Synthesis and anticancer activity of chalcones derived from vanillin and isovanillin

Article abstract of DOI:10.1007/s00044-015-1453-2

An array of chalcones from vanillin/isovanillin and differently substituted acetophenones were synthesized and assessed for their anticancer activity against A549, MCF7 and MIA PaCa-2 cell lines using MTT assay. Some of the chalcones exhibited good antica

Full text of DOI:10.1007/s00044-015-1453-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1453-2
ORIGINAL RESEARCH
Synthesis and anticancer activity of chalcones derived
from vanillin and isovanillin
Saiharish Raghavan1 Prasath Manogaran2
•
•
Balasubramanian Kalpattu Kuppuswami1 Ganesh Venkatraman2
Krishna Kumari Gadepalli Narasimha1
•
•
Received: 5 December 2014 / Accepted: 24 August 2015
Ó Springer Science+Business Media New York 2015
Abstract An array of chalcones from vanillin/isovanillin
and differently substituted acetophenones were synthesized
and assessed for their anticancer activity against A549, MCF7
and MIA PaCa-2 cell lines using MTT assay. Some of the
chalcones exhibited good anticancer activity with IC50 values
below 10 lM. Compound 5f with IC50 values 5.4 0.7,
10.45 2.15 and 13.0 1.68 lM on MIA PaCa-2, A549
and MCF7, respectively, was more potent than curcumin and
hencewasanalyzedforchangesincellmorphology,inhibition
of cell migration, mechanism of cell death and arrest of cell
cycle progression on MIA PaCa-2 cells.
study involves synthesis and screening of vanillin- and
isovanillin-based chalcones (Fig. 1b) which can be dubbed
as mimics of curcumin due to structural similarity such as
presence of two phenyl rings and a,b-unsaturated ketone
moiety and presence of hydroxyl and methoxy groups in
ring B (vanillin ring). Chalcones (1,3-diaryl-2-propen-1-
one) exhibit large number of biological activities including
anticancer activity (Kamal et al., 2010; Rao et al., 2009;
Vogel et al., 2010). Chalcones can be synthesized easily
using conventional methods and relish the stature of good
safety profile and is pertinent for oral administration. There
are recent reports on anticancer activity of chalcones, teth-
ered chalcones, chalcone hybrids, bis-chalcones and
polyphenol chalcones (Van Luu et al., 2014; Sharma et al.,
2014; Winter et al., 2014; Zhang et al., 2015). However,
reports on anticancer activity and biological studies of
vanillin- and isovanillin-based chalcones are relatively
sparse. Hence, a detailed investigation into the anticancer
activity of these compounds was carried out in this study.
Keywords Chalcones Á Vanillin and isovanillin Á
MTT assay Á MIA PaCa-2 Á Apoptosis Á Cell cycle analysis
Introduction
Curcumin [diferuloylmethane or (1E,6E)-1,7-bis-(4-hy-
droxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (Fig. 1a)
is affirmed to have striking anticancer activity against dif-
ferent cell lines (Agarwal et al., 2003; Yamakoshi et al.,
2010; Han et al., 2011; Wang et al., 2011). Curcumin is
reputed to be safe even at high doses, and its efficacy in
humans is also manifested (Anand et al., 2007). This present
Materials and methods
Chemistry
Melting points were recorded on Veego (WMP-DS) cap-
illary melting point apparatus and are uncorrected. All the
reagents were procured from Sigma-Aldrich, Rankem,
Avra and Spectrochem and used without further purifica-
tion. All commercial solvents were purchased from Avra
and used after distillation. Purity of the compounds was
checked by TLC on Merck silica gel 60 F254 plates, and
compounds were purified using column chromatography
with silica gel (100–200, 230–400 mesh sizes) as stationary
phase and hexane: ethyl acetate as eluent. FTIR spectra
& Ganesh Venkatraman
ganeshv@sriramachandra.edu.in
& Krishna Kumari Gadepalli Narasimha
drkrishna2011@gmail.com
1
Department of Medicinal Chemistry, Sri Ramachandra
University, Porur, Chennai, Tamil Nadu 600 116, India
2
Department of Human Genetics, Sri Ramachandra
University, Porur, Chennai, Tamil Nadu 600 116, India
123

Med Chem Res
Fig. 1 a Curcumin, b chalcones
H
O
O
R3
A
O
B
H
O
H
O
R2
R4
O
O
R1
a
b
H
O
veratraldehyde (3,4-dimethoxybenzaldehyde) 8 was con-
densed with differently substituted acetophenones to yield
corresponding chalcones 8a–8c (Table 1). The chalcones
were either purified by recrystallization from appropriate
solvents or by column chromatography using hexane: ethyl
acetate as eluent. The percentage yields of all chalcones
reported in this study were around 80–90 % including the
recovery of starting materials. The structures of these com-
pounds were confirmed by IR, NMR spectroscopic studies
and are found to be in accordance with the literature (Arty
et al., 2000; Lahtchev et al., 2008; Bakht et al., 2013; Juvale
et al., 2012; Kumari et al., 2014; Sharma et al., 2012).
