Synthesis and biological screening of a combinatorial library of β-chlorovinyl chalcones as anticancer, anti-inflammatory and antimicrobial agents

Article abstract of DOI:10.1016/j.bmc.2009.12.077

A combinatorial library of β-chlorovinyl chalcones (4) were synthesized by Claisen-Schmidt condensation reaction. Catalytic reaction of substituted 3-chloro-3-phenyl-propenal (2) and 1-(2,4-dimethoxy-phenyl)-ethanone or 1-(4-methoxy-phenyl)-ethanone (3) i

Full text of DOI:10.1016/j.bmc.2009.12.077

Bioorganic & Medicinal Chemistry 18 (2010) 2060–2065
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological screening of a combinatorial library of b-chlorovinyl
chalcones as anticancer, anti-inflammatory and antimicrobial agents
b
Babasaheb P. Bandgar a,b, , Shrikant S. Gawande
*
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
a r t i c l e i n f o
a b s t r a c t
Article history:
A combinatorial library of b-chlorovinyl chalcones (4) were synthesized by Claisen–Schmidt condensa-
tion reaction. Catalytic reaction of substituted 3-chloro-3-phenyl-propenal (2) and 1-(2,4-dimethoxy-
phenyl)-ethanone or 1-(4-methoxy-phenyl)-ethanone (3) in alkaline conditions furnished the target
compound 5-chloro-1-(2,4-dimethoxy-phenyl)-5-phenyl-penta-2,4-dien-1-one (4). The synthesized
compounds were screened for their biological activity viz. anticancer, anti-inflammatory and antimicro-
bial activities. Synthesized compounds 4g and 4h revealed promising anti-inflammatory activity (66–67%
Received 17 December 2009
Revised 29 December 2009
Accepted 31 December 2009
Available online 11 January 2010
This article is dedicated to my grandfather
late Pralhadrao G. Gawande.
TNF-
cell lines and found to be nontoxic to slightly toxic. Furthermore, the anticancer activity (30–40%) was
shown by compounds 4d, 4e, 4h and 4b at 10 M concentrations against ACHN followed by Calu 1, Panc1,
HCT116 and H460 cell lines. Some of the compounds 4d, 4e, 4a, 4i and 4b revealed promising antimicro-
bial activity at MIC 50–100 g/mL against selected pathogenic bacteria and fungi.
Ó 2010 Published by Elsevier Ltd.
a and 95–97% IL-6 inhibitory activity at 10 lM). Cytotoxicity of the compounds checked using CCK-8
l
Keywords:
b-Chlorovinyl chalcones
Anticancer
Anti-inflammatory activity
Antibacterial activity
Antifungal activity
l
1. Introduction
more acute in developing countries due to nonavailability of de-
sired medicines and emergence of widespread drug resistance.8
Chalcone derivatives have received a great deal of attention due
to their relatively simple structures and wide variety of pharmaco-
logical activities reported for these compounds including antican-
cer, anti-inflammatory, antioxidant, cytotoxicity, antimicrobial,
analgesic, antipyretic, anti-anginal, anti-hepatotoxic, antimalarial
and anti-allergic activities. These activities are largely attributed
due to the unsaturated ketone moiety.9 Moreover, introduction of
substituents into the two aryl rings also a subject of interest be-
cause it leads to useful structure–activity relationship (SAR) con-
clusions and thus helps to synthesize pharmacologically active
chalcones.10 Many of the combinatorial libraries are synthesized
and studied for their biological activities.11,12
Combinatorial organic synthesis is still of great interest because
it promises to accelerate the drug discovery process as well as
speed up the development of new catalysts.1,2 The synthesis of
unbiased compound libraries offers the advantage of rapidly pro-
ducing a considerable number of potentially active compounds
with diverse structures and thus enabling medicinal chemistry
projects to more rapidly identify potentially interesting drug can-
didates to be further pursued.3 Cancer is one of the leading causes
of death in the present society.4 Over 1 million cases of cancer oc-
cur in the United States annually and cancer-related deaths are
estimated to reach 12 million worldwide, by the year 2015.5 Acute
and chronic inflammation and different types of arthritis are the
inflammatory disorders, which are a big blow to humanity and
continual search for newer non-steroidal anti-inflammatory agents
is the only way to fortify against this awful threat.6 There has been
a tremendous burst of new development activity during the 1990s
that has culminated in the launch of several new classes of anti-
inflammatory drugs such as the leukotriene antagonists, cyclooxy-
genase 2 (COX-2) inhibitors and tumor necrosis factor (TNF) block-
ers.7 Moreover, infectious diseases caused by bacteria, fungi,
viruses and parasites are also a major threat to public health, de-
spite tremendous progress in medicinal chemistry. The impact is
In the previous communications, we reported the pyrazole chal-
cones and simple chalcones as anticancer, anti-inflammatory,
antioxidant and antimicrobialagents.13 Here, we reporta combinato-
rial synthesis and biological evaluation of substituted b-chlorovinyl
chalcones asanticancer, anti-inflammatoryand antimicrobialagents.
