Synthesis and structure elucidation of 1-(2,5/3,5-difluorophenyl)-3-(2,3/2,4/2,5/3,4-dimethoxyphenyl)-2-propen-1-ones as anticancer agents

Article abstract of DOI:10.1007/s00044-017-1911-0

The compounds titled 1-(2,5/3,5-difluorophenyl)-3-(2,3/2,4/2,5/3,4-dimethoxyphenyl)-2-propen-1-ones (1–8) were synthesized via Claisen-Schmidt condensation under basic condition. The chemical structure of the compounds were identified using several spectroscopic techniques such as ¹H nuclear magnetic resonance (NMR), ¹³C NMR, ¹⁹F NMR, DEPT 90, DEPT 135, COSY, HMBC, and HMQC. Cytotoxic activities of the compounds were investigated towards several human tumour cell lines [gingival carcinoma (Ca9-22), oral squamous cell carcinoma derived from tongue (HSC-2)] and human normal oral cells [gingival fibroblasts (HGF), periodontal ligament fibroblasts?(HPLF)]. Most of these compounds presented higher cytotoxicity than reference drug 5-fluorouracil while the compounds 7, [1-(3,5-difluorophenyl)-3-(2,5-dimethoxyphenyl)-2-propen-1-one)], and 2, [1-(2,5-difluorophenyl)-3-(2,4-dimethoxyphenyl)-2-propen-1-one], were presenting the best activity according to potency selectivity expression values. Type of cell death induced by compound 7 in both HSC-2 and Ca9-22 cells was investigated to understand mechanism of action of the compounds. The compound 7 produced cleaved products of PARP and caspase-3 were produced, suggesting the induction of apoptosis as a possible mechanism of action of the compounds characterized via activation of caspase-3 in both human oral squamous cell carcinomas.

Full text of DOI:10.1007/s00044-017-1911-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2017) 26:2015–2023
DOI 10.1007/s00044-017-1911-0
ORIGINAL RESEARCH
Synthesis and structure elucidation of 1-(2,5/3,5-difluorophenyl)-3-
(2,3/2,4/2,5/3,4-dimethoxyphenyl)-2-propen-1-ones as anticancer
agents
Cem Yamali1 Dilan Ozmen Ozgun2 Halise Inci Gul1 Hiroshi Sakagami3
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Cavit Kazaz4 Noriyuki Okudaira3
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Received: 12 March 2017 / Accepted: 27 April 2017 / Published online: 9 May 2017
© Springer Science+Business Media New York 2017
Abstract The compounds titled 1-(2,5/3,5-difluorophenyl)-
3-(2,3/2,4/2,5/3,4-dimethoxyphenyl)-2-propen-1-ones (1–8)
were synthesized via Claisen-Schmidt condensation under
basic condition. The chemical structure of the compounds
were identified using several spectroscopic techniques such
carcinoma (Ca9-22), oral squamous cell carcinoma derived
from tongue (HSC-2)] and human normal oral cells [gin-
gival fibroblasts (HGF), periodontal ligament fibro-
blasts (HPLF)]. Most of these compounds presented higher
cytotoxicity than reference drug 5-fluorouracil while the
compounds 7, [1-(3,5-difluorophenyl)-3-(2,5-dimethox-
yphenyl)-2-propen-1-one)], and 2, [1-(2,5-difluorophenyl)-
3-(2,4-dimethoxyphenyl)-2-propen-1-one], were presenting
the best activity according to potency selectivity expression
values. Type of cell death induced by compound 7 in both
HSC-2 and Ca9-22 cells was investigated to understand
mechanism of action of the compounds. The compound 7
produced cleaved products of PARP and caspase-3 were
produced, suggesting the induction of apoptosis as a pos-
sible mechanism of action of the compounds characterized
via activation of caspase-3 in both human oral squamous
cell carcinomas.
1
as H nuclear magnetic resonance (NMR), 13C NMR, 19F
NMR, DEPT 90, DEPT 135, COSY, HMBC, and HMQC.
Cytotoxic activities of the compounds were investigated
towards several human tumour cell lines [gingival
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-1911-0) contains supplementary material,
which is available to authorized users.
* Halise Inci Gul
incigul1967@yahoo.com
Cem Yamali
c.yamali@yahoo.com
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Keywords Anticancer Cytotoxicity Chalcone Fluorine
Methoxy PARP
Dilan Ozmen Ozgun
eczdilan@hotmail.com
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Hiroshi Sakagami
sakagami@dent.meikai.ac.jp
Introduction
Cavit Kazaz
ckazaz@atauni.edu.tr
Noriyuki Okudaira
noku@dent.meikai.ac.jp
Cancer is the second cause of death in the World after
cardiovascular diseases from which 22 million people will
be affected by 2030 (Mahapatra et al. 2015). Although
several cancer chemotherapeutics are available in markets,
side effects related to the drugs in clinical use lead the
researchers working in this field to investigate new drug
candidates which are more safe, potent and selective than
the available ones (Mahapatra et al. 2015).
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ataturk University, Erzurum, Turkey
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Agri Ibrahim Cecen University, Agri, Turkey
3
Meikai University Research Institute of Odontology (M-RIO),
Meikai University School of Dentistry, Sakado, Saitama 350-
0283, Japan
Chalcones and their derivatives are very well known with
cytotoxic/anticancer activities (Bilginer et al. 2013; Gul
et al. 2009, 2008; Hossain et al. 2016; Karki et al. 2016;
4
Department of Chemistry, Faculty of Science, Ataturk University,
Erzurum, Turkey

2016
Med Chem Res (2017) 26:2015–2023
Tugrak et al. 2016; Yerdelen et al. 2015; Yamali et al.