R1
R
R2
= -
= -
1
2
3
4
7
8
R1
R1
R1
R1
R1
R1
H
H
O
OC
;
= -
O
= -
OC
= -
= -
=
2 = -
3
H
;
R2
H
OC
3
= -
H
R
THP
O
;
3
2
= -
THP R
H
O
OC
;
2 = -
H
R
H3
H
OC
OC 2COO
;
2
= -3
R2
H
OC
3
Fig. 2 Vanillin (1), isovanillin (2) and their derivatives (3, 4 and 7)
and veratraldehyde (8)
Biology
were recorded in KBr pellet using Bruker FTIR Alpha
spectrometer in the range 500–4000 cm-1 1H and 13C
.
Growth medium Dulbecco’s Modified Eagle’s Medium
(DMEM), Fetal Bovine Serum (FBS), Acridine Orange
(AO) and Ethidium Bromide (EB) were purchased from
Himedia Laboratories. MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide] and propidium iodide
(PI) were purchased from Sigma. Dimethyl sulfoxide
(DMSO) (spectroscopic grade) was purchased from Spec-
trochem, and 5-FU (5-fluorouracil) was purchased from
United Biotech. Cisplatin was purchased from Cipla.
NMR were run on Bruker 400 MHz and 100 MHz spec-
trometers, respectively, using TMS (tetramethylsilane) as
internal standard in CDCl3.
General procedure for the synthesis of chalcones
In the present study, vanillin (1) and isovanillin (2) (Fig. 2)
were chosen as starting materials for the preparation of
chalcones and the hydroxyl groups of vanillin and isovanillin
were protected using dihydropyran to form corresponding
tetrahydropyranyl ethers (3 and 4) using standard procedures
available in the literature (Greene and Wuts, 1999; Miyashita
et al., 1977) in order to increase the reactivity of carbonyl
group toward Claisen Schmidt condensation. Then, the
tetrahydropyranyl ethers of vanillin and isovanillin were
condensed with differently substituted acetophenones (Arty
et al., 2000) to yield chalcones of tetrahydropyranyl ethers.
After completion of condensation reaction, the THP (te-
trahydropyranyl) ethers were deprotected using methanol/
4 N HCl or methanol/para toluene sulfonic acid under mild
conditions (Corey et al., 1978) to yield chalcones 5a–5f and
6a–6d (Table 1). Vanillin was converted to corresponding
aryloxyacetic acid derivative 7 by known method (Abraham
et al., 1986) which was then condensed with acetophenone to
yield corresponding chalcone 7a (Table 1) according to the
literature procedure (Bakht et al., 2013). Similarly,
Biological assays
MTT assay
The cells A549, MCF7 and MIA PaCa-2 were cultured in
DMEM containing 10 % FBS at 37 °C and 5 % CO2. In
total, 20,000 cells were seeded in each well (in 96-well
plates) containing 200 ll of DMEM medium. After 24 h of
incubation, different concentrations of the test compounds
were added, and again after 24 h of treatment, 20 ll per
well of MTT (5 mg/ml stock solution) was added. The
plates were incubated at 37 °C for additional 4 h. The
medium was discarded, and the formazan, which formed in
the cells, was dissolved in 200 ll of DMSO. The intensity
of color production was measured at 570 and 695 nm in a
spectrophotometer.
IC50 values were calculated using Microsoft Excel
software for semilog curve fitting with regression analysis.
123

Med Chem Res
Table 1 Synthesized chalcones and their IC50 values
S.
no.
Compound
R1
R2
R3
R4
IC50 values (lM) SD
MIA PaCa-2
MCF7
A549
1.
5a
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OCH3
–OH
–OH
–H
–H
–H
–H
–H
–Cl
–H
–H
–H
–Cl
–H
–H
–H
–H
–
–H
25.1 2.4
[100
36.45 4.4
74.0 0.5
17.1 0.32
11.95 0.74
8.5 0.50
13.0 1.68
45.7 0.50
74.15 0.15
16.8 3.58
10.0 0.04
46.8 0.37
37.8 0.20
40.74 0.26
17.8 0.50
17 2.21
18.8 3.18
28.1 4.1
15.4 0.40
31.3 1.30
34.1 4.11
10.45 2.15
25.05 0.75
33.65 1.35
23.5 1.75
10.1 0.04
42.4 4.11
25.8 1.50
28.83 0.88
17.6 1.20
21 1.6
2.
