS (321.40) Calcd.: C 67.27; H 4.70; N 13.07%, Found: C
67.13; H 4.77; N 13.36%.
4.1.7. N-[(4Z)-3-Methyl-1-phenyl-1H-furo [2,3-c]pyrazol-4(5H)-
ylidene]-1H-benzimidazol-2-amine (7)
This compound was prepared from 2-aminobenzimidazole
(1.33 g, 10 mmol) by heating for 5 h under reflux and recrystallized
from a mixture of EtOH/DMF (1:1).
4.1.3. 4-(2H-1,4-benzoxazin-3-yl)-3-methyl-1-phenyl-1H-pyrazol-
5-ol (2c)
Pale yellow crystals; Yield 82%; mp 180e181 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼3320 (NH), 2920 (CeH aliph.),1627 (C]N). 1H NMR (DMSO-
d6): dppm ¼ 2.39 (s, 3H, CH3), 5.33 (s, 2H, OCH2), 7.21e8.18 (m, 9H,
AreH), 9.15 (s,1H, NH). 13C NMR (DMSO-d6): dppm ¼ 12.6 (CH3), 68.6
(CH2O), 95.2 (C-3a), 114.6, 118.9, 121.3, 122.6, 123.2, 126.6, 129.8,
136.7, 137.9 (AreC), 135.5 (C-6a), 144.8 (C-3), 156.4 (C-4), 157.8
(C-6a), 167.1 (benzimidazole C-2). MS m/z (%): 329 (Mþ, 19.2), 252
(10.6), 224 (10.2), 198 (58.8), 131 (43.8), 105 (16.6), 91 (49.9), 77
(100). Anal.; For C19H15N5O (329.36) Calcd.: C 69.29; H 4.59; N
21.26%, Found: C 69.24; H 4.63; N 21.27%.
This compound was prepared from o-aminophenol (1.09 g,
10 mmol) by heating for 10 h under reflux and recrystallized from
DMF.
Yellow crystals; Yield 80%; mp 200e201 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼3431 (OH), 2945 (CeH aliph.), 1616 (C]N). 1H NMR (DMSO-
d6): dppm ¼ 2.22 (s, 3H, CH3), 4.75 (s, 2H, OCH2), 7.21e8.14 (m, 9H,
AreH), 12.72 (s, 1H, OH). 13C NMR (DMSO-d6): dppm ¼ 12.6 (CH3),
65.5 (CH2O), 102.0 (pyrazole C-4), 117.4, 122.3, 123.5, 124.2, 125.6,
126.4, 130.1, 136.4, 137.0, 148.0 (AreC), 147.2 (pyrazole C-3), 148.5
(benzoxazine C-3), 164.9 (pyrazole C-5). MS m/z (%): 305 (Mþ, 35.8),
229 (20.9), 200 (15.4),173 (8.6),105 (18.3), 91 (12.3), 77 (100). Anal.;
For C18H15N3O2 (305.33) Calcd.: C 70.81; H 4.95; N 13.76%, Found: C
70.72; H 4.99; N 13.91%.
4.2. Synthesis of 3-methyl-1-phenyl-1H-furo [2,3-c]pyrazol-4-
(5H)-one (5)
A solution of compound 1 (1.25 g, 5 mmol) and triethylamine
(1 mL) in absolute ethanol (25 mL) was heated to 50 ꢂC for 3 h. After
cooling the resultant precipitate was filtered and crystallized from
ethanol.
4.1.4. N-[(4Z)-3-Methyl-1-phenyl-1H-furo [2,3-c]pyrazol-4(5H)-
ylidene]pyridin-2-amine (3a)
This compound was prepared from 2-aminopyridine (0.94 g,
10 mmol) by heating for 6 h under reflux and recrystallized from
DMF.
Colorless crystals; Yield 78%; mp 152e153 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼ 2923 (CeH aliph.), 1631 (C]O). 1H NMR (CDCl3):
dppm ¼ 2.52 (s, 3H, CH3), 5.05 (s, 2H, OCH2), 7.27e7.81 (m, 5H,
AreH). 13C NMR (DMSO-d6): dppm ¼ 15.4 (CH3), 73.5 (CH2O),
100.3 (C-3a), 122.5, 127.8, 128.5, 136.4 (AreC), 149.6 (C-3), 159.8
(C-6a), 191.5 (C]O). MS m/z (%): 214 (Mþ, 40.1), 186 (22.2), 137
(19.3), 109 (18.5), 105 (53.2), 77 (100). Anal.; For C12H10N2O2
(214.22) Calcd.: C 67.28; H 4.71; N 13.08% Found: C 67.52; H 4.64;
N 12.91%.
