Synthesis and biological evaluation of simple methoxylated chalcones as anticancer, anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1016/j.bmc.2009.11.066

Chalcones have been identified as interesting compounds with cytotoxicity, anti-inflammatory and antioxidant properties. In the present study, simple methoxychalcones were synthesized by Claisen-Schmidt condensation reaction and evaluated for above biological activities. The structures of the compounds were established by IR, ¹H NMR and mass spectral analysis. The data revealed that compound 3s (99-100% at 10 μM concentration) completely inhibit the selected five human cancer cell lines as compared to standard flavopiridol and gemcitabine (70-90% at 700 nM and 500 nM concentrations, respectively), followed by 3a, 3n, 3o, 3p, 3q, 3r. Among the tested compounds 3l, 3m, 3r, and 3s exhibited promising anti-inflammatory activity against TNF-α and IL-6 with 90-100% inhibition at 10 μM concentration. DPPH free radical scavenging activity was given by the compounds 3o, 3n, 3l, 3r, 3m, 3a, 3p, 3c and 3s at 1 mM concentration. Overall, 3s was obtained as lead compound with promising anticancer, anti-inflammatory and antioxidant activities. Bioavailability of compounds were checked by in vitro cytotoxicity study and confirmed to be nontoxic. The structure activity relationship (SAR) and in silico drug relevant properties (HBDs, HBAs, PSA, c Log P, ionization potential, molecular weight, E_HOMO and E_LUMO) further confirmed that the compounds were potential candidates for future drug discovery study.

Full text of DOI:10.1016/j.bmc.2009.11.066

Bioorganic & Medicinal Chemistry 18 (2010) 1364–1370
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of simple methoxylated chalcones
as anticancer, anti-inflammatory and antioxidant agents
b
c
d
Babasaheb P. Bandgar a,b, , Shrikant S. Gawande , Ragini G. Bodade , Jalinder V. Totre ,
*
Chandrahas 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 Institute for Drug Research, Deptartment of Medicinal Chemistry and Natural Products, School of Pharmacy, The Hebrew University of Jerusalem, 91120, Israel
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcones have been identified as interesting compounds with cytotoxicity, anti-inflammatory and anti-
oxidant properties. In the present study, simple methoxychalcones were synthesized by Claisen–Schmidt
condensation reaction and evaluated for above biological activities. The structures of the compounds
were established by IR, 1H NMR and mass spectral analysis. The data revealed that compound 3s (99–
Received 17 November 2009
Revised 25 November 2009
Accepted 26 November 2009
Available online 6 December 2009
100% at 10 lM concentration) completely inhibit the selected five human cancer cell lines as compared
to standard flavopiridol and gemcitabine (70–90% at 700 nM and 500 nM concentrations, respectively),
followed by 3a, 3n, 3o, 3p, 3q, 3r. Among the tested compounds 3l, 3m, 3r, and 3s exhibited promising
Dedicated to my research colleague late Ms.
Sunita B. Bandgar
anti-inflammatory activity against TNF-
a and IL-6 with 90–100% inhibition at 10 lM concentration.