2. Results and discussion
2.1. Chemistry
In the present investigation 5-chloro-1-(2,4-dimethoxy-phe-
nyl)-5-phenyl-penta-2,4-dien-1-one (4) have been prepared by
* Corresponding author. Tel./fax: +91 217 2351300.
E-mail address: bandgar_bp@yahoo.com (B.P. Bandgar).
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.12.077

B. P. Bandgar, S. S. Gawande / Bioorg. Med. Chem. 18 (2010) 2060–2065
2061
Table 1
the Claisen–Schmidt condensation of 1-(2,4-dimethoxy-phenyl)-
Structure–analytical data of b-chlorovinyl chalcone derivatives
ethanone or 1-(4-methoxy-phenyl)-ethanone (3) and substituted
3-chloro-3-phenyl-propenal (2) by the known literature method14
(Scheme 1). Substituted b-chlorovinyl aldehydes (4) were synthe-
sized using the method described in the literature with minor
modifications.15 The residue was purified on column chromatogra-
phy using silica gel with 10% ethyl acetate in hexane. The struc-
tures of compounds were confirmed by spectral data (IR, 1H NMR
and MS). The chemical profile of the compounds is as shown in Ta-
ble 1.
O
Cl R1
R2
R3
R2'
R1'
R4
Entry R01
R20
R1
R2 R3
R4 Reaction time
(h)
Yield
(%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
H
H
H
H
H
Br
Cl
F
H
OCH3
OCH3
Br
Cl
F
H
H
H
H
H
H
H
H
H
H
H
H
2
85
89
75
78
91
69
88
84
77
64
93
70
H
H
Cl
H
H
H
H
H
Cl
H
H
2.2
2.4
2.1
2
2.2. Biological evaluation
Anti-inflammatory activity of the synthesized compounds was
determined in terms of TNF-
4a–l does not revealed any acceptable TNF-
cept compounds 4g and 4h at 10 M concentration. As compared
to the standard dexamethasone (73% at 1 M), both the com-
pounds 4g (66% at 10 M) and 4h (67% at 10 M) equally inhibit
the TNF- activity therefore are potent. All the synthesized com-
a
and IL-6 inhibition. The compounds
OCH3 OCH3 OCH3
3
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
2.2
2.3
2.5
2.9
2.1
2.8
a
inhibitory activity ex-
l
l
4j
4k
4l
H
OCH3
OCH3
l
l
a
OCH3 OCH3
pounds were found effective in IL-6 inhibitory activity. Compounds
4g and 4h showed promising IL-6 inhibitory activity (95–97% at
10 lM) as compared to standard dexamethasone (84% at 1 lM).
structure of chalcones that possess antibacterial activity of synthe-
sized or modified natural chalcones.16 The antibacterial activity of
the compounds is summarized in Table 4 and found to be compa-
rable with the standard drug tetracycline. MIC50 were recorded as
the minimum concentration of a compound that inhibits the 50%
growth of the tested microorganisms. The compounds 4a, 4b, 4d,
4e, 4i and 4j showed 100% inhibition of all the selected bacterial
Compounds 4e, 4c, 4b, 4l and 4a showed moderate activity (36–
49% inhibition), while compounds 4f, 4k, 4d, 4i and 4j have negli-
gible activity. Cytotoxicity of the compounds was tested for their
bioavailability and evaluated using CCK-8 cells. The compounds
4a–f, 4i and 4k were completely nontoxic while 4g, 4h, 4j and 4l
were slightly toxic (Table 2).
