Synthesis and anticancer activity of chalcone analogues with sulfonyl groups

Article abstract of DOI:10.1007/s00044-018-02283-4

Three series of sulfonyl esters were synthesized in reactions of sulfonyl chlorides with three different phenolic chalcone analogues (dehydrozingerone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one), (E)-1-(4-hydroxy-3-methoxyphenyl)pent-1-en-3-one, and (E)-1-(4-hydroxy-3-methoxyphenyl)-5-methylhex-1-en-3-one). The structures of the new compounds were determined by IR, MS, and NMR methods. Screening of the new sulfonyl esters’ in vitro cytotoxic activities against human epithelial cervical carcinoma (HeLa) and normal human fibroblast (MRC-5) cell lines by the MTT method was performed. The five most active were selected and further tested on HeLa, MRC-5, and MCF-7 (breast carcinoma) cell lines. The examined compounds exhibit strong in vitro anticancer activities with moderate-to-high selectivity, inducing apoptotic cell death and cell cycle arrest in both HeLa and MCF-7 cell lines, but have little to no effect on the non-cancerous MRC-5 cell line.

Full text of DOI:10.1007/s00044-018-02283-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-02283-4
ORIGINAL RESEARCH
Synthesis and anticancer activity of chalcone analogues with
sulfonyl groups
1 ●
1 ●
2,3 ●
3 ●
4 ●
Jovana M. Muškinja Adrijana Z. Burmudžija Dejan D. Baskić
Suzana L. Popović Danijela V. Todorović
Milan M. Zarić5 Zoran R. Ratković
1
●
Received: 13 July 2018 / Accepted: 21 December 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Three series of sulfonyl esters were synthesized in reactions of sulfonyl chlorides with three different phenolic chalcone
analogues (dehydrozingerone (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one), (E)-1-(4-hydroxy-3-methoxyphenyl)pent-1-
en-3-one, and (E)-1-(4-hydroxy-3-methoxyphenyl)-5-methylhex-1-en-3-one). The structures of the new compounds were
determined by IR, MS, and NMR methods. Screening of the new sulfonyl esters’ in vitro cytotoxic activities against human
epithelial cervical carcinoma (HeLa) and normal human fibroblast (MRC-5) cell lines by the MTT method was performed.
The five most active were selected and further tested on HeLa, MRC-5, and MCF-7 (breast carcinoma) cell lines. The
examined compounds exhibit strong in vitro anticancer activities with moderate-to-high selectivity, inducing apoptotic cell
death and cell cycle arrest in both HeLa and MCF-7 cell lines, but have little to no effect on the non-cancerous MRC-5 cell
line.
●
●
●
●
Keywords Apoptosis Chalcones Cell death Cytotoxicity Sulfonyl ester
Introduction
discovering new anticancer agents from naturally sourced
products (Kinghorn et al. 2003). Many natural products and
dietary components have been evaluated as potential che-
mopreventive agents; herbal remedies used in traditional
medicine are a promising source of potentially active drugs
Cancer is one of the most human widespread diseases, so
there is considerable scientific and commercial interest in
(Rajasekar et al. 2012). Although many anticancer drugs of
natural origin have been discovered, the search for new
anticancer agents is still of great importance, particularly in
terms of increasing their bioavailability and finding less
toxic and more effective drugs.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-018-02283-4) contains supplementary
material, which is available to authorized users.
*
Jovana M. Muškinja
Chalcones (trans-1,3-diaryl-2-propen-1-ones) are natural
products of the flavonoid family; they are considered as
intermediates in flavonoid biosynthesis, and are widespread
in plants. These compounds are characterized by the pre-
sence of two phenyl rings linked by a three-carbon bridge
that contains an α,β-unsaturated ketone fragment, forming
the central core in a variety of important biological com-
pounds (Mahapatra et al. 2015; Tseng et al. 2015). Chal-
cones and their derivatives have fascinated chemists due to
their numerous useful biological and pharmacological
properties, such as antioxidant (Vasil’ev et al. 2010;
Sivakumar et al. 2011; Vogel et al. 2008), anti-
inflammatory (Yadav et al. 2010; Zhang et al. 2010),
antibacterial (Nielsen et al. 2004; Batovska et al. 2009;
Alcaradz et al. 2000), antifungal (Lahtchev et al. 2008;
jovana.muskinja@gmail.com
1
2
3
Department of Chemistry, Faculty of Science, University of
Kragujevac, Radoja Domanovića 12, 34000 Kragujevac, Serbia
Public Health Institute Kragujevac, Nikole Pašića 1, 34000
Kragujevac, Serbia
Department of Microbiology and Immunology, Faculty of Medical
Sciences, University of Kragujevac, Center for Molecular
Medicine and Stem Cell Research, Svetozara Markovića 67,
3
4000 Kragujevac, Serbia
4
5
Department of Genetics, Faculty of Medical Sciences, University
of Kragujevac, Svetozara Markovića 67, 34000
Kragujevac, Serbia
Department of Biochemistry, Faculty of Medical Sciences,
University of Kragujevac, Svetozara Markovića 67, 34000
Kragujevac, Serbia

Medicinal Chemistry Research
and 50 MHz for 13C), using CDCl as the solvent and TMS
as the internal standard. H– and C–NMR chemical shifts
O
O
O
3
13
1
H
were reported in parts per million (p.p.m.) and were refer-
1
enced to the solvent peak; CDCl (7.26 p.p.m. for H and
7
3
Fig. 1 Synthesis of chalcones
13
6.90 p.p.m. for C). Multiplicities were represented by s
(singlet), d (doublet), t (triplet), q (quartet), and m (multi-
ElSohly et al. 2001; Gafner et al. 1996), antiviral (Trivedi
et al. 2007; Phrutivorapongkul et al. 2003; Park et al. 2011),
antiparasitic (Nielsen et al. 1995, 1998), anticancer
plet). Coupling constants (J) were in Hertz (Hz).
Mass spectrometry was performed using a Waters
Micromass ZQ mass spectrometer and MassLynx software
for control and data processing. Electro spray ionization in
the positive mode was used. The electro spray capillary was
set at 4.3 kV and the cone at 40 V. The ion source tem-
perature was 125 °C, and the nitrogen flow rates were 400
and 50 l/h, for desolvation and cone gas flow, respectively.
The collision energy was 40 eV. The melting point of the
products was determined using a MelTemp1000 apparatus.
(
Zsoldos-Mady et al. 2006; Anto et al. 1995), antileishma-
nial (Chen et al. 1993, 1994; Liu et al. 2001; Li et al. 1995;
Narender et al. 2005), and antitubercular activities (Hans
et al. 2010; Sivakumar et al. 2007).
Classical synthesis of chalcones involves the Claisen–
Schmidt condensation reaction of aryl methyl ketones with
aromatic aldehydes (Fig. 1).
A well-known natural phenolic product, dehydrozinger-
one (4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, 3), is
isolated from ginger root (Zingiber officinale), and is a very
important chalcone analog, which exhibits anti-inflamma-
tory, antioxidant, and antitumor-promoting activities
Chemical synthesis
General procedure for synthesis of (E)-2-methoxy-4-(3-
oxoprop-1-enyl-3-alkyl)phenyl benzenesulfonates
(
Motohashi et al. 1998).
Dehydrozingerone and its semi-chalcone analogs, with
Solutions of selected methyl ketones (2 mmol) in metha-
nol (6 mL) were stirred and corresponding sulfonyl
chlorides (2.4 mmol) were added. Then, triethylamine
(0.6 mL) was added dropwise. The mixtures were stirred
overnight at room temperature. After completion of the
reactions, mixtures were poured into iced water with
stirring. In some cases (for our compounds 7f, 7g, 8e, 8f,
8g, and 9g; see below), the formed precipitate was filtered
and washed with cold diethyl ether. In other cases, when
no precipitate formed, the organic layer was extracted
with dichloromethane (3 × 50 mL), dried over anhydrous
Na SO and evaporated under vacuum. The residue was
purified by short column chromatography on silica gel,
using dichloromethane as eluent. The oil obtained usually
crystallized; if not, the oily residue was dissolved in
diethyl ether from which products crystallized on standing
in a deepfreeze. Crude products were washed with cold
ether, and 21 pure, mostly white crystals were obtained, as
follows:
alkyl groups linked to the carbonyl groups instead of to the
aromatic groups (as in standard chalcones), were prepared.
These compounds have well-established biological activ-
ities, as was previously reported (Muškinja et al. 2016).
Furthermore, the free phenolic groups reacted with corre-
sponding aryl sulfonyl chlorides to form esters (Loewenthal
1
959). Although aryl sulfonates are widely used as pro-
tective agents for hydroxy groups, they are highly stable
and usually require drastic deprotection conditions. Com-
pounds containing aryl sulfonate moieties have received
considerable attention during the last two decades, as they
are endowed with a variety of biological activities (anti-
papillomavirus (Christensen et al. 2001), anti-HIV-1
Rusconi et al. 1996), antineoplastic (Hanna et al. 1991),
as well as having activities against various kinds of cancers
Betts et al. 2006; Cyr et al. 2008)).
2
4
(
(
Materials and methods
(
E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl benzenesulfonate
Chemistry
(7a) Yellowish white crystals; yield: 88.7%; m.p. 84–85 °C;
IR (KBr): 3075, 3009, 1666, 1626, 1585, 1371, 1198, 1116,
−
1
1
All starting chemicals were commercially available and
used as received, except the solvents, which were purified
by distillation.
Column chromatography: silica gel 60 (Merck, 230-400
mesh ASTM); TLC: silica gel 60 F254-precoated plates
1091, 1031, 861 cm ; H–NMR (200 MHz, CDCl3):
δ = 2.38 (s, 3H, CH CO), 3.56 (s, 3H, OCH ), 6.63 (d, 1H,
3
3
J = 16.2 Hz, CH), 6.97–7.28 (m, 3H, Ar–H), 7.42 (d, 1H,
J = 16.2 Hz, CH), 7.49–7.71 (m, 3H, Ar–H), 7.86–7.89 (m,
3H, Ar–H); C–NMR (50 MHz, CDCl ): δ = 27.5, 55.6,
13
3
(
Merck); IR spectra: Perkin-Elmer Spectrum One FT-IR
111.5, 121.1, 124.5, 127.9, 128.5, 128.8, 134, 134.5, 136.1,
−
1
spectrometer with a KBr disc, ν in cm ; NMR spectra:
Varian Gemini 200 MHz spectrometer (200 MHz for H
139.7, 141.9, 152, 197.9 (CO). ESI-MS (40 eV): m/z
1
+
(%) = 332.37 (100%) [M] , 141.17 (17%).

