Synthesis and biological evaluation of a novel series of pyrazole chalcones as anti-inflammatory, antioxidant and antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2009.10.035

A novel series of 1-(2,4-dimethoxy-phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-propenone (3) have been prepared by the Claisen-Schmidt condensation of 1-(2,4-dimethoxy-phenyl)-ethanone (1) and substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2). Substit

Full text of DOI:10.1016/j.bmc.2009.10.035

Bioorganic & Medicinal Chemistry 17 (2009) 8168–8173
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a novel series of pyrazole chalcones
as anti-inflammatory, antioxidant and antimicrobial agents
b
c
d
Babasaheb P. Bandgar a,b, , Shrikant S. Gawande , Ragini G. Bodade , Nalini M. Gawande ,
*
Chandrahasya N. Khobragade c
a Organic Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur 413 255, India
b Organic Chemistry Research Laboratory, School of Chemical Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431 606, India
c Biochemistry Research Laboratory, School of Life Sciences, Swami Ramanand Teerth Marathawada University, Nanded 431 606, India
d Bilcare Clinical Research Academy, Sai Capital, Senapati Bapat Road, Pune 411 016, 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 1-(2,4-dimethoxy-phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-propenone (3) have been
prepared by the Claisen–Schmidt condensation of 1-(2,4-dimethoxy-phenyl)-ethanone (1) and substi-
tuted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2). Substituted 1,3-diphenyl-1H-pyrazole-4-carbalde-
hydes (2) were prepared by Vilsmeir–Haack reaction on acetophenonephenylhydrazones to offer the
target compounds. The structures of the compounds were established by IR, 1H NMR and mass spectral
Received 9 October 2009
Revised 16 October 2009
Accepted 17 October 2009
Available online 22 October 2009
analysis. All the compounds were evaluated for their anti-inflammatory (TNF-
assays), antioxidant (DPPH free radical scavenging assay) and antimicrobial activities (agar diffusion
method) against some pathogenic bacteria and fungi. Of 10 compounds screened, compounds 3a, 3c
a and IL-6 inhibitory
Keywords:
Pyrazole chalcones
Anti-inflammatory activity
Antioxidant activity
Antimicrobial activity
and 3g exhibited promising IL-6 inhibitory (35–70% inhibition, 10
lM), free radical scavenging (25–
35% DPPH activity) and antimicrobial activities (MIC 100 g/mL and 250
l
l
g/mL) at varied concentrations.
The structure–activity relationship (SAR) and in silico drug relevant properties (HBD, HBA, PSA, c Log P,
molecular weight, EHOMO and ELUMO) further confirmed that the compounds are potential lead compounds
for future drug discovery study. Toxicity of the compounds was evaluated theoretically and experimen-
tally and revealed to be nontoxic except 3d and 3j.
Ó 2009 Published by Elsevier Ltd.
1. Introduction
estingly a number of therapeutically useful NSAID’s have been
shown to act by virtue of their free radical scavenging activity.6–8
Non-steroidal anti-inflammatory drugs (NSAIDs) are therapeu-
tically important in the treatment of rheumatic arthritis and in var-
ious types of inflammatory conditions, but they are found to be
limited because of their frequently observed gastrointestinal side
effects. Thus, there is an urgent need for new targets that are re-
quired for the design and development of novel anti-inflammatory
agents as an alternative to NSAIDs.1 Tumor necrosis factor alpha
Antioxidants are the compounds that prevent oxidative damage in-
duced by free radicals and ROS. Thus, antioxidant therapy has also
gained immense importance in the treatment of the above-men-
tioned diseases.9
Flavonoids are widely distributed phytochemicals classified as
anthocyanidins, flavonols, chalcones, aurones, flavanone, isoflav-
ones, flavans, flavanonols, flavanols and flavones differencing in
their structural group arrangements.10 Chalcone, an important
intermediate of flavonoid synthetic pathway exerts a great deal
