Selected novel 5’-amino-2’-hydroxy-1, 3-diaryl-2- propen-1-ones arrest cell cycle of HCT-116 in G0/G1 phase

Article abstract of DOI:10.17179/excli2015-610

A series of 5’-amino-2’-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt condensation of 5'-acetamido-2’-hydroxy acetophenone with various substituted aromatic aldehydes. The syn- thesized compounds were characterized by FTIR, 1H NMR and mass spectrometry and evaluated for their selec- tive cytotoxicity using MTT assay on two cancer cell lines namely breast cancer cell line (MCF-7), colon cancer cell line (HCT-116) and one normal kidney epithelial cell line (Vero). Among the tested compounds, AC-10 showed maximum cytotoxic effect on MCF-7 cell line with IC₅₀ value 74.7 ± 3.5 μM. On HCT-116 cells, AC-13 exhibited maximum cytotoxicity with IC₅₀ value 42.1 ± 4.0 μM followed by AC-14 and AC-10 with IC₅₀ values 62 ± 2.3 μM and 95.4 ± 1.7 μM respectively. All tested compounds were found to be safe on Vero cell line with IC50 value more than 200 μM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treatment (31%). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G₀/G₁ phase and by AC-13 in G₀/G₁ and G₂/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the lead molecules for further optimization.

Full text of DOI:10.17179/excli2015-610

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Original article:
SELECTED NOVEL 5’-AMINO-2’-HYDROXY-1, 3-DIARYL-2-
PROPEN-1-ONES ARREST CELL CYCLE OF HCT-116
IN G0/G1 PHASE
Lalitha Simon1, K. K. Srinivasan2, Nitesh Kumar3, Neetinkumar D. Reddy3,
Subhankar Biswas3, C. Mallikarjuna Rao3*, Sudheer Moorkoth4
1
Department of Chemistry, Manipal Institute of Technology, Manipal University, Manipal,
576 104, India
2
Department of Chemistry, Shri Madhwa Vadiraja Institute of Technology & Management,
(affiliated to Visvesvaraya Technological University, Belgaum), Bantakal, Udupi, 574115,
India
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal
3
University, 576 104, India
Department of Pharmaceutical Quality Assurance, Manipal College of Pharmaceutical
4
Sciences, Manipal University, 576 104, India
* Corresponding author: Dr. C. Mallikarjuna Rao, Principal & Professor of Pharmacology,
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, 576 104,
Karnataka, India, Tel: +91 8202922482,
E-mail: mallikin123@gmail.com, mallik.rao@manipal.edu
http://dx.doi.org/10.17179/excli2015-610
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
A series of 5’-amino-2’-hydroxy-1,3-diaryl-2-propen-1-ones (AC1-AC15) were synthesized by Claisen-Schmidt
condensation of 5'-acetamido-2’-hydroxy acetophenone with various substituted aromatic aldehydes. The syn-
thesized compounds were characterized by FTIR, 1H NMR and mass spectrometry and evaluated for their selec-
tive cytotoxicity using MTT assay on two cancer cell lines namely breast cancer cell line (MCF-7), colon cancer
cell line (HCT-116) and one normal kidney epithelial cell line (Vero). Among the tested compounds, AC-10
showed maximum cytotoxic effect on MCF-7 cell line with IC50 value 74.7 ± 3.5 µM. On HCT-116 cells, AC-13
exhibited maximum cytotoxicity with IC50 value 42.1 ± 4.0 µM followed by AC-14 and AC-10 with IC50 values
62 ± 2.3 µM and 95.4 ± 1.7 µM respectively. All tested compounds were found to be safe on Vero cell line with
IC50 value more than 200 µM. Based on their highest efficacy on HCT-116, AC-10, AC-13 and AC-14 were
selected for mechanistic study on this cell line by evaluating changes nucleomorphological characteristics using
acridine orange-ethidium bromide (AOEB) dual stain and by analyzing cell cycle with flow cytometry using
propidium iodide stain. In AOEB staining, all three tested compounds showed significant (p < 0.05) increase in
percentage apoptotic nuclei compared to control cells, with highest increase in apoptotic nuclei by AC-13 treat-
ment (31 %). Flow cytometric studies showed cell cycle arrest by AC-10 and AC-14 treatment in G0/G1 phase
and by AC-13 in G0/G1 and G2/M phase. The study reflected the potential of AC-10, AC-13 and AC-14 to be the
lead molecules for further optimization.