Medicinal Chemistry Research
−
1
1
(
E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl 4-methylbenzene-
1194, 1114, 1091, 1027, 857 cm ; H–NMR (200 MHz,
CDCl ): δ = 2.38 (s, 3H, CH CO), 3.59 (s, 3H, OCH ), 6.64
sulfonate (7b) White crystals; yield: 97.4%; m.p. 130–131
3
3
3
°
1
C; IR (KBr): 3010, 2945, 1667, 1626, 1597, 1362, 1149,
114, 1086, 1032, 856 cm ; H–NMR (200 MHz, CDCl ):
3
(d, 1H, J = 16.4 Hz, CH), 6.99–7.28 (m, 3H, Ar–H), 7.42 (d,
1H, J = 16.4 Hz, CH), 7.48–7.52 (m, 2H, Ar–H), 7.79–7.84
−1 1
1
3
δ = 2.37 (s, 3H, CH CO), 2.45 (s, 3H, CH ), 3.59 (s, 3H,
(m, 2H, Ar–H); C–NMR (50 MHz, CDCl ): δ = 27.6,
3
3
3
OCH ), 6.63 (d, 1H, J = 16.2 Hz, CH), 6.99–7.09 (m, 2H,
55.5, 111.6, 121.1, 124.5, 128, 129.1, 129.9, 134.5, 134.6,
3
Ar–H), 7.17 (d, 1H, J = 8.2 Hz, Ar–H), 7.28–7.33
139.5, 140.8, 141.7, 151.8, 197.8 (CO). ESI-MS (40 eV):
m/z (%) = 366.82 (100%) [M] , 175.61 (36%).
+
(
m, 2H, Ar–H), 7.42 (d, 1H, J = 16.4 Hz, CH), 7.73–7.77
(
m, 2H, Ar–H); 13C–NMR (50 MHz, CDCl ): δ = 21.6,
3
2
1
7.5, 55.6, 111.6, 121, 124.3, 127.8, 128.5, 129.4, 133.1,
(E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl 4-nitrobenzene
sulfonate (7g) Light beige powder; yield: 83.8%; m.p.
149–151 °C; IR (KBr): 3113, 2979, 1674, 1651, 1590,
34.3, 139.8, 141.9, 145.2, 152.1, 197.9 (CO). ESI-MS
+
(
40 eV): m/z (%) = 346.40 (100%) [M] , 155.19 (31%).
−
1 1
1376, 1350, 1199, 1112, 1085, 1029, 842 cm ; H–NMR
(E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl 4-methoxybenze-
(200 MHz, CDCl ): δ = 2.38 (s, 3H, CH CO), 3.59 (s, 3H,
3
3
nesulfonate (7c) Yellowish white crystals; yield: 82.2%;
m.p. 128–129 °C; IR (KBr): 3091, 3014, 1670, 1645, 1598,
OCH ), 6.65 (d, 1H, J = 16.2 Hz, CH), 7.00–7.28 (m, 3H,
3
Ar–H), 8.10 (d, 2H, J = 8.8 Hz, Ar–H), 8.38 (d, 2H, J =
−1
1
13
1
501, 1357, 1173, 1092, 1024, 852 cm ; H–NMR (200
8.8 Hz, Ar–H), 7.42 (d, 1H, J = 16.2 Hz, CH); C–NMR
MHz, CDCl ): δ = 2.37 (s, 3H, CH CO), 2.62 (s, 3H,
(50 MHz, CDCl ): δ = 27.6, 55.6, 111.7, 121.2, 123.9,
3
3
3
OCH ), 3.89 (s, 3H, OCH ), 6.63 (d, 1H, J = 16.2 Hz, CH),
124.5, 128.3, 129.9, 135.1, 139.2, 141.5, 141.8, 150.9,
3
3
6
7
.94–7.29 (m, 5H, Ar–H), 7.42 (d, 1H, J = 16.4 Hz, CH),
151.6, 197.7 (CO). ESI-MS (40 eV): m/z (%) = 377.37
(100%) [M] , 186.17 (7%).
.77–7.82 (m, 3H, Ar–H); 13C–NMR (50 MHz, CDCl3):
+
δ = 27.5, 55.6, 55.7, 111.6, 113.9, 121.1, 124.4, 127.4, 127.8,
30.8, 134.3, 139.9, 141.9, 152.1, 164.1, 197.9 (CO). ESI-MS
1
(E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl benzenesulfo-
nate (8a) Yellowish crystals; yield: 93.1%; m.p. 80–82 °C;
IR (KBr): 2979, 2876, 1669, 1629, 1400, 1287, 1188, 1113,
+
(40 eV): m/z (%) = 362.40 (46%) [M] , 171.19 (100%).
−
1 1
(E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl 4-tert-butylbenze-
1088, 1035, 867 cm ; H–NMR (200 MHz, CDCl ): δ =
3
nesulfonate (7d) Yellowish white crystals; yield: 92%; m.
p. 92–94 °C; IR (KBr): 3079, 2964, 1689, 1609, 1507, 1370,
1.16 (t, 3H, J = 7.4 Hz, CH CH ), 2.69 (q, 2H, J = 7.4 Hz,
3
2
CH CH ), 3.56 (s, 3H, OCH ), 6.66 (d, 1H, J = 16.2 Hz,
3
2
3
−
1
1
1
154, 1109, 1087, 1028, 852 cm ; H–NMR (200 MHz,
CH), 6.97–7.28 (m, 3H, Ar–H), 7.42–7.67 (m, 4H, CH,
CDCl ): δ = 1.35 (s, 9H, (CH ) –C), 2.38 (s, 3H, CH CO),
Ar–H), 7.46 (d, 1H, J = 16.2 Hz, CH), 7.85–7.90 (m, 2H,
3
3 3
3
1
3
3
7
2
.54 (s, 3H, OCH ), 6.64 (d, 1H, J = 16.2 Hz, CH), 6.97–
Ar–H); C–NMR (50 MHz, CDCl ): δ = 8.1, 34.1, 55.6,
3
3
.28 (m, 3H, Ar–H), 7.43 (d, 1H, J = 16.4 Hz, CH), 7.53 (d,
H, J = 8.8 Hz, Ar–H), 7.79 (d, 2H, J = 8.8 Hz, Ar–H);
111.6, 120.9, 124.4, 126.8, 128.5, 128.8, 134, 134.6, 136.1,
139.6, 140.8, 151.9, 200.4 (CO). ESI-MS (40 eV): m/z
1
3
+
C–NMR (50 MHz, CDCl ): δ = 27.5, 30.9, 35.3, 55.5,
(%) = 346.40 (100%) [M] , 141.17 (24%).
3
1
1
=
11.4, 121.1, 124.6, 125.7, 127.8, 128.4, 133, 134.4, 139.8,
41.9, 152.1, 158.2, 197.9 (CO). ESI-MS (40 eV): m/z (%)
(E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl 4-methylbenzene-
sulfonate (8b) Yellowish white crystals; yield: 90.5%; m.p.
+
388.48 (100%) [M] , 197.27 (39%).
7
2–73 °C; IR (KBr): 2968, 2935, 1689, 1612, 1598, 1369,
−1
1
(E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl 4-bromobenzene-
1177, 1119, 1090, 1032, 851 cm ; H–NMR (200 MHz,
CDCl ): δ = 1.16 (t, 3H, J = 7.2 Hz, CH CH ), 2.45 (s, 3H,
sulfonate (7e) Yellowish crystals; yield: 71.9%; m.p. 123-
3
3
2
1
1
24 °C; IR (KBr): 3090, 2945, 1676, 1655, 1571, 1374,
199, 1168, 1113, 1067, 831 cm ; H–NMR (200 MHz,
CH ), 2.69 (q, 2H, J = 7.2 Hz, CH CH ), 3.60 (s, 3H, OCH ),
3 3 2 3
−
1
1
6.66 (d, 1H, J = 16.2 Hz, CH), 6.98–7.33 (m, 5H, Ar–H),
7.46 (d, 1H, J = 16.2 Hz, CH), 7.73–7.79 (m, 2H, Ar–H);
CDCl ): δ = 2.38 (s, 3H, CH CO), 3.59 (s, 3H, OCH ), 6.64
3
3
3
1
3
(
d, 1H, J = 16.2 Hz, CH), 6.99–7.28 (m, 3H, Ar–H), 7.42 (d,
C–NMR (50 MHz, CDCl ): δ = 8.1, 21.6, 34.1, 55.6, 111.7,
3
13
1
H, J = 16.4 Hz, CH), 7.64–7.76 (m, 4H, Ar–H); C–NMR
120.9, 124.4, 126.8, 128.6, 129.4, 133.2, 134.5, 139.8, 140.8,
(
50 MHz, CDCl ): δ = 27.6, 55.6, 111.6, 121.1, 124.5, 128,
29.4, 129.9, 132.1, 134.7, 135.1, 139.5, 141.7, 151.8,
97.8 (CO). ESI-MS (40 eV): m/z (%) = 411.27 (29%) [M]
145.2, 152.1, 200.5 (CO). ESI-MS (40 eV): m/z (%) = 360.42
(100%) [M] , 155.19 (44%).
3
+
1
1
+
,
220.06 (4%), 155.19 (100%).
(E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl 4-methoxybenzen-
esulfonate (8c) Yellowish white crystals; yield: 83.1%; m.p.
108–109 °C; IR (KBr): 2982, 1674, 1585, 1366, 1191, 1117,
(
E)-2-methoxy-4-(3-oxobut-1-enyl)phenyl 4-chlorobenzene-
sulfonate (7f) Yellowish powder; yield: 88%; m.p. 110–
12 °C; IR (KBr): 3091, 2985, 1667, 1641, 1586, 1372,
−1
1
1088, 1025, 833 cm ; H–NMR (200 MHz, CDCl ): δ =
3
1
1.16 (t, 3H, J = 7.2 Hz, CH CH ), 2.69 (q, 2H, J = 7.2 Hz,
3
2

