Synthesis of methoxy-substituted chalcones and in vitro evaluation of their anticancer potential

Article abstract of DOI:10.1111/cbdd.12184

Methoxy-substituted chalcones, 3 were obtained using simple, efficient method from 2-naphtylethanone, 1 and aromatic aldehydes, 2. The in vitro cytotoxicity activities of the chalcones against a panel of three human cancer cell lines were explored. The tested compounds were found to possess significant cytotoxic activity. The DNA strand break and damage was quantified through alkaline comet assay, flow cytometric analysis, and chromatin condensation studies, which revealed the apoptotic nature of the compounds. Compound 3c, (3-(3,4,5-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) showed highest cytotoxicity of 0.019 μm against HeLa, 0.020 μm against HCT15 and 0.022 μm against A549. Compound 3e, (3-(3,5-dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) showed better IC50 values against all the three cell lines employed for the study. Simple, highly efficient procedure from 2-naphtylethanone, 1 and aromatic aldehydes, 2 is reported. The in vitro cytotoxicity activity of the chalcones against a panel of three human cancer cell lines was explored. The tested compounds were found to possess significant cytotoxic activity. DNA stand break and damage was quantified by alkaline comet assay. Flow cytometric analysis and chromatin condensation studies revealed the apoptotic nature of the compounds.

Full text of DOI:10.1111/cbdd.12184

Received Date : 29-Oct-2012
Revised Date : 26-Apr-2013
Accepted Date : 07-May-2013
Article type
: Research Article
Title: Synthesis of methoxy substituted chalcones and in vitro evaluation of their anticancer
potential.
Running title: Cytotoxicity of synthesized novel methoxychalcones
Authors: K R. Ethiraj1,3, Jesil Mathew. A2, F. Nawaz Khan3*
Affiliation of authors:
1Pharmaceutical Chemistry Division, School of Advance Sciences, VIT University,
Vellore 632 014, Tamil Nadu, India
2Department of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal
University, Udupi, Karnataka, India 576104, Fax: 91-0820-2571998
3Organic and Medicinal Chemistry Research Laboratory, School of Advance Sciences,
VIT University, Vellore 632 014, Tamil Nadu, India. Phone no. 0416-2202334
*Corresponding Author, Prof. F. Nawaz Khan Email: nawaz_f@yahoo.co.in
Abstract:
Methoxy substituted chalcones, 3 were obtained using simple, efficient method from 2-naphtylethanone,
1 and aromatic aldehydes, 2. The in vitro cytotoxicity activities of the chalcones against a panel of three
human cancer cell lines were explored. The tested compounds were found to possess significant cytotoxic
activity. The DNA strand break and damage was quantified through alkaline comet assay, flow
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10.1111/cbdd.12184
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cytometric analysis and chromatin condensation studies which revealed the apoptotic nature of the
compounds. Compound 3c, (3-(3,4,5-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) showed highest
cytotoxicity of 0.019µM against HeLa, 0.020µM against HCT15 and 0.022µM against A549. Compound
3e, (3-(3,5-dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) showed better IC50 values against all the
three cell lines employed for the study.
Keywords:
Apoptosis, Chromatin condensation, Cytotoxicity, DNA-damage, Flow cytometry, Chalcones..
Introduction
Combretastain A-4 (Figure 1) isolated from the bark of South African tree Combretum caffrum, possesses
potent antimitoic agents (1, 2), which are highly active cytotoxic agents against a variety of cancer cell
lines (3). The structure activity studies revealed that three essential components are responsible for its
cytotoxicity, which includes 3,4,5-trimethoxyphenyl moiety (A ring), unsaturation, and a phenolic ring
(B ring). Chalcones possessing structural similarity with combretastatin are known to possess a wide
range of biological activities such as antiangiogenic (4), cytotoxic, antioxidant (5), antimalarial (6),
antileishmanial (7), anti-inflammatory (8), anti-tumor and antibacterial properties (9).
The relative ease in the synthesis of chalcones and the wide range of their biological activities (10-15),
encouraged our intention and efforts, directed us to synthesize methoxy substituted chalcones and to
investigate their cytotoxic effects on selected cancer cells. This is achieved by carbon carbon bond
formation which is the most important reaction in organic chemistry (16-19). These compounds were also
analysed for their ability to induce DNA damage through alkaline comet assay. The flow cytometric
analysis using propedium iodide along with flourescent staining of condensed chromatin revealed the
induction of apoptosis in cells exposed to these compounds.