of biological and pharmacological activities viz. antimicrobial,
anti-anginal, anti-hepatotoxic, anti-inflammatory, antioxidant,
anticancer, antimalarial and anti-allergic.11,12 On the other hand,
pyrazoles are of interest as potent bioactive molecules. They are
known to exhibit pharmaceutical activities such as CNS depressant,
neuroleptic, tuberculostatic, antihypertensive, antileishmanial,
analgesic, antidiabetic, antitumor and antimicrobial.13,14 Moreover,
pyrazole derivatives find applications as dyestuffs, analytical re-
agents and agrochemicals.15
(TNF-a) and interleukin-6 (IL-6), the two important multifunc-
tional proinflammatory cytokines, are involved in the pathogenesis
of autoimmune, inflammatory, cardiovascular, neurodegenerative
and cancer diseases through a series of cytokine signaling path-
ways.2,3 IL-6 contributes to the initiation and extension of the
inflammatory process, therefore it is considered as a central medi-
ator in a range of inflammatory diseases but has not received the
desired attention in drug discovery.4 TNF-
a and IL-6 are thus phar-
maceutically important molecular targets for the treatment of the
above-mentioned diseases. Reactive oxygen species are implicated
in the induction and prolongation of inflammatory process.5 Inter-
Anti-inflammatory activity of the chalcones was confirmed in
several in vivo clinical trials by the inhibition of cell migration
and TNF-a synthesis, while antioxidant activity is related with its
* Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.10.035

B. P. Bandgar et al. / Bioorg. Med. Chem. 17 (2009) 8168–8173
8169
free radical scavenging ability attributed to phenolic –OH group at-
tached to the ring structure.16,17
stabilize the DPPH in the range of 15–35% except 3b and 3h. Anti-
oxidant activity of the compounds is related with their electron or
hydrogen radical releasing ability to DPPH so that they become sta-
ble diamagnetic molecules. This might be the reason for the higher
antioxidant activity of the above-mentioned compounds. Antioxi-
dant activity of the compounds is also well explained by EHOMO
and ELUMO, as the electron donation property has been strongly
attributed to EHOMO (electron donation capability) and ELUMO (elec-
tron accepting capability). The compounds possessing higher EHO-
Synthesis of flavonols having pyrazole moiety at C-2 position
was recently reported as potent antifungal and antibacterial
agents.18,19 Furthermore, the presence of enone function in chal-
cone moiety with pyrazole ring also enhanced the biological
activity.20
Prompted by all these observations, we report here the synthe-
sis and biological activity of pyrazole chalcones as anti-inflamma-
tory, antioxidant and antimicrobial agents. The structure–activity
relationship (SAR) was studied to explore its biological activities
using pharmacological parameters such as EHOMO, ELUMO, c Log P,
hydrogen bond acceptors (HBAs) and hydrogen bond donors
(HBDs), calculated using computational softwares. The toxicity of
the compounds was evaluated theoretically and experimentally
to determine their potential as safe leading compounds for
bioavailability.
and ELUMO were found to be effective agents for stabilizing the
MO
DPPH radicals.
A broad spectrum antifungal activity of the compounds 3c, 3b,
3a, 3j, 3f and 3e was obtained against all the fungi at MIC
250 lg/mL, while other compounds revealed a slight antifungal
activity. Fungi Aspergillus flavus was observed to be more sensitive
than Trichoderma viridae and Aspergillus niger. Of the five tested
bacterial strains, Escherichia coli, Bacillus subtilis and Staphylococcus
aureus were inhibited mostly by compounds 3c, 3d, 3b, 3e, 3i and
3j at MIC 100 g/mL as listed in Table 4. Regarding the SAR study
from the analysis data, it is suggested that compounds substituted
with electron-releasing groups (OCH3 > CH3 > F > Br > Cl) increase
the antimicrobial activity. It is also reported that a part from the
active moiety (1-(2,4-dimethoxy-phenyl)-propenone), substituent
position in the phenyl ring is also found to be biologically relevant.