DPPH free radical scavenging activity was given by the compounds 3o, 3n, 3l, 3r, 3m, 3a, 3p, 3c and
3s at 1 mM concentration. Overall, 3s was obtained as lead compound with promising anticancer, anti-
inflammatory and antioxidant activities. Bioavailability of compounds were checked by in vitro cytotox-
icity study and confirmed to be nontoxic. The structure activity relationship (SAR) and in silico drug rel-
Keywords:
Chalcones
Anticancer activity
Anti-inflammatory activity
Antioxidant activity
Cytotoxicity
evant properties (HBDs, HBAs, PSA, c Log P, ionization potential, molecular weight, EHOMO and ELUMO
)
further confirmed that the compounds were potential candidates for future drug discovery study.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
cells. They are also effective in vivo as cell proliferating inhibitors,
anti-tumor promoting and chemopreventing agents. Since a num-
ber of clinically useful anticancer drugs have genotoxic effects due
to interaction with the amino groups of nucleic acids, chalcones
may be devoid of this important side effect.6–8
Chalcones (1,3-diaryl-2-propen-1-ones) constitute an impor-
tant class of natural products belonging to the flavonoids family,
display interesting biological activities including anticancer, anti-
inflammatory, antioxidant, cytotoxic, antimicrobial, analgesic and
antipyretic, anti-anginal, anti-hepatotoxic, antimalarial and anti-
allergic. Chemically they consist of open-chain flavonoids in which
Interleukin-6 (IL-6) and Tumor necrosis factor alpha (TNF-a) are
two important multifunctional proinflammatory cytokines involved
in pathogenesis of cardiovascular, neurodegenerational diseases
and cancer through a series of cytokine signaling pathways.9
Recently it is also suggested that tumor necrosis factor alpha
the two aromatic rings are joined by a three carbon
a,b-unsatu-
rated carbonyl system.1,2 Cancer, the uncontrolled, rapid and path-
ological proliferation of abnormal cells, is the second leading cause
of human death after cardiovascular diseases in developing as well
as advanced countries.3,4 Although there are many therapeutic
strategies including chemotherapy and radiotherapy, high sys-
temic toxicity and drug resistance limit the successful outcomes
in most cases. Therefore, novel diagnosis, treatment and preven-
tion approaches are urgently needed for cancer therapy.5
(TNF-a
) is act target for cancer therapy.10 Literature survey de-
scribed dimethoxy and trimethoxychalcone derivatives as effective
anti-inflammatory agents.11–13 A number of therapeutically useful
NSAID’s have been shown to act by virtue of their free radical scav-
enging activity, which is implicated in the induction and prolonga-
tion of inflammatory process.14
Reactive oxygen species (ROS) in the form of superoxide anion
(O2ꢀꢁ), hydroxyl radical (OHÅ) and hydrogen peroxide (H2O2) attack
various biological macromolecules (proteins, enzymes, DNA, etc.)
under ‘oxidative stress’ conditions, give rise to a number of inflam-
matory, metabolic disorders, cellular aging, reperfusion damage
and cancer.15,16 Cytotoxic effects of antioxidant flavonoids (includ-
ing chalcones) are associated with their pro-oxidant effects.8 The
Among the naturally occurring hydroxy chalcones and their
synthetic analogues, several compounds displayed cytotoxic activ-
ity (antimitotic, cell growth inhibitor) towards cultured tumor
* Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.066

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 1364–1370
1365
O
O
O
H
CH3
NaOH, C2H5OH
R
+
R'
R
R'
A
A
B
B
rt.
1
2
3
Scheme 1.
antioxidant properties of chalcones are known to be influenced to a
great extent by the two aryl structures, that is, the substitutions on
two aryl rings of chalcone molecule and their substitution patterns.
Especially, the hydroxyl substituent is one of the key groups to en-
hance greatly the antioxidant activity of chalcone mainly due to its
easy conversion to phenoxy radicals through the hydrogen atom
transfer mechanism.17
In continuation of our research work, in order to find the broad
spectrum biologically active chalcones, we have previously de-
scribed the anti-inflammatory, antimicrobial and antioxidant
activity of pyrazole chalcones.18 Here, we report the synthesis
and biological activity of methoxychalcones as an anticancer,
anti-inflammatory and antioxidant agents. In the structure activity
relationship (SAR) studies, the biological properties of these mole-
cules were compared with several theoretical parameters such as
EHOMO, ELUMO, c Log P, PSA, ionization potential, molecular weight,
hydrogen bond acceptors (HBA) and hydrogen bond donors
(HBD), calculated using computational softwares. Since the com-
pounds are considered for oral delivery, they were also submitted
to the analysis of Lipinski rule of five.19 Finally, toxicity of com-
pounds was experimentally and theoretically evaluated to deter-
mine their potential as safe leading compounds.
nyl)-propenone (3) have been prepared by the Claisen–Schmidt
condensation of substituted 1-phenyl-ethanone (2) and 2,4-dime-
thoxy-benzaldehyde or 3,4,5-trimethoxy-benzaldehyde (1) by
known literature method18 (Scheme 1). The residue was purified
on column chromatography using silica gel with 10% ethyl acetate
in hexane. The products were characterized by comparison of their
spectral and physical data with those of authentic samples. All
authentic chalcones were prepared from the corresponding reac-
tants according to the method described.20 The chemical profile
of the compounds is as shown in Table 1.
