Synthesis and in vitro evaluation of novel coumarin-chalcone hybrids as potential anticancer agents

Article abstract of DOI:10.1016/j.bmcl.2010.10.116

A series of coumarin-chalcone hybrids have been synthesized and evaluated for their in vitro cytotoxicity against a panel of four human cancer cell lines and normal fibroblasts (NIH3T3). Among 21 compounds screened, three compounds (23, 25 and 26) showed IC₅₀ range from 3.59 to 8.12 μM. The most promising compound 26 showed around 30-fold more selectivity towards C33A (cervical carcinoma) cells over normal fibroblast NIH3T3 cells with an IC ₅₀ value of 3.59 μM.

Full text of DOI:10.1016/j.bmcl.2010.10.116

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7205–7211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro evaluation of novel coumarin–chalcone hybrids
as potential anticancer agents q
Koneni V. Sashidhara a, , Abdhesh Kumar a, Manoj Kumar a, Jayanta Sarkar b, Sudhir Sinha b
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute, (CDRI-CSIR), Lucknow 226 001, India
b Drug Target Discovery and Development Division, Central Drug Research Institute, (CDRI-CSIR), Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of coumarin–chalcone hybrids have been synthesized and evaluated for their in vitro cytotoxicity
against a panel of four human cancer cell lines and normal fibroblasts (NIH3T3). Among 21 compounds
Received 21 June 2010
Revised 29 September 2010
Accepted 22 October 2010
Available online 27 October 2010
screened, three compounds (23, 25 and 26) showed IC50 range from 3.59 to 8.12
compound 26 showed around 30-fold more selectivity towards C33A (cervical carcinoma) cells over
normal fibroblast NIH3T3 cells with an IC50 value of 3.59 M.
lM. The most promising
l
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Synthesis
Coumarin
Chalcone
In vitro
Cytotoxicity
Cancer, a diverse group of diseases characterized by uncontrolled
growth of abnormal cells, is a major worldwide problem. It is a fatal
disease standing next to the cardiovascular disease in terms of mor-
bidity and mortality. Although the cancer research has led to a num-
ber of new and effective solutions, the medicines used as treatments
have clear limitations and unfortunately cancer is projected as the
primary cause of death in the future.1,2 Currently there is a huge
scientific and commercial interest in the discovery of potent, safe
and selective anticancer drugs.
Coumarins form an important class of compounds, which occupy
a special role in nature. They belong to the flavonoid class of plant
secondary metabolite, which have been found to exhibit a variety
of biological activities, usually associated with low toxicity and have
raised considerable interest because of their potential beneficial
effects on human health.3,4 They have attracted intense interest in
recent years because of their diverse pharmacological properties
like anti-HIV,5 anticoagulant,6 antibacterial,7 antioxidant,8 and
dyslipidemic.9 Among these properties, cytotoxic effects were most
extensively examined.10,11 Recently, Lee et al. reported that neo-
tanshinlactone, (Fig. 1) a coumarin containing compound, showed
significant inhibition against two ER+ human breast cancer cell lines
and was 10-fold more potent and 20-fold more selective than
Tamoxifen.12
On the other hand, chalcones (1,3-diaryl-2-propen-1-ones)
constitute an another important class of natural products belonging
to the flavonoid family, which display interesting biological activi-
ties including anti-inflammatory,13 antibacterial,14 antioxidant,15
antimalarial16 and anticancer.17 Due to their abundance in plants
and ease of synthesis, this class of compounds has generated great
interest for possible therapeutic uses. They are also effective
in vivo as cell proliferating inhibitors, anti-tumor promoting and
chemopreventing agents (Fig. 1). Since a number 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.18
In the design of new drugs, the development of hybrid mole-
cules through the combination of different pharmacophores may
lead to compounds with interesting biological profiles. In recent
years, combination chemotherapy with agents possessing different
mechanisms of action is one of the methods, that is, being adopted
to treat cancer. Therefore, a single molecule containing more than
one pharmacophore, each with different mode of action could be
beneficial for the treatment of cancer.19,20 Adopting this approach,
several research groups have recently reported hybrid molecules
by coupling coumarins with different bioactive molecules like:
resveratrol, maleimide and alpha-lipoic acid; these studies resulted
in new compounds showing antiplatelet, antioxidant and anti-
inflammatory activities.21–23 Recently, Belluti et al. explored anti-
cancer activities of stilbene–coumarin hybrid compounds.24
q
Part of this work has been filed for provisional Indian patent vide Application No.
1843-DEL2010.
⇑
Corresponding author. Tel.: +91 9919317940; fax: +91 522 2623405.
E-mail addresses: sashidhar123@gmail.com, kv_sashidhara@cdri.res.in (K.V.
Sashidhara).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.116