Antiproliferative activity of compounds in cell lines from multi-
ple cancer origin is described in Table 3. For the activity, growing
cultures of each cell lines were exposed to 10 lM concentration
strains at MIC value of the range of 50 lg/mL, while compounds
4c, 4k, 4h, 4l, 4g and 4f were found to have moderate to negligible
antibacterial activity. Out of five selected bacterial strains Esche-
richia coli was affected more followed by Proteus vulgaris, Klebsiella
pneumoniae, Staphylococcus aureus, and Bacillus subtilis by all the
compounds. The crude mortality from opportunistic fungal infec-
tions still exceeds 50% in most human studies and has been re-
ported to be as high as 95% in a bone marrow transplant
recipients infected with Aspergillus spp.17 All the tested compounds
(4a, 4d, 4e, 4h, 4i and 4k) illustrated significant antifungal activity
as compared to the reference drug nystatin followed by the com-
of the different chalcones. In particular, the compounds 4d and
4e were active against all the selected cell lines (30–40% inhibition
at 10
lM concentration). Further the compounds 4h, 4b, 4c and 4a
(30–39% inhibition at 10
lM concentration) gave moderate inhib-
itory activity and the remaining compounds (4g, 4j, 4k, 4l, 4f and
4i) were less effective. Moreover, if the cell lines consider, ACHN
was affected more followed by Calu 1, Panc1, HCT116 and H460.
The obtained results were found comparable with the standard
flavopiridol and gemcitabine.
pounds 4b, 4g, 4j and 4l at MIC value 100 lg/mL. Compounds 4c
and 4f inhibit only Aspergillus flavus and Aspergillus niger,
respectively.
Antibacterial activity of chalcones is being increasingly docu-
mented. Many research groups either isolated or identified the
R1
O
R1
Cl
R2
R3
R2
R3
CHO
a
R4
R4
1
2
O
Cl
R1
O
Cl
R1
R4
OHC
R2
R3
R2
R3
b
+
R'2
R'1
R2'
R1'
R4
3
2
4
Scheme 1. General synthetic route for the synthesis of b-chlorovinyl chalcone derivatives. Reagents and conditions: (a) DMF, POCl3, 0 °C–80 °C, 1 h (b) 40% NaOH, EtOH,
10 °C, 2–3 h.

2062
B. P. Bandgar, S. S. Gawande / Bioorg. Med. Chem. 18 (2010) 2060–2065
Table 2
O
O
Anti-inflammatory activity of chalcone derivatives
2
4
Compounds
% Inhibition at 10
l
M
Toxicity
1
3
TNF-a
IL-6
O
O
Cl
Cl
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
0
0
0
0
0
36
43
44
7
49
27
97
95
6
0
0
0
0
0
(5h)
Cl
4
0
0
2
66
67
0
1
2
30
28
2
35
0
1
3
3
11
40
84
0
73
35
0
(4h)
Dexamethasone (1
lM)
Figure 1. Comparative anticancer and anti-inflammatory activity of 4h and 5h.
Table 3
Anticancer activity of chalcone derivatives
only (Fig. 1). This can be evaluated by comparing the anticancer
and anti-inflammatory activity of the synthesized compounds
(4h) with that of the commercially available chalcone (5h) as sum-
marized in Table 5. The results revealed that the compound 4h
have an increased activity than the regular chalcones (Fig. 2);
hence a new chalcone library with enhanced biological activity
was confirmed.
Compounds
Anticancer activity at 10
l
M concn
ACHN
Panc1
Calu 1
H460
HCT116
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
39
37
30
32
33
26
39
35
17
22
28
33
68
70
33
32
23
32
31
23
25
30
1
18
16
7
75
71
27
32
36
40
31
24
29
32
0
20
38
7
68
70
16
21
26
31
33
18
20
33
14
26
19
15
84
68
22
26
14
37
31
29
20
29
0
31
16
0
71
76
3. Conclusion
A new series of b-chlorovinyl chalcones exhibiting anticancer,
anti-inflammatory and antimicrobial activity was synthesized.
