Design, synthesis and characterization of fluoro substituted novel pyrazolylpyrazolines scaffold and their pharmacological screening

Article abstract of DOI:10.1016/j.ejmech.2014.07.008

A novel series of fluoro substituted pyrazolylpyrazolines 7a-l was synthesized in good to excellent yield (77-88%) from pyrazole chalcones 5a-d and substituted phenyl hydrazine hydrochlorides 6a-c under microwave irradiation. The newly synthesized compounds were screened for their preliminary in vitro antibacterial activity against a panel of pathogenic stains of bacteria and fungi, antituberculosis activity against Mycobacterium tuberculosis H37Rv and antimalarial activity against Plasmodium falciparum. Compounds 7a, 7b, 7g, 7h, 7j and 7k displayed excellent activity against P. falciparum stain as compared to quinine IC₅₀ 0.268. Good antitubercular activity was exhibited by compounds 7a, 7e, 7h and 7k. Some of them also exhibited superior antibacterial activity as compared to the first line drugs.

Full text of DOI:10.1016/j.ejmech.2014.07.008

European Journal of Medicinal Chemistry 84 (2014) 51e58
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and characterization of fluoro substituted novel
pyrazolylpyrazolines scaffold and their pharmacological screening
Sharad C. Karad*, Vishal B. Purohit, Dipak K. Raval
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, 388 120 Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of fluoro substituted pyrazolylpyrazolines 7ael was synthesized in good to excellent yield
(77e88%) from pyrazole chalcones 5aed and substituted phenyl hydrazine hydrochlorides 6aec under
microwave irradiation. The newly synthesized compounds were screened for their preliminary in vitro
antibacterial activity against a panel of pathogenic stains of bacteria and fungi, antituberculosis activity
against Mycobacterium tuberculosis H37Rv and antimalarial activity against Plasmodium falciparum.
Compounds 7a, 7b, 7g, 7h, 7j and 7k displayed excellent activity against P. falciparum stain as compared
to quinine IC50 0.268. Good antitubercular activity was exhibited by compounds 7a, 7e, 7h and 7k. Some
of them also exhibited superior antibacterial activity as compared to the first line drugs.
© 2014 Elsevier Masson SAS. All rights reserved.
Received 15 May 2014
Received in revised form
2 July 2014
Accepted 3 July 2014
Available online 4 July 2014
Keywords:
Microwave irradiation
Pyrazolylpyrazolines
Antimicrobial activity
Antituberculosis activity
Antimalarial activity
1. Introduction
have occupied a distinct place due to a range of bioactivities such as
antiproliferative [15], antimicrobial [16e18], antidepressant [19],
The most imperative vector-borne infectious diseases malaria is
caused by the protozoan parasite Plasmodium falciparum [1]. The
most recent report from the WHO states that malaria is responsible
for the death of over 1 million persons every year including chil-
dren under the age of five. It is most common in subtropical and
tropical areas and 90% of the cases are originated in sub-Saharan
Africa [2]. Tuberculosis is the second most common lethal infec-
tious disease subsequent to AIDS and HIV [3]. About one-third of
the world's population is infected by Mycobacterium tuberculosis
every year and more than 2 million deaths are reported [4]. In this
context, it was thought worth to synthesize novel compounds
which may exhibit synergistic potency to be employed as antimi-
crobial, antituberculosis and antimalarial agents.
The substitution of fluorine in to a potential drug molecule can
improve efficacy of drugs by extending pharmacokinetic and
pharmacodynamics properties [5]. Trifluoromethyl group is a well-
known substituent of unique qualities. Its high lipophilicity enables
to improve pharmacological activities of the molecule [6,7]. Pyr-
azoles and their derivatives possess numerous medicinal applica-
tions because of their versatile biological activities [8e14]. They
antipyretic [20], anti-inflammatory [21] and anticonvulsant [22].
Pyrazoline is also an important nitrogenous heterocyclic moiety in
many drugs. Literature survey revealed that various pyrazoline
derivatives have displayed significant biological roles [23e29].
Microwave irradiation as a source of energy leads to environ-
mentally benign protocols in terms of reduction in reaction time,
energy saving with high efficiency, improved yields and selectivity
[30]. In context of the above consequences and in continuation to
our previous studies directed toward the synthesis of biologically
active novel heterocyclic scaffolds [30e37], herein we report mi-
crowave assisted synthesis of some fluorinated novel pyr-
azolylpyrazoline derivatives. The synthesized compounds
exhibited an interesting profile as antimalarial, antitubercular and
antimicrobial agents.
2. Chemistry
The synthesis of novel series of pyrazolylpyrazolines 7ael was
performed as outlined in Scheme 1. The starting material 5-chloro-
3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 1 was prepared
according to VilsmeiereHaack reaction of 3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one [38]. 3-methyl-5-substituted aryloxy-1-phenyl-
1H-pyrazole-4-carbaldehydes 3aed were prepared by refluxing
compound 1 and substituted phenols 2aed in presence of anhy-
drous K2CO3 as basic catalyst in DMF as solvent. 3-methyl-5-
* Corresponding author.
E-mail addresses: krdsharad1126@gmail.com (S.C. Karad), dipanalka@yahoo.
com (D.K. Raval).
http://dx.doi.org/10.1016/j.ejmech.2014.07.008
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

52
S.C. Karad et al. / European Journal of Medicinal Chemistry 84 (2014) 51e58
Scheme 1. Synthesis of 5-(4-fluorophenyl)-30-methyl-50-substituted aryloxy-10,2-diphenyl-3,4-dihydro-10H,2H-3,40-bipyrazole (7ael) (i) DMF, K2CO3, Reflux 2 h. (ii) 20% ethanolic
NaOH, room temperature. (iii) Ethanol, catalytic glacial acetic acid, MW, 8e10 min, 350 W.
substituted aryloxy-1-phenyl-1H-pyrazole-4-carbaldehydes 3aed
were subjected to base catalysed ClaiseneSchmidt condensation
reaction with 4-Fluoro acetophenone 4 generating the required (E)-
1-(4-fluorophenyl)-3-(3-methyl-5-substituted aryloxy-1-phenyl-
1H-pyrazol-4-yl)prop-2-en-1-ones 5aed. Finally pyrazolyl-
pyrazolines 7ael were obtained by the condensation of 5aed and
substituted phenyl hydrazine hydrochlorides 6aec in ethanol
containing catalytic amount of glacial acetic acid under microwave
irradiation at 350 W power level for 8e10 min.