2016a; Yerdelen et al. 2015a). The reported mechanisms for
their anticancer activities include inhibition of angiogenesis,
inhibition of tubulin polymerization, induction of apoptosis,
inhibition of kinases (Karthikeyan et al. 2015; Mahapatra
et al. 2015), inhibition of topoisomerase enzyme (Gul et al.
2009) and thiol alkylation (Dimmock et al. 1998).
In the light of the literature survey, the aims of this study
include the followings: (i) The synthesis of 1-(2,5/3,5-
difluorophenyl)-3-(2,3/2,4/2,5/3,4-dimethoxyphenyl)-2-pro-
pen-1-ones, 1–8. (ii) The structure elucidation of these com-
pounds 1–8 with various spectroscopic methods. (iii) To
investigate their cytotoxic/anticancer properties and mechan-
ism of action on several cancer cell lines and non-cancer cells.
Methoxylated chalcones have structural similarity to
combretastatin and colchicine which are biologically active
and have cytotoxic properties. They have similar spatial
orientation between two aromatic rings (Kong et al. 2010)
(Fig. 1). Similar to combretastatin and colchicine, methoxy-
lated chalcone and their derivatives were reported with the
binding ability to the tubulin effectively (Kong et al. 2010).
Studies on methoxylated chalcones suggested that the number
and the position of methoxy groups on the aromatic rings
were critical for their cytotoxicity activities (Boumendjel et al.
2008; Ethiraj et al. 2013; Karthikeyan et al. 2015).
Fluorine bearing compounds or drugs have been reported
with several bioactivities including anticancer activities
(Fig. 1) (Nakamura et al. 2002; Purser et al. 2008; Zuo et al.
2012). The bioisosteric substitution of fluorine in place of
hydrogen in many bioactive molecules has led to potent
compounds without extensive stereochemical changes due
to its small size (Nakamura et al. 2002; Purser et al. 2008).
However, substitution of fluorine is known to modulate
overall reactivity and stability of the compounds due to
resistance of the carbon-fluorine bond toward metabolic
transformations (Kirk 2000; Nakamura et al. 2002). In
addition, incorporation of fluorine in a ring system may
increase biological half life of molecules by delaying oxi-
dative biotransformation and can increase bioabsorption by
lipophilic effects (Nakamura et al. 2002; Purser et al. 2008).
Recently, some fluorinated chalcones were reported as
anticancer agents (Burmaoglu et al. 2016). In the study at
issue the compound 10 (Fig.1) was found as the most
cytotoxic one comparing others towards A549, A498, and
A375 cancer cell lines with IC50 values as 0.120, 0.030, and
0.060l M, respectively (Burmaoglu et al. 2016).
Materials and methods
The nuclear magnetic resonance (NMR) spectra (1H NMR,
13C NMR, 19F NMR), distortionless enhancement by
polarization transfer (DEPT) (DEPT 90, DEPT 135) cor-
relation spectroscopy (COSY), heteronuclear multiple bond
correlation (HMBC), heteronuclear multiple-quantum cor-
relation (HMQC) in CDCl3 were recorded on a Bruker
AVANCE III 400 MHz (Bruker, Karlsruhe, Germany)
spectrometer [400 MHz (1H), 400 MHz (19F) and 100 MHz
(13C)]. For 1D, 2D NMR, in Topspin 2.1 NMR program
was used. Chemical shifts are given as δ values in ppm
against tetramethylsilane as the internal standard and J
values were expressed in Hz. Mass spectra of the com-
pounds were taken using a liquid chromatography ion trap-
time of flight tandem mass spectrometer (Shimadzu, Kyoto,
Japan) equipped with an electrospray ionization (ESI)
source, operating in both positive and negative ionization
mode. Shimadzu’s LCMS Solution software was used for
data analysis. Melting points were determined using an
Electrothermal 9100 instrument (IA9100, Bibby Scientific
Limited, Staffordshire, UK) and are uncorrected.
General procedure for the preperation of chalcones, 1–8,
Scheme 1
The title compounds were synthesized by Claisen-Schmidt
condensation (Dimmock et al. 1998; Gul et al. 2008; Mete
et al. 2016; Yamali et al. 2016a, b; Bilginer et al. 2013;
Yerdelen et al. 2015a, b). A mixture of fluorinated acet-
ophenone (6.4 mmol) and methoxylated aldehyde (6.4
mmol) was dissolved in ethanol (5 ml). Aqueous sodium
hydroxide solution (30%, 10 ml) was added into the mixture
under cold condition (0–5 °C). After overnight stirring at
room temperature, the reaction mixture was poured into ice-
water mixture and acidified with HCl solution (10%) to pH
= 3 (Scheme 1). The solids obtained were crystallized from
suitable solvents [It was ethanol-water (2 and 3) or ethanol
(5–8)]. On the other hand, the compounds 1 and 4 were
purified by passing through a column of silica gel using
chloroform as the eluent.