5b
–OH
–OCH3
–Cl
–F
3.
5c
–OH
17.8 0.87
32.7 3.6
41.25 1.7
5.4 0.7
47.2 7.02
64.2 1.20
11.43 0.01
7.5 0.01
[100
4.
5d
–OH
5.
5e
–OH
–NO2
–Cl
–H
6.
5f
–OH
7.
6a
–OCH3
–OCH3
–OCH3
–OCH3
–OCH2COOH
–OCH3
–OCH3
–OCH3
–
8.
6b
–OH
–OCH3
–Cl
–Cl
–H
9.
6c
–OH
10.
11.
12.
13.
14.
15.
16.
6d
–OH
7a
–OCH3
–OCH3
–OCH3
–OCH3
–
8a
–H
[100
8b
–OH
–Cl
–
[100
8c
18.6 1.5
–
Cisplatin
5-Fluorouracil
–
–
–
–
38.8 0.8
–
–
Morphological changes
Percentage of migration ¼ fðWidth at 0 h
À Width at 24 h)=Width at 0 hg
 100
MIA PaCa-2 cells were seeded in six-well plates (5 9 105)
and incubated in DMEM at 37 °C in an atmosphere of 5 %
CO2 for 24 h. Cells were treated with different concen-
trations of the most potent compound 5f. After incubation
for 24 h, cellular morphology was observed using phase
contrast microscopy (Optica view). Images were captured
at a magnification of 109.
The average data point was used to calculate the percent-
age migration.
AO/EB assay
The most potent compound 5f was analyzed for the mode
of cell death induced in MIA PaCa-2 cell line by AO/EB
assay. 1 9 105 cells were seeded onto 12-well tissue cul-
ture plates and incubated overnight at 37 °C in an atmo-
sphere of 5 % CO2. Once the cells reached 80 %
confluency, the cells were treated with IC50 concentration
of compound 5f as well as 29IC50 concentration for 24 h.
Cells treated with 0.1 % DMSO were maintained as vehi-
cle control and cells treated with 5-fluorouracil (500 lg/
ml) as positive control. Following treatment, the cells were
detached, pelleted and resuspended in complete media.
Twenty microliters of cell suspension was treated with
10 ll of AO (1 mg/ml) and 10 ll of EB (1 mg/ml) and
In vitro scratch assay
3 9 105 cells were seeded in six-well plates and incubated
overnight at 37 °C in an atmosphere of 5 % CO2 for cell
attachment. Following treatment with mitomycin C (5 mg/
ml) for 2 h, a scratch was induced using a sterile pipette tip.
The cells were then treated with increasing concentrations
(1, 2, 3 and 4 lM) of the most potent compound 5f for
24 h. Cells treated with 10 % FBS were maintained as
positive control. Following incubation, the migration of
cells into the marked region was observed and rate of
migration was calculated using the formula:
123

Med Chem Res
loaded onto a clean glass slide and analyzed under a flu-
orescence microscope. The cells were divided into four
categories as live cells (green nucleus), early apoptotic
cells (bright green nucleus with condensed or fragmented
chromatin), late apoptotic cells (orange-stained nuclei with
chromatin condensation or fragmented) and necrotic cells
(uniform orange-stained cell nuclei).
cell lines. Introduction of electron-withdrawing groups such
as –NO2 and –F increased the activity in MCF7, whereas it
decreased the activity in A549 and MIA PaCa-2. But –Cl was
found to increase cytotoxicity in all the three cell lines, viz.
A549, MCF7 and MIA PaCa-2. Since –Cl increased cyto-
toxicity consistently on all three cell lines, dichloro-substi-
tuted chalcones were synthesized and, as predicted, these
dichloro-substituted chalcones 5f and 6d were found to be
more potent than 5c and 6c, respectively. Further biological
studies were carried out with most potent compound 5f on
MIA PaCa-2 cell line. However, extensive studies are
required to establish the complete SAR of chalcones.
Cell cycle analysis
Analysis of inhibition of cell cycle progression on MIA
PaCa-2 cells treated with compound 5f and vehicle control
was determined using flow cytometry. Using hypo PI
method, the DNA of the cells was stained with PI, and the
percentage of cells in each phase of the cell cycle was
determined. Briefly, 4 9 105 cells were seeded in 35 mm
plate and incubated for 24 h for attachment. Following this,
the cells were treated with IC50 and 29IC50 concentrations
of compound 5f. The cells were maintained at 37 °C with
5 % CO2 for 24 h. At the end of this incubation period, the
cells were scraped, collected and centrifuged at 1500 rpm
for about 5 min. Following this, 1 ml of hypotonic solution
was added and the cells were vortexed vigorously. Samples
were analyzed on BD C6 Accuri flow cytometer with
10,000 events acquired for each sample.
