Synthesis of chalcones with anticancer activities

Article abstract of DOI:10.3390/molecules17066179

Several chalcones were synthesized and their in vitro cytotoxicity against various human cell lines, including human breast adenocarcinoma cell line MCF-7, human lung adenocarcinoma cell line A549, human prostate cancer cell line PC3, human adenocarcinoma cell line HT-29 (colorectal cancer) and human normal liver cell line WRL-68 was evaluated. Most of the compounds being active cytotoxic agents, four of them with minimal IC₅₀ values were chosen and studied in detail with MCF-7 cells. The compounds 1, 5, 23, and 25 were capable in eliciting apoptosis in MCF-7 cells as shown by multiparameter cytotoxicity assay and caspase-3/7, -8, and -9 activities (p < 0.05). The ROS level showed 1.3-fold increase (p < 0.05) at the low concentrations used and thus it was concluded that the compounds increased the ROS level eventually leading to apoptosis in MCF-7 cells through intrinsic as well as extrinsic pathways.

Full text of DOI:10.3390/molecules17066179

Molecules 2012, 17, 6179-6195; doi:10.3390/molecules17066179
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Chalcones with Anticancer Activities
Suvitha Syam 1, Siddig Ibrahim Abdelwahab 2,*, Mohammed Ali Al-Mamary 3 and Syam Mohan 4
1
UPM-MAKNA Cancer Research Lab, Institute of Bioscience, University Putra Malaysia,
43400 Serdang, Selangor, Malaysia; E-Mail: suvithaavs@gmail.com
2
Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur,
Malaysia; E-Mail: siddigroa@yahoo.com
3
Department of Chemistry, Faculty of Science and Arts in Al-Mukhwah, Al-Baha University,
Al-Baha 65431, Saudi Arabia; E-Mail: almamarym@hotmail.com
4
Centre for Natural Products and Drug Discovery (CENAR), Department of Pharmacology,
Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia;
E-Mail: syammohanm@yahoo.com
* Author to whom correspondence should be addressed; E-Mail: siddigroa@yahoo.com;
Tel.: +603-7967-4909; Fax: +603-7967-4964.
Received: 28 March 2012; in revised form: 20 April 2012 / Accepted: 27 April 2012/
Published: 25 May 2012
Abstract: Several chalcones were synthesized and their in vitro cytotoxicity against
various human cell lines, including human breast adenocarcinoma cell line MCF-7, human
lung adenocarcinoma cell line A549, human prostate cancer cell line PC3, human
adenocarcinoma cell line HT-29 (colorectal cancer) and human normal liver cell line
WRL-68 was evaluated. Most of the compounds being active cytotoxic agents, four of
them with minimal IC50 values were chosen and studied in detail with MCF-7 cells. The
compounds 1, 5, 23, and 25 were capable in eliciting apoptosis in MCF-7 cells as shown by
multiparameter cytotoxicity assay and caspase-3/7, -8, and -9 activities (p < 0.05). The
ROS level showed 1.3-fold increase (p < 0.05) at the low concentrations used and thus it
was concluded that the compounds increased the ROS level eventually leading to apoptosis
in MCF-7 cells through intrinsic as well as extrinsic pathways.
Keywords: chalcone; synthetic; cytotoxicity; MCF-7 cells; apoptosis; high content
screening; caspase; ROS

Molecules 2012, 17
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1. Introduction
The burden of cancer is increasing across the World and thus it is the leading cause of deaths in
economically developed countries and second leading cause of deaths in developing countries [1].
Cancer is considered to be one of the most intractable diseases because of the innate characteristics of
cancer cells to proliferate uncontrollably, avoid apoptosis, invade and metastasize [2]. Despite the
advances in chemotherapy, there are no sufficient clinically useful cytotoxic agents that selectively
targets cancer cells.
Most of the chemotherapeutic agent have anticancer activity thanks to their capacity to elicit
apoptosis [3]. The physiologically determined cell death, apoptosis, is necessary to maintain tissue
homeostasis; where homeostasis refers to the balance between cell proliferation and cellular loss. Since
it provides a mechanism of autodigestion for cells that are not functioning properly, the screening of
anticancer agents for chemotherapy is designed to identify agents that selectively kill tumor cells.
Many natural as well as synthetic agents have demonstrated to elicit apoptosis in cancer cells [4–7].
Synthetic derivatives are often found to be more active than parent compounds [6–9].
Chalcones, considered as the precursors of flavonoids and isoflavonoids are widely present in
edible plants. Chemically, they consist of open-chain flavonoids in which the two aromatic rings are
joined by a three-carbon α,β-unsaturated carbonyl system. Among the flavonoids, chalcones are an
interesting target class of compounds which are extensively investigated due to their broad spectrum
of biological activities, including anti-inflammatory [10], anti-invasive [11] and antitumour [12] and
antibacterial [13] properties. They are regarded as promising anticancer agents against most human
cancers. Previous literature suggests that chalcones are capable of inducing apoptosis [14,15] and also
have the ability to uncouple mitochondrial respiration and thus collapse mitochondrial membrane
potential [16]. Since a number of clinically useful anticancer drugs have genotoxic effects due to their
interaction with the amino groups of nucleic acids, chalcones may be devoid of this important side
effects [17]. In the ongoing search for potent and selective cytotoxic chalcones, we aimed to synthesize
some chalcones and investigate their cytotoxic potential against some cancer cell lines, and also study
the detailed mechanism of their cytotoxic activity. The synthesis of chalcones mentioned in this report
can be done in simple steps with most of the compounds being produced in >50% overall yield.
2. Results and Discussion
2.1. Synthesis of Chalcones
Different chalcone derivatives were obtained as indicated in Figure 1. The reactants used, chemical
namea and formulae of the compounds are detailed in Table 1. The melting points, IR spectra (KBr),
cm−1 and 1H-NMR spectra (δ, ppm) of the compounds were determined (refer to Table 2). Compounds
1, 5, 11–13, 15–17, 20 and 25 were obtained in high yields ranging between 75–90%; amongst which
compound 15 and 20 had the maximum yield, reaching up to 90%. Compounds 3, 4, 7–9, 19 and 21
had yields ranging from 50–74% whilst compounds 2, 6, 10, 14, 18, 22–24 showed low yields of less
than 50%.