Our antimicrobial activity data confirmed that the substitution of
Cl at m position is more relevant for increasing its biological activ-
ity than at o and p positions. This is also supported by the previous
report.23,24
To qualify the compound as a drug candidate, it is analyzed by
the parameters set by Lipinski’s rule of five using Osiris property
explorer. The c Log P is the important physiochemical property
indicating the lipophilicity and the ability of molecule to cross
the various biological membranes. According to Lipinski’s rule of
five the c Log P value below 5 is feasible for a compound to be a fu-
ture drug. The synthesized compounds showed a marginal lipo-
philicity within the range of 4.0–5.0. The molecular weight
property of the compound is related to its in vivo administration.
All the synthesized compounds have the molecular weight within
the acceptable range, that is, 400–500. The compounds showed the
HBA below 10 and HBD below 5, which is also within the limit set
by Lipinski’s rule. The polar surface area (PSA) > 140 A2 is thought
to have low oral bioavailability, which is also revealed within the
range for these compounds. Interestingly the compounds also pre-
sented a better drug likeness values. Overall, the compounds 3a, 3c
and 3g showed a good drug score, calculated by combining all
parameters. Drug toxicity is a factor of great importance for a po-
tential commercial drug, since a significant number of drugs are
disapproved in clinical trials based on their high toxicity profile.
The toxicity of the compounds is calculated in terms of mutagenic-
ity, tumorigenic, reproductively effective and irritant. All the com-
pounds were confirmed as non-mutagenic and therefore were
2. Results and discussion
2.1. Chemistry
In the present investigation 1-(2,4-dimethoxy-phenyl)-3-(1,3-
diphenyl-1H-pyrazol-4-yl)-propenones (3) have been prepared by
the Claisen–Schmidt condensation of 1-(2,4-dimethoxy-phenyl)-
ethanone (1) and substituted 1,3-diphenyl-1H-pyrazole-4-carbal-
dehydes (2) by the known literature method21 (Scheme 1).
Substituted 1,3-diphenyl-1H-pyrazole-4-carbaldehydes (2) were
prepared by Vilsmeir–Haack reaction on acetophenonephenylhyd-
razones.22 The residue was purified on column chromatography
(silica gel with 10% ethyl acetate in hexane). The compounds struc-
ture (Fig. 1) was confirmed by spectral data (IR, 1H NMR and MS).
The chemical profile of the compounds is as shown in Table 1.
2.2. Biological evaluation
All the synthesized compounds were evaluated for anti-inflam-
matory activity by TNF-
compounds showed a promising TNF-
3d and 3e up to 1–2% at 10 M concentration, while a promising
IL-6 inhibitory activity was shown by 3a, 3c, 3g, 3i and 3j up to
35–70% inhibition at 10 M concentration. As compared to the
a
and IL-6 inhibition assays. None of the
a
inhibitory activity except
l
l
standard dexamethasone, the activity results are revealed to be
comparable as summarized in Table 2. Most of the compounds
did not show significant cytotoxicity at 10 lM concentration ex-
cept 3d and 3j.
DPPH is the well-known method for determining antioxidant
activity of plant flavonoids. Antioxidant activity of the compounds
is summarized in Table 3. DPPH radical scavenging activity of the
compounds was found to be good to moderate as compared to
the standard BHA. The compounds 3a, 3c, 3j, 3f, 3d, 3g, 3i and 3e
R
O
O
N
R
N
NaOH,C2H5OH
N
+
rt
O
O
CHO
O
O
N
1-(2,4-Dimethoxy-phenyl)-
ethanone
1-(2,4-Dimethoxy-phenyl)-3-(1,3-diphenyl-
1,3-Diphenyl-1H-pyrazole-
1H-pyrazol-4-yl)-propenone
4-carbaldehyde
1
3a-j
2
Scheme 1.

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B. P. Bandgar et al. / Bioorg. Med. Chem. 17 (2009) 8168–8173
H
CH3O
OCH3
CH3
a
j
b
Cl
Cl
Cl
CH3O
O
O
O
i
c
R
R
Introduction of Different
Substituents 3a-j
N
N
Cl
d
h
Cl
Br
F
g
e
f
Figure 1. Structure of pyrazole chalcones.