Medicinal Chemistry Research
CH CH ), 3.62 (s, 3H, OCH ), 3.88 (s, 3H, OCH ), 6.66 (d,
2.69 (q, 2H, J = 7.4 Hz, CH CH ), 3.59 (s, 3H, OCH ), 6.68
3
2
3
3
3
2
3
1
H, J = 16.2 Hz, CH), 6.94–7.28 (m, 5H, Ar–H), 7.46 (d, 1H,
(d, 1H, J = 16.2 Hz, CH), 7.00–7.28 (m, 3H, Ar–H), 7.46
(d, 1H, J = 16.2 Hz, CH), 8.07–8.12 (m, 2H, Ar–H), 8.35–
13
J = 16.2 Hz, CH), 7.75–7.83 (m, 2H, Ar–H); C–NMR (50
MHz, CDCl ): δ = 8.1, 34.1, 55.6, 55.7, 111.6, 113.9, 120.9,
1
3
8.40 (m, 2H, Ar–H); C–NMR (50 MHz, CDCl ): δ = 8,
3
3
1
2
1
24.4, 126.7, 127.4, 130.8, 134.5, 139.8, 140.8, 152.1, 164,
00.4 (CO). ESI-MS (40 eV): m/z (%) = 376.42 (58%) [M] ,
71.19 (100%).
34.2, 55.6, 111.8, 121.1, 123.9, 124.4, 127.2, 129.9, 135.2,
+
139.1, 140.3, 141.8, 150.9, 151.6, 200.3 (CO). ESI-MS (40
+
eV): m/z (%) = 391.40 (100%) [M] , 186.17 (18%).
(
E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl 4-(tert-butyl)be
(E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl benze-
nesulfonate (9a) Yellow crystals; yield: 96.2%; m.p. 59–
61 °C; IR (KBr): 2952, 2869, 1658, 1618, 1598, 1369,
nzenesulfonate (8d) White crystals; yield: 78.7%; m.p.
1
1
20–121 °C; IR (KBr): 2972, 1662, 1595, 1370, 1190,
−
1
1
−1
1
110, 1087, 1030, 853 cm ; H–NMR (200 MHz, CDCl ):
1188, 1116, 1089, 1036, 841 cm ; H–NMR (200 MHz,
CDCl ): δ = 0.97 (d, 6H, J = 6.8 Hz, 2xCH ), 2.15–2.29
3
δ = 1.16 (t, 3H, J = 7.2 Hz, CH CH ), 1.35 (s, 9H, 3xCH ),
3
2
3
3
3
2
.69 (q, 2H, J = 7.4 Hz, CH CH ), 3.54 (s, 3H, OCH ), 6.67
(m, 1H, CH), 2.53 (d, 2H, J = 7.0 Hz, CH ), 3.56 (s, 3H,
3
2
3
2
(
7
d, 1H, J = 16.2 Hz, CH), 6.97–7.28 (m, 3H, Ar–H), 7.43–
.56 (m, 3H, CH, Ar–H), 7.77–7.81 (m, 2H, Ar–H); C–
OCH ), 6.65 (d, 1H, J = 16.2 Hz, CH), 6.97–7.27 (m, 3H,
3
1
3
Ar–H), 7.44 (d, 1H, J = 16.2 Hz, CH), 7.52–7.67 (m, 3H,
1
3
NMR (50 MHz, CDCl ): δ = 8.1, 30.9, 34.1, 35.3, 55.5,
Ar–H), 7.86–7.89 (m, 2H, Ar–H); C–NMR (50 MHz,
3
1
1
4
11.5, 120.9, 124.6, 125.8, 126.8, 128.4, 133, 134.5, 139.7,
CDCl ): δ = 22.6, 25.1, 49.9, 55.6, 111.6, 120.9, 124.5,
3
40.8, 152, 158.1, 200.5 (CO). ESI-MS (40 eV): m/z (%) =
128.5, 128.8, 134, 134.6, 136.1, 139.6, 140.9, 151.9, 199.9
+
+
02.49 (100%) [M] , 346.38 (14%), 197.27 (37%).
(CO). ESI-MS (40 eV): m/z (%) = 374.46 (100%) [M] ,
141.17 (36%).
(
E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl 4-bromobenzen
esulfonate (8e) White powder; yield: 84.2%; m.p. 110–
(E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl 4-methy
lbenzenesulfonate (9b) Yellowish powder; yield: 87.9%;
m.p. 96–97 °C; IR (KBr): 2958, 2873, 1687, 1612, 1598,
1
1
11 °C; IR (KBr): 2984, 1668, 1587, 1375, 1176, 1115,
085, 1027, 830 cm ; H–NMR (200 MHz, CDCl3):
−1
1
−1
1
δ = 1.16 (t, 3H, J = 7.2 Hz, CH CH ), 2.69 (q, 2H, J = 7.4
1370, 1177, 1064, 1030, 856 cm ; H–NMR (200 MHz,
3
2
Hz, CH CH ), 3.59 (s, 3H, OCH ), 6.67 (d, 1H, J = 16.2
CDCl ): δ = 0.97 (d, 6H, J = 6.8 Hz, 2xCH ), 2.16–2.29 (m,
3
2
3
3
3
Hz, CH), 6.98–7.27 (m, 3H, Ar–H), 7.46 (d, 1H, J = 16.2
1H, CH), 2.45 (s, 3H, CH ), 2.53 (d, 2H, J = 7.0 Hz, CH ),
3 2
Hz, CH), 7.64–7.76 (m, 4H, Ar–H); 13C–NMR (50 MHz,
3.60 (s, 3H, OCH ), 6.65 (d, 1H, J = 16.2 Hz, CH), 6.98–7.33
3
CDCl ): δ = 8.1, 34.2, 55.6, 111.6, 121, 124.5, 126.9,
(m, 5H, Ar–H), 7.44 (d, 1H, J = 16.2 Hz, CH), 7.73–7.78 (m,
3
13
1
29.4, 130, 132.1, 134.9, 135.1, 139.4, 140.6, 151.8, 200.4
2H, Ar–H); C–NMR (50 MHz, CDCl ): δ = 21.6, 22.6,
3
+
(
CO). ESI-MS (40 eV): m/z (%) = 425.29 (24%) [M] ,
20.06 (4%), 175.59 (33%), 155.18 (100%).
25.1, 49.9, 55.6, 111.7, 120.9, 124.4, 127.4, 128.5, 129.4,
2
133.1, 134.5, 139.8, 141, 145.2, 152.1, 199.9 (CO). ESI-MS
+
(
40 eV): m/z (%) = 388.48 (100%) [M] , 155.19 (79%).
(
E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl 4-chlorobenzen
esulfonate (8f) White powder; yield: 87.9%; m.p. 122–
(E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl 4-meth
oxybenzenesulfonate (9c) White crystals; yield: 88.5%;
m.p. 74–75 °C; IR (KBr): 2954, 2873, 1687, 1610, 1597,
1
1
24 °C; IR (KBr): 2972, 1667, 1629, 1585, 1375, 1198,
−
1 1
115, 1091, 1024, 845 cm ; H–NMR (200 MHz, CDCl ):
3
−
1
1
δ = 1.16 (t, 3H, J = 7.4 Hz, CH CH ), 2.69 (q, 2H, J = 7.2
1368, 1170, 1094, 1030, 856 cm ; H–NMR (200 MHz,
CDCl ): δ = 0.97 (d, 6H, J = 6.6 Hz, 2xCH ), 2.15–2.29
3
2
Hz, CH CH ), 3.60 (s, 3H, OCH ), 6.67 (d, 1H, J = 16.0
3
2
3
3
3
Hz, CH), 6.99–7.27 (m, 3H, Ar–H), 7.46 (d, 1H, J = 16.2
Hz, CH), 7.42–7.52 (m, 2H, Ar–H), 7.78–7.84 (m, 2H, Ar–
(m, 1H, CH), 2.53 (d, 2H, J = 6.8 Hz, CH ), 3.62 (s, 3H,
2
OCH ), 3.88 (s, 3H, OCH ), 6.65 (d, 1H, J = 16.2 Hz, CH),
3
3
1
3
H); C–NMR (50 MHz, CDCl ): δ = 8.1, 34.2, 55.6, 111.6,
6.94–7.27 (m, 5H, Ar–H), 7.44 (d, 1H, J = 16.2 Hz, CH),
3
1
3
1
1
3
21, 124.5, 126.9, 129.1, 129.9, 134.6, 134.9, 139.4, 140.6,
7.77–7.82 (m, 2H, Ar–H); C–NMR (50 MHz, CDCl ): δ
3
40.8, 151.8, 200.4 (CO). ESI-MS (40 eV): m/z (%) =
= 21.6, 22.6, 25.1, 49.9, 55.6, 111.7, 120.9, 124.4, 127.4,
+
80.84 (90%) [M] , 175.61 (100%).
128.5, 129.4, 133.1, 134.5, 139.8, 141, 145.2, 152.1, 199.9
+
(
CO). ESI-MS (40 eV): m/z (%) = 404.48 (44%) [M] ,
(
E)-2-methoxy-4-(3-oxopent-1-enyl)phenyl 4-nitrobenzene
171.19 (100%).
sulfonate (8g) Light beige powder; yield: 85.7%; m.p.
1
1
40–141 °C; IR (KBr): 2978, 2877, 1662, 1589, 1536,
379, 1199, 1188, 1111, 1089, 1024, 848 cm ; H–NMR
(E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl 4-tert-
butylbenzenesulfonate (9d) Yellowish crystals; yield:
86.9%; m.p. 86–87 °C; IR (KBr): 2957, 2900, 1658, 1628,
−
1 1
(
200 MHz, CDCl ): δ = 1.16 (t, 3H, J = 7.2 Hz, CH CH ),
3 3 2

