Synthesis of new 3-aryl-4-(3-aryl-4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H- pyrazole derivatives as potential antimicrobial agents

Article abstract of DOI:10.1007/s00044-012-0267-8

New (E)-1-aryl-3-(3-aryl-1-phenyl-1H-pyrazol-4-yl)prop-2-en-1-ones 6 (pyrazolic chalcones) were synthesized from Claisen-Schmidt reaction of 3-aryl-1-phenylpyrazol-4-carboxaldehydes 4 with several acetophenone derivatives 5. Subsequently, the cyclocondensation reaction of chalcones 6 with phenylhydrazine in acetic acid medium afforded the new 3-aryl-4-(3-aryl-4,5- dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazoles 7. The synthesized compounds were characterized by spectral studies and evaluated for their in vitro antibacterial activity against three pathogenic bacterial strains, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and in vitro antifungal activity against three pathogenic fungal strains Aspergillus flavus, Chrysosporium keratinophilum, and Candida albicans.

Full text of DOI:10.1007/s00044-012-0267-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2654–2664
DOI 10.1007/s00044-012-0267-8
ORIGINAL RESEARCH
Synthesis of new 3-aryl-4-(3-aryl-4,5-dihydro-1H-pyrazol-5-yl)-1-
phenyl-1H-pyrazole derivatives as potential antimicrobial agents
•
Shridhar Malladi Arun M. Isloor
•
•
S. K. Peethambar Hoong Kun Fun
Received: 16 April 2012 / Accepted: 28 September 2012 / Published online: 9 October 2012
Ó Springer Science+Business Media New York 2012
Abstract New (E)-1-aryl-3-(3-aryl-1-phenyl-1H-pyrazol-4-
yl)prop-2-en-1-ones 6 (pyrazolic chalcones) were synthesized
from Claisen–Schmidt reaction of 3-aryl-1-phenylpyrazol-4-
carboxaldehydes 4 with several acetophenone derivatives 5.
Subsequently, the cyclocondensation reaction of chalcones 6
with phenylhydrazine in acetic acid medium afforded the new
3-aryl-4-(3-aryl-4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-
pyrazoles 7. The synthesized compounds were characterized
by spectral studies and evaluated for their in vitro antibacterial
activity against three pathogenic bacterial strains, Staphylo-
coccus aureus, Escherichia coli, and Pseudomonas aerugin-
osa, and in vitro antifungal activity against three pathogenic
fungal strains Aspergillus flavus, Chrysosporium keratino-
philum, and Candida albicans.
Introduction
Antimicrobial resistance is a global public health concern
that is impacted by both human and non-human antimi-
crobial use. The consequences of antimicrobial resistance
are particularly important when pathogens are resistant to
antimicrobials that are critically important in the treatment
of human disease. The treatment of microbial infections
still remains an important and challenging therapeutic
problem because of factors that include emerging infec-
tious diseases and the increasing number of multidrug-
resistant microbial pathogens. In spite of the large number
of antibiotics and chemotherapeutics available for medical
use, the emergence of old and new antibiotic resistant
bacterial strains in the last decades constitutes a substantial
need for new classes of antibacterial agents (Chopra et al.,
2008). It has been postulated that the development of
resistance to known antibiotics could be overcome by
identifying new drug targets via genomics, improving
the existing nature of antibiotics, and identifying new
antibacterial agents with novel mode of action and struc-
ture (Walsh, 2000; Ritter and Wong, 2001; Wijkmans and
Beckett, 2002).
Keywords Claisen–Schmidt reaction ꢀ
Pyrazolic chalcones ꢀ Pyrazolines ꢀ Antimicrobial
S. Malladi ꢀ A. M. Isloor (&)
Medicinal Chemistry Laboratory, Department of Chemistry,
National Institute of Technology Karnataka, Surathkal,
Mangalore 575025, India
e-mail: isloor@yahoo.com
1,2-Diaryl substituted heterocyclic system occurs in so
many diverse classes of biologically interesting low
molecular-weight compounds that it would be an under-
statement to link it to the ease of synthesis of the vicinal
diaryl system (Kubinyi and Muller, 2004). Drugs based on
a pyrazole ring bearing two adjacent aryl groups in a vic-
inal relation have often been occupying a position in the
list of best selling pharmaceutical products since the
beginning of this decade (Kubinyi and Muller, 2004).
Recently, there has been considerable amount of progress
in 1,3-diarylpyrazole chemistry because of the recognition
of importance of the pyrazole structure in biological
S. K. Peethambar
Department of Bio-Chemistry, Kuvempu University,
Jnanasahyadri, Shankaraghatta 577451,
Karnataka, India
H. K. Fun
X-Ray Crystallography Unit, School of Physics, Universiti Sains
Malaysia, 11800 Georgetown, Penang, Malaysia
H. K. Fun
Deparment of Pharmaceutical Chemistry, College of Pharmacy,
King Saud University, P.O. Box 2457, Riyadh 11451,
Saudi Arabia
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Med Chem Res (2013) 22:2654–2664
2655
NH
Scheme 1 Synthesis of the novel
pyrazolic chalcones 6a–k
2
O
HN
CH3
N
R
ZnCl2 / EtOH
RT
CH3
R
NH
+
3
1
2
1. POCl3 / DMF
R
2. NaHCO3 / H2O
A
O
O
H
1
3
CH
R
3
O
6
N
5
N
N
N 1
EtOH, NaOH
7
5
R1
R
B
6a-k
4
6
a
b
c
d
e
f
g
h
i
R
Cl
Br
OCH3
H
Cl
Br
OCH3
H
Cl
Br
R1
biphenyl
biphenyl
biphenyl
4-SCH3-C6H4
4-SCH3-C6H4
4-SCH3-C6H4
4-SCH3-C6H4
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
j
k
OCH3
processes such as antimicrobial (Thumar and Patel, 2011),
anti-inflammatory (Kumar et al., 2011), antitubercular
(Chovatia et al., 2007), antitumor (Joksovic et al., 2009;
Insuasty et al., 2010), antiangiogenesis (Abadi et al., 2003),
antiparasitic (Rathelot et al., 2002), antiviral (Hashem
et al., 2007) and also possesses analegesic and anxiolytic
activity (Shetty and Bhagat, 2008). Motivated by these
findings, and in continuation of our interest in the synthesis
of a variety of heterocyclic systems for biological and
pharmacological evaluations (Vijesh et al., 2011a, b), we
set out to synthesize some novel 3-aryl-4-(3-aryl-4,5-
dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole deriva-
tives and evaluate their antimicrobial activities. It must be
noted that this scaffold provides vicinal diaryl substitution
pattern on both the pyrazole as well as pyrazoline nucleus.