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Materials and methods
Melting points were recorded with an open capillary tube and are uncorrected. Infrared spectra were
1
recorded on a Perkin–Elmer 1720 FT IR, H NMR spectra were recorded on a Bruker AC 400 MHz,
spectrometer using Tetra Methyl Silane (TMS) as an internal standard in CDCl3. The HRMS mass spectra
were recorded on a JEOLSX 102/DA-6000 Mass Spectrometer.
Thin layer chromatography (TLC) was performed using glass plates coated with silica gel G, and the
spots visualization was done using iodine vapor.
The chemicals were purchased from Sigma Aldrich, India, Sd-Fine Chemicals, India and were used
without furthur purification. All the solvents were purchased from Sd- Fine Chemicals, India and used
without furthur purification.
The synthetic pathway of methoxy substituted chalcones is depicted in Scheme1 (20) . The sodium
hydroxide, base catalysed Claisen-Schmidt condensation of equimolar mixture of 2-acetylnaphtalene, 1
and various methoxy substituted benzaldehydes, 2 under stirring for 3h in alkaline ethanol solution and
kept in ice bath overnight afforded the respective chalcones, 3 (a-f) in excellent yields (Table 1). The
transformation was clean and efficient.
The reactions were carried out by dissolving the equimolar quantity of appropriate ketones and aldehydes
in a minimum amount of ethanol and stirred in the presence of 10 % sodium hydroxide (NaOH) under
room temperature for an appropriate time period. The reaction after completion [as monitored by thin
layer chromatography, using ethyl acetate: petroleum ether (EtOAc: Pet. Ether) solvent system] was kept
aside overnight, at room temperature, for the precipitation of crude chalcones, which was filtered off and
recrystallized from methanol. The differently methoxy substituted chalcones required for the present
study are tabulated in table 1. The synthesized samples are characterized by FT infrared (FTIR), Nuclear
Magnetic Resonance (NMR) and mass spectrometry (MS).
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3-(2,5-Dimethoxyphenyl)-1-(2-naphthyl)prop-2-en-1-one, 3a.
Pale yellow solid (84.6%), mp 75-77 °C: 1H NMR (400 MHz, CDCl3, ppm): δ= 3.84 (s, 3H, OCH3), 3.88
(s, 3H, OCH3), 6.88 (d, J = 8 Hz, 1H, CH), 6.94 (d, J = 3Hz, 1H, CH), 7.22-8.11 (m, 10H, Ar H);13C
NMR (100 MHZ, CDCl3, ppm) δ= 190.8, 153.5, 153.4, 135.8, 135.4, 132.6, 129.9, 129.5, 128.4, 128.2,
127.8, 126.7, 2x 124.6, 123.2, 117. 19, 113.8, 112.5, 56.1, 55.9; FTIR (KBr, cm-1) 3022, 2825, 1651,
1280; MS m/z: 319.14 [M+H]+, HRMS calcd for C21H18O3 318.3658, found m/z: 318.1256
3-(2,3,4-Trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3b.
Pale yellow solid (78.7%), mp 117-120 °C: 1H NMR (400 MHz, CDCl3, ppm) δ 3.90 (s, 3H, OCH3), 3.92
(s, 3H, OCH3), 3.96 (s, 3H, OCH3) 6.73 (d, J = 8.76 Hz, 1H, CH), 7.26, 1H, CH), 7.43-8.11 (m, 9H, Ar
13
H); C NMR (100 MHz, CDCl3, ppm) δ 190.7, 155.8, 153.8, 142.5, 140.1, 135.9, 135.4, 132.6, 129.7,
129.5, 128.4, 128.2, 127.8, 126.7, 124.6, 123.9, 122.1, 107.1, 61.4, 60.9, 56.1; FTIR (KBr, cm-1) 3055,
2831, 1651, 1259; MS m/z: 349.18 [M+H]+, HRMS calcd for C22H20O4 348.3918, found m/z: 348.1362
3-(3,4,5-Trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3c
1
Whitish yellow solid (79.4%), mp 109-111 °C: H NMR (400 MHz, CDCl3, ppm) δ 3.79 (s, 3H, OCH3),
3.91 (s, 6H, OCH3), 6.91, (1H, CH), 7.53 (d, J = 15.6 Hz, 1H, CH), 7.59-8.10 (m, 9H, Ar H);13C NMR
(100 MHz, CDCl3, ppm) δ 190.3, 153.5, 145.0, 140.5, 135.6, 135.4, 132.5, 130.4, 129.8, 129.4, 128.5,
128.3, 127.8, 126.8, 124.5, 121.5, 105.8, 61.0, 56.3; FTIR (KBr, cm-1) 2996, 2830, 1656, 1278; MS m/z:
349.16 [M+H]+, HRMS calcd for C22H20O4348.3918, found m/z: 348.136.