Table 1
Characterization of compounds and in silico pharmacological parameters for bioavailability
Entry
Product (3)
Molecular
formula
Molecular
weight
Yield (%)
mp (°C)
EHOMO
ELUMO
HBD
HBA
Mol. PSA
c Log P
Solubility
Drug
likeness
Drug
score
3a
3b
3c
3d
3e
3f
3g
3h
3i
R = 4-H
C26H20Cl2N2O3
C27H24N2O3
C27H24N2O4
410
424
440
444
488
428
444
444
478
470
67
72
70
84
58
62
72
70
68
64
157
168
120
149
171
132
127
121
162
110
ꢀ8.6
ꢀ0.745
ꢀ0.728
ꢀ0.731
ꢀ0.802
ꢀ0.824
ꢀ0.826
ꢀ0.793
ꢀ0.745
ꢀ0.796
ꢀ0.65
0
0
0
0
0
0
0
0
0
0
4
4
5
4
4
4
4
4
4
6
44.22
44.22
53.08
44.22
44.22
44.22
44.22
44.22
44.22
61.05
4.16
4.48
4.06
4.78
4.86
4.22
4.78
4.78
5.39
3.95
ꢀ5.39
ꢀ5.73
ꢀ5.41
ꢀ6.13
ꢀ6.22
ꢀ5.7
ꢀ0.3
ꢀ1.56
0.05
0.37
0.27
0.38
0.34
0.2
R = 4-CH3
R = 4-OCH3
R = 4-Cl
R = 4-Br
R = 4-F
R = 3-Cl
R = 2-Cl
R = 2,4-Cl
R = 2,4-OCH3
ꢀ8.59
ꢀ8.43
ꢀ8.62
ꢀ8.78
ꢀ8.78
ꢀ8.68
ꢀ8.84
ꢀ8.89
ꢀ8.65
c26h21n2o3
0.76
C26H21BrN2O3
C26H21FN2O3
C26H21CW2O3
C26H21ClN2O3
C26H20CL2N2O3
C28H26N2O5
ꢀ2.11
ꢀ0.99
ꢀ0.31
0.32
0.93
ꢀ0.4
0.3
ꢀ6.13
ꢀ6.13
ꢀ6.86
ꢀ5.43
0.29
0.32
0.27
0.34
3j
Table 2
3. Conclusion
Anti-inflammatory activity against TNF-
a and IL-6
Entry
% Inhibition at 10
IL-6
lM
The synthesized novel series of pyrazole chalcones were studied
for their in vitro biological activity. The compounds were also
tested for their bioavailability by in silico study. SAR study con-
firmed that 3a, 3c and 3g are potent lead compounds for drug dis-
covery with negligible toxicity.
TNF-
a
Toxicity
3a
3b
3c
3d
3e
3f
3g
3h
3i
0
0
0
1.13
1.81
0
0
0
0
0
64.72
0
38.16
2.71
11.3
0
67.48
14.5
40.78
37.94
90
0
0
0
11.488
0
0
0
0
0
4. Experimental
4.1. General
3j
2.5107
0
Melting points were recorded in open capillaries with electrical
melting point apparatus and were uncorrected. IR spectra (KBr
disks) were recorded using a Perkin–Elmer 237 spectrophotome-
Dexamethasone (1
lM/mL)
69
ter. 1H NMR spectra were recorded on
a Bruker Avance
biologically safe for intake. The toxicity of the compounds was thus
confirmed theoretically and experimentally.
(400 MHz) spectrometer in CDCl3 solutions, with TMS as an inter-
nal reference. Mass spectra were recorded on a Shimadzu GCMS-

B. P. Bandgar et al. / Bioorg. Med. Chem. 17 (2009) 8168–8173
8171
Table 3
% DPPH activity
J = 2 Hz, 2 Hz), 6.47 (d, 1H, J = 2 Hz), 3.86 (s, 3H), 3.84 (s, 3H); m/
z = 411 (M+1).