Molecules 2012, 17
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Figure 1. Structures of different chalcones synthesized in this study. ‘Cpd’ stands for an
abbreviation of “Compound”.
Table 1. Chemical data for compounds 1–25.
No
1
Reactants
Chalcone (Chemical name)
1,3-Diphenylpropenone
Formula
Acetophenone + Benzaldehyde
4H Acetophenone + Benzaldehyde
4-Methylacetophenone + Benzaldehyde
4-Methoxyacetophenone + Benzaldehyde
Acetophenone + 2-Chloro-benzaldehyde
4H Acetophenone + 2-Chloro-
benzaldehyde
C15H12O
C15H12O2
C16H14O
2
1-(4-Hydroxyphenyl)-3-phenylpropenone
3-Phenyl-1-p-tolylpropenone
3
4
1-(4-Methoxyphenyl)-3-phenylpropenone
3-(2-Chlorophenyl)-1-phenylpropenone
3-(2-Chlorophenyl)-(4-hydroxyphenyl)-
propenone
C16H14O2
C15H11OCl
C15H11O2Cl
5
6
7
8
4-Methylacetophenone + 2-Chloro-
benzaldehyde
3-(2-Chlorophenyl)-1-P-tolylpropenone
C16H13OCl
C16H13O2Cl
4-Methoxyacetophenone + 2-Chloro-
benzaldehyde
3-(2-Chlorophenyl)-1-(4-methoxy-
phenyl)propenone
9
Acetophenone + 3-Methyl-benzaldehyde
4H Acetophenone + 3-Methyl-
benzaldehyde
1-Phenyl-3-m-tolylpropenone
1-(4-Hydroxyphenyl)-3-m-tolylpropenone
C16H14O
C16H14O2
10
11
4-Methylacetophenone + 3-Methyl-
benzaldehyde
3-m-Tolyl-1-p-tolylpropenone
C17H16O