The chemical structures of the compounds were con-
1
firmed by H NMR, 13C NMR, 19F NMR and HRMS. The
Fig. 1 Several most potent compounds as anticancer agents
proton and carbon atoms of the compounds were

Med Chem Res (2017) 26:2015–2023
2017
O
F
O
O
O
+
F
H
CH
H
+
3
CH
R
1
3
R
1
R
R
2
2
F
F
i
i
OCH
O
3
OCH
O
OCH
OCH
F
F
O
3
F
O
O
3
3
F
OCH
F
F
3
OCH3
OCH
3
2
OCH
1
3
6
F
5
F
F
F
F
OCH
OCH
OCH
3
3
F
O
O
3
3
O
OCH
OCH
3
OCH
OCH
F
3
3
3
4
3
F
8
7
OCH
F
F
Scheme 1 General procedure for the synthesis of the chalcones 1–8. Reagents and conditions: i: Ethanol, NaOH solution (30%), rt. R1 and R2: Suitable
substitutions for the compounds 1–8. R1 and R2 are methoxy groups at different position of phenyl ring as shown at the compounds 1–8’s formula
completely assigned by one and two-dimensional (1D and
2D) homonuclear and heteronuclear experiments (DEPT
H), 7.56 (d, J = 8.6 Hz, 1H, C12-H), 7.50 (ddd, J = 8.5, 5.4,
3.1 Hz, 1H, C4-H), 7.39 (dd, J = 15.8, 2.7 Hz, 1H, C9-H),
7.21–7.11 (m, 2H, C2-H, C1-H), 6.54 (dd, J = 8.6, 2.3 Hz,
1H, C13-H), 6.47 (d, J = 2.3 Hz, 1H, C15-H), 3.89 (s, 3H,
C17-H, –OCH3), 3.87 (s, 3H, C18-H, –OCH3). 13C NMR
(100 MHz, δ, ppm, CDCl3) 188.2 (d, JCO–F = 2 Hz, C=O,
C7), 163.5 (C14), 160.6 (C16), 158.6 (dd, JCF = 245.0, 2.0
Hz, C6 or C3), 156.9 (dd, JCF = 245.0, 2.0 Hz, C6 or C3),
141.5 (C10), 131.1 (C12), 128.8 (dd, JCF = 16.0, 6.0 Hz,
C5), 123.1 (d, JCF = 7 Hz, C9), 119.8 (dd, JCF = 24.0, 9.0
Hz, C2), 117.8 (dd, JCF = 26.0, 8.0 Hz, C1), 116.9 (dd, JCF
= 25.0, 3.0 Hz, C4), 116.7 (C11), 105.6 (C15), 98.4 (C13),
55.6 (C17), 55.5 (C18). 19F NMR (400 MHz, δ, ppm,
CDCl3) −117.1 (d, JFF = 20 Hz), −118.0 (d, JFF = 20 Hz)
HRMS (ESI-MS) calc. for C17H15O3F2 [M+H]+ 305.0984;
found 305.0966.
1
1
90–135, H-1H COSY, H-13C HMQC and HMBC, See
Supplementary Material for representative spectra).
1-(2,5-Difluorophenyl)-3-(2,3-dimethoxyphenyl)-2-propen-
1-one (1)
1
A yellow solid, yield 55%. Mp: 81–83 °C. H NMR (400
MHz, δ, ppm, CDCl3) 8.09 (dd, J = 16.0, 1.9 Hz, 1H, C10-H),
7.53 (ddd, J = 8.5, 5.4, 3.2 Hz, 1H, C4-H), 7.45 (dd, J = 16.0,
2.8 Hz, 1H, C9-H), 7.26 (d, J = 1.3 Hz, 1H, C12-H),
7.25–7.14 (m, 2H, C2-H, C1-H), 7.11 (t, J = 8.0 Hz, 1H, C13-
H), 7.00 (dd, J = 7.4, 1.3 Hz, 1H, C14-H), 3.91 (s, 3H, C18-H,
–OCH3), 3.90 (s, 3H, C17-H, –OCH3). 13C NMR (100 MHz,
δ, ppm, CDCl3) 188.0 (d, JCO–F = 3 Hz, C=O, C7), 158.7 (dd,
JCF = 246.0, 2.0 Hz, C6 or C3), 157.2 (dd, JCF = 246.0, 2.0
Hz, C6 or C3), 153.2 (C15), 149.2 (C16), 140.5 (C10), 128.7
(C11), 128.3 (dd, JCF = 17.0, 7.0 Hz, C5), 126.2 (d, JCF = 7
Hz, C9), 124.3 (C13), 120.4 (dd, JCF = 25.0, 9.0 Hz, C2),
119.7 (C12), 117.9 (dd, JCF = 27.0, 8.0 Hz, C1), 117.1 (dd,
JCF = 25.0, 3.0 Hz, C4), 114.6 (C14), 61.4 (C17), 55.9 (C18).
19F NMR (400 MHz, δ, ppm, CDCl3) −116.5 (d, JFF = 20
Hz), −117.7 (d, JFF = 20 Hz) HRMS (ESI-MS) calc. for
C17H15O3F2 [M+H]+ 305.0984; found 305.0983.
1-(2,5-Difluorophenyl)-3-(2,5-dimethoxyphenyl)-2-propen-
1-one (3)
1
A yellow solid, yield 52%. Mp: 80–82 °C. H NMR (400
MHz, δ, ppm, CDCl3) 8.07 (dd, J = 15.9, 1.8 Hz, 1H, C10-
H), 7.52 (ddd, J = 8.5, 5.4, 3.1 Hz, 1H, C4-H), 7.45 (dd, J
= 15.9, 2.8 Hz, 1H, C9-H), 7.25–7.18 (m, 2H, C2-H, C1-
H), 7.16 (d, J = 2.8 Hz, 1H, C12-H), 6.98 (dd, J = 9.0, 3.0
Hz, 1H, C14-H), 6.89 (d, J = 9.0 Hz, 1H, C15-H), 3.88 (s,
3H, C17-H, –OCH3), 3.87 (s, 3H, C18-H, –OCH3). 13C
NMR (100 MHz, δ, ppm, CDCl3) 188.1 (d, JCO–F = 2 Hz,
C=O, C7), 158.6 (d, JCF = 245.0 Hz, C6 or C3), 157.1 (d,
JCF = 245.0 Hz, C6 or C3), 153.5 (d, J = 4 Hz, C13, C16),
140.9 (C10), 128.4 (dd, JCF = 16.0, 6.0 Hz, C5), 125.7 (d,
1-(2,5-Difluorophenyl)-3-(2,4-dimethoxyphenyl)-2-propen-
1-one (2)
1
A yellow solid, yield 50 %. Mp: 91–93 °C. H NMR (400
MHz, δ, ppm, CDCl3) 8.03 (dd, J = 15.8, 1.8 Hz, 1H, C10-

2018
Med Chem Res (2017) 26:2015–2023
JCF = 7 Hz, C9), 124.0 (C11), 120.3 (dd, JCF = 24.0, 9.0
Hz, C2), 118.1 (C12), 117.9 (dd, JCF = 18.0, 12.0 Hz, C1),
117.0 (dd, JCF = 25.0, 4.0 Hz, C4), 113.6 (C14), 112.5
(C15), 56.1 (C17), 55.8 (C18). 19F NMR (400 MHz, δ,
ppm, CDCl3) −116.7 (d, JFF = 20 Hz), −117.8 (d, JFF = 20
Hz) HRMS (ESI-MS) calc. for C17H15O3F2 [M+H]+
305.0984; found 305.0970.