Entry
% DPPHb
4.2.2. 1-(2,4-Dimethoxy-phenyl)-3-(1-phenyl-3-p-tolyl-1H-
pyrazol-4-yl)-propenone (3b)
3a
3b
3c
3d
3e
3f
3g
3h
3i
31.79 0.22
13.5 0.21
34.64 0.18
18.68 0.16
17.24 0.22
26.68 0.25
29.68 0.19
13.81 0.23
18.25 0.15
30.33 0.20
66.78 0.21
Light yellow solid, mp: 168 °C, IR (KBr):
mmax: 3027, 2944, 1670,
1604, 1414, 1231, 822 cmꢀ1 1H NMR (300 MHz, CDCl3):d 8.24 (s,
.
1H), 7.78 (d, 1H, J = 7.6 Hz), 7.74 (m, 2H), 7.62 (d, 2H, J = 15.6 Hz),
7.45 (m, 2H), 7.31 (m, 2H), 7.21 (d, 1H, J = 15.6 Hz), 6.55 (dd, 1H,
J = 2 Hz, 2 Hz), 6.46 (d, 1H, J = 2 Hz), 3.98 (s, 3H), 3.84 (s, 3H),
2.46 (s, 3H); m/z = 425 (M+1).
3j
BHAa
4.2.3. 1-(2,4-Dimethoxy-phenyl)-3-[3-(4-methoxy-phenyl)-1-
(1-methyl-buta-1,3-dienyl) -1H-pyrazol-4-yl]-propenone (3c)
a
Standard substance.
Mean SD, n = 3.
b
Yellow solid, mp: 120 °C, IR (KBr):
mmax: 3092, 2930, 1675, 1617,
1420, 1235, 835 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.24 (s, 1H),
.
7.77 (d, 1H, J = 7.6 Hz), 7.72 (m, 2H), 7.64 (d, 1H, J = 15.6 Hz),
7.48 (m, 3H), 7.30 (m, 2H), 7.00 (d, 1H, J = 15.6 Hz), 6.56 (dd, 1H,
J = 2 Hz, 2 Hz), 6.47 (d, 1H, J = 2 Hz), 3.86 (s, 3H), 3.88 (s, 3H),
3.84 (s, 3H) ; m/z = 440 (M+).
QP 1000 EX. All the reagents and solvents used were of analytical
grade, and were used as supplied unless otherwise stated. TLC
was performed on silica gel coated plates for monitoring the
reactions.
4.2.4. 3-[3-(4-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dimethoxy-phenyl)-propenone (3d)
4.2. Synthesis of pyrazole chalcones (3a–j)
Yellow solid, mp: 149 °C, IR (KBr):
mmax: 3056, 2937, 1673, 1605,
A mixture of 1-(2,4-dimethoxy-phenyl)-ethanone 1 (0.180 g,
1 mmol) and 1,3-diphenyl-1H-pyrazole-4-carbaldehyde 2 (0.248 g,
1 mmol) was dissolved in 15 mL ethanol. To this mixture, sodium
hydroxide (40%, 2 mL) was added at 0–5 °C. The reaction mixture
was stirred at room temperature for 2.5 h. Then this reaction mix-
ture was poured over crushed ice and acidified with dil HCl. The yel-
low solid thus obtained was filtered, washed with water and dried.
The residue was purified on column chromatography (silica gel with
10% ethyl acetate in hexane) to afford pure 1-(2,4-dimethoxy-phe-
nyl)-3-(1,3-diphenyl-1H-pyrazol-4-yl)-propenone (3a) (Scheme 1).
The physical and spectral data of new 1-(2,4-dimethoxy-phe-
nyl)-ethanone chalcone analogues of pyrazoles (3a–j) are given
below.
1425, 1209, 826 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.23 (s, 1H),
.
7.79–7.75 (m, 4H), 7.66 (d, 1H, J = 1 5.9 Hz), 7.50 (m, 2H), 7.48
(m, 1H), 7.45 (d, 1H, J = 15.9 Hz), 7.35 (m, 3H), 6.57 (dd, 1H,
J = 2 Hz, 2 Hz), 6.48 (d, 1H, J = 2 Hz), 3.87 (s, 3H), 3.85 (s, 3H) ; m/
z = 445 (M+).
4.2.5. 3-[3-(4-Bromo-phenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dimethoxy-phenyl)-propenone (3e)
Light yellow solid, mp: 171 °C, IR (KBr):
mmax: 3059, 2939, 1676,
1609, 1427, 1211, 829 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.19 (s,
.