3-(2,4-Dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3d
Dark yellow solid (82.6%), mp 87-89 °C: 1H NMR (400 MHz, CDCl3, ppm) δ 3.86 (s, 3H, OCH3), 3.91
(s, 3H, OCH3), 6.49 (d, J = 2.24 Hz, 1H, CH), 6.54 (d, J = 2.32 Hz, 1H, CH), 7.25-8.0 (m, 10H, Ar H);13C
NMR (100 MHz, CDCl3, ppm) δ 190.9, 163.1, 160.4, 140.4, 136.6, 135.3, 132.6, 130.8, 129.6, 129.5,
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128.3, 128.0, 127.8, 126.6, 124.7, 120.5, 117. 2, 105.5, 98.4, 55.6, 55.5; FTIR (KBr, cm-1) 3017, 2829,
1654,1274; MS m/z: 319.3650 [M+H]+, HRMS calcd for C21H18O3 318.3658, found m/z: 318.1256
3-(3,5-Dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3e
1
Off white solid (85.7%), mp 128-131 °C: H NMR (400 MHz, CDCl3, ppm) δ 3.84 (s, 6H, OCH3), 6.54
(d, J = 2.2 Hz,1H, CH), 6.55 (d, J = x Hz,1H, CH), 7.25-8.08 (m, 10H, Ar H);13C NMR (100 MHz,
CDCl3, ppm) δ 192.1, 163.1, 161.7.4, 136.7, 136.1, 135.2, 132.6, 129.5, 129.4, 128.1, 127.8, 127.7, 126.4,
124.9, 122.2, 106.7. 19, 90.5, 55.8, 55.4; FTIR (KBr, cm-1) 3057, 2835, 1658, 1283; MS m/z: 319.16
[M+H]+, HRMS calcd for C21H18O3 318.3656, found m/z: 318.1256.
3-(2,4,6-Trimethoxyphenyl)1-(2-naphthyl)-prop-2-en-1-one, 3f
Pale yellow solid (82.5%), mp 151-153 °C: 1H NMR (400 MHz, CDCl3, ppm) δ 3.87 (s, 3H, OCH3), 3.92
13
(s, 6H, OCH3), 6.15 (s, 2H, CH), 7.25-8.51 (m, 9H, Ar H); C NMR (100 MHz, CDCl3, ppm) δ 190.3,
161.1, 144.7, 136.9, 135.5, 132.6, 129.9, 129.5, 128.6, 128.4, 127.8, 126.8, 124.5, 122.9, 106.4, 102.7,
55.5; FTIR (KBr, cm-1) 2940, 2833, 1642, 1292; MS m/z: 349.14 [M+H]+, HRMS calcd for
C22H20O4318.3918, found m/z: 318.1362.
Cell lines
The different cell lines employed in this study are carcinomic human alveolar basal epithelial cell line (A-
549), Human cervix epithelial carcinoma cell line (HeLa) and human colorectal adenocarcinoma (HCT-
15). The cells were obtained from The National Centre for Cell Sciences, Pune, India and were
maintained at our institute. Hela and A-549 were maintained in Dulbecco's modified Eagle's medium
(DMEM), supplemented with 10% Fetal bovine serum (FBS) whereas HCT was maintained in RPMI
1640 supplimented with 10% FBS. The cells were grown at 37ºC in the presence of 5% Carbon dioxide
(HF-90, Heal force).