Results and discussion
Chemistry
In order to obtain the pyrazolic chalcones, the corresponding
4-formylpyrazolic precursors 4 were initially synthesized
from the phenylhydrazones 3 (prepared by condensation of
the respective acetophenones 1 and phenylhydrazine 2); fol-
lowing a reported procedure (Kira et al., 1969; Insuasty et al.,
2010). In this sense, the Vilsmeier–Haack reagent (POCl3/
DMF) was employed affording compounds 4. Subsequently,
the Claisen–Schmidt condensation of the obtained aldehydes
4 with the acetophenones 5 afforded the corresponding pyr-
azolic chalcones 6 (Scheme 1). Continuing with the synthetic
approach, the cyclocondensation of the chalcones 6a–k with
123

2656
Med Chem Res (2013) 22:2654–2664
R
Scheme 2 Synthesis of the new
pyrazoles 7a–k
R1
3'
4'
N
NH
1'
N
2
R
5'
HN
3
O
N
N
AcOH
+
5
N
1
N
Δ
R1
2
7a-k
6a-k
7
a
b
c
d
e
f
g
h
i
R
Cl
Br
OCH3
H
Cl
Br
OCH3
H
Cl
Br
R1
biphenyl
biphenyl
biphenyl
4-SCH3-C6H4
4-SCH3-C6H4
4-SCH3-C6H4
4-SCH3-C6H4
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
j
k
OCH3
phenylhydrazine in the presence of acetic acid as solvent,
afforded the desired products 7a–k (Scheme 2). The struc-
turesofthesynthesizedcompounds(7a–k) wereconfirmedby
spectral data (IR, 1H NMR, Mass and elemental analysis).
The structure elucidation of compounds 6a–k was based
on the spectral data (IR, mass spectrometry, and Elemental
analysis). The IR spectrum of compound 6a showed
absorption bands at 3,122–3,055, 1,659, and 1,593 which
were due to the presence of C–H, C=O, and C=N, respec-
tively. In the 1H NMR spectrum of compound 6a the H-6 and
H-7 each one appears as a doublet at d = 7.61 and d =
7.52 ppm, respectively, with coupling constant between
doublet of doublet at d 4.08–4.01 ppm with coupling con-
stants of 12 and 17.2 Hz and a doublet of doublet at d
3.31–3.25 ppm with coupling constants of 7.6 and 17.6 Hz.
Methine proton (50-H) resonated as doublet of doublet at d
5.54–5.49 ppm with coupling constants of 7.6 and 12.2 Hz.
The pyrazole 5-H resonated as singlet at d 8.36. In some cases,
the doublet of doublet of methylene proton around d 3.31 has
notbeenobservedclearlybecauseofmergingofthisparticular
protonwiththewater signalfromDMSO-d6. Themainsignals
in the 13C NMR for 7a corresponds to C-40 at d 42.8, C-50 d
56.4 ppm, and the quaternary C-30 at d 148.9 ppm. The mass
spectrum of 7a showed a molecular ion peak at m/z =
551(M?1), which is in agreement with the molecular for-
mulaC36H27ClN4. Furthermore, the structure ofcompound7c
was confirmed by single crystal X-ray analysis. 1,4-Dioxane
was used as crystallization solvent for 7c, which is in associ-
ation with the crystal (Fig. 2). The crystallographic data of
compounds 6h, 6j, and 7c have been summarized in Table 1.
3
them of J = 15.2 and 14.6 Hz, which agrees with a trans
configuration. The mass spectrum of 6a showed a molecular
ion peak at m/z = 461 (M?1), which is in agreement with
the molecular formula C30H21ClN2O. Furthermore, the
compounds 6h and 6j were confirmed by single crystal X-ray
analysis (Fig. 1).
Similarly, the IR spectrum of compound 7a showed
absorptionbandsat3,042–2,923,1,590,and1,492whichwere
Antimicrobial activity
1
due to C–H, C=N, and C=C, respectively. In the H NMR
spectrum of compounds 7a, the two methylenic 40-H protons
and the stereogenic 50-H proton of the pyrazolinic moiety
displayed a typical ABX type pattern of doublet of doublet.
Thus, the two methylene protons (40-H) displayed two signals;
The newly synthesized compounds 7a–k were tested fortheir
antibacterial activity (in vitro) against Escherichia coli,
Staphylococcus aureus, and Pseudomonas aeruginosa and
their activity was compared to a well-known commercial
123

Med Chem Res (2013) 22:2654–2664
2657
Table 1 Crystallographic data for compounds 6h, 6j and 7c
Crystal data 6h 6j 7c
Empirical
formula
C24H16Cl2N2O C24H15BrCl2N2O C37H30N4O.C4H8O2
Formula
weight
419.29
498.19
296
634.75
100
Temperature 296
(K)
Wavelength 0.71073
˚
(A)
0.71073
Monoclinic
P21/c
0.71073
Triclinic
P1
Crystal
system
Triclinic
Space group P1
Cell dimensions
˚
a (A)
9.6185 (8)
10.6596 (9)
11.4203 (14)
9.9357 (13)
11.1189 (2)
13.0541 (2)
13.0852 (2)
1,662.48
2
˚
b (A)
˚
c (A)
11.8537 (10) 19.656 (3)
3
Volume (A ) 1,044.64
˚
2,222.9
4
Z
2
Density (Mg/ 1.333
m3)
1.489
1.268
F (000)
432
1,000
672
H range for 2.4–28.8
data
2.9–22.2
2.3–29.9
collection
(°)
against all the three bacterial strains. Six compounds 7a, 7b,
7e, 7f, 7i, and 7j showed good broad spectrum activity
against all the tested bacterial strains. Compounds 7c, 7g, and
7k were also relatively active against tested bacterial strains.
All the synthesized compounds were also tested for their
antifungal activity (in vitro) against Aspergillus flavus,
Chrysosporium keratinophilum, and Candida albicans by
measuring its average zone of inhibition (Table 3). Fluco-
nazole was used as standard for antifungal activity. Among
the tested compounds, 7a, 7b, 7e, 7f, 7i, and 7j were found to
be moderately active against Aspergillus flavus and Chry-
sosporium keratinophilum. These compounds are also found
to be slightly active against Candida albicans.
Fig. 1 Single crystal X-ray structures of compounds 6h and 6j
Based on these preliminary results, it can be seen that all
the six compounds 7a, 7b, 7e, 7f, 7i, and 7j showing good
antimicrobial activity have a halogen, Cl or Br as one of the
substituents. However, in general, compounds containing a
halogen substituents showed better antibacterial activity
than the compounds with other substituent’s which is also
supported by the previous report (Sharma et al., 2011). From
the antimicrobial results we can conclude that synthesized
compounds are specific antibacterial agents. Hence they are
ideally suited for further modifications to obtain more effi-
cacious antibacterial compounds.
