Synthesis, molecular docking and biological evaluation of novel bis-pyrazole derivatives for analgesic, anti-inflammatory and antimicrobial activities

Article abstract of DOI:10.1007/s00044-015-1467-9

A new series of bis-pyrazoles were synthesized by Michael addition of hydrazine to chalcones. The starting-material-substituted acetophenones required for the synthesis of chalcones were prepared from itaconic anhydride. The newly synthesized compounds we

Full text of DOI:10.1007/s00044-015-1467-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2015) 24:4191–4206
DOI 10.1007/s00044-015-1467-9
ORIGINAL RESEARCH
Synthesis, molecular docking and biological evaluation of novel
bis-pyrazole derivatives for analgesic, anti-inflammatory
and antimicrobial activities
Prakash S. Nayak1 B. Narayana1 B. K. Sarojini2
•
•
•
Jennifer Fernades3 B. R. Bharath4 L. N. Madhu5
•
•
Received: 21 April 2015 / Accepted: 4 September 2015 / Published online: 11 September 2015
Ó Springer Science+Business Media New York 2015
Abstract A new series of bis-pyrazoles were synthesized
by Michael addition of hydrazine to chalcones. The start-
ing-material-substituted acetophenones required for the
synthesis of chalcones were prepared from itaconic anhy-
dride. The newly synthesized compounds were character-
ized by IR, 1H-NMR, 13C-NMR, mass spectral and
analytical data. All the synthesized compounds were
evaluated for in vivo analgesic, anti-inflammatory and
in vitro antimicrobial activities. Among the tested com-
pounds, 5a, 5b and 5d showed potential anti-inflammatory
and analgesic activities. Further anti-inflammatory results
were supported by in silico docking study, in which tested
bis-pyrazoles were found to be more selective toward
COX-2 (PDB ID: 1CX2) rather than COX-1 (PDB ID:
1CQE). The LD50 values for these products 5(a–l) showed
a high safety margin with a dose level [2000 mg/kg.
Among all synthesized compounds, N-[4-(5-(4-bro-
mophenyl)-1-phenyl-1H-pyrazol-3-yl)phenyl-2-(3-hydroxy-
1-phenyl-1H-pyrazol-4-yl)] acetamide (5b) emerged as
most potent molecule with anti-inflammatory, analgesic and
antimicrobial properties.
Graphical Abstract
Keywords Bis-pyrazoles Á Analgesic activity Á
Anti-inflammatory activity Á Antimicrobial activity Á
Molecular docking study
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1467-9) contains supplementary
material, which is available to authorized users.
Introduction
& B. Narayana
nbadiadka@yahoo.co.uk
The development of an effective therapeutic agent for
management of inflammation has undergone continual
evolution leading to the emergence of more efficacious
class of drugs. Since the discovery of aspirin, many efforts
have been devoted to the development of nonsteroidal anti-
inflammatory drugs (NSAIDs). Their mechanism of action
involves inhibition of the enzyme cyclooxygenase (COX),
which catalyzes the rate-limiting step in the formation of
prostanoids, prostaglandins (PGs) and thromboxane A2
(TxA2) (Vane, 1971). It is predicted that COX-2 enzyme is
responsible for some aspects of pain and inflammation in
arthritis, while constitutive COX-1 enzyme appears to be
1
Department of Chemistry, Mangalore University,
Mangalagangothri, Mangalore, Karnataka 574 199, India
2
Department of Studies in Industrial Chemistry, Mangalore
University, Mangalagangothri, Mangalore, Karnataka
574 199, India
3
Department of Pharmachemistry, N G S M institute of
Pharma Sciences, Deralakatte, Mangalore, Karnataka
575 018, India
4
Department of Biotechnology, NMAM Institute of
Technology, Nitte, Karkala, Karnataka 574 110, India
5
Post Graduation Department of Biochemistry, St. Aloysius
College, Mangalore, Karnataka 575 003, India
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Med Chem Res (2015) 24:4191–4206
responsible for most of gastroprotective prostaglandin
synthesis in the stomach and duodenum. It is also sug-
gested that COX-2 inhibitors are not only promising agents
for treatment of pain and inflammation, but also used for
prevention of cancer (Marnett and Kalgutkar, 1998). Thus,
search for novel anti-inflammatory drugs with minimal
gastrointestinal side effects and high safety margin is still
warranted.
potential for acting synergistically as analgesic, anti-in-
flammatory and antimicrobial agents with minimal gas-
trointestinal side effects and high safety margin.
Results and discussion
Chemistry
Pyrazole derivatives have a long history of application
in agrochemicals and pharmaceutical industries as herbi-
cides and active pharmaceuticals. The recent success of
pyrazole-derived COX-2 inhibitors further highlights the
importance of pyrazole as biodynamic ring system. Many
useful clinical agents, such as difenamizole (Kameyama
and Nabeshima, 1978; Kameyama et al., 1978), celecoxib
(Penning et al., 1997) and tepoxalin (Anderson et al., 1990)
do possess pyrazole ring system. The prevalence of pyra-
zole cores in biologically active molecules has stimulated
the need for elegant and efficient ways to make these
heterocyclic leads. Microbial infections often produce pain
and inflammation. Analgesic and anti-inflammatory drugs
are prescribed simultaneously in normal practice under
chemotherapy. The compound possessing all three activi-
ties is not common. It has been reported that certain
pyrazole derivatives possess analgesic, anti-inflammatory
(Amir and Kumar, 2005; Zelenin et al., 1999; Adnan et al.,
2005), antimicrobial (Susant et al., 2007; Akihiko et al.,
2005; Goda et al., 2003), antitumor, antioxidant, antiviral,
antiparasitic, antitubercular and insecticidal activities
(Amir et al., 2008; Hes et al., 1978; Grosscurt et al., 1979).
In addition, pyrazoles have also played a crucial part in the
development of theory in heterocyclic chemistry and are
also used extensively in organic synthesis (Klimova et al.,
1999).
A new series of bis-pyrazole derivatives were prepared
starting form itaconic anhydride (ITA) and 3/4-aminoace-
tophenone. The resultant oxobutanoic acids 3(a–
b) (Scheme 1) were treated with 4-substituted aryl alde-
hydes to yield chalcone derivatives 4(a–l). These inter-
mediate chalcones were converted into bis-pyrazole
derivatives by condensing with phenyl hydrazine according
to the reaction sequence depicted in Scheme 2. The for-
mation of bis-pyrazole products and other intermediates
was confirmed by spectral and elemental analysis.
In the first step, the intermediate c-oxobutanoic acids
(3a, 3b) required for the synthesis of chalcones were pre-
pared by the reaction of nucleophilic reagents with ITA.
This formation of the products was confirmed by single-
crystal XRD data (Fig. 1) (Narayana et al., 2014; Nayak
et al., 2014a, b, c, d, e). During single-crystal X-ray
diffraction study of compounds 3a (CCDC No. 1005968),
3b (CCDC No. 949575), a twist between the mean planes
of methylidene group and amide group to an extent of
approximately 100° was found, which confirmed E (trans)
form of the oxobutanoic acids (3a, 3b) (Nayak et al.,
2014c).
The Claisen–Schmidt condensation of oxobutanoic
acids (3a, 3b) with differently substituted aldehydes yiel-
ded intermediate chalcones. The IR spectra of intermediate
chalcones exhibited stretching bands in the region of
3275–3345, 3059–3136, 1678–1688 and 1652–1665 cm-1
representing NH, OH, amide C=O and acid C=O functional
Motivated by these findings, coupled with our ongoing
research on pyrazoles and other heterocyclic compounds
(Nayak et al., 2014a, b, c, d, e), it was decided to synthe-
size, some novel series of bis-pyrazole derivatives with a
e
R
f
O
c
O
O
e
f
d
O
O
O
R-C H -CHO
6 4
MDC
+
H
O
A
NaOH/EtOH+H O
2
NH
H
O
H
A
B
NH
NH
2
H
B
OH
O
OH
O
1(a-b)
2
3(a-b)
4(a-l)
Scheme 1 Synthetic pathways to chalcone derivatives 4(a–l)
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Med Chem Res (2015) 24:4191–4206
4193
Scheme 2 Synthetic pathways
to bis-pyrazole derivatives
5(a–l)
R
e
R
f
c
1
N
5
4
O
e
A
N
2
f
d
3
Ph-NH-NH
2
H
O
A
EtOH, HCl
O
NH
H
B
5
NH
1
4
N
B
OH
N
2
O
3
HO
5(a-l)
4(a-l)
Fig. 1 ORTEP drawing of the molecules, showing the labeling scheme with 30 % (3a) and 40 % (3b) probability displacement ellipsoids
groups, respectively. These data suggested the formation of
1
intermediate chalcones. The H-NMR spectrum of repre-
observed around 1670–1709 cm-1 was due to the stretch-
ing vibration of amide carbonyl group, suggesting that acid
group of oxobutanoic acid was also involved in the reaction
with phenyl hydrazine to form an additional pyrazole ring.
This suggested the formation of bis-pyrazole products. The
1H-NMR spectrum of representative bis-pyrazole 5a
exhibited two singlets at d 6.58 ppm and d 8.15 ppm,
representing the protons attached to C-4 of pyrazole ring A
and C-5 of pyrazole ring B, respectively. Similarly, a sin-
glet observed at d 5.43 ppm could be due to OH proton of
pyrazolyl alcohol. The upfield shift of this signal suggested
that enol form predominated over keto form. A singlet
found at d 10.10 ppm was attributed to NH protons.