From the results of the tested compounds, 4g and 4h showed
promising activity against TNF-a and IL-6. The promising antipro-
liferative activity was given by the compounds 4d and 4e followed
by 4h, 4b, 4a and 4c as compared to standard drug. ACHN was af-
fected more followed by Calu 1, Panc1, HCT116 and H460. All the
compounds revealed good to moderate antimicrobial activity as
compared to the reference drug tetracycline and nystatin at MIC
Flavopiridol (700 nM)
Gemcitabine (500 nM)
50–100 lg/mL.
Table 4
Antibacterial activity of b-chlorovinyl chalcones (zone of inhibition in mm)
4. Experimental
4.1. Synthesis
Compound
Bacteria (MIC at 50
l
g/mL)
Fungi (MIC at 100 lg/mL)
EC
PV
KN
SA
BS
AN
AF
TV
CA
PC
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
13
10
11
12
14
—
10
18
16
13
15
13
—
18
15
—
16
12
—
11
10
15
17
12
—
12
13
—
16
12
—
12
—
16
13
12
—
16
14
10
18
20
—
12
14
—
14
15
13
—
13
8
—
18
12
10
15
12
—
16
12
—
13
14
—
12
—
—
10
8
—
—
10
8
—
11
—
16
14
—
10
12
—
12
12
15
—
15
—
4.1.1. General procedure for synthesis of substituted b-
chlorovinyl aldehyde (2a–f)
Substituted acetophenone 1 (10 mmol) was dissolved in DMF
(75 mL) at room temperature and then POCl3 (5.58 mL, 60 mmol)
was added dropwise at 0 °C. After complete addition of POCl3,
the reaction mixture was warmed to room temperature and then
heated at 80 °C for 1 h. The reaction mass was poured onto the
crushed ice and then neutralized with a 10% aqueous NaOH solu-
tion. The product was extracted with dichloromethane
(3 ꢀ 100 mL) and extract was washed with water (5–6 times) to re-
move the excess DMF. The organic layer was dried over anhydrous
Na2SO4 and evaporated. Finally the crude material was purified by
column chromatography using petroleum ether and ethyl acetate
(9:1).
—
—
—
—
8
8
—
12
15
8
14
10
—
12
14
11
14
12
—
11
10
17
10
15
—
16
15
—
—
16
—
15
15
—
12
18
—
14
11
—
Tetracycline
Nystatin
Control
20
—
18
—
—
—
13
16
12
11
14
—
—
—
Data represent is 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); CA—Candida albicans (MTCC 227); PC—
4.1.2. Synthesis of b-chlorovinyl chalcones (4a–l)
Penicillium chrysogenum (MTCC 1996); not detected —; trace activity
.
A mixture of 1-(2,4-dimethoxy-phenyl)-ethanone 3 (0.180 g,
1 mmol) and 3-(4-bromophenyl)-3-chloro-propenal 2a (0.247 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 h. Then this reaction mixture
was poured over crushed ice and acidified with dil HCl. The yellow
We also tried to predict that whether the promising activity
(anticancer and anti-inflammatory) of 4h was due to the Cl substi-
tution at fourth position or its due to the enone system of chalcone

B. P. Bandgar, S. S. Gawande / Bioorg. Med. Chem. 18 (2010) 2060–2065
2063
Table 5
Comparative study of anticancer and anti-inflammatory activity of 4h and 5h compounds
Compounds
Anticancer activity at 10
l
M concn
H460
% Inhibition at 10
l
M
Toxicity
ACHN
Panc1
Calu 1
HCT116
TNF-a
IL-6
4h
5h
35
30
30
24
32
27
33
23
29
24
67
45
95
62
28
32
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 5-(4-bromo-phenyl)-
J = 6 Hz), 7.71 (m, 1H), 7.40 (m, 3H), 6.61 (d, 1H, J = 6 Hz). EIMS
(70 eV) m/z: 201 (M+1).
5-chloro-1-(2,4-dimethoxy-phenyl)-penta-2,4-dien-1-one
(Scheme 1).
