Synthesis and anti-proliferative activity of fluoro-substituted chalcones

Article abstract of DOI:10.1016/j.bmcl.2016.04.096

A series of novel fluoro-substituted chalcone derivatives have been synthesized. All synthesized compounds were characterized by ¹H nuclear magnetic resonance (NMR), ¹³C NMR, and elemental analysis. Their anti-proliferative activities were evaluated against five cancer cells lines, namely, A549, A498, HeLa, A375, and HepG2 using the MTT method. Most of the compounds showed moderate to high activity with IC₅₀ values in the range of 0.029-0.729 μM. Of all the synthesized compounds, 10 and 19 exhibited the most potent anti-proliferative activities against cancer cells, and 10 was identified as the most promising compound.

Full text of DOI:10.1016/j.bmcl.2016.04.096

Accepted Manuscript
Synthesis and Anti-proliferative Activity of Fluoro-substituted Chalcones
Serdar Burmaoglu, Oztekin Algul, Derya Aktas Anıl, Arzu Gobek, Gulay
Gulbol Duran, Ronak Haj Ersan, Nizami Duran
PII:
S0960-894X(16)30496-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.04.096
BMCL 23871
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2016
27 April 2016
28 April 2016
Please cite this article as: Burmaoglu, S., Algul, O., Anıl, D.A., Gobek, A., Duran, G.G., Ersan, R.H., Duran, N.,
Synthesis and Anti-proliferative Activity of Fluoro-substituted Chalcones, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.04.096
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and Anti-proliferative Activities of
Fluoro-substituted Chalcones
Leave this area blank for abstract info.
Serdar Burmaoglu, Oztekin Algul, Derya Aktas Anıl, Arzu Gobek, Gulay Gulbol Duran, Ronak Haj Ersan, and
Nizami Duran
0.030 μM A498 cancer cell line

1
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Synthesis and Anti-proliferative Activity of Fluoro-substituted Chalcones
b
c
c
d
Serdar Burmaoglua,c *, Oztekin Algul , Derya Aktas Anıl , Arzu Gobek , Gulay Gulbol Duran , Ronak Haj

Ersanb, and Nizami Durane
a Tercan Vocational High School, Erzincan University, Erzincan 24800, Turkey
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin 33169, Turkey
c Department of Chemistry, Faculty of Science, Ataturk University, Erzurum 25240, Turkey
d Department of Medical Biology, Medical Faculty, Mustafa Kemal University, Antakya-Hatay 31100, Turkey.
e Department of Medical Microbiology, Medical Faculty, Mustafa Kemal University, Antakya-Hatay 31100, Turkey.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel fluoro-substituted chalcone derivatives have been synthesized. All synthesized
compounds were characterized by 1H nuclear magnetic resonance (NMR), 13C NMR, and
elemental analysis. Their anti-proliferative activities were evaluated against five cancer cells lines,
namely, A549, A498, HeLa, A375, and HepG2 using the MTT method. Most of the compounds
showed moderate to high activity with IC50 values in the range of 0.029-0.729 µM. Of all the
synthesized compounds, 10 and 19 exhibited the most potent anti-proliferative activities against
cancer cells, and 10 was identified as the most promising compound.
Keywords:
Chalcone
Synthesis
2009 Elsevier Ltd. All rights reserved.
Anti-proliferative activity
Organofluorine
Claisen-Schmidt condensation
Fluorine-containing compounds have been widely researched in
the literature for their use as drugs or drug candidates. Fluorine as
a substituent affects the bonding interactions and metabolic
stabilities of drugs and has an impact on their physical features and
selective reactivities.1The introduction of fluorine or fluorine-
containing group to a molecule significantly changes its features.
There are no elements or groups that cause similar effects on
molecules.2 In general, the effects of the addition of a fluorine atom
to a molecule can be divided into two categories: chemical or
biological. Table 1 shows the features of organofluorine
molecules.2
Chalcones, one of the major classes of natural products, are
open-chain flavanoids in which two aromatic rings are linked by a
three carbon α,β–unsaturated/saturated carbonyl system. In
general, they include polyhydroxy groups in their aromatic rings.
The free radical quenching properties of these phenolic groups
increase the interest in the use of these compounds or chalcone-
rich plant extracts as drugs or food preservatives.3 In addition,
chalcones show many biological activities such as anti-diabetic,4
anti-neoplastic,5 anti-hypertensive,6 anti-retroviral,7 anti-
inflammatory,8 anti-histaminic,9 anti-oxidant,10 anti-malarial11 and
anti-cancer4 activities.
Table 1. Properties of organofluorine compounds.
Recently, many studies have been conducted on the anti-cancer
properties of chalcones. In the last thirty years, research on the
effects of chalcones on different types of cancer has attracted
significant interest despite the considerable increase in the number
of studies on the subject. The anti-cancer properties of chalcones
result from the different functional groups on both aromatic rings
(Figure 1).4Chalcone derivatives still attract much attention as
drug candidates because of their wide-ranging biological activities
as well as their accessibility from the appropriate benzaldehyde
and acetophenone derivatives. Here, we report the syntheses and
anticancer activities of ten chalcones with fluorine substituents on
their B-rings and two non-substituted chalcones.
Chemical
Small size
Biological
Electronegative effect of neighboring
functionalities
Lipophilic
Strong C–F bonds resistant to metabolic
processes
High
Increases lipid solubility (bioavailability)
electronegativity
Low reactivity
Synthesis of isosteric analogues of drugs
Useful for studying biochemical processes
———
 Corresponding authors. These authors contributed equally. Tel.: +90-324-341-2815; fax: +90-324-341-3022 (O.A); +90-446-441-3627; fax:
+90-446-441-3672 (S.B)
e-mail: oztekinalgul@mersin.edu.tr, sburmaoglu@erzincan.edu.tr