1H), 7.79–7.77 (m, 4H), 7.62 (d, 1H, J = 15.8 Hz), 7.59 (m, 2H),
7.46–7.41 (m, 1H), 7.31 (d, 1H, J = 15.9 Hz), 7.29–7.25 (m, 3H),
6.58 (dd, 1H, J = 2 Hz, 2 Hz), 6.41 (d, 1H, J = 2 Hz), 3.84 (s, 3H),
3.83 (s, 3H); m/z = 490 (M+).
4.2.1. 1-(2,4-Dimethoxy-phenyl)-3-(1,3-diphenyl-1H-pyrazol-4-
yl)-propenone (3a)
4.2.6. 1-(2,4-Dimethoxy-phenyl)-3-[3-(4-fluoro-phenyl)-1-
Light yellow solid, mp: 157 °C, IR (KBr):
mmax: 3025, 2941, 1668,
phenyl-1H-pyrazol-4-yl]-propenone (3f)
1601, 1411, 1234, 825 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.26 (s,
.
Light yellow solid, mp: 132 °C, IR (KBr):
1611, 1426, 1211, 829 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.24 (s,
1H), 7.79–7.76 (m, 4H), 7.69 (d, 1H, J = 16.1 Hz), 7.51–7.49 (m,
mmax: 3058, 2940, 1676,
1H), 7.79 (d, 1H, J = 7.5 Hz), 7.75 (m, 2H), 7.57 (d, 2H, J = 15.7 Hz),
7.46 (m, 3H), 7.32 (m, 3H), 7.31 (d, 1H, J = 15.5 Hz), 6.56 (dd, 1H,
.
Table 4
Antimicrobial activity of pyrazole chalcone derivatives (zone of inhibition in mm)
Compound
Bacteria (MIC at 100
PV
l
g/mL)
Fungi (MIC at 250
l
g/mL)
EC
KP
SA
BS
AN
TV
AF
3a
3b
3c
3d
3e
3f
3g
3h
10
11
8
11
9
12
9
12
11
10
—
—
12
—
—
7
12
—
10
8
—
—
—
20
—
—
8
9
10
8
10
13
7
14
8
7
9
6
—
—
—
12
14
15
11
11
13
9
11
8
12
14
15
16
13
13
14
11
12
10
14
—
15
12
14
10
13
12
10
9
11
11
8
15
6
8
10
7
—
10
—
—
—
8
32
—
3i
3j
10
13
—
Tetracycline
Nystatin
Control
17
—
—
—
11
12
11
Data represent the mean of three replicates.
EC—Escherichia coli (MTCC 1650); PV—Proteus vulgaris (MTCC 1771); KN—Klebsiella pneumoniae (NCIM 2957), SA—Staphylococcus aureus (MTCC 96); BS—Bacillus subtilis (MTCC
1789); AN—Aspergillus niger (MTCC 1781); AF—Aspergillus flavus (MTCC 2501); TV—Trichoderma viridae (MTCC 167); not detected, — ; trace activity,
.

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B. P. Bandgar et al. / Bioorg. Med. Chem. 17 (2009) 8168–8173
3H), 7.37–7.35 (m, 3H), 7.10 (d, 1H, J = 16.1 Hz), 6.59 (dd, 1H,
J = 2 Hz, 2 Hz), 6.50 (d, 1H, J = 2 Hz), 3.84 (s, 3H), 3.82 (s, 3H) ; m/
z = 429 (M+1).
Stock solutions of different compounds (1 mM) were mixed
with DPPH methanol solution (0.5 mL, 0.3 mM) in 3 mL of total
reaction mixture and allowed to react at room temperature. After
30 min, absorbance values were measured at 520 nm and con-
verted to % antioxidant activity. For a comparative study the Butyl-
ated hydroxyl anisole (BHA) was used as the standard. The
percentage inhibition activity was calculated by using a formula.
The data are summarized in Table 3.