Absence of a broad singlet at d 12.56 due to carboxylic OH
of the precursor 4a confirmed the formation of 5a. Further
signals for methylidene protons were also absent in the bis-
pyrazole spectrum, which confirmed the formation of
product 5a. In 13C-NMR spectrum, peaks observed at d
157.64, 149.56, 147.45, 122.67, 119.43 and 104.49 ppm
were attributed to the number of pyrazole carbons (pyra-
zole A C-3, pyrazole A C-5, pyrazole B C-3, pyrazole B
C-5, pyrazole B C-4 and pyrazole A C-4). Further
sentative chalcone 4k displayed two doublets at d
7.48 ppm and d 7.79 ppm due to Hc and Hd protons. The
coupling constant (J) for Hc and Hd was 15.6 Hz charac-
teristic of (E)-isomer of the chalcone. Similarly, two more
doublets appeared at d 5.57 ppm and d 6.50 ppm due to HA
and HB of methylidene protons of oxobutanoic acid group.
The coupling constant (J) for HA and HB was found to be
1.6 Hz characteristic of geminal coupling of methylidene
group. The signals for aromatic protons appeared as mul-
tiplets in the range of d 6.95–7.96 ppm. The two singlets
observed at d 10.50 ppm and d 11.55 ppm confirmed the
presence of NH and OH protons. Further formation of
chalcone products 4(a–i) was confirmed by mass spectral
data and elemental analysis.
The formation of bis-pyrazole derivatives 5(a–l) was
explained via formation of hydrazones followed by
cyclization and dehydration process on the chalcone end,
while at the other end, phenyl hydrazine condenses with
oxobutanoic acid followed by the cyclization and dehy-
dration. In the IR spectra of 5(a–l), stretching band
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Med Chem Res (2015) 24:4191–4206
formation of bis-pyrazole products 5(a–i) was confirmed
by mass spectral studies and elemental analysis.
ANOVA followed by Dunnett’s t test. Compound 5b
(68.87 %), N-[4-(5-(4-bromophenyl)-1-phenyl-1H-pyra-
zol-3-yl)phenyl-2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)
acetamide] conferred maximum anti-inflammatory activity
followed by 5a (68.42 %) bearing fluoro substitution.
Other compounds 5(c–i) showed moderate-to-less activity
at the dosage of 10 mg/kg p.o. Interestingly, it was also
observed that para amido bis-pyrazoles 5(a–f) showed
better activity in comparison with the meta amido bis-
pyrazoles 5(g–i) similar to that of analgesic activity.
In general, para amido bis-pyrazoles [N-(4-(5-(4-
substituted-phenyl)-1-phenyl-1H-pyrazol-3-yl)phenyl-2-
(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide)] 5(a–f)
showed good analgesic and anti-inflammatory activity.
Among these, halogen-substituted compounds 5(a–c) ex-
hibited better activities than nonhalogenated compounds
5(d–f).
Pharmacology
Analgesic activity
Abdominal constriction induced by acetic acid is used to
screen the peripheral analgesic effect (nonsteroidal type of
analgesic action) (Vogel and Vogel, 2002a, b) mediated by
local peritoneal receptors (Gene et al., 1998). The results of
analgesic activity as shown in Table 1 indicated that para
amido bis-pyrazoles 5(a–f) showed better activity in
comparison with the meta amido bis-pyrazoles 5(g–i).
Among the para amido bis-pyrazoles, halogen-substituted
compounds 5a (66.66 %), 5b (66.66 %) and 5c (64.58 %)
showed good percentage of inhibition in comparison with
that of the standard drug aspirin (68.75 %). Other com-
pounds exhibited comparably less activity. The enhanced
activity of these compounds might be due to the presence
of halogen-substituted aryl moieties and para amido pyra-
zole functional group.
Acute toxicity
There was no incidence of mortality among groups of
animals which were fed with compounds 5(a–i), and they
did not exhibit any toxicity or behavioral changes at a dose
level of 2000 mg/kg. The compounds showed a high safety
margin when screened at dose of 2000 mg/kg, p.o., and
lethal dose (LD50) values were found to be [2000 mg/kg.
Anti-inflammatory activity
The anti-inflammatory activity of the newly synthesized
compounds 5(a–i) was evaluated by carrageenan-induced
paw edema model in rats using indomethacin as a reference
drug. Results were expressed as mean SD and given in
Table 2. Difference between control and treatment groups
was evaluated for statistical significance using one-way
Molecular docking study
The synthesized bis-pyrazole derivatives 5(a–i) were
screened for in silico molecular docking with COX-1 (PDB
Table 1 Analgesic activity compounds 5(a–l) by acetic acid-induced writhing test
Compounds
R
Dose (mg/kg)
Number of writhings
% inhibition
Control
Aspirin
5a
–
0.5
10
25
25
25
25
25
25
25
25
25
25
25
25
48.00 0.90
15.00 0.72*
16.00 0.68*
16.00 0.79*
17.00 0.59*
19.00 0.81*
19.00 0.81*
18.00 0.72*
19.00 0.78*
19.00 0.67*
20.00 0.79*
22.00 0.63*
21.00 0.62*
20.00 0.75*
–
–
68.75
66.66
66.66
64.58
60.41
60.41
62.50
60.41
60.41
58.33
54.16
56.25
58.33
F
5b
Br
5c
Cl
5d
CH3
OCH3
CH(CH3)2
F
5e
5f
5g
5h
Br
5i
Cl
5j
CH3
OCH3
CH(CH3)2
5k
5l
* The mean difference is significant at the 0.001 level, when compared to the control group
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Med Chem Res (2015) 24:4191–4206
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Table 2 Anti-inflammatory activity compounds 5(a–l) by carrageenan-induced paw edema model
Compounds
R
Dose (mg/kg) % decrease in paw volume at 3 h
0 h 1 h
0.073 0.005 0.113 0.005
% decrease in paw
volume at 3 h
2 h
3 h
Control
–
0.1
10
10
10
10
10
10
0.160 0.006
0.221 0.008
–
Indomethacin
–
0.025 0.009 0.031 0.007* 0.033 0.005* 0.043 0.005* 80.63
5a
5b
5c
5d
5e
5f
F
0.048 0.005 0.056 0.006
0.042 0.003 0.051 0.006
0.040 0.005 0.050 0.004
0.044 0.005 0.052 0.006
0.048 0.005 0.058 0.006
0.051 0.004 0.065 0.006
0.050 0.005 0.058 0.006
0.450 0.006 0.059 0.005
0.050 0.006 0.058 0.004
0.046 0.005 0.059 0.006
0.050 0.004 0.059 0.005
0.047 0.005 0.058 0.004
0.065 0.004
0.060 0.002
0.061 0.005
0.066 0.007
0.067 0.004
0.072 0.005
0.069 0.004
0.068 0.003
0.068 0.005
0.071 0.008
0.069 0.007
0.067 0.006
0.070 0.005* 68.42
0.069 0.004* 68.87
0.072 0.005* 67.52
0.073 0.006* 67.07
0.079 0.008* 64.36
0.073 0.006* 67.07
0.078 0.005* 64.70
0.077 0.005* 65.51
0.080 0.007* 63.80
0.085 0.006* 61.53
0.083 0.006* 62.44
0.081 0.007* 63.34
Br
Cl
CH3
OCH3
CH(CH3)2 10
5g
5h
5i
F
10
10
10
10
10
Br
Cl
5j
CH3
OCH3
5k
5l
CH(CH3)2 10
* The mean difference is significant at the 0.001 level, when compared to the control group
ID: 1CQE) and COX-2 (PDB ID: 1CX2) enzymes to test
the putative interaction. Automated docking was used to
determine the orientation of inhibitors bound in the active
site of COX-1 and COX-2 enzymes. A Lamarckian genetic
algorithm method, implemented in the program AutoDock
4.2, was employed.
From the docking study results (Table 3), it could be
concluded that ligand–receptor fitting of all screened
compounds was best with highest binding energy for
COX-2 than for COX-1 enzyme. The compound 5b
showed better fitting with minimum total energy value
-11.92 kcal/mol followed by 5a (energy value
-11.21 kcal/mol), 5f and 5c (energy value -10.33 kcal/
mol) with COX-2 enzyme. The compound 5b showed
binding energy with total energy value -3.04 kcal/mol
with COX-1 enzyme followed by 5c (energy value
-2.41 kcal/mol) and 5a (energy value -2.36 kcal/mol).
These in silico study results are complementing with the
other biological experimental results and support the
overall outcome of the study. The selective binding of
tested molecules to COX-2 in preference to COX-1 is an
encouraging result in favor of anti-inflammatory activity.
Docking results between the screened bis-pyrazole
derivatives 5(a–i) and selected receptors, i.e., the
cyclooxygenase-1 (PDB ID: 1CQE) and cyclooxygenase-2
(PDB ID: 1CX2) enzymes from mouse, are tabulated in
Table 3. The structure of receptor, ligand and ligand–re-
ceptor complex is shown in Figs. 2, 3. Among bis-pyra-
zoles 5(a–i), meta amido bis-pyrazoles 5(g–i) could not be
docked against COX-1 and COX-2 enzymes as they were
showing some mathematical error. This might be due to the
size or geometry of the molecules resisting the docking.