(d, J = 15.7 Hz, 1H, C10-H), 7.58 (d, J = 8.6 Hz, 1H, C12-
H), 7.52 (dd, J = 8.0, 2.3 Hz, 2H, C4-H, C6-H), 7.45 (d, J
= 15.7 Hz, 1H, C9-H), 7.02 (tt, J = 8.7, 2.3 Hz, 1H, C2-H),
6.56 (dd, J = 8.6, 2.4 Hz, 1H, C13-H), 6.50 (d, J = 2.4 Hz,
1H, C15-H), 3.94 (s, 3H, C17-H, –OCH3), 3.88 (s, 3H,
C18-H, –OCH3). 13C NMR (100 MHz, δ, ppm, CDCl3)
188.3 (t, JCO–F = 4.1 Hz, C=O, C7), 163.5 (C14), 163.0 (d,
JCF = 249.0 Hz, C1 or C3), 162.9 (d, JCF = 249.0 Hz, C1 or
C3), 160.7 (C16), 142.2 (C10), 141.9 (t, JCF = 8.0 Hz, C5),
1-(2,5-Difluorophenyl)-3-(3,4-dimethoxyphenyl)-2-propen-
1-one (4)
131.5 (C12), 119.1 (C9), 116.7 (C11), 111.3 (dd, JCF
=
19.0, 7.0 Hz, C4, C6), 107.5 (t, JCF = 25.0 Hz, C2), 105.6
(C13), 98.5 (C15), 55.6 (C17), 55.5 (C18). 19F NMR (400
MHz, δ, ppm, CDCl3) −108.6 (s, 2F) HRMS (ESI-MS)
calc. for C17H15O3F2 [M+H]+ 305.0984; found 305.0955.
1
A yellow solid, yield 45%. Mp: 79–81 °C. H NMR (400
MHz, δ, ppm, CDCl3) 7.72 (dd, J = 15.7, 1.8 Hz, 1H, C10-
H), 7.50 (ddd, J = 8.5, 5.4, 3.2 Hz, 1H, C4-H), 7.27–7.15
(m, 4H, C1-H, C2-H, C9-H, C12-H), 7.14 (d, J = 1.6 Hz,
1H, C16-H), 6.90 (d, J = 8.3 Hz, 1H, C13-H), 3.95 (s, 3H,
C18-H, –OCH3), 3.94 (s, 3H, C17-H, –OCH3). 13C NMR
(100 MHz, δ, ppm, CDCl3) 187.5 (d, JCO–F = 2 Hz, C=O,
1-(3,5-Difluorophenyl)-3-(2,5-dimethoxyphenyl)-2-propen-
1-one (7)
C7), 158.7 (d, JCF = 246.0 Hz, C6 or C3), 157.0 (d, JCF
246.0 Hz, C6 or C3), 151.8 (C15), 149.3 (C14), 145.9
(C10), 128.4 (dd, JCF = 17.0, 7.0 Hz, C5), 127.5 (C11),
123.5 (C12), 122.9 (d, JCF = 7 Hz, C9), 120.2 (dd, JCF
24.0, 9.0 Hz, C2), 117.9 (dd, JCF = 26.0, 8.0 Hz, C1), 116.9
(dd, JCF = 25.0, 4.0 Hz, C4), 111.1 (C13), 110.1 (C16),
56.0 (C18), 55.9 (C17). 19F NMR (400 MHz, δ, ppm,
CDCl3) −116.7 (d, JFF = 20 Hz), −117.8 (d, JFF = 20 Hz)
HRMS (ESI-MS) Calc. for C17H15O3F2 [M+H]+ 305.0984;
found 305.0980.
=
A yellow solid, yield 50%. Mp: 110–112 °C. 1H NMR (400
MHz, δ, ppm, CDCl3) 8.13 (d, J = 15.8 Hz, 1H, C10-H),
7.56–7.53 (m, 2H, C4-H, C6-H), 7.50 (d, J = 15.8 Hz, 1H,
C9-H), 7.17 (d, J = 3 Hz, 1H, C12-H), 7.04 (tt, J = 8.5, 2.4
Hz, 1H, C2-H), 6.99 (dd, J = 9.0, 3.0 Hz, 1H, C14-H), 6.91
(d, J = 9.0 Hz, 1H, C15-H), 3.91 (s, 3H, C17-H, –OCH3),
3.84 (s, 3H, C18-H, –OCH3). 13C NMR (100 MHz, δ, ppm,
CDCl3) 188.3 (t, JCO–F = 4.1 Hz, C=O, C7), 163.1 (d, JCF
= 249.0 Hz, C1 or C3), 162.9 (d, JCF = 249.0 Hz, C1 or
C3), 153.5 (C13, C16), 141.8 (C10), 141.5 (t, JCF = 8.0 Hz,
C5), 123.9 (C9), 121.9 (C11), 117.9 (C12), 114.0 (C14),
112.5 (C15), 111.4 (dd, JCF = 18.0, 7.0 Hz, C4, C6), 107.8
(t, JCF = 25.0 Hz, C2), 56.1 (C17), 55.9 (C18). 19F NMR
(400 MHz, δ, ppm, CDCl3) −108.3 (s, 2F) HRMS (ESI-
MS) calc. for C17H15O3F2 [M+H]+ 305.0984; found
305.0956.
