Novel 1-(7-ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives: Synthesis, characterization and anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2017.05.017

Cancer treatment still requires new compounds to be discovered. Chalcone and its derivatives exhibit anticancer potential in different cancer cells. A new series of benzofuran substituted chalcone derivatives was synthesized by the base-catalyzed Claisen-

Full text of DOI:10.1016/j.ejmech.2017.05.017

Accepted Manuscript
Novel 1-(7-ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives: Synthesis,
characterization and anticancer activity
Demet Coskun, Merve Erkisa, Engin Ulukaya, Mehmet Fatih Coskun, Ferda Ari
PII:
S0223-5234(17)30378-1
10.1016/j.ejmech.2017.05.017
EJMECH 9447
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 March 2017
Revised Date: 2 May 2017
Accepted Date: 4 May 2017
Please cite this article as: D. Coskun, M. Erkisa, E. Ulukaya, M.F. Coskun, F. Ari, Novel 1-(7-ethoxy-1-
benzofuran-2-yl) substituted chalcone derivatives: Synthesis, characterization and anticancer activity,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.05.017.
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Novel 1-(7-ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives: synthesis,
characterization and anticancer activity
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Demet Coskun1*, Merve Erkisa2, Engin Ulukaya3, Mehmet Fatih Coskun1, Ferda Ari2
1Firat University, Faculty of Science, Department of Chemistry, 23119, Elazig, Turkey.
2Uludag University, Faculty of Science and Arts, Department of Biology, 16059, Bursa,
Turkey.
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3Istinye University, Faculty of Medicine, Department of Clinical Biochemistry, 34010,
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Istanbul, Turkey.
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* Corresponding Author
E-Mail: dcoskun@firat.edu.tr
Abstract
Cancer treatment still requires new compounds to be discovered. Chalcone and its derivatives
exhibit anticancer potential in different cancer cells. A new series of benzofuran substituted
chalcone derivatives was synthesized by the base-catalyzed Claisen-Schmidt reaction of the 1-
(7-ethoxy-1-benzofuran-2-yl) ethanone with different aromatic aldehydes to yield 1-(7-
ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives 3a-j. The derivatives were
characterized by elemental analysis, FT-IR, 1H-NMR and 13C-NMR spectroscopy techniques.
The anti-growth effect of chalcone compounds was tested in breast cancer (MCF-7), non-
small cell lung cancer (A549) and prostate cancer (PC-3) cell lines by the SRB and ATP cell
viability assays. Apoptosis was detected by mitochondrial membrane potential, Annexin V
staining and caspase 3/7 activity. Formation of reactive oxygen species was determined by
DCFDA. The results revealed that chalcone derivatives have anticancer activity with
especially chalcone derivative 3a showing cytotoxic effects on cancer cells. In addition,
chalcone derivative 3a induced apoptosis through caspase dependent pathways in prostate,
lung and breast cancer cells.
Key words: Benzofuran, chalcone, cytotoxicity, anticancer activity, apoptosis
1. Introduction
Recently, benzofuran derivatives attracted natural product researchers due to their
valuable biological activities including anticancer, antimicrobial, immunomodulatory,
neuroprotective, antioxidant and anti-inflammatory properties [1-4]. The cyclopenta [b]
benzofuran silvestrol, shown in scheme 1, is a very potent cytotoxic natural product against

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several human cancer cell lines [5,6]. Benzofurans with substituents at C-2 and C-3 positions
have been extremely investigated for their biological and pharmacological properties [7-9],
and considerable, antimicrobial activities [10].
Recently, there are many studies related to the cytotoxic activity of chalcone
derivatives in various cancer cell lines [11-13]. Chalcones were first isolated from flavonoid
biosynthesis in plants [14] and since then many studies have focused on structural
modifications of the chalcone scaffold and the variety of its biological activities [15].
Chalcones consist of two aromatic rings connected by an α,β-unsaturated carbonyl group. It
has been shown that the removal of α,β-unsaturated carbonyl system could hinder their
biological activities [16]. A number of synthetic modifications, such as heterocyclic infused
[17], biphenyl based [18], coumarin based chalcones [19] or other substitutions [20-24], have
also been reported to affect the biological activities including anticancer activities of
chalcones [25-27].
Furthermore, we observed that anticancer properties of benzofuran substituted chalcone
derivatives have not been yet studied. This case has encouraged us to synthesize benzofuran
substituted chalcone compounds 3a-j and to investigate anticancer properties of these
compounds. With these views in mind, herein we report the synthesis and characterization of
a series of some new aryl-1-(7-ethoxy-1-benzofuran-2-yl)-2-propenones 3a-j and their
anticancer properties in different cancer cells.
2. Results and discussion
2.1. Synthesis and characterization
First, new 1-(7-ethoxy-1-benzofuran-2-yl) ethanone was obtained from the reaction of
3-ethoxy salicylaldehyde and 1-chloroacetone. A series of benzofuran substituted chalcones
(3a-j) were synthesized by condensation of 1-(7-eethoxy-1-benzofuran-2-yl) ethanone and
different aromatic aldehydes (2a-j) (Scheme 1). The synthesized compounds (3a-j) were
characterized by elemental analysis, FT-IR, 1H-NMR and 13C-NMR spectroscopy techniques.