Keywords: 5’-amino-2’-hydroxy-1,3-diaryl-2-propen-1-ones, cytotoxicity, MTT, acridine orange-ethidium
bromide nuclear staining, flow cytometry
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EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
INTRODUCTION
anti-parasitic, estrogenic, antimalarial and
antitumor activities (Khatib et al., 2005; Mi-
randa et al., 2000; Modzelewska et al.,
2006). Xanthohumol, a prenylated chalcone
isolated from the hop cones (Humuluslupu-
lus L.) is suggested to exhibit broad spec-
trum anticancer properties against different
types of human cancer cells primarily
through inhibition of proliferation and induc-
tion of human cancer cell apoptosis
(Mosmann, 1983; Nowakowska, 2007). Fla-
vokawain A, B, and C (Go et al., 2005; Gold
and Moellering, 1996; Kawabata et al.,
2003) in kava extracts have been shown to
possess strong antiproliferative and apoptotic
effect in human bladder cancer cells (Palkar
and Master, 2000).
Cancer is rapid and uncontrolled growth
of abnormal cells. It is the major cause of
death, after cardiovascular diseases all over
the world (Bandgar et al., 2010). Most of the
drugs available today for the treatment of
cancer are cytotoxic in nature, which act by
interfering with the operation of the cell's
DNA in some way. These cytotoxic drugs
are very harmful to the body unless the drugs
are specific to target cancer cells. The cancer
specific targeting by cytotoxic agents is dif-
ficult to achieve because the modifications,
which convert healthy cell into cancerous
one are very subtle. Thus a major challenge
is always present in designing new drugs
with more selectivity for cancer cells.
Introduction of amino groups into vari-
ous heterocyclic systems had led to very ef-
fective therapeutic agents. 5-Amino flavones
showed antitumor activity highly selective to
the ER-positive breast cancer cell line (Pan
et al., 2005). 6-Aminoflavone inhibited
mammalian intestinal α-glucosidase (Srini-
vasan et al., 2009). 5,4’-Diamino-6,8,3’-tri-
fluoroflavone (Wu et al., 2011) exhibited
strong growth inhibitory activity against
MCF-7 cells. But very little has been report-
ed on the synthesis and biological activities
of chalcones with a free amino substitution
in the ring A. Studies on the anticancer po-
tential of chalcones having amino group in
the 5th position, and hydroxyl group at the
2nd positions of ring A of chalcones have not
been reported as of our knowledge. In view
of the wide spectrum of medicinal applica-
tions of chalcone derivatives, our research
continues to explore the anticancer potential
of flavonoids having free amino group/s. The
present study includes the synthesis, charac-
terization and mechanistic insight into anti-
cancer activities of some new 5’-amino-2’-
hydroxy-1,3-diaryl-2-propen-1-ones.
1,3-Diaryl-2-propen-1-ones, commonly
known as chalcones, belong to the flavonoid
family (Bennett et al., 1980; Chiaradia et al.,
2008; Chimenti et al., 2009). Structurally,
they contain an open-chain flavonoid skele-
ton in which two aromatic rings are linked
by a three-carbon α,β-unsaturated carbonyl
system. Chalcones exhibit a wide range of
biological activities, which include anti-
cancer (Chen et al., 1994; Dimmock et al.,
1999), anti-inflammatory (Chiaradia et al.,
2008; Domınguez et al., 2001; Fu et al.,
́
2004), antioxidant (Go et al., 2005), antimi-
crobial, anti-tubercular (Gold and Moeller-
ing, 1996; Kawabata et al., 2003), antimalar-
ial (Lust et al., 2005) and anti-allergic activi-
ties (Kouskoura et al., 2008).The biological
activities of chalcones are considered to be
due to the presence of a reactive α,β-
unsaturated keto function, while the presence
of a double bond allows these molecules to
exist as cis or trans geometric isomers. The
trans-isomer has been proven to be thermo-
dynamically as well as biologically favoura-
ble.
Many naturally occurring chalcones with
potent anticancer efficacy against a variety
of cancer cell lines have been found. Lico-
MATERIALS AND METHODS
chalcone A, an oxygenated chalcone found Experimental
in the roots of the Chinese liquorice (Glycyr-
The chemicals required for the synthesis
rhiza uralensis), has been demonstrated to were purchased from Sigma Aldrich, Hi-
possess many bioactive properties including Media, Loba Chemicals and Nice Fine
22

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Chemical India. Melting points were record- General procedure for the synthesis of 5’-
ed by open capillary method and purity was amino-2’-hydroxy -1,3-diaryl-2-propen-1-
assessed using Rf value in thin layer chroma- ones (AC1 –AC15)
tography (TLC) on pre-coated silica gel alu-
A mixture of concentrated hydrochloric
minium backed plates (Kieselgel 60 F254 acid and water (1:1) was added to 5’-
Merck (Germany)). The IR spectra in KBr acetamido-2’-hydroxy chalcone, and boiled
pellets were recorded using Shimadzu FTIR for one hour. The reaction progress was
1
8400S spectrophotometer. H NMR spectra monitored with TLC using petroleum ether-
were recorded by Bruker AV400 (400MHz) ethyl acetate solvent system in the ratio 2:3.
spectrometer in deuterated dimethyl sulphox- When the reaction was complete, the reac-
ide using tetramethyl silane as internal tion mixture was cooled to room temperature
standard. Mass spectra were scanned on a and ice cold water was added. The solution
Shimadzu LCMS (ESI) 2010A spectrometer. was basified by adding 10 % sodium bicar-
5’-Acetamido-2’-hydroxy acetophenone (2) bonate solution. The product obtained was
(Figure 1) was prepared according to the filtered and purified by recrystallization from
known procedure. 5’-acetamido-2’-hydroxy- ethanol to get amino chalcones (Figure 1).