2.2. Biological evaluation
Among the currently identified anti-tumor agents, chalcones
represents an important class of molecules that are abundant in
edible plants (fruits and vegetables). The anticancer activity of cer-
tain chalcones is believed to be a result of binding to tubulin and
preventing it from polymerizing into microtubules.21,22 Here, we
synthesized 2,4-dimethoxy (3a–k) and 3,4,5-trimethoxy (3l–s)
chalcone derivatives and evaluated for their anticancer activity
against the five human cancer cell lines including ACHN (renal cell
carcinoma), Pancc1 (pancreatic carcinoma), Calu1 (non-small cell
lung carcinoma), H460 (non-small cell lung carcinoma), and
HCT116 (colon carcinoma). 2,4-Dimethoxychalcone (3a) gives
promising anticancer activity with 90–95% of cell line inhibition.
Substitution pattern in B-ring was changed simultaneously with
CH3, OCH3, Cl, Br, F and NO2 groups (3b–k), showed only moderate
2. Results and discussion
2.1. Chemistry
cell lines inhibition (15–50%) at 10 lM concentration as per Table
2. Introduction of one more methoxy group in A-ring (3,4,5-tri-
In the present investigation substituted 3-(2,4-dimethoxy-phe-
nyl)-1-phenyl-propenone or 1-phenyl-3-(3,4,5-trimethoxy-phe-
Table 1
Synthesis of simple methoxylated chalcones
O
β
6'
6
5'
4'
5
α
B
A
4
2'
2
3
3'
S.NO.
B-ring
30-
A-ring
4-
Product (3)
Yielda (%)
Reaction time (min)
20-
H
40
H
2-
3-
H
5-
O
a
b
c
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
91
96
86
90
35
30
45
O
O
O
H
H
H
CH3
OCH3
Cl
OCH3
OCH3
OCH3
H
H
H
CH3
O
O
O
O
O
O
O
d
48
Cl
O
O
(continued on next page)

1366
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 1364–1370
Table 1 (continued)
S.NO.
20-
B-ring
30-
A-ring
4-
Product (3)
Yielda (%)
Reaction time (min)
40
2-
3-
H
5-
H
O
e
f
H
H
H
H
Br
OCH3
OCH3
OCH3
94
96
32
45
Br
O
O
O
F
OCH3
OCH3
H
H
H
H
F
O
O
O
g
H
Cl
H
OCH3
84
60
O
O
O
Cl
O
h
i
Cl
Cl
H
H
H
OCH3
OCH3
H
H
OCH3
OCH3
H
H
80
92
70
45
Cl
O
O
Cl
Cl
Cl
O
O
O
j
OCH3
H
H
OCH3
NO2
OCH3
OCH3
H
H
OCH3
OCH3
H
H
94
95
75
25
O
O
O
O
O
k
H
O2N
O
O
O
O
l
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
96
91
89
85
30
40
55
50
H
O
O
O
O
O
m
n
o
CH3
OCH3
OH
CH3
O
O
O
O
O
O
O
O
O
O
HO
Cl
O
O
p
H
H
Cl
H
OCH3
OCH3
OCH3
92
30
O
O

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 1364–1370
1367
Table 1 (continued)
S.NO.
20-
B-ring
30-
A-ring
Product (3)
Yielda (%)
Reaction time (min)
40
2-
H
3-
4-
5-
O
O
O
q
r
H
H
H
H
Br
OCH3
OCH3
OCH3
OCH3
OCH3
95
25
75
45
Br
O
O
O
OCH3
OCH3
H
H
OCH3
OCH3
OCH3
OCH3
94
85
O
O
O
O
O
O
s
H
NO2
NO2
O
O
a
Isolated yield.
methoxychalcone), 3l shows negligible anticancer activity. Further
substitution of NO2 in B-ring (3s) increases the inhibitory activity
up to 100% as compared to the standard flavopiridol (700 nM)
and gemcitabine (500 nM). It is reported that some nitro com-
pounds are afraid to may possess carcinogenicity and mutagenic-
ity.23 This found to be in contrast in case of compound (3s),
which is revealed potent anticancer agent without inducing any
drug toxicity. Remaining compounds 3n, 3o, 3p, 3q and 3r substi-
tuted with methoxy, hydroxyl and halo groups showed consider-
able anticancer activity (50–95%) in the order of
OCH3 > OH > Cl > Br. Edwards et al. confirmed that, more the meth-
oxy groups in a compound, is beneficial for antimitotic activity
against HeLa cells.24 This found contrast in case of compound
(3r), which might be due to the more bulky nature of the com-
pound.22 Fluorinated organic molecules are known to perform a
wide rang of biological functions and fluorinated anticancer agents
have become a focus in the development of new therapies for can-
cer.25 Although compound (3f) is with fluorine, it doesn’t show any
remarkable anticancer activity. In general, cell line HCT116 was af-
fected more by all the compounds followed by Pancc1 > ACHN >
Calu1 > H460 cell lines (Table 2).