3. Pharmacology
3.1. In vitro antimicrobial activity
The synthesized pyrazolylpyrazoline derivatives 7ael were
evaluated for their antimicrobial activity by broth micro dilution
method according to National Committee for Clinical Laboratory
Standards (NCCLS) [39]. The compounds were screened for anti-
bacterial activity employing three Gram positive (Clostridium tetani
MTCC 449, Bacillus subtilis MTCC 441, and Streptococcus pneumoniae
MTCC 1936) and three Gram negative (Escherichia coli MTCC 443,
Salmonella typhi MTCC 98 and Vibrio cholerae MTCC 3906) bacteria
against ampicillin, norfloxacin, ciprofloxacin and chloramphenicol
as the reference drugs. Antifungal activity was screened against
two fungal species (Candida albicans MTCC 227 and Aspergillus
fumigats MTCC 3008) where nystatin and griseofulvin were used as
the standard drugs. The result of the antimicrobial screening data is
shown in Table 1.
Table 1
In vitro antimicrobial activity (MIC,
m
g/mL) of compounds 7ael.
Comp. Gram positive bacteria
Gram negative bacteria
Fungi
S.P.
B.S.
C.T.
E.C.
S.T.
V.C.
C.A.
A.F.
MTCC MTCC MTCC MTCC
MTCC
98
MTCC
3906
MTCC MTCC
227 3008
1000 250
1936
441
449
443
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
A
500
500
100
200
500
200
100
500
125
250
250
500
100
10
500
250
200
250
250
100
500
500
62.5
500
200
100
250
100
50
500
250
200
500
250
200
250
125
200
250
500
500
250
50
200
500
500
200
250
100
500
200
500
200
100
500
100
10
250
200
500
200
200
100
250
200
500
250
200
500
100
10
125
125
200
250
250
250
200
500
100
250
500
500
100
10
3.2. In vitro antituberculosis activity
250
>1000
1000
250
>1000
1000
A primary in vitro antituberculosis activity of novel pyr-
azolylpyrazolines 7ael was conducted at 250
tuberculosis H37Rv stain by using LowensteineeJensen medium as
described by Rattan [40]. The obtained result is presented in Table 2
in the form of % inhibition. Rifampicin and Isoniazid were employed
as the standard drugs.
1000 500
mg/mL against M.
250
200
500
500
1000
250
500
n. t.a
n. t.
n. t.
n. t.
100
500
500
>1000
>1000
250
100
1000
250
n. t.
n. t.
n. t.
n. t.
Table 2
In vitro antituberculosis activity (% inhibition) of compounds 7ael against
M. tuberculosis H37Rv (at concentration 250 mg/mL).
B
C
D
E
50
50
50
50
25
n. t.
n. t.
50
25
n. t.
n. t.
Comp.
% Inhibition
Comp.
% Inhibition
25
n. t.
n. t.
50
n. t.
n. t.
100
n. t.
n. t.
25
n. t.
n. t.
100
100
7a
7b
7c
7d
7e
7f
90
56
56
65
91
52
40
7h
7i
7j
7k
96
74
10
94
22
98
99
F
S.P.: Streptococcus pneumoniae, B.S.: Bacillus subtilis, C.T.: Clostridium tetani, E.C.:
Escherichia coli S.T.: Salmonella typhi, V.C.: Vibrio cholerae, C.A.: Candida albicans, A.F.:
Aspergillus fumigatus, MTCC: Microbial Type Culture Collection. A: Ampicillin, B:
Norfloxacin, C: Chloramphenicol, D: Ciprofloxacin, E: Nystatin, F: Griseofulvin. The
bold values indicate comparable/superior potency as compared to the reference
drugs.
7l
Rifampicin
Isoniazid
7g
The bold values indicate comparable/superior potency as compared to the reference
drugs.
a
n.t.: not tested.

S.C. Karad et al. / European Journal of Medicinal Chemistry 84 (2014) 51e58
53
3.3. In vitro antimalarial activity
to that of ampicillin. Against C. tetani, compounds 7b (R ¼ 3-F,
R1 ¼ 4-Br), 7e (R ¼ 3-CF3, R1 ¼ 4-Br), 7g (R ¼ 4-F, R1 ¼ 4-F), and 7j
(R ¼ 2-F, R1 ¼ 4-F) displayed same influence as that of ampicillin.
Compounds 7h (R ¼ 4-F, R1 ¼ 4-Br), 7c (R ¼ 3-F, R1 ¼ 4-OCH3), 7f
(R ¼ 3-CF3, R1 ¼ 4-OCH3), 7i (R ¼ 4-F, R1 ¼ 4-OCH3) were found to
exhibit superior activity as compared to ampicillin against C. tetani.
In case of inhibiting gram negative bacteria compounds 7f (R ¼ 3-
CF3, R1 ¼ 4-OCH3) and 7k (R ¼ 2-F, R1 ¼ 4-Br) were found equi-
potent against E. coli as compared to ampicillin. Compounds 7f
(R ¼ 3-CF3, R1 ¼ 4-OCH3) and 7i (R ¼ 4-F, R1 ¼ 4-OCH3) also
In vitro antimalarial activity of the novel pyrazolylpyrazolines
derivatives 7ael against P. falciparum stain was performed using
chloroquine and quinine as the reference drugs. The consequence
of antimalarial screening is expressed as the drug concentration
resulting in 50% inhibition of parasite growth (IC50) and is listed in
Table 3.
4. Results and discussion
exhibited the same power as that of ampicillin i.e. 100
mg/mL
against S. typhi and V. cholera respectively.
4.1. Analytical results
4.2.2. In vitro antifungal activity
The structures of the synthesized compounds were confirmed
by 1H and 13C NMR, FT-IR, mass spectrometry and elemental
analysis. The IR spectra of compounds 7ael exhibited characteristic
absorption band in the range of 1260e1253 cmꢀ1 due to the
presence of ether linkage. The absorption band around
3062e3051 cmꢀ1 is due to aromatic CeH stretching. The absorp-
tion band observed for all the compounds in between of
1626e1598 cmꢀ1 corresponds to eC]N stretching. The strong
absorption band is also observed in the range of 1368e1357 cmꢀ1
due to eCH3 rocking. The 1H NMR spectra of the target pyr-
azolylpyrazolines displayed a typical ABX type pattern of doublet of
doublet due to three pyrazoline protons. Methine proton of pyr-
azoline was found at around 5.39e5.17 ppm as a doublet of doublet
with coupling constants of nearly 12.8 Hz and 5.6 Hz. Two meth-
ylene protons displayed two signals; a doublet of doublet at around
3.74e3.62 ppm with coupling constants of nearly 17 Hz and 12 Hz
and a doublet of doublet at around 3.32e3.22 ppm merging with
the water signal from DMSO-d6. At around 3.67 ppm C4eH pyr-
azoline proton got merged with the methoxy signal in most of the
cases. The data from 13C NMR spectral studies is also in accordance
with the suggested structures.