Table 3 Docking study results of screened active compounds with COX-1 and COX-2 enzymes
Compounds Binding energy (BE)
(kcal/mol)
Ligand efficiency (LE) Inhibition constant (IC)
Electrostatic energy
(EE)
H-bond
COX-1
COX-2
COX-1
COX-2
COX-1 (mM) COX-2 (nM) COX-1
COX-2
COX-1 COX-2
5a
5b
5c
5d
5e
5f
-2.36
-3.04
-2.41
-2.19
-2.04
-2.13
-11.21
-11.92
-10.33
-10.27
-9.31
-0.06
-0.08
-0.06
-0.05
-0.03
-0.04
-0.28
-0.29
-0.26
-0.25
-0.17
-0.26
6.8
5.9
6.3
7.1
7.9
7.3
6.11
5.81
-0.02
-0.03
-0.02
-0.01
0.02
-0.32
-0.35
-0.20
-0.12
-0.08
-0.18
0
1
0
0
0
0
2
2
2
2
1
1
26.97
27.01
33.41
26.72
-10.46
0.01
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Med Chem Res (2015) 24:4191–4206
Fig. 2 Compound 5b in the active pocket of COX-1 (PDB ID: 1CQE) and interaction with COX-1 residues in an active pocket
Fig. 3 Compound 5b in the active pocket of COX-2 (PDB ID: 1CX2) and interaction with COX-2 residues in an active pocket
Antimicrobial activity
It could also be noticed that zone of inhibition by the
compounds containing halogen substituents 5a, 5b, 5c, 5g,
5h and 5i was better than that of nonhalogenated
derivatives.
The results are depicted in Table 4. The results revealed
that the synthesized compounds displayed variable inhibi-
tory effects on the growth of the tested gram-positive
(Staphylococcus aureus NCIM 2079) and gram-negative
(Escherichia coli NCIM 2931) bacterial strains. In general,
most of the tested compounds exhibited moderate activity
against both the gram-positive and gram-negative bacteria.
The minimum inhibitory concentration (MIC) values of
compounds were tested to measure antimicrobial activity
of compounds (5a–i) (Table 5). The MIC values for the
compounds 5a, 5b and 5g were found to be 125 lg/mL
against both the tested strains. MIC values for other
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4197
Table 4 Results of antimicrobial activity of the compounds 5(a–l)
Compounds
R
Zone of inhibition (mm)
Gram-positive
S. aureus
Gram-negative
E. coli
5a
F
10.00 0.57
11.00 0.28
09.00 0.28
09.00 0.57
09.00 0.00
08.00 0.57
11.00 0.00
11.00 0.28
10.00 0.57
09.00 0.57
07.00 0.28
09.00 0.28
19.00 0.28
12.00 0.00
11.00 0.57
11.00 0.00
12.00 0.28
11.00 0.00
09.00 0.28
11.00 0.28
10.00 0.57
11.00 0.28
11.00 0.00
09.00 0.57
08.00 0.28
21.00 0.57
5b
Br
5c
Cl
5d
CH3
5e
OCH3
CH(CH3)2
F
5f
5g
5h
Br
5i
Cl
5j
CH3
5k
OCH3
CH(CH3)2
5l
Streptomycin (10 lg/mL)
in KBr (mmax in cm-1). The H-NMR (400 MHz) spectra
were recorded on a BRUKER AV400 NMR spectrometer,
with 5-mm PABBO BB-1H TUBES. The 13C-NMR
(100 MHz) spectra were recorded for approximately
0.03 M solutions in DMSO at 100 MHz with TMS as the
internal standard. LCMS was completed using the Agilent
1200 series LC and Micromass ZQ spectrometer. Ele-
mental analysis was carried out with the VARIO EL-III
(Elementar Analysensysteme, GmBH). Crystallographic
data were collected on a Agilent Eos Gemini diffractometer
1
Table 5 MIC values of compounds 5(a–l)
Compounds
R
Minimum inhibitory concentration
(MIC) (lg/mL)
S. aureus
E. coli
5a
F
125
125
125
125
5b
Br
5c
Cl
250
250
5d
CH3
OCH3
CH(CH3)2
F
250
125
5e
250
250
˚
with a CuKa radiation (k = 1.54184 A) for compound 3a
and Bruker SMART APEXII CCD area-detector diffrac-
5f
250
250
5g
125
125
tometer with a graphite-monochromated MoKa radiation
˚
5h
Br
125
250
(k = 0.71073 A) for compound 3b equipped with an
5i
Cl
250
125
X’Calibur CCD area-detector diffractometer. The geome-
try of the molecule was calculated using the PLATON,
PARST and OLEX2 software. The structure was solved by
direct method and refined by least squares using the
SHELX97, SHELXL2012 software package. CCDC
1005968 and 949575 contain the supplementary crystallo-
graphic data for 3a and 3b. These data can be obtained free
of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.
html (or from the Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge CB2 1EZ, UK; fax: 44 1223
336033: email: deposit@ccdc.cam.ac.uk).
5j
CH3
OCH3
CH(CH3)2
[400
[400
[400
\12.5
125
5k
250
5l
[400
\12.5
Streptomycin
compounds were found to be in the range from 250 to
[400 lg/mL, revealing their lower activity against S.
aureus and E. coli bacterial strains.
Experimental protocol
Synthesis of 4-[(3/4-acetylphenyl)amino]-2-methylidene-4-
Chemistry
oxobutanoic acid (3a–b)
The melting points were measured in open capillary tubes
and were not corrected. Thin-layer chromatography was
performed on Merck silica gel 60 F254. The IR spectra
were recorded on a Shimadzu-FTIR infrared spectrometer
4-[(4-Acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3a) ITA (2) (0.112 g, 1 mmol) was dissolved in
acetone (30 mL), and 4-amino acetophenone (0.135 g,
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Med Chem Res (2015) 24:4191–4206
1 mmol) was added in small portions under stirring at room
temperature over time span of 30 min. The mixture became
yellow slurry. Stirring was continued for 1.5 h, after which
the solution was filtered. The solid obtained was washed
with acetone and dried. The crude product was further
purified by recrystallization method, and single crystals
were grown from methanol by the slow evaporation. The
product was obtained as a light yellow solid with a yield of
84 %, m.p. 188–190 °C; IR (KBr): mmax (cm-1), 3282
(OH), 3055 (NH), 2900 (Ar–H), 2634 (aliphatic C-H) 1685
(amide C=O), 1650 (acid C=O), 1591 (C=C). 1H-NMR
(400 MHz, DMSO-d6): d ppm, 2.53 (3H, s, CH3), 3.35
(2H, s, O=C-CH2), 5.75 (1H, d, JAB = 1.6 Hz, HA), 6.18
(1H, d, JAB = 1.6 Hz, HB), 7.69–7.93 (4H, m, Ar–H),
10.37 (1H, s, NH), 12.57 (1H, s, OH). LCMS (m/z): 248
(M? ? 1). Calcd. for C13H13NO4: C, 63.15; H, 5.30; N,
5.67; Found: C, 63.13; H, 5.32; N, 5.65 %. XRD data:
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 68 % yield. m.p.
170–172 °C; IR (KBr): mmax (cm-1), 3340 (OH), 3103 (NH),
2922 (Ar–H), 2706 (aliphatic C-H) 1678 (amide C=O), 1662
1
(acid C=O), 1517 (C=C). H-NMR (400 MHz, DMSO-d6):
d ppm, 3.41 (2H, s, O=C-CH2), 5.97 (1H, d, JAB = 1.6 Hz,
HA), 6.50 (1H, d, JAB = 1.6 Hz, HB), 7.26 (2H, dt,
J = 2.0 Hz, 5.2 Hz, Ar–He), 7.65 (1H, d, J = 16.4 Hz, Hc),
7.92–8.30 (7H, m, Ar–H), 10.36 (1H, s, NH),12.56 (1H, s,
OH). LCMS (m/z): 354 (M? ? 1). Calcd. for C20H16FNO4:
C, 67.98; H, 4.56; N, 3.96; Found: C, 67.96; H, 4.58; N,
3.94 %.
4-({4-[3-(4-Bromophenyl)prop-2-enoyl]phenyl}amino)-2-
methylidene-4-oxobutanoic acid (4b) To a mixture of
4-[(4-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3a) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 65 % yield. m.p.
175–177 °C; IR (KBr): mmax (cm-1), 3344 (OH), 3100
(NH), 2927 (Ar–H), 2716 (aliphatic C-H) 1687 (amide
C=O), 1652 (acid C=O), 1520 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 3.40 (s, 2H, O=C-CH2), 5.93 (d, 1H,
HA, JAB = 1.6 Hz), 6.45 (d, 1H, HB, JAB = 1.6 Hz), 7.27
(dt, 2H, Ar–He, J = 2.0 Hz, 5.2 Hz), 7.67 (d, 1H, Hc,
J = 16.4 Hz), 7.40–8.30 (m, 7H, Ar–H), 10.35 (s, 1H,
NH),12.54 (s, 1H, OH). LCMS (m/z): 415 (M? ? 1).
Calcd. for C20H16BrNO4: C, 57.99; H, 3.89; N, 3.38;
Found: C, 57.97; H, 3.91; N, 3.36 %.
ꢀ ˚ ˚
1; a = 5.0164(5) A, b = 5.2908(4) A,
triclinic,
P
3
c = 21.8464(18) A, V = 575.67(8) A , Z = 2.