4.2.7. 3-[3-(3-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dimethoxy-phenyl)-propenone (3g)
Light yellow solid, mp: 127 °C, IR (KBr):
mmax: 3060, 2941, 1674,
1608, 1430, 1212, 825 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.19 (s,
.
1H), 7.77–7.75 (m, 4H), 7.65 (d, 1H, J = 16.4 Hz), 7.59–7.57 (m,
2H), 7.45–7.40 (m, 1H), 7.35 (m, 3H), 7.2 (d, 1H, J = 16.4 Hz), 6.49
(dd, 1H, J = 2 Hz, 2 Hz), 6.40 (d, 1H, J = 2 Hz), 3.81 (s, 3H), 3.80 (s,
3H) ; m/z = 445 (M+), 447 (M+2).
% antioxidant activity
¼ ½1 ꢀ OD of test compound=OD of control compoundꢁ ꢂ 100
4.5. Antimicrobial activity (agar diffusion method)
4.2.8. 3-[3-(2-Chloro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dimethoxy-phenyl)-propenone (3h)
Antimicrobial activity of all synthesized compounds was deter-
mined by agar diffusion method.27,28 All human pathogenic bacte-
ria viz. Bacillus megaterium (MTCC 1684), Bacillus subtilis (MTCC
1789), Klebsiella pneumoniae (NCIM 2957), Staphylococcus aureus
(MTCC 96), Proteus vulgaris (MTCC 1771), Escherichia coli (MTCC
1650) and fungi viz. Trichoderma viridae (MTCC 167), Aspergillus fla-
vus (MTCC 2501) and Aspergillus niger (MTCC 1781) were procured
from the Institute of Microbial Technology (IMTech), Chandigarh,
India and the National Collection of Industrial Microorganisms
(NCIM), Pune, India. Stock solutions of compounds were diluted
in dimethyl sulfoxide (1% DMSO) to give a final concentration rang-
Light yellow solid, mp: 121 °C, IR (KBr):
mmax: 3062, 2943, 1679,
1610, 1431, 1214, 832 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.43 (s,
.
1H), 7.79–7.77 (m, 4H), 7.65 (d, 1H, J = 16.4 Hz), 7.58–7.56 (m,
3H), 7.41–7.39 (m, 1H), 7.35 (m, 3H), 7.1 (d, 1H, J = 15.7 Hz), 6.43
(dd, 1H, J = 2 Hz, 2 Hz), 6.39(d, 1H, J = 2 Hz), 3.85 (s, 3H), 3.84 (s,
3H) ; 445 (M+).
4.2.9. 3-[3-(2,4-Dichloro-phenyl)-1-phenyl-1H-pyrazol-4-yl]-1-
(2,4-dimethoxy-phenyl)-propenone (3i)
Yellow solid, mp: 162 °C, IR (KBr):
mmax: 3061, 2957, 1680, 1632,
1427, 1212, 830 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.22 (s, 1H),
.
ing from 50 to 500 lg/mL for determining the MIC value. Minimum
7.79–7.76 (m, 3H), 7.59 (d, 1H, J = 16.2 Hz), 7.58–7.57 (m, 2H),
7.44–7.41 (m, 1H), 7.35 (m, 3H), 7.19 (d, 1H, J = 16.0 Hz), 6.59
(dd, 1H, J = 2 Hz, 2 Hz), 6.44 (d, 1H, J = 2 Hz), 3.83 (s, 3H), 3.81 (s,
3H) ; m/z = 479 (M+).
inhibitory concentration (MIC) was defined as the lowest concen-
tration of compound required for a complete inhibition of the fun-
gal and bacterial growth after incubation time. For antifungal
activity, different fungal spore suspensions in sterile distilled water
were adjusted to give a final concentration of 106 cfu/mL. An inoc-
ulum of 0.1 mL spore suspension of each fungus was spread on
Sabouraud’s Dextrose agar plates (HiMedia). For antibacterial
activity Muller Hinton agar was used (HiMedia) seeded with
0.1 mL of the respective bacterial culture strains suspension pre-
pared in a sterile saline (0.85%) of 105 cfu/mL dilution. The wells
of 6 mm diameter were filled with 0.1 mL of each compound dilu-
tion separately for each test of fungi and bacterial strain. The
DMSO (1%) alone was used as a control. The antibiotic nystatin
4.2.10. 1-(2,4-Dimethoxy-phenyl)-3-[3-(2,4-dimethoxy-
phenyl)-1-phenyl-1H-pyrazol-4-yl]-propenone (3j)
Yellow solid, mp: 110 °C, IR (KBr):
mmax: 3096, 2935, 1680, 1619,
1428, 1240, 838 cmꢀ1 1H NMR (300 MHz, CDCl3): d 8.22 (s, 1H),
.