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Table 1. Cont.
No
Reactants
Chalcone (Chemical name)
Formula
12
4-Methoxyacetophenone + 3-Methyl-
benzaldehyde
1-(4-Methoxyphenyl)-3-m-tolylpropenone
C17H16O2
13
14
Acetophenone + Anisaldehyde
4H Acetophenone + Anisaldehyde
3-(4-Methoxyphenyl)-1-phenylpropenone
1-(4-Hydroxyphenyl)-3-(4-methoxyphenyl)-
propenone
C16H14O2
C16H15O3
15
16
17
4-Methylacetophenone + Anisaldehyde
4-Methoxyacetophenone + Anisaldehyde
Acetophenone + 4-Dimethylamino-
benzaldehyde
3-(4-Methoxyphenyl)-1-p-tolylpropenone
1,3-Bis-(4-Methoxyphenyl)-propenone
3-(4-Dimethylaminophenyl)-1-phenyl-
propenone
C17H16O2
C17H16O3
C17H17NO
18
19
20
4H Acetophenone + 4-Dimethylamino-
benzaldehyde
3-(4-Dimethylaminophenyl)-1-(4-hydroxy-
phenyl)-propenone
C17H17NO2
C18H19NO
C18H19NO2
4-Methylacetophenone + 4-Dimethylamino-
benzaldehyde
3-(4-Dimethylaminophenyl)-1-p-tolyl-
propenone
4-Methoxyacetophenone + 4-Dimethylamino-
benzaldehyde
3-(4-Dimethylaminophenyl)-1-(4-methoxy-
phenyl)-propenone
21
22
Acetophenone + Salicylaldehyde
4H Acetophenone + Salicylaldehyde
3-(2-Hydroxyphenyl)-1-phenylpropenone
3-(2-Hydroxyphenyl)-1-(4-hydroxyphenyl)-
propenone
C15H12O2
C15H12O3
23
24
4-Methylacetophenone + Salicylaldehyde
4-Methoxyacetophenone + Salicylaldehyde
3-(2-Hydroxyphenyl)-1-p-tolylpropenone
3-(2-Hydroxyphenyl)-1-(4-methoxyphenyl)-
propenone
C16H14O2
C16H14O3
25
Acetophenone + 3-Chlorobenzaldehyde
3-(3-Chlorophenyl)-1-phenylpropenone
C15H11OCl
Table 2. Spectral data, yield and melting point of compounds 1–25.
Cpd.
IR-Spectrum (KBr), сm−1
3030 (C-H aromatic), 1664 (C=O),
1598 (C=C)
1H-NMR spectrum (δ, ppm)
7.77 (d, 1Hα), 8.08 (d, 1Hβ),
Yield (%) m.p. (°C)
1
70–80
35–45
70–73
70–74
75–80
40–43
70–75
60–65
68–70
56–57
120–121
70–72
7.4–8 (m, 10H, Ar-H)
3083 (C-H aromatic), 1680 (C=O),
1580 (C=C), 3380 (-OH)
7.56 (d, 1Hα), 7.99 (d, 1Hβ), 7.04–7.9
(m, 9H, Ar-H), 12.9 (s, 1H, -OH)
7.38 (d, 1Hα), 8.1 (d, 1Hβ), 7.5–7.9
(m, 9H, Ar-H), 2.29 (s, 3H, -CH3)
7.19 (d, 1Hα), 8.2 (d, 1Hβ), 7.29–7.9
(m, 9H, Ar-H), 3.79 (s, 3H, -OCH3)
7.9 (d,1Hα), 8.2 (d, 1Hβ), 7.39–8
(m, 9H, Ar-H)
2
3
4
5
6
7
8
9
3030 (C-H aromatic), 1649 (C=O),
1598 (C=C)
3047 (C-H aromatic), 1649 (C=O),
1603 (C=C), 1128 (-OCH3)
3047 (C-H aromatic), 1664 (C=O),
1608(C=C), 829 (Ar-Cl)
108–110
53–54
3452 (-OH), 1690 (C=O), 1613 (C=C),
829 (Ar-Cl)
7.5 (d, 1Hα), 8.19 (d, 1Hβ), 6.4–8
(m, 8H, Ar-H), 12.7 (s, 1H, -OH)
7.9 (d, 1Hα), 8.19 (d, 1Hβ), 7.3–8.1
(m, 8H, Ar-H), 2.3 (s, 3H, -CH3)
7 (d, 1Hα), 7.7 (d, 1Hβ), 6.9–8.1
(m, 8H, Ar-H), 3.79 (s, 3H, -OCH3)
7.19 (d,1Hα), 8.2 (d, 1Hβ), 7.2–8
(m, 9H, Ar-H), 2.3 (s, 3H, -CH3)
191–193
50–51
3052 (C-H aromatic), 1664 (C=O),
1598 (C=C), 829 (Ar-Cl)
3034 (C-H aromatic), 1664 (C=O),
1590 (C=C), 1130 (-OCH3), 828 (Ar-Cl)
3057 (C-H aromatic), 1664 (C=O),
1590 (C=C)
124–126
68–70