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Determination of Cytotoxicity
The cytotoxic effect of synthesized chalcones were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assay (21), against the three cancer cell lines viz., A-549, HeLa and
HCT-15. In the procedure of Moasmann, acid isopropyl alcohol was used as the solvent to dissolve the
formazan crystals. If there are solubility problems, DMSO was found to be an appropriate solvent to
dissolve the crystals of formazan (22). However, in our study we followed the original method of
Mosmann and the solvent used was isopropyl alcohol and a wave length of 570 nm was utilized as
reported (23-26)
Briefly, 100µl of cell suspension (containing 5 × 104 cells/ml for Hela and 8 × 104 cells/ml for other two cell
lines) were plated on each well of a 96 well tissue culture plate. After 24 h, upon attachment of the cells, they
were exposed to different concentrations of the chalcones ranging from 0.1 to 100µg/mL and incubated
for 72 h. After 72 h, the medium was flicked off and the cells were incubated with 50 µl of MTT reagent
(2 mg/mL) for 4 h at 37 °C. The purple coloured formazan crystals formed by the reduction of the soluble
3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide (MTT) were quantified after dissolving
them in 50µl isopropyl alcohol. After mixing with a mechanical plate mixer, the absorbance of each well
was measured by a microplate reader using a test wavelength of 570 nm. The results were expressed as
IC50, which is the concentration of the drugs inducing a 50% inhibition of cell growth of treated cells
when compared to the growth of control cells. The percentage cytotoxicity was calculated using the
formula
=
DNA damaging activity (alkaline comet assay)
The genotoxic effects of chalcones, 3a-f were assessed by comet assay as described by protocol (27), with
slight modifications. Briefly, one million A-549 cells were seeded in T-25 flasks of 5 mL DMEM
medium supplemented with 10% FBS and allowed to form a monolayer for 24 h. The monolayer was
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trypsinised and the cell count was adjusted to 50,000 cells/ml. 2.5 mL of cell suspension was added in
each of the 6 well plates and incubated for 24 h. After 24 h, to this exponentially growing monolayer,
the chalcones to be tested were added in their corresponding wells giving a final concentration of half the
IC50 value of the compounds as indicated by MTT assay. The cells were incubated at 37 °C for a period
of 24 h in an atmosphere of 5% carbon dioxide. Upon incubation, the cells were harvested by
trypsinisation. 20,000 cells in 50 ml medium was suspended in 150 ml of low melting agarose (0.75%)
and layered onto slides pre-coated earlier with a layer of agarose (1.5%). The slides were kept in lysing
solution (2.5 M NaCl, 100 mM (Ethylene diamine tetra acetate) EDTA, 10 mMTris; pH 10–10.5; 1%
Triton X-100 and 10% DMSO) overnight at 4°C in darkness, these were subjected to unwinding under
alkaline conditions (pH 13) for 1 h to allow DNA super coils to relax and express DNA single-strand
breaks and alkali-labile sites. Electrophoresis (E3300 midi horizontal unit consort, Belgium) was then
carried out under highly alkaline (pH 13) conditions for 20 min at 16 V and 300 mA. After neutralization,
staining was done using ethidium bromide. Slides were prepared in triplicate. About 100 cells were
captured per slide using a fluorescent microscope (CKX-41, Olympus, Japan) and the images were
analyzed using Comet Score (Tritekcorp) software.
Cell cycle analysis by Flow cytometry
For cell cycle analysis (28, 29), A549 cells were treated with the selected compounds for 48 h at IC50
concentration. Control comprised of cells without any treatmen; positive control included cells treated
with doxorubicin hydrochloride at a concentration of 2.0 μM and the vehicle control was equal volume of
DMSO (final concentration 0.5 %). At the end of the treatment period, cells were monitored using a phase
contrast microscope (CKX-41 Olympus), and the supernatant floating dead cells were collected; the
adherent cells were trypsinised to get single cell suspension. Pooled cell suspension was centrifuged at
1200 rpm for 5 min at room temperature, washed with 1X phosphate buffered saline (PBS) and then
resuspended in 1.0 mL of 1X PBS. The cells were then fixed with 70 % ice-cold ethanol overnight at -20
°C. The cells were centrifuged at 1200 rpm for 5 min and washed twice with 1mL of PBS. The Cells were
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stained at 37 °C with PBS containing 50 μg/mL of RNase A and 25 μg/mL of propidium iodide for 3 h. A
cell cycle analysis was performed in BD Biosciences LSR II and the data analysis was done in WIN MDI
2.8. A dot plot of forward scatter versus propidium iodide and the histogram of propidium iodide were
plotted. Cells in different phases of the cell cycle were computed for the controls and cell lines were
treated with selected chalcones.