Non-steroidal anti-inflammatory drugs (NSAIDs) are therapeu-
tically important in the treatment of rheumatic arthritis and vari-
ous types of inflammatory conditions but found to be limited
because of their frequently observed gastrointestinal side effects.14
Being a natural origin, chalcones may be devoid of toxicity and
hence beneficial for drug discovery as active compound.21
Anti-inflammatory activity of all the synthesized compounds was
evaluated in terms of TNF-
3,4,5-trimethoxychalcones (3l–s) inhibit 90–100% at 10
a
and IL-6 inhibitory activity. Entire
M con-
l
centration as compared to standard dexamethasone and
2,4-dimethoxychalcones (3a–k). Compounds 3l, 3m, 3r, and 3s
found to be promising anti-inflammatory agents without toxicity
(Table 3). Interestingly, several studies have demonstrated endog-
enous TNF-a
as a tumor promoter and metastatic factor.31–33 Sig-
Table 3
Anti-inflammatory and % antioxidant activity of chalcone derivatives
Compounds
% Inhibition at
10
% Antioxidant
Toxicity
Table 2
l
M
activityb (1 mM/mL)
Anticancer activity of chalcone derivatives
TNF-
a
IL-6
Compounds
Anti-cancer activity at 10 lM concd
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
98
0
0
0
0
20
0
0
0
2
100
29
39
12
0
61
27
18
6
38.00 0.24
25.27 0.22
32.33 0.25
26.39 0.15
0
79
16
29
0
15
45
0
0
2
35
0
73
29
89
70
79
68
39
18
0
ACHN
Panc1
Calu 1
H460
HCT116
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
93
44
45
28
25
35
36
30
35
48
28
33
17
92
83
57
48
79
99
68
70
94
45
36
29
30
36
36
37
35
43
16
7
91
37
31
28
21
30
37
38
30
32
38
7
92
34
38
29
32
35
22
26
29
38
19
15
14
89
74
54
44
57
100
84
68
95
31
37
22
32
37
27
25
26
31
16
0
0
19.21 0.19
10.12 0.27
11.28 0.22
31.32 0.29
18.23 0.29
47.23 0.46
43.21 0.66
48.29 0.45
52.00 0.43
36.53 0.32
23.32 0.23
44.91 0.33
30.21 0.25
81
0
0
100
97
100
99
98
99
99
65
73
100
99
100
100
100
100
100
93
84
1
0
0
93
76
66
57
62
100
75
71
93
76
67
57
58
100
68
70
97
94
92
86
84
99
71
76
3q
3r
3s
3q
3r
3s
Dexamethasone (1
BHA
lM)
a
74.00 0.53
Flavopiridol (700 nM)
Gemcitabine (500 nM)
a
Standard substance.
Mean SD, n = 3.