1,3-diaryl-2-propen-1-ones (3) (Figure 1)
were prepared by procedure given in the lit-
erature (Wu et al., 2003).
Figure 1: Scheme for the synthesis of 5’-amino-2’-hydroxy-1,3-diaryl-2-propen-1-ones
23

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Characterization
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-
1H,J = 16.2 Hz), 8.01 (d, 1H, J = 16.2 Hz),
8.26 (t, 2H, J = 8.9 Hz), 8.30 (d, 1H, J = 2.5
phenylprop-2-en-1-one (AC-1): Light Brown Hz), 11.86 (s,1H,OH); LCMS (m/z): 284
solid, yield: 72 %; m.p. 126 °C; IR (KBr) (M+).
ν
max/cm−1: 3425, 3340 (NH2), 3565 (OH),
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-
1690 (α,β-unsaturated C=O), 3026 (Ar-CH), ethoxyphenyl)prop-2-en-1-one
(AC-5):
1
1136 (C-N ), 1510 (C=C); H NMR (400 Brown solid, yield: 68 %; m.p. 135 °C;
MHz, DMSO-d6): δ ppm 5.43 (s,2H,NH2), IR(KBr) νmax/cm−1: 3420, 3329 (NH2), 3568
6.60 (d, 1H,J = 2.5 Hz), 6.71 (d, 1H, J = 15.7 (OH), 1692 (α,β-unsaturated C=O), 3060
1
Hz), 6.90 (d, 1H, J = 2.5 Hz), 7.47 (m, 3H), (Ar-CH), 1130 (C-N), 1510 (C=C); HNMR
7.51 ( d, 1H, J = 15.7 Hz), 7.71 (dd,1H, J = (400 MHz, DMSO-d6): δppm: 1.40
2.5 Hz, 8.9 Hz), 7.79 (m, 2H), 8.17 (d, 1H, J (t,3H,CH3), 4.23 (q,2H,OCH2), 5.43
= 2.5 Hz), 11.70 (s, OH); LCMS (m/z): 239 (s,2H,NH2), 6.75 (d,1H,J=8.4Hz), 6.86
(M+).
(d,1H,J=16Hz), 6.98 (d,1H,J=8.0Hz), 7.06
(d,2H,J=8.0Hz), 7.46 (d,2H,J=8.0Hz), 7.771
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-
chlorophenyl)prop-2-en-1-one (AC-2): Dark (d,1H,J=16Hz), 7.84 (d,1H,J=8.8Hz), 11.8
Brown solid, yield: 70 %; m.p. 138 °C; (s,1H,OH); LCMS (m/z): 283 (M+).
IR(KBr) νmax/cm−1 : 3450, 3341 (NH2), 3588
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(3-
(AC-6):
(OH), 1683 (α,β-unsaturated C=O), 3070 methoxyphenyl)prop-2-en-1-one
(Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR Brown solid, yield: 79 %; m.p. 110 °C; IR
(400MHz, DMSO-d6): δppm 5.34 (s, 2H, (KBr) νmax/cm−1: 3415, 3329 (NH2), 3588
NH2), 6.97 (d, 1H, J = 8.9 Hz), 7.24 (t, 2H,J (OH), 1672 (α,β-unsaturated C=O), 3060 (Ar
1
= 8.9 Hz), 7.63 (dd, 1H, J = 2.5, 8.9 Hz), -CH), 1130(C-), 1510(C=C); H NMR (400
7.70 (d, 1H,J = 15.7 Hz), 7.78 (d, 1H, J = MHz, DMSO-d6): δppm: 3.81 (s, OCH3),
15.7 Hz), 7.84 (d, 2H, J = 8.9 Hz), 8.18 (d, 5.43 (s,2H,NH2), 6.96 (d, 1H, J = 8.9 Hz),
1H, J = 2.5 Hz), 11.81 (s, 1H,OH); LCMS 7.04 (m,1H), 7.37 (m, 3H), 7.71 (dd, 1H, J =
(m/z): 273 (M+).
8.9, 2.5 Hz), 7.72 (d, 1H, J = 15.7 Hz), 7.79
(d, 1H, J = 15.7 Hz), 8.14(d, 1H, J = 2.5 Hz,
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-
fluorophenyl)prop-2-en-1-one (AC-3): Pale 11.71 (s, OH); LCMS (m/z): 269 (M+).
brown solid, yield: 68 %; m.p. 154 °C;
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-
IR(KBr) νmax/cm−1: 3430, 3329 (NH2), 3572 hydroxy-3-methoxyphenyl)prop-2-en-1-one
(OH), 1690 (α,β-unsaturated C=O), 3062 (Ar (AC-7): Brown solid, yield: 61 %; m.p.