2
Figure 1. Anti-cancer activity properties of chalcones according to their different functional groups.
The condensation of trimethoxyacetophenone (1) with 2 in
an aqueous solution of KOH afforded chalcone 3 in 90% yield
through the adoption of a general synthesis protocol (Scheme
1).12 Hydrogenation of compound 3 on Pd/C afforded 4 in 80%
yield. After the successful synthesis of chalcone 4, compounds
8, 9, and 10 were obtained from the reaction of 1 with fluoro-
substituted benzaldehyde (5, 6, 7) in 80%–95% yield by
employing the developed procedure. Hydrogenation of
chalcones 5, 6, and 7 on Pd/C afforded 11, 12, and 13 in
excellent yields (70%–90%) (Scheme 1).
Padhye and co-workers13 prepared 2’-hydroxychalcones with
hydroxyl/fluoro groups in their B rings and evaluated their
radical scavenging potential and anti-proliferative activities
against human pancreatic and breast cancer cells. They found
that the B ring fluoro-substituted chalcones exhibited superior
anti-proliferative activities than their hydroxylated derivatives
toward these cell lines.
Scheme 2. Preparation of fluoro-substituted trihydroxy-
chalcones.
Scheme 1. Preparation of non- and fluoro-substituted
trimethoxychalcones.
Having successfully synthesized the trimethoxychalcones, we
turned our attention to trihydroxychalcones. Condensation of
trihydroxyacetophenone
(14)
with
fluoro-substituted
benzaldehydes (5 and 7) did not afford compounds 16 and 17
and only the starting material was recovered. After this setback,
we strategically decided to protect the hydroxyl groups with
MOMCl, which is stable under basic conditions and can be
deprotected easily under mild acidic conditions. The base-
catalyzed reaction of 15 with both 5 and 7 afforded MOM-
protected chalcones 16 and 17 in good yields. After the removal
of MOM with HCl in MeOH, the α,β-unsaturated double bond
of 18 and 19 was reduced to afford target compounds 20 and 21
in 80% and 85% yield, respectively (Scheme 2).
Scheme 3. Structures of the synthesized molecules.
In this study, the anti-proliferative activities of twelve
chalcone derivatives (3, 4, 8–13, and 18–21) were evaluated.
The primary objective of the synthesis of the fluoro-substituted
chalcone derivatives was to determine their potency and
specificity against five different cancer cells compared with
those of methotrexate (MTX). To assess the anti-proliferative
properties, the synthesized compounds were grouped into two
main groups (methoxy- or hydroxyl-substituted) and three
subgroups (chalcone, semi-saturated, or saturated), and their
effects on cancer cell proliferation were assessed in vitro using
human lung adenocarcinoma epithelial cells (A549), human
renal cancer cells (A498), human cervical cancer cells (HeLa),
human skin malignant melanoma cells (A375), and human
hepatocellular carcinoma cells (HepG2).
The structures of the molecules synthesized in this study are
shown in Scheme 3. To the best of our knowledge, this is the
first report on the preparation of compounds 8–13 and 19–21,
and of these compounds only 18 has been reported previously in
the literature.13 All the compounds were isolated as either solids
or liquids and characterized by 1H NMR, 13C NMR, and
elemental analysis (for detailed information see the
supplementary data).
Several studies have been conducted on the anti-cancer
properties of chalcones; however, only few studies on chalcones
with fluoro groups in their B rings exist.13,14 In a similar study,