Medicinal Chemistry Research
−
1 1
1
596, 1370, 1179, 1111, 1087, 1032, 859 cm ; H–NMR
Cytotoxicity
(
200 MHz, CDCl ): δ = 0.97 (d, 6H, J = 6.6 Hz, 2xCH ),
3
3
1
.35 (s, 9H, 3xCH ), 2.16–2.29 (m, 1H, CH), 2.53 (d, 2H,
Cell cultures
3
J = 7.0 Hz, CH ), 3.54 (s, 3H, OCH ), 6.66 (d, 1H, J =
2
3
1
1
6.2 Hz, CH), 6.97–7.28 (m, 3H, Ar–H), 7.45 (d, 1H, J =
6.2 Hz, CH), 7.50–7.55 (m, 2H, Ar–H), 7.77–7.81 (m, 2H,
Epithelial cervical carcinoma (HeLa), breast carcinoma
(MCF-7), and the normal human fibroblast (MRC-5) cell
lines were obtained from the American Type Culture Col-
lection (Rockville, MD, USA). The cells were maintained in
high glucose Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% heat-inactivated fetal
bovine serum (all from Sigma, Germany). Antibiotics were
omitted from the medium to avoid undesired interactions.
The cells were cultivated at 37 °C in absolute humidity in an
1
3
Ar–H); C–NMR (50 MHz, CDCl ): δ = 22.6, 25.1, 30.9,
3
3
5.3, 49.9, 55.5, 111.5, 120.9, 124.6, 125.8, 127.3, 128.4,
1
33, 134.5, 139.7, 141, 152, 158.1, 199.9 (CO). ESI-MS
+
(
40 eV): m/z (%) = 430.56 (100%) [M] , 197.27 (96%).
(
E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl 4-brom
obenzenesulfonate (9e) Yellowish powder; yield: 83.9%;
m.p. 114–115 °C; IR (KBr): 2952, 2868, 1659, 1598, 1586,
atmosphere containing 5% CO and were subcultured two
2
−
1 1
1
377, 1187, 1115, 1086, 1033, 839 cm ; H–NMR (200
to three times a week.
MHz, CDCl ): δ = 0.98 (d, 6H, J = 6.6 Hz, 2xCH ), 2.16–
3
3
2
3
3
4
5
1
.29 (m, 1H, CH), 2.53 (d, 2H, J = 7.0 Hz, CH ), 3.59 (s,
Preparation of sulfonyl ester and control solutions
2
H, OCH ), 6.66 (d, 1H, J = 16.2 Hz, CH), 6.98–7.27 (m,
3
H, Ar–H), 7.44 (d, 1H, J = 16.2 Hz, CH), 7.64–7.76 (m,
All compounds examined for cytotoxicity, including cis-
platin (used as a control), were dissolved in dimethyl sulf-
oxide (DMSO; Fisher Scientific, UK) at a concentration of
20 mM, filtered through 0.22 mm Millipore filters and
diluted appropriately with DMEM to working concentra-
tions of 0.3, 1, 3, 10, 30, and 100 μM. The final con-
centration of DMSO in DMEM never exceeded 0.5% (vol/
vol). All solutions were freshly prepared on the day of cell
line treatment.
1
3
H, Ar–H); C–NMR (50 MHz, CDCl ): δ = 22.6, 25.1,
3
0, 55.6, 111.7, 121.1, 124.5, 127.6, 129.4, 130, 132.1,
34.9, 135.1, 139.4, 140.8, 151.8, 199.8 (CO). ESI-MS (40
+
eV): m/z (%) = 453.35 (21%) [M] , 220.07 (6%), 155.18
(
100%).
(
E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl 4-chlor
obenzenesulfonate (9f) Yellowish crystals; yield:
9
1
0.9%; m.p. 115-117 °C; IR (KBr): 2956, 2871, 1659,
621, 1585, 1377, 1187, 1116, 1090, 1033, 844 cm ;
−
1
MTT assay
1
H–NMR (200 MHz, CDCl ): δ = 0.98 (d, 6H, J = 6.6 Hz,
3
2
xCH ), 2.16–2.29 (m, 1H, CH), 2.53 (d, 2H, J = 7.0 Hz,
The
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
3
CH ), 3.59 (s, 3H, OCH ), 6.66 (d, 1H, J = 16.2 Hz, CH),
bromide (MTT) assay was used to determine the cytotoxicity
of the compounds as previously described (Zaric et al.
2015). The cells were harvested from the culture flasks
during the exponential growth phase, counted and 5 × 103
cells per well were seeded into 96-well culture plates. The
cells were allowed to adhere overnight in a humidified
2
3
6
7
.99–7.27 (m, 3H, Ar–H), 7.40–7.52 (m, 3H, CH, Ar–H),
.79–7.84 (m, 2H, Ar–H); 13C–NMR (50 MHz, CDCl3):
δ = 22.6, 25.1, 50, 55.6, 111.7, 121, 124.5, 127.6, 129.1,
29.9, 134.6, 134.9, 139.4, 140.7, 140.8, 151.8, 199.8
1
+
(
CO). ESI-MS (40 eV): m/z (%) = 408.9 (59%) [M] ,
75.61 (100%).
1
incubator with 5% CO . Afterwards, the supernatants were
2
removed and the remaining cell monolayers were treated
with 200 µL volumes of dilutions of the investigated com-
pounds in fresh DMEM, as described above. Fresh DMEM
was used as a control. A positive control containing cisplatin
was used as reference drug for comparison. Controls wells
were treated the same as test wells. All cells were incubated
(
E)-2-methoxy-4-(5-methyl-3-oxohex-1-enyl)phenyl 4-nitrob
enzenesulfonate (9g) Beige powder; yield: 80.5%; m.p.
1
1
38–139 °C; IR (KBr): 2962, 2870, 1664, 1540, 1378, 1186,
−1 1
113, 1089, 1025, 848 cm ; H–NMR (200 MHz, CDCl ):
3
δ = 0.97 (d, 6H, J = 6.8 Hz, 2xCH ), 2.16–2.29 (m, 1H,
3
CH), 2.53 (d, 2H, J = 7.0 Hz, CH ), 3.59 (s, 3H, OCH ),
at 37 °C in an atmosphere of 5% CO and absolute humidity
2
3
2
6
7
8
.67 (d, 1H, J = 16.2 Hz, CH), 7.00–7.28 (m, 3H, Ar–H),
.44 (d, 1H, J = 16.2 Hz, CH), 8.08–8.12 (m, 2H, Ar–H),
.36–8.40 (m, 2H, Ar–H); 13C–NMR (50 MHz, CDCl3):
for 24 and 48 h. Then, the cell culture media (with the
investigated compounds) was removed, and 100 µL of MTT
(0.5 mg/mL) was added to each well. After 2 h incubation at
37 °C, MTT solution was removed, and 150 µL of DMSO
was added to dissolve the formazan crystals. Absorbance
was measured with a multiplate reader (Zenyth 3100,
Anthos Labtec Instruments GmbH, Austria) at 595 nm.
δ = 22.5, 25, 48.9, 55.5, 111.7, 121.1, 123.8, 124.4, 127.7,
29.8, 135.2, 139, 140.4, 141.8, 150.8, 151.5, 199.6 (CO).
1
+
ESI-MS (40 eV): m/z (%) = 419.45 (56%) [M] , 186.17
47%).
(