Spectral data of chalcones
(E)-3-(4-hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-
one (5a) mp 92–94 °C; IR (KBr) mmax (cm-1): 3454
(–OH, H-bonded), 1690 (C=O), 1550 (Ar C–C); H NMR
1
(CDCl3, 400 MHz, TMS = 0): 3.78 (3H, s, –OCH3), 6.53
(1H, d, J = 8.4 Hz, H-5), 7.03 (1H, d, J = 2 Hz, H-2), 7.04
(1H, dd, J = 8.4 Hz, J0 = 2 Hz, H-6), 7.23 (1H, d,
J = 15.2 Hz, =CH), 7.45 (3H, m, H-30, H-40, H-50), 7.68
(1H, d, J = 15.2 Hz, =CH), 7.93 (2H, m, H-20, H-60). 13C
NMR (CDCl3, 100 MHz, TMS = 0): 193.0 (C=O), 161.4
(C-40), 152.0 (C-3), 149.5 (C-4), 140.4 (C-b), 133.5 (C-10),
129.7 (C-30, C-50), 129.4 (C-20, C-60), 127.5 (C-1), 123.3 (C-
6), 118.8 (C-a), 116.0 (C-5), 111.4 (C-2), 56.1 (–OCH3).
Results and discussion
(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl)
prop-2-en-1-one (5b) mp 148–151 °C; IR (KBr) mmax
(cm-1): 3411 (–OH, H-bonded), 1653 (C=O), 1277 (C–O);
1H NMR (CDCl3, 400 MHz, TMS = 0): 3.89 (3H, s,
–OCH3), 3.97 (3H, s, –OCH3), 6.95 (1H, d, J = 8 Hz, H-
5), 6.99 (2H, d, J = 8.8 Hz, H-30, H-50), 7.13 (1H, d,
J = 1.6 Hz, H-2), 7.22 (1H, dd, J = 8 Hz, J0 = 1.6 Hz,
H-6), 7.39 (1H, d, J = 15.6 Hz, =CH), 7.74 (1H, d,
J = 15.6 Hz, =CH), 8.03 (2H, d, J = 8.8 Hz, H-20, H-60);
13C NMR (CDCl3, 100 MHz, TMS = 0): 188.8 (C=O),
163.3 (C-40), 148.3 (C-3), 146.8 (C-4), 144.3 (C-b), 131.3
(C-10), 130.7 (C-20, C-60), 128.3 (C-1), 127.7 (C-6), 119.6
(C-a), 114.8 (C-5), 113.8 (C-30, C-50), 110.0 (C-2), 56.0
and 55.5 (–OCH3).
Analysis of cytotoxicity using MTT assay
The cytotoxicity of the synthesized chalcones was studied
using MTT assay on three human cancer cell lines—A549,
MCF7 and MIA PaCa-2. IC50 values were identified using
Microsoft Excel software for semilog curve fitting with
regression analysis. The variation in cytotoxicity by modi-
fying functional groups in both rings was studied. Protection
of –OH group of ring B as aryloxy acetic acid (7a) or
methoxy (8a) resulted in decreased activity of chalcones 7a
and 8a. This is in accordance with the literature report
wherein the presence of hydroxyl group in para position of
ring B is reported to be essential for activity (Venkateswar-
arao et al., 2012). Variation in substituents in ring A did not
lead to results corroborative with the literature which reports
that presence of electron-withdrawing group in ring A
decreases cytotoxicity whereas electron-donating group
increases cytotoxicity in all the five tested cell lines (Bai
et al., 2014). Also there is another report which says intro-
duction of any substituent (either electron donating or elec-
tron withdrawing) decreases the cytotoxicity as compared to
unsubstituted chalcones on the tested cell lines (Prabhakar
et al., 2014). In our present study, introduction of electron-
donating group in ring A decreasedthe activity in all the three
(E)-1-(4-chlorophenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-
2-en-1-one (5c) mp 195–196 °C; IR (KBr) mmax (cm-1):
3429 (–OH, H-bonded), 1645 (C=O), 1513 (Ar C–C), 1275
(C–O), 1032 (Ar–Cl); 1H NMR (CDCl3, 400 MHz,
TMS = 0): 3.98 (3H, s, –OCH3), 6.05 (1H, s, –OH), 6.98
(1H, d, J = 8 Hz, H-5), 7.14 (1H, d, J = 1.6 Hz, H-2),
7.23 (1H, dd, J = 8 Hz, J0 = 1.6 Hz, H-6), 7.34 (1H, d,
J = 15.6 Hz, =CH), 7.48 (2H, d, J = 8.8 Hz, H-30, H-50),
7.77 (1H, d, J = 15.6 Hz, =CH), 7.97 (1H, d, J = 8.8 Hz,
H-20, H-60’); 13C NMR (CDCl3, 100 MHz, TMS = 0):
123

Med Chem Res
189.3 (C=O), 148.5 (C-3), 146.9 (C-4), 145.8 (C-b), 139.0
(C-40), 136.8 (C-10), 129.8 (C-20, C-60), 128.9 (C-30, C-50),
127.3 (C-1), 123.5 (C-6), 119.2 (C-a), 114.9 (C-5), 110.1
(C-2), 56.1 (–OCH3).