The antifungal screening data (Table 1) revealed that, against
C. albicans, compounds 7g (R ¼ 4-F, R1 ¼4-F), 7b (R ¼ 3-F, R1 ¼4-Br),
7d (R ¼ 3-F, R1 ¼ 4-F), 7f (R ¼ 3-CF3, R1 ¼ 4-OCH3) and 7k (R ¼ 2-F,
R1 ¼ 4-Br) were found to possess significant activity as compared to
griseofulvin. Compounds 7h (R ¼ 4-F, R1 ¼ 4-Br), 7i (R ¼ 4-F, R1 ¼ 4-
OCH3) and 7l (R ¼ 2-F, R1 ¼ 4-OCH3) exhibited equivalent potency
against C. albicans as compared to griseofulvin. Compound 7j
(R ¼ 2-F, R1 ¼ 4-F) showed equal potency against A. fumigates as
compared to griseofulvin as well as nystatin.
4.2.3. In vitro antituberculosis activity
Antituberculosis screening of all the synthesized compounds
7ael was conducted at 250 mg/mL concentrations against tuber-
culosis H37Rv stains. Compounds 7a (R ¼ 3-CF3, R1 ¼ 4-F), 7e
(R ¼ 3-CF3, R1 ¼ 4-Br), 7h (R ¼ 4-F, R1 ¼ 4-Br) and 7k (R ¼ 2-F,
R1 ¼ 4-Br) found to possess brilliant activity (i.e. 90%, 91%, 96 and
94% at 250
mg/mL) against M. tuberculosis H37Rv (Table 2).
Remaining all other compounds showed poor inhibition against
M. tuberculosis growth.
4.2.4. In vitro antimalarial activity
Compounds 7ael were also evaluated for their antimalarial
screening against chloroquine and quinine sensitive stains of
P. falciparum. All experiments were performed in duplicate and a
mean value of IC50 is mentioned in Table 3. As shown in Table 3,
they were found to have IC50 between 0.022 and 0.088 upon
P. falciparum stain. It is important to note that compounds 7a
(R ¼ 3-CF3, R1 ¼ 4-F), 7b (R ¼ 3-F, R1 ¼ 4-Br), 7g (R ¼ 4-F, R1 ¼ 4-F),
7h (R ¼ 4-F, R1 ¼ 4-Br), 7j (R ¼ 2-F, R1 ¼ 4-F) and 7k (R ¼ 2-F, R1 ¼ 4-
Br) displayed excellent activity against P. falciparum stain as
compared to quinine IC50 0.268. From the above results, it can be
concluded that compound 7g, 7h and 7j may prove themselves as
new antimalarial agents in future.
4.2. Biological section
4.2.1. In vitro antibacterial activity
Upon investigation of antimicrobial activity data (Table 1), it has
been observed that against Bacillus subtillis, compound 7i (R ¼ 4-F,
R1 ¼ 4-OCH3) was found to possess excellent potency i.e. 62.5
mg/
mL as compared to ampicillin i.e. 250
i.e. 100
g/mL. Compounds 7f (R ¼ 3-CF3, R1 ¼ 4-OCH3) and 7l
(R ¼ 2-F, R1 ¼ 4-OCH3) were found to be more potent against
B. subtillis i.e. 100 g/mL as compared to ampicillin. Compounds 7c
(R ¼ 3-F, R1 ¼ 4-OCH3) and 7k (R ¼ 2-F, R1 ¼ 4-Br) were found to be
g/mL) against B. subtillis as compared to
mg/mL as well as norfloxacin
m
m
more effective (MIC ¼ 200
m
ampicillin. Compounds 7b (R ¼ 3-F, R1 ¼ 4-Br), 7d (R ¼ 3-F, R1 ¼ 4-
F), 7e (R ¼ 3-CF3, R1 ¼ 4-Br) were equipotent as that of ampicillin
against B. subtillis. Against S. pneumonia compounds 7c (R ¼ 3-F,
R1 ¼4-OCH3) and 7g (R ¼ 4-F, R1 ¼ 4-F) showed comparable activity
4.3. Structureeactivity relationship (SAR)
The results of the biological evaluation revealed that the activity
was significantly affected by introducing fluorinated aryloxy nu-
cleus at the C-50 position in pyrazoline scaffold (Scheme 1). It has
been observed that electron donating group (4-OCH3) existing at R1
position and 3-CF3 group on aryloxy ring on pyrazoline induced the
greatest antibacterial activity against B. subtilis and C. tetani (Fig. 1).
The presence of electron withdrawing group (4-F, 4-Br) at R1
position and eCF3 group at meta position of aryloxy ring illustrated
superior antimalarial activity as well as antituberculosis activity.
The presence of bromo group at R1 position and fluoro group at
ortho, meta, and para positions of aryloxy ring showed enhanced
antituberculosis activity as well as remarkable antimalarial activity.
The presence of fluoro group at R1 position and the ortho, meta, and
para positions of aryloxy ring displayed excellent antimalarial ac-
tivity. It can be concluded that the presence of electron
Table 3
In vitro antimalarial activity of compounds 7ael.
Comp.
IC50
(m
g/mL)
Comp.
IC50 (mg/mL)
7a
7b
7c
7d
7e
7f
0.034
0.060
0.57
0.82
1.46
7h
7i
7j
7k
0.025
0.25
0.025
0.088
0.65
7l
0.30
Chloroquine
Quinine
0.020
0.268
7g
0.022
The bold values indicate comparable/superior potency as compared to the reference
drugs.

54
S.C. Karad et al. / European Journal of Medicinal Chemistry 84 (2014) 51e58
Fig. 1. Structureeactivity relationships for antimicrobial, antituberculosis and antimalarial activity of the synthesized compounds 7ael.
withdrawing groups (4-F, 4-Br) at R1 position and fluoro group at
different positions of aryloxy ring are responsible for increasing
antimalarial activity.
sensor for temperature control through attachment of reflux
condenser with constant stirring. Microwaves are generated by
magnetron at a frequency of 2450 MHz having an adjustable output
power levels (i.e. 1 to 10 levels from 140 to 700 Watts).