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7205–7211
Figure 1. Chemical structure of some naturally occurring coumarin and chalcones with potent anticancer activity and general structure of our synthesized hybrids.
Scheme 1. Synthesis of coumarins (3–5) and novel coumarin–chalcone hybrids (7–15). Reagents and conditions: (a) (1) hexamethylenetetramine/TFA, 120 °C, 3 h; (2)10%
H2S04, 90–100 °C, 2 h; (b) CH3COCH2COOC2H5, EtOH, piperidine, reflux, 30 min; (c) CH2(COOR)2, ROH, piperidine, reflux, 30 min; (d) concd HCl, p-R1C6H4COCH3, dioxane,
80–90 °C, 2.5–3.5 h.

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7205–7211
7207
Furthermore, Bombardelli and Valenti have synthesized a series of
coumarin–chalcone hybrids (prototype shown in Fig. 1) wherein,
these hybrids have shown significant tumor inhibition, in taxol
resistant cancers.25 Inspired by this study, we have designed and
synthesized a series of novel compounds that have both coumarin
and chalcones entities in one molecule and have evaluated them
for their anti-tumor activity. Figure 1 shows the chemical struc-
tures of some naturally occurring potent anticancer molecules that
either contain a coumarin or chalcone in their molecular makeup
and form the basis of our designed prototype.
The new compounds were evaluated for their in vitro anticancer
activity using Sulforhodamine B assays.28,29 The growth-inhibitory
effects was undertaken in four human cancer cell lines, KB (oral
squamous cell carcinoma), C33A (cervical carcinoma), MCF-7
(breast adenocarcinoma), A549 (lung) and one normal fibroblast
NIH3T3 (mouse embryo fibroblast) in order to determine their
cyto-selective nature. The results are presented in Table 1. IC50 val-
ues were based on dose–response curves. Each test compound dis-
played a concentration-dependent cytotoxic profile in all four cell
lines. Out of all the compounds evaluated, three compounds
The route followed for the preparation of coumarin derivatives
and coumarin–chalcone hybrids is illustrated in Scheme 1. The
Duff reaction on naphthalen-1-ol 1 gave compound 2 which was
engaged in a Knoevenagel-type reaction with different active
methylene compounds resulted in the formation of coumarinic
compounds (3–5). Alternatively, compound 2 on reaction with dif-
ferent acetophenone in refluxing dioxane in the presence of a cat-
alytic amount of concd HCl gave regioselective para-condensed
chalcones26 (6a–6c) in good yields. These chalcone derivatives on
subsequent Knoevenagel-type condensation with different active
methylene compounds furnished coumarinic–chalcone hybrids27
(7–15) (Scheme 1). Similarly, another series of coumarinic–
chalcones were prepared starting from 2-sec-butylphenol 16 which
was subjected to same series of above mentioned transformations
resulting in coumarinic compounds (18–20) and coumarinic–
chalcone hybrids (22–27) (Scheme 2). In all the chalcones synthe-
sized the trans double bond (on the basis of coupling constant) was
obtained exclusively. All compounds were characterized using 1H
NMR, 13C NMR, mass spectrometry and IR spectroscopy. The purity
of these compounds was ascertained by TLC and spectral analysis.
showed IC50 range from 3.59 to 8.12
lM. The compounds having
IC50 value more than 200 M, were considered inactive.
l
A closure look into the structure activity relationship indicates
that of the two series of coumarinic–chalcones hybrids synthesized
(7–15) and (22–27), the former were inactive with just two excep-
tions (3 and 4) that exhibited very limited activity, while the latter
were found to be more active against one or the other cell lines.
Furthermore, in the second series of compounds, as far as pharma-
cophore 1 (coumarin core) is considered, it revealed that the sub-
stitution at position 3 play a pivotal role, it is interesting to note
that the ester- containing members all posses interesting activity,
while the ketone (22) does not. Furthermore, the ethyl esters seem
to be less effective against MCF-7 cell line compared to the methyl
esters (23 and 24). A cursory look at the second pharmacophore 2
(chalcone core) reveals that the para-chloro substituent signifi-
cantly diminishes selectivity (24 and 27) for cancer versus non-
cancer cell lines.
In conclusion, we report here a series of new coumarin–
chalcones hybrids (23–27) prepared by a novel method and their
ability to kill tumor cells in vitro. Though the mechanisms
Scheme 2. Synthesis of coumarins (18–20) and novel coumarin–chalcone hybrid (22–27). Reagents and conditions: (a) (1) hexamethylenetetramine/TFA, 120 °C, 3 h; (2)10%
H2S04, 90–100 °C, 2 h; (b) CH3COCH2COOC2H5, EtOH, piperidine, reflux, 30 min; (c) CH2(COOR)2, ROH, piperidine, reflux, 30 min; (d) concd HCl, p-R1C6H4COCH3, dioxane,
80–90 °C, 1.0–1.5 h.