(C-40), 128.6, 128.4 (C-1, C-20, C-30, C-50 and C-60), 122.7
(C-a), 120.3 (C-6), 113.0 (C-5), 110.6 (C-2), 56.0 (–OCH3).
(E)-3-(3-hydroxy-4-methoxyphenyl)-1-(4-methoxyphenyl)
prop-2-en-1-one (6b) mp 150–152 °C; IR (KBr) mmax
(cm-1): 3461(–OH, H-bonded), 3010 (Ar C–H), 1655(C=O),
(E)-1-(4-flurophenyl)-3-(4-hydroxy-3-methoxyphenyl)prop-
2-en-1-one (5d) mp 90–92 °C; IR (KBr) mmax (cm-1):
3415 (–OH, H-bonded), 1662 (C=O), 1259 (C–O), 1064
(Ar–F); 1H NMR (CDCl3, 400 MHz, TMS = 0): 3.97 (3H,
s, –OCH3), 5.95 (1H, bs, –OH), 6.96 (1H, d, J = 8 Hz, H-
5), 7.2 (2H, m, H-2, H-6), 7.34 (1H, d, J = 15.6 Hz, =CH),
7.75 (1H, d, J = 15.6 Hz, =CH), 8.05 (4H, m, H-20, H-30,
H-50, H-60); 13C NMR (CDCl3, 100 MHz, TMS = 0):
189.1 (C=O), 148.5 (C-3), 147.0 (C-4), 145.6 (C-b), 131.2
(C-40), 131.1 (C-10), 127.5 (C-20, C-60), 123.6 (C-1), 119.4
(C-6), 115.9 (C-30, C-50), 115.7 (C-a), 115.1 (C-5), 110.2
(C-2), 56.2 (–OCH3).
1
1459 (Ar C–C), 1277 (C–O); H NMR (CDCl3, 400 MHz,
TMS = 0): 3.91 (3H, s, –OCH3), 3.92 (3H, s, –OCH3), 7.00
(1H, d, J = 8.4 Hz, H-5), 7.07 (2H, d, J = 8.8 Hz, H-30, H-
50), 7.21 (1H, dd, J = 8.4 Hz, J0 = 2 Hz, H-6), 7.25 (1H, d,
J = 2 Hz, H-2), 7.57(1H, d, J = 15.6 Hz, =CH), 7.69(1H, d,
J = 15.6 Hz, =CH), 8.09(2H, d, J = 8.8 Hz, H-20, H-60);13C
NMR (CDCl3, 100 MHz, TMS = 0): 191.2 (C=O), 165.3 (C-
40), 151.9 (C-4), 148.1 (C-3), 146.1 (C-b), 132.2, 132.1 (C-20,
C-60), 129.5 (C-10), 127.4 (C-1), 123.6 (C-6), 120.4 (C-a),
115.2 and 115.1 (C-30, C-50), 112.6 (C-5), 110.2 (C-2), 56.5,
56.2 (–OCH3).
(E)-3-(4-hydroxy-3-methoxyphenyl)-1-(4-nitrophenyl)prop-
2-en-1-one (5e) mp 170–175 °C; IR (KBr) mmax (cm-1):
3465 (–OH, H-bonded), 1665 (C=O), 1546 (Ar C–C), 1283
(N–O); 1H NMR (CDCl3, 400 MHz, TMS = 0): 3.98 (3H,
s, –OCH3), 5.98 (1H, bs, –OH), 6.98 (1H, d, J = 8 Hz, H-5),
7.28 (2H, m, H-2, H-6), 7.31 (1H, d, J = 15.6 Hz, =CH),
7.78 (1H, d, J = 15.6 Hz, =CH), 8.22 (1H, d, J = 7.6 Hz,
H-20, H-60), 8.35 (2H, d, J = 7.6 Hz, H-30, H-50); 13C NMR
(CDCl3, 100 MHz, TMS = 0): 189 (C=O), 148 (C-40), 147
(C-3), 145 (C-4), 131 (C-b), 130 (C-10), 127 (C-20, C-60),
123 (C-1), 119 (C-30, C-50), 115.5 (C-6), 115 (C-a), 114 (C-
5), 110 (C-2), 56 (–OCH3).