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Table 2. Cont.
Cpd.
IR-Spectrum (KBr), сm−1
1H-NMR spectrum (δ, ppm)
Yield (%) m.p. (°C)
3457 (-OH), 3011 (C-H aromatic),
1685 (C=O), 1591 (C=C)
7.2 (d, 1Hα), 7.7 (d, 1Hβ), 7.1–8 (m, 8H,
Ar-H), 12.8 (s, 1H, -OH), 2.3 (s, 3H, CH3)
7.8 (d, 1Hα), 8.2 (d, 1Hβ), 7.1–8
(m, 8H, Ar-H), 2.3 (s, 3H, -CH3)
7.19 (d, 1Hα), 8.21 (d, 1Hβ), 6.9–8 (m, 8H,
Ar-H), 3.8 (s, 3H, -OCH3), 2.96 (s, 3H, CH3)
7 (d, 1Hα), 8.2 (d, 1Hβ), 6.9–8.2
(m, 9H, Ar-H), 3.8 (s, 3H, -OCH3)
6.9 (d, 1Hα), 7.9 (d, 1Hβ), 6.7–8.1
(m, 8H, Ar-H), 12.8 (s, 1H, -OH),
3.8 (s, 3H, -OCH3)
10
30
84
80
80
114–116
3027 (C-H aromatic), 1654 (C=O),
1593 (C=C)
11
12
13
89–91
74–75
77–78
3011 (C-H aromatic), 1659 (C=O),
1588 (C=C), 1126 (-OCH3)
3057 (C-H aromatic), 1659 (C=O),
1588 (C=C), 1168 (-OCH3)
3380 (-OH), 3035 (C-H aromatic)
14
15
35
179–181
99–100
1660 (C=O), 1590 (C=C), 1166 (-OCH3)
7.7 (d, 1Hα), 8.2 (d, 1Hβ), 6.9–8.2
(m, 8H, Ar-H), 2.29 (s, 3H, -CH3),
3.8 (s, 3H, -OCH3)
3080 (C-H aromatic), 1649 (C=O),
1588 (C=C), 1170 (-OCH3)
85–90
7.7 (d, 1Hα), 8.2 (d, 1Hβ), 6.9–8.2
(m, 8H, Ar-H), 3.8(s, 3H, -OCH3),
3.8 (s, 3H, OCH3)
3033 (C-H aromatic), 1659 (C=O),
1588 (C=C), 1168 (-OCH3)
16
17
18
80–88
80
102–103
111–113
76–78
3010 (C-H aromatic), 1654(C=O),
1562(C=C), 1340 (C-N)
6.7 (d, 1Hα), 8.21 (d, 1Hβ), 7.3–7.8
(m, 9H, Ar-H), 2.9 (s, 6H, N-(CH3)2)
6.8 (d, 1Hα), 8.7 (d, 1Hβ), 6.8–8.8
(m, 8H, Ar-H), 9.7 (s, 1H, -OH),
2.99 (s, 6H, N-(CH3)2)
3380 (-OH), 3012 (C-H aromatic),
47
1664 (C=O), 1598 (C=C), 1372 (C-N)
6.7 (d, 1Hα), 8.21 (d, 1Hβ), 7.3–8
(m, 8H, Ar-H), 2.98 (s, 6H, N-(CH3)2),
2.3 (s, 3H, -CH3)
3083 (C-H aromatic), 1649 (C=O),
1603 (C=C), 1360 (C-N)
19
20
70
124–125
129–131
6.7 (d, 1Hα), 8.2 (d, 1Hβ), 6.7–8.1
(m, 8H, Ar-H), 2.9 (s, 6H, N-(CH3)2),
3.79 (s, 3H, -OCH3)
3010 (C-H aromatic), 1650 (C=O),
86–90
1593 (C=C), 1161 (-OCH3), 1330 (C-N)
1639 (C=O), 1557 (C=C), 3206 (-OH),
3010 (C-H aromatic)
7.6 (d, 1Hα), 8.2 (d, 1Hβ), 6.7–8.2
(m, 9H, Ar-H), 10.3 (s, 1H, -OH)
7.8 (d, 1Hα), 7.5 (d, 1Hβ), 6.3–8.3 (m, 8H,
Ar-H), 10.2 (s, 1H, -OH), 10.2 (s, 1H, -OH)
6.8 (d, 1Hα), 7.8 (d, 1Hβ), 6.7–8.2 (m, 8H,
Ar-H), 10.2 (s, 1H, -OH), 2.28 (s, 3H, -CH3)
7.96 (d, 1Hα), 8.19 (d, 1Hβ), 6.7–8.2
(m, 8H, Ar-H), 10.19 (s, 1H, -OH),
3.69 (s, 3H, OCH3)
21
22
23
50
154–155
287–289
165–167
1669 (C=O), 1590 (C=C), 3467 (-OH),
3005 (C-H aromatic)
8–10
37–40
1644 (C=O), 1577 (C=C) , 3216 (-OH),
3030 (C-H aromatic)
3252 (-OH), 3000 (C-H aromatic),
24
25
45
80
151–153
78–80
1640 (C=O), 1603 (C=C), 1165 (-OCH3)
1654 (C=O), 1603 (C=C),
7.9 (d, 1Hα), 8.2 (d, 1Hβ),
3052 (C-H aromatic), 823 (Ar-Cl)
7.2–8 (m, 9H, Ar-H)