˚
˚
4-[(3-Acetylphenyl)amino]-2-methylidene-4-oxobutanoic acid
(3b) ITA (2) (0.112 g, 1 mmol) was dissolved in acetone
(30 mL), and 3-amino acetophenone (0.135 g, 1 mmol)
was added in small portions under stirring at room tem-
perature over time span of 30 min. The mixture became
yellow slurry. Stirring was continued for 1.5 h, after which
the solution was filtered. The obtained solid was washed
with acetone and dried. The crude product was further
purified by recrystallization method, and single crystals
were grown from methanol by the slow evaporation
method. The product was obtained as a light yellow solid
with 80 % yield. m.p. 180–182 °C; IR (KBr): mmax (cm-1),
3282 (OH), 3055 (NH), 2900 (Ar–H), 2634 (aliphatic C-H)
1685 (amide C=O), 1591 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 2.55 (3H, s, –CH3), 3.35(2H, s, O=C-
CH2), 5.75 (1H, d, JAB = 1.6 Hz, HA), 6.18 (1H, d,
JAB = 1.6 Hz, HB), 7.43–8.17 (4H, m, Ar–H), 10.23 (1H,
s, NH), 12.55 (1H, s, OH). LCMS (m/z): 248 (M? ? 1).
Calcd. for C13H13NO4: C, 63.15; H, 5.30; N, 5.67; Found:
C, 63.13; H, 5.32; N, 5.65 %. XRD data: triclinic, P-1,
4-({4-[3-(4-Chlorophenyl)prop-2-enoyl]phenyl}amino)-2-
methylidene-4-oxobutanoic acid (4c) To a mixture of
4-[(4-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3a) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tempera-
ture for 3 h. The precipitate formed was collected by fil-
tration and recrystallized in ethanol. The product was
obtained as a yellow solid with 65 % yield. m.p. 159–16 °C.
IR (KBr): mmax (cm-1), 3345 (OH), 3105 (NH), 2972 (Ar–
H), 2761 (aliphatic C-H) 1684 (amide C=O), 1658 (acid
C=O), 1528 (C=C). 1H-NMR (400 MHz, DMSO-d6): d
ppm, 3.40 (s, 2H, O=C-CH2), 5.95 (1H, d, JAB = 1.6 Hz,
HA), 6.55 (1H, d, JAB = 1.6 Hz, HB), 7.25 (2H, dt,
J = 2.0 Hz, 5.2 Hz, Ar–He), 7.64 (1H, d, J = 16.4 Hz, Hc),
7.91–8.32 (7H, m, Ar–H), 10.34 (1H, s, NH),12.52 (1H, s,
OH). LCMS (m/z): 370 (M? ? 1). Calcd. for C20H16ClNO4:
C, 64.96; H, 4.36; N, 3.79; Found: C, 64.94; H, 4.38; N,
3.77 %.
˚
˚
a = 4.9485 (3) A, b = 5.3614 (6) A, c = 22.457 (2) A,
˚
3
V = 592.77(9) A , Z = 2.
˚
Synthesis of chalcones (4a–l)
4-({4-[3-(4-Fluorophenyl)prop-2-enoyl]phenyl}amino)-2-
methylidene-4-oxobutanoic acid (4a) To a mixture of
4-[(4-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3a) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
123

Med Chem Res (2015) 24:4191–4206
4199
2-Methylidene-4-({4-[3-(4-methylphenyl)prop-2-enoyl]
phenyl}amino)-4-oxobutanoic acid (4d) To a mixture
JAB = 1.6 Hz, HA), 6.52 (1H, d, JAB = 1.6 Hz, HB), 7.65
(1H, d, J = 16.4 Hz, Hc), 7.42–8.15 (9H, m, Ar–H), 10.36
(1H, s, NH),12.56 (1H, s, OH). LCMS (m/z): 378
(M? ? 1). Calcd. for C23H23NO4: C, 73.19; H, 6.14; N,
3.71; Found: C, 73.17; H, 6.16; N, 3.69 %.
of
4-[(4-acetylphenyl)amino]-2-methylidene-4-oxobu-
tanoic acid (3a) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 63 % yield. m.p.
168–170 °C. IR (KBr): mmax (cm-1), 3295 (OH), 3103
(NH), 2972 (Ar–H), 2760 (aliphatic C-H) 1683 (amide
C=O), 1654 (acid C=O), 1526 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 2.35 (3H, s, CH3), 3.43 (2H, s, O=C-
CH2), 5.98 (1H, d, JAB = 1.6 Hz, HA), 6.45 (1H, d,
JAB = 1.6 Hz, HB), 7.25 (2H, dt, J = 2.0 Hz, 5.2 Hz, Ar–
He), 7.64 (1H, d, J = 16.4 Hz, Hc), 7.93–8.30 (7H, m, Ar–
H), 10.35 (1H, s, NH),12.67 (1H, s, OH). LCMS (m/z): 350
(M? ? 1). Calcd. for C21H19NO4: C, 72.19; H, 5.48; N,
4.01; Found: C, 72.17; H, 5.50; N, 3.99 %.
4-({3-[3-(4-Fluorophenyl)prop-2-enoyl]phenyl}amino)-
2-methylidene-4-oxobutanoic acid (4g) To a mixture of
4-[(3-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3b) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 60 % yield. m.p.
161–163 °C. IR (KBr): mmax (cm-1), 3273 (OH), 3136
(NH), 3057 (Ar–H), 2924 (aliphatic C-H) 1685 (amide
C=O), 1656 (acid C=O), 1585 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 3.33 (2H, s, O=C-CH2), 5.75 (1H, d,
JAB = 1.6 Hz, HA), 6.17 (1H, d, JAB = 1.6 Hz, HB),
6.99–8.30 (m, 8H, Ar–H), 7.85 (1H, d, J = 16.4 Hz, Hd),
10.36 (1H, s, NH), 12.56 (1H, s, OH). LCMS (m/z): 354
(M? ? 1). Calcd. for C20H16FNO4: C, 67.98; H, 4.56; N,
3.96; Found: C, 67.96; H, 4.58; N, 3.94 %.
4-({4-[3-(4-Methoxyphenyl)prop-2-enoyl]phenyl}amino)-
2-methylidene-4-oxobutanoic acid (4e) To a mixture of
4-[(4-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3a) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 60 % yield. m.p.
195–197 °C. IR (KBr): mmax (cm-1), 3340 (OH), 3103
(NH), 2922 (Ar–H), 2706 (aliphatic C-H) 1678 (amide
C=O), 1662 (acid C=O), 1517 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 3.37 (2H, s, O=C-CH2), 3.36 (3H, s,
CH3), 5.76 (1H, d, JAB = 1.6 Hz, HA), 6.18 (1H, d,
JAB = 1.6 Hz, HB), 7.01 (1H, d, J = 15.6 Hz, Hc),
7.47–8.22 (9H, m, Ar–H), 10.25 (1H, s, NH), 12.72 (1H, s,
OH). LCMS (m/z): 366 (M? ? 1). Calcd. for C21H19NO5:
C, 69.03; H, 5.24; N, 3.83; Found: C, 69.01; H, 5.26; N,
3.81 %.
4-({3-[3-(4-Bromophenyl)prop-2-enoyl]phenyl}amino)-
2-methylidene-4-oxobutanoic acid (4h) To a mixture
of 4-[(3-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3b) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 65 % yield. m.p.
176–178 °C. IR (KBr): mmax (cm-1), 3274 (OH), 3135
(NH), 3058 (Ar–H), 2942 (aliphatic C-H) 1686 (amide
1
C=O), 1665 (acid C=O), 1587 (C=C). H-NMR (400 MHz,
DMSO-d6): d ppm, 3.31(2H, s, O=C-CH2), 5.72 (1H, d,
JAB = 1.6 Hz, HA), 6.15 (1H, d, JAB = 1.6 Hz, HB),
6.99–8.33 (8H, m, Ar–H), 7.84 (1H, d, J = 16.4 Hz, Hd),
10.35 (1H, s, NH), 12.55 (1H, s, OH). LCMS (m/z): 415
(M? ? 1). Calcd. for C20H16BrNO4: C, 57.99; H, 3.89; N,
3.38; Found: C, 57.97; H, 3.91; N, 3.36 %.
4-(4-(3-(4-Isopropylphenyl)acryloyl)phenylamino)-2-
methylene-4-oxobutanoic acid (4f) To a mixture of
4-[(4-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3a) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 69 % yield. m.p.
165–167 °C. IR (KBr): mmax (cm-1), 3295 (OH), 3104
(NH), 2972 (Ar–H), 2760 (aliphatic C-H) 1683 (amide
C=O), 1664 (acid C=O), 1516 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 1.29 [6H, d, JAB = 6.8 Hz, (CH3)2],
3.23 (2H, s, O=C-CH2), 3.34 (1H, m, CH) 5.95 (1H, d,
4-({3-[3-(4-Chlorophenyl)prop-2-enoyl]phenyl}amino)-2-
methylidene-4-oxobutanoic acid (4i) To a mixture of
4-[(3-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3b) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 63 % yield. m.p.