Although the starting material 1-(7-ethoxy-1-benzofuran-2-yl) ethanone showed a
carbonyl stretching vibration at 1682 cm-1 in the FTIR spectrum, the chalcones showed the
characteristic bands between 1642 and 1660 cm-1. The reason for the observation observed at
of lower wave numbers of C=O stretching vibrations of chalcones was delocalization of π-
electrons between α and β carbons which decreases the C=O bond order and thus, increasing
the bond order between the carbonyl carbon and α carbon atom. Another characteristic band
shown by chalcones was C=C stretching vibration between 1574 and 1613 cm-1, at which

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wave numbers were changed according to the structure of chalcones. One of the most
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characteristic signals in H-NMR spectra of the benzofuran substituted chalcones were
observed a singlet in the region 8.35-7.87 ppm belong to 8-H at benzofuran ring. The 1H
NMR spectra of all the synthesized chalcones exihibited two doublets in the region 7.48–7.80
and 8.11–7.76 ppm with characteristric coupling constants (J) of 15.6 and 15.2 Hz, which
confirms the formation of chalcones. The higher value of coupling constant confirmed the E-
geometry of double bond in chalcones and also assigned the purity of all the synthesized
chalcones (3a-j). The signal of β-H was at a lower field than that about α-H due to resonance
of π-electrons between α and β carbons with carbonyl group. In the 13C NMR spectra, the
peaks of Cα and Cβ carbon atoms of the α,β-unsaturated carbonyl system resonated at 118.82–
123.01 and 130.44–145.13 ppm, respectively. The peak in the region 178.37–178.95 ppm was
attributed to the carbonyl carbon atom.
2.2. Anti-growth activity of chalcone derivatives by the SRB and ATP viability assays
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The anti-growth effects of chalcone derivatives (3a-j) were investigated against human
breast (MCF-7), lung (A549) and prostate (PC-3) cell lines by the SRB assay. Cancer cells
were treated for 72 h with 20 µM dose of derivatives 3a-j. It was found that various chalcone
derivatives exhibited anti-growth effects in different cell lines (Figure 1). Among the ten
compounds tested, 3a exhibited the most potent anti-growth effect in A549 cell line. Unlike
A549 cells, the growth of human breast cancer cell (MCF-7) was inhibited at 20 µM of 3a, 3c,
3d, 3e, 3h, 3i and 3j after 72 h treatment (Figure 1). At 20 µM, 3e showed the most growth-
inhibitory effect on PC-3 cell line for 72 h (Figure 1).
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The cytotoxic effect of the chalcone derivatives was confirmed by the ATP viability
assay. Chalcone derivatives significantly inhibited the growth of the different cells at 20 µM
for 72 h (Figure 3). As shown in Figure 3, it was determined that 3a (20 µM) significantly
inhibited the growth of A549 cells after 72 h. On the other hand, 3a, 3c, 3d, 3e, 3h, 3i and 3j
(20 µM) significantly inhibited the growth of MCF-7 cells during the same period. A greater
inhibition of the growth of prostate cancer cells (PC-3) was obtained with 3a and 3e chalcone
derivatives at 20 µM for 72 h, as shown in Figure 2. The growth of these cancer cells was
significantly inhibited by 3a, 3e, 3h and 3j at 20 µM for 72 h.
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IC50 values (50% inhibition of cell growth) were calculated on the basis of the ATP
assay results as shown in Table 1. The chalcone derivative 3a was more powerful than the
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others derivatives with the lowest IC50 value against cancer cells. IC50 values were 9, 2 and 10
µM for A549, MCF-7 and PC-3 cell lines, respectively. It was clearly shown that only 3a
demonstrated the most efficient anti-growth effect on A549, MCF-7 and PC-3 cancer cell
lines. The morphological evaluation by phase contrast microscopy showed that chalcone
derivatives caused a reduction in cell density, which confirmed by the viability assays. As
shown in Figure 3, among chalcone derivatives, 3a (20 µM, for 72 h) has anti-growth effects
on A549, MCF-7 and PC-3 and cancer cells.
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2.3. Apoptosis-inducing effect of chalcone derivative (3a) on cancer cells
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The apoptosis inducing effects of 3a were investigated by caspase 3/7 and Anneksin V
assays. Caspases (cysteinyl-directed aspartate-specific proteases) [28] are cysteine proteases
that play a central role in propagating the process of programmed cell death (apoptosis) in
response to proapoptotic signals. While some caspases primarily act to initiate intracellular
event cascade, other caspases that are called effector caspases act further downstream and
direct cellular breakdown through cleavage of structural proteins (Caspase-3, and Caspase-7).
Activation of Caspase-3/7 is thus a hallmark of apoptosis [29]. Figure 4 shows that 3a induces
caspase-dependent apoptosis not only in breast cancer cell lines but also in lung and prostate
cancer cells. 3a was increased in the percentage of total apoptotic cells compared with the
controls at 48 h of treatment with 20 µM dose.
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Cytofluorimetric analysis of apoptotic events was carried out through the exposure of
Annexin V marker in the breast, lung and prostate cancer cell lines. Figure 5 shows that 48 h
of treatment with (20 µM) 3a stimulated increase in the percentage of total apoptotic cells
compared with the controls. Chalcone derivative (3a) at 20 µM was in the late apoptotic stage
approximately more than 90% of the cells in A549 and MCF-7. As shown in PC-3 cell line,
we found that 6.45 % were already in the early apoptotic stage and 59.70% in the late
apoptotic stage.