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2.2. Cytotoxic Screening
The cytotoxicity of the synthesized chalcones were studied using the MTT assay in five human
cancer cell lines, including MCF-7 (breast), A549 (lung), PC3 (prostate), HT-29 (colorectal) and
WRL68 (liver). The results are listed in Table 3. Compounds 2, 7, 8, 14, 18, 20, 22 showed low
cytotoxic effects on all the cell lines. Compounds 1, 5, 10, 23, 24, 25 showed high cytotoxicity in all
cell lines with IC50 concentrations lines, except for the A549 cell line. It is already well known that
chalcones (both natural and synthetic of less than 20 µg/mL. Compounds 3, 11, 12, 13, and 21 also
showed IC50 values of less than 20 µg/mL in all cell origin) are cytotoxic to cancer cells [18–20] and
this was confirmed by the data of the current study.
Table 3. Cytotoxicity of compounds on different cell lines.
Cell lines/IC50 values (µg/mL)
Compound
A549
16.76 ± 1.08
>100
PC3
9.108 ± 0.9
>100
MCF-7
6.875 ± 0.219
>100
HT-29
10.1 ± 1.01
>100
WRL68
10.55 ± 0.89
>100
1
2
3
36.58 ± 1.76
77.04 ± 2.1
19.94 ± 1.66
25.22 ± 1.21
>100
17.30 ± 1.1
21.13 ± 1.24
13.84 ± 1.2
10.99 ± 1.8
>100
13.62 ± 1.01
19.15 ± 1.0
7.992 ± 0.81
10.01 ± 1.4
>100
19.10 ± 1.00
37.28 ± 2.81
13.24 ± 1.2
15.52 ±
21.34 ± 3.01
57.29 ± 3.8
11.46 ± 1.1
13.22 ± 1.3
>100
4
5
6
7
>100
8
>100
>100
>100
>100
>100
9
24.74 ± 2.0
19.68 ± 1.22
24.89 ± 1.9
31.76 ± 1.5
32.57 ± 2.5
>100
9.40 ± 1.9
13.71 ±1.1
8.73 ± 0.21
9.87 ± 0.71
12.09 ± 0.99
>100
9.34 ± 0.56
8.343 ± 0.472
10.16 ± 1.89
9.53 ± 1.99
11.62 ± 0.101
>100
18.96 ± 1.5
12.1 ± 3.0
16.30 ± 1.9
15.76 ± 2.1
22.79 ± 2.3
>100
9.78 ± 1.01
13.45 ± 2.09
13.95 ± 1.3
11.13 ± 1.8
21.46 ± 1.7
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
>100
43.27 ± 4.01
>100
32.37 ± 1.88
41.44 ± 1.91
99.29 ± 6.16
>100
29.55 ± 2.8
67.07 ± 4.1
64.26 ± 5.02
>100
50.94 ± 3.51
60.79 ± 4.22
>100
>100
>100
>100
>100
>100
>100
58.54 ± 3.2
78.85 ± 4.4
22.61 ± 1.1
>100
>100
57.28 ± 3.1
>100
69.54 ± 4.23
>100
39.78 ± 3.9
>100
>100
11.07 ± 0.4
>100
9.353 ± 1.2
>100
19.84 ± 2.01
>100
17.02 ± 1.7
>100
16.79 ± 1.76
14.16 ± 1.0
14.49 ± 0.2
9.492 ± 0.7
5.584 ± 0.2
6.936 ± 0.61
6.873 ± 1.2
7.149 ± 0.4
5.251 ± 0.67
12.98 ± 0.54
11.43 ± 1.0
7.772 ± 1.1
9.533 ± 1.5
8.722 ± 1.0
7.72 ± 1.6

Molecules 2012, 17
Figure 2. Representative images of MCF7 cells treated with medium alone and
6185
compounds, and stained with nuclear staining dye, cell permeability dye, mitochondrial
membrane potential dye and cytochrome c. The images from each row are obtained from
the same field of the same treatment sample. MCF7 cells produced a marked reduction in
mitochondrial membrane potential, and marked increases in membrane permeability and
cytochrome c. (Magnification 20X). (1A- 3 µg/mL; 1B- 6 µg/mL), (23A- 3 µg/mL; 23B-
6 µg/mL), (25A- 2 µg/mL; 25B- 4 µg/mL) and (5A- 3 µg/mL; 5B- 6 µg/mL). The positive
control used in the analysis was tamoxifen (0.04 µg/mL). “Cpd” refers to compound.
Hoechst 33342
Cell Permeability
Mitochondrial
Cytochrome c
Composite image
Membrane Potential
Control
Tamoxife
Cpd 1
Cpd 23
Cpd 25
Cpd 5

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2.3. Multiparameter Cytotoxic Analysis
Though the chalcones produced growth inhibition, the mode of cell death was yet to be studied.
Different modes of cell death include autophagy, apoptosis and necrosis; of which apoptosis is the
desired mode of cell death in chemotherapy [21]. As such, studies are extensively focused on the
apoptotic potential of a compound to be anticancer agent [5,22]. In the current study, we therefore
investigated the involvement of apoptosis in the cytotoxicity produced by select compounds (1, 5, 23
and 25) in MCF-7 cells by examining the cellular changes associated with apoptosis using HCS
analysis. The treated cells exhibited a concentration-dependent increase in nuclear chromatin staining
with Hoechst 33342 (Figures 2 and 3) with all compounds, suggesting an increase in nuclear
condensation. The increase in nuclear chromatin staining to Hoechst 33342 dye was also observed in
tamoxifen positive control cells whereas the fluorescent intensity was significantly lower in the control.
Figure 3. Quantitative analysis of compounds mediated apoptosis parameter. Changes in
(A) total nuclear intensity; (B) cell permeability; (C) mitochondrial membrane potential
and (D) cytochrome c localization were all measured simultaneously in MCF7 cells.
Following treatment with compounds 1, 23, 25 and 5, there was statistically significant cell
loss (data not shown), increased total nuclear intensity, increased cell permeability, loss of
mitochondrial membrane potential and cytochrome c release from mitochondria with good
p values. (1A- 3 µg/mL; 1B- 6 µg/mL), (23A- 3 µg/mL; 23B- 6 µg/mL), (25A- 2 µg/mL;
25B- 4 µg/mL) and (5A- 3 µg/mL; 5B- 6 µg/mL). The positive control used in the analysis
was tamoxifen (0.04 µg/mL). Each experiment was performed at least two times. Results
are expressed as the means value ± standard deviation (SD). Statistical analysis was
performed with one-way analysis of variance (ANOVA) using GraphPad Prism software
(version 4.0; GraphPad Software Inc.). Statistical significance is expressed as *, p < 0.05.