=
1-(3,5-Difluorophenyl)-3-(2,3-dimethoxyphenyl)-2-propen-
1-one (5)
1
A yellow solid, yield 52%. Mp: 96–98 °C. H NMR (400
MHz, δ, ppm, CDCl3) 8.15 (d, J = 15.8 Hz, 1H, C10-H),
7.54 (dd, J = 8.0, 2.4 Hz, 2H, C4-H, C6-H), 7.50 (d, J =
15.8 Hz, 1H, C9-H), 7.30 (dd, J = 8.0, 1.3 Hz, 1H, C12-H),
7.13 (t, J = 8.0 Hz, 1H, C13-H), 7.07–7.04 (m, 1H, C2-H),
7.01 (dd, J = 8.0, 1.3 Hz, 1H, C14-H), 3.92 (s, 6H, C17-H,
C18-H, –OCH3). 13C NMR (100 MHz, δ, ppm, CDCl3)
188.1 (d, JCO–F = 2 Hz, C=O, C7), 163.1 (d, JCF = 246.0
Hz, C1 or C3), 162.9 (d, JCF = 246.0 Hz, C1 or C3), 153.3
(C15), 149.2 (C16), 141.4 (C10), 141.3 (t, JCF = 8.0 Hz,
C5), 128.6 (C11), 124.3 (C13), 122.3 (C9), 119.7 (C12),
114.7 (C14), 111.4 (dd, JCF = 19.0, 7.0 Hz, C4, C6), 107.9
(t, JCF = 25.0 Hz, C2), 61.4 (C17), 55.9 (C18). 19F NMR
(400 MHz, δ, ppm, CDCl3)-108.0 (s, 2F). HRMS (ESI-MS)
calc. for C17H15O3F2 [M+H]+ 305.0984; found 305.0955.
1-(3,5-Difluorophenyl)-3-(3,4-dimethoxyphenyl)-2-propen-
1-one (8)
A yellow solid, yield 56%. Mp: 122–123 °C. 1H NMR (400
MHz, δ, ppm, CDCl3) 7.81 (d, J = 15.6 Hz, 1H, C10-H),
7.53 (dd, J = 7.9, 2.4 Hz, 2H, C4-H, C6-H), 7.28 (d, J =
15.6 Hz, 1H, C9-H), 7.26 (d, J = 8.3 Hz, 1H, C12-H), 7.17
(d, J = 1.9 Hz, 1H, C16-H), 7.04 (tt, J = 8.4, 2.4 Hz, 1H,
C2-H), 6.93 (d, J = 8.3 Hz, 1H, C13-H), 3.99 (s, 3H, C17-
H, –OCH3), 3.96 (s, 3H, C18-H, –OCH3). 13C NMR (100
MHz, δ, ppm, CDCl3) 187.2 (t, JCO-F = 2.2 Hz, C=O, C7),
163.1 (d, JCF = 249.0 Hz, C1 or C3), 162.9 (d, JCF = 249.0
Hz, C1 or C3), 151.9 (C15), 149.4 (C14), 146.6 (C10), 141.5
(d, JCF = 7.0 Hz, C5), 127.4 (C11), 123.7 (C12), 118.7 (C9),
111.3 (dd, JCF = 19.0, 7.0 Hz, C4, C6), 111.1 (C13), 110.1
(C16), 107.8 (t, JCF = 25.0 Hz, C2), 56.1 (C18), 56.0 (C17).
19F NMR (400 MHz, δ, ppm, CDCl3)-108.2 (s, 2F). HRMS
1-(3,5-Difluorophenyl)-3-(2,4-dimethoxyphenyl)-2-propen-
1-one (6)
A yellow solid, yield 54%. Mp: 147–148 °C, 144–149 °C
(Wu et al. 2012). 1H NMR (400 MHz, δ, ppm, CDCl3) 8.09

Med Chem Res (2017) 26:2015–2023
2019
(ESI-MS) calc. for C17H15O3F2 [M+H]+ 305.0984; found
305.0975.
Results and discussion
Chemistry
Biological activity
The general procedure for the preparation of targeted
chalcones 1–8 are outlined in Scheme 1. A suitable acet-
ophenone [2,5-difluoroacetophenone (for compounds 1–4)
or 3,5-difluoroacetophenone (for compounds 5–8)] in
ethanol and aqueous solution of NaOH were reacted with a
Cytotoxicity assay
The cytotoxicity of the compounds were assayed towards
oral squamous cell carcinoma cell lines (Ca9-22 and HSC-
2) and human oral normal cells (HGF and HPLF) as
described with some minor modifications (Gul et al. 2016a,
b, c, 2017a, b; Yamali et al. 2016a; Tugrak et al. 2016;
Yerdelen et al. 2015; Sakagami et al. 2015; Bilginer et al.