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It has been previously reported that, several different chalcone derivatives have
anticancer activity in different cancer cell lines. Jovanovic et al. (2016) showed that the
Ruthenium(II)-DMSO-chalcone complexes have cytotoxic and apoptotic activity in HeLa
cells [30]. Canela et al. (2016) demonstrated that the newly synthesized chalcone derivative
TUB091 ((E)-3-(3’’-amino-4’’-methoxyphenyl)-1-(5’-methoxy-3’,4’-methylendioxyphenyl)-
2-methylprop-2-en-1-one) has anticancer activity of breast cancer cells in vitro (1-100 nM)

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and in vivo (10 mg/kg) [31]. In addition, they showed anti metastatic activity of this
compound. Xu et al. (2015) reported that chalcone derivative (L2H17) was a selectivity agent
for colon cancer cell lines (IC50 value of 11.65 µM in SW620, 25.95 µM in HCT116, and
3.36 µM in CT26.WT) and induced apoptotic cell death [32]. In another study, it was found
that quinazolinone chalcone derivative (3-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)-2-
propenoyl) phenyl)-2-methyl-3,4-dihydro-4-quinazolinone) has antiproliferative effects on
HCT-116 colon cancer cells with IC50 values of 600, 140, 26 and 16 µM in 6, 12, 24 and 48 h
and induces mitochondrial dependent apoptosis [33].
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2.4. Chalcone derivative (3a)-induced apoptosis through the altered mitochondrial
membrane potential and increased ROS production
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Mitochondria are important cellular organelles that are key regulators of cell death
processes such as apoptosis. Loss of the mitochondrial inner transmembrane potential is often
[34-40] observed to be associated with the early stages of apoptosis. Chalcone derivative (3a)
causes a decrease in mitochondrial membrane potential, inducing apoptosis in breast, lung and
prostate cancer cell lines. Compared to untreated cells, the 3a-treated cells showed a
significant decrease in mitochondrial membrane potential from 85.84% to 15.29 on A549 at
48 h, from 78.59% to 24.30% on MCF-7 at 48 h, and from 83.15% to 31.75% on PC-3 at 48 h
(Figure. 6). These results show that 3a can cause a reduction in the mitochondrial membrane
potential, which may induce apoptosis in lung, breast and prostate cancer cells via the
mitochondrial pathway.
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The mitochondria are primarily responsible for the generation of intracellular ROS
levels. Our results show that 3a increased ROS level from 48 h on A549 and MCF-7 cell lines
(Figure 7). Other research groups have reported increased ROS in different cell lines
stimulated by chalcones [41,42]; and the antioxidant effect of chalcones has also been
reported [43-46]. Various chalcones have been showed to induce apoptosis via c-Myc-
mediated ROS generation [47-49].
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Table 1. Anti-growth parameters for 3a determined by the ATP assay following 72 h of treatment
against cancer cell lines.
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*IC50 is defined as the dose inhibiting 50% of viability
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Scheme 1. General synthesis of benzofuran ketone (1) with chalcone derivatives (3a-j).
Reagants and conditions (i). K2CO3, acetone, reflax; (ii). NaOH, MeOH, r.t.
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Fig. 1. Viability screening of the cancer cells treated with 3a-3j (20 µM) for 72 h. The SRB assay was
performed as viability assay. *** denotes statistically significant differences in comparison with
negative control (NC) (p<0.0001).
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Fig. 2. Viability screening of the cancer cells treated with 3a-3j (20 µM) for 72 h. The ATP assay was
performed as viability assay. *** denotes statistically significant differences in comparison with
negative control (NC) (p<0.0001).
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Fig. 3. Images of Phase Contrast Microscopy. Cells were treated with 20 µM 3a for 72 h. Controls
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included untreated cells. A scale bar represents 10 µm.

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Fig. 4. Induction of caspase 3/7 activity in response to 3a. Cells were treated with 3a (20 µM) and
were stained using caspase 3/7 kit and counted with a flow cytometer as described in Materials and
methods. Representative plots for cells following 48 h drug exposure are shown in the figure.
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Fig. 5. Chalcone-derivative induces apoptosis in A549, MCF-7 and PC-3 cells as determined by
Annexin V/PI staining. Cells were treated with the 3a (20 µM) and were stained with Annexin V/dead
cell marker and counted with a flow cytometer as described in Materials and methods. Representative
plots for cells following 48 h drug exposure are shown in the figure.
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Fig. 6. Change in MMP in response to Chalcone-derivative. Cells were treated with the 3a (20 µM)
and were stained using MitoPotential kit and counted with a flow cytometer as described in Materials
and methods. Representative plots for cells following 48 h drug exposure are shown in the figure.
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Fig. 7. Increase in ROS in response to Chalcone derivative. (A) Cells were treated with the 3a (20
µM) and were stained using Oxidative stress kit and counted with a flow cytometer as described in
Materials and methods. (B) Cells were pretreated with DCFDA with the 3a (20 µM) for 48 h and ROS
was measured as described in Materials and methods.