Molecules 2012, 17
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Figure 3. Cont.
The high fluorescence intensity for the permeability dye in the cytoplasm of chalcone treated and
tamoxifen treated cells evidenced the increase in membrane permeability compared to the control cells.
A reduction in mitochondrial membrane potential dye retention was observed with increasing
concentrations of the compounds used for treatment and with the tamoxifen positive control whereas
the control cells had intact mitochondrial membrane potential dye retention. The specific apoptosis
indicator, release of cytochrome c was found to increase in concentration dependent manner with all
compounds used in the treatment and with the tamoxifen treated cells. Chromatin condensation [23],
increased cell permeability [24], decline in mitochondrial membrane potential [25] and cytochrome c
release [26] are key events of apoptosis which were obviously observed when the selected compounds
were treated on MCF-7 cells. There are previous studies demonstrating that chalcones were good
candidates in producing apoptosis in MCF-7 cells [22,27].
2.4. Caspase-3/7, -8, and -9 Activity
The caspases, a family of cysteine-dependent aspartate-directed proteases, are prominent among
the death proteases [28]. There are two type of caspases, namely upstream (initiator) caspases
and downstream (effector) caspases [29]. Upstream caspases, which include caspase-8 and caspase-9
cleave inactive pro-forms of downstream caspases like caspase-3 and caspase-7; which in turn cleave
proteins involved in programmed cell death events [30]. In our analysis, following treatment with the
selected synthesized compounds, the caspase-3/7, caspase-8, and caspase 9 activity was increased in
MCF-7 cells as compared to control. Caspase-3/7 activity of all compounds was more than 100,000
RLU, where the maximum caspase-3/7 activity (210430 RLU) was achieved at highest concentration
of compound 1. The control cells, on the other hand, had only caspase-3/7 activity of 30,140 RLU.
When the caspase-8 activity of control cells were just 50892 RLU, the highest concentrations of
compound 1, 23, 25 and 5 showed significant activity (p < 0.05) of 17,568 RLU, 18,342 RLU, 18,356
RLU and 10,435 RLU respectively. Similarly, caspase-9 activity was also elevated in all treatments of

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the compounds used to analyze. All the compounds except compound 5 showed caspase-9 activity of
more than 12,000 RLU. On the whole, all the four compounds showed increase in caspase-3/7,
caspase-8, and caspase-9 activity with increase in concentration suggesting that there is the activation
of both intrinsic and extrinsic apoptotic pathways [31]. It is also important to note that MCF-7 cells do
not have caspase-3 [32], so the activity represented is of caspase-7. Recently, Deeb et al., found
that xanthohumol, a chalcone, elicited apoptosis in cancer cells by both intrinsic and extrinsic
pathways [33]. Similarly, another study proved that chalcones are capable in inducing both pathways
in MCF-7 and MDA-MB-231 breast cancer cell lines [27].
Figure 4. Relative luminescence expression (RLU) of caspase 3/7, 8 and 9 in the MCF7
cells treated with different concentrations compounds. (1A- 3 µg/mL; 1B- 6 µg/mL), (23A-
3 µg/mL; 23B- 6 µg/mL), (25A- 2 µg/mL; 25B- 4 µg/mL) and (5A- 3 µg/mL; 5B- 6 µg/mL).
Triplicates of each treatment group were used in each independent experiment. The
statistical significance is expressed as *, p < 0.05.
2.5. Intracellular ROS Level
There was loss of mitochondrial membrane potential (mitochondrial damage) in MCF-7 cells upon
treatment with the selected compounds. This could probably be due to the reactive oxygen species
(ROS) produced within the mitochondria, which primarily affect the mitochondria itself [34]. The
intracellular ROS level was therefore studied with DCFDA and the resulting data showed that all the