3
Table 2. IC50 value of normal cells and cancer cell lines, cLog P, and the specificity of the synthesized compounds.
Compound
No
HEK293 A549 A498 HeLa A375 HepG2
Specificity*
cLog P**
IC50 (µM)
A549 A498 HeLa A375 HepG2
3
4
8
9
10
11
12
13
18
19
20
21
0.335
1.665
0.632
0.632
0.598
1.643
0.657
1.551
0.365
1.711
1.810
0.340
0.022
0.134 0.067 0.134 0.067
0.533 0.533 0.533 0.533
0.126 0.126 0.126 0.126
0.253 0.126 0.506 0.506
0.120 0.030 0.060 0.060
0.526 0.526 0.263 0.526
0.263 0.526 0.066 0.263
0.248 0.248 0.248 0.496
0.146 0.729 2.334 0.146
0.137 0.274 0.068 0.274
0.145 0.145 0.579 0.579
0.136 0.136 0.068 0.068
0.022 0.022 0.022 0.022
0.134
0.533
0.126
0.253
0.060
0.263
0.263
0.496
0.029
0.068
0.290
0.136
0.022
2.5
3.125
5.0
2.5
5.0
3.125
2.5
6.3
5.0
3.1
5.0
5.0
20.0
3.1
1.3
6.3
0.5
6.3
2.5
5.0
2.5
3.125
5.0
3.21
3.23
3.36
4.74
3,36
4.74
3.52
4.90
2.57
3.95
2.73
4.11
0.94
3.125 3.125
5.0
5.0
1.25
10.0
1.25
10.0
6.25 3.125
10.0 2.5
6.25 3.125
2.5
10.0
6.25
2.5
3.125
12.5
25.0
6.25
2.5
2.5
0.156
25.0
2.5
6.25
12.5
12.5
2.5
12.5 3.125 3.125
2.5
1.0
5.0
1.0
5.0
1.0
Methotrexate
1.0
1.0
HEK293, human embryonic kidney cells; A549, human lung adenocarcinoma epithelial cells; A498, human renal cancer cells; HeLa, human cervical cancer cells;
A375, human skin malignant melanoma cells; HepG2, Human hepatocellular carcinoma cell lines.
* Specificity of the compounds: the ratio for each compound of its IC50 value for normal cells (HEK293) to its IC50 value for the cancer cell.
**cLogP value of the synthesized compounds (calculated from ChemBioDrawUltra 12.0.3)
To assess the specificity of the compounds, their toxicity was
tested against normal cells using human embryonic kidney cells
(HEK 293). The specificity of the compounds was calculated as
their IC50 values for normal cells divided by their IC50 values for
the specific cancer cells, according to Eq. 1. The specificity of
the compounds appeared to be cell-dependent with good
specificity toward HepG2 cells but a relatively moderate
specificity toward other cancer cells. Table 2 shows the
specificity of all 12 compounds toward normal cells and cancer
cells.
and the amount of live cells was lower than that observed with
the compounds. The anti-proliferative activity of all the tested
compounds increased as the exposure time was extended, as
proven by the decline in the number of surviving cells. After a
longer exposure, all tested compounds had an activity equal to
that of MTX.
The synthesized compounds were methoxy- or hydroxyl-
substituted analogs of chalcone with varying components:
fluorine atoms, ketone groups, or alkyl chains. Accordingly, a
structure–activity relationship (SAR) was established by
comparing the anti-proliferative activities of the different
chalcone groups. Among these compounds, 3, 8–10, 18, and 19
exhibited the most potent anti-proliferative activity. In contrast,
compounds 4, 11–13, 20, and 21 with semi-saturated or
saturated linkers showed the weakest anti-proliferative activity
against all the tested cancer cells except HeLa and A375. The
resulting data is presented in Table 2 and shows that all the
tested compounds generally had significant activity against
cancer cell lines with 0.029–0.729 µM. The most active agent
against the HepG2 cells was 18, 0.029 µM. Hydroxy-substituted
compounds (18–21) displayed improved anti-proliferative
activity against the A549, HeLa, and A375 cancer cells. In
addition, the saturated chalcone derivatives (11–13, 20, and 21)
did not significantly improve the activities against the cancer
cell lines except for the A549 cancer cell line. The fluoro-
substituted compounds (8–13 and 18–21) exhibited improved
anti-proliferative activity against almost all cancer cell lines
compared with MTX and the non-fluoro compounds (3 and 4).
On the other hand, non-fluoro-substituted chalcone 3 exhibited
moderate anti-proliferative activity (0.067 µM) against the
A375 and A498 cell lines compared with all fluoro-substituted
compounds except 10.
The non-toxic concentrations of all compounds were
determined on the HEK-293 cell line. Then, anti-proliferative
activity studies were performed at these non-toxic
concentrations. The compounds with the best selectivity values
for each cancer cell line are shown in Table 3. The selected
compounds and MTX were exposed for a period of 0–96 h for
each cancer cell line. At the end of the incubation, all cells were
evaluated and subjected to an assessment of their cytotoxic
responses using an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay (Figure 2).15,16
Table 3. The compounds with the best selectivity values and
non-toxic concentrations for each cancer cell line.
Cancer cell
line
The highest selective
Non-toxic
concentration (μg/mL)
compound
A549
A498
HeLa
A375
19 and 20
0.16 and 0.08
0.02
10
19
10
19
0.16
0.02
0.16
The mono fluoro substituted compounds showed activity
toward A549 and HepG2. However, considering the difluoro-
substituted compounds, 10 exhibited activity toward A549,
A498, HeLa and A375 whereas compound 19 showed activity
toward HeLa and HepG2. Compared with all tested cell lines,
the difluoro-substituted compounds exhibited high anti-
proliferative activity toward HeLa cell lines, as indicated in
Table 2.
HepG2
As shown in Figure 2, the proliferation of cancer cells
treated with a non-toxic and in lower concentration dose of
compounds and MTX was inhibited in a time-dependent
manner. After a short treatment (24 h), MTX was more effective