172–174 °C. IR (KBr): mmax (cm-1), 3277 (OH), 3130
123

4200
Med Chem Res (2015) 24:4191–4206
(NH), 3054 (Ar–H), 2945 (aliphatic C-H) 1685 (amide
1
C=O), 1655 (acid C=O), 1585 (C=C). H-NMR (400 MHz,
30 mL ethanol, 10 mL of 10 % sodium hydroxide solution
was added and stirred at room temperature for 3 h. The
precipitate formed was collected by filtration and recrys-
tallized in ethanol. The product was obtained as a yellow
solid with 52 % yield. m.p. 168–170 °C. IR (KBr): mmax
(cm-1), 3274 (OH), 3109 (NH), 2962 (Ar–H), 2853 (ali-
phatic C-H) 1686 (amide C=O), 1656 (acid C=O), 1584
DMSO-d6): d ppm, 3.33 (2H, s, O=C-CH2), 5.75 (1H, d,
JAB = 1.6 Hz, HA), 6.17 (1H, d, JAB = 1.6 Hz, HB),
6.89–8.30 (8H, m, Ar–H), 7.85 (1H, d, J = 16.4 Hz, Hd),
10.36 (1H, s, NH), 12.56 (1H, s, OH). LCMS (m/z): 370
(M? ? 1). Calcd. for C20H16ClNO4: C, 64.96; H, 4.36; N,
3.79; Found: C, 64.94; H, 4.38; N, 3.77 %.
1
(C=C). H-NMR (400 MHz, DMSO-d6): d ppm, 1.25 (6H,
d, JAB = 6.8 Hz), 3.33 (2H, s, O=C-CH2), 3.45 (1H, m,
CH), 5.75 (1H, d, JAB = 1.6 Hz, HA), 6.18 (1H, d,
JAB = 1.6 Hz, HB), 7.03–8.30 (8H, m, Ar–H), 7.85 (1H, d,
J = 16.4 Hz, Hd), 10.43 (s, 1H, NH), 11.56 (s, 1H, OH).
LCMS (m/z): 378 (M? ? 1). Calcd. for C23H23NO4: C,
73.19; H, 6.14; N, 3.71; Found: C, 73.17; H, 6.16; N,
3.69 %.
2-Methylidene-4-({3-[3-(4-methylphenyl)prop-2-enoyl]-
phenyl}amino)-4-oxobutanoic acid (4j) To a mixture of
4-[(3-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3b) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 48 % yield. m.p.
170–172 °C. IR (KBr): mmax (cm-1), 3272 (OH), 3129
(NH), 3045 (Ar–H), 2954 (aliphatic C-H) 1688 (amide
C=O), 1665 (acid C=O), 1586 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 2.35 (3H, s, CH3), 3.25 (2H, s, O=C-
CH2), 5.76 (1H, d, JAB = 1.6 Hz, HA), 6.19 (1H, d,
JAB = 1.6 Hz, HB), 6.39–8.30 (8H, m, Ar–H), 7.88 (1H, d,
J = 16.4 Hz, Hd), 10.36 (1H, s, NH), 12.56 (1H, s, OH).
LCMS (m/z): 350 (M? ? 1). Calcd. for C21H19NO4: C,
72.19; H, 5.48; N, 4.01; Found: C, 72.17; H, 5.50; N,
3.99 %.
Synthesis of bis-pyrazole derivatives (5a–l)
N-(4-(5-(4-flurophenyl)-1-phenyl-1H-pyrazol-3-yl)phenyl-
2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide (5a)
mixture of 4-({4-[3-(4-fluorophenyl)prop-2-enoyl]pheny-
l}amino)-2-methylidene-4-oxobutanoic acid (4a)
A
(0.01 mol) and excess of phenyl hydrazine (0.023 mol) in
ethanol (20 mL) with the 2–3 drops of HCl were refluxed
for 48 h. The reaction mixture was cooled and poured into
ice-cold water (50 mL). The precipitate was collected by
filtration and purified by recrystallization from ethanol. The
product was obtained as a yellowish brown solid with 42 %
yield. m.p. 112–114 °C. IR (KBr): mmax (cm-1), 3296
(OH), 3062 (NH), 2924 (Ar–H), 2854 (aliphatic C-H) 1670
4-({3-[3-(4-Methoxyphenyl)prop-2-enoyl]phenyl}amino)-
2-methylidene-4-oxobutanoic acid (4k) To a mixture of
4-[(3-acetylphenyl)amino]-2-methylidene-4-oxobutanoic
acid (3b) (0.1 mol) and 4-substituted benzaldehyde
(0.1 mol) in 30 mL ethanol, 10 mL of 10 % sodium
hydroxide solution was added and stirred at room tem-
perature for 3 h. The precipitate formed was collected by
filtration and recrystallized in ethanol. The product was
obtained as a yellow solid with 55 % yield. m.p.
158–160 °C. IR (KBr): mmax (cm-1), 3275 (OH), 3059
(NH), 2926 (Ar–H), 2837 (aliphatic C-H) 1685 (amide
C=O), 1656 (acid C=O), 1585 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 3.44 (2H, s, O=C-CH2), 3.86 (3H, s,
CH3), 5.57 (1H, d, JAB = 1.6 Hz, HA), 6.50 (1H, d,
JAB = 1.6 Hz, HB), 6.95 (2H, dt, J = 2.0 Hz, 5.2 Hz, Ar–
He), 7.48 (1H, d, J = 15.6 Hz, Hc), 7.79 (1H, d,
J = 15.6 Hz, Hd), 6.95–7.96 (6H, m, Ar–H), 10.50 (1H, s,
NH), 11.55 (1H, s, OH). LCMS (m/z): 366 (M? ? 1).
Calcd. for C21H19NO5: C, 69.03; H, 5.24; N, 3.83; Found:
C, 69.01; H, 5.26; N, 3.81 %.
1
(amide C=O), 1597 (C=C). H-NMR (400 MHz, DMSO-
d6): d ppm, 3.34 (2H, s, O=C-CH2), 5.43 (1H, s, OH), 6.58
(1H, s, pyrazole A H-4), 6.97–8.05 (20H, m, Ar–H); 8.15
(1H, s, pyrazole B H-5), 10.10 (1H, s, NH). 13C-NMR
(100 MHz, DMSO-d6): d ppm,169.76 (amide C=O),
157.64 (pyrazole A C-3), 149.56 (pyrazole A C-5), 147.45
(pyrazole B C-3), 143.23, 140.12, 139.76, 134.66, 132.55,
130.43, 129.54, 128.31, 127.54, 126.43, 125.66, 119.67,
118.33, 114.58, 113.76, 112.25 (aromatic C’s), 122.67
(pyrazole B C-5), 119.43 (pyrazole B C-4), 104.49 (pyra-
zole A C-4), 23.65 (O=C-CH2). LCMS (m/z): 530
(M? ? 1). Calcd. for C32H24FN5O2: C, 72.58; H, 4.57; N,
13.22; Found: C, 72.56; H, 4.59; N, 13.20 %.
N-(4-(5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)phe-
nyl-2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide
(5b)
A mixture of 4-({4-[3-(4-bromophenyl)prop-2-
enoyl]phenyl}amino)-2-methylidene-4-oxobutanoic acid
(4b) (0.01 mol) and excess of phenyl hydrazine
(0.023 mol) in ethanol (20 mL) with the 2–3 drops of HCl
were refluxed for 48 h. The reaction mixture was cooled
and poured into ice-cold water (50 mL). The precipitate
was collected by filtration and purified by recrystallization
4-(3-(3-(4-Isopropylphenyl)acryloyl)phenylamino)-2-
methylene-4-oxobutanoic acid (4l) To a mixture of
4-[(3-acetylphenyl)amino]-2-methylidene-4-oxobutanoic acid
(3b) (0.1 mol) and 4-substituted benzaldehyde (0.1 mol) in
123

Med Chem Res (2015) 24:4191–4206
4201
from ethanol. The product was obtained as a brown solid
with 46 % yield. m.p. 132–134 °C. IR (KBr): mmax (cm-1),
3269 (OH), 3026 (NH), 2942 (Ar–H), 2845 (aliphatic C-H)
1674 (amide C=O), 1596 (C=C). 1H-NMR (400 MHz,
DMSO-d6): d ppm, 3.35 (2H, s, O=C-CH2), 5.38 (1H, s,
OH), 6.65 (1H, s, pyrazole A H-4), 6.96–8.06 (20H, m, Ar–
H); 8.14 (1H, s, pyrazole B H-5), 10.16 (1H, s, NH). 13C-
NMR (100 MHz, DMSO-d6): d ppm, 168.79 (amide C=O),
157.74 (pyrazole A C-3), 149.88 (pyrazole A C-5), 147.57
(pyrazole B C-3), 143.94, 140.43, 139.82, 134.45, 132.56,
130.23, 129.17, 128.10, 127.50, 126.32, 125.65, 119.73,
118.73, 114.85, 113.36, 112.51 (aromatic C’s), 122.76
(pyrazole B C-5), 119.24 (pyrazole B C-4), 104.93 (pyra-
zole A C-4), 23.54 (O=C-CH2). LCMS (m/z): 591
(M? ? 1). Calcd. for C32H24BrN5O2: C, 65.09; H, 4.10; N,
11.86; Found: C, 65.07; H, 4.12; N, 11.84 %.