Fig. 2 The single crystal X-ray structure of compound 7c
antibiotic, streptomycin. Antibacterial activity was carried
out by well plate method by measuring its zone of inhibition.
The compounds 7a–k were screened for their antibacterial
activity in triplicate against the above-mentioned bacterial
strains at two different concentrations of 1,000 and 500
lg/mL as shown in (Table 2). Results revealed that in gen-
eral, most of the tested compounds showed good activity
123

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Med Chem Res (2013) 22:2654–2664
Table 2 Antibacterial activity of compounds 7a–k
Compound code
Zone of inhibition (mm)
Escherichia coli
Staphylococcus aureus
Pseudomonas aeruginosa
Concentration (lg/mL)
1,000
500
1,000
500
1,000
500
7a
07 0.1
09 0.2
04 0.2
03 0.1
05 0.1
07 0.1
02 0.2
00
05 0.2
06 0.2
02 0.1
01 0.2
04 0.2
06 0.1
01 0.1
00
08 0.2
06 0.1
04 0.1
02 0.2
08 0.2
09 0.1
04 0.2
00
06 0.2
04 0.2
02 0.2
00
04 0.2
07 0.2
05 0.1
03 0.1
07 0.1
08 0.2
04 0.1
00
06 0.1
05 0.2
02 0.1
02 0.1
04 0.1
06 0.1
02 0.2
00
7b
7c
7d
7e
05 0.1
07 0.2
03 0.2
00
7f
7g
7h
7i
06 0.2
08 0.1
03 0.2
16 0.2
04 0.2
05 0.1
02 0.1
10 0.1
07 0.2
08 0.1
04 0.1
15 0.2
04 0.1
06 0.1
03 0.1
10 0.2
08 0.1
05 0.1
04 0.2
16 0.2
06 0.1
05 0.1
01 0.1
13 0.2
7j
7k
Streptomycin (Std.)
Table 3 Antifungal activity of compounds 7a–k
Compound code
Zone of inhibition (mm)
Aspergillus flavus
Chrysosporium keratinophilum
Candida albicans
1,000
Concentration (lg/mL)
1,000
500
1,000
500
500
7a
05 0.1
06 0.1
00
03 0.1
05 0.2
00
04 0.2
04 0.1
00
03 0.1
03 0.1
00
05 0.1
05 0.1
00
02 0.1
04 0.1
00
7b
7c
7d
00
00
00
00
00
00
7e
03 0.1
04 0.1
00
02 0.1
03 0.2
00
03 0.1
06 0.1
00
01 0.1
05 0.2
00
03 0.1
04 0.1
00
01 0.1
02 0.1
00
7f
7g
7h
00
00
00
00
00
00
7i
04 0.1
04 0.2
00
02 0.1
01 0.1
00
05 0.1
04 0.1
00
02 0.1
02 0.1
00
04 0.1
03 0.2
00
02 0.1
01 0.2
00
7j
7k
Fluconazole (Std.)
13 0.2
10 0.1
17 0.2
15 0.2
22 0.2
20 0.2
Conclusion
keratinophilum whereas slightly active against Candida
albicans.
The present study highlights the synthesis and investigation of
antimicrobial properties of a new series of pyrazolylpyrazo-
lines with the hope of discovering new structure leads are
served as antimicrobial agents. The structures of intermediate
compounds 6h and 6j and the final compound 7c hve been
solved by single crystal X-ray analysis. Antimicrobial results
showed that the compounds 7a, 7b, 7e, 7f, 7i, and 7j showed
good broad spectrum activity against all the tested bacterial
strains. Antifungal results showed that among the tested
compounds, 7a, 7b, 7e, 7f, 7i, and 7j were found to be mod-
erately active against Aspergillus flavus and Chrysosporium
Experimental procedure
Chemistry
Melting points were determined by open capillary method and
are uncorrected. The IR spectra were recorded on a Thermo
Nicolet avatar 330-FT-IR spectrophotometer. 1H-NMR spectra
were recorded (DMSO-d6) on a Bruker (400 MHz) spectrom-
eter. Chemical shift values are given in d scales. The mass
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Med Chem Res (2013) 22:2654–2664
2659
(2E)-3-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-
1-[4-biphenyl]prop-2-en-1-one (6c)
spectra were recorded on LC–MS-Agilent 1100 series. Ele-
mental analyses were performed on a Flash EA 1112 series
CHNS-O analyzer. The completion of the reaction was checked
by thin layer chromatography (TLC) on silica gel coated alu-
minum sheets (silica gel 60 F254). Single crystal X-ray analysis
was performed using Bruker APEXII CCD diffractometer.
Commercial grade solvents and reagents were used without
further purification.
Yield 80.8 %; mp 183–185 °C; IR (KBr, tmax cm-1):
3,118–3,047 (C–H), 1,654 (C=O), 1,580 (C=N); H-NMR
1
(DMSO-d6): d 8.34 (s, 1H, pyrazole-5H), 7.82 (d, 2H,
J = 7.5 Hz, Ho biphenyl), 7.69 (d, 1H, J = 15.6 Hz, H-6),
7.64 (d, 2H, J = 7.79 Hz, Hm biphenyl), 7.54 (d, 1H,
J = 15.2 Hz, H-7), 7.44 (m, 2H, Ho biphenyl), 7.37 (d, 2H,
J = 7.2 Hz, HoA), 7.34 (m, 5H, Ar–H of B), 7.32 (dd, 2H,
J = 7.5 Hz, Hm biphenyl), 7.25 (m, 1H, Hp biphenyl), 6.89
(d, 2H, J = 7.6 Hz, HmA). MS: m/z = 457 (M?1). Anal.
calcd. for C31H24N2O2: C, 81.56; H, 5.30; N, 6.14. Found:
C, 81.49; H, 5.26; N, 6.11 %.
General procedure for the preparation of chalcones
(6a–k)
To a cold, stirred mixture of methanol (20 mL) and sodium
hydroxide (12.09 mmol), appropriate acetophenone (5,
4.03 mmol) was added. The reaction mixture was stirred for
10 min. To this appropriate formyl pyrazole (4, 4.03 mmol)
was added followed by tetrahydrofuran (30 mL). The
solution was further stirred for 2 h at 0 °C and then at room
temperature for 5 h. It was then poured into ice cold water.
The resulting solution was neutralized with dil. HCl. The
solid that separated out was filtered, washed with water,
dried and crystallized from ethanol to afford product 6a–k.