(E)-1-(4-chlorophenyl)-3-(3-hydroxy-4-methoxyphenyl)prop-
2-en-1-one (6c) mp 195–196 °C; IR (KBr) mmax (cm-1):
3531 (–OH, H-bonded), 1650 (C=O), 1513 (Ar C–C), 1264
(C–O), 1032 (Ar–Cl); 1H NMR (CDCl3, 400 MHz,
TMS = 0): 3.88 (3H, s, –OCH3), 5.63 (1H, s, –OH), 6.81
(1H, dd, J = 8.4 Hz, J0 = 2 Hz, H-6), 7.07 (1H, d,
J = 8.4 Hz, H-5), 7.21 (1H, d, J = 2 Hz, H-2), 7.27 (1H,
d, J = 15.6 Hz, =CH), 7.48 (2H, d, J = 8.8 Hz, H-30,
H-50), 7.67 (1H, d, J = 15.6 Hz, =CH), 7.88 (2H, d,
J = 8.8 Hz, H-20, H-60); 13C NMR (CDCl3, 100 MHz,
TMS = 0): 189.3 (C=O), 148.5 (C-4), 146.9 (C-3), 145.8
(C-b), 139.0 (C-40), 136.8 (C-10), 129.8 (C-20, C-60), 128.9
(C-30, C-50), 127.3 (C-1), 123.5 (C-a), 119.2 (C-6), 114.9
(C-5), 110.1 (C-2), 56.1 (–OCH3).
(E)-1-(2,4-dichlorophenyl)-3-(4-hydroxy-3-methoxyphenyl)
prop-2-en-1-one (5f) mp 66–68 °C; IR (KBr) mmax
(cm-1): 3397 (–OH, H-bonded), 1642 (C=O), 1585 (Ar C–
C), 1290 (C–O), 1031 (Ar–Cl); 1H NMR (CDCl3, 400 MHz,
TMS = 0): 3.86 (3H, s, –OCH3), 5.97 (1H, s, –OH), 6.86
(1H, d, J = 8 Hz, H-5), 6.87 (1H, d, J = 16 Hz, =CH), 6.99
(1H, d, J = 2 Hz, H-2), 7.04 (1H, dd, J = 8 Hz, J0 = 2 Hz,
H-6), 7.27 (1H, dd, J = 8.4 Hz, J0 = 2 Hz, H-50), 7.29 (1H,
d, J = 16 Hz, =CH), 7.33 (1H, d, J = 8.4 Hz, H-60), 7.40
(1H, d, J = 2 Hz, H-30); 13C NMR (CDCl3, 100 MHz,
TMS = 0): 192.8 (C=O), 148.8 (C-3), 147.3 (C-4), 146.9
(C-20), 137.7 (C-b), 136.6 (C-10), 132.2 (C-60), 130.3 (C-40),
130.1 (C-30), 127.2 (C-1), 126.8 (C-50), 124.1 (C-6), 123.7
(C-a), 114.9 (C-5), 109.8 (C-2), 56.0 (–OCH3).
(E)-1-(2,4-dichlorophenyl)-3-(3-hydroxy-4-methoxyphenyl)
prop-2-en-1-one (6d) mp 92–94 °C; IR (KBr) mmax
(cm-1):3417 (–OH, H-bonded), 1643 (C=O), 1270 (C–O);
1H NMR (CDCl3, 400 MHz, TMS = 0): 3.87 (3H, s,
–OCH3), 5.60 (1H, s, –OH), 6.79 (1H, d, J = 8 Hz, H-5),
6.89 (1H, d, J = 16 Hz, =CH), 7.00 (1H, dd, J = 8.4 Hz,
J0 = 2 Hz, H-6), 7.12 (1H, d, J = 2 Hz, H-2), 7.27 (1H,
dd, J = 8 Hz, J0 = 2 Hz, H-50), 7.31 (1H, d, J = 16 Hz,
=CH), 7.35 (1H, d, J = 8 Hz, H-60), 7.41 (1H, d,
J = 2 Hz, H-30); 13C NMR (CDCl3, 100 MHz, TMS = 0):
192.6 (C=O), 149.2 (C-4), 146.7 (C-3), 146.0 (C-20), 137.7
(C-b), 136.7 (C-10), 132.3 (C-60), 130.4 (C-40), 130.2 (C-
30), 127.9 (C-1), 127.2 (C-50), 124.2 (C-6), 122.9 (C-a),
113.3 (C-2), 110.6 (C-5), 56.1 (–OCH3).