(NH), 2929 (Ar–H), 2837 (aliphatic C-H) 1670 (amide
C=O), 1595 (C=C), 1H-NMR (400 MHz, DMSO-d6): d ppm,
1.99 (3H, s, Ar-CH3), 3.51 (2H, s, O=C-CH2), 6.11 (1H, s,
OH), 7.00–8.14 (20H, m, Ar–H); 10.70 (1H, s, NH). 13C-
NMR (100 MHz, DMSO-d6): d ppm, 168.78 (amide C=O),
159.74 (pyrazole A C-3), 149.86 (pyrazole A C-5), 147.54
(pyrazole B C-3), 144.94, 140.46, 139.82, 134.95, 132.56,
130.27, 129.27, 128.15, 127.55, 126.22, 125.67, 119.83,
118.75, 114.85, 113.36, 112.51 (aromatic C’s), 122.76
(pyrazole B C-5), 119.24 (pyrazole B C-4), 104.93 (pyrazole
A 4C), 27.45 (CH3) 24.56 (O=C-CH2). LCMS (m/z): 526
(M? ? 1). Calcd. for C33H27N5O2: C, 75.41; H, 5.18; N,
13.32; Found: C, 75.39; H, 5.20; N, 13.30 %.
2-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-N-(4-(5-(4-meth-
oxyphenyl)-1-phenyl-1H-pyrazol-3-yl) phenyl)acetamide
(5e) A mixture of 4-({4-[3-(4-methoxyphenyl)prop-2-
enoyl]phenyl}amino)-2-methylidene-4-oxobutanoic acid
(4e) (0.01 mol) and excess of phenyl hydrazine
(0.023 mol) in ethanol (20 mL) with the 2–3 drops of HCl
were refluxed for 48 h. The reaction mixture was cooled
and poured into ice-cold water (50 mL). The precipitate
was collected by filtration and purified by recrystallization
from ethanol. The product was obtained as a yellowish
brown solid with 45 % yield. m.p. 102–104 °C. IR (KBr):
mmax (cm-1), 3294 (OH), 3035 (NH), 2929(Ar–H), 2835
N-(4-(5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)phenyl-
2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide (5c)
A
mixture of 4-({4-[3-(4-chlorophenyl)prop-2-enoyl]pheny-
l}amino)-2-methylidene-4-oxobutanoic acid (4c) (0.01 mol)
and excess of phenyl hydrazine (0.023 mol) in ethanol
(20 mL) with the 2–3 drops of HCl were refluxed for 48 h.
The reaction mixture was cooled and poured into ice-cold
water (50 mL). The precipitate was collected by filtration
and purified by recrystallization from ethanol. The product
was obtained as a yellowish brown solid with 47 % yield.
m.p. 136–138 °C. IR (KBr): mmax (cm-1), 3279 (OH), 3046
(NH), 2943 (Ar–H), 2844 (aliphatic C-H) 1672 (amide
C=O), 1597 (C=C). 1H-NMR (400 MHz, DMSO-d6): d
ppm, 3.35 (2H, s, O=C-CH2), 5.45 (1H, s, OH), 6.56 (1H, s,
pyrazole A H-4), 6.96–8.15 (20H, m, Ar–H); 8.25 (1H, s,
pyrazole B H-5), 10.21 (1H, s, NH). 13C-NMR (100 MHz,
DMSO-d6): d ppm, 168.79 (amide C=O), 157.74 (pyrazole
A C-3), 149.88 (pyrazole A C-5), 147.57 (pyrazole B C-3),
143.94, 140.43, 139.82, 134.45, 132.56, 130.23, 129.17,
128.10, 127.50, 126.32, 125.65, 119.73, 118.73, 114.85,
113.36, 112.51 (aromatic C’s), 122.76 (pyrazole B 5C),
119.34 (pyrazole B C-4), 105.93 (pyrazole A C-4), 24.54
(O=C-CH2). LCMS (m/z): 547 (M? ? 1). Calcd. for C32-
H24ClN5O2: C, 70.39; H, 4.43; N, 12.83; Found: C, 70.37;
H, 4.45; N, 12.81 %.
1
(aliphatic C-H) 1672 (amide C=O), 1597 (C=C), H-NMR
(400 MHz, DMSO-d6): d ppm, d 3.33 (2H, s, O=C-CH2),
3.76 (3H, s, O-CH3), 5.34 (1H, s, OH), 6.57 (1H, s, pyra-
zole A H-4), 6.68–7.75 (20H, m, Ar–H); 7.82 (1H, s,
pyrazole B H-5), 10.09 (1H, s, NH). 13C-NMR (100 MHz,
DMSO-d6): d ppm, 168.78 (amide C=O), 164.07 (C-O-
CH3), 159.74 (pyrazole A C-3), 149.86 (pyrazole A C-5),
147.54 (pyrazole B C-3), 144.90, 140.41, 139.82, 134.98,
130.27, 129.27, 128.15, 127.55, 126.22, 125.67, 119.85,
118.77, 114.80, 113.37, 112.54 (aromatic C’s), 122.78
(pyrazole B C-5), 119.25 (pyrazole B C-4), 104.95 (pyra-
zole A C-4), 55.65 (O-CH3), 24.55 (O=C-CH2). LCMS (m/
z): 542 (M? ? 1). Calcd. for C33H27N5O3: C, 73.18; H,
5.02; N, 12.93; Found: C, 73.16; H, 5.04; N, 12.91 %.
2-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-N-(4-(5-(4-isopropyl-
phenyl)-1-phenyl-1H-pyrazol-3-yl) phenyl)acetamide (5f)
A
2-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-N-(4-(1-phenyl-
mixture of 4-(4-(3-(4-isopropylphenyl)acryloyl)pheny-
lamino)-2-methylene-4-oxobutanoic acid (4f) (0.01 mol)
and excess of phenyl hydrazine (0.023 mol) in ethanol
(20 mL) with the 2–3 drops of HCl were refluxed for 48 h.
The reaction mixture was cooled and poured into ice-cold
water (50 mL). The precipitate was collected by filtration
and purified by recrystallization from ethanol. The pro-
duct was obtained as a yellowish brown solid with 48 %
yield. m.p. 114–116 °C. IR (KBr): mmax (cm-1), 3290
(OH), 3065 (NH), 2927(Ar–H), 2835 (aliphatic C-H) 1671
5-p-tolyl-1H-pyrazol-3-yl)phenyl) acetamide (5d)
A
mixture of 2-methylidene-4-({4-[3-(4-methylphenyl)prop-
2-enoyl]phenyl}amino)-4-oxobutanoic acid (4d) (0.01 mol)
and excess of phenyl hydrazine (0.023 mol) in ethanol
(20 mL) with the 2–3 drops of HCl were refluxed for 48 h.
The reaction mixture was cooled and poured into ice-cold
water (50 mL). The precipitate was collected by filtration
and purified by recrystallization from ethanol. The product
was obtained as a yellowish brown solid with 43 % yield.
m.p. 108–110 °C. IR (KBr): mmax (cm-1), 3415 (OH), 3081
1
(amide C=O), 1596 (C=C). H-NMR (400 MHz, DMSO-
123

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Med Chem Res (2015) 24:4191–4206
d6): d ppm, 1.35 (6H, d, JAB = 6.8 Hz, (CH3)2), 3.36 (2H,
s, O=C-CH2), 3.48 (1H, m, CH), 5.47 (1H, s, OH), 6.56
(1H, s, pyrazole A H-4), 6.89–8.15 (20H, m, Ar–H); 8.25
(1H, s, pyrazole B H-5), 10.19 (1H, s, NH). 13C-NMR
(100 MHz, DMSO-d6): d ppm, 168.75 (amide C=O),
159.74 (pyrazole A C-3), 149.88 (pyrazole A C-5), 147.56
(pyrazole B C-3), 144.84, 140.41, 139.72, 134.94, 132.56,
130.21, 129.26, 128.17, 127.55, 126.24, 125.65, 119.82,
118.75, 114.83, 113.35, 112.51 (aromatic C’s), 122.77
(pyrazole B C-5), 119.23 (pyrazole B C-4), 104.95
(pyrazole A C-4), 36.30 (CH), 27.45 ((CH3)2), 24.56
(O=C-CH2). LCMS (m/z): 554 (M? ? 1). Calcd. for
C35H31N5O2: C, 75.93; H, 5.64; N, 12.65; Found: C,
75.91; H, 5.66; N, 12.63 %.
DMSO-d6): d ppm, 3.34 (2H, s, O=C-CH2), 5.54 (1H, s, –
OH), 6.72 (1H, s, pyrazole A H-4), 6.73–8.05 (20H, m, Ar–
H); 8.15 (1H, s, pyrazole B H-5), 10.09 (1H, s, NH). 13C-
NMR (100 MHz, DMSO-d6): d ppm, 168.85 (amide C=O),
151.47 (pyrazole A C-3), 147.67 (pyrazole A C-5), 144.48
(pyrazole B C-3), 143.44, 142.26, 140.25, 140.06, 139.91,
133.39, 132.38, 129.83, 128.67, 125.80, 122.80, 122.43,
121.23, 120.46, 119.98, 119.29, 116.16, 113.42 (aromatic
C’s), 122.10 (pyrazole B C-5), 120.93 (pyrazole B C-4),
105.93 (pyrazole A C-4), 24.51 (O=C-CH2). LCMS (m/z):
591 (M? ? 1). Calcd. for C32H24BrN5O2: Calcd. for C32-
H24BrN5O2: C, 65.09; H, 4.10; N, 11.86; Found: C, 65.07;
H, 4.12; N, 11.84 %.