(2E)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-1-[4-
(methylsulfanyl)phenyl]prop-2-en-1-one (6d)
Yield 74.6 %; mp 136–138 °C; IR (KBr, tmax cm-1):
3,122–3,051 (C–H), 1,662 (C=O), 1,586 (C=N); H-NMR
1
(DMSO-d6): d 8.33 (s, 1H, pyrazole-5H), 7.71 (d, 2H,
J = 7.65 Hz, Hm SCH3-phenyl), 7.65 (d, 1H, J = 15.7 Hz,
H-6), 7.56 (d, 1H, J = 15.4 Hz, H-7), 7.48 (m, 2H, HoA),
7.37 (d, 2H, J = 7.6 Hz, Ho SCH3-phenyl),7.34 (m, 5H,
Ar–H of B), 7.32 (dd, 2H, J = 7.8 Hz, HmA), 7.22 (m, 1H,
HpA), 2.49 (s, 3H, SCH3). MS: m/z = 397 (M?1). Anal.
calcd. for C25H20N2OS: C, 75.73; H, 5.08; N, 7.07. Found:
C, 75.68; H, 5.01; N, 7.02 %.
(2E)-3-[3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
[4-biphenyl]prop-2-en-1-one (6a)
Yield 79.3 %; mp 158–160 °C; IR (KBr, tmax cm-1):
3,122–3,055 (C–H), 1,659 (C=O), 1,593 (C=N); H-NMR
1
(DMSO-d6): d 8.32 (s, 1H, pyrazole-5H), 7.87 (d, 2H,
J = 7.5 Hz, Ho biphenyl), 7.67 (d, 2H, J = 7.8 Hz, Hm
biphenyl), 7.61 (d, 1H, J = 15.2 Hz, H-6), 7.52 (d, 1H,
J = 14.6 Hz, H-7), 7.48 (m, 2H, Ho biphenyl), 7.42 (d, 2H,
J = 7.3 Hz, HoA), 7.34 (d, 2H, J = 7.4 Hz, HmA), 7.32
(dd, 2H, J = 7.8 Hz, Hm biphenyl), 7.3 (m, 5H, Ar–H of
B), 7.22 (m, 1H, Hp biphenyl). MS: m/z = 461 (M?1).
Anal. calcd. for C30H21ClN2O: C, 78.17; H, 4.59; N, 6.08.
Found: C, 78.11; H, 4.54; N, 6.01 %.
(2E)-3-[3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
[4-(methylsulfanyl)phenyl]prop-2-en-1-one (6e)
Yield 73.3 %; mp 160–162 °C; IR (KBr, tmax cm-1):
3,118–3,057 (C–H), 1,647 (C=O), 1,583 (C=N); H-NMR
1
(DMSO-d6): d 8.35 (s, 1H, pyrazole-5H), 7.72 (d, 2H,
J = 7.6 Hz, Hm SCH3-phenyl), 7.68 (d, 1H, J = 15.4 Hz,
H-6), 7.58 (d, 1H, J = 15.6 Hz, H-7), 7.42 (d, 2H,
J = 7.4 Hz Ho A), 7.38 (d, 2H, J = 7.6 Hz, Ho SCH3-
phenyl), 7.35 (m, 5H, Ar–H of B), 7.33 (d, 2H, J = 7.7 Hz,
HmA), 2.49 (s, 3H, SCH3). MS: m/z = 431 (M?1). Anal.
calcd. for C25H19ClN2OS: C, 69.68; H, 4.44; N, 6.50.
Found: C, 69.62; H, 4.38; N, 6.46 %.
(2E)-3-[3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
[4-biphenyl]prop-2-en-1-one (6b)
Yield 77.7 %; mp 165–167 °C; IR (KBr, tmax cm-1):
3,116–3,052 (C–H), 1,656 (C=O), 1,596 (C=N); H-NMR
1
(2E)-3-[3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
[4-(methylsulfanyl)phenyl] prop-2-en-1-one (6f)
(DMSO-d6): d 8.37 (s, 1H, pyrazole-5H), 7.86 (d, 2H,
J = 7.51 Hz, Ho biphenyl), 7.63 (d, 1H, J = 15.2 Hz, H-6),
7.65 (d, 2H, J = 7.82 Hz, Hm biphenyl), 7.55 (d, 1H,
J = 14.8 Hz, H-7), 7.49 (d, 2H, J = 7.7 Hz, HmA), 7.46 (m,
2H, Ho biphenyl), 7.36 (d, 2H, J = 7.6 Hz, HoA), 7.34 (dd,
2H, J = 7.8 Hz, Hm biphenyl), 7.31 (m, 5H, Ar–H of B),
7.23 (m, 1H, Hp biphenyl). MS: m/z = 507 (M?2). Anal.
calcd. for C30H21BrN2O: C, 71.29; H, 4.19; N, 5.54. Found:
C, 71.23; H, 4.14; N, 5.48 %.
Yield 73.5 %; mp 156–158 °C; IR (KBr, tmax cm-1):
3,116–3,060 (C–H), 1,648 (C=O), 1,583 (C=N); H-NMR
1
(DMSO-d6): d 8.34 (s, 1H, pyrazole-5H), 7.75 (d, 2H,
J = 7.7 Hz, Hm SCH3-phenyl), 7.64 (d, 1H, J = 15.7 Hz,
H-6), 7.54 (d, 1H, J = 15.4 Hz, H-7), 7.49 (d, 2H,
J = 7.6 Hz, HmA), 7.39 (d, 2H, J = 7.5 Hz HoA), 7.36 (m,
5H, Ar–H of B), 7.33 (d, 2H, J = 7.8 Hz, Ho SCH3-
123

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(2E)-1-(2,4-dichlorophenyl)-3-[3-(4-methoxyphenyl)-1-
phenyl-1H-pyrazol-4-yl]prop-2-en-1-one (6k).
phenyl), 2.49 (s, 3H, SCH3). MS: m/z = 477 (M?2). Anal.
calcd. for C25H19BrN2OS: C, 63.16; H, 4.03; N, 5.89.
Found: C, 63.11; H, 3.98; N, 5.80 %.
Yield 80.55 %; mp 174–176 °C; IR (KBr, tmax cm-1):
3,118–3,071 (C–H), 1,687 (C=O), 1,610 (C=N); H-NMR
1
(2E)-3-[3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl]-
1-[4-(methylsulfanyl)phenyl]prop-2-en-1-one (6g)
(DMSO-d6): d 8.35 (s, 1H, pyrazole-5H), 7.67 (d, 1H,
J = 7.4 Hz, Ho 2,4-Cl2-phenyl), 7.63 (d, 1H, J = 15.5 Hz,
H-6), 7.59 (d, 1H, J = 15.2 Hz, H-7), 7.45 (s, 1H, Hm 2,4-
Cl2-phenyl), 7.39 (d, 1H, Hm J = 7.4 2,4-Cl2-phenyl), 7.36
(m, 5H, Ar–H of B), 7.32 (d, 2H, J = 7.6 Hz, HoA), 6.83
(d, 2H, J = 7.3 Hz, HmA). MS: m/z = 449 (M?). Anal.
calcd. for C25H18Cl2N2O: C, 66.83; H, 4.04; N, 6.23.