Medicinal Chemistry Research
All compounds were examined in triplicate and repeated
in three independent series. The percentage of cytotoxicity
was calculated using the formula: ((Acontrol − Atest)/Acon-
trol)) × 100, where A is the absorbance determined for
untreated (control) and treated (test) cells. The selectivity
indexes (SI) for all examined compounds after 48 h
screening were calculated using the formula: SI = (%
cytotoxicity HeLa/% cytotoxicity MRC-5) × % cytotoxicity
HeLa. Also, using Microsoft Office Excel 2010, 50%
inhibitory concentration (IC ), i.e., the concentration
flow cytometer, Cytomics FC500 (Beckman Coulter, USA),
and the percentages of viable, apoptotic, and necrotic cells
were evaluated using Flowing Software (http://www.flow
ingsoftware.com/). The results were presented as dot plots.
Cell cycle analysis
HeLa and MCF-7 cells were treated with the examined
compounds in concentrations corresponding to their IC50
values for 48 h at 37 °C in an atmosphere of 5% CO and
50
2
required to cause toxic effects in 50% of intact cells, was
calculated. Selectivity index (SI) values to identify selective
anticancer cell activity were calculated by dividing the IC50
values of normal MRC-5 cells by the IC50 of HeLa cells
absolute humidity. The control was DMEM alone, treated
the same as the examined compounds. After incubation,
cells were collected into tubes, washed in PBS, and finally
suspended in 1 mL of ice-cold 70% ethanol. After overnight
incubation at 4 °C, cells were washed in PBS and treated
with RNase A (500 μg/mL PBS) for 30 min at 37 °C. Pro-
pidium iodide (5 μL of 10 mg/mL PBS) was added to each
tube, and after 15 min’ incubation in the dark, cells were
assayed using a flow cytometer Cytomics FC500. The data
were analyzed using FlowJo Software and the results were
presented as histograms.
(
Joksimović et al. 2016).
Flow cytometric analysis
The Annexin V-FITC/7-AAD kit (Beckman Coulter, USA)
was used according to the manufacturer’s instructions to
determine the type of cell death induced by the examined
compounds. Briefly, HeLa and MCF-7 cells were treated
with the examined compounds in concentrations corre-
sponding to their IC50 values for 48 h at 37 °C in an
Results and discussion
atmosphere of 5% CO and absolute humidity. The control
2
was DMEM alone, treated the same as the examined
compounds. Then, cells were collected, washed in phos-
phate buffered saline (PBS), and finally suspended in ice-
Chemical synthesis
In continuation of our work on the synthesis of chalcone
analogs, we decided to synthesize some new compounds
with various sulfonyl groups and vanillin’ fragments
incorporated in the same molecule, starting from the easily
accessible, natural product, vanillin. In the first step, using
Claisen–Schmidt condensation, vanillin (1) was condensed
with three different non-aromatic methyl ketones (acetone
5
cold binding buffer (1 × 10 cells/100 μL binding buffer).
Cells were stained with 10 μL of Annexin V-FITC and 20
μL 7-AAD, and after 15 min’ incubation in the dark, 400 μL
of binding buffer was added. Cells were analyzed using a
(
2a), 4-methyl-2-pentanone (2b), and 2-butanone (2c)).
These condensation reactions produced chalcone analogs 3,
, and 5, according to Scheme 1. Chalcone analog 3 was
synthesized following a previously published procedure
Smith 1996), whereas chalcone analogs 4 and 5 were
synthesized following a different method (Muškinja et al.
016).
4
(
2
Scheme 1 Synthesis of chalcone analogs 3, 4, and 5 from vanillin 1
Scheme 2 Synthesis of sulfonyl
esters 7, 8, and 9 in reactions of
chalcone analogs with sulfonyl
chlorides 6a–g