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7205–7211
Table 1
Anticancer activity (IC50, lM) of coumarins and novel coumarin–chalcone hybrids
Compounds
Structure
KB
C33A
72.82
MCF-7
146.99
A549
NA
NIH3T3
NA
O
O
O
O
O
O
3
109.21
CHO
O
O
O
O
4
5
57.91
30.07
93.55
61.70
75.67
65.96
CHO
O
O
123.75
155.24
NA
NA
CHO
O
O
7
NA
NA
NA
NA
NA
O
O
O
O
8
NA
NA
NA
NA
NA
O
O
O
O
9
NA
NA
NA
NA
NA
O
Cl
O
O
O
O
10
90.76
62.40
102.73
NA
NA
O
O
O
O
O
11
70.38
52.04
117.79
NA
NA
O

K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7205–7211
7209
Table 1 (continued)
Compounds
Structure
KB
C33A
NA
MCF-7
A549
NA
NIH3T3
O
O
O
O
12
NA
NA
NA
O
O
Cl
O
O
O
O
O
O
O
O
O
13
NA
NA
NA
NA
NA
O
O
14
NA
NA
NA
NA
NA
O
O
15
NA
NA
NA
NA
NA
O
Cl
O
O
O
O
O
O
18
19
20
176.36
145.73
131.59
70.04
87.99
64.30
141.40
NA
NA
NA
NA
CHO
O
O
O
O
NA
NA
CHO
O
O
112.89
146.82
CHO
O
O
22
NA
NA
NA
NA
NA
O
Cl
(continued on next page)

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K. V. Sashidhara et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7205–7211
Table 1 (continued)
Compounds
Structure
KB
C33A
6.28
MCF-7
11.41
A549
10.69
NIH3T3
O
O
O
O
23
13.41
NA
O
O
O
O
O
24
28.25
5.90
10.80
16.67
42.41
O
O
Cl
O
O
O
O
O
O
O
O
O
25
10.47
8.12
88.09
12.87
NA
O
O
26
17.97
3.59
81.10
32.80
NA
O
O
27
14.29
0.22
4.54
0.82
11.07
0.61
12.85
0.52
38.42
ND
O
Cl
Doxorubicin
The compounds having IC50 value more than 200
ND = Not determined.
lM, were considered Not Active (NA).
underlying this process remain to be fully elucidated, previous
literature studies reveal that both coumarin and chalcone are
known microtubule inhibitor with antimitotic activity.30–38
Detailed mechanistic studies and lead optimization of these
coumarin–chalcone hybrids are under investigation. It is intended
that results from these studies will assist in elucidating their pre-
cise mechanisms of action and provide an approach to develop
new potent coumarin–chalcone hybrid prototypes for further
optimization and development to get new leads for the treatment
of cancer.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.116.
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