2013). In brief, all cells were cultured in Dulbecco’s Mod-
ified Eagle Medium (GIBCO BRL, Grand Island, NY,
USA) supplemented with 10% fetal bovine serum (Sigma-
Aldrich Inc., St. Louis, MO, USA). The following con-
centrations of the compounds in dimethylsulfoxide
(DMSO) were added to the medium and incubated at 37 °C
for 48 h: compounds 1–8 (0.32, 1, 3.2, 10, 31.6, 100, 316,
1000 µM) and 5-fluorouracil (5-FU) (Kyowa, Tokyo, Japan)
(3.12, 6.25, 12.5, 25, 50, 100, 200, 400 µM). The media that
contained the same concentration of DMSO (0.0078, 0.156,
0.03125, 0.0625, 0.125, 0.25, 0.5 or 1%) were used as
controls, since DMSO above 0.25% is cytotoxic. The viable
cell numbers were determined by the MTT [3-(4,5-
suitable
ybenzaldehyde (for compounds 1 and 5), 2,4-dimethox-
ybenzaldehyde (for compounds and 6), 2,5-
benzaldehyde
derivatives
[2,3-dimethox-
2
dimethoxybenzaldehyde (for compounds 3 and 7), 3,4-
dimethoxybenzaldehyde (for compounds 4 and 8)] at room
temperature. Then, the mixture was poured on ice-water and
acidified with HCl solution to pH = 3. The compounds were
purified by crystallization or column chromatography.
Except compound 6, all compounds were reported here
for the first time with detailed spectroscopic analyzses and
cytotoxicity activities. In addition, this study is the first
report on the detailed structure elucidation of this type
fluorinated chalcones. The chemical structures of the chal-
cones were identified by 1H NMR, 13C NMR, 19F NMR and
HRMS. The full characterisation of the chalcones 1–8 were
presented in the experimental part. Unambiguous chemical
shift assignments were made by one and two-dimensional
(1D and 2D) homonuclear and heteronuclear experiments
(DEPT 90–135, 1H-1H COSY, 1H-13C HMQC and HMBC,
See Supplementary Material for representative spectra).
Chalcones, one of the major classes of natural products,
are open-chain flavanoids in which two aromatic rings are
linked by a three carbon and α,β-unsaturated carbonyl
system. They have the tendency to exist both in cis (Z) or
trans (E) forms (Singh et al. 2014). The compounds syn-
thesized in this study were seen as E izomer on the basis of
coupling constants. The C9-H and C10-H resonances were
easily identified by their large trans coupling constants (J) in
the range of 15–16 Hz. In addition, it was observed that C9-
H is more shielded than C10-H because of resonance effects
of the carbonyl group.
The chemical shift and splitting patterns of the protons
and carbon atoms on the acetophenone moiety and carbon
atom of the carbonyl group were affected by the C-F and/or
H-F coupling. Interesting splitting patterns were observed in
the 1H NMR and 13C NMR spectra for the fluorinated
compounds. For compound 1, which substituted 2,5-
difluoro, C4-H was resonated at 7.53 ppm as doublet of
doublet of doublet (ddd) splitted by fluorine atoms and C2-
H proton, with coupling constants 8.5, 5.4, 3.2 Hz values,
actually C4-H should be observed as singlet (s) or doublet
(d) with meta coupling. In 13C NMR, for the compound 1,
the carbonyl carbon, which is away from the fluoro group,
was splitted into a doublet (d) at δ 188.0 ppm with a
dimethlthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)]
method. CC50 value was determined from the growth curves
plotted at different concentrations of each compounds in
triplicate wells.
Western blot analysis
The cells were washed with phosphate buffer solution and
processed for western blot analysis, as described previously
(Sakagami et al. 2017). In brief, antibodies against cleaved
caspase-3 (Cell Signaling Technology Inc., Beverly, MD,
USA), poly(ADP-ribose) polymerase (PARP) (Cell Sig-
naling Technology Inc.) and glyceraldehyde 3-phosphate
dehydrogenase (Trevigen, Gaithersburg, MD, USA) were
used as primary antibodies. As secondary antibodies, we
used α-rabbit IgG (DAKO Japan) antibodies which were
conjugated with horseradish peroxidase. HSC-2 and Ca9-22
cells were treated with compound 7 for 24 h at the con-
centrations 8–32 µM. The detached and attached cells were
combined and subjected to western blot analysis. Activation
of caspase-3 as evidenced by appearance of cleaved
caspase-3 and cleavage of PARP, which is one of the
caspase-3 substrates, were determined according to litera-
ture (Sakagami et al. 2017). Actinomycin D (Act D) was
used as a positive control.