Brown crystals; Yield 76%; mp 245e246 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼ 2935 (CeH aliph.), 1635 (C]N). 1H NMR (DMSO-d6):
dppm ¼ 2.33 (s, 3H, CH3), 5.04 (s, 2H, OCH2), 7.13e7.82 (m, 9H,
AreH). 13C NMR (DMSO-d6): dppm ¼ 12.6 (CH3), 69.9 (CH2O), 104.7
(C-3a), 118.9 (pyridine C-5), 120.5, 126.6, 129.6, 136.6 (AreC), 123.4
(pyridine C-3), 136.0 (pyridine C-4), 146.3 (C-3), 146.9 (pyridine
C-6), 153.2 (C-4), 159.4 (pyridine C-2), 161.2 (C-6a). MS m/z (%): 290

2560
S. Bondock et al. / European Journal of Medicinal Chemistry 46 (2011) 2555e2561
4.3. Alternative method for the synthesis of compounds 3aeb
(benzofuran C-2), 155.7 (pyrazole C-3), 157.5 (pyrazole C-5), 164.3
(C]O). MS m/z (%): 318 (Mþ, 15), 278 (31.5), 261 (12.1), 201 (45),
174 (28.8), 118 (96.1), 91(89.6), 77 (100). Anal.; For C19H14N2O3
(318.33) Calcd.: C 71.69; H 4.43; N 8.80%, Found: C 71.62; H 4.32;
N 8.69%.
A mixture of 2-aminopyridine (0.47 g, 5 mmol) or 2-amino-
pyrazine (0.48 g, 5 mmol) and 3-methyl-1-phenyl-1H-furo[2,3-c]
pyrazol-4(5H)-one (5) (1.07 g, 5 mmol) in ethanol (25 mL) was
refluxed for 4 h. The solution was allowed to cool and poured on
cold water (50 mL). The solid that obtained was collected, dried,
and crystallized from DMF to give compounds 3aeb.
4.5.2. (5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-(3-methyl-
1-benzofuran-2-yl)methanone (11)
Pale yellow crystals (EtOH/DMF); Yield 88%; mp 195e196 ꢂC, IR
(KBr) nmax/cmꢀ1 ¼ 3390 (OH), 2924 (CeH aliph.), 1635 (CO). 1H
NMR (DMSO-d6): dppm ¼ 2.31 (s, 3H, CH3), 2.50 (s, 3H, CH3),
6.93e8.01 (m, 9H, AreH), 12.80 (s, 1H, OH). MS m/z (%): 333 (Mþ,
24.32), 201 (100), 134 (56.4), 91 (26.4), 77 (6.7). 13C NMR (DMSO-
d6): dppm ¼ 12.3 (CH3), 16.4 (CH3), 96.3 (benzofuran C-3), 114.0,
117.4, 118.1, 123.1, 123.8, 125.9, 128.6, 130.2, 136.7, 138.8 (AreC),
144.1 (benzofuran C-2),146.7 (pyrazole C-3), 154.2 (pyrazole C-5),
165.5 (C]O). Anal.; For C20H16N2O3 (332.35) Calcd.: C 72.28; H
4.85; N 8.43%, Found: C 72.32; H 4.77; N 8.51%.
4.4. General procedure for reaction of compound 1 with secondary
amines
To a solution of compound 1 (1.25 g, 5 mmol) in absolute ethanol
(20 mL) was added piperidine (0.6 g, 7 mmol) or morpholine
(0.65 g, 7 mmol) at room temperature. Immediately a deep colored
solution (usually red) was obtained and the temperature of the
reaction mixture was rose to 60e65 ꢂC. After standing for 30 min,
the reaction mixture was run into cold water (100 mL). The
precipitated solid was collected by filtration, washed with water,
dried, and recrystallized from ethanol.
4.6. General procedure for reaction of compound 1 with
acetylacetone and 4-hydroxycoumarin
4.4.1. 1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
(piperidin-1-yl)ethanone (9a)
To an ethanolic solution of NaOEt (prepared from sodium metal
(0.11 g, 5 mg atom) and 25 mL of absolute ethanol) was added
(5 mmol) of the appropriate acetylacetone or 4-hydroxycoumarin.
After stirring for 15 min at room temperature, compound 1 (1.25 g,
5 mmol) was added and stirring continued for 24 h. The solvent was
evaporated under reduced pressure, and the remainder was poured
onto ice-water (50 mL) and neutralized with diluted HCl. The solid
products obtained were collected by filtration, washed with water,
dried, and crystallized from an appropriate solvent.