Found: C, 66.78; H, 3.97; N, 6.18 %.
Yield 74.1 %; mp 140–142 °C; IR (KBr, tmax cm-1):
3,114–3,053 (C–H), 1,657 (C=O), 1,592 (C=N); H-NMR
1
(DMSO-d6): d 8.31 (s, 1H, pyrazole-5H), 7.78 (d, 2H,
J = 7.6 Hz, Hm SCH3-phenyl), 7.62 (d, 1H, J = 15.8 Hz,
H-6), 7.52 (d, 1H, J = 15.6 Hz, H-7), 7.38 (d, 2H,
J = 7.5 Hz HoA), 7.36 (m, 5H, Ar–H of B), 7.34 (d, 2H,
J = 7.6 Hz, Ho SCH3-phenyl), 6.83 (d, 2H, J = 7.4 Hz,
HmA), 3.82 (s, 3H, OCH3), 2.5 (s, 3H, SCH3). MS:
m/z = 427 (M?1). Anal. calcd. for C26H22N2O2S: C, 73.21;
H, 5.20; N, 6.57. Found: C, 73.17; H, 5.15; N, 6.51 %.
General procedure for the synthesis of pyrazolines
(7a–k)
A mixture of chalcone 6a–k (1.0 mmol) and phenylhydr-
azine 2 (1.5 mmol) was refluxed in glacial acetic acid
(8 mL) for 4–5 h. Reaction progress was monitored by
TLC and the precipitate formed was filtered off and
recrystallized from ethanol, affording compounds 7a–k.
(2E)-1-(2,4-dichlorophenyl)-3-(1,3-diphenyl-1H-pyrazol-
4-yl)prop-2-en-1-one (6h)
Yield 76.19 %; mp 133–135 °C; IR (KBr, tmax cm-1):
3,121–3,066 (C–H), 1,687 (C=O), 1,586 (C=N); MS:
m/z = 419 (M?). Anal. calcd. for C24H16Cl2N2O: C,
68.75; H, 3.85; N, 6.68. Found: C, 68.71; H, 3.79; N,
6.60 %. Crystal structure of 6h has been presented in Fig. 1
(Fun et al., 2011a).
3-(4-chlorophenyl)-1-phenyl-4-(1-phenyl-3-biphenyl-4,5-
dihydro-1H-pyrazol-5-yl)-1H-pyrazole (7a)
Yield 76.0 %; mp 211–213 °C; IR (KBr, tmax cm-1):
3,042 (Ar–C–H), 2,923 (C–H), 1,590 (C=N), 1,492 (C=C);
1H-NMR (DMSO-d6): d 8.36 (s, 1H, pyrazole-5H),
7.83–7.69 (m, 10H, Ar–H), 7.55 (d, 2H, J = 8.4 Hz, Ar–
H), 7.48–7.41 (m, 5H, Ar–H), 7.27 (dd, 1H, J = 7.2 Hz,
Ar–H), 7.15 (dd, 2H, J = 8 Hz, Ar–H), 7.03 (d, 2H,
J = 7.6 Hz, Ar–H), 6.73 (dd, 1H, J = 7.2 Hz, Ar–H),
5.58–5.53 (dd, 1H, J = 7.6 Hz, 12.2 Hz, 50-H of pyrazo-
line), 4.08–4.01 (dd, 1H, J = 12 Hz, 17.2 Hz, 40-H of
pyrazoline), 3.31-3.25 (dd, 1H, J = 7.6 Hz, 17.6 Hz, 40-H
of pyrazoline). 13C-NMR (DMSO-d6): d 148.9, 147.8,
145.0, 140.6, 139.9, 139.5, 130.1, 129.9, 129.4, 129.3,
129.2, 127.0, 126.8, 119.5, 118.7, 113.8, 56.4, 42.8. MS:
m/z = 551 (M?1). Anal. calcd. for C36H27ClN4: C, 78.46;
H, 4.94; N, 10.17. Found: C, 78.39; H, 4.88; N, 10.13 %.
(2E)-3-[3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dichlorophenyl)prop-2-en-1-one (6i)
Yield 70.83 %; mp 165–167 °C; IR (KBr, tmax cm-1):
3,118–3,088 (C–H), 1,658 (C=O), 1,585 (C=N). H-NMR
1
(DMSO-d6): d 8.45 (s, 1H, pyrazole-5H), 7.7 (d, 1H,
J = 7.6 Hz, Ho 2,4-Cl2-phenyl), 7.68 (d, 1H, J = 14.9 Hz,
H-6), 7.53 (d, 1H, J = 15.6 Hz, H-7), 7.47 (s, 1H,
J = 7.5 Hz, Hm 2,4-Cl2-phenyl), 7.43 (d, 2H, J = 7.6 Hz
HoA), 7.38 (d, 1H, J = 7.2 Hz, Hm 2,4-Cl2-phenyl), 7.35 (d,
2H, J = 7.8 Hz, HmA), 7.3 (m, 5H, Ar–H of B). MS:
m/z = 454 (M?1). Anal. calcd. for C24H15Cl3N2O: C,
63.53; H, 3.33; N, 6.17. Found: C, 63.47; H, 3.29; N, 6.11 %.
3-(4-bromophenyl)-1-phenyl-4-(1-phenyl-3-biphenyl-4,5-
dihydro-1H-pyrazol-5-yl)-1H-pyrazole (7b)
(2E)-3-[3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dichlorophenyl)prop-2-en-1-one (6j)
Yield 85.0 %; mp 224–226 °C; IR (KBr, tmax cm-1): 3,041
(Ar–C–H), 2,920 (C–H), 1,590 (C=N), 1,492 (C=C); H-
1
Yield 80.0 %; mp 184–186 °C; IR (KBr, tmax cm-1):
3,122–3,060 (C–H), 1,683 (C=O), 1,584 (C=N). MS:
m/z = 499 (M?1). Anal. calcd. for C24H15BrCl2N2O: C,
57.86; H, 3.03; N, 5.62. Found: C, 57.81; H, 2.97; N,
5.58 %. Crystal structure of 6j has been presented in Fig. 1
(Fun et al., 2011b).