b

1368
B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 1364–1370
Table 4
In silico pharmacological parameters for bioavailability
Compounds
Molecular
formula
Molecular
Weight
Mp (°C)
EHOMO
ELUMO
Ionization
potential (eV)
HBD
HBA
PSA (A2)
c log P
Drug
likeness
Drug
score
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
C17H16O3
C18H18O3
C18H18O4
C17H15O3Cl
C17H15O3Br
C17H15O3F
C17H15O3Cl
268.313
282.342
298.341
302.757
347.208
286.302
302.757
302.757
337.202
328.364
315.325
313.311
312.365
328.364
314.337
332.783
377.234
358.39
60
74
82
120
152
136
86
ꢁ9.092
ꢁ9.396
ꢁ8.877
ꢁ9.47
ꢁ0.453
ꢁ0.458
ꢁ0.358
ꢁ0.636
ꢁ0.725
ꢁ0.555
ꢁ0.609
ꢁ0.564
ꢁ0.65
8.987
9.354
8.879
9.452
9.408
9.515
9.219
9.133
9.192
9.087
9.129
8.711
8.697
8.703
8.714
8.753
8.771
9.081
9.157
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
3
3
4
3
3
3
3
3
3
5
5
4
4
5
5
4
4
6
6
29.71
29.71
38.1
3.3
ꢁ0.48
ꢁ0.75
0.14
2.12
ꢁ2.97
0.58
ꢁ1.87
ꢁ0.07
0.96
0.91
ꢁ0.53
4
3.95
4.81
5.19
6.51
1.74
3.11
4.08
0.33
0.29
0.36
0.38
0.2
0.37
0.23
0.5
3.61
3.19
3.91
4
29.71
29.71
29.71
29.71
29.71
29.71
46.23
63.11
37.62
37.62
46.01
54.88
37.62
37.62
54.14
71.02
ꢁ9.125
ꢁ9.182
ꢁ9.184
ꢁ9.121
ꢁ9.179
ꢁ9.057
ꢁ9.153
ꢁ9.118
ꢁ9.124
ꢁ9.141
ꢁ9.069
ꢁ9.221
ꢁ9.174
ꢁ9.117
ꢁ9.077
3.36
3.91
3.91
4.52
3.09
3.31
3.19
3.51
3.09
2.89
3.81
3.89
2.89
3.2
C17H15O3Cl
79
C17H14O3Cl2
C19H20O5
C17H15NO5
C18H18O4
C19H20O4
C19H20O5
125
148
134
173
122
129
98
105
102
127
136
0.46
0.5
ꢁ0.292
ꢁ0.651
ꢁ0.957
ꢁ0.917
ꢁ0.921
ꢁ0.947
ꢁ1.05
0.14
0.47
0.43
0.46
0.49
0.4
0.35
0.74
0.21
C18H18O5
C18H17O4Cl
C18H17O4Br
C20H22O6
ꢁ1.078
ꢁ0.696
ꢁ0.97
C18H17NO6
343.351
nificant levels of TNF-
a
was found in tumor microenvironment of
obtained by the same compounds, which is beneficial for cancer
treatment also. Bioavailability and toxicity of the compounds re-
vealed the compounds to be nontoxic with drug properties. The re-
sults of this study may find a lead (3s) toward the development of
new therapeutic agent to fight cancer.
various human cancers, including those of breast, ovarian, prostate,
lymphoma, melanoma and leukemia.34 We demonstrated that
TNF-a libration in human THP-1 cells was inhibited by synthesized
compounds .This effect revealed that they have dual activity as
anti-inflammatory as well as more effective against cancer treat-
ment since, it can act by two mechanisms, directly by killing tumor
cells and indirectly, resolving the inflammatory environment that
supports tumor development.
4. Experimental
4.1. General
Reactive oxygen species and nitrogen specie contribute to the
pathophysiology of anti-inflammatory conditions.16 Antioxidant
are the compounds capable of scavenging the free radicals; for this
antioxidant therapy is one of the recent options.26 The antioxidant
activity of the compounds was determined by DPPH free radical
scavenging activity. Free radical scavenging activity was measured
in terms of %antioxidant activity as represented in Table 3. Com-
pounds 3o, 3l, 3n, 3r, 3p, 3m, 3a, 3c and 3s showed 30–55% inhi-
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-
ter. 1H NMR spectra were recorded on Bruker Avance (400 MHz)
Spectrometer in CDCl3 solutions, with TMS as an internal reference.
Mass spectra were recorded on a Shimadzu GCMS-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.
bition as compared to standard BHA (74%).
A moderate
antioxidant activity of the compound is related with their electron
or hydrogen radical releasing ability to DPPH so that it became sta-
ble diamagnetic molecule. Hydroxy and methoxy groups are more
electron releasing atoms, therefore induces more antioxidant
activity.17 Bioavailability of the compounds was checked by
in vitro cytotoxicity assay using CCK-8 cells in RPMI 1640 culture
media. Dimethoxylated chalcones (3a–k) were found to be non-
toxic as compared to trimethoxylated chalcones (3l–s), except 3l,
3m, 3r and 3s. All the synthesized compounds were also tested
by Lipinski rule of five to find new potential drug candidates. The
results reveled that all compounds are within the range set by
Lipinski rule of five as per Table 4. These data have significance
for the biological activity for interaction with the receptor/enzyme,
penetration through the cell membrane, chemical properties of the
molecule during drug metabolism.27
4.2. Synthesis of chalcones (3a–s)
A
mixture of 1-(4-methoxy-phenyl)-ethanone 2 (0.150 g,
1 mmol) and 2,4-dimethoxy-benzaldehyde 1 (0.166 g, 1 mmol)
was dissolved in 10 mL ethanol. To this mixture, sodium hydroxide
(40%, 1 mL) was added at 0–5 °C. The reaction mixture was stirred
at room temperature for 45 min. Then this reaction mixture was
poured over crushed ice and acidified with dil HCl. The light yellow
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 3-(2,4-dimethoxy-phe-
nyl)-1-(4-methoxy-phenyl)-propenone (3c) (Scheme 1).