Medicinal Chemistry Research
Table 1 Synthesized sulfonyl esters

Medicinal Chemistry Research
Synthesized chalcone analogs were used as substrates for
further transformations. In reactions of their free phenolic
groups with sulfonyl chlorides in ethanolic solution, trie-
thylamine was used as a base, and corresponding sulfonyl
esters 7a–g, 8a–g, and 9a–g were obtained (Scheme 2).
These reactions produced very high yields of sulfonyl esters
using the MTT assay (Fig. 2). Sulfonyl esters 8c and 9b–e
displayed very low cytotoxic activity against HeLa cells.
These compounds (at concentrations of 100 µM) were toxic
to <70% of cancer cells after 48 h, and therefore, they were
not eligible candidates for further investigation. All other
examined compounds (100 µM) had very high cytotoxic
activity, i.e., up to 96%. Additionally, we screened the
cytotoxic activity of the sulfonyl esters against non-
cancerous MRC-5 cells, and calculated the selectivity
index (SI), in order to select the most effective sulfonyl
esters that, at the same time, had activity against cancer cells
but were the least damaging to non-cancerous cells.
Among compounds 7a–g, sulfonyl ester 7g had the
highest SI (Table S1, supplementary materials). Also,
among the other two groups of sulfonyl esters, compounds
8b, 8e, 8d, 9a, and 9g displayed very high selective cyto-
toxicity against cancer cells as demonstrated by their SI
values. Therefore, we decided to perform a more detailed
analysis of the cytotoxic activity of these six compounds
and to calculate their IC50 values.
(
Table 1), and the products were characterized by their
1
13
spectral data (IR, H–NMR and C–NMR). The aim of this
study was to obtain compounds which had enone systems
and sulfonyl groups, as these groups are mostly responsible
for cytotoxic activity against different cell lines.
Cytotoxic activities
Sulfonyl esters 7g, 8b, 8e, 9a, and 9g demonstrate high
cytotoxicity against tumor cell lines
The cytotoxic activities of the 21 examined sulfonyl esters
from three groups (7a–g, 8a–g, and 9a–g) and cisplatin
(
each at 100 µM concentration) against HeLa cancer cells
and MRC-5 non-cancerous cells were screened after 48 h
All six examined sulfonyl esters (7g, 8b, 8e, 8d, 9a and
9g) showed dose-dependent cytotoxic activity toward can-
cer cells after 24 and 48 h (the results of MTT assay after 24
and 48 h are shown in Fig. S1, supplementary materials).
Their IC50 values against cancer cell lines (HeLa and MCF-
7) and normal human fibroblast MRC-5 cells are shown in
Table 2. All compounds showed high cytotoxic activity
against cancer cells lines and moderate to low or no cyto-
toxicity against non-cancerous cells.
Compound 7g displayed very low selectivity and high
cytotoxic activity against both normal and cancer cells.
Compound 8e had the highest cytotoxicity and was four
times more selective towards MCF-7 cancer cells than cis-
platin, 8b and 8d, with an IC50 value of 12.5 ± 1.3 µM and
SI of 8. Also, compound 9a was the most cytotoxic and the
most selective of all the examined compounds against HeLa
cells, with an IC50 value of 11.3 ± 0.8 µM and SI of 8.9 after
Fig. 2 Cytotoxicity of 21 sulfonyl esters and cisplatin at 100 µM
concentrations against HeLa and MRC-5 cells after 48 h. The data
show the percentage of cytotoxicity and are means of three indepen-
dent replicates
a
Table 2 Cytotoxicity (IC50 in
µM) of six sulfonyl esters and
Compound HeLa
MCF-7
MRC-5
cisplatin against HeLa, MCF-7,
and MRC-5 cells and selectivity
index (SI) values
IC50 (24
h)
IC50 (48
h)
SI (48
h)
IC50 (24
h)
IC50 (48
h)
SI (48
h)
IC50 (24
h)
IC50 (48
h)
b
7
8
8
8
9
9
g
b
d
e
58.8 ± 4.2 11.6 ± 0.9 1.6
13.8 ± 0.6 14.5 ± 1.3 3.9
44.7 ± 2.7 18.4 ± 0.9
1
>100
18.7 ± 2.1
84.4 ± 5.5 30.6 ± 1.9 1.9
91.1 ± 7.1 21.5 ± 1.6 4.7
71.4 ± 5.2 57.1 ± 3.3
43.9 ± 2.7 14 ± 1.1
7.1
>100
>100
>100
>100
>100
>100
98.7 ± 6.3 56.9 ± 4.2 1.7
13.5 ± 0.9 11.3 ± 0.8 8.9
>100
>100
>100
12.5 ± 1.3
8
>100
a
18.1 ± 1.2 5.5
41.5 ± 2.7 2.4
>100
g
>100
38.6 ± 4.2 2.6
4.9
>100
Cisplatinc 25.7 ± 1.8 8.7 ± 1
94.1 ± 8.3 24.5 ± 2.2 1.7
43.1 ± 3.8
aIC50 is defined as the concentration that causes a 50% cell proliferation inhibition
bThe data represent the mean ± SD of three independent replicates
cUsed as a positive control

Medicinal Chemistry Research
Fig. 3 Flow cytometric analysis of Annexin V-FITC/7-AAD staining.
Graphs show percentages of early apoptotic (EA), late apoptotic (LA),
and necrotic cells (N) in untreated (control) and treated HeLa a and
MCF-7 b cells. Results are means ± SD of three independent repli-
cates. Dot plots (HeLa c and MCF-7 d) are representative of two
independent analyses
4
8 h. Furthermore, compounds 7g, 8e, and 9a showed
IC50 value of 43.1 ± 3.8 µM. In contrast to cisplatin, com-
pounds 8d, 8e, 9a, and 9g had no cytotoxic activity against
normal MRC-5 cells with IC50 values > 100 µM. Therefore,
even though these sulfonyl esters, particularly 8d and 9a,
higher cytotoxic activity against MCF-7 cells than did cis-
platin under the same conditions. Also, cisplatin displayed
moderate cytotoxicity against normal MRC-5 cells, with an

Medicinal Chemistry Research
Fig. 4 Cell cycle analysis. Graphs show cell cycle distribution in untreated (control) and treated HeLa a and MCF-7 b cells. Results are the average
of three independent replicates. Histograms (HeLa c and MCF-7 d) are representative of three independent analyses
had higher IC50 values against HeLa cells than cisplatin,
they might have promising roles in the treatment of human
epithelial cervical carcinoma considering their highly
selective cytotoxicity toward cancer cells only.
Our synthesized sulfonyl esters were more potent antic-
ancer agents than similar sulfonamide derivatives described
previously (Ghorab et al. 2015). Esters 7g, 8b, 8d, and 9a
showed high cytotoxic activity toward cancer HeLa cells