N-(3-(5-(4-chlorophenyl)-1-phenyl-1H-pyrazol-3-yl)phe-
nyl-2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide
N-(3-(5-(4-fluorophenyl)-1-phenyl-1H-pyrazol-3-yl)phenyl-
2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide (5g)
A
(5i)
A
mixture of 4-({3-[3-(4-cholrophenyl)prop-2-
mixture of 4-({3-[3-(4-fluorophenyl)prop-2-enoyl]phenyl}
amino)-2-methylidene-4-oxobutanoic acid (4g) (0.01 mol)
and excess of phenyl hydrazine (0.023 mol) in ethanol
(20 mL)withthe2–3dropsofHClwererefluxedfor48 h.The
reaction mixture was cooled and poured into ice-cold water
(50 mL). The precipitate was collected by filtration and
purified by recrystallization from ethanol. The product was
obtained as a yellowish brown solid with 38 % yield. m.p.
102–104 °C; IR (KBr): mmax (cm-1), 3375 (OH), 3057 (NH),
2987 (Ar–H), 2942 (aliphatic C-H) 1706 (amide C=O), 1596
(C=C). 1H-NMR (400 MHz, DMSO-d6): d ppm, 3.32 (2H, s,
O=C-CH2), 5.59 (1H, s, –OH), 6.78 (1H, s, pyrazole A H-4),
7.03–8.15 (20H, m, Ar–H); 8.25 (1H, s, pyrazole B H-5),
10.19 (1H, s, NH). 13C-NMR (100 MHz, DMSO-d6): d ppm,
168.58 (amide C=O), 151.74 (pyrazole A 3C), 147.76 (pyra-
zole A C-5), 144.84 (pyrazole B C-3), 143.44, 142.62, 140.52,
140.36, 139.97, 133.89, 132.68, 129.73, 128.77, 125.80,
123.80, 122.13, 121.23, 120.46, 119.98, 119.92, 116.61,
113.24 (aromatic C’s), 122.90 (pyrazole B C-5), 120.93
(pyrazoleBC-4), 105.93(pyrazoleA C-4), 24.51(O=C-CH2).
LCMS (m/z): 530 (M? ? 1). Calcd. for C32H24FN5O2: C,
72.58; H, 4.57; N, 13.22; Found: C, 72.56; H, 4.59; N,
13.20 %.
enoyl]phenyl}amino)-2-methylidene-4-oxobutanoic acid
(4i) (0.01 mol) and excess of phenyl hydrazine (0.023 mol)
in ethanol (20 mL) with the 2–3 drops of HCl were
refluxed for 48 h. The reaction mixture was cooled and
poured into ice-cold water (50 mL). The precipitate was
collected by filtration and purified by recrystallization from
ethanol. The product was obtained as a yellowish brown
solid with 43 % yield. m.p. 130–132 °C. IR (KBr): mmax
(cm-1), 3376 (OH), 3055 (NH), 2977(Ar–H), 2925 (ali-
phatic C-H) 1709 (amide C=O), 1595 (C=C). 1H-NMR
(400 MHz, DMSO-d6): d ppm, 3.36 (2H, s, O=C-CH2),
5.45 (1H, s, -OH), 6.76 (1H, s, pyrazole A H-4), 7.07–8.15
(20H, m, Ar–H); 8.23 (1H, s, pyrazole B H-5), 10.14 (1H,
s, NH). 13C-NMR (100 MHz, DMSO-d6): d ppm, 168.78
(amide C=O), 158.69 (pyrazole A C-3), 151.42 (pyrazole A
C-5), 144.54 (pyrazole B C-3), 142.15, 140.63, 140.54,
134.28, 133.56, 133.16, 130.30, 129.32, 128.17,
127.54,125.67, 123.87, 121.16, 119.22, 116.37, 114.98,
114.57, 113.58 (aromatic C’s), 122.64 (pyrazole B C-5),
120.60 (pyrazole B C-4), 105.15 (pyrazole A C-4), 24.36
(O=C-CH2). LCMS (m/z): 547 (M? ? 1). Calcd. for C32-
H24ClN5O2: C, 70.39; H, 4.43; N, 12.83; Found: C, 70.37;
H, 4.45; N, 12.81 %.
N-(3-(5-(4-bromophenyl)-1-phenyl-1H-pyrazol-3-yl)phe-
nyl-2-(3-hydroxy-1-phenyl-1H-pyrazol-4-yl)acetamide
2-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-N-(3-(1-phenyl-
5-p-tolyl-1H-pyrazol-3-yl)phenyl) acetamide (5j)
A
(5h)
A
mixture of 4-({3-[3-(4-bromophenyl)prop-2-
mixture of 2-methylidene-4-({3-[3-(4-methylphenyl)prop-
2-enoyl]phenyl}amino)-4-oxobutanoic acid (4j) (0.01 mol)
and excess of phenyl hydrazine (0.023 mol) in ethanol
(20 mL) with the 2–3 drops of HCl were refluxed for 48 h.
The reaction mixture was cooled and poured into ice-cold
water (50 mL). The precipitate was collected by filtration
and purified by recrystallization from ethanol. The product
was obtained as a yellowish brown solid with 33 % yield.
m.p. 116–118 °C; IR (KBr): mmax (cm-1), 3367 (OH), 3065
(NH), 2976(Ar–H), 2926 (aliphatic C-H) 1701 (amide
C=O), 1596 (C=C); 1H-NMR (400 MHz, DMSO-d6): d
enoyl]phenyl}amino)-2-methylidene-4-oxobutanoic acid
(4h) (0.01 mol) and excess of phenyl hydrazine
(0.023 mol) in ethanol (20 mL) with the 2–3 drops of HCl
were refluxed for 48 h. The reaction mixture was cooled
and poured into ice-cold water (50 mL). The precipitate
was collected by filtration and purified by recrystallization
from ethanol. The product was obtained as a brown solid
with 46 % yield. m.p. 112–114 °C. IR (KBr): mmax (cm-1),
3373 (OH), 3057 (NH), 2978(Ar–H), 2924 (aliphatic C-H)
1708 (amide C=O), 1595 (C=C); 1H-NMR (400 MHz,
123

Med Chem Res (2015) 24:4191–4206
4203
1
ppm, 1.89 (3H, s, Ar-CH3), 3.34 (2H, s, O=C-CH2), 5.54
(1H, s, OH), 6.72 (1H, s, pyrazole A H-4), 6.73–8.05 (20H,
m, Ar–H); 8.20 (1H, s, pyrazole B C-5), 10.09 (1H, s, NH).
13C-NMR (100 MHz, DMSO-d6): d ppm, 168.87 (amide
C=O), 158.59 (pyrazole A C-3), 151.24 (pyrazole A C-5),
144.45 (pyrazole B C-3), 141.95, 140.65, 140.50, 134.28,
133.56, 133.16, 130.30, 129.32, 128.17, 127.54, 125.67,
123.87, 121.15, 119.12, 116.31, 114.94, 114.54, 113.54
(aromatic C’s), 122.65 (pyrazole B C-5), 120.64 (pyrazole
B C-4), 105.18 (pyrazole A C-4), 55.65 (O-CH3), 27.52
(CH3), 24.35 (O=C-CH2). LCMS (m/z): 526 (M? ? 1).
Calcd. for C33H27N5O2: C, 75.41; H, 5.18; N, 13.32;
Found: C, 75.39; H, 5.20; N, 13.30 %.
(aliphatic C-H) 1679 (amide C=O), 1595 (C=C). H-NMR
(400 MHz, DMSO-d6): d ppm, 1.36 (6H, d, JAB = 6.8 Hz,
(CH3)2), 3.34 (2H, s, O=C-CH2), 3.48 (1H, m, CH), 5.54
(1H, s, OH), 6.72 (1H, s, pyrazole A H-4), 6.73–8.05 (20H,
m, Ar–H); 8.15 (1H, s, pyrazole B H-5), 10.09 (1H, s, NH).
13C-NMR (100 MHz, DMSO-d6): d ppm, 168.87 (amide
C=O), 158.59 (pyrazole A C-3), 151.24 (pyrazole A C-5),
144.45 (pyrazole B C-3), 141.95, 140.56, 140.05, 134.82,
133.65, 133.26, 130.33, 129.23, 128.71, 127.45, 125.77,
124.56, 121.51, 119.21, 116.31, 114.94, 114.45, 113.54
(aromatic C’s), 122.56 (pyrazole B C-5), 120.62 (pyrazole
B C-4), 105.16 (pyrazole A C-4), 55.56 (O-CH3), 36.34
(CH), 27.54 ((CH3)2), 24.53 (O=C-CH2). LCMS (m/z): 554
(M? ? 1). Calcd. for C35H31N5O2: C, 75.93; H, 5.64; N,
12.65; Found: C, 75.91; H, 5.66; N, 12.63 %.