The physical and spectral data of selective methoxylated chal-
cones are given below.
3. Conclusion
4.2.1. 3-(2,4-Dimethoxy-phenyl)-1-(4-methoxy-phenyl)-
propenone (3c)
In summary, we have prepared and evaluated a series of
2,4-dimethoxy and 3,4,5-trimethoxy chalcones for their biological
activities. 3,4,5-Trimethoxychalcones inhibit the growth of five hu-
man cancer cell lines. Some of them are exciting, as they preferen-
tially inhibit the cell lines more or similar with the standard
anticancer agents (flavopiridol and gemcitabine). Moreover prom-
ising anti-inflammatory and antioxidant activities were also
Pale yellow solid, mp 82 °C, IR (KBr disk): 3062, 2933, 1644,
1699, 1279 cmꢁ1
; d 8.05 (d,
1H NMR (300 MHz, CDCl3):
J = 15.6 Hz, 1H), 8.03 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 15.6 Hz, 1H),
7.57 (d, J = 8.6 Hz, 1H), 6.96 (d, J = 8.6 Hz, 2H), 6.53 (dd, J1 = 8.4,
J2 = 2.3 Hz, 1H), 6.47 (d, J = 2.3 Hz, 1H), 3.89 (s, 3H), 3.87 (s, 3H),
3.85 (s, 3H); MS (ESI) m/z = 299 (M+1).

B. P. Bandgar et al. / Bioorg. Med. Chem. 18 (2010) 1364–1370
1369
4.2.2. 1-(4-Bromo-phenyl)-3-(2,4-dimethoxy-phenyl)-
4.4. Anti-inflammatory and cytotoxicity assay
propenone (3e)
Light Yellow, mp: 152 °C, IR (KBr) 2920, 2827, 1648, 1597, 1443,
Proinflammatory cytokine production by lipopolysaccharide
(LPS) in THP-1 cells was measured according to the method de-
scribed by Hwang et al.29 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 phor-
bol myristate acetate (PMA, Sigma). Following cell plating, the test
compounds 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
1357, 1263, 1157, 1022, 813 cmꢁ1 1H NMR (CDCl3, 300 MHz) d
;
7.95 (d, J = 7.1 Hz, 2H), 7.67 (d, J = 15.5 Hz, 1H), 7.52 (d,
J = 15.5 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.22 (s, 1H), 6.93 (d,
J = 8.4 Hz, 1H), 6.87 (d, J = 7.1 Hz, 2H), 3.91 (s, 3H), 3.87 (s, 3H);
MS (ESI) m/z = 347 (M+1).
4.2.3. 1-(2,4-Dichloro-phenyl)-3-(2,4-dimethoxy-phenyl)-
propenone (3i)
Yellow solid, mp: 125 °C, IR (KBr) 2968, 2832, 1653, 1512, 1469,
1315, 1262, 1142, 1025, 801 cmꢁ1
;
1H NMR (CDCl3, 300 MHz) d
concentration of 1
bated at 37 °C for 24 h in 5% CO2. After incubation, supernatants
were harvested, and assayed for TNF- and IL-6 by ELISA as de-
lg/mL in each well. Plates were further incu-
8.10 (s, 1H), 7.85 (m, 1H), 7.79 (d, J = 15.6 Hz, 1H), 7.59 (d,
J = 8.3 Hz, 1H), 7.30 (d, J = 15.6 Hz, 1H), 7.26 (d, J = 8.9 Hz, 1H),
7.17 (s, 1H), 6.91 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H); MS
(ESI) m/z = 339.1 (M+2).
a
scribed by the manufacturer (BD Biosciences). The cells were
simultaneously evaluated for cytotoxicity using CCK-8 from Dojin-
do Laboratories. Percent inhibition of cytokine release compared to
4.2.4. 3-(2,4-Dimethoxy-phenyl)-1-(4-nitro-phenyl)-propenone
(3k)
the control was calculated. The 50% inhibitory concentration (IC50
values were calculated by a nonlinear regression method.