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3. Conclusions
In summary, a class of novel 1-(7-ethoxy-1-benzofuran-2-yl) substituted chalcone
derivatives were designed, synthesized and their cytotoxic activity was evaluated on breast,
lung and prostate cancer cell lines. Among the chalcone derivatives, compound 3a showed
strong inhibitory effects on cancer cells with IC50 values of 9, 2 and 10 µM for A549, MCF-7
and PC-3 cell lines, respectively. The apoptosis-inducing activity was measured by Annexin
V staining, caspase 3/7 activity, ROS production and mitochondrial membrane potential.
Molecular analysis indicates that the chalcone derivative 3a induced apoptosis through
caspase dependent pathways in prostate, lung and breast cancer cells. Given these promising
results, we suggest that this chalcone derivative makes further studies undoubtedly necessary,
to be developed as an anticancer agent.

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4. Experimental
4.1. Characterization Techniques
Melting points were recorded using a differential scanning calorimeter (Shimadzu
DSC-50) and were uncorrected. Elemental analyses were performed with a Leco CHNS-932
apparatus. NMR spectra were determined on a Bruker Avance (400-MHz) spectrometer, with
tetramethylsilane (TMS) as the internal standard in DMSO-d6 as solvent. IR spectra (KBr
disk) were recorded using a Perkin-Elmer Spectrum One FT-IR spectrometer. All the reagents
and solvents used were of analytical grade and were used as supplied unless otherwise stated.
TLC was performed on silica gel coated plates for monitoring the reactions.
4.2. Synthesis of the 1-(7-ethoxy-1-benzofuran-2-yl) ethanone (1)
A mixture of 3-ethoxysaliciylaldehyde (1 g, 6.02 mmol) and potassium carbonate
(0.83 g, 6.02 mmol) in dry acetone (10 ml) was stirred at room temperature for 1 h and the
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reaction mixture was cooled to 0-5 C, and then chloroacetone (0.48 ml, 6.02 mmol) was
added dropwise. The reaction mixture was stirred at room temperature for ten minutes and
refluxed. Progress of the reaction was monitored by TLC. Upon completion, the reaction
mixture was poured into crushed ice. The precipitated solid was filtered, washed with water
and dried. The product was crystallized from ethanol.
Yield: 70%, mp 84-86 C; FT-IR (KBr, cm-1): 3071 (Arom. C-H), 2978-2877 (Aliph. C-H),
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1682 (C=O); H NMR (400 MHz, DMSO-d6): δ (ppm) 7.86 (s, 1H, 3-H), 7.34 (d, 1H, J=7.6
Hz, 4-H), 7.25 (dd, 1H, J=7.6 and 7.2 Hz, 5-H), 7.08 (d, 1H, J=7.6 Hz, 6-H), 4.22 (q, 2H,
J=6.8 Hz, OCH2), 2.56 (s, 3H, COCH3), 1.42 (t, 3H, J=5.8 Hz, CH3); 13C-NMR (DMSO-d6) δ
(ppm) 188.26 (C=O), 152.52 (7-C), 145.15 (2-C), 145.04 (8-C), 128.88 (9-C), 125.22 (5-C),
115.46 (4-C), 115.16 (6-C), 111.00 (3-C), 64.54 (OCH2), 26.88 (COCH3), 15.07 (CH3). Anal.
calcd. for C12H12O3: C, 70.57; H, 5.92; found: C, 70.66; H, 5.87.
4.3. General Procedure for synthesis of chalcones (3a-j)
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A mixture of 1-(7-ethoxy-1-benzofuran-2-yl) ethanone (0.41 g, 2 mmol) and one of
the aldehyde derivatives (2a-i, 2 mmol) in MeOH (10 ml) was cooled to 0-5 oC and then 3 ml
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of aqueous NaOH (1 mol/L) was added to this mixture and stirred at 25 C for 3 h. The
reaction mixture was poured into crushed ice and acidified with HCl. The precipitated solid
was filtered, washed with water and dried. The residue was crystallized from ethanol.
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4.3.1. 3-[4-(dimethylamino)phenyl]-1-(7-ethoxy-1-benzofuran-2-yl)prop-2-en-1-one (3a)
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Yield: 50%, mp 122-124 oC; FT-IR (KBr, cm-1): 3053 (Arom. C-H), 2989-2888 (Aliph. C-H),
1660 (C=O), 1603 (C=C); 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.18 (s, 1H, Br3-H), 7.76
(d, 1H, J=15.2 Hz, β-H), 7.73 (d, 2H, J=7.6 Hz, Ar2-H, Ar6-H), 7.58 (d, 1H, J=15.2 Hz, α-H),
7.39 (d, 1H, J=7.6 Hz, Br4-H), 7.28 (dd, 1H, J=7.6 and 8.0 Hz, Br5-H), 7.12 (d, 1H, J=8.0 Hz,
Br6-H), 6.77 (d, 2H, J=9.2 Hz, Ar3-H, Ar5-H), 4.26 (q, 2H, J=6.8 Hz, OCH2), 3.03 (s, 6H,
N(CH3)2), 1.46 (t, 3H, J=6.8 Hz, CH3); 13C-NMR (DMSO-d6): δ (ppm) 178.52 (C=O), 154.28
(7-C), 152.64 (Ar4-C), 145.15 (Br2-C), 145.13 (8,β-C), 131.46 (Ar2,6-C), 129.18 (9-C),
125.21 (Br5-C), 122.05 (α-C), 116.27 (Ar1-C), 115.40 (Br4-C), 114.43 (Br6-C), 112.22
(Ar3,5-C), 110.84 (Br3-C), 64.58 (OCH2), 26.88 (N(CH3)2), 15.14 (CH3). Anal. calcd. for
C21H21O3N: C, 75.20; H, 6.31; N, 4.18; found: C, 75.15; H, 6.25; N, 4.13.