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compounds used in this study significantly increased the production of ROS in MCF-7 cells compared
to untreated cells. (Figure 5). All the compounds showed more than 1.3-fold increase (p < 0.05) at low
concentration and more than 2.0-fold increase (p < 0.05) at high concentration as compared to control.
Amongst the compounds used, compound 5 showed the highest ROS level (2.7-fold higher) (p < 0.05)
at the highest concentration used (6 µg/mL). It is thus concluded that ROS production is involved in
induction of apoptosis by chalcones, which is consistent with previous findings [35–37]. ROS-dependent
apoptosis induction by chalcones was observed previously in similar studies where flavokawain B, a
novel chalcone, increased the intracellular ROS levels in colon cancer HCT116 cells [35] and oral
carcinoma HSC-3 cells [36].
Figure 5. Fold increase of reactive oxygen species (ROS) in MCF-7 cells after treatment
with compounds 1, 23, 25, and 5 compared to control (untreated MCF-7 cells). (1A-
3 µg/mL; 1B- 6 µg/mL), (23A- 3 µg/mL; 23B- 6 µg/mL), (25A- 2 µg/mL; 25B- 4 µg/mL)
and (5A- 3 µg/mL; 5B- 6 µg/mL). Triplicates of each treatment group were used in each
independent experiment. The statistical significance is expressed as * p < 0.05.
3. Experimental
3.1. Synthesis of Chalcones
Procedure A (compounds 1, 3, 4, 5, 7–9, 11–13, 15–17, 19–20): Substituted acetophenone (0.01 mol)
and substituted aldehyde (0.01 mol) were mixed in ethanol (40 mL) in a round bottom flask placed in
an ice bath. To this NaOH solution (10 mL, 60%) were added dropwise with continuous stirring for
30 min. The mixing was continued for another 2–3 h at room temperature. The mixture was kept in a
refrigerator for overnight when it became quite thick. Then it was diluted with ice-cold distilled water
(40 mL), filtered, washed well with cold water, dried in air and recrystallized from rectified methanol.
Procedure B (compounds 2, 6, 10, 14, 18, 21, 23–24): Substituted acetophenone (0.01 mol) and
substituted aldehyde (0.01 mol) were mixed in ethanol (40 mL) in a round bottom flask placed in an
ice bath. To this NaOH solution (10 mL, 60%) were added dropwise with continuous stirring for
30 minutes. The mixing was continued for another 2–3 h at room temperature. Turbidity appeared in
the mixture, which was then diluted with cold distilled water (40 mL), and neutralized to litmus paper

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with 2 N HCL. The product (ppt) was filtered, washed well with cold water, dried in air and
recrystallized from rectified methanol.
Procedure, C (compound 22): Substituted acetophenone (0.01 mol) and substituted aldehyde
(0.01 mol) were mixed in ethanol (40 mL) in a round bottom flask placed in an ice bath. To this NaOH
solution (10 mL, 60%) were added dropwise with continuous stirring for 30 min. The mixture was
refluxed overnight. The stirring of the mixture was continued for 2 days at room temperature (turbidity
appears in the mixture), then the mixture was diluted with cold distilled water (40 mL), and neutralized
to litmus paper with 2 N HCL. The product (ppt) was filtered, washed well with cold water, dried in air
and recrystallized from rectified methanol.
3.2. Identification of Chalcones
The IR spectra were obtained using a Shimadzu IR-FTIR-8300 spectrophotometer in a disk of
1
potassium bromide, over the range of 4,000–400 cm−1. The H-NMR spectra were recorded on a
Varian Mercury-VX-200 (300 MHz) spectrophotometer using DMSO-d6 as solvent and TMS as the
internal standard. The melting temperatures were determined in sealed capillaries on a SMP3 melting
point apparatus (Stuart Scientific, UK).
3.3. Cell Culture and Cell Viability Assay
A549, PC3, MCF-7, HT-29, WRL68 and MCF-7 cells were obtained from the ATTC (Rockville,
MD, USA) and grown in RPMI-1640 medium, 10% fetal bovine serum (FBS) and 1% pen-strep, in
humidified conditions at 5% CO2 and 37 °C. The different cell lines mentioned above were used to
determine the inhibitory effects of compounds on cell growth using the MTT assay. This colorimetric
assay is based on the conversion of the yellow tetrazolium bromide (MTT) to a purple formazan
derivative by mitochondrial succinate dehydrogenase in viable cells. Cells were seeded in 96-well
plate and incubated for 24 h at 37 °C with 5% CO2 saturation. After incubation, the cells were treated
with different concentration of compounds and incubated for another 24 h. After 24 h of drug
treatment, 20 µL of MTT solution at 5 mg/mL was added and incubated for 4 h. Dimethyl sulfoxide
(DMSO) in volume of 100 µL is added into each well to dissolve the purple formazan formed. The
colorimetric assay is measured and recorded at absorbance of 570 nm.
3.4. Cell Treatment
Due to the observation of significant cytotoxicity of selected compounds towards MCF-7 cells, the
following analyses were done on them only. Cells were treated with compound 1 (1A- 3 µg/mL; 1B-
6 µg/mL), 23 (23A- 3 µg/mL; 23B- 6 µg/mL), 25 (25A- 2 µg/mL; 25B- 4 µg/mL) and 5 (5A-
3 µg/mL; 5B- 6 µg/mL).
3.5. Multiparametric High Content Screening (HCS) Assays
The toxic effects of Cpd 1, 5, 23 and 25 at different doses on MCF-7 cells were assayed using the
high content screening (HCS) KineticScan Reader (Cellomics Inc, Pittsburgh, PA, USA) with the
Multiparameter Cytotoxicity Kit 3. The kit contains four fluorescent dyes, i.e., blue fluorescent