Chromatin condensation studies
Cells undergoing apoptosis exhibit distinct morphologic changes (30). Among these changes, chromatin
condensation and DNA fragmentation of dying cells are the most recognized markers of apoptosis (31,
32). These changes in chromatin DNA mark the point of no return for execution of the cellular apoptotic
pathway (33). In order to confirm that the cytotoxicity action of tested chalcones was processed through
growth inhibition mediated by a DNA replication defect followed by apoptosis, chromatin condensation
studies were conducted. Condensation of chromatin is usually the late event in apoptosis. A-549 cells
were grown in 96-well plates in duplicates and subjected to the treatment of drugs at IC-50 concentration
and incubated for 24 h. About 0.05 ml of the medium was aspirated; same amount of diluted Hoechst
33342 dye was added and incubated for 5 min. 0.05 mL of the medium was removed and viewed under
fluorescence microscope. Hoechst dye binds at the adenine–thymine-rich regions of DNA and emits blue
fluorescence when excited by UV light about 350 nm. DMSO-treated wells were used as control. The
presence of condensed apoptotic chromatin confirms the apoptotic action of drugs.
Results and Discussion
Chemistry
The 2-acetynapthalene (1) was treated with appropriate methoxy substituted benzaldehyde, (2) dissolved
in alkaline ethanol (20 % NaOH), at room temperature for 3h with constant stirring. The chalcones
formed overnight were filtered, dried and dissolved in methanol and recrystallized to get the desired
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products (Table 1), in high purity. The experimental procedure is relatively easy as high yield
transformation did not form any undesirable by-products. Further the products were obtained with a
higher degree of purity that no further purification was needed. The significant advantages of this
procedure are operational simplicity, short reaction time, purity of the products, inexpensive nature ,
nontoxicity of the reagents used and the high yields.
Melting points were measured on an open capillary melting point apparatus and the readings were
1
uncorrected. The structural assignments of compounds, 3 were based on their spectroscopic (IR, H
NMR, 13CNMR and HRMS) data (kindly refer supporting documents).
In FTIR spectrum, the subtituted chalcones (3a-f) shows the α,β-unsaturated carbonyl frequency at
around 1658-1648 cm-1 and the alkenic C=C frequency at around 1259-1292 cm-1. The IR spectra of
compound 3a revealed a characteristic band between 3,022 and 1,280 cm-1 confirming the presence of
(C=C) groups and a characteristic band between 1,584 and 1,651 cm-1, confirming the presence of C=O
groups (Figure.2). The olefinic proton Ha being placed in β-pisition of α-β-unsaturated carbonyl group
was deshielded from the normal alkenyl region. And the signal δ 8.13 split into doublet by Hb with J =
15.84 Hz. In the same way, the proton Ha deshielded with Hb and appeared as doublet of 7.70 ppm with J
13
= 15.84 Hz (Figure.3). In C NMR spectrum the chalcones (3a-f), the α,β-unsatutated carbonyl peaks
appeared at δ 192.10-190.03. The compound 3a showed a characteristic peak around 190.8, 153.5, 153.4,
135.8, 135.4, 132.6, 129.9, 129.5, 128.4, 128.2, 127.8, 126.7, 2x 124.6, 123.2, 117.19, 113.8, 112.5, 56.1,
55.9 ppm in the 13C NMR spectrum (Figure. 4). The HRMS of title compound 3a showed a molecular ion
peak M+ in the positive mode. The molecular ion peak of 3a was observed at m/z 318.1256.