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4.3.2. 1-(7-ethoxy-1-benzofuran-2-yl)-3-phenylprop-2-en-1-one (3b)
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Yield: 71%, mp 93-95 C; FT-IR (KBr, cm-1): 3093 (Arom. C-H), 2978-2816 (Aliph. C-H),
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1642 (C=O), 1613 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 8.35 (s, 1H, Br3-H),
7.93-7.81 (m, 4H, α-H, β-H, Ar2,6-H), 7.50-7.49 (m, 3H, Ar3,4,5-H), 7.42 (d, 1H, J=7.2 Hz,
Br4-H), 7.30 (dd, 1H, J=7.6 and 8.0 Hz, Br5-H), 7.15 (d, 1H, J=8.0 Hz, Br6-H), 4.26 (q, 2H,
J=6.8 Hz, OCH2), 1.45 (t, 3H, J=6.8 Hz, CH3); 13C-NMR (DMSO-d6): δ (ppm) 178.76 (C=O),
153.62 (7-C), 145.42 (Br2-C), 145.19 (Br8-C), 143.97 (β-C), 134.80 (Ar1-C), 131.38 (Ar2,6-
C), 129.47 (9-C), 128.99 (Ar3-C), 128.67 (Ar5-C), 125.39 (Br5-C), 122.29 (α-C), 116.24
(Br4-C), 115.62 (Br6-C), 111.27 (Br3-C), 64.60 (OCH2), 15.12 (CH3). Anal. calcd. for
C19H16O3: C, 78.06; H, 5.52; found: C, 77.95; H, 5.63.
4.3.3. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(4-methylphenyl)prop-2-en-1-one (3c)
Yield: 73%, mp 122-124 oC; FT-IR (KBr, cm-1): 3125-3014 (Arom. C-H), 2974-2935 (Aliph.
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C-H), 1657 (C=O), 1599 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 8.32 (s, 1H, Br3-
H), 7.82-7.80 (m, 4H, α,β-H, Ar2,6-H), 7.40 (d, 1H, J=8.0 Hz, Br4-H), 7.30 (d, 2H, J=8.0 Hz,
Ar3,5-H), 7.29 (t, 1H, J=8.0 Hz, Br5-H), 7.13 (d, 1H, J=8.0 Hz, Br6-H), 4.24 (q, 2H, J=6.8
Hz, OCH2), 2.36 (s, 3H, R3=CH3), 1.45 (t, 3H, J=6.8 Hz, CH3); 13C-NMR (DMSO-d6): δ
(ppm) 178.78 (C=O), 153.70 (7-C), 145.38 (Br2-C), 145.18 (8-C), 144.03 (β-C), 141.53 (Ar4-
C), 132.10 (Ar1-C), 130.09 (Ar2,6-C), 129.50(9-C), 129.03 (Ar3,5-C), 125.36 (Br5-C),
121.26 (α-C), 115.96 (Br4-C), 115.58 (Br6-C), 111.21 (Br3-C), 64.60 (OCH2), 21.60

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(R3=CH3), 15.12 (CH3). Anal. calcd. for C20H18O3: C, 78.41; H, 5.92; found: C, 78.56; H,
5.89.
4.3.4. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (3d)
Yield: 70%, mp 104-106 oC; FT-IR (KBr, cm-1): 3089 (Arom. C-H), 2961-2830 (Aliph. C-H),
1649 (C=O), 1585 (C=C); 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.28 (s, 1H, Br3-H), 7.88
(d, 2H, J=8.8 Hz, Ar2,6-H), 7.81 (d, 1H, J=15.6 Hz, β-H), 7.74 (d, 1H, J=15.6 Hz, α-H), 7.40
(d, 1H, J=8.0 Hz, Br4-H), 7.28 (dd, 1H, J=8.0 and 7.6 Hz, Br5-H), 7.14 (d, 1H, J=7.6 Hz,
Br6-H), 7.05 (d, 2H, J=8.8 Hz, Ar3,5-H), 4.25 (q, 2H, J=6.8 Hz, OCH2) 3.84 (s, 3H, OCH3),
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1.45 (t, 3H, J=6.8 Hz, CH3); C-NMR (DMSO-d6): δ (ppm) 178.75 (C=O), 162.06 (Ar4-C)
153.83 (7-C), 145.31 (Br2-C), 145.18 (8-C), 143.97 (β-C), 131.44 (Ar2,6-C), 129.05 (9-C),
127.44 (Ar1-C), 125.34 (Br5-C), 119.78 (α-C), 115.60 (Br4-C), 115.54 (Br6-C) 114.96
(Ar3,5-C), 111.11 (Br3-C), 64.59 (OCH2), 55.89 (OCH3), 15.13 (CH3). Anal. calcd. for
C20H18O4: C, 74.52; H, 5.63; found: C, 74.66; H, 5.58.