Yellow crystals; Yield 76%; mp 165e166 ꢂC; IR (KBr) ymax
/
cmꢀ1 ¼3380 (OH), 2924 (CeH aliph.),1660 (C]O). 1H NMR (DMSO-
d6): dppm ¼ 1.13e1.45 (m, 6H, 3CH2), 2.27 (t, J ¼ 6.5 Hz, 4H, 2NCH2),
2.33 (s, 3H, CH3), 3.43 (s, 2H, NCH2CO), 7.12e7.65 (m, 5H, AreH),
12.92 (s, 1H, OH). 13C NMR (DMSO-d6): dppm ¼ 15.0 (CH3), 24.7
(piperidine C-4), 26.3 (piperidine C-3 and C-5), 55.6 (piperidine C-2
and C-6), 61.5 (CH2N), 102.8 (pyrazole C-4), 121.3, 126.4, 128.9, 136.1
(AreC), 146.2 (pyrazole C-3), 158.6 (pyrazole C-5), 188.3 (C]O). MS
m/z (%): 299 (Mþ, 34.9), 284 (8.6), 201 (20.2), 187 (12.8), 174 (9.6),
125 (7.2), 98 (100), 84 (10.1). Anal.; For C17H21N3O2 (299.37) Calcd.:
C 68.20; H 7.07; N 14.04%, Found: C 68.39; H 7.14; N 14.16%.
4.6.1. 1-(5-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
methylfuran-3-yl)ethanone (12)
Pale yellow crystals (EtOH); Yield 82%; mp 220e221 ꢂC; IR (KBr)
ymax/cmꢀ1 ¼ 3440 (OH), 2923 (CeH aliph.), 1695 (C]O). 1H NMR
(DMSO-d6): dppm ¼ 2.33 (s, 3H, CH3), 2.43 (s, 3H, CH3), 2.61 (s, 3H,
4.4.2. 1-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-2-
(morpholin-4-yl)ethanone (9b)
Yellow crystals; Yield 80%; mp 145e146 ꢂC; IR (KBr) ymax
/
CH3), 7.15e7.84 (m, 6H, AreH and furan H-4), 12.82 (s, 1H, OH). 13
C
cmꢀ1 ¼3395 (OH), 2935 (CeH aliph.),1665 (C]O). 1H NMR (DMSO-
d6): dppm ¼ 2.29 (t, J ¼ 6.8 Hz, 4H, 2NCH2), 2.36 (s, 3H, CH3), 3.47
(s, 2H, NCH2CO), 3.75 (t, J ¼ 6.8 Hz, 4H, 2CH2O), 7.16e7.75 (m, 5H,
AreH), 12.85 (s, 1H, OH). MS m/z (%): 301 (Mþ, 36.7), 284 (7.9), 201
(26.2), 173 (19.8), 128 (15.3), 100 (100), 86 (14.3). Anal.; For
C16H19N3O3 (301.34) Calcd.: C 63.77; H 6.36; N 13.94%, Found: C
63.86; H 6.40; N 13.99%.
NMR (DMSO-d6): dppm ¼ 12.4 (CH3), 14.3 (CH3), 28.9 (CH3), 100.5
(furan C-3), 103.2 (pyrazole C-4), 110.2 (furan C-4), 120.8, 126.7,
128.6, 137.2 (AreC), 144.4 (pyrazole C-3), 159.1 (pyrazole C-5), 161.8
(furan C-2), 166.2 (furan C-5), 195.4 (C]O). MS m/z (%): 296 (Mþ,
15), 281 (16.9), 253 (17.9), 219 (19.8), 201 (13.8), 173 (100), 105 (39),
77 (56.2). Anal.; For C17H16N2O3 (296.12) Calcd.: C 68.91; H 5.44; N
9.45%, Found: C 68.79; H 5.29; N 9.53%.
4.5. General procedure for reaction of compound 1 with
salicyaldehyde and 2-hydroxyacetophenone
4.6.2. 2-(5-Hydroxy-3-methyl-1-phenyl-1H-pyrazol-4-yl)-4H-furo
[3,2-c]chromen-4-one (13)
Yellow crystals (EtOH/CHCl3); Yield 80%; mp 195e196 ꢂC; IR
(KBr) nmax/cmꢀ1 ¼3380 (OH), 2925 (CeH aliph.),1715 (CO). 1H NMR
(DMSO-d6): dppm ¼ 2.30 (s, 3H, CH3), 7.12e7.94 (m, 10H, AreH and
furan H-3), 12.98 (s, 1H, OH). MS m/z (%): 358 (Mþ, 12.1), 282 (6.8),
253 (16), 214 (42), 198 (17), 173 (100), 144 (6.3), 105 (40), 91 (45), 77
(56.2). Anal.; For C21H14N2O4 (358.35) Calcd.: C 70.39; H 3.94; N
7.82% Found: C 70.24; H 3.90; N 7.88%.