Medicinal Chemistry Research
with IC50 values < 15 µM, and esters 7g, 8e, and 9a also
showed high anticancer activity toward MCF-7 cancer cells
with IC50 values < 19 µM. Also, these sulfonyl esters had no
cytotoxic activity toward normal MRC-5 cells. Therefore,
the examined sulfonyl esters synthesized in the current
study might be better candidates for future anticancer
research compared to sulfonamide compounds researched
earlier.
be removed, and so the apoptotic program is activated.
Conversely, in cancer cells, altered regulatory molecules
allow cells to proceed through the cell cycle and proliferate,
despite cellular damage. Altered apoptosis and dysfunc-
tional cell cycle regulation give cancer cells a survival
advantage, irrespective of intended lifespan, absence of
survival factors or presence of injury.
The results of our study showed that all five examined
sulfonyl esters induced perturbance in the cell cycles of
both cancer cell lines. Given that the selected cell lines have
different characteristics and bear mutations in distinctive
DNA regions, it can be presumed that different substances
have differing effects on the two cancer cell lines. In HeLa
cells, sulfonyl esters 7g, 8b, and 9a arrested cells in the G2/
M phase, while treatment with 8e and 9g resulted in
increased percentages of cells in the S phase of the cell
cycle. In MCF-7 cells, sulfonyl esters 7g, 8e, and 9a
induced G2/M arrest, whereas esters 8b and 9g induced
higher percentages of cells in both G0/G1 and G2/M pha-
ses, indicating impediment of DNA synthesis and cell
division (Fig. 4). Altogether, experimental results showed
that all examined sulfonyl esters were both cytotoxic and
cytostatic towards HeLa and MCF-7 cell lines, and there-
fore, these novel compounds should be seriously considered
as potential anticancer agents.
Benzofuran-substituted chalcone derivates (Coskun et al.
2
017) and our newly synthesized chalcone derivates 7g, 8b,
8
e, 8d, 9a, and 9g had similar anticancer activities against
MCF-7 cancer cells. The cytotoxic activity of benzofuran-
substituted chalcone against normal cells has not been
reported, to our knowledge. On the other hand, compounds
8
d, 8e, 9a, and 9g had no cytotoxic activity against normal
MRC-5 cells.
Based on the results obtained, showing compounds 7g,
8
b, 8e, 9a, and 9g had high cytotoxicity and the best
selectivity (Table 2), we examined the compounds’ effects
with regard to the predominant type of cell death they
caused and to the cell cycle progression of HeLa and MCF-
7
cells.
Sulfonyl esters 7g, 8b, 8e, 9a, and 9g induce apoptosis and
cell cycle arrest in HeLa and MCF-7 cells
Flow cytometric analysis showed the five examined sulfo-
nyl esters, at concentrations corresponding to their IC50
values, induced apoptosis in both HeLa and MCF-7 cells
Conclusion
(
Fig. 3). The majority of nonviable cells were early or late
Three chalcone analogs, starting from the natural product,
vanillin, were prepared. The free phenolic groups in these
compounds were modified by simple synthesis procedures,
giving very high yields of sulfonyl esters. These esters show
high in vitro anticancer activity, inducing apoptosis in
human epithelial carcinoma HeLa cells and breast carci-
noma MCF-7 cell lines. Importantly, these new sulfonyl
esters show low or no cytotoxicity against a human fetal
lung fibroblast cell line (MRC-5). Also, the examined
compounds exert cytostatic action, inducing cell cycle arrest
in HeLa and MCF-7 cells. The novel sulfonyl esters can be
considered as a significant starting point for the develop-
ment of new anticancer drugs.
apoptotic, whereas only minor percentages of cells were
necrotic. While cell death due to necrosis induces the
inflammatory response and, therefore, injury of surrounding
tissue, apoptosis is limited to the dying cell and cause no
damage to the neighboring cells. Hence, induction of
apoptosis in cancer cells is the main objective of anticancer
therapy. However, in cancer cells, the apoptotic machinery
is deregulated. Alterations of both extrinsic and intrinsic
apoptosis pathways and disturbance of the ratio of proa-
poptotic and antiapoptotic molecules facilitate survival of
cancer cells and tumor formation and progression (Pistritto
et al. 2016).
Therefore, the efficacy of antitumor therapy depends on
the ability of the therapeutic agent to induce apoptosis.
Except for the direct cytotoxic action of antitumor drugs,
their cytostatic effects are also advantageous. It should be
emphasized that in cancer cells, mechanisms that regulate
cell cycle progression are often dysfunctional (Shapiro and
Harper 1999). In normal cells, defects emerging from
extracellular or intracellular insults result in arrest of cell
cycle progression, giving repair mechanisms time to fix the
defect. If the damage is irreparable, the damaged cell must
Acknowledgements The authors are grateful to the Ministry of Edu-
cation, Science and Technological Development of the Republic of
Serbia (Grant 172034) for financial support.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