In vitro cytotoxicity assay
The cytotoxicity of the synthesized chalcones towards different cell lines viz., HCT-15 (Humancolon
carcinoma), HeLa (Cervical carcinoma), A549 (Lung adenocarcinoma) were determined by MTT ([3-
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(4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)) assay. The formazan produced during the
MTT assay formed by the viable cells was solubilized by adding 100 mL isopropanol. The absorbance
was recorded at 570 nm in a micro plate reader. The minimum concentration required for 50 %
cytotoxicity and inhibitory concentration required for 50 % cytotoxicity (IC50) values were tabulated
(Table 2). The datas are represented as mean ( ) SD for all cell lines. However, the data presented in our
paper are not a comparison between the synthesized molecules and existing drugs as standard. The IC 50
values of standard anticancer drugs such as doxorubicin or cisplatin are available in the literature for any
reader to access. The reported values are concentrations at which the synthesized compounds are
inhibiting cells proliferation similar to literature reports (34, 35). In all the three cell lines tested, the
highest cytotoxicity was shown by 3c of 0.019 µM against HeLa, 0.020 µM against HCT 15 and 0.022
µM against A549. Compound 3e (3-(3,5-Dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) showed
better IC50 of 0.018 µM against HeLA and 0.022 and 0.025 against HCT15 and A549 respectively. The
order of cytotoxicity was found to be 3c > 3e > 3b > 3a > 3f > 3d, indicating that methoxy group in the
R2 position is critical for the activity. As seen from Table 2, all the compounds tested, showed potential
cytotoxicity against all the three human cancer cell lines employed for the study. There was no significant
difference between the activity of 3c and 3e indicating that methoxy substitution in the R3 position is not
critical in cytotoxicity. The Hela cells were found to be more resistant when compared with the other two
cells used in the study. Similarly substitution at R1 position also did not contribute to the cytotoxic effect
of the chalcones.
Alkaline comet assay
The tail moment has been regarded as one of the best indices of DNA damage among the other
parameters,which was calculated using the formula,
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To evaluate the extent of DNA damage offered by the tested chalcone molecules, alkaline comet assay
was performed and the tail moment was calculated. The highest tail moment was shown by chalcone, 3c
which was found to produce extensive DNA damage, followed by 3a and 3e. The results (Table.3) were
in agreement with that of MTT assay. Similarly, other chalcones also produced significant DNA damage
and the results are comparable with that of the standard drug cisplatin.
Cell cycle analysis
Considering the total number of cells taken into account in every instance as 100%, the percentage of
cells found in different phases of cell the cycle were analyzed and compared with vehicle (DMSO) at a
concentration of drug solutions used (control). The number of cells arrested in each phase of the cell
division cycle was calculated. The DMSO treated cells showed a typical histogram (Figure 5) of cell
cycle pattern with minimum cells in the subG0 phase and two peaks for G0/G1 and G2/M transitions
separated by S phase. The population of Sub G0 cells was very less in vehicle treated cells indicating an
absence of apoptosis. The histogram of chalcones, 3b and 3c (Figure 6, 7) suggest the arrest of cells in
sub G0 phase which is a characteristic of apoptotic mode of death. Hence, the DNA damage indicated in
comet assay was proven to be due to apoptosis and not due to necrosis. A similar characteristic arrest of
cells was found in all cells treated with chalcones except 3e. In using chalcones 3e, the accumulation was
found to be in G2/M phase when compared to the other three phases. This indicates an incomplete mitosis
and could be due to the arrest of tubulin polymerization. This is in agreement with the findings of Ducki
et al (36-38) and Lee (39). The results were compared with that of the standard drug doxorubicin (Table
4). Doxorubicin-induced Apoptosis is reported by many workers (40-43).
Chromatin condensation studies
Combretastatin like chalcones can act as inhibitors of microtubule polymerization leading to chromatin
condensation and apoptosis. Nuclear condensation was found to be one of the hall marks of apoptosis.
Cells that undergo apoptosis demonstrate a nuclear condensation and DNA fragmentation, which can be
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detected by staining with Hoechst 33342 and fluorescence microscopy. The cells exposed to compounds
3a, 3b, 3c and 3f were stained with Hoechst 33342 exhibited numerous cells with condensed or
fragmented nuclei. Hoechst 33342 is a "vital" DNA stain that binds preferentially to A-T base-pairs. The
cells require no permeabilization for labeling but do require physiologic conditions since the dye
internalization is an active transport process. Apoptotic events were found to be maximum in cells treated
with 3c followed by 3e, 3b and 3a (Figure 8-11). There is a good correlation between the results of
nuclear staining with that of flow cytometry analysis.
Compared to the classic methods of DNA ladder formation by gel electrophoresis and of morphologic
examination by microscopy for determination of apoptosis, flow cytometry permits rapid and quantitative
measurements on apoptotic cells (44). The work reported here is a preliminary study on the compounds
synthesized on their ability to inhibit the cell proliferation by flow cytometric analysis which gave us an
insight on possible targets of drug action. From the morphology studies and fluorescent staining studies,
condensation of the nucleus and fragmentation of nucleus is seen. These are symptoms of apoptosis.