(4a)
4.2.5. 3-(4-Methoxyphenyl)-3-chloro-propenal (2e)
Yellow solid; mp: 78 °C, IR (KBr) 2912, 2721, 1656, 1601, 1257,
1278, 829 cmꢁ1 1H NMR (300 MHz, CDCl3): d 10.19 (d, 1H,
.
4.2. Physico-chemical data of synthesized compounds
J = 6.8 Hz), 7.72 (d, 2H, J = 8.8 Hz), 6.79 (d, 2H, J = 8.8 Hz), 6.62 (d,
2H, J = 6.8 Hz), 3.89 (s, 3H). EIMS (70 eV) m/z: 197 (M+1).
The purified chalcones were obtained in yields of 70–93%. Their
structures were identified using infrared spectroscopy (IR), nuclear
magnetic resonance spectroscopy (NMR) and mass analyses. Melt-
ing points were determined with a Microquimica MG APF-301
apparatus and are uncorrected. IR spectra were recorded with a
FT Perkin Elmer 16 PC spectrometer on KBr disks. 1H NMR spectra
were recorded on a Brucker Ac-200 F (300 MHz) with tetramethyl-
silane as an internal standard. Mass spectra were obtained on a
JMX-HX 100 mass spectrometer. The purity of the synthesized sub-
stances was analyzed by thin-layer chromatography (TLC) using
4.2.6. 3-(2,4-Dimethoxyphenyl)-3-chloro-propenal (2f)
Black solid; mp = 78 °C; IR (KBr) 2943, 2734, 1634, 1589, 1253,
1050, 956 cmꢁ1 1H NMR (300 MHz, CDCl3): d 10.173 (d, 1H,
.
J = 6.9 Hz), 7.372 (dd, 1H, J = 2.1 Hz and 2.1 Hz), 7.168 (t, 1H),
6867 (d, 1H, J = 8.6 Hz), 6.573 (d, 1H, J = 6.9 Hz), 3.880 (s, 1H),
3.869 (s, 1H). EIMS (70 eV) m/z: 227 (M+1).
4.2.7. 5-(4-Bromo-phenyl)-5-chloro-1-(2,4-dimethoxy-phenyl)-
penta-2,4-dien-1-one (4a)
Merck silica pre-coated aluminum plates 200
l
m in thickness with
Light yellow solid; mp = 132 °C; IR (KBr) 3018 (Ar-H), 1610
several solvent systems of different polarities. Compounds were
visualized with ultraviolet light (254 nm) and purified on column
chromatography (silica gel with 10% ethyl acetate in hexane).
The physical and spectral data of new b-chlorovinyl chalcones
(4a–l) are given below.
(C@O), 1591 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.79 (dd,
.
1H, J = 12.6 Hz and 10.4 Hz), 7.57–7.49 (m, 2H), 7.39 (d, 1H,
J = 12.6 Hz), 7.24 (s, 1H), 7.19–7.10 (m, 2H), 6.94 (d, 1H,
J = 10.4 Hz), 6.54 (d, 1H, J = 8.1 Hz), 6.47 (d, 1H, J = 8.1 Hz), 3.86
(s, 3H), 3.87 (s, 3H). EIMS (70 eV) m/z: 406 (M+).
4.2.1. 3-(4-Bromophenyl)-3-chloro-propenal (2a)
4.2.8. 5-Chloro-5-(4-chloro-phenyl)-1-(2,4-dimethoxy-phenyl)-
penta-2,4-dien-1-one (4b)
Light yellow solid; mp: 67 °C, IR (KBr) 2986, 2731, 1640, 1599,
1210, 1034, 934 cmꢁ1
.
1H NMR (300 MHz, CDCl3): d 10.21 (d, 1H,
Light yellow solid; mp = 141 °C; IR (KBr) 3010 (Ar-H), 1615
J = 6.8 Hz), 7.62 (d, 2H, J = 8.8 Hz), 7.60 (d, 2H, J = 8.8 Hz), 6.65 (d,
2H, J = 6.8 Hz). EIMS (70 eV) m/z: 247 (M+1).
(C@O), 1599 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.72 (dd,
.