5. Conclusion
6.1.1. General procedure for synthesis of 3-methyl-5-substituted
aryloxy-1-phenyl-1H-pyrazole-4-carbaldehyde (3aed)
The novel series of fluoro substituted pyrazolylpyrazoline de-
rivatives have been synthesized in excellent yield using microwave
irradiation and examined for their pharmacological screening. The
results demonstrated that some analogues of this series were found
to have more potency against C. tetani and B. subtilis. The com-
pounds 7b, 7d, 7f, 7g and 7k illustrated remarkable antifungal ac-
tivity as compared to griseofulvin. Amongst the tested compounds,
7a, 7e, 7h, and 7k showed pronounced antituberculosis activity
against M. tuberculosis H37Rv. Whereas, compounds 7a, 7b, 7g, 7h,
7j and 7k displayed superior antimalarial activity against
P. falciparum stain as compared to quinine. Compound 7h with an
excellent dual profile as antimalarial and antituberculosis agent
was recognized as the most active member among the prepared
series.
5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde
1
(1 mmol), substituted phenols 2aed (1 mmol) and anhydrous po-
tassium carbonate (2 mmol) in dimethylformamide (10 mL) were
charged in a 100 mL round bottom flask equipped with a me-
chanical stirrer and a condenser. The reaction mixture was heated
at 90 ꢁC for 2 h. The progress of the reaction was monitored by TLC.
After the completion of reaction as confirmed by TLC, the reaction
mixture was poured in to 100 mL ice-water, filtered, washed
thoroughly with water, dried and recrystallized from ethanol to
obtain a white solid.
6.1.1.1. 5-(2-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (3a). Yield 85%; m.p. 225e227 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
CHO).
d 2.46 (s, 3H, CH3), 7.13e7.64 (m, 9H, AreH), 9.56 (s, 1H,
6. Experimental section
6.1.1.2. 5-(3-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (3b). Yield 78%; m.p. 210e212 ꢁC; 1H NMR (400 MHz,
6.1. Chemistry
DMSO-d6):
CHO).
d 2.47 (s, 3H, CH3), 6.92e7.63 (m, 9H, AreH), 9.61 (s, 1H,
Melting points in ꢁC were determined in open capillaries using
ThermoCal10 melting point apparatus (Analab Scientific Pvt. Ltd,
m
India) and are uncorrected. Precoated silica gel plates (silica gel
0.25 mm, 60 G F 254; Merck, Germany) were used for thin layer
chromatography. The IR spectra were recorded on Shimadzu FTIR
8401 spectrophotometer using potassium bromide pellets in the
range 4000e400 cmꢀ1 and frequencies of only characteristic peaks
are expressed in cmꢀ1. Electron impact Mass Spectra were recorded
on Shimadzu LCMS 2010 spectrometer (Shimadzu, Tokyo, Japan)
purchased under PURSE program of DST at Sardar Patel University,
Vallabh Vidyanagar, India. NMR spectra (in DMSO-d6) were recor-
ded on Bruker Avance 400F NMR Spectrometer at 400 MHz using
TMS as the internal standard. The elemental analysis was per-
formed on PerkineElmer 2400 series-II elemental analyzer (Per-
kineElmer, USA) at Sophisticated Instrumentation Centre for
Applied Research & Training (SICART), Vallabh Vidyanagar, India.
All compounds were found within 0.4% of their theoretical values.
All the reactions were carried out at atmospheric pressure using a
multimode microwave reactor (Microwave Synthesis System,
Model: Cata-R, Catalyst Systems, Pune-India) with an individual
6.1.1.3. 5-(4-Fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazole-4-
carbaldehyde (3c). Yield 82%; m.p. 245e247 ꢁC; 1H NMR (400 MHz,
DMSO-d6):
CHO).
d 2.46 (s, 3H, CH3), 7.17e7.64 (m, 9H, AreH), 9.54 (s, 1H,
6.1.2. General procedure for synthesis of (E)-1-(4-fluorophenyl)-3-
(3-methyl-5-substituted aryloxy-1-phenyl-1H-pyrazol-4-yl)prop-2-
en-1-ones(5aed)
To a mixture of 3-methyl-5-substituted aryloxy-1-phenyl-1H-
pyrazole-4-carbaldehydes 3aed (5.0 mmol) and 4-fluoro aceto-
phenone 4 (5.0 mmol), 20% ethanolic NaOH (5 mL) was added. The
reaction mixture was stirred at room temperature until formation
of precipitate. The solid obtained was isolated by filtration, washed
with cold ethanol and recrystallized from CHCl3.
6.1.2.1. (E)-3-(5-(2-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-1-(4-fluoro phenyl)prop-2-en-1-one (5a). Yield 78%; m.p.

S.C. Karad et al. / European Journal of Medicinal Chemistry 84 (2014) 51e58
55
229e231 ꢁC; IR (KBr, nmax, cmꢀ1): 1362 (eCH3 rocking.), 1257
(CeOeC ether str.), 1660 (C]O and eC]N), 1582 (C]C), 3060
6.1.3.2. 2-(4-Bromophenyl)-50-(3-fluorophenoxy)-5-(4-
fluorophenyl)-30-methyl-10-phenyl-3,4-dihydro-10H,2H-3,40-bipyr-
azole (7b). Yield 79%; m.p. 176e178 ꢁC; IR (KBr, nmax, cmꢀ1): 1366
(eCH3 rocking.), 1259 (CeOeC ether str.), 1618 (eC]N), 3061 (eCH
aromatic); 1H NMR (400 MHz, DMSO-d6): 7.68e6.56 (17H, m, ArH),
5.35 (1H, dd, J ¼ 5.6, 12.4 Hz, C5eH pyrazoline), 3.72 (1H, dd,
J ¼ 12.8, 17.6 Hz, C4eH pyrazoline), 3.32 (1H, dd C4eH pyrazoline
merged with peak of H2O), 2.19 (3H, s, CH3); 13C NMR: (400 MHz,
(eCH aromatic); 1H NMR (400 MHz, DMSO-d6):
d 2.52 (s, 3H, CH3),
6.94e7.86 (m, 15H, AreH þ eCH]CHe). ESI-MS (m/z): 417.3 (Mþ);
Anal. Calcd (%) for C25H18F2N2O2: C, 72.11; H, 4.36; N, 6.73. Found: C,
71.90; H, 4.14; N, 6.49.