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Yield: 74%, mp 144-146 oC; FT-IR (KBr, cm-1): 3115-3061 (Arom. C-H), 2978-2902 (Aliph.
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C-H), 1657 (C=O), 1606 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 8.25 (s, 1H, Br3-
H), 8.11 (d, 1H, J=15.6 Hz, β-H), 7.70 (d, 1H, J=15.6 Hz, α-H), 7.52 (s, 1H, Ar6-H), 7.40 (d,
1H, J=8.0 Hz, Br4-H), 7.28 (t, 1H, J=8.0 Hz, Br5-H), 7.13 (d, 1H, J=8.0 Hz, Br6-H), 6.76 (s,
1H, Ar3-H), 4.26 (q, 2H, J=6.8 Hz, OCH2), 3.93 (s, 3H, R1=OCH3), 3.90 (s, 3H, R3=OCH3),
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3.85 (s, 3H, R4=OCH3), 1.46 (t, 3H, J=6.8 Hz, CH3); C-NMR (DMSO-d6): δ (ppm) 178.79
(C=O), 155.03 (Ar2-C), 154.03 (Ar4-C) 153.66 (7-C), 145.24 (Br2-C), 145.18 (8-C), 143.51
(β-C), 138.36 (Ar5-C), 129.06 (9-C), 125.31 (Br5-C), 118.93 (α-C), 115.44 (Br4-C), 115.17
(Br6-C) 114.23 (Ar6-C), 111.24 (Ar1-C), 111.02 (Br3-C), 97.83 (Ar3-C), 64.60 (OCH2),
56.83 (R1=OCH3), 56.74 (R3=OCH3), 56.30 (R4=OCH3), 15.13 (CH3). Anal. calcd. for
C22H22O6: C, 69.10; H, 5.80; found: C, 68.97; H, 5.91.
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4.3.6. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (3f)
Yield: 60%, mp 123-125 oC; FT-IR (KBr, cm-1): 3093-3043 (Arom. C-H), 2981-2845 (Aliph.
1
C-H), 1649 (C=O), 1592 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 8.16 (s, 1H, Br3-
H), 8.05 (d, 1H, J=15.6 Hz, β-H), 7.91 (d, 1H, J=9.2 Hz, Ar6-H), 7.71 (d, 1H, J=15.6 Hz, α-
H), 7.37 (d, 1H, J=8.0 Hz, Br4-H), 7.28 (t, 1H, J=8.0 Hz, Br5-H), 7.10 (d, 1H, J=8.0 Hz, Br6-

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H), 6.66-6.64 (m, 2H, Ar3,5-H), 4.23 (q, 2H, J=6.8 Hz, OCH2), 3.92 (s, 3H, R1=OCH3), 3.85
(s, 3H, R3=OCH3), 1.44 (t, 3H, J=6.8 Hz, CH3); 13C-NMR (DMSO-d6): δ (ppm) 178.95
(C=O), 163.87 (Ar2-C), 160.65 (Ar4-C), 153.97 (7-C), 145.23 (Br2-C), 145.16 (8-C), 138.70
(β-C), 130.78 (Ar6-C), 129.09 (9-C), 125.27 (Br5-C), 119.23 (α-C), 115.98 (Br4-C), 115.48
(Br6-C), 115.01 (Ar1-C), 110.99 (Br3-C), 106.94 (Ar5-C), 98.70 (Ar3-C), 64.58 (OCH2),
56.28 (R1=OCH3), 56.02 (R3=OCH3), 15.11 (CH3). Anal. calcd. for C21H19O3: C, 71.58; H,
5.72; found: C, 71.72; H, 5.83.
4.3.7. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(4-bromophenyl)prop-2-en-1-one (3g)
Yield: 80%, mp 145-147 oC; FT-IR (KBr, cm-1): 3100-3020 (Arom. C-H), 2981-2891 (Aliph.
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C-H), 1649 (C=O), 1585 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 8.33 (s, 1H, Br3-
H), 7.90 (d, 1H, J=15.6 Hz, β-H), 7.85 (d, 2H, J=8.8 Hz, Ar3,5-H), 7.77 (d, 1H, J=15.6 Hz, α-
H), 7.68 (d, 2H, J=8.4 Hz, Ar2,6-H), 7.39 (d, 1H, J=8.0 Hz, Br4-H), 7.27 (t, 1H, J=8.0 Hz,
Br5-H), 7.12 (d, 1H, J=8.0 Hz, Br6-H), 4.24 (q, 2H, J=6.8 Hz, OCH2), 1.44 (t, 3H, J=6.8 Hz,
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CH3); C-NMR (DMSO-d6): δ (ppm) 178.59 (C=O), 153.54 (7-C), 145.44 (Br2-C), 145.17
(8-C), 142.54 (β-C), 134.08 (Ar1-C), 132.40 (Ar3,5-C), 131.30 (Ar2,6-C), 128.94 (9-C),
125.38 (Br5-C), 124.75 (Ar4-C), 123.01 (α-C), 116.44 (Br4-C), 115.61 (Br6-C), 111.28 (Br3-
C), 64.58 (OCH2), 15.11 (CH3). Anal. calcd. for C19H15O3Br: C, 61.47; H, 4.07; found: C,
61.39; H, 4.15.