NMR (DMSO-d6): d 8.38 (s, 1H, pyrazole-5H), 7.85–7.69 (m,
12H, Ar–H), 7.51–7.37 (m, 5H, Ar–H), 7.29 (dd, 1H,
J = 7.4 Hz, Ar–H), 7.17 (dd, 2H, J = 8 Hz, Ar–H), 7.05 (d,
2H, J = 7.6 Hz),6.76(dd,1H, J = 7.2 Hz, Ar–H), 5.61–5.56
(dd, 1H, J = 7.6 Hz, 12.2 Hz, 50-H of pyrazoline), 4.11–4.03
(dd, 1H, J = 12 Hz, 17.2 Hz, 40-H of pyrazoline), 3.33–3.27
123

Med Chem Res (2013) 22:2654–2664
2661
(m, 1H, 40-H of pyrazoline merged with peak of HOD). 13C-
NMR (DMSO-d6): d 148.9, 147.8, 145.0, 140.6, 139.9, 139.5,
132.1, 130.4, 129.9, 129.4, 129.3, 128.1, 127.8, 127.2, 127.0,
126.8, 119.5, 118.7, 113.8, 56.4, 42.8. MS: m/z = 595
(M?1). Anal. calcd. for C36H27BrN4: C, 72.61; H, 4.57; N,
9.41. Found: C, 72.57; H, 4.52; N, 9.37 %.
7.83–7.78 (m, 4H, Ar–H), 7.67 (d, 2H, J = 8.8, Ar–H),
7.54 (d, 2H, J = 8.8 Hz, Ar–H), 7.43 (dd, 2H, J = 8.0 Hz,
Ar–H), 7.30–7.27 (m, 3H, Ar–H), 7.13 (dd, 2H,
J = 8.0 Hz, Ar–H), 6.99 (d, 2H, J = 7.6 Hz, Ar–H), 6.72
(d, 1H, J = 7.4 Hz, Ar–H), 5.53–5.48 (dd, 1H,
J = 7.6 Hz, 12.4 Hz, 50-H of pyrazoline), 4.03–3.96 (dd,
1H, J = 12.0 Hz, 17.2 Hz, 40-H of pyrazoline), 3.25–3.19
(dd, 1H, J = 7.6 Hz, 17.2 Hz, 40-H of pyrazoline), 2.49 (s,
3H, –SCH3, merged with DMSO-d6 peak). 13C-NMR
(DMSO-d6): d 148.9, 147.8, 145.1, 139.5, 130.1, 129.9,
129.3, 129.2, 127.8, 126.9, 126.6, 126.1, 119.4, 118.7,
113.8, 56.4, 42.8, 14.9. MS: m/z = 521 (M?1). Anal.
calcd. for C31H25ClN4S: C, 71.45; H, 4.84; N, 10.75.
Found: C, 71.39; H, 4.80; N, 10.71 %.
3-(4-methoxyphenyl)-1-phenyl-4-(1-phenyl-3-biphenyl-
4,5-dihydro-1H-pyrazol-5-yl)-1H-pyrazole (7c)
Yield 87.0 %; mp 163–165 °C; IR (KBr, tmax cm-1): 3,031
(Ar–C–H), 2,939 (C–H), 1,593 (C=N), 1,491 (C=C); 1H-NMR
(DMSO-d6): d 8.31 (s, 1H, pyrazole-5H), 7.83–7.80 (m, 4H,
Ar–H), 7.74–7.70 (m, 6H, Ar–H), 7.49–7.35 (m, 5H, Ar–H),
7.25 (dd, 1H, J = 7.4 Hz, Ar–H), 7.14 (dd, 2H, J = 8 Hz, Ar–
H), 7.07–7.0 (m, 4H, Ar–H), 6.73 (dd, 1H, J = 7.2 Hz, Ar–H),
5.54–5.49 (dd, 1H, J = 7.6 Hz, 12.4 Hz, 50-H of pyrazoline),
4.08–4.01 (dd, 1H, J = 12.4 Hz, 17.2 Hz, 40-H of pyrazoline),
3.80 (s, 3H, –OCH3), 3.32–3.26 (m, 1H, 40-H of pyrazoline
merged with peak of HOD). 13C-NMR (DMSO-d6): d 159.7,
150.0, 147.7, 145.0, 140.5, 139.9, 139.7, 131.9, 129.9, 129.7,
129.4, 129.3, 128.1, 127.4, 127.2, 127.0, 126.8, 126.6, 125.5,
122.9, 119.4, 118.5, 114.6, 113.8, 56.5, 55.6, 42.8. MS:
m/z = 547 (M?1). Anal. calcd. for C37H30N4O: C, 81.29; H,
5.53; N, 10.25. Found: C, 81.24; H, 5.49; N, 10.20 %. Crystal
structure of 7c has been presented in Fig. 2 (Fun et al., 2012).
3-(4-bromophenyl)-4-(3-(4-(methylthio)phenyl)-1-phenyl-
4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole (7f)
Yield 76.0 %; mp 206–208 °C; IR (KBr, tmax cm-1):
3,034 (Ar–C–H), 2,916 (C–H), 1,586 (C=N), 1,490 (C=C);
1H-NMR (DMSO-d6): d 8.34 (s, 1H, pyrazole-5H), 8.03 (d,
1H, J = 8.4 Hz, Ar–H), 7.94 (d, 1H, J = 7.6 Hz, Ar–H),
7.87–7.56 (m, 5H, Ar–H), 7.45–7.41 (m, 3H, Ar–H),
7.30–7.25 (m, 3H, Ar–H), 7.13 (dd, 2H, J = 8.0 Hz, Ar–
H), 6.99 (d, 2H, J = 7.6 Hz, Ar–H), 6.72 (dd, 1H,
J = 7.2 Hz, Ar–H), 5.53–5.48 (dd, 1H, J = 7.6 Hz,
12.2 Hz, 50-H of pyrazoline), 4.03–3.96 (dd, 1H,
J = 12.0 Hz, 17.2 Hz, 40-H of pyrazoline), 3.25–3.19 (dd,
1H, J = 7.6 Hz, 17.2 Hz, 40-H of pyrazoline), 2.49 (s, 3H,
–SCH3). MS: m/z = 565 (M?). Anal. calcd. for
C31H25BrN4S: C, 65.84; H, 4.46; N, 9.91. Found: C, 65.79;
H, 4.41; N, 9.88 %.