2-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-N-(3-(5-(4-meth-
oxyphenyl)-1-phenyl-1H-pyrazol-3-yl) phenyl)acetamide
(5k) A mixture of 4-({3-[3-(4-methoxyphenyl)prop-2-
enoyl]phenyl}amino)-2-methylidene-4-oxobutanoic acid
(4k) (0.01 mol) and excess of phenyl hydrazine
(0.023 mol) in ethanol (20 mL) with the 2–3 drops of HCl
were refluxed for 48 h. The reaction mixture was cooled
and poured into ice-cold water (50 mL). The precipitate
was collected by filtration and purified by recrystallization
from ethanol. The product was obtained as a yellowish
brown solid with 39 % yield. m.p. 106–108 °C. IR (KBr):
mmax (cm-1), 3332 (OH), 3059 (NH), 2926 (Ar–H), 2854
Pharmacology
Analgesic activity (acetic acid-induced writhing method)
The male albino mice weighing between 20 and 25 g were
selected and divided into 10 groups of six animals each
(Vogel and Vogel, 2002a, b). All the animals received
0.1 mL acetic acid 0.6 % v/v. i.p., and the first group
served as the control. The second group served as positive
control and received aspirin. The third–tenth groups were
administrated test compounds 4(a–d) and 5(a–d) at a dose
of 25 mg kg-1 bodyweight (suspended in 0.5 % CMC
given p.o.) 30 min prior to the administration of acetic acid
injection. The writhing effect was indicated by the
stretching of the abdomen with simultaneous stretching of
at least one hind limb. This was observed for 30 min and
the change in the number of writhings in the test group
compared to the standard treated and control treated
groups.
1
(aliphatic C-H) 1670 (amide C=O), 1597 (C=C). H-NMR
(400 MHz, DMSO-d6): d ppm, 3.43 (2H, s, O=C-CH2),
3.76 (3H, s, –O–CH3), 5.41 (1H, s, OH), 6.68 (1H, s,
pyrazole A H-4), 6.78–7.92 (20H, m, Ar–H); 7.82 (1H, s,
pyrazole B H-5), 10.10 (1H, s, NH). 13C-NMR (100 MHz,
DMSO-d6): d ppm, 168.78 (amide C=O), 165.07 (C-O-
CH3), 158.95 (pyrazole A C-3), 151.22 (pyrazole A C-5),
144.49 (pyrazole B C-3), 140.95, 140.36, 140.05, 134.80,
133.62, 133.24, 130.30, 129.32, 128.17, 127.54, 125.77,
121.15, 119.12, 116.13, 114.94, 114.54, 113.45 (aromatic
C’s), 122.65 (pyrazole B C-5), 120.82 (pyrazole B C-4),
105.13 (pyrazole A C-4), 55.65 (O-CH3), 24.50 (O=C-
CH2). LCMS (m/z): 542 (M? ? 1). Calcd. for
C33H27N5O3: C, 73.18; H, 5.02; N, 12.93; Found: C, 73.16;
H, 5.04; N, 12.91 %.
Anti-inflammatory activity (Carrageenan-induced rat paw
edema model)
In carrageenan-induced rat paw edema method (Winter
et al., 1962), 1 % carrageenan was used in a dose of
0.1 mL as a phlogistic agent (irritant), injected subcuta-
neously into the plantar aspect of the left hind paw of the
rats. A line was drawn at the tibio–tarsal junction, so that
every time the paw was dipped in the mercury column up
to the fixed mark to ensure constant paw volume. The paw
volume was measured immediately after the carrageenan
injection, which served as the initial volume, both in con-
trol and drug-treated animals. The paw volume was mea-
sured every 30-min interval up to 3 h. The second group
was treated with indomethacin 10 mg/kg bodyweight
(p.o.). The third–tenth groups were administered test
2-(3-Hydroxy-1-phenyl-1H-pyrazol-4-yl)-N-(3-(5-(4-iso-
propylphenyl)-1-phenyl-1H-pyrazol-3-yl)
etamide (5l) A mixture of 4-(3-(3-(4-isopropylphenyl)
acryloyl)phenylamino)-2-methylene-4-oxobutanoic acid
phenyl)-ac-
(4l) (0.01 mol) and excess of phenyl hydrazine (0.023 mol)
in ethanol (20 mL) with the 2–3 drops of HCl were
refluxed for 48 h. The reaction mixture was cooled and
poured into ice-cold water (50 mL). The precipitate was
collected by filtration and purified by recrystallization from
ethanol. The product was obtained as a yellowish brown
solid with 35 % yield. m.p. 108–110 °C. IR (KBr): mmax
(cm-1), 3330 (OH), 3059 (NH), 2925 (Ar–H), 2855
123

4204
Med Chem Res (2015) 24:4191–4206
compounds 5(a–l) at a dose 10 mg kg-1 bodyweight
(suspended in 0.5 % CMC given p.o.). After 30 min of the
drug administration, the rats were treated with 0.1 mL of
1 % carrageenan by subcutaneous route to the right hind
paw. Immediately after the injection, the paw volumes
were measured in a mercury plethysmograph. Thereafter,
the paw volume was measured at 60, 120 and 180 min. The
amount of edema in the drug-treated groups was compared
in relation to the control group with the corresponding time
intervals. The results were expressed as % inhibition of
edema over the untreated control group.
procured from National Chemical Laboratory, Pune, India.
The bacterial strains were maintained on nutrient agar
slants.
Antimicrobial assay was carried out by the disk diffu-
sion method (Vardar-Unlu et al., 2003) For in vitro
antimicrobial activity, 200 lL of overnight grown culture
of each bacteria was dispensed into 20 mL sterile nutrient
broth and incubated for 4–5 h at 37 °C to standardize the
culture to 10-5 CFU/mL. For this, 0.1 mL (10-5 CFU/mL)
of 24-h-old bacterial culture was placed on Muller–Hinton
agar medium and spread throughout the plate by spread
plate technique. Synthesized compounds (10 mg) were
dissolved in 1 mL of DMSO (dimethyl sulphoxide), and 25
lL of respective samples was added to the sterile disks
(6 mm diameter) purchased from HIMEDIA laboratories
individually and aseptically transferred to the inoculated
petri plates and incubated for 24 h. Antimicrobial activity
was recorded by measuring the diameter of zone of inhi-
bition. Streptomycin (HIMEDIA) was used as positive
standard against bacterial strains.
Acute toxicity study
Acute toxicity study of selected compounds was deter-
mined in Wistar albino rats (150–200 g) according to
OECD guidelines No. 425. The animals fasted overnight,
and the selected compounds were administered orally with
a starting dose of 2000 mg/kg to different animals. Ani-
mals were observed continuously for first 3 h and moni-
tored for 14 days for mortality and general behavior of
animals, signs of discomfort and nervous manifestations.
Minimum inhibitory concentration (MIC)
Molecular docking study
MIC of the DMSO extracts was determined by using dif-
ferent concentrations of extracts in Mueller–Hinton broth
for bacteria and potato dextrose broth for fungi by macro-
dilution method (National Committee for Clinical Labo-
ratory Standards, 2000; Akinyemi et al., 2005). The lowest
concentration of the DMSO extract inhibiting the visible
growth of microorganisms was considered as MIC.
Automated docking was used to determine the orientation
of inhibitors bound at the active site of COX-1 (PDB ID:
1CQE) and COX-2 (PDB ID: 1CX2). A Lamarckian
genetic algorithm method implemented in the AutoDock
4.2 was employed to identify appropriate binding modes
and conformations of the ligand. For docking calculations,
Gasteiger–Marsili partial charges (Gasteiger and Marsili,
1980) were assigned to the ligands, and nonpolar hydrogen
was merged. All torsions were allowed to rotate during
docking. The grid maps were generated with Autogrid
using grid values X = 28.313, Y = 9.701, Z = 187.515 for
COX-1 and X = 31.564, Y = -0.979, Z = -8.786 for
COX-2, respectively. The Lamarckian genetic algorithm
and the pseudo-Solis and Wets methods were applied for
energy minimization using default parameters. Binding
free energy was calculated using the following equation
(Sanner et al., 1996).
Freshly prepared nutrient broth was used as diluents.
The 24-h-old culture of test bacteria S. aureus and E. coli
was diluted 100-fold in nutrient broth (NB) (100 lL bac-
terial cultures in 10 mL NB). The stock solution of syn-
thesized compounds was prepared in DMSO by dissolving
10 mg of compound in 1 mL of DMSO. Increasing con-
centrations of test samples (1.25, 2.5, 5, 10, 20, 40 lL of
stock solution contains 12.5, 25, 50, 100, 200, 400 lg of
the compounds) were added to test tubes containing the
bacterial cultures. All tubes were incubated at 37 °C for
24 h for bacteria. Tubes were examined for visible tur-
bidity and using NB as control. Control without test sam-
ples and with solvent was assayed simultaneously. The
lowest concentration that inhibited visible growth of the
tested organisms was recorded as MIC.
Binding Energy ¼ ½ð1Þ þ ð2Þ þ ð3Þ þ ð4ފ
where (1)—final intermolecular energy, (2)—final total
internal energy, (3)—torsional free energy, (4)—unbound
system’s energy.
Conclusions
Antimicrobial activity (disk diffusion method)
A new series of N-[4-(5-(4-substituted-phenyl)-1-phenyl-
1H-pyrazol-3-yl)phenyl-2-(3-hydroxy-1-phenyl-1H-pyra-
zol-4-yl)] acetamides 5(a–i) were synthesized by Michael
addition reaction of hydrazine to chalcones 4(a–i). All
Microbial isolates The microorganisms used for the
microbial sensitivity assay were gram-positive S. aureus
(NCIM 2079) and gram-negative E. coli (NCIM 2931)
123

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and evaluated for in vivo analgesic, anti-inflammatory
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halogens 5a, 5b and 5c possess better activity in com-
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through BSR one-time grant for the purchase of chemicals. P.S.N.
thanks Manipal Institute of Technology for NMR data and DST-
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