4.3.8. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(2-furyl)prop-2-en-1-one (3h)
Yield: 75%, mp 121-123 oC; FT-IR (KBr, cm-1): 3140-3040 (Arom. C-H), 2985-2877 (Aliph.
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C-H), 1653 (C=O), 1595 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 8.13 (s, 1H, Br3-
H), 7.97 (s, 1H, Ar4-H), 7.66 (d, 1H, J=15.2 Hz, β-H), 7.48 (d, 1H, J=15.2 Hz, α-H), 7.37 (d,
1H, J=8.0 Hz, Br4-H), 7.27 (t, 1H, J=8.0 Hz, 5-H), 7.14-7.11 (m, 2H, Ar2-H, 6-H), 6.73 (s,
1H, Ar3-H), 4.25 (q, 2H, J=6.8 Hz, OCH2), 1.44 (t, 3H, J=6.8 Hz, CH3); 13C-NMR (400 MHz,
DMSO-d6): δ (ppm) 178.46 (C=O), 153.55 (7-C), 151.36 (Ar1-C), 147.07 (Ar4-C), 145.28
(Br2-C), 145.15 (8-C), 130.44 (β-C), 129.07 (9-C), 125.36 (5-C), 118.82 (α-C), 118.43 (Ar2-
C), 115.55 (Br4-C), 115.28 (6-C), 113.79 (Ar3-C), 111.16 (Br3-C), 64.60 (OCH2), 15.11
(CH3). Anal. calcd. for C17H14O4: C, 72.33; H, 5.00; found: C, 72.36; H, 5.04.
4.3.9. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(2-thienyl)prop-2-en-1-one (3i)
Yield: 79%, mp 79-81 oC; FT-IR (KBr, cm-1): 3133-3111 (Arom. C-H), 2978-2837 (Aliph. C-
H), 1653 (C=O), 1588 (C=C); 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.23 (s, 1H, Br3-H),

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8.01 (d, 1H, J=15.6 Hz, β-H), 7.83 (d, 1H, J=4.4 Hz, Ar4-H), 7.72 (d, 1H, J=3.2 Hz, Ar2-H),
7.51 (d, 1H, J=15.2 Hz, α-H), 7.38 (d, 1H, J=7.6 Hz, Br4-H), 7.28 (dd, J=8.0 and 7.6 Hz, 5-
H), 7.22 (dd, 1H, J=4.8 and 3.6 Hz, Ar3-H), 7.12 (d, 1H, J=7.6 Hz, 6-H), 4.25 (q, 2H, J=6.8
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Hz, OCH2), 1.45 (t, 3H, J=6.8 Hz, CH3); C-NMR (400 MHz, DMSO-d6): δ (ppm) 178.37
(C=O), 153.51 (7-C), 145.33 (Br2-C), 145.16 (8-C), 139.90 (β-C), 136.81 (Ar1-C), 134.01
(Ar2-C), 131.45 (Ar4-C), 129.31 (Ar3-C), 129.06 (9-C), 125.35 (5-C), 120.49 (α-C), 115.66
(Br4-C), 115.57 (6-C), 111.20 (Br3-C), 64.60 (OCH2), 15.11 (CH3). Anal. calcd. for
C17H14O3S: C, 68.44; H, 4.73; found: C, 68.38; H, 4.79.
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4.3.10. 1-(7-ethoxy-1-benzofuran-2-yl)-3-(1H-pyrrol-2-yl)prop-2-en-1-one (3j)
o
Yield: 50%, mp 187-189 C; FT-IR (KBr, cm-1): 3248 (N-H), 3125 (Arom. C-H), 2978-2931
1
(Aliph. C-H), 1649 (C=O), 1574 (C=C); H NMR (400 MHz, DMSO-d6): δ (ppm) 11.83 (s,
1H, NH), 7.87 (s, 1H, Br3-H), 7.68 (d, 1H, J=15.2 Hz, β-H), 7.48 (d, 1H, J=15.6 Hz, α-H),
7.40 (d, 1H, J=7.6 Hz, Br4-H), 7.27 (t, 1H, J=7.6 Hz, 5-H), 7.20 (s, 1H, Ar4-H), 7.12 (d, 1H,
J=7.6 Hz, 6-H), 6.80 (s, 1H, Ar2-H), 6.26 (s, 1H, Ar3-H), 4.26 (q, 2H, J=6.8 Hz, OCH2), 1.45
13
(t, 3H, J=6.8 Hz, CH3); C-NMR (400 MHz, DMSO-d6): δ (ppm) 178.56 (C=O), 154.33 (7-
C), 145.13 (Br2-C), 145.07 (8-C), 134.34 (β-C), 129.43 (Ar1-C), 129.11 (9-C), 125.39 (5-C),
125.24 (α-C), 117.29 (Ar4-C), 115.40 (6-C), 114.87 (Br6-C), 113.63 (Ar2-C) 111.40 (Br3-C),
110.86 (Ar3-C), 64.59 (OCH2), 15.14 (CH3). Anal. calcd. for C17H15O3N: C, 72.58; H, 5.37;
found: C, 72.56; H, 5.44.