6.1.2.2. (E)-3-(5-(3-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-1-(4-fluoro phenyl)prop-2-en-1-one (5b). Yield 83%; m.p.
201e203 ꢁC; IR (KBr, nmax, cmꢀ1): 1358 (eCH3 rocking.), 1254
(CeOeC ether str.), 1659 (C]O and eC]N), 1580 (C]C), 3062
DMSO-d6):
d, 13.5 (eCH3 of pyrazole), d, 53.4 (eCH2 of pyrazoline),
d, 108.3, 110.3, 115.2, 116.4, 117.3, 122.2, 124.8, 125.4, 127.3, 128.3,
128.7, 129.7, 132.0, 137.5, 143.5,143.6,144.8, 147.0, 147.6, 150.1, 152.4,
161.2, 164.8 (23 signals, aromatic carbons, pyrazole carbons, C3 and
C4 of pyrazoline); ESI-MS (m/z): 585.4 (Mþ); 587.4 (Mþ2); Anal.
Calcd (%) for C31H23BrF2N4O: C, 63.60; H, 3.96; N, 9.57. Found: C,
63.36; H, 3.73; N, 9.31.
(eCH aromatic); 1H NMR (400 MHz, DMSO-d6):
d 2.51 (s, 3H, CH3),
6.87e7.85 (m, 15H, AreH þ eCH]CHe). ESI-MS (m/z): 417.3 (Mþ);
Anal. Calcd (%) for C25H18F2N2O2: C, 72.11; H, 4.36; N, 6.73. Found: C,
71.91; H, 4.12; N, 6.48.
6.1.3.3. 50-(3-Fluorophenoxy)-5-(4-fluorophenyl)-2-(4-
methoxyphenyl)-30-methyl-10-phenyl-3,4-dihydro-10H,2H-3,40-bipyr-
azole (7c). Yield 88%; m.p. 166e168 ꢁC; IR (KBr, nmax, cmꢀ1): 1367
(eCH3 rocking.), 1253 (CeOeC ether str.), 1616 (eC]N), 3060 (eCH
aromatic); 1H NMR (400 MHz, DMSO-d6): 7.66e6.61 (17H, m, ArH),
5.22 (1H, dd, J ¼ 7.2, 12.8 Hz, C5eH pyrazoline), 3.64 (4H, s, C4eH
pyrazoline merged with eOCH3), 3.27 (1H, dd, J ¼ 7.2, 17.6 Hz, C4eH
6.1.2.3. (E)-1-(4-fluorophenyl)-3-(3-methyl-1-phenyl-5-(3-(tri-
fluoromethyl)phenoxy)-1H-pyrazol-4-yl)prop-2-en-1-one
(5c).
Yield 80%; m.p. 258e260 ꢁC; IR (KBr, nmax, cmꢀ1): 1362 (eCH3
rocking.), 1257(CeOeC ether str.), 1660 (C]O and eC]N), 1576
(C]C), 3060 (eCH aromatic); 1H NMR (400 MHz, DMSO-d6):
d 2.52
(s, 3H, CH3), 7.11e7.86 (m, 15H, AreH þ eCH]CHe). ESI-MS (m/z):
467.3 (Mþ); Anal. Calcd (%) for C26H18F4N2O2: C, 66.95; H, 3.89; N,
6.01. Found: C, 66.74; H, 3.69; N, 5.79.
pyrazoline), 2.18 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
d,
13.9 (eCH3 of pyrazole),
d, 54.9 (eCH2 of pyrazoline), d, 55.6
(eOCH3), , 109.1, 110.7, 110.9, 111.5, 114.7, 115.0, 115.8, 116.0, 122.0,
d
6.1.2.4. (E)-3-(5-(4-fluorophenoxy)-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-1-(4-fluoro phenyl)prop-2-en-1-one (5d). Yield 75%; m.p.
238e240 ꢁC; IR (KBr, nmax, cmꢀ1): 1358 (eCH3 rocking.), 1255
(CeOeC ether str.), 1662 (C]O and eC]N), 1578 (C]C), 3060
127.4, 127.8, 129.8, 131.6, 137.9, 138.8, 144.9, 146.1, 147.2, 153.4, 157.6,
164.3 (23 signals, aromatic carbons, pyrazole carbons, C3 and C4 of
pyrazoline); ESI-MS (m/z): 537.5 (Mþ); Anal. Calcd (%) for
C32H26F2N4O2: C, 71.63; H, 4.88; N, 10.44. Found: C, 71.36; H, 4.66;
(eCH aromatic); 1H NMR (400 MHz, DMSO-d6):
d 2.51 (s, 3H, CH3),
N, 10.21.
7.09e7.90 (m, 15H, AreH þ eCH]CHe). ESI-MS (m/z): 417.3 (Mþ);
Anal. Calcd (%) for C25H18F2N2O2: C, 72.11; H, 4.36; N, 6.73. Found: C,
71.91; H, 4.10; N, 6.47.
6.1.3.4. 50-(3-fluorophenoxy)-2,5-bis(4-fluorophenyl)-30-methyl-10-
phenyl-3,4-dihydro-10H,2H-3,40-bipyrazole (7d). Yield 80%; m.p.
144e146 ꢁC; IR (KBr, nmax, cmꢀ1): 1361 (eCH3 rocking.), 1256
(CeOeC ether str.), 1616 (eC]N), 3062 (eCH aromatic); 1H NMR
(400 MHz, DMSO-d6): 7.68e6.57 (17H, m, ArH), 5.30 (1H, dd, J ¼ 6.4,
12.4 Hz, C5eH pyrazoline), 3.69 (1H, dd, J ¼ 12.8, 17.6 Hz, C4eH
pyrazoline), 3.28 (1H, dd, C4eH pyrazoline merged with peak of
6.1.3. General procedure for synthesis of 5-(4-fluorophenyl)-30-
methyl-50-substituted aryloxy-10,2-diphenyl-3,4-dihydro-10H,2H-
3,40-bipyrazoles (7ael)
Pyrazole chalcones 5aed (1.0 mmol) and substituted phenyl
hydrazine hydrochlorides 6aec (1.0 mmol) were thoroughly mixed
in ethanol (5 mL) with catalytic amount of glacial acetic acid (2e3
drops) in a 50 mL round bottom flask. The reaction mixture was
subjected to microwave irradiation at 350 W (50% of output power)
for 8e10 min. After the completion of the reaction as monitored by
TLC (ethyl acetate:hexane: 1:1), the reaction mixture was cooled to
room temperature. The solid separated was filtered, washed with
cold ethanol (10 mL), dried and recrystallized from ethanol,
affording compounds 7ael.