2020
Med Chem Res (2017) 26:2015–2023
Table 1 Cytotoxic activities of the chalcones 1–8 towards human OSCC cell lines and human oral normal cells
CC50 (μM)
Compounds
Human OSCC cell lines
Human oral normal cells
Ca9-22
A
HSC-2
Mean Mean
HGF
C
HPLF
Mean
D
TS
PSE
SI
SI
B
SI (E)
D/B
C/A
8.6
1
2
3
4
5
6
7
8
5.4
8.7
6.1
9.7
4.5
4.7
8.3
6.5
4.0
6.8
5.0
11.3
6.0
46.3
19.3
32.8
4.8
73
149
90
12.9
7.5
7.6
6.7
88.9
41.7
79.9
33.7
84.4
37.7
11.1
5.6
10.2
5.0
8.3
5.0
42.1
5.1
32.9
7.6
6.1
37.5
6.4
5.4
200.0
76.5
200.0
34.8
200.0
55.6
5.3
4.7
14
10.9
12.6
7.3
9.1
8.8
15.0
12.6
20.3
26.3
>30.4
143
145
442
312
15.9
3.4
8.7
23.1
15.2
23.7
>26.5
12.3
3.4
17.8
15.0
22.5
>28.5
200.0
69.6
200.0
31.9
200.0
50.8
16.3
15.0
22.4
>28.3
14.8
3.3
7.6
21.2
6.8
7.2
200.0
122.6
161.3
5-Fluorouracil 32.9
>30.4
37.7
35.3
>1000
>1000
>1000
80.7
CC50 values refer to the concentrations of the compounds in μM which reduce the viable cell number by 50%. Oral squamous cell carcinoma
(OSCC) cell lines used are Ca9-22 (derived from gingiva) and HSC-2 (derived from tongue). Normal oral cells used are human gingival fibroblasts
(HGF) and human periodontal ligament fibroblasts (HPLF). Tumour-specificity (TS) value is calculated by dividing the mean CC50 value of each
compound against normal cells to mean CC50 value against OSCC (D/B or C/A). Selectivity index (SI) figures were generated which are quotients
of the average CC50 values of non-malignant cells and CC50 figure of a compound towards a specific cell line. A potency selectivity expression
(PSE) was devised, which is reciprocal of the average CC50 value (a measure of potency) and the average SI figure (a determination of tumour
selectivity) (Column E/Column B × 100) CC50 value was determined from the growth curves plotted at different concentrations of each
compounds in triplicate wells
coupling constant of 3 Hz. In addition all carbon atoms on
acetophenone moiety were splitted into a doublet of doublet
(dd). The splitting of these carbon atoms was because of
resonance effects of the fluorine atom. For compound 6,
which substituted 3,5-difluoro, C2-H proton was resonated
at 7.02 ppm as tirplet of triplet (tt) splitted by two protons
(C4-H and C6-H) and two fluorine atoms, with coupling
constants 8.7, 2.3 Hz. Signal of C4-H and C6-H were
observed at 7.52 ppm as a doublet of doublet (dd) with
coupling constants 8.0 and 2.3 Hz. The carbon of carbonyl
group of compound 6 was splitted into a triplet (t) at δ 188.3
ppm with a coupling constant of 4.1 Hz. (See Supplemen-
tary Material for representative spectra).
than 5-FU towards HSC-2 cell line [The compound and
times potency 1 (4.6), 2 (5.8), 3 (7.5), 4 (1.1), 5 (5.0), 6
(4.3), 7 (11.3), 8 (5.5)]. The conclusion to be drawn is that
all compounds in series possess noteworthy antineoplastic
potencies.
The second aspect of these compounds to be considered
is whether they are tumour specific cytotoxins since
tumours are surrounded by different types of normal cells.
Hence selectivity index (SI) figure of a compound which
reflects whether a compound is tumour specific cytotoxine
is very important. SI figures which are quotients of the
average CC50 values of non-malignant cells and CC50 figure
of a compound towards a specific cell line were generated
(Yamali et al. 2016a; Robles-Escajeda et al. 2016; Tugrak
et al. 2016; Bilginer et al. 2013). The results in Table 1
reveal that SI values of greater than 1 were obtained for 1–8.
This suggested that the compounds 1–8 are tumour specific
antineoplastic agents based on the literature (Robles-Esca-
jeda et al. 2016). SI values changed in the range of 4.5–21.2
towards Ca9-22 cells while it was in the range of 4.0–23.7
towards HSC-2 cell line. The order of SI values for the
compounds towards Ca9-22 cells was 8 (21.2) > 7 (14.8) >
6 (12.6) > 5 (10.9) > 2 (9.7) > 1 (6.1) > 4 (4.7) > 3 (4.5)
while it was 8 (23.7) > 6 (23.1) > 7 (15.2) > 2 (12.9) > 3
(7.5) > 5 (7.3) > 4 (6.1) > 1 (4.0) towards HSC-2 cell line.
Tumour selectivity (TS) was also calculated for the
compounds by dividing the average CC50 value towards
normal cells into the average CC50 value towards cancer
cell lines. (Column D/Column B, Table 1). According to
Cytotoxic activity
Compounds synthesized 1–8 were screened for their in vitro
cytotoxic effects against human tumour cell lines [gingival
carcinoma (Ca9-22), oral squamous cell carcinoma (HSC-
2)] and human normal oral cells [gingival fibroblasts
(HGF), periodontal ligament fibroblasts (HPLF)] via MTT
test. The results presented in Table 1 reveal that CC50
values of the compounds are in low micro molar range
(5.0–42.1 µM) towards cancer cell lines used.
Except 4 towards Ca9-22 cell line, all compounds had
2.1–9.7 times higher cytotoxicity than reference compound
5-FU [The compound and times potency 1 (6.1), 2 (3.8), 3
(4.0), 5 (6.5), 6 (2.1), 7 (9.7), 8 (4.3)] against Ca9-22 cell
line. The compounds had 1.1–11.4 times higher cytotoxicity

Med Chem Res (2017) 26:2015–2023
2021
this calculation the compound having the highest TS value
was compound 8 (TS: 22.4). The order of TS values of the
compounds was as follows: the compound (TS): 8 (22.4) >
6 (16.3) > 7 (15.0) > 2 (11.1) > 5 (8.8) > 3 (5.6) > 4 (5.3) >
1 (4.8). If any compound having TS value over 10, it can be
considered as possible drug candidate for further studies.
Hence, it can be said that the compounds 2, 6, 7, and 8 can
be considered as lead molecules according to this calcula-
tion. By considering the fact that HGF is the corresponding
normal cell of cancer cell Ca9-22 having the same origin,
TS values were also generated for a compound by dividing
the average CC50 value towards HGF cells into the CC50
value towards Ca9-22 cells. According to this calculation
(Column C/Column A, Table 1), the order of TS values was
as follows: the compound (TS): 8 (26.3) > 7 (20.3) > 5
(15.0) > 6 (12.6) > 2 (10.2) > 1 (8.6) > 3 (5.0) > 4 (4.7).
