Synthesis, biological evaluation and molecular docking studies of bis-chalcone derivatives as xanthine oxidase inhibitors and anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2019.103149

In this study, a series of B-ring fluoro substituted bis-chalcone derivatives were synthesized by Claisen-Schmidt condensation reactions and evaluated for their ability to inhibit xanthine oxidase (XO) and growth inhibitory activity against MCF-7 and Caco

Full text of DOI:10.1016/j.bioorg.2019.103149

Journal Pre-Proof
Synthesis, biological evaluation and molecular docking studies of bis-chalcone
derivatives as xanthine oxidase inhibitors and anticancer agents
Serdar Burmaoglu, Seyda Ozcan, Sevgi Balcioglu, Melis Gencel, Samir Abbas
Ali Noma, Sebnem Essiz, Burhan Ates, Oztekin Algul
PII:
S0045-2068(19)30350-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.103149
YBIOO 103149
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 March 2019
9 July 2019
23 July 2019
Please cite this article as: S. Burmaoglu, S. Ozcan, S. Balcioglu, M. Gencel, S. Abbas Ali Noma, S. Essiz, B. Ates,
O. Algul, Synthesis, biological evaluation and molecular docking studies of bis-chalcone derivatives as xanthine
oxidase inhibitors and anticancer agents, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.
2019.103149
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Synthesis, biological evaluation and molecular docking studies of bis-chalcone derivatives
as xanthine oxidase inhibitors and anticancer agents
Serdar Burmaoglua,b*, Seyda Ozcanb, Sevgi Balciogluc, Melis Genceld, Samir Abbas Ali Nomac,
Sebnem Essizd, Burhan Atesc, Oztekin Algule*
aTercan Vocational High School, Erzincan Binali Yildirim University, Erzincan 24800, Turkey
bDeparment of Chemistry, Faculty of Science, Ataturk University, Erzurum 25240, Turkey
cDepartment of Chemistry, Faculty of Science and Arts, İnönü University, Malatya 44280, Turkey
dBioinformatics and Genetics Department, Faculty of Engineering and Natural Sciences, Kadir
Has University, Istanbul 34083, Turkey
eDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin
33169, Turkey.
Corresponding authors. Tel.: +90-324-341-2815; fax: +90-324-341-3022; e-mail:
oztekinalgul@mersin.edu.tr (OA), Tel.: +90-446-441-3627; fax: +90-446-441-3672; e-mail:
sburmaoglu@erzincan.edu.tr (SB). *These authors contributed equally.

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ABSTRACT
In this study, a series of B-ring fluoro substituted bis-chalcone derivatives were synthesized by
Claisen-Schmidt condensation reactions and evaluated for their ability to inhibit xanthine oxidase
(XO) and growth inhibitory activity against MCF-7 and Caco-2 human cancer cell lines, in vitro.
According to the results obtained, the bis-chalcone with fluoro group at the 2 (4b) or 2,5-position
(4g) of B-ring were found to be potent inhibitors of the enzyme with IC50 values in the low
micromolar range. The effects of these compounds were about 7 fold higher than allopurinol. The
binding modes of the bis-chalcone derivatives in the active site of xanthine oxidase were explained
using molecular docking calculations. Also, compound 4g and 4h showed in vitro growth
inhibitory activity against a panel of two human cancer cell lines 1,9 and 6.8ꢀμM of IC50 values,
respectively.
Keywords: Bis-chalcone, synthesis, Claisen-Schmidt condensation, inhibition, cytotoxicity
GRAPHICAL ABSTRACT:
OH
O
F
F
F
F
O
O
OH
O
Claisen-Schmidt Condensation
MeO
OMe
MeO
OMe
XO inhibitor; 7 fold higher than allopurinol
MCF-7 IC50: 1.9 µM
HIGHLIGHTS:
 Eight bis-chalcone derivatives were synthesized by Claisen-Schmidt condensation reaction.
 Target compounds were assessed in vitro against MCF-7 and Caco-2 tumor cell lines.
 Target compounds were evaluated for their ability to inhibit xanthine oxidase.
 Molecular Modeling studies have been carried out.