4-(3-(4-(methylthio)phenyl)-1-phenyl-4,5-dihydro-1H-
pyrazol-5-yl)-1,3-diphenyl-1H-pyrazole (7d)
Yield 81.0 %; mp 208–210 °C; IR (KBr, tmax cm-1):
3,042 (Ar–C–H), 2,917 (C–H), 1,590 (C=N), 1,492 (C=C);
1H-NMR (DMSO-d6): d 8.33 (s, 1H, pyrazole-5H),
7.83–7.77 (2d, 4H, J = 8 Hz, J = 7.2 Hz, Ar–H), 7.67 (d,
2H, J = 8.4, Ar–H), 7.52–7.41 (m, 5H, Ar–H), 7.30–7.24
(m, 3H, Ar–H), 7.12 (dd, 2H, J = 7.8 Hz, Ar–H), 6.98 (d,
2H, J = 7.6 Hz, Ar–H), 6.71 (dd, 1H, J = 7.2 Hz, Ar–H),
5.51–5.46 (dd, 1H, J = 7.6 Hz, 12.2 Hz, 50-H of pyrazo-
line), 4.05–3.97 (dd, 1H, J = 12.4 Hz, 17.0 Hz, 40-H of
pyrazoline), 3.27–3.21 (dd, 1H, J = 8.0 Hz, 17.0 Hz, 40-H
of pyrazoline), 2.49 (s, 3H, –SCH3, merged with DMSO-d6
peak). 13C-NMR (DMSO-d6): d 150.1, 147.8, 145.1, 139.6,
139.5, 129.9, 129.3, 129.2, 128.6, 128.4, 126.8, 126.6,
126.1, 119.3, 118.6, 113.7, 56.49, 42.9, 14.9. MS:
m/z = 487 (M?1). Anal. calcd. for C31H26N4S: C, 76.51;
H, 5.39; N, 11.51. Found: C, 76.47; H, 5.34; N, 11.47 %.
3-(4-methoxyphenyl)-4-(3-(4-(methylthio)phenyl)-1-
phenyl-4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-
pyrazole (7g)
Yield 72.0 %; mp 210–212 °C; IR (KBr, tmax cm-1):
3,011 (Ar–C–H), 2,917 (C–H), 1,591 (C=N), 1,491 (C=C);
1H-NMR (DMSO-d6): d 8.30 (s, 1H, pyrazole-5H), 7.81 (d,
2H, J = 7.6 Hz, Ar–H), 7.71 (d, 2H, J = 8.8 Hz, Ar–H),
7.65 (d, 2H, J = 8.4 Hz, Ar–H), 7.42 (dd, 2H, J = 8.0 Hz,
Ar–H), 7.30–7.23 (m, 4H, Ar–H), 7.12 (dd, 2H,
J = 8.0 Hz, Ar–H), 7.06 (d, 2H, J = 8.8 Hz, Ar–H), 6.98
(d, 2H, J = 7.6 Hz, Ar–H), 6.71 (dd, 1H, J = 7.2 Hz, Ar–
H), 5.48–5.43 (dd, 1H, J = 7.6 Hz, 12.2 Hz, 50-H of pyr-
azoline), 4.03–3.95 (dd, 1H, J = 12.0 Hz, 17.2 Hz, 40-H of
pyrazoline), 3.8 (s, 3H, –OCH3), 3.25–3.19 (dd, 1H,
J = 7.6 Hz, 17.2 Hz, 40-H of pyrazoline), 2.49 (s, 3H, –
SCH3). MS: m/z = 517 (M?1). Anal. calcd. for
C32H28N4OS: C, 74.39; H, 5.46; N, 10.84. Found: C, 74.34;
H, 5.41; N, 10.80 %.
3-(4-chlorophenyl)-4-(3-(4-(methylthio)phenyl)-1-phenyl-
4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole (7e)
Yield 78.0 %; mp 219–221 °C; IR (KBr, tmax cm-1):
3,038 (Ar–C–H), 2,916 (C–H), 1,586 (C=N), 1,488 (C=C);
1H-NMR (DMSO-d6): d 8.35 (s, 1H, pyrazole-5H),
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4-(3-(2,4-dichlorophenyl)-1-phenyl-4,5-dihydro-1H-
pyrazol-5-yl)-1,3-diphenyl-1H-pyrazole (7h)
4-(3-(2,4-dichlorophenyl)-1-phenyl-4,5-dihydro-1H-
pyrazol-5-yl)-3-(4-methoxyphenyl)-1-phenyl-1H-
pyrazole (7k)
Yield 71.0 %; mp 196–198 °C; IR (KBr, tmax cm-1):
3,058 (Ar–C–H), 2,871 (C–H), 1,590 (C=N), 1,494 (C=C);
1H-NMR (DMSO-d6): d 8.39 (s, 1H, pyrazole-5H), 7.83 (d,
2H, J = 7.6 Hz, Ar–H), 7.78–7.72 (m, 3H, Ar–H), 7.67 (d,
1H, J = 2.4 Hz, Ar–H), 7.51–7.4 (m, 6H, Ar–H), 7.28 (dd,
1H, J = 7.4 Hz, Ar–H), 7.14 (dd, 2H, J = 8.0 Hz, Ar–H),
7.0 (d, 2H, J = 8.0 Hz, Ar–H), 6.75 (dd, 1H, J = 7.4 Hz,
Ar–H), 5.59–5.54 (dd, 1H, J = 7.6 Hz, 12.2 Hz, 50-H of
pyrazoline), 4.15–4.08 (dd, 1H, J = 12.0 Hz, 17.4 Hz, 40-
H of pyrazoline), 3.42–3.36 (dd, 1H, J = 7.6 Hz, 17.2 Hz,
40-H of pyrazoline). MS: m/z = 508 (M?). Anal. calcd. for
C30H22Cl2N4: C, 70.73; H, 4.35; N, 11.00. Found: C,
70.69; H, 4.31; N, 10.97 %.
Yield 68.0 %; mp 160–162 °C; IR (KBr, tmax cm-1):
3,064 (Ar–C–H), 2,926 (C–H), 1,594 (C=N), 1,489 (C=C);
1H-NMR (DMSO-d6): d 8.36 (s, 1H, pyrazole-5H), 7.82,
7.80, 7.78 (d, 3H, J = 8 Hz, Ar–H), 7.67–7.64 (d, 3H,
J = 12 Hz, Ar–H), 7.48–7.42 (m, 3H, Ar–H), 7.26 (dd, 1H,
J = 6 Hz, Ar–H), 7.15 (t, 2H, J = 6 Hz, Ar–H), 7.05–7.0
(m, 4H, Ar–H), 6.76 (dd, 1H, J = 8 Hz, Ar–H), 5.57–5.52
(dd, 1H, J = 8 Hz, 12 Hz, 50-H of pyrazoline), 4.13-4.06
(dd, 1H, J = 12 Hz, 16 Hz, 40-H of pyrazoline), 3.80 (s,
3H, –OCH3), 3.40–3.34 (dd, 1H, J = 8 Hz, 16 Hz, 40-H of
pyrazoline). Anal. calcd. for C31H24Cl2N4O: C, 69.02; H,
4.48; N, 10.39. Found: C, 68.97; H, 4.41; N, 10.34 %.