1
-CH), 1130 (C-N), 1510 (C=C); H NMR 136 °C; IR (KBr) νmax/cm−1: 3446, 3301
(400MHz, DMSO-d6): δppm; 5.48 (s, 2H, (NH2), 3591 (OH), 1680 (α,β-unsaturated
NH2), 6.97 (d, 1H, J = 8.9 Hz), 7.32 (d, 2H,J C=O), 3078 (Ar-CH), 1138 (C-N), 1520
1
= 8.9 Hz), 7.71 (dd, 1H, J = 2.5, 8.9 Hz), (C=C); H NMR (400 MHz, DMSO-d6):
7.73 (d, 1H,J = 15.7 Hz), 7.78 (d, 1H, J = δppm: 3.89 (s,3H,OCH3), 5.37 (s,2H,NH2),
15.7 Hz), 7.90 (t, 2H, J = 8.9 Hz), 8.18 (d, 7.84 (d,1H,J=8.8Hz), 7.77 (s,1H), 7.46
1H, J = 2.5 Hz), 11.73 (s, 1H,OH); LCMS (d,2H,J=8.0Hz), 7.06 (d,2H,J=8.0Hz), 6.98
(m/z): 257 (M+).
(d,1H,J=8.0Hz), 6.86 (d,1H,J=16Hz), 6.75
(d,1H,J=8.4Hz), 11.8 (s,1H,OH); LCMS
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(3-
nitrophenyl)prop-2-en-1-one (AC-4): Brown (m/z): 285 (M+).
solid, yield: 50 %; m.p. 112 °C; IR (KBr)
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(3-
νmax/cm−1: 3420, 3319 (NH2), 3570 (OH), hydroxy-4-methoxyphenyl)prop-2-en-1-one
1676 (α,β-unsaturated C=O) 3060 (Ar-CH), (AC-8): Pale brown solid, yield: 61 %; m.p.
1
1130 (C-N), 1510 (C=C); H NMR (400 136 °C; IR (KBr) νmax/cm−1: 3446, 3301
MHz, DMSO-d6): δppm: 5.40 (s, 2H, NH2), (NH2), 3589 (OH), 1680 (α,β-unsaturated
6.97 (d, 1H, J = 8.9 Hz), 7.46 (d, 2H,J = 8.9 C=O), 3078 (Ar-CH), 1138 (C-N), 1520
1
Hz), 7.88 (dd, 1H, J = 2.5, 8.9 Hz), 7.93 (d, (C=C); H NMR (400 MHz, DMSO-d6):
24

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
δppm: 3.81 (s,3H,OCH3), 5.40 (s,2H,NH2), (OH), 1690 (α,β-unsaturated C=O), 3070
7.84 (d,1H,J=8.8Hz), 7.77 (s,1H), 7.46 (Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR
(d,2H,J=8.0Hz), 7.06 (d,2H,J=8.0Hz), 6.98 (400MHz, DMSO-d6): δppm: 5.34 (s, 2H,
(d,1H,J=8.0Hz), 6.86 (d,1H,J=16Hz), 6.75 NH2), 7.01 (d, 1H, J = 8.9 Hz), 7.24 (d, 2H,J
(d,1H,J=8.4Hz), 11.5 (s,1H,OH); LCMS = 8.9 Hz), 7.63 (d, 1H, J = 2.5, 8.9 Hz), 7.70
(m/z): 285 (M+).
(d, 1H,J = 15.7 Hz), 7.78 (d, 1H, J = 15.7
Hz), 7.86 (s,1H Hz), 8.18 (d, 1H, J = 2.5
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-
(3,4-dimethoxyphenyl)prop-2-en-1-one (AC- Hz), 11.75 (s, 1H,OH); LCMS (m/z): 308
9): Brown solid, yield: 73 %; m.p. 90 °C; (M+).
IR(KBr) νmax/cm−1: 3430, 3329 (NH2), 3588
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-
(OH), 1690 (α,β-unsaturated C=O), 3062 (3,4-methylenedioxyphenyl)prop-2-en-1-one
(Ar-CH), 1130 (C-N), 1510 (C=C); 1H NMR (AC-13): Pale brown solid, yield: 81 %; m.p.
(400 MHz, DMSO-d6): δppm: 3.78 (6H,s, 23 128 °C; IR (KBr) νmax/cm−1: 3446, 3301
x OCH3), 5.48 (s, 2H, NH2), 6.97 (d, 1H, J = (NH2), 3591 (OH), 1680 (α,β-unsaturated
8.9 Hz), 7.32 (d, 2H, J = 8.9 Hz), 7.71 (d, C=O), 3078 (Ar-CH), 1138 (C-N), 1520
1
1H, J = 2.5, 8.9 Hz), 7.73 (d, 1H,J = 15.7 (C=C); H NMR (400 MHz, DMSO-d6):
Hz), 7.78 (d, 1H, J = 15.7 Hz), 7.90 (s, 1H), δppm: 5.40 (s,2H,NH2), 6.034 (s,2H,CH2-O),
8.18 (d, 1H, J = 2.5 Hz), 11.73 (s, 1H,OH); 6.90 (d,1H,J=8.4Hz), 6.96 (d,1H,J=16Hz),
LCMS (m/z): 299 (M+).