1H, J = 12.6 Hz and 10.4 Hz), 7.56–7.47 (m, 2H), 7.37 (d, 1H,
J = 12.6 Hz), 7.20 (s, 1H), 6.95 (d, 1H, J = 10.4 Hz), 6.55 (d, 2H,
J = 8.0 Hz), 6.45 (d, 2H, J = 8.0 Hz), 3.86 (s, 3H), 3.85 (s, 3H). EIMS
(70 eV) m/z: 362 (M+).
4.2.2. 3-(4-Chlorophenyl)-3-chloro-propenal (2b)
Yellow solid; mp: 88 °C, IR (KBr) 2954, 2722, 1641, 1590, 1230,
1023, 915 cmꢁ1 1H NMR (300 MHz, CDCl3): d 10.12 (d, 1H,
.
J = 6.8 Hz), 7.71 (d, 2H, J = 8.8 Hz), 7.63 (d, 2H, J = 8.8 Hz), 6.69 (d,
2H, J = 6.8 Hz). EIMS (70 eV) m/z: 201 (M+1).
4.2.9. 5-Chloro-1-(2,4-dimethoxy-phenyl)-5-(4-fluoro-phenyl)-
penta-2,4-dien-1-one (4c)
Yellow solid; mp = 121 °C; IR (KBr) 3025 (Ar-H), 1622 (C@O),
4.2.3. 3-(4-Fluorophenyl)-3-chloro-propenal (2c)
1588 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.81 (dd, 1H,
.
Colorless solid; mp: 93 °C, IR (KBr) 2918, 2716, 1653, 1600,
J = 12.8 Hz and 10.8 Hz), 7.73 (d, 1H, J = 12.8 Hz), 7.70–7.65 (m,
2H), 7.24 (s, 1H), 7.17–7.03 (m, 2H), 6.88 (d, 1H, J = 10.8 Hz), 6.54
(d, 1H, J = 8.0 Hz), 6.46 (d, 1H, J = 8.0 Hz), 3.88 (s, 3H), 3.86 (s,
3H). EIMS (70 eV) m/z: 346 (M+).
1250, 1276, 824 cmꢁ1 1H NMR (300 MHz, CDCl3): d 10.26 (d, 1H,
.
J = 6 Hz), 7.81 (m, 2H, J = 8 Hz), 7.62 (d, 2H, J = 8 Hz), 6.69 (d, 2H,
J = 6 Hz). EIMS (70 eV) m/z: 185 (M+1).
4.2.4. 3-(3-Chlorophenyl)-3-chloro-propenal (2d)
4.2.10. 5-Chloro-5-(3-chloro-phenyl)-1-(2,4-dimethoxy-
phenyl)-penta-2,4-dien-1-one (4d)
Light yellow solid; mp: 78 °C, IR (KBr) 2923, 2712, 1643, 1599,
1267, 1056, 756 cmꢁ1
.
1H NMR (300 MHz, CDCl3): d 10.10 (d, 1H,
Light yellow solid; mp = 113 °C; IR (KBr) 3014 (Ar-H), 1619
(C@O), 1581 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.81 (dd,
.
1H, J = 12.6 Hz and 10.4 Hz), 7.71 (d, 1H, J=12.6 Hz), 7.64 (d, 1H,
J = 8.7 Hz), 7.63 (d, 1H, J = 8.7 Hz), 7.24 (s, 1H), 7.14 (d, 1H,
J = 10.8 Hz), 6.91–6.87 (m, 2H), 6.56–6.45 (m, 2H), 3.88 (s, 3H),
3.86 (s, 3H). EIMS (70 eV) m/z: 362 (M+).
100
4h
80
5h
60
40
20
0
4.2.11. 5-Chloro-1-(2,4-dimethoxy-phenyl)-5-(4-methoxy-
phenyl)-penta-2,4-dien-1-one (4e)
Light yellow solid; mp = 148 °C; IR (KBr) 3018 (Ar-H), 1616
(C@O), 1594 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.83 (dd,
.
1H, J = 12.3 Hz and 10.8 Hz), 7.72 (d, 1H, J = 12.3 Hz), 7.64 (d, 1H,
J = 8.7 Hz), 7.53 (d, 1H, J = 8.7 Hz), 7.24 (s, 1H), 7.10 (d, 1H,
Figure 2. Graphical representation of anticancer and anti-inflammatory activity of
4h and 5h.