Further studies using advanced models to understand the mode of action, systemic toxicity and anticancer
activity is in progress, which is beyond the scope of this paper.
Conclusions
In summary, the syntheses and biological evaluations of methoxy substituted chalcones, which are
derivatives of Combretastatin were performed. The scheme of synthesis is very simple and has only
limited number of steps which make this process economical. The synthesized chalcons were tested for
thir relative cytotoxicity and apoptosis inducing ability. Cytotoxic analysis was performed against three
important cancer cell lines of lung, cervical and colon origin. The synthesized molecules proved to
possess potent cyctotoxicity against human cancer cells. Among the compounds tested, compound 3c (3-
(3,4,5-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) and 3e (3-(3,5-dimethoxyphenyl)-1-(2-
naphthyl) prop-2-en-1-one, have been found to be most effective. Compound 3d, (3-(2, 4-
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dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one) is found to be least effective. The DNA damage
following alkaline comet, flow cytometric analysis and chromatin condensation studies confirmed the
mode of cell death to be apoptosis and arrest of tubulin polymerization. Further presence of condensed
apoptotic chromatin confirms the apoptotic action.
Acknowledgements
The authors wish to express their gratitude to VIT- University, Vellore and Manipal university, Manipal
and for their support and facilities. We are thankful to Dr. Vijayalakshmi, School of Social Sciences and
Launguages, VIT University for the technical support
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Legends of Schemes, Figures and Tables
Scheme 1. Synthesis of chalcones 3 (a-f)
Figure 1: Structure of combretastain A12
Figure 2: FTIR spectrum of compound 3a
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Figure 3: 1H spectrum of compound 3a
Figure 4: 13C NMR spectrum of compound 3ª
Figure 5: Histogram of DMSO treated cells
Figure 6: Histogram of flow cytometric analysis of compounds (1-(2-naphthyl)-3-(3, 4, 5-
trimethoxyphenyl) prop-2-en-1-one, 3c.
Figure 7: Histogram of flow cytometric analysis of compounds 3-(2,3,4-Trimethoxyphenyl)-1-(2-
naphthyl) prop-2-en-1-one, 3b.
Figure 8: Control
Figure 9: Cells treated with 3-(2,5-Dimethoxyphenyl)-1-(2-naphthyl)prop-2-en-1-one, 3a
Figure 10: Cells treated with 3-(2,3,4-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3b
Figure 11: Cells treated with 3-(3,4,5-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3c
Figure 12: Cells treated with 3-(3,5-dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3e
Table 1: Synthesised chalcones
Table 2: Cytotoxic analysis of methoxy substited chalcones against various cell lines using MTT assay
(mean SD)
Table 3: DNA Damage analysis by Comet assay. (mean SD)
Table 4: Percentage of cells in different stages of the cell cylce – flow cytometric analysis.
R2
CHO
CH3
R1
R3
R4
R5
R1
R2
a
O
+
R4
O
R5
R3
3 (a-f)
2 (a-f)
1
Scheme 1. Synthesis of chalcones 3 (a-f)
Reagents and conditions: a)NaOH, ethanol, 3 h, RT.