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1.
Introduction
Cancer is one of the most challenging diseases that have been struggled despite the
many clinical and basic studies conducted on it over the years. It is reported that the chance
of cytotoxic agents to destroy tumor cells in convential chemotherapy is higher than
compared with normal cells. However, since these agents do not have too much specificity,
they may result in systemic toxicity caused by undesirable side effects. Therefore, obtaining
novel anticancer agents specific to tumor would be a move to significantly improve the
efficacy of cancer chemotherapy [1].
In recent years, numerous FDA-supported fluorine-containing compounds come to
the fore, indicating that fluorine atom plays an important role in medical chemistry,
chemical biology and drug discovery. In the current studies of drug design and discovery,
the fluorine atom is the second most favourite hetero atom after nitrogen. Replacement of
a C-H or C-O bond with a C-F bond on a biologically active molecule has been reported to
generally show many desired pharmacological properties such as metabolic stability,
binding to target molecules, and enhanced membrane permeability. Moreover, the
properties that make fluorine appealing may include the small atomic radius, high
electronegativity and low polarization of the C-F bond [2]. In light of all this information,
it can be said that the anticancer agents containing fluorine can act as targeted molecular
missile warheads [1].
The chalcones are simple-structure compounds found in the structure of many
natural products and have a very wide distribution in vegetables, fruits, teas and many other
plants. The chalcones have always aroused researchers' interest not only from synthetic or
biosynthetic point of view, but also due to their interesting biological characteristics.
Because the therapeutic applications of chalcones by means of using plants and medicinal
herbs have been utilized in conventional medicine to treat numerous diseases, such as
cancer, inflammation and diabetes, for thousands of years [3].
Several compounds in the chalcone structure have been approved for their clinical
use. Figure 1 illustrates the structures of metochalcone, once sold as a choleretic drug, and
sofalcone compounds used as anti-ulcer and mucoprotective drugs [3].

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O
A
B
Chalcone
O
O
OH
O
OMe O
O
MeO
OMe
O
Metochalcone
Sofalcone
Figure 1. Structures of chalcone and two clinically approved chalcone-based drugs.
The compounds containing two chalcone units in a single structure are called bis-chalcones.
The bis-chalcones also exhibit a broad variety of biological properties. Some bis-chalcone analogs
have been reported to be potent NO production inhibitors and cytotoxic agents against four human
cancer cell lines (A549, DU145, KB and KB-VIN) [4]. The biphenyl-based bis-chalcones have
been reported to have an anticancer activity against MCF-7 and MDA-MB 231 human breast
cancer, HeLa, human embryonic kidney (HEK-293) cells [5].
XO is an enzyme that plays a role in the degradation of purines. During the
degradation process of purines, XO produces reactive oxygen species (ROS). The excess
activity of XO can lead to oxidative stress, mutagenesis and possibly cancer. In addition,
inhibition of XO causes to reduced oxidative stress and subsequently to reduced inflammation and
reduced tissue wasting and improved outcome during treatment of disease. Therefore, the
inhibition of XO might be strategy for cancer therapy [6,7].
The aim of this study was to investigate the compounds obtained by strategically
including the fluorine atom in the compounds in the bis-chalcone structure in terms of
anticancer and XO inhibition. For this, XO inhibition activity studies and cytotoxic
properties on MCF-7 and Caco-2 cancer cells lines were conducted by synthesizing a series
of bis-chalcone derivatives containing fluorine in different positions of both B-rings. The
general structure of synthesized compounds is shown in Figure 2.

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O
OH
O
A
R
R
B
B
MeO
OMe
bis-chalcone
Figure 2. General structure of bis-chalcone.
2. Results and discussion
2.1. Synthesis of bis-chalcone derivatives
Compound 2 was prepared from trimethoxybenzene (1) and acetyl chloride in the
presence of AlCl3. Bis-chalcones were prepared from compound 2 and appropriate
benzaldehydes (3a-h) in the presence of 50% KOH solution in MeOH (Scheme 1). After a
standard work-up (addition of NH4Cl solution and extraction with ethyl acetate), the
resulting solid was purified by column chromatography to yield the desired products.
OMe
O
OH
O
O
i
H
R
MeO
OMe
MeO
OMe
3a; R = H, 3b; R = 2-F
3c; R = 3-F, 3d; R = 4-F
3e: R = 2,3-diF, 3f; R = 2,4-diF
3g; R = 2,5-diF, 3h; R = 3,4-diF
1
2
ii
O
OH
O
R
R
MeO
OMe
4a; R = H, 4b; R = 2-F
4c; R = 3-F, 4d; R = 4-F
4e: R = 2,3-diF, 4f; R = 2,4-diF
4g; R = 2,5-diF, 4h; R = 3,4-diF
Scheme 1. General synthetic method. Reagents; (i) CH3COCl, AlCl3; (ii) 50% KOH,
MeOH, rt.