Medicinal Chemistry Research
References
Li R, Kenyon GL, Cohen FE, Chen X, Gong B, Dominguez JN,
Davidson E, Kurzban G, Miller RE, Nuzum EO, Rosenthal PJ,
McKerrow JH (1995) In vitro antimalarial activity of chalcones
and their derivatives. J Med Chem 38:5031–5037
Liu M, Wilairat P, Go ML (2001) Antimalarial alkoxylated and
hydroxylated chalcones: structure-activity relationship analysis. J
Med Chem 44:4443–4452
Alcaradz LE, Blanco SE, Puig ON, Tomads F, Ferretti FH (2000)
Antibacterial activity of flavonoids against methicillin-resistant
Staphylococcus aureus strains. J Theor Biol 205:231–240
Anto RJ, Sukumaran K, Kuttan G, Rao MNA, Subbaraju V, Kuttan R
(
1995) Anticancer and antioxidant activity of synthetic chalcones
Loewenthal HJE (1959) Selective reactions and modification of
functional groups in steroid chemistry. Tetrahedron 6:269–303
Mahapatra DM, Bharti SK, Asati V (2015) Anti-cancer chalcones:
structural and molecular target perspectives. Eur J Med Chem
and related compounds. Cancer Lett 97:33–37
Batovska D, Parushev S, Stamboliyska B, Tsvetkova I, Ninova M,
Najdenski H (2009) Examination of growth inhibitory properties
of synthetic chalcones for which antibacterial activity was pre-
dicted. Eur J Med Chem 44:2211–2218
9
8:69–114
Motohashi N, Yamagami C, Tokuda H, Konoshima T, Okuda Y,
Okuda M, Mukainaka T, Nishino H, Saito Y (1998) Inhibitory
effects of dehydrozingerone and related compounds on 12-O-
tetradecanoylphorbol-13-acetate induced Epstein-Barr virus early
antigen activation. Cancer Lett 134:37–42
Muškinja J, Ratković Z, Ranković B, Kosanić M (2016) Synthesis of
o-alkyl derivatives of dehydrozingeronean alogues Kragujevac J
Sci 38:97–106
Narender T, Tanvir K, Shweta N, Goyal N, Gupta S (2005) Synthesis
of chromenochalcones and evaluation of their in vitro antil-
eishmanial activity. Bioorg Med Chem 13:6543–6550
Nielsen SF, Boesen T, Larsen M, Schønning K, Kromann H (2004)
Antibacterial chalcones-bioisosteric replacement of the
Betts LM, Tam NC, Kabir SMH, Langler RF, Crandall I (2006) Ether
aryl sulfonic acid esters with improved antimalarial/anticancer
activities. Aust J Chem 59:277–282
Chen M, Christensen SB, Blom J, Lemmich E, Nadelmann L, Fich K,
Theander TG, Kharazmi A (1993) Licochalcone A, a novel
antiparasitic agent with potent activity against human pathogenic
protozoan species of Leishmania. Antimicrob Agent Chemother
3
7:2550–2556
Chen M, Christensen SB, Theader TG, Kharazmi A (1994) Antil-
eishmanial activity of licochalcone A in mice infected with
Leishmania major and in hamsters infected with Leishmania
donovani. Antimicrob Agent Chemother 38:1339–1344
Christensen ND, Reed CA, Culp TD, Hermonat PL, Howett MK,
Anderson RA, Zaneveld LJ (2001) Papillomavirus microbicidal
activities of high molecular weight cellulose sulphate, dextrane
sulphate and polystyrene sulfonate. Antimicrob Agents Che-
mother 45:3427–3432
4
′-hydroxy group. Bioorg Med Chem 12:3047–3054
Nielsen SF, Chen M, Theander TG, Kharazmi A, Christensen SB
1995) Synthesis of antiparasitic licorice chalcones. Bioorg Med
(
Chem Lett 5:449–452
Nielsen SF, Kharazmi A, Christensen SB (1998) Modifications of the
alpha,beta-double bond in chalcones only marginally affect the
antiprotozoal activities. Bioorg Med Chem 6:937–945
Park JY, Jeong HJ, Kim YM, Park SJ, Rho MC, Park KH, Ryu YB,
Lee WS (2011) Characteristic of alkylated chalcones from
Angelica keiskei on influenza virus neuraminidase inhibition.
Bioorg Med Chem Lett 21:5602–5604
Phrutivorapongkul A, Lipipun V, Ruangrungsi N, Kirtikara K, Nishi-
kawa K, Maruyama S, Watanabe T, Ishikawa T (2003) Studies on
the chemical constituents of stem bark of Millettia leucantha: Iso-
lation of new chalcones with cytotoxic, anti-herpes simplex virus
and anti-inflammatory activities. Chem Pharm Bull 51:187–190
Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D’Orazi G (2016) Apop-
tosis as anticancer mechanism: function and dysfunction of its
modulators and targeted therapeutic strategies. Aging 8:603–619
Rajasekar S, Park DJ, Park C, Park S, Park YH, Kim ST, Choi YH,
Choi YW (2012) In vitro and in vivo anticancer effects of
Lithospermum erythrorhizon extract on B16F10 murine mela-
noma. J Ethnopharmacol 144:335–345
Coskun D, Erkisa M, Ulukaya E, Coskun MF, Ari F (2017) Novel 1-
(
7-ethoxy-1-benzofuran-2-yl) substituted chalcone derivatives:
synthesis, characterization and anticancer activity. Eur J Med
Chem 136:212–222
Cyr L, Langler R, Lavigne C (2008) Cell cycle arrest and apoptosis
responses of human breast epithelial cells to the synthetic orga-
nosulfur compound p-methoxyphenyl p-toluenesulfonate. Antic-
ancer Res 28:2753–2764
ElSohly HN, Joshi AS, Nimrod AC, Walker LA, Clark AM (2001)
Antifungal chalcones from Maclura tinctoria. Planta Med 67:87–89
Gafner S, Wolfender JL, Mavi S, Hostettmann K (1996) Antifungal and
antibacterial chalcones from Myrica serrata. Planta Med 62:67–69
Ghorab MM, Ragab FA, Heiba HI, El-Gazzar MG, Zahran SS (2015)
Synthesis, anticancer and radiosensitizing evaluation of some
novel sulfonamide derivatives. Eur J Med Chem 92:682–692
Hanna MA, Girges MM, Berghot MA (1991) Sulfonate ester con-
taining (imidazol-1-yl) N-substituted benzenesulfonamides of
anticipated antineoplastic activity. Phosphorus Sulfur Silicon
Relat Elem 61:239–246
Rusconi S, Moonis M, Merrill DP, Pallai PV, Neidhardt EA, Singh
SK, Osburne MS, Profy AT, Jenson JC, Hirsch MS (1996)
Naphthalene sulfonate polymers with CD-4 blocking and anti-
human immunodeficiency virus type 1 activities. Antimicrob
Agents Chemother 40:234–236
Hans RH, Guantai EM, Lategan C, Smith PJ, Wan B, Franzblau SG,
Gut J, Rosenthal PJ, Chibale K (2010) Synthesis, antimalarial and
antitubercular activity of acetylenic chalcones. Bioorg Med Chem
Lett 20:942–944
Joksimović N, Baskić D, Popović S, Zarić M, Kosanić M, Ranković B,
Stanojković T, Novaković SB, Davidović G, Bugarčić Z, Janković
N (2016) Synthesis, characterization, biological activity, DNA and
BSA binding study: novel copper (II) complexes with 2-hydroxy-
Shapiro GI, Harper JW (1999) Anticancer drug targets: cell cycle and
checkpoint control. J Clin Invest 15:1645–1653
Sivakumar PM, Geetha Babu SM, Mukesh D (2007) QSAR studies on
chalcones and flavonoids as anti-tuberculosis agents using genetic
function approximation (GFA) method. Chem Pharm Bull 55:44–49
Sivakumar PM, Prabhakar PK, Doble M (2011) Synthesis, antioxidant
evaluation, and quantitative structure-activity relationship studies
of chalcones. Med Chem Res 20:482–492
Smith LR (1996) Rheosmin (“raspberry ketone”) and zingerone, and
their preparation by crossed aldol-catalytic hydrogenation
sequences. Chem Educ 1:1–18
4
-aryl-4-oxo-2-butenoate. Dalton Trans 45:15067–15077
Kinghorn A, Farnsworth N, Soejarto D, Cordell G, Swanson S, Pez-
zuto J, Wani M, Wall M, Oberlies N, Kroll D, Kramer R, Rose
W, Vite G, Fairchild C, Peterson R, Wild R (2003) Novel stra-
tegies for the discovery of plant derived anticancer agents. Pharm
Biol 41:53–67
Lahtchev KL, Batovska DI, Parushev SP, Ubiyvovk VM, Sibirny AA
(
2008) Antifungal activity of chalcones: a mechanistic study
Trivedi JC, Bariwal JB, Upadhyay KD, Naliapara YT, Joshi SK,
Pannecouque CC, Clercq ED, Shah AK (2007) Improved and
using various yeast strains. Eur J Med Chem 43:2220–2228

Medicinal Chemistry Research
rapid synthesis of new coumarinyl chalcone derivatives and their
antiviral activity. Tetrahedron Lett 48:8472–8474
Tseng CH, Tzeng CC, Hsu CY, Cheng CM, Yang CN, Chen YL
Yadav HL, Gupta P, Pawar PS, Singour PK, Patil UK (2010) Synth-
esis and biological evaluation of anti-inflammatory activity of
1,3-diphenyl propenone derivatives. Med Chem Res 19:1–8
Zaric M, Mitrovic M, Nikolic I, Baskic D, Popovic S, Djurdjevic P,
Milosavljevic Z, Zelen I (2015) Chrysin induces apoptosis in
peripheral blood lymphocytes isolated from human chronic lym-
phocytic leukemia. Anti-Cancer Agents Med Chem 15:189–195
Zhang XW, Zhao DH, Quan YC, Sun LP, Yin XM, Guan LP (2010)
Synthesis and evaluation of anti-inflammatory activity of sub-
stituted chalcone derivatives. Med Chem Res 19:403–412
(
2015) Discovery of 3-phenylquinolinyl chalcone derivatives as
potent and selective anticancer agents against breast cancers. Eur
J Med Chem 97:306–319
Vasil’ev RF, Kancheva VD, Fedorova GF, Batovska DI, Trofimov AV
(
2010) Antioxidant activity of chalcones: the chemiluminescence
determination of the reactivity and the quantum chemical calcu-
lation of the energies and structures of reagents and intermediates.
Kinet Catal 51:507–515
Zsoldos-Mady V, Csampai A, Szabo R, Meszaros-Alapi E, Pasztor J,
Hudecz F, Sohar P (2006) Synthesis, structure, and in vitro anti-
tumor activity of some glycoside derivatives of ferrocenyl‐chal-
cones and ferrocenyl‐pyrazolines. Chem Med Chem 1:1119–1125
Vogel S, Ohmayer S, Brunner G, Heilmann J (2008) Natural and non-
natural prenylated chalcones: synthesis, cytotoxicity and anti-
oxidative activity. Bioorg Med Chem 16:4286–4293