6.99 (d,1H,J=8.0Hz), 7.06 (d,2H,J=8.0Hz),
7.46 (d,2H,J=8.0Hz), 7.70 (s,1H), 7.84
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(2-
hydroxyphenyl)prop-2-en-1-one
(AC-10): (d,1H,J=8.8 Hz), 11.5 (s,1H,OH); LCMS
Dark brown solid, yield: 68 %; m.p. 130- (m/z): 283 (M+).
132 °C; IR(KBr) νmax/cm−1: 3448, 3320
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-
(NH2), 3576 (OH), 1692 (α,β-unsaturated (3,4,5-trimethoxyphenyl)prop-2-en-1-
C=O), 3062 (Ar -CH), 1138 (C-N), 1510 one(AC-14): Light Brown solid, yield: 81 %;
(C=C); 1H NMR (400MHz, DMSO-d6): m.p. 136-138 °C; IR (KBr) νmax/cm−1: 3430,
δppm.; 5.39 (s, 2H, NH2), 6.97 (d, 1H, J = 3329 (NH2), 3588 (OH), 1690 (α,β-
8.9 Hz), 7.32 d, 2H,J = 8.9 Hz), 7.64 (d, 1H, unsaturated C=O), 3062 (Ar-CH), 1130 (C-
J = 2.5, 8.9 Hz), 7.75 (d, 1H,J = 15.7 Hz), N), 1510 (C=C); 1H NMR (400 MHz,
7.78 (d, 1H, J = 15.7 Hz), 7.90 (s, 1H), 8.18 DMSO-d6): δppm: 3.85 (9H,s, 3 x OCH3),
(d, 1H, J = 2.5 Hz), 11.80 (s, 1H,OH); 5.48 (s, 2H, NH2), 7.50 (d, 1H, J = 8.9 Hz),
LCMS (m/z): 255 (M+).
7.32 (d, 1H,J = 8.9 Hz), 7.71 (d, 1H, J = 2.5,
8.9 Hz), 7.73 (d, 1H,J = 15.7 Hz), 7.78 (d,
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-
methoxyphenyl)prop-2-en-1-one
Brown solid, yield: 72 %; m.p. 102-104 °C; (s,1H,OH); LCMS (m/z): 329 (M+).
IR (KBr) νmax/cm−1: 3450, 3344 (NH2),
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-(4-
(AC-11): 1H, J = 15.7 Hz), 7.93 (s, 2H), 11.73
3591(OH), 1678 (α,β-unsaturated C=O), benzyloxyphenyl)prop-2-en-1-one (AC-15):
1
3070 (Ar-CH), 1138 (C-N), 1515 (C=C); H Dark Brown solid, yield: 73 %; m.p. 136-
NMR (400 MHz, DMSO-d6): δppm: 3.84 138 °C; IR (KBr) νmax/cm−1: 3425, 3340
(s,3H, OCH3), 5.43 (s,2H,NH2), 7.84 (NH2), 3565 (OH), 1690 (α,β-unsaturated
(d,1H,J=8.8Hz), 7.771 (d,1H,J=16Hz), 7.46 C=O), 3026 (Ar-CH), 1136 (C-N), 1510
1
(d,2H,J=8.0Hz), 7.06 (d,2H,J=8.0Hz), 6.98 (C=C); H NMR (400 MHz, DMSO-d6):
(d,1H,J=8.0Hz), 6.86 (d,1H,J=16Hz), 6.75 δppm: 5.25 (s, 2H, Ar-CH2-), 5.50
(d,1H,J=8.4Hz), 11.8 (s,1H,OH); LCMS (s,2H,NH2), 7.11 (dd, 1H, J =2.5Hz, 8.9 Hz),
(m/z): 269(M+).