H2O), 2.20 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
(eCH3 of pyrazole), , 54.7 (eCH2 of pyrazoline), , 109.3, 112.8,
d, 13.5
d
d
114.4, 114.6, 115.2, 115.4, 115.6, 116.0, 119.4, 120.5, 122.0, 127.7, 127.9,
128.3, 129.4, 129.9, 137.6, 137.8, 141.4, 144.8, 147.5, 147.8, 156.7, 164.6
(23 signals, aromatic carbons, pyrazole carbons, C3 and C4 of pyr-
azoline); ESI-MS (m/z): 525.4 (Mþ); Anal. Calcd (%) for
C31H23F3N4O: C, 70.98; H, 4.42; N,10.68. Found: C, 70.73; H, 4.20; N,
10.41.
6.1.3.1. 2,5-Bis(4-fluorophenyl)-30-methyl-10-phenyl-50-(3-(tri-
6.1.3.5. 2-(4-Bromophenyl)-5-(4-fluorophenyl)-30-methyl-10-phenyl-
50-(3-(trifluoromethyl) phenoxy)-3,4-dihydro-10H,2H-3,40-bipyrazole
(7e). Yield 81%; m.p. 138e140 ꢁC; IR (KBr, nmax, cmꢀ1): 1361 (eCH3
rocking.), 1259 (CeOeC ether str.), 1620 (eC]N), 3059 (eCH aro-
matic); 1H NMR (400 MHz, DMSO-d6): 7.63e6.80 (17H, m, ArH),
5.39 (1H, dd, J ¼ 6.0,12.8 Hz C5eH pyrazoline), 3.74 (1H, dd, J ¼ 12.4,
17.6 Hz, C4eH pyrazoline) 3.27 (1H, dd, C4eH pyrazoline merged
with peak of H2O), 2.27 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-
fluoromethyl)phenoxy)-3,4-dihydro-10H,2H-3,40-bipyrazole
(7a).
Yield 82%; m.p. 146e148 ꢁC; IR (KBr, nmax, cmꢀ1): 1368 (eCH3
rocking.), 1260 (CeOeC ether str.), 1608 (eC]N), 3059 (eCH aro-
matic); 1H NMR (400 MHz, DMSO-d6): 7.63e6.84 (17H, m, ArH),
5.34 (1H, dd, J ¼ 6.4, 12.8 Hz, C5eH pyrazoline), 3.71 (1H, dd,
J ¼ 12.8, 17.6 Hz, C4eH pyrazoline), 3.27 (1H, dd, J ¼ 6.4, 17.6 Hz,
C4eH pyrazoline), 2.27 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-
d6):
d
, 13.7 (eCH3 of pyrazole),
d, 54.4 (eCH2 of pyrazoline),
d
, 109.0,
d6): d, 14.0 (eCH3 of pyrazole), d, 55.1 (eCH2 of pyrazoline), d, 108.9,
112.3, 114.3, 114.4, 115.6, 115.8, 115.9, 116.0, 119.3, 120.4, 122.0, 127.5,
127.9, 128.0, 129.1, 129.7, 137.5, 137.7, 141.1, 144.6, 147.0, 147.3, 156.5,
164.3 (24 signals, aromatic carbons, pyrazole carbons, C3, C4 of
pyrazoline and eCF3 of aryloxy ring of pyrazole); ESI-MS (m/z):
575.5 (Mþ); Anal. Calcd (%) for C32H23F5N4O: C, 66.90; H, 4.03; N,
9.75. Found: C, 66.69; H, 3.80; N, 9.50.
111.9, 114.1, 114.2, 115.1, 115.3, 115.8, 116.8, 119.5, 120.6, 122.2, 127.6,
127.9,128.7,129.4,129.9,136.9,137.2,141.0,144.3,146.8,147.3,156.7,
164.7 (24 signals, aromatic carbons, pyrazole carbons, C3, C4 of
pyrazoline and eCF3 of aryloxy ring of pyrazole); ESI-MS (m/z):
635.5 (Mþ); 637.4 (Mþ2); Anal. Calcd (%) for C32H23BrF4N4O: C,
60.48; H, 3.65; N, 8.82. Found: C, 60.25; H, 3.44; N, 8.57.

56
S.C. Karad et al. / European Journal of Medicinal Chemistry 84 (2014) 51e58
6.1.3.6. 5-(4-Fluorophenyl)-2-(4-methoxyphenyl)-30-methyl-10-
phenyl-50-(3-(trifluoromethyl) phenoxy)-3,4-dihydro-10H,2H-3,40-
6.1.3.10. 50-(2-Fluorophenoxy)-2,5-bis(4-fluorophenyl)-30-methyl-10-
phenyl-3,4-dihydro-10H,2H-3,40-bipyrazole (7j). Yield 85%; m.p.
162e164 ꢁC; IR (KBr, nmax, cmꢀ1): 1361 (eCH3 rocking.), 1260
(CeOeC ether str.), 1622 (eC]N), 3052 (eCH aromatic); 1H NMR
(400 MHz, DMSO-d6): 7.66e6.84 (17H, m, ArH), 5.32 (1H, dd,
J ¼ 6.4, 12.8 Hz, C5eH pyrazoline), 3.71 (1H, dd, J ¼ 12.4, 17.2 Hz,
C4eH pyrazoline), 3.27 (1H, dd, J ¼ 6.4, 17.6 Hz, C4eH pyrazoline),
bipyrazole (7f). Yield 85%; m.p. 134e136 ꢁC; IR (KBr, nmax, cmꢀ1):
1363 (eCH3 rocking.), 1259 (CeOeC ether str.), 1598 (eC]N), 3062
(eCH aromatic); 1H NMR (400 MHz, DMSO-d6): 7.61e6.75 (17H, m,
ArH), 5.27 (1H, dd, J ¼ 7.2, 12.8 Hz, C5eH pyrazoline), 3.66 (4H, s,
C4eH pyrazoline merged with eOCH3), 3.24 (1H, dd, J ¼ 6.8,17.2 Hz,
C4eH pyrazoline), 2.24 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-
2.27 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
d, 14.0 (eCH3 of
d6):
d
, 13.9 (eCH3 of pyrazole),
d
, 54.0 (eCH2 of pyrazoline),
d
, 55.5
pyrazole), d, 54.1 (eCH2 of pyrazoline), d, 109.5, 109.9, 115.7, 116.5,
(eOCH3),
d
, 109.8, 112.8, 114.1, 114.9, 115.2, 115.6, 115.9, 116.0, 119.7,
117.9, 123.5, 124.9, 125.8, 127.7, 128.5, 128.9, 129.0, 132.9, 137.7,
143.2, 143.9, 144.8, 147.9, 148.9, 150.3, 152.6, 162.6, 164.7 (23 signals,
aromatic carbons, pyrazole carbons, C3 and C4 of pyrazoline); ESI-
MS (m/z): 525.4 (Mþ); Anal. Calcd (%) for C31H23F3N4O: C, 70.98; H,
4.42; N, 10.68. Found: C, 70.73; H, 4.19; N, 10.45.