To a solution of compound 1 (0.71 g, 2.84 mmol) and anhydrous
potassium carbonate (1.0 g) in 15 mL DMSO was added an equi-
molar amount of salicyaldehyde or 2-hydroxyacetophenone and
the reaction mixture was stirred at room temperature for 8 h. The
reaction mixture was then poured into water (25 mL) and the
precipitate obtained was filtered, dried, and crystallized from an
appropriate solvent.
5. Antimicrobial evaluation
4.5.1. 1-Benzofuran-2-yl(5-hydroxy-3-methyl-1-phenyl-1H-
pyrazol-4-yl)methanone (10)
The disks of Whatman filter paper were prepared with standard
size (5.0 mm diameter) and kept into 1.0 Oz screw capped wide
mouthed containers for sterilization. These bottles are kept into hot
air oven at a temperature of 150 ꢂC. Then, the standard sterilized
filter paper disks impregnated with a solution of the test compound
in DMF (1 mg/mL) were placed on nutrient agar plate seeded with
the appropriate test organism in triplicates. Standard concentrations
Yellow crystals (EtOH/DMF); Yield 85%; mp 185e186 ꢂC; IR
(KBr) nmax/cmꢀ1 ¼ 3380 (OH), 2924 (CeH aliph.), 1635 (CO). 1H
NMR (DMSO-d6): dppm ¼ 2.31 (s, 3H, CH3), 7.12e8.01 (m, 10H,
AreH and benzofuran H-3), 12.92 (s, 1H, OH). 13C NMR (DMSO-d6):
dppm ¼ 15.6 (CH3), 104.3 (pyrazole C-4), 108.2 (benzofuran C-3),
117.1, 119.8, 123.9, 125.7, 126.8, 128.0, 129.3, 138.6 (AreC), 143.7

S. Bondock et al. / European Journal of Medicinal Chemistry 46 (2011) 2555e2561
2561
of 106 CFU/mL (Colony Forming U/mL) and 104 CFU/mL were used for
antibacterial and antifungal assay, respectively. Pyrex glass Petri
dishes (9 cm in diameter) were used and two disks of filter paper
were inoculated in each plates. The utilized test organisms were: B.
subtilis and B. thuringiensis as examples of Gram-positive bacteria
and E. coli and P. aeruginosa as examples of Gram-negative bacteria.
They were also evaluated for their in vitro antifungal potential
against F. oxysporum and B. fabae fungal strains. Chloramphenicol,
cephalothin and cycloheximide were used as standard antibacterial
and antifungal agents, respectively. DMF alone was used as control at
the same above-mentioned concentration and due this there was no
visible change in bacterial growth. The plates were incubated at
37 ꢂC for 24 h for bacteria and for 48 h for fungi. Compounds that
showed significant growth inhibition zones (>14 mm) using the
twofold serial dilution technique, were further evaluated for their
minimal inhibitory concentrations (MICs).
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The microdilution susceptibility test in MüllereHinton Broth
(Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for the
determination of antibacterial and antifungal activity, respectively.
Stock solutions of the tested compounds, chloroamphenicol,
cephalothin and cycloheximide were prepared in DMF at concen-
tration of 1000
dard method broth (Difco) to prepare serial twofold dilutions in the
range of 500e3.125 g/mL 10 mL of the broth containing about
mg/mL. Each stock solution was diluted with stan-
m
106 CFU/mL of test bacteria was added to each well of 96-well
microtiter plate. The sealed microplates were incubated at 37 ꢂC for
24 h for antibacterial activity and at 37 ꢂC for 48 h for antifungal
activity in a humid chamber. At the end of the incubation period,
the minimal inhibitory concentrations (MIC) values were recorded
as the lowest concentrations of the substance that had no visible
turbidity. Control experiments with DMF and uninoculated media
were run parallel to the test compounds under the same conditions.
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Acknowledgments
[31] A. Khalaj, M. Nakhjiri, A.S. Negahbani, M. Samadizadeh, L. Firoozpour,
S. Rajabalian, N. Samadi, M.A. Faramarzi, N. Adibpour, A. Shafiee,
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(2008) 1329e1335.
The authors are very grateful to Dr. Ehab Ali, Department of
Botany, Faculty of Science, Mansoura University, for performing the
antimicrobial evaluation.
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