7.29 (d, 1H,J=17Hz), 7.47 (dd, 2H, J =2.5Hz,
8.9 Hz), 7.71 (d, 2H, J = 2.5 Hz), 7.77 (d,
(E)-1-(5-Amino-2-Hydroxyphenyl)-3-
(3,4-dichlorophenyl)prop-2-en-1-one (AC- 2H, J = 2.5 Hz), 7.79 (d,2H, J = 2.5 Hz), 7.82
12): Brown solid, yield: 70 %; m.p. 138 °C; (d,1H, J = 2.5 Hz), 7.87 (d,1H,J=17Hz), 8.01
IR(KBr) νmax/cm−1: 3450, 3341 (NH2), 3580
25

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
(d,1H, J = 2.5 Hz), 8.17 (d, 1H, J = 2.5 Hz), washed with phosphate buffer saline (PBS,
11.70 (s, OH); LCMS (m/z): 345 (M+).
pH 7.4). Cells were fixed in ice-cold metha-
nol for 20 min, washed with PBS again and
stained with acridine orange and ethidium
Evaluation of cytotoxicity by MTT assay
Cytotoxicity of the synthesized com- bromide stain (20/30 µg/ml). After incuba-
pounds was evaluated in cancer cells namely tion for 20 min at 37 °C, and washing with
human colon cancer (HCT-116) cells and PBS thrice, the plate was observed under a
human breast cancer (MCF-7) cells and fluorescent microscope for morphological
normal kidney epithelial cells (Vero). These changes in nucleus such as condensed chro-
cells were originally procured from National matin and fragmented nuclei. The apoptotic
Cancer Center for Cell Science, Pune, India index (AI) was calculated as % of apoptotic
and cultured in our lab using Dulbecco’s cells from randomly counted 100 cells in
modified Eagles medium (DMEM) contain- each treatment group (Reddy et al., 2015).
ing 10 % fetal bovine serum (FBS) at 37 °C
in an atmosphere containing 5 % CO2. All Cell cycle analysis
other chemicals used in this study were pur-
chased from Sigma-Aldrich, USA.
Flow cytometric analysis technique eval-
uates the effect of test compounds on cell
Cell suspension containing 1x104 cells in cycle progression and check-points. Cells (1
0.1 mL was seeded in a 96 well plate for x 106) were seeded in 25 cm2 flasks and, af-
24 h. Test compounds were serially diluted ter overnight adherence, incubated with test
with the medium to get a stock solution of compounds. Then cells were detached by
400 µM, 200 µM, 100 µM, 50 µM. After trypsinization and mixed with floating cells,
24 h of incubation, 100 µL of test solution centrifuged and washed with PBS. The cell
from respective stocks was added in tripli- pellets were fixed in 70 % ice-cold methanol
cate and incubated for 48 h. After the treat- and stored at -20 °C for 24 h. After that cell
ment, drug-containing media was removed pellets were washed with PBS and isotonic
and 20 µL of MTT reagent (5 mg/mL in PI solution [25 µM propidium iodide, 0.03 %
PBS) was added to each well. After 4 h of NP-40 and 40 µg /ml RNase A] was added.
incubation at 37 °C, the MTT reagent was The stained cells were analyzed using Accuri
removed and 100 µL of 100 % DMSO was C6 flow cytometer (BD Biosciences, San
added to each well to solubilize formazan Jose, CA, USA) with excitation at 488 nm
crystals. The optical density was measured and emission at 575/40 nm (Reddy et al.,
using an ELISA plate reader at 540 nm and 2015).
percentage cytotoxicity was calculated.
(Kumar et al., 2012; Zi and Simoneau, Statistical analysis
2005).
Statistical analysis of data were per-
formed by one way ANOVA followed by
Tukey’s post hoc using Graph Pad Prism v
5.03 (demo version), CA, USA, where
p<0.05 was considered to be significant.
Acridine orange (AO) and Ethidium
bromide double staining using fluorescent
microscopy
DNA-binding dyes Acridine orange
(AO) and Ethidium bromide (EB) (Sigma,
USA) were used for the morphological de-
tection of apoptotic cells. 5 x 103 cells were
seeded per well in 24-well plates with
DMEM, containing 10 % FBS. After 24 h,
cells were treated with selected concentra-
tions of test compounds and incubated for
24 h. The media was removed and plate was
RESULTS
Chemistry
A series of chalcones having amino sub-
stituent at the 5th position and hydroxyl sub-
stituent at the 2nd position of ring A and dif-
ferent substituents in the 2nd, 3rd, 4th, 5th posi-
tions of ring B were synthesized. The acet-
amido chalcones were synthesized by
26

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Table 1: Cytotoxicity of 5’-amino-2’-hydroxy
chalcones by MTT assay after 48 h incubation
Claisen–Schmidt condensation of substituted
benzaldehydes and 5’-acetamido-2’-hydroxy
acetophenone. All the compounds were ob-
tained in excellent yields (70-90 %). The ac-
etamido chalcones were hydrolysed in acidic
medium to obtain amino chalcones in good
yields (50-81 %) (Figure 2).