4.4. Determination of cytotoxicity
4.4.1. Cell culture and chemicals
Breast cancer (MCF-7), prostate cancer (PC-3) and non-small cell lung cancer (A549)
cell lines were cultured in RPMI 1640 medium supplemented with penicillin G (100 U/mL),
streptomycin (100 µg/mL), L-glutamine, and 10% fetal bovine serum. All cell lines were
maintained at 37 °C in a humidified atmosphere containing 5% CO2.
4.4.2. The SRB viability assay
The sulforhodamine B (SRB) assay was used to evaluate the drug sensitivity of cancer
cells (MCF-7, A549, PC-3) based on the measurement of the cellular protein content. The
cells were seeded at a density of 5×103 cells per well of a 96-well plate in 200 µL culture
medium. The chalcone compounds (3a-j) were dissolved in DMSO (the concentration of

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DMSO was equal to 0.2% v/v at 100 µM) while the final concentration (20 µM) was prepared
in culture medium.
Cells were fixed by using 50 µL of 50% (w/v) ice-cold trichloroacetic acid for 1 hour
at 4 °C and then stained with 50 µL of SRB solution (0.4% w/v in 1% aqueous acetic acid) for
30 min at room temperature. The dye was washed with 1% (v/v) acetic acid and added 10 mM
tris base solution (pH: 10, 150 µL) to dissolve the protein-bound dye. The plate was
subsequently shaked for 10 min at 150 rpm. After solubilization, the absorbance (Abs) was
read by using a spectrophotometer (FLASH Scan S12, Analytik Jena) at 564 nm. Viability of
cells was calculated with reference to the untreated control cells using the following formula:
Viability (%) = [100 × (Sample Abs)/(Control Abs)].
4.4.3. The ATP viability assay
The seeding of cells and treatment conditions as well as the calculation of viability
was similar to those of the SRB assay (see above). The ATP assay uses the highly sensitive
reaction to determine the level of cellular ATP as a surrogate indicator of the number of
viable (ATP-producing) cells [50]. ATP content was determined according to the
manufacturer’s recommendations (ATP Bioluminescence Assay, Sigma, St. Louis, MO,
USA). Briefly, following the treatment, ATP was extracted from the cells, and then luciferin-
luciferase solution was added. Luminescence was determined in a luminometer (BioTek,
USA). The results were expressed in U/L.
The cells were also morphologically observed under a phase contrast microscope (Olympus
CKX41, Japan) after exposure to 20 µM chalcone compounds 72 h.
4.4.4. Determination of caspase 3/7 activity and Annexin-V staining
3×105 cells (A549, MCF-7 and PC-3) were seeded in 6-well plates and cultured
overnight. The cells were treated with 3a at the 20 µM for 48 h. At the end of the treatment,
cells were washed with PBS, harvested by trypsinisation, and analyzed for the detection of
early/late apoptosis and cell death mode using Annexin V/Dead Cell (kit MCH100105,
Millipore, Darmstadt, Germany) and caspase 3/7 kit (MCH100108, Millipore, Darmstadt,
Germany) respectively, according to the manufacturer's instructions. The live, dead, early and
late apoptotic cells were counted with the Muse Cell Analyzer (Millipore, USA) based on the
presence/absence of caspase 3/7 activity and Annexin V positivity due to PS translocation
during apoptosis. In addition, 7-aminoactinomycin D (7-AAD) dye was also included in both
assays as a dead cell marker.

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4.4.5. Measurement of the mitochondrial potential (MMP)
A549, MCF-7 and PC-3 cells were treated with 3a (20ꢀµM) for 48h and then harvested
by trypsinisation. Mitochondrial membrane depolarization was measured using the Muse
MitoPotential kit (Millipore). Briefly, the cells were incubated with Muse MitoPotential dye
for 20ꢀmin in a 37ꢀ°C CO2 incubator. At the end of the incubation, 7-AAD was added as a
dead cell marker for an additional 5 min. Cells were analyzed by using the Muse Cell
Analyzer (Millipore).
4.4.6. Induction of Oxidative Stress and ROS Measurement
To evaluate the effect of 3a (20 µM) on ROS generation, the assay was conducted on
the Muse Cell Analyzer (Millipore, Bedford, MA, USA) using the ROS Kit (Cat no MCH
100111-2; Millipore). Staining procedure was followed as per the manufacturer’s protocol.
To confirm ROS levels with DCFDA (Sigma, cat # D6883) 5 × 103 cells were seeded in 96-
well plates, and incubated at 37 °C in 5% CO2. The following day, cells were pretreated with
5 µM of DCFDA (dissolved in PBS) for 2 h. Following pretreatment, 5 µM of DCFDA and
20 µM of freshly prepared 3a were simultaneously added to cells. After 48h of treatments,
plates were read on a micro-plate fluorometer (FLx800 Bio-Tek, Vermont, USA) using
excitation and emission wavelengths of 485 and 535 nm, respectively. The experiment was
done using three biological repeats in duplicate wells.
Acknowledgments
The authors thank Firat University for financial support of this work. Project Number:
FF.16.15
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Highlights
• Novel benzofuran substituted chalcone derivatives (3a-j) was synthesized and
characterized
• Chalcone derivative 3a showed potent cytotoxicity on prostate, lung and breast cancer
cells
• The chalcone derivative 3a induced apoptosis through caspase dependent pathways