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2.2. Cytotoxicity Evaluation
The cytotoxicity evaluation of the compounds was performed by MTT test against
MCF-7 and Caco-2 cell lines. Cisplatin was used as standard for comparison. As shown in
Figure 3 and Figure 4, all complexes showed much higher cytotoxicity than Cisplatin
against Caco-2 and MCF-7 cell lines. As seen from the Figure 3, compound 4g showed the
best IC50 value against MCF-7 cell line as 1.9 µM which is at least three-fold higher than
the other compounds. Furthermore, among all complexes, 4a and 4b showed higher toxicity
than 4c, 4d, 4e, 4f and 4h. Compound 4g showed the lowest toxicity result against MCF-7
cell line as 103.7 µM. Figure 4 shows the toxicity values of the compounds against Caco-2
cell line. In this cell line, results are generally parallel to MCF-7. Also, compound 4g and
4h showed the highest toxicity, and compound 4d and 4e showed the lowest toxicity.
Generally, there are no major differences between cell lines in terms of toxicity of the
compounds, indicating that there is no cell specific toxicity. The images monitored by invert
microscope at the end of 24 h confirmed the results (Figure 5, 6, 7 and 8, see supplementary
information). The morphological changes in MCF-7 cells after treatment with 4g for 24 h
demonstrated that cell confluent level dramatically decreased in compared with the other
compounds (Figure 5 and 6, see supplementary information). In addition, growth rate of
Caco-2 cell from the morphological images is parallel with IC50 values (Figure 7 and 8, see
supplementary information).
Although more detailed studies are needed to understand the mechanism of action,
except for compound 4e, high cytotoxicity results of the compounds make them a promising
candidate for the treatment of colorectal and breast cancer.

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Figure 3. % Inhibition graphics and IC50 values of the compounds against MCF-7 cell line.
Figure 4. % Inhibition and IC50 values of the compounds against Caco-2 cell line.

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2.3. Xanthine Oxidase (XO) Inhibitory Activity
The experimental results indicated that all the compounds showed remarkable
inhibition activity toward XO as compared with the standard allopurinol. In this study, we
investigated the XO inhibitor effects of eight different compounds, the results are given as
half maximal inhibitory micromolar concentrations (IC50) values calculated by equation
prepared from the different concentrations of compounds. All compounds showed
inhibition behaviour on XO enzyme. The range of IC50 value for XO inhibition was
determined from (0.728-6.058 μM) as shown in (Table 1). Compound 4b showed the lowest
IC50 value as 0.728 μM while compound 4c showed the highest IC50 value as 6.058 μM.
The IC50 for allopurinol was determined 5.43 μM as a positive control. In addition,
compounds 4a, 4b, 4d, and 4g have non-competitive inhibition, while compounds 4c, 4e,
4f, and 4h have competitive inhibition like allopurinol (Table 1). It means that non-
competitive inhibition is effective mechanism in the low IC50 values compared with
competitive inhibition.
In a previous study, the range of IC50 value for XO inhibition was determined from
4.608 to 7.084 μM for some pyrrole carboxamide derivatives by Kibriz et al. [8]. In other
study, Zhang et al. founded the range from (6.7-45 μM) for seventeen compounds of
benzonitrile derivatives as IC50 value for XO inhibition [9]. Nile et al., investigated natural
plant flavonoids effect on XO enzyme and found IC50 range between (4.5-21.3 μg/mL) [10].
According to our results, all compounds exhibited more inhibition activity than allopurinol
except for compounds 4c and 4e. In addition, 4b and 4g have almost 7-fold higher inhibition
activity on XO than allopurinol.

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Table 1. The IC50 values and inhibition types of the compounds on XO activity.
Compound No
IC50 (μM)
2.010 ± 0.046
0.728 ± 0.009
6.058 ± 0.095
3.592 ± 0.044
5.516 ± 0.077
4.001 ± 0.089
0.764 ± 0.008
3.995 ± 0.059
5.430 ± 0.029
r2
Type of Inhibition
Non-competitive
Non-competitive
Competitive
4a
0.938
0.980
0.993
0.904
0.999
0.997
0.954
0.993
0.986
4b
4c
4d
Non-competitive
Competitive
4e
4f
Competitive
4g
4h
Non-competitive
Competitive
Allopurinol
Competitive
2.4. Binding pose analyses
In Figure 9A, the binding pose of quercetin and in Figure 9B the binding pose of
salicylic acid are inspected. X-Ray conformations are displayed on the left column and
docked positions are displayed on the right column. Also, RMSD of the best docked position
from crystalized ligand is calculated. The RMSD values for quercetin and salicylic acid are
0.9 Å and 1.2 Å, respectively. If the RMSD is lower than 2Å, docking process count as
successful [11]. The visual and RMSD inspection of the binding site and known binding
poses validated that docking scoring function is able to pick the correct pose for these
molecules.
Before moving to the compounds of this study, allopurinol and oxypurinol which are
clinically known inhibitors of XO, are also docked to the binding site, and their binding energies
are 4.48  0.01 and 6.60  0.00 kcal/mol, respectively.