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Hoechst 33342, membrane permeability, mitochondrial membrane potential, and cytochrome c dyes.
They respectively detect changes in nuclear morphology (nuclear condensation), membrane
permeability, mitochondrial membrane potential and expression of cytochrome c. The multiparametric
apoptosis kit quantifies three fundamental parameters related to the process of apoptosis, i.e., nuclear
condensation detected by the blue fluorescent nuclear dye, Hoechst 33342, cell permeability detected
by permeability dye, mitochondrial damage detected by mitochondrial membrane potential dye and
cytochrome c release by DyLight 649 Conjugates. Following treatment with different concentrations
of compounds 1, 23, 25 and 5 as mentioned before, the fixation and staining for imaging analysis of
the MCF-7 cells were performed according to the manufacturer’s instructions. Cells treated with
tamoxifen (0.04 µg/mL) were used as positive control and untreated cells were used as negative
control. Plates were analyzed using a Thermo Scientific ArrayScanHVTI HCS Reader (Cellomics Inc,
Pittsburgh, PA, USA). This is a computerized automated fluorescence imaging microscope that
automatically identifies stained cells and measures the intensity and distribution of fluorescence in
individual cells. Images for each fluoroprobe were acquired at different channels using suitable filters
with 20x objective at fixed exposure time. The Cell Health Profiling BioApplication software was used
for image acquisitions and analysis. For each well, at least 25 fields, corresponding to at least 500 cells
were automatically acquired and analyzed. All experiments were performed in triplicates. Cell average
intensity (Mean) under the modified object mask within selected range in each channel was used as
assay indicator, and reported as average fluorescence intensity.
3.6. Caspase Activity
Activity of cellular caspases was measured using the Caspase-Glo-9, Caspase-Glo-3/7 and Caspase-
Glo-8 assay reagents (Promega), according to the manufacturer's instructions. The assays provide a
luminogenic caspase-3/caspase-7 or caspase-8 or caspase-9 substrate, respectively, in a buffer system
optimized for caspase activity measurement in cell lysates. The luciferase signal generated is
proportional to the amount of caspase activity present. MCF-7 cells have no caspase-3 activity due to
functional deletion of the CASP-3 gene [38]. Therefore, the activity measured in this test is that of
caspase-7. Briefly, cells were plated in opaque 96-well plates in triplicate and treated with the. After
24 h treatment with compounds 1, 23, 25 and 5 as mentioned before, the assay reagents were added to
the plates at room temperature and incubated for 60 min, and the luminescence was measured using a
plate reader. All assays were done in triplicate, and means ± SD were calculated for each condition.
3.7. Intracellular ROS Level
The production of ROS was estimated by fluorescence using the redox-sensitive fluorescent
probe DCFDA (Sigma). The oxidation of DCFDA by ROS converts the molecule to
2′,7′-dichlorodihydrofluorescein (DCF), which is highly fluorescent. Thus, ROS production by
stimulated cells causes an increase in the fluorescence signal over time. ROS assays were performed as
previously described (13). Briefly, the MCF-7 cells were seeded in 96-well plates (5 × 104 cells/well)
and allowed to attach for 24 h. After 24 h treatment with compounds 1, 23, 25 and 5 as mentioned
before, the cells were washed three times with PBS and incubated for 60 min with five DCFDA at
room temperature. Again the wells were washed with PBS. Fresh PBS was added (100 μL/well) and

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DCF fluorescence was detected using a Tecan Infinite M200 fluorescence microplate reader at
excitation and emission wavelengths of 485 and 528 nm, respectively. The fluorescence intensity unit
of treatments was compared with control.
3.8. Statistical Analysis
All assays were conducted in at least three separate experiments. Results are expressed as the mean
value ± standard deviation (SD). Statistical analysis was performed with one-way analysis of variance
(ANOVA) using GraphPad Prism software (version 4.0; GraphPad Software Inc., San Diego, CA, USA).
4. Conclusions
In summary, we have synthesized some chalcones and identified them from their spectral data. We
have nhat most of the compounds were very active cytotoxic agents towards several cancer cell lines.
Four active compounds chosen in the study showed apoptosis induction in MCF-7 cells with the
involvement of caspase-7, caspase-8, and caspase-9. We also demonstrated that all four compounds used
in the study induced ROS generation and loss of mitochondrial membrane potential to cause the release
of cytochrome c to result in apoptosis. The apoptosis was through both intrinsic and extrinsic pathways.
Acknowledgments
The authors thank University of Malaya (Kuala Lumpur, Malaysia) for its financial support through
the UMRG grant (RG043/11BIO).
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Sample Availability: Samples of the compounds are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).