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OMe
MeO
OMe
MeO
HO
OH
Figure 2: Structure of combretastain A12
Table 5: Synthesised chalcones
O
R1
R4
R2
R3
R5
R1
R2
H
R3
H
R4
OCH3
H
R5
H
%Yield
85.6
OCH3
OCH3
H
3a
3b
3c
3d
3e
3f
OCH3
OCH3
H
OCH3
OCH3
OCH3
H
H
78.7
OCH3
H
H
79.4
OCH3
H
H
82.6
OCH3
H
OCH3
H
H
85.7
OCH3
OCH3
OCH3
82.5
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Table 6: Cytotoxic analysis of methoxy substited chalcones against various cell lines using MTT
Assay (mean SD)
IC50 values in µM
code
A549
HeLa
HCT-15
3a
3b
3c
3d
3e
3f
0.047 0.002
0.037 0.0007
0.022 0.001
0.149 0.008
0.025 0.002
0.072 0.0012
0.126 0.003
0.095 0.002
0.019 0.002
0.204 0.002
0.018 0.006
0.144 0.001
0.050 0.004
0.043 0.004
0.020 0.001
0.116 0.002
0.022 0.003
0.022 0.002
Table 7: DNA Damage analysis by Comet assay. (mean SD)
Compounds
% DNA in Head % DNA in tail
Tail moment
Control
3a
89.7 1.63
33.6 1.3
37.2 1.8
22.4 3.1
41.2 2.8
27.8 3.4
26.8 2.4
37.28 1.5
10.3 1.63
66.4 1.3
62.8 1.8
77.6 3.1
58.8 2.8
72.2 3.4
73.2 2.4
62.72 1.5
3.24 0.65
88.6 2.46
86.4 3.12
93.1 3.18
80.6 5.91
88.3 3.76
82.5 4.28
87.58 6.28
3b
3c
3d
3e
3f
Cisplatin
(Standard)
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Table 8: Percentage of cells in different stages of the cell cylce – flow cytometric analysis.
Compounds Percentage of cells in each phases
G0/G1
73.13
54.56
39.92
26.68
16.62
40.55
59.59
S phase
9.87
G2/M Sub G0
16.33 1.04
27.1 21.63
2.74 55.95
9.11 61.53
76.72 2.26
25.39 28.14
17.46 11.87
28.37 21.63
Control
3a
14.38
1.95
3b
2.22
3c
4.01
3d
5.79
3e
11.7
3f
Doxorubicin 36.8
(Standard)
14.1
Legends for supporting documents
S1-IR Spectrum- 3a
S2-HNMR- 3a
S3-HNMR- 3a- Expanded
S4-13C NMR Spectrum 3a
S5-13C NMR Spectrum 3a (Intrepreted)
S6-Mass Spectrum 3a
S7-IR SPECTRUM- 3b
S8-HNMR- 3b
S9-HNMR- 3b (expanded)
S10-13C NMR Spectrum 3b
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S11-13C NMR Spectrum 3b (Intrepreted)
S12-Mass Spectrum – 3b
S13-IR Spectrum of 3c
S14-1H NMR Spectrum of 3c
S15-1H NMR Expanded Spectrum of 3c
S16-13C NMR Spectrum 3c (Intrepreted)
S17-13C NMR Spectrum 3c
S18- MASS Spectrum of 3c
S19-IR SPECTRUM 3d
S20-HNMR- 3d
S21-HNMR- 3d- Expanded
S22-13C NMR Spectrum 3d (Intrepreted)
S23-13C NMR Spectrum 3d
S24-Mass- 3d
S25-IR spectrum 3e
S26-HNMR-3e
S27-HNMR- 3e -Expanded
S28-13C NMR Spectrum 3e (Intrepreted)
S29-13C NMR Spectrum 3e
S30-Mass- 3e
S31-IR Spectrum of 3f
S32-1H NMR Spectrum of 3f
S33-1H NMR Expanded Spectrum of 3f
S34-13C NMR Spectrum 3f (Intrepreted)
S35-13C NMR Spectrum 3e
S36-MASS Spectrum of 3f
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100
80
60
40
20
0
2823
1262
1661 1500
1600
4000
3500
3000
2500
2000
1500
1000
500
W avenumber
Figuree 2: FTIR spectrum of compound 3a
Figure 3: 1H spectrum of compound 3a

Figure 4: 13C NMR spectrum of compound 3a
Figure 5: Histogram of DMSO treated cells
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Figure 6: Histogram of flow cytometric analysis of compounds (1-(2-naphthyl)-3-(3, 4, 5-
trimethoxyphenyl) prop-2-en-1-one, 3c.
Figure 7: Histogram of flow cytometric analysis of compounds 3-(2,3,4-Trimethoxyphenyl)-1-(2-
naphthyl) prop-2-en-1-one, 3b.
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Figure 8: Control
Figure 9: Cells treated with 3-(2,5-Dimethoxyphenyl)-1-(2-naphthyl)prop-2-en-1-one, 3a
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Figure 10: Cells treated with 3-(2,3,4-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3b
Figure 11: Cells treated with 3-(3,4,5-trimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3c
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Figure 12: Cells treated with 3-(3,5-dimethoxyphenyl)-1-(2-naphthyl) prop-2-en-1-one, 3e
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