120.4, 122.5, 127.8, 127.9, 128.5, 129.5, 129.9, 137.3, 137.9, 141.7,
144.5, 147.6, 147.9, 156.8, 164.4 (24 signals, aromatic carbons, pyr-
azole carbons, C3, C4 of pyrazoline and eCF3 of aryloxy ring of
pyrazole); ESI-MS (m/z): 587.4 (Mþ); Anal. Calcd (%) for
C
9.27.
33H26F4N4O2: C, 67.57; H, 4.47; N, 9.55. Found: C, 67.34; H, 4.24; N,
6.1.3.11. 2-(4-Bromophenyl)-50-(2-fluorophenoxy)-5-(4-
fluorophenyl)-30-methyl-10-phenyl-3,4-dihydro-10H,2H-3,40-bipyr-
azole (7k). Yield 77%; m.p. 165e167 ꢁC; IR (KBr, nmax, cmꢀ1): 1357
(eCH3 rocking.), 1258 (CeOeC ether str.), 1623 (eC]N), 3054 (eCH
aromatic); 1H NMR (400 MHz, DMSO-d6): 7.67e6.62 (17H, m, ArH),
5.32 (1H, dd, J ¼ 6.4, 12.8 Hz, C5eH pyrazoline), 3.70 (1H, dd,
J ¼ 12.8, 17.6 Hz, C4eH pyrazoline) 3.27 (1H, dd, C4eH pyrazoline
merged with peak of H2O), 2.18 (3H, s, CH3); 13C NMR: (400 MHz,
6.1.3.7. 50-(4-Fluorophenoxy)-2,5-bis(4-fluorophenyl)-30-methyl-10-
phenyl-3,4-dihydro-10H,2H-3,40-bipyrazole (7g). Yield 83%; m.p.
148e150 ꢁC; IR (KBr, nmax, cmꢀ1): 1357 (eCH3 rocking.), 1254
(CeOeC ether str.), 1610 (eC]N), 3059 (eCH aromatic); 1H NMR
(400 MHz, DMSO-d6): 7.82e6.84 (17H, m, ArH), 5.34 (1H, dd,
J ¼ 6.0, 12.4 Hz, C5eH pyrazoline), 3.72 (1H, dd, J ¼ 12.4, 17.2 Hz,
C4eH pyrazoline), 3.27 (1H, dd, J ¼ 6.0, 17.2 Hz, C4eH pyrazoline),
DMSO-d6):
d, 13.7 (eCH3 of pyrazole), d, 53.7 (eCH2 of pyrazoline),
d, 108.5, 110.3, 115.0, 116.0, 117.4, 122.0, 124.8, 125.3, 127.6, 128.1,
2.27 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
d
, 13.5 (eCH3 of
128.9, 129.7, 131.9, 137.7, 143.4, 143.9, 144.7, 147.0, 147.9, 150.0, 152.5,
161.6, 164.9 (23 signals, aromatic carbons, pyrazole carbons, C3 and
C4 of pyrazoline); ESI-MS (m/z): 585.4 (Mþ); 587.4 (Mþ2); Anal.
Calcd (%) for C31H23BrF2N4O: C, 63.60; H, 3.96; N, 9.57. Found: C,
63.32; H, 3.73; N, 9.36.
pyrazole), d, 54.9 (eCH2 of pyrazoline), d, 109.7, 113.7, 114.4, 115.3,
116.9, 117.2, 122.2, 126.3, 127.8, 129.2, 136.9, 138.9, 145.4, 146.2,
147.5, 151.9, 152.3, 157.4, 159.9, 162.3, 164.8, (21signals, aromatic
carbons, pyrazole carbons, C3 and C4 of pyrazoline); ESI-MS (m/z):
525.4 (Mþ); Anal. Calcd (%) for C31H23F3N4O: C, 70.98; H, 4.42; N,
10.68. Found: C, 70.73; H, 4.21; N, 10.43.
6.1.3.12. 50-(2-Fluorophenoxy)-5-(4-fluorophenyl)-2-(4-
methoxyphenyl)-30-methyl-10-phenyl-3,4-dihydro-10H,2H-3,40-bipyr-
azole (7l). Yield 87%; m.p. 141e143 ꢁC; IR (KBr, nmax, cmꢀ1): 1359
(eCH3 rocking.), 1259 (CeOeC ether str.), 1626 (eC]N), 3055 (eCH
aromatic); 1H NMR (400 MHz, DMSO-d6): 7.66e6.68 (17H, m, ArH),
5.18 (1H, dd, J ¼ 7.2, 12.4 Hz C5eH pyrazoline), 3.67 (4H, s, C4eH
pyrazoline merged with eOCH3), 3.62 (1H, dd, J ¼ 12.8, 17.6 Hz,
C4eH pyrazoline), 3.23 (1H, dd, J ¼ 7.2, 17.6 Hz C4eH pyrazoline),
6.1.3.8. 2-(4-Bromophenyl)-50-(4-fluorophenoxy)-5-(4-
fluorophenyl)-30-methyl-10-phenyl-3,4-dihydro-10H,2H-3,40-bipyr-
azole (7h). Yield 79%; m.p. 179e181 ꢁC; IR (KBr, nmax, cmꢀ1): 1361
(eCH3 rocking.), 1254 (CeOeC ether str.), 1615 (eC]N), 3051 (eCH
aromatic); 1H NMR (400 MHz, DMSO-d6): 7.68e6.79 (17H, m, ArH),
5.30 (1H, dd, J ¼ 6.0, 12.4 Hz, C5eH pyrazoline), 3.69 (1H, dd,
J ¼ 12.8, 17.6 Hz, C4eH pyrazoline), 3.27 (1H, dd, J ¼ 6.0, 17.6 Hz,
C4eH pyrazoline), 2.16 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
2.17 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
d
, 14 (eCH3 of
pyrazole), d, 55 (eCH2 of pyrazoline), d, 55.6 (eOCH3), d, 108.8,114.7,
115.1, 115.8, 116.0, 116.8, 117.3, 117.5, 122, 124.9, 125, 125.4, 127.5,
127.8, 127.9, 129.7, 137.8, 138.9, 144.9, 146.2, 147.2, 153.4, 164.7 (23
signals, aromatic carbons, pyrazole carbons, C3 and C4 of pyrazo-
line); ESI-MS (m/z): 537.5 (Mþ); Anal. Calcd (%) for C32H26F2N4O2: C,
71.63; H, 4.88; N, 10.44. Found: C, 71.38; H, 4.65; N, 10.19.