Compound IC50 (µM)
Code
HCT-116
MCF-7
Vero
>200
AC-1
AC-2
AC-3
AC-4
AC-5
AC-6
AC-7
AC-8
AC-9
AC-10
AC-11
AC-12
AC-13
AC-14
AC-15
299.2±49.79 1956±776
205.3±3.15
174±3.53
309.3±74.1
No activity
No activity
392.7±77.9
123.3±7.2
185.7±7.3
95.4±1.7
No activity
169.4±66.7
42.1± 4.0
62±2.3
No activity >200
541.5±38.5 >200
No activity >200
No activity >200
No activity >200
247.9±4.31 >200
170.6±12.9 >200
190.2±10.5 >200
3
3'
OH
2'
1'
2
4'
5'
4
R'
1
5
H2N
6
6'
O
74.7±3.5
>200
1033±60.6 >200
236.5±9.8
130.3±3.8
196.6±7.9
>200
>200
>200
Figure 2: Structure of 5’-amino-2’-hydroxy chal-
cone
211.4±26
No activity >200
In vitro cytotoxicity by MTT assay method
Fifteen 5’-amino-2’-hydroxy chalcones
were evaluated for their cytotoxicity against
two human tumor cell lines. Among the
compounds analyzed, seven (viz., AC-2,
AC-3, AC-8, AC-9, AC-10, AC-13 and AC-
14) displayed high cytotoxicity (close to
200 µM including SEM) against HCT-116
and five compounds (AC-8, AC-9, AC-10,
AC-13, AC-14) against MCF-7 cells. Most
of the compounds were free from cytotoxici-
ty to normal kidney epithelial cells (Vero) at
< 200 µM concentration, which indicated the
selectivity of the compounds towards tested
tumor cells. The results are presented in Ta-
ble 1.
All values are mean ± SEM of three independent
triplicates. IC50 were determined by nonlinear
regression using Graph Pad Prism v 5.03 (demo
version), CA, USA.
AO/EB (dual) nuclear staining
HCT-116 cells treated with compounds
show changes in cellular morphology, in-
cluding chromatin condensation and frag-
mented nuclei, which are characteristic fea-
tures of an early apoptotic cell death (green
colour) but not necrosis (orange colour)
(Nayak et al., 2013; Reddy et al., 2015). AC-
10, AC-13 and AC-14 were tested at their
IC50 value i.e, 100, 50 and 50 µM, respec-
tively. All tested compounds produced mor-
phological changes in the nuclei and showed
significant (p < 0.05) increase in apoptotic
nuclei. AC-13 showed maximum increase in
percentage apoptotic nuclei (31 ± 4.16 %)
followed by AC-10 (27 ± 3.21 %) and AC-
14 (24.3 ± 3.53 %) (Figures 3 and 4).
27

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Figure 3: Nuclear staining. The representative images for induction of apoptosis by different treat-
ments for 48 h in HCT-116 cells by AO/EB. Apoptotic index was calculated by counting specific pat-
tern of condensed and fragmented nuclear morphology.
and 23.0 % cells in G0/G1, S and G2/M
phase, respectively. The standard, doxorubi-
cin (1 µM), accumulated 7.4 % cells in G2/M
phase as compared with that of normal con-
trol suggesting cell cycle arrest at G2/M
phase. The test compounds AC-10 (100 µM),
AC-13 (50 µM), and AC-14 (50 µM), caused
accumulation of cells (70.5 %, 66.5 % and
70.8 %), in G0/G1, phase which indicated the
arrest of cell cycle in this phase. In addition
AC-13 also showed accumulation of cells in
G2/M phase (25.1 %) (Figure 5).
DISCUSSION
Figure 4: Apoptotic index. All values are mean
± SEM of three readings in triplicate. Data are
analysed by one way ANOVA followed by Tuk-
ey’s post hoc test, where p<0.05 compared to
medium control
Several reports are available which state
that chalcone, both derived from nature and
synthetic sources, exhibit cytotoxic and anti-
tumor activities (Ducki et al., 1998). Fla-
vokawain B, a chalcone of plant origin sig-
nificantly prevents colon cancer cells
growth. It produces ROS generation and
GADD153 up-regulation leading to mito-
chondria-dependent apoptosis through re-
lease of cytochrome C and the translocation
of Bak (Kuo et al., 2010).
a
Cell cycle analysis
The effect of the compounds on cell cy-
cle phase was assessed and the results are
shown as % cells in G0/G1, S, and G2/M
phase. The normal control showed 62.3, 15.3
28

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Figure 5: Effect on the cell cycle of HCT-116 after 48 h treatment with AC-10, AC-13 and AC-14. The
concentrations used for cell cycle analysis were 100 µM for AC-10, 50 µM for AC-13 and AC-14.