2064
B. P. Bandgar, S. S. Gawande / Bioorg. Med. Chem. 18 (2010) 2060–2065
J = 10.8 Hz), 6.90–6.85 (m, 2H), 6.55–6.52 (m, 2H), 3.87 (s, 3H), 3.85
(s, 3H), 3.83 (s, 3H). EIMS (70 eV) m/z: 359 (M+1).
2H), 6.40–6.56 (m, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H). EIMS
(70 eV) m/z: 358 (M+).
4.2.12. 5-Chloro-1,5-bis-(2,4-dimethoxy-phenyl)-penta-2,4-
dien-1-one (4f)
4.3. Anti-inflammatory and cytotoxicity assay
Light yellow solid; mp = 123 °C; IR (KBr) 3011 (Ar-H), 1613
Proinflammatory cytokine production by lipopolysaccharide
(LPS) in THP-1 cells was measured according to the method de-
scribed by Hwang et al.18 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 separately and
the plate was incubated for 30 min at 37 °C. Finally, LPS (E. coli
0127:B8, Sigma Chemical Co., St. Louis, MO) was added, at a final
(C@O), 1597 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.80 (dd,
.
1H, J = 12.2 Hz and 10.8 Hz), 7.73 (d, 1H, J = 12.2 Hz), 7.63 (d, 1H,
J = 8.7 Hz), 7.61 (d, 1H, J = 8.7 Hz), 7.26 (s, 1H), 7.10 (d, 1H,
J = 10.8 Hz), 6.90–6.85 (m, 2H), 6.54–6.51 (m, 1H), 3.87 (s, 3H),
3.86 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H). EIMS (70 eV) m/z: 388 (M+).
4.2.13. 5-(4-Bromo-phenyl)-5-chloro-1-(4-methoxy-phenyl)-
penta-2,4-dien-1-one (4g)
Light yellow solid; mp = 132 °C; IR (KBr) 3015 (Ar-H), 1610
(C@O), 1591 (C@C) cmꢁ1
.
1H NMR (300 MHz, CDCl3): d 7.76 (dd,
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-
1H, J = 12.8 Hz and 10.8 Hz), 7.56–7.48 (m, 2H), 7.38 (d, 1H,
J = 12.8 Hz), 7.20–7.12 (m, 2H), 6.95 (d, 1H, J = 10.8 Hz), 6.53 (d,
2H, J = 8.0 Hz), 6.46 (d, 2H, J = 8.0 Hz), 3.86 (s, 3H). EIMS (70 eV)
m/z: 376 (M+).
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
4.2.14. 5-Chloro-5-(4-chloro-phenyl)-1-(4-methoxy-phenyl)-
penta-2,4-dien-1-one (4h)
the control was calculated. The 50% inhibitory concentration (IC50
values were calculated by a nonlinear regression method.
)
Yellow solid; mp = 138 °C; IR (KBr) 3021 (Ar-H), 1617 (C@O),
1586 (C@C) cmꢁ1
.
1H NMR (300 MHz, CDCl3): d 7.74 (dd, 1H,
4.4. Anticancer activity
J = 12.8 Hz and 10.8 Hz), 7.58–7.46 (m, 2H), 7.36 (d, 1H, 12.8 Hz),
7.20–7.15 (m, 2H), 6.90 (d, 1H, J = 10.8 Hz), 6.56 (d, 2H,
J = 8.1 Hz), 6.49 (d, 2H, J = 8.1 Hz), 3.86 (s, 3H). EIMS (70 eV) m/z:
332 (M+).
Cytotoxic assay is performed on ACHN (human renal cell carci-
noma), Panc1 (human pancreatic carcinoma), Calu 1 (human non-
small cell lung carcinoma), H460 (human non-cell lung carcinoma)
and HCT116 (human colon carcinoma) cell line using propidium io-
dide fluorescence assay.19 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
permeabilized. Seed cells of 3000–7500 cells/well were placed in
4.2.15. 5-Chloro-5-(4-fluoro-phenyl)-1-(4-methoxy-phenyl)-
penta-2,4-dien-1-one (4i)
Yellow solid; mp = 108 °C; IR (KBr) 3022 (Ar-H), 1623 (C@O),
1596 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.81 (dd, 1H,
.