(E)-3-(3-hydroxy-4-methoxyphenyl)-1-phenylprop-2-en-1-
one (6a) mp 92–94 °C; IR (KBr) mmax (cm-1): 3431
(–OH, H-bonded), 3060 (Ar C–H), 1650 (C=O), 1270 (C–
(E)-2-(2-methoxy-4-(3-oxo-3-phenylprop-1-en-1-yl)phenoxy)
acetic acid (7a) mp 148–149 °C (dc); IR (KBr) mmax
(cm-1): 3377 (–OH), 1740 (C=O, acid), 1640 (C=O), 1262
(C–O); 1H NMR (CDCl3, 400 MHz, TMS = 0): 3.86 (3H,
s, –OCH3), 4.48 (2H, s, –OCH2COOH), 6.83 (1H, d,
J = 8.4 Hz, H-5), 7.32 (1H, dd, J = 8 Hz, J0 = 1.6 Hz, H-
6), 7.50 (1H, d, J = 1.6 Hz, H-2), 7.57 (2H, m, H-30, H-50),
1
O); H NMR (CDCl3, 400 MHz, TMS = 0): 3.9 (3H, s,
–OCH3), 6.86 (1H, d, J = 8.4 Hz, H-5), 7.36 (1H, d,
J = 15.6 Hz, =CH), 7.33–7.38 (1H, m), 7.48–7.6 (4H, m),
7.71 (1H, d, J = 15.6 Hz, =CH), 7.98 (2H, d, J = 7.6 Hz);
13C NMR (CDCl3, 100 MHz, TMS = 0): 191.6 (C=O),
148.8 (C-4), 145.9 (C-3), 144.8 (C-b), 138.4 (C-10), 132.6
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Med Chem Res
7.67 (1H, m, H-40), 7.68 (1H, d, J = 16 Hz, =CH), 7.78
(1H, d, J = 16 Hz, =CH), 8.12 (2H, m, H-20, H-60); 13C
NMR (CDCl3, 100 MHz, TMS = 0): 189.0 (C=O), 170.0
(–COOH), 149.7 (C-3), 149.0 (C-4), 144.4 (C-b), 137.8 (C-
10), 132.9 (C-40), 128.7 (C-30, C-50), 128.4 (C-20, C-60),
127.9 (C-1), 123.6 (C-6), 119.8 (C-a), 112.6 (C-5), 111.2
(C-2), 65.0 (–OCH2-COOH), 55.8 (–OCH3).
with compound 5f at IC50 concentration (5.4 lM) and
29IC50 concentration (10.8 lM). After 24 h of treatment,
cell shrinkage was observed along with the development of
bubble like blebs on the membrane. These characteristic
morphological features are indicative of cytotoxicity. Fig-
ure 3 shows the morphology of treated cells when observed
under bright field 109.
(E)-3-(3,4-dimethoxyphenyl)-1-phenylprop-2-en-1-one
(8a) mp 68–69 °C; IR (KBr) mmax (cm-1): 3059 (Ar C–
H), 1659 (C=O), 1591 (Ar C–C), 1260 (C–O); H NMR
Effect on cell migration by in vitro scratch assay
1
Cell migration is a key property of cancer metastasis and
inflammation. Hence, compound 5f was evaluated for its
effect on cell migration on MIA PaCa-2 cells by in vitro
scratch assay. The cells were treated with increasing con-
centrations (1, 2, 3 and 4 lM) of the compound for 24 h.
Positive control (10 % FBS) was maintained simultane-
ously. After 24 h, a progressive inhibition in cell migration
was observed with successive increase in the concentra-
tions of compound 5f when compared to the control (10 %
FBS). Figure 4 shows the progressive cell migration of
MIA PaCa-2 cells on treatment with compound 5f. Inhi-
bition of cell migration was found to be dose dependent as
shown in Fig. 4 with a slight decrease in wound closure
with increasing concentrations of compound 5f.
(CDCl3, 400 MHz, TMS = 0): 3.94 (6H, s, –OCH3), 6.9
(1H, d, J = 8.4 Hz, H-5), 7.16 (1H, d, J = 2 Hz, H-2),
7.23 (1H, dd, J = 8.4 Hz, J0 = 2 Hz, H-6), 7.39 (1H, d,
J = 15.6 Hz, =CH), 7.5 (2H, m, H-30, H-50), 7.58 (1H, m,
H-40), 7.76 (1H, d, J = 15.6 Hz, =CH), 8.01 (2H, dd,
J = 8.4 Hz, J0 = 1.2 Hz, H-20, H-60); 13C NMR (CDCl3,
100 MHz, TMS = 0): 190.6 (C=O), 151.5 (C-3), 149.3 (C-
4), 145.0 (C-b), 138.5 (C-10), 132.6 (C-40), 128.6 (C-30,
C-50), 128.4 (C-20, C-60), 127.9 (C-1), 123.2 (C-6), 120.1
(C-a), 111.2 (C-5), 110.2 (C-2), 56.0 (–OCH3).