4
As shown in Table 2, a correlation is observed between the
lipophilicities of the compounds and their anti-proliferative
activities, especially for the HeLa, A375 and HepG2 cell lines.
As log P increases, the specificity value increases and thus the
anti-proliferative activity increases, but it has an upper limit.
Note: p values: 24.hours: I-II: p<.000; I-III: p<.000; I-IV: p<.000; I-V; p<.000 II-III: NS; II-IV: NS; II-V: NS; III-IV: NS; III-V: NS; IV-V; p<.05 48.hours: I-II:
p<.000; I-III: p<.000; I-IV: p<.000; I-V; p<.000 II-III: NS; II-IV: p<.05; II-V: NS; III-IV: p<.05; III-V: NS; IV-V; p<.05 72.hours:I-II: p<.000; I-III: p<.000; I-
IV: p<.000; I-V; p<.000 II-III: NS; II-IV: p<.05; II-V: NS; III-IV: p<.05; III-V: NS; IV-V; p<.05 96.hours: I-II: p<.000; I-III: p<.000; I-IV: p<.000; I-V; p<.000
II-III: NS; II-IV: NS; II-V: NS; III-IV: NS; III-V: NS ;IV-V; NS NS: Non-significant.
Figure 2. MTT assay on Human Hepatocellular Carcinoma Cells (HepG2 cell line) lines after 96 hours incubation with methotrexate
(0,022 µM) and compound 10 (0,060 µM), compound 18 (0,029 µM), compound 19 (0,068 µM). The absorbance values were selected
as 570 nm for the MTT method. Control cells (I) not containing compounds and MTX were incubated same conditions. Cell viability was
calculated as the ratio of absorbance of treated cells with chemical synthesis or MTX to untreated cells. Given values show the mean ±
standard deviations from three independent experiments carried out in triplicate.
In summary, a range of chalcone derivatives was synthesized
successfully using Claisen–Schmidt condensation followed by
the hydrogenation of either the unsaturated double bond or the
carbonyl and unsaturated double bond. We discovered that the
reduction of the carbonyl group to a hydrocarbon occurred only
in the case of the methoxy groups on the A-ring. Conversely,
hydroxyl groups caused the selective reduction of the
unsaturated double bond without further reduction of the
carbonyl group. Biological assays showed that difluoro-
methoxy/hydroxyl-substituted chalcone derivatives exhibited
higher anti-proliferative activities against the tested cancer cells.
We found that 10, in particular, was more effective than the
other studied compounds against the A549, A498, and A375
cancer cell lines with following IC50 values; 0.120, 0.030, and
0.060 µM, respectively. Based on our results, 10 is considered
to be the most promising compound toward the development of
new therapeutic agents, specifically for the treatment of human
renal cancer. The current studies13,17 and our findings indicate
that the bioisosteric substitution of fluorine instead of hydrogen
yields more potent compounds in the case of many biologically
active molecules.
cytometry. Thus, we are currently working on the syntheses and
mechanism of actions of these compounds regarding cell
survival, apoptotic index, telomerase activity and cell
proliferation. The results will be published in due course.
Acknowledgements
We thank the Scientific and Technological Research Council
of Turkey (TUBITAK, Grant Number: 114Z554) and Erzincan
University for their financial support. Serdar Burmaoglu
especially thanks Bilal Altundas for his critical reading of this
article.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://.....................
References and notes
1. Hagmann, K. W., J. Med. Chem. 2008, 51 (15), 4359.
2. Fluorine in Medicinal Chemistry and Chemical Biology;
Ojima, I., Blackwell Publishing, Ltd.; 2009; p 3; Part I.
3. Nowakowska, Z., Eur. J. Med. Chem. 2007, 42, 125.
4. Mahapatra, D. K., Bharti, S. K., Asati, V., Eur. J. Med.
Chem. 2015, 98, 69.
In conclusion, flouro-substituted chalcones could provide an
effective solution to improve the bioavailability and cell-growth
inhibitory activity of chalcones against cancer cells. While
control compound, MTX, was found most effective in S phase
of cell cycle, our compounds were determined to be effective
mostly in G0/1 phase of these cancer cells by using flow
5. Dimmock, J. R. , Elias, D. W., Beazely, M. A., Kandepu,
N. M., Curr. Med. Chem. 1999, 6, 1125.