The structures of synthesized compounds trimethoxy substitution (AC-14) on ring B of
1
were confirmed with FT-IR and H NMR the chalcone increase the cytotoxic activity.
and mass spectroscopy. The FT-IR spectra of Regarding the structure activity relationship,
the newly synthesized chalcones displayed a it appeared that the number of methoxy
strong absorption band due to α,β-unsatur- groups in ring B is important for cytotoxici-
ated C=O stretching at 1692–1672 cm-1, ty. AC-14 with three methoxy groups at 3,4
3591-3565 cm-1 (Ar-OH), 3078-3026 cm-1 and 5 positions of ring B showed good activ-
(Ar–H), 3344-3319 cm-1 (NH2). Inspection ity with IC50 62 ± 2.3 µM and 196.6 ±
1
of the H-NMR spectra suggested that the 7.9 µM against HCT-116 and MCF-7 respec-
chalcones presented trans configurations (J tively whereas compounds having one meth-
= 15-17 Hz). The other expected peaks were oxy group at 3 or 4 positions of ring B (AC-
in the region of 11.50-11.81 ppm (s, Ar-OH), 6, AC-11) and two methoxy substituents at 3
5.34-5.50 ppm (s, Ar-NH2), 6.90-8.18 ppm and 4 positions of ring B (AC-1) were inac-
(Ar-H). The OCH3 protons resonated at tive. It is observed that AC-9 with no sub-
3.85 ppm and were observed as a singlet. stituent on ring B was more active than com-
The mass spectra of all newly synthesized pounds having electron withdrawing groups
amino chalcones showed molecular ion like NO2, F or Cl at the 4 position of ring B.
peaks, which were in accordance with their The compound AC-8 with OH substitution at
respective molecular masses.
3 and OMe substitution at 4 positions of ring
The results revealed that the most potent B showed significant cytotoxicity. However,
compounds were AC-13, AC-10 and AC-14. when the positions of OH and OMe were
The 3,4-methylene substitution (AC-13), 2- interchanged, as in AC-7, the cytotoxicity
hydroxy substitution (AC-10), and 3,4,5- was reduced.
29

EXCLI Journal 2016;15:21-32 – ISSN 1611-2156
Received: October 01, 2015, accepted: December 22, 2015, published: January 13, 2016
Nuclear staining method using fluores- G0/G1 and G2/M phase. These accumulations
cent dye is an ideal method for detection of of cells indicated the arrest of cell cycle by
morphonuclear changes viz., nuclear con- AC-10, and AC-14 in G0/G1 phase and by
densation, fragmentation and a disrupted AC-13 in G0/G1 and G2/M phase. The results
membrane with cytoplasmic disintegration obtained were in accordance with the earlier
(Reddy et al., 2015). AO/EB stain can easily reports on chalcones, which state that cell
permeate to the cell and stain the cycle arrest by chalcones happens in either
DNA/RNA. It is also helpful in identifying G0/G1 phase or G2/M phase (Shen et al.,
apoptotic changes in the cell. Compound 2007).
with lowest IC50 values namely, AC-10, AC-
13 and AC-14, were selected for evaluation
of apoptotic changes on the colon cancer
cells (HCT-116) using acridine or-
ange/ethidium bromide double staining tech-
nique. Treatments showed increased apopto-
sis compared to untreated control cells. The
nucleomorphological changes such as apop-
totic nuclei in the form of nuclear condensa-
tion and fragmentation were significantly
(p<0.05) high in the treated cells compared
to control cells. AC-13 treatment was found
to be more effective with about five times
increase in apoptotic nuclei compared to me-
dium control (Figures 3 and 4).
CONCLUSION
In this study, a series of fifteen 5’-amino-
2’-hydroxy-1,3-diaryl-2-propen-1-ones were
synthesized and characterized. The com-
pound AC-13 exhibited maximum cytotoxi-
city in HCT-116 and moderate cytotoxicity
in MCF-7. AC-10 exhibited best cytotoxicity
in MCF-7. The AO/EB staining showed the
extent of apoptotic damage. AC-13 displayed
highest % of apoptotic nuclei and arrested
cell cycle in G0/G1 and G2/M phase and AC-
10 and AC-14 arrested G0/G1 phase of the
cell cycle.
Flow cytometry analysis is one of the
important methods to identify the effect of
cytotoxic drug on cell cycle analysis
(Riccardi and Nicoletti, 2006). The healthy
cells undergo mitotic phase and divide into
two distinct daughter cells. The cells pass
through different phases of cycle namely
G0/G1, S and G2/M to ensure proper division
of the cells. Any abnormality detected in the
cell cycle leads to arrest further growth with
the help of check-points. In the present
study, the regulation of cell cycle check-
points in HCT cancer cells was studied by
cell cycle analysis using propidium iodide
staining. The effect of the compounds on cell
cycle phase was assessed and the results of
the same were shown as the percentage of
cells in G0/G1, S, and G2/M phase. The
standard, doxorubicin (1 µM), showed 7.4 %
more accumulation of cells in G2/M phase as
compared to normal control. This suggests
that cell cycle arrest is at G2/M phase. The
test compounds, AC-10 and AC-14, caused
accumulation of cells in G0/G1 phase while
AC-13 produced accumulation of cells in
Acknowledgement
The authors acknowledge Manipal Insti-
tute of Technology and Manipal College of
Pharmaceutical Sciences for providing re-
search facilities. We thank Dr. N. Gopalan
Kutty, Professor, Department of Pharmacol-
ogy, MCOPS, Manipal University Manipal,
for his support in drafting manuscript.
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