3-(4-chlorophenyl)-4-(3-(2,4-dichlorophenyl)-1-phenyl-
4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole (7i)
Antimicrobial activity
The following bacteria and fungi were used for the experi-
ment. Bacteria: Staphylococcus aureus ATCC 25923,
Escherichia coli ATCC 25922, and Pseudomonas aerugin-
osa ATCC 27853. All bacterial strains were maintained on
nutrient agar medium at 37 °C. Fungi: Aspergillus flavus,
Chrysosporium keratinophilum, and Candida albicans
MTCC 227 are used in this study. These cultures are obtained
from the Department of Microbiology, Kuvempu University,
Shimoga. All fungi strains were maintained on potato dex-
trose agar (PDA) at 25 °C.
Yield 69.0 %; mp 185–187 °C; IR (KBr, tmax cm-1): 3,047
(Ar–C–H), 2,904 (C–H), 1,590 (C=N), 1,496 (C=C); H-
1
NMR (DMSO-d6): d 8.5 (s, 1H, pyrazole-5H), 7.83 (d, 2H,
J = 7.6 Hz, Ar–H), 7.77–7.72 (m, 3H, Ar–H), 7.67 (d, 1H,
J = 2.4 Hz, Ar–H), 7.53–7.43 (m, 5H, Ar–H), 7.28 (dd, 1H,
J = 7.4 Hz, Ar–H), 7.16 (dd, 2H, J = 8.0 Hz, Ar–H), 7.02
(d, 2H, J = 8.0 Hz, Ar–H), 6.76 (dd, 1H, J = 7.4 Hz, Ar–
H), 5.61–5.56 (dd, 1H, J = 7.6 Hz, 12.0 Hz, 50-H of pyr-
azoline), 4.13–4.06 (dd, 1H, J = 12.4 Hz, 17.2 Hz, 40-H of
pyrazoline), 3.40–3.33 (dd, 1H, J = 7.6 Hz, 17.6 Hz, 40-H
of pyrazoline). MS: m/z = 543 (M?1). Anal. calcd. for
C30H21Cl3N4: C, 66.25; H, 3.89; N, 10.30. Found: C, 66.20;
H, 3.83; N, 10.25 %.
Antibacterial activity
The antibacterial activity of newly synthesized compounds
(7a–k) were determined by well plate method in Mueller–
Hinton Agar (Arthington-Skaggs et al., 2000; Rocha et al.,
1995). The compounds were tested against a panel of patho-
genic microorganisms, including Escherichia coli, Staphylo-
coccus aureus, and Pseudomonas aeruginosa. Microorganism
strains were maintained on nutrient agar medium at 37 °C. The
cultures were inoculated in fresh 10 mL nutrient broth to yield
an initial suspension of approximately 10-100 cfu/mL. All
broths were then incubated statically at the aforementioned
temperatures for microorganisms, for 18–24 h so that all cells
were in the stationary phase. Susceptibility of the test organism
to the compounds was determined by employing in the well
plate technique. The bacterial suspensions were diluted tenfold
in distilled water, and 0.1 mL from the appropriate dilution was
spread plated on nutrient agar to give a population of approxi-
mately 106 cfu/plate. Six millimeter diameter well was then
punched carefully using a sterile cork borer and 30 lL of test
solutions of different concentrations (1 and 0.5 mg/mL) were
added into each labeled well. The same procedure was repeated
3-(4-bromophenyl)-4-(3-(2,4-dichlorophenyl)-1-phenyl-
4,5-dihydro-1H-pyrazol-5-yl)-1-phenyl-1H-pyrazole (7j)
Yield 70.0 %; mp 163–165 °C; IR (KBr, tmax cm-1):
3,035 (Ar–C–H), 2,922 (C–H), 1,589 (C=N), 1,495 (C=C);
1H-NMR (DMSO-d6): d 8.4 (s, 1H, pyrazole-5H), 7.83 (d,
2H, J = 7.6 Hz, Ar–H), 7.77 (d, 1H, J = 8.4 Hz, Ar–H),
7.69–7.64 (m, 4H, Ar–H), 7.48–7.43 (m, 4H, Ar–H), 7.28
(dd, 1H, J = 7.4 Hz, Ar–H), 7.16 (dd, 2H, J = 8.0 Hz,
Ar–H), 7.02 (d, 2H, J = 8.0 Hz, Ar–H), 6.76 (dd, 1H,
J = 7.2 Hz, Ar–H), 5.61–5.56 (dd, 1H, J = 7.6 Hz,
12.2 Hz, 50-H of pyrazoline), 4.13–4.05 (dd, 1H,
J = 12.4 Hz, 17.4 Hz, 40-H of pyrazoline), 3.39–3.33 (dd,
1H, J = 7.6 Hz, 17.2 Hz, 40-H of pyrazoline). MS:
m/z = 587 (M?1). Anal. calcd. for C30H21BrCl2N4: C,
61.25; H, 3.60; N, 9.52. Found: C, 61.21; H, 3.57; N,
9.48 %.
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for different microorganisms. Each experiment was carried out
in triplicate. After the inoculation of the organism and com-
pound, the Petri plates were incubated for 24 h at 37 °C. After
the incubation, the inhibition zone was measured and the values
for Dimethylsulfoxide (DMSO) were subtracted to get the
actual values. Streptomycin was used as standard drug.
Antifungal activity
Antifungal studies of newly synthesized compounds (7a–k)
were carried out against Aspergillus flavus, Chrysosporium
keratinophilum, and Candida albicans. Sabourands agar
media were prepared by dissolving peptone (10 g), D-glucose
(40 g), and agar (20 g) in distilled water (1,000 mL) and
adjusting the pH to 5.7. Normal saline was used to make a
suspension of spore of fungal strains for lawning. A loopful of
particular fungal strain was transferred to 3 mL saline to get a
suspension of corresponding species. Twenty milliliters of
agar media was poured into each Petri dish. Excess of sus-
pension was decanted and plates were dried by placing in
incubator at 37 °C for 1 h. Wells were made on these seeded
agar plates using sterile cork borer punched carefully, and
different concentrationsof the test compounds in DMSO were
added into each labeled well. A control was also prepared for
the plates in the same way using solvent DMSO. The Petri
dishes were prepared in triplicate and maintained at 25 °C for
72 h. Antifungal activity was determined by measuring the
diameter of inhibition zone. The activity of each compound
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funding the work through the research group project No. RGP-VPP-
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