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Figure 9. X-Ray (yellow) and docked (orange) positions A) Quercetin and B) Salicylic
Acid.
All eight compounds showed similar binding poses and their binding energies range from
-9.8 kcal/mol to -8.8 kcal/mol which are all better than oxy or allopurinol binding energies. Three
dominant interaction poses observed are summarized in Table 2 with their corresponding binding
energies. In Figure 10 panels A, B and C, the binding energy of each compound is displayed in the
graph with the pose on the bottom of the panel respectively for the three poses.
Table 2. Binding energies of each pose and percentage of the pose observed in 1000
conformations.
Compounds
Lowest Binding Energies (kcal/mol)
Number in Cluster (1000 run)
Pose 1
Pose 2
Pose 3
Pose 1
24%
25%
29%
29%
28%
33%
26%
30%
Pose 2
24%
24%
24%
23%
21%
23%
22%
21%
Pose 3
29%
24%
22%
3%
19%
5%
19%
3%
4a
4b
4c
4d
4e
4f
-9.82±0.05
-9.70±0.02
-9.83±0.03
-9.11±0.04
-9.80±0.04
-8.98±0.05
-9.70±0.09
-9.30±0.05
-8.55±0.01
-8.84±0.05
-8.63±0.04
-8.54±0.08
-8.76±0.03
-8.63±0.05
-8.83±0.04
-8.47±0.04
-8.44±0.01
-8.54±0.01
-8.49±0.01
-7.90±0.06
-8.60±0.02
-8.17±0.09
-8.44±0.03
-7.98±0.04
4g
4h
Namely, first pose is the cluster which contains the lowest binding energy conformation for each
compound (Figure 10 A). Bis-chalcone derivatives show a T-shaped π-π stacking interaction with
PHE1009 (Figure 10 B and C). This interaction is shown to be important in substrate binding and
activation [12]. Another T-shaped π-π interaction is seen between ligand and PHE649 in addition
to three hydrogen bonds between ligand and protein residues ASN768, LYS771, GLU802 (Figure
10 B and C). In this pose, compounds 4c, 4e and 4g display the lowest binding energy (Figure 10
A).

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The second pose of the compounds (Figure 10 E and H) shows single T-shaped π-π stacking with
PHE1009 and three hydrogen bonds with ASN768, LYS771, GLU802 residues. Formation of
halogen bond between; amino group of LYS771 with compounds of 4e and 4g and hydroxyl group
of THR1010 with compound 4e has been observed. Binding energies for this pose varies between
-8.84 to -8.47 kcal/mol (Figure 10B).
When pose 3 is considered, it resembles a pose similar to pose 1. Both of the phenyl rings are
curved though binding pocket. One of the phenyl rings is sandwiched between PHE1009 and
PHE914 and makes parallel π-π stacking with PHE914. Two hydrogen bonds are recognized
between molecules and target protein (GLU802, SER876). Hydroxyl group of THR1010 forms a
halogen bond to compound 4h, carboxyl group of GLU802 forms halogen bond with 4b and 4g.
For pose 2 and 3, the binding energy shows a similar trend, namely compounds 4b, 4e and 4g show
lowest binding energies (Figure 10 B and C respectively).

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A)
B)
C)
D)
E)
F)
G)
H)
I)
Figure 10. Binding Free Energies for eight compounds A) Pose 1, B) Pose 2, C) Pose 3.
3D view of each pose in binding site D) Pose 1, E) Pose 2, F) Pose 3 (Pictures were prepared
using VMD) [13]. 2D interaction graphs of each pose G) Pose 1, H) Pose 2, I) Pose 3 (2D
interaction maps were taken from Maestro) [14]. Compound 4d was used as a representation
in figures.
3.
Conclusions
Two series of bis-chalcone functionalized in B-ring were synthesized to evaluate
their effects as inhibitors of xanthine oxidase and anticancer activities. Eight bis-chalcone
derivatives were synthesized by Claisen-Schmidt condensation reactions. The bis-chalcone