(E)-3-(3,4-dimethoxyphenyl)-1-(4-hydroxyphenyl)prop-2-en-
1-one (8b) mp 201–203 °C (dc); IR (KBr) mmax (cm-1):
3230 (–OH), 1635 (C=O), 1602 (Ar C–C), 1263 (C–O); 1H
NMR (CDCl3, 400 MHz, TMS = 0): 3.84 (6H, s, –OCH3),
6.9 (2H, d, J = 8.4 Hz, H-30, H-50), 7.01 (1H, d,
J = 8.2 Hz, H-5), 7.36 (1H, dd, J = 8.2 Hz, J0 = 1.6 Hz,
H-6), 7.52 (1H, d, J = 1.6 Hz, H-2), 7.64 (1H, d,
J = 15.6 Hz, =CH), 7.8 (1H, d, J = 15.6 Hz, =CH), 8.08
(2H, d, J = 8.4 Hz, H-20, H-60); 13C NMR (CDCl3,
100 MHz, TMS = 0): 187.0 (C=O), 162.0 (C-40), 151.0
(C-3), 148.9 (C-4), 143.2 (C-b), 131.0 (C-20, C-60), 129.3
(C-10), 127.7 (C-1), 123.6 (C-6), 119.6 (C-a), 115.3 (C-30,
C-50), 111.5 (C-5), 110.5 (C-2), 55.6 and 55.5 (–OCH3).
Analysis of mode of cell death by AO/EB staining
To assess the type of cell death induced by chalcones in
MIA PaCa-2 cells, the morphological changes after double-
staining cells with AO and EB were investigated. These
dyes emit different shades of fluorescence after intercala-
tion in DNA and can be used to detect nuclear changes that
characterize apoptosis. Compound 5f was tested on MIA
PaCa-2 cells and observed for changes in morphology after
24 h. Fluorescence microscopic analysis of treated cells
revealed characteristic features of apoptosis such as chro-
matin condensation, nuclear fragmentation, alteration in
the size and shape of cells. Figure 5 shows the fluorescence
microscopy images of MIA PaCa-2 cells treated with 5.4
and 10.8 lM concentrations of compound 5f. Treatment
with 10.8 lM concentration was seen to result in visibly
higher number of apoptotic cells suggesting a dose-de-
pendent relationship.
(E)-1-(4-chlorophenyl)-3-(3, 4-dimethoxyphenyl)prop-2-
en-1-one (8c) Mp 103–105 °C; IR (KBr) mmax (cm-1):
1658 (C=O), 1591 (Ar C–C), 1271 (C–O), 1030 (Ar–Cl);
1H NMR (CDCl3, 400 MHz, TMS = 0): 3.94 (6H, s,
–OCH3), 6.9 (2H, d, J = 8.8 Hz, H-30, H-50), 7.01 (1H, d,
J = 8 Hz, H-5), 7.36 (1H, dd, J = 8 Hz, J0 = 1.6 Hz, H-
6), 7.52 (1H, d, J = 1.6 Hz, H-2), 7.64 (1H, d,
J = 15.6 Hz, =CH), 7.8 (1H, d, J = 15.6 Hz, =CH), 8.08
(2H, d, J = 8.8 Hz, H-20, H-60); 13C NMR (CDCl3,
100 MHz, TMS = 0): 189.3 (C=O), 151.6 (C-3), 149.3 (C-
4), 145.6 (C-b), 139.0 (C-40), 136.8 (C-10), 129.9 (C-20,
C-60), 128.9 (C-30, C-50), 127.7 (C-1), 123.3 (C-6), 119.5
(C-a), 111.1 (C-5), 110.1 (C-2), 56.0 and 55.9 (–OCH3).
Cell cycle analysis of MIA PaCa-2 cells treated
with compound
MIA PaCa-2 cells were treated with IC50 concentration of
compound 5f for 24 h. Cell cycle analysis was performed
using BD C6 Accuri flow cytometer. On treatment with
IC50 concentration of compound 5f, an increase in the
percentage of cells in the G2/M phase of the cell cycle was
observed. Percentage of cells in the G2/M phase of the cell
Effect on cellular morphology
Morphological changes in MIA PaCa-2 cells were identi-
fied on treatment with compound 5f. Cells were treated
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Med Chem Res
Fig. 3 Morphology of cells treated with compound 5f for 24 h as observed under bright field
Fig. 4 Images showing cell migration of MIA PaCa-2 cells on treatment with increasing concentrations of compound 5f at 0 h and after 24 h
Fig. 5 Fluorescence microscopy images of MIA PaCa-2 cells treated with compound 5f
123

Med Chem Res
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