J = 12.8 Hz and 10.8 Hz), 7.73 (d, 1H, J = 12.8 Hz), 7.73 (d, 2H,
J = 8.4 Hz), 7.22 (d, 2H, J = 8.4 Hz), 7.02 (d, 2H, J = 8.0 Hz), 6.89 (d,
1H, J = 10.8 Hz), 6.52 (d, 2H, J = 8.0 Hz), 3.86 (s, 3H). EIMS (70 eV)
m/z: 316 (M+).
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
4.2.16. 5-Chloro-5-(3-chloro-phenyl)-1-(4-methoxy-phenyl)-
penta-2,4-dien-1-one (4j)
triplicate wells with 10 lM concentrations. 0.1% DMSO alone was
used as control. After 48 h in humidified 5% CO2 incubator at
Light yellow solid; mp = 98 °C; IR (KBr) 3010 (Ar-H), 1616
37 °C condition, the medium was removed and treated with
25 lL of propidium iodide (50 lg/mL in water/medium) per well.
(C@O), 1586 (C@C) cmꢁ1
.
1H NMR (300 MHz, CDCl3): d 7.82 (dd,
1H, J = 12.7 Hz and 10.8 Hz), 7.72 (d, 1H, J = 12.7 Hz), 7.64 (d, 2H,
J = 8.0 Hz), 7.25 (d, 2H, J = 8.0 Hz), 7.15 (d, 2H, J = 10.8 Hz), 6.90–
6.87 (m, 2H), 6.56–6.40 (m, 2H), 3.87 (s, 3H). EIMS (70 eV) m/z:
332 (M+).
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.
4.2.17. 5-Chloro-1,5-bis-(4-methoxy-phenyl)-penta-2,4-dien-1-
one (4k)
Reading of controlꢁReading of treated cells
Percent cytotoxicity ¼
Reading of control
Light yellow solid; mp = 152 °C; IR (KBr) 3025 (Ar-H), 1629
ꢀ100
(C@O), 1594 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.81 (dd,
.
1H, J = 12.8 Hz and 10.8 Hz), 7.70 (d, 1H, J = 12.8 Hz), 7.64 (d, 2H,
J = 8.7 Hz), 7.20 (s, 1H), 7.12 (d, 1H, J = 10.8 Hz), 6.75–6.60 (m,
2H), 6.40–6.56 (m, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H). EIMS
(70 eV) m/z: 329 (M+1).
The results were compared with the standard drug inhibitors
flavopiridol (700 nM) and gemcitabine (500 nM).
4.5. Antimicrobial activity (agar diffusion method)
Antimicrobial activity of all synthesized compounds was deter-
mined by agar diffusion method.20,21 All human pathogenic bacte-
ria viz. B. subtilis (MTCC 1789), K. pneumoniae (NCIM 2957), S.
aureus (MTCC 96), P. vulgaris (MTCC 1771), E. coli (MTCC 1650)
and fungi viz. Trichoderma viridae (MTCC 167), A. flavus (MTCC
2501), A. niger (MTCC 1781), Candida albicans (MTCC 227) and
4.2.18. 5-Chloro-5-(2,4-dimethoxy-phenyl)-1-(4-methoxy-
phenyl)-penta-2,4-dien-1-one (4l)
Light yellow solid; mp = 129 °C; IR (KBr) 3012 (Ar-H), 1617
(C@O), 1592 (C@C) cmꢁ1 1H NMR (300 MHz, CDCl3): d 7.81 (dd,
.
1H, J = 12.8 Hz and 10.8 Hz), 7.70 (d, 1H, J = 12.8 Hz), 7.64 (d, 2H,
J = 8.7 Hz), 7.20 (s, 1H), 7.12 (d, 1H, J = 10.8 Hz), 6.75–6.60 (m,

B. P. Bandgar, S. S. Gawande / Bioorg. Med. Chem. 18 (2010) 2060–2065
2065
References and notes
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The authors are thankful and acknowledge Mr. Mahesh Namb-
iar and Mrs. Asha Almeida, Piramal Life Sciences Ltd., Mumbai for
screening of the compounds for anticancer and anti-inflammatory
activities.