Lead compounds should possess both marked cytotoxic
potencies and selective toxicity for tumour. In order to
identify such molecules, a potency selectivity expression
value (PSE) was devised which is reciprocal of the average
CC50 value (a measure of potency) and the average SI figure
(determination of tumour selectivity) (Column E/Column
B × 100, Table 1) (Gul et al. 2016a; Yamali et al. 2016a;
Bilginer et al. 2013; Tugrak et al. 2016). The order of PSE
values of the compounds was as follows: the compound
(PSE): 7 (442) > 8 (312) > 2 (149) > 6 (145) > 5 (143) > 3
(90) > 1 (73) > 4 (14). The compound having the highest
PSE value (442) was the compound 7, [1-(3,5-difluor-
ophenyl)-3-(2,5-dimethoxyphenyl)-2-propen-1-one], can be
considered as the leader compound of the study (Fig. 2).
Another point considered was whether compound 7 can
induce apoptosis in two human oral squamous cell carci-
noma cell lines (HSC-2, Ca9-22), derived from both tongue
and gingiva, by western blot analysis (Fig. 3). The apoptosis
markers which were used in this study were activation of
caspase 3 (detected by the active form of caspase 3, that is,
cleaved caspase) and the cleavage of PARP, one of the
substrates of caspase 3) (Bressenot et al. 2009). When HSC-
2 and Ca9-22 cells were treated for 24 h with the increasing
concentrations of compound 7, caspase-3 was dose-
dependently cleaved, up to the levels attained by Act D
(used as positive apoptosis inducer) in both of these cells,
suggesting the activation of caspase-3. This was confirmed
by the finding that PARP was cleaved dose-dependently.
Fig. 3 Apoptosis induction by compound 7 in both human oral
squamous cell carcinoma cell lines. HSC-2 or Ca9-22 cells were
treated for 24 h with either vehicle (DMSO 0.08%), the indicated
concentrations of compound 7 or 1 μM Actinomycin D (Positive
control). Detached and attached cells were combined for western blot
analysis
All of these data suggest that compound 7 induced apop-
tosis in both human oral squamous cell carcinomas.
Structure–activity relationships (SAR)
In this study, it was designed and tested a series of difluoro-
dimethoxy substituted chalcones (1–8) in order to investi-
gate their anticancer effects and also to understand how the
positions of fluorine and methoxy groups effect the bioac-
tivity. The compounds 1 and 5, 2 and 6, 3 and 7, 4 and 8 are
the couples of compounds which permit comparison the
compounds in terms of the effect of fluorine positions to
PSE value i.e., the effect of replacement of second fluorine
atom on phenyl ring from position 2 to position 3 while the
other fluorine which is available at position 5 was kept
constant.
When the compound 1 was converted to the compound
5, the PSE value increased 2.0 times. In the case of con-
version of the compound 3 to 7, PSE value increased 4.9
times. On the other hand, PSE value increased 22.3 times by
conversion of 4 to 8. This suggests that 3,5-positions of
fluorine atoms were better positions than 2,5- positions in
terms of PSE values in compound couples having the
methoxy groups at the same positions which allow
comparison.
The effects of methoxy groups to PSE value were con-
sidered among compounds 1–4 and compounds 5–8 with
each others. The compound 2 had the highest PSE value
(149) among 1–4. This suggests that the best positions of
methoxy groups were 2,4 positions comparing to 2,3-; 2,5-;
3,4- positions of methoxy groups while two fluorines were
kept constant at 2,5- positions. On the other hand, the
compound 7 had the highest PSE value (442) among
compounds 5–8. This suggested that the best positions for
methoxy groups were 2,5 positions comparing to 2,3-; 2,4-;
3,4- positions while two fluorines were kept constant at 3,5-
positions.
Fig. 2 Chemical structure and general numbering of leader compound
7 (PSE = 442)

2022
Med Chem Res (2017) 26:2015–2023
Gul HI, Tugrak M, Sakagami H (2016b) Synthesis of some acrylo-
phenones with N-methylpiperazine and evaluation of their cyto-
toxicities. J Enzyme Inhib Med Chem 31:147–151
Conclusion
In summary, all compounds had higher cytotoxicity than
reference drug 5-FU. The compounds 2, 5, 6, 7 and 8 had
higher PSE than 5-FU and their PSE values were over value
of 100. Judging from the PSE values, (i) the fluorine
positions 3.5- were better than 2,5-positions, (ii) the best
positions for methoxy groups were 2,4-positions among
2,5-difluoro derivatives while they were 2,5-dimethoxy
position among 3,5-difluoro derivatives. It can be concluded
that compound 7 with having the highest PSE value (442)
can be used as the leader compound for further studies
based on its cytotoxic potency and apoptosis-inducing
activity against human oral squamous cell carcinoma cell
lines.
Gul HI, Tugrak M, Sakagami H, Taslimi P, Gulcin I, Supuran CT
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Gul HI, Mete E, Eren SE, Sakagami H, Yamali C, Supuran CT
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Acknowledgements This research work was supported by Ataturk
University Research Found, Turkey (Project No: 2015/078). Gul HI
thanks to Dr. Yusuf Ozkay (Faculty of Pharmacy, Anadolu University,
Eskisehir, Turkey) and MSc. Serkan Levent (Faculty of Pharmacy,
Anadolu University, Eskisehir, Turkey) for HRMS analyses.
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Bis(3-alkylaminomethyl-4-hydroxybenzylidene)-4-piperidones: a
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Conflict of interest The authors declare that they have no competing
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