d, 13.8 (eCH3 of pyrazole), d, 53.8 (eCH2 of pyrazoline), d, 108.3,
110.2, 115.9, 116.5, 117.1, 121.9, 127.4, 128.4, 128.9, 129.7, 131.9, 137.8,
143.4, 145.5, 147.1, 147.8, 152.8, 157.1, 159.5, 161.3, 164.0 (21 signals,
aromatic carbons, pyrazole carbons, C3 and C4 of pyrazoline); ESI-
MS (m/z): 585.4 (Mþ); 587.4 (Mþ2); Anal. Calcd (%) for
C
31H23BrF2N4O: C, 63.60; H, 3.96; N, 9.57. Found: C, 63.33; H, 3.72;
7. Biological evaluation
N, 9.35.
7.1. In vitro antimicrobial assay
6.1.3.9. 50-(4-Fluorophenoxy)-5-(4-fluorophenyl)-2-(4-
methoxyphenyl)-30-methyl-10-phenyl-3,4-dihydro-10H,2H-3,40-bipyr-
azole (7i). Yield 84%; m.p. 158e160 ꢁC; IR (KBr, nmax, cmꢀ1): 1361
(eCH3 rocking.), 1258 (CeOeC ether str.), 1619 (eC]N), 3053 (eCH
aromatic); 1H NMR (400 MHz, DMSO-d6): 7.67e6.78 (17H, m, ArH),
5.17 (1H, dd, J ¼ 7.2, 12.4 Hz, C5eH pyrazoline), 3.63 (4H, s, C4eH
pyrazoline merged with eOCH3), 3.22 (1H, dd, J ¼ 7.2, 17.6 Hz, C4eH
The antimicrobial activity of pyrazolylpyrazolines derivatives
7ael was carried out by broth micro dilution method. DMSO was
used as the diluent to get the desired concentration of compounds
to test upon standard bacterial stains. Mueller-Hinton broth was
used as nutrient medium to grow and dilute the compound sus-
pension for the test bacteria. Sabouraud Dextrose broth was used
for fungal nutrition. Inoculum size for test stain was adjusted to
108 CFU mLꢀ1 by comparing the turbidity. Serial dilutions were
prepared in primary and secondary screening. Each synthesized
pyrazoline), 2.15 (3H, s, CH3); 13C NMR: (400 MHz, DMSO-d6):
d,
13.9 (eCH3 of pyrazole),
d, 55.0 (eCH2 of pyrazoline), d, 55.6
(eOCH3), , 108.7, 114.7, 115.4, 116.3, 116.9, 117.2, 121, 127.3, 127.8,
d
129.5, 137.9, 138.9, 145.6, 146.1, 147.1, 152.9, 153.3, 157.2, 159.5, 161.3,
163.8, (21signals, aromatic carbons, pyrazole carbons, C3 and C4 of
pyrazoline); ESI-MS (m/z): 537.5 (Mþ); Anal. Calcd (%) for
compound and the standard drugs were diluted obtaining 2000
mL concentration as a stock solution. The drugs which were found
to be active in primary screening (i.e. 500, 250 and 200 g/mL
concentrations) were further screened in their second set of dilu-
tion at 100, 50, 25 and 12.5 g/mL concentration against all
mg/
m
C
32H26F2N4O2: C, 71.63; H, 4.88; N, 10.44. Found: C, 71.37; H, 4.60;
N, 10.17.
m

S.C. Karad et al. / European Journal of Medicinal Chemistry 84 (2014) 51e58
57
microorganisms. 10
m
L suspensions were further inoculated on
software. The standard drugs chloroquine and quinine were used
as the reference antimalarial agents, blood smears were read
blind and each duplicate experiment was repeated thrice. The
results are summarized in Table 3.
appropriate media and growth was noted after 24 and 48 h. The
control tube containing no antibiotic was instantaneously subcul-
ture (before inoculation) by evenly spreading a loopful over an area
of plate of medium suitable for the growth of the test organism. The
tubes were then put overnight for incubation at 37 ꢁC. The highest
dilution preventing appearance of turbidity after spot subculture
Acknowledgement
was considered as minimal inhibitory concentration (MIC, mg/mL).
The authors are thankful to Head, Department of Chemistry,
Sardar Patel University for providing necessary research facilities.
We are also thankful for the assistance in the form of concessional
analysis by SICART, Vallabh Vidyanagar-388 120, India. The anti-
microbial, antimalarial and antituberculosis screenings of the
compounds were performed by Dhanji P. Rajani, Microcare Labo-
ratory, Surat-395 007, India. SCK and VBP gratefully acknowledge
the University Grants Commission, New Delhi, India for UGC Basic
Scientific Research Fellowship to Meritorius Students awarded to
them during 2013e2015.
All the tubes showing no visible growth (same as the control tube)
were subcultured and incubated overnight at 37 ꢁC. The amount of
growth from the control tube before incubation was compared. In
this study ampicillin, norfloxacin, chloramphenicol and ciproflox-
acin were used as the standard antibacterial drugs. Nystatin and
Griseofulvin were used as the standard antifungal drugs. The re-
sults are summarized in Table 1.
7.2. In vitro antituberculosis assay
All pyrazolylpyrazolines derivatives 7ael were screened for
their antitubercular activity against M. tuberculosis H37Rv per-
formed by LowensteineeJensen method with minor modification
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.008.
where 250 mg/mL dilution of each compound was added to Low-
ensteineeJensen medium and then media was uncontaminated by
inspissation method. A culture of M. tuberculosis H37Rv grown on
LowensteineeJensen medium was harvested in 0.85% saline in
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at which complete inhibition of colonies occurred was taken as
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tuberculosis H37Rv was also tested with the known drugs isoniazid
and rifampicin for comparison. The results are summarized in
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