)
Yellow solid, mp: 134 °C, IR (KBr) 2930, 1650, 1523, 1342, 1257,
1161, 1016, 905 cmꢁ1
;
1H NMR (CDCl3, 300 MHz) d 8.00 (d,
4.5. In vitro antioxidant activity (DPPH method)
J = 15.6 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H),
7.70–7.52 (m, 3H), 7.37 (d, J = 15.6 Hz, 1H), 6.58 (dd, J = 8.6,
2.1 Hz, 1H), 6.50 (d, J = 2.1 Hz, 1H), 3.90 (s, 3H), 3.88 (s, 3H); MS
(ESI) m/z = 315.1 (M+1).
The compounds (3a–s) were evaluated for their in vitro free
radical scavenging activity by the 2,20diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging method described by Blois .30 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 mix-
ture and allowed to react at room temperature. After 30 min,
absorbance values were measured at 520 nm and converted to
%antioxidant activity. For a comparative study the Butylated hy-
droxyl anisole (BHA) was used as the standard. The percentage
inhibition activity was calculated by using a formula.
4.2.5. 1-Phenyl-3-(3,4,5-trimethoxy-phenyl)-propenone (3l)
Light yellow solid, mp: 173 °C, IR (KBr disk): 3069, 2932, 1640,
1696, 1272 cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.90 (d, J = 16.4 Hz,
.
1H), 7.81 (d, J = 8.2 Hz, 2H), 7.56 (d, J = 16.4 Hz, 1H), 7.57 (d,
J = 8.6 Hz, 1H), 6.96 (d, J = 8.6 Hz, 2H), 6.50 (dd, J1 = 8.4,
J2 = 2.3 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 3.88 (s, 3H), 3.86 (s, 3H),
3.88 (s, 3H); MS (ESI) m/z = 299 (M+1).
% Activity
¼ ½1 ꢁ OD of test compound=OD of control compoundꢃ ꢂ 100:
4.3. Anticancer activity
Cytotoxic assay is performed on ACHN (human renal cell carci-
noma), Panc 1 (human pancreatic carcinoma), Calu 1 (human non
small cell lung carcinoma), H460 (human non cell lung carcinoma)
and HCT 116 (human colon carcinoma) cell line using propidium
iodide fluorescence assay.28 Dyes such as propidium iodide (PI),
which bind DNA, provide a rapid and accurate means for quantitat-
ing total nuclear DNA. The fluorescence signal intensity of the PI is
directly proportional to the amount of DNA in each cell, PI is not
able to penetrate an intact membrane, and so cells must first be
permeabilised. Seed cells of 3000–7500 cells/well were placed in
2000 lL of tissue culture grade 96 well plates and allowed them
to recover for 24 h in humidified 5% CO2 incubator at 37 °C. After
culturing for 24 h compounds (in 0.1% DMSO) were added onto
triplicate wells with 10 lM concentrations. 0.1% DMSO alone was
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
windows ME 9820000 and XP operating system. Other parameters
like HBA, HBD, molecular PSA, ionization potential, drug score and
drug likeness of the compounds were also studied using online
Osiris property explorer for drug bioavailability of chemical
compounds.19
used as control. After 48 h in humidified 5% CO2 incubator at
37 °C condition, the medium was removed and treated with
25 lL of propidium iodide (50 lg/mL in water/medium) per well.
Acknowledgements
The plates were freeze at ꢁ80 °C for 24 h then thawed and allowed
it to come at room temperature and the plate absorbance was read
on fluorometer (Polar-Star BMG Tech), using 530 nm excitation
and 620 nm emission wavelength. Lastly percent cytotoxicity of
the compounds was calculated by using following formula.
The authors are thankful to Mr. Mahesh Nambiar and Mrs. Asha
Almeida, Piramal Life Sciences Ltd, Mumbai for anti-inflammatory,
anticancer activities and to the Director, School of Life Sciences,
Swami Ramanand Teerth Marathawada University, Nanded, for
antioxidant activity.
Percent Cytotoxicity
Reading of control ꢁ Reading of treated cells
References and notes
¼
ꢂ 100
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