5
6. Detsi, A., Majdalani, M., Kontogiorgis, C. A.,
Hadjipavlou-Litina, D., Kefalas, P., Bioorg. Med. Chem.
2009, 17, 8073.
12. March, J.; Advanced Organic Chemistry, Reactions,
Mechanisms, and Structure, Fourth edition, A Wiley-
Interscience Publication, 1992; pp 939-943.
7. Mahapatra, D. K., Asati, V., Bharti, S. K., Eur. J. Med.
Chem. 2015, 92, 839.
13. Padhye S., Ahmad, A., Oswal, N., Dandawate, P., Rub,
A. R., Deshpande, J., Swamy, K. V., Sarkar, F. H.,
Bioorg. Med. Chem. Lett. 2010, 20, 5818.
14. Zhao, S. L., Peng, Z., Zhen, X. H., Jin, H. G., Han,
Y., Qu, Y. L., Guan, L. P., Med. Chem. Res. 2015, 24,
2573.
15. McGahon A. J., Martin S. J., Bissonnette R. P. Methods
Cell Biol. 1995, 46, 153.
16. Mosmann T. J., Immunol Methods, 1983, 65, 55.
17. Sun, L. P., Gao, L. X., Ma, W. P., Nan, F. J., Li, J., Piao,
H. R. Chem. Biol. Drug. Des. 2012, 80, 584.
8. Chen, M., Christensen, S. B., Zhai, L., Rasmussen, M. H.,
Theander, T. G., Frøkjaer, S., Steffansen, B., Davidsen,
J., Kharazmi, A., J. Infect. Dis. 1997, 176, 1327.
9. Kantevari, S., Addla, D., Bagul, P. K., Sridhar, B.,
Banerjee, S. K., Bioorg. Med. Chem. 2011, 19, 4772.
10. Mishra, L., Itokawa, H., Bastow, K. F., Tachibana, Y.,
Nakanishi, Y., Kilgore, N., Lee, K. H., Bioorg. Med.
Chem. 2001, 9, 1667.
11. Israf, D. A., Khaizurin, T. A., Syahida, A., Lajis, N. H.,
Khozirah, S., Mol Immunol. 2007, 44, 673.