7.76 (d, 1H), 7.75–7.73 (m, 3H), 7.64 (d, 1H, J = 16.2 Hz), 7.48–
7.47 (m, 3H), 7.30–7.28 (m, 2H), 7.21 (d, 1H, J = 16.2 Hz), 6.59
(dd, 1H, J = 2 Hz, 2 Hz), 6.45 (d, 1H, J = 2 Hz), 3.86 (s, 3H), 3.85 (s,
3H), 3.84 (s, 3H), 3.83 (s, 3H) ; m/z = 471 (M+1).
(30 lg/mL) and tetracycline (10 lg/mL) are used as reference anti-
4.3. Anti-inflammatory and cytotoxicity assay
fungal and antibacterial agents, respectively, for comparison. Inoc-
ulated plates in duplicate were then incubated at 37 0.5 °C for
antibacterial activity for 24 h and 48 h at 28 0.2 °C for antifungal
activity. After incubation the antimicrobial activity was measured
in terms of the zone of inhibition in mm as shown in Table 4.
Proinflammatory cytokine production by lipopolysaccharide
(LPS) in THP-1 cells was measured according to the method de-
scribed by Hwang et al. During assay, THP-1 cells were cultured in
RPMI 1640 culture medium (Gibco BRL, Pasley, UK) containing
100 U/mL penicillin and 100 mg/mL streptomycin containing 10%
fetal bovine serum (FBS, JRH). Cells were differentiated with phorbol
myristate acetate (PMA, Sigma). Following cell plating, the test com-
pounds in 0.5% DMSO were added to each well and the plate was
incubated for 30 min at 37 °C. Finally, LPS (Escherichia coli 0127:B8,
Sigma Chemical Co., St. Louis, MO) was added, at a final concentra-
4.6. In silico pharmacological property and SAR study
The pharmacological properties of the compounds, such as
molecular weight, c Log P and quantum chemical descriptors such
as EHOMO (Energy of highest occupied molecular orbital) and ELUMO
(Energy of lowest unoccupied molecular orbital) of the synthesized
compounds were calculated using a BioMed CaChe 6.1 (FujiSuit
Ltd), a computer aided molecular design modeling tool for win-
dows ME 9820000 and XP operating system. Other parameters
such as HBA, HBD, molecular PSA, drug score and drug likeness
of the compounds were also studied using online Osiris property
explorer for drug bioavailability of chemical compounds. Since
compounds are considered for oral delivery, they were also as-
sessed for toxicity using in silico ADME prediction methods.23,24
tion of 1
for 24 h in 5% CO2. After incubation, supernatants were harvested,
and assayed for TNF- and IL-6 by ELISA as described by the manu-
lg/mL in each well. Plates were further incubated at 37 °C
a
facturer (BD Biosciences). The cells were simultaneously evaluated
for cytotoxicity using CCK-8 from Dojindo Laboratories. Percent
inhibition of cytokine release compared to the control was calcu-
lated.25 The 50% inhibitory concentration (IC50) values were calcu-
lated by a nonlinear regression method as shown in Table 2.
4.4. In vitro antioxidant activity (DPPH method)
Acknowledgements
The compounds (3a–j) were evaluated for their in vitro free rad-
ical scavenging activity by the 2,20diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging method.26
The authors are thankful to Dr. Somesh Sharma, Piramal Life
Sciences, Mumbai, for anti-inflammatory activity and to the

B. P. Bandgar et al. / Bioorg. Med. Chem. 17 (2009) 8168–8173
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