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with substituted at 2- and 2,5- position (compounds 4b and 4g) were found to be more potent
as compared to the other substituted derivatives 4a, 4c, 4d, 4e, 4f and 4 h. The nature of
substituents at the 3- and 4-position of compounds 4c, 4d, and disubstituted compounds 4e,
4f and 4h do not influence sufficiently their inhibitory activity. Docking of inhibitors to the
active site of XO allows to explain possible binding modes of the two groups of bis-
chalcone derivatives.
There is generally a compatibility between enzyme inhibition assay and docking
results. Molecules 4b, 4e and 4g which form a halogen bond, in addition to the other
interaction types, displayed stronger binding in general. Only compound 4e results showed
some incompatibility. This is due to problems with the dissolution of compound 4e in vitro
enzyme inhibition studies. Structure-activity relationship analysis in conjunction with
molecular docking indicated that the most active XO inhibitors carried a minimum of one
fluoro group in 2 position. These data reveal that fluoro substituted bis-chalcone scaffold
may be useful for designing new inhibitors that target XO.
In parallel, the ability of the synthesized bis-chalcones to cytotoxicity was
determined. In vitro activity of the compounds was performed by MTT test agains MCF-7
and Caco-2 cell lines. Compound 4g showed the best IC50 value against MCF-7 (1.9 µM)
and 4g and 4h showed for Caco-2 cell line as 7.3 and 6.8 µM, respectively. Also, compound
4g and 4h showed the highest toxicity, and the images monitored by invert microscope at
the end of 24 h confirmed the results. These results provided an important basis for further
optimization of compound 4g as a potential anticancer agent.
In the future, it is planned to carry out the designs for the solution of the existing
dissolution problem in the compounds and to reveal the mechanisms of actions.
4.
4.1. General
All reagents used were commercially available unless otherwise specified. Melting
Experimental Part
points were measured with Gallenkamp melting point devices. IR Spectra: PerkinElmer
Spectrum One FT-IR spectrometer. 1H- and 13C-NMR Spectra: Varian 400 and Bruker 400
spectrometers. Elemental analysis results were obtained on a Leco CHNS-932 instrument.

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4.1.1. 1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)diethanone (2)
To a solution of trimethoxybenzene (1) (5 g, 29.72 mmol) in DCM (50 mL) AlCl3
(12.209 g, 91.56 mmol) and acetyl chloride (6.15 mL 86.50 mmol) were added sequentially
and stirred at -15 oC for 1,5 h. The reaction was then allowed to warm to room temperature
and stirred for an additional 20 h at this temperature. Completion of reaction was monitored
by TLC analysis. After that, water (50 mL) was slowly added to the mixture at 0 oC. The
mixture was then extracted with DCM (3 x 100 mL). The combined extracts were dried
over Na2SO4 and the solvent was removed in vacuo. The crude product purified via coloumn
chromatography over silica gel using gradient elution with EtOAc and hexanes to yield
1
compound 2 as a white solid (55% yield). H NMR (400 MHz, CDCl3) δ 14.20 (s, 1H),
1
5.93 (s, 1H), 3.89 (s, 6H), 2.53 (s, 6H). The H NMR spectrum is in agreement with the
reported data of [15].
4.1.2. General procedure for preparation of bis-chalcones (4a-h)
To a solution of compound 2 in MeOH (30 mL/1 mmol of substrate) benzaldehyde
derivatives (3a-h) (3 eq) and 50% KOH solution (15 mL/1 mmol of substrate) were added
sequentially and stirred for 24 h at room temperature. Completion of reaction was monitored
by TLC analysis. The resulting mixture was concentrated in vacuo, diluted with NH4Cl
solution (50 mL) and extracted with ethyl acetate (2 x 50 mL). The organic layer was dried
over Na2SO4 and concentrated in vacuo. The residual solid was purified by column
chromatography to yield the desired products.
4.1.3. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-phenylprop-2-en-1-one) (4a)
The above general procedure was followed with benzaldehyde (3a) to yield 4a as a
o
1
dark yellow solid (62% yield). Rf (EtOAc/Hexanes 60:40) = 0,50; mp = 163-164 C; H
NMR (400 MHz, CDCl3) δ 14.26 (s, 1H), 7.64 (d, 2H, J = 15.8 Hz), 7.61 – 7.53 (m, 6H),
7.45 (d, 2H, J = 15.8 Hz), 7.41 – 7.34 (m, 4H), 6.06 (s, 1H), 3.97 (s, 6H); 13C NMR (100
MHz, CDCl3) δ 193.2, 164.8, 163.9, 143.9, 135.4, 130.5, 129.1, 128.7, 128.1, 108.9, 86.8,
56.2; Anal. calcd for C26H22O5: C, 75.35; H, 5.35; Found: C,75.75; H, 5.36.

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4.1.4. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(2-fluorophenyl)prop-2-en-
1-one) (4b)
The above general procedure was followed with 2-fluorobenzaldehyde (3b) to yield 4b as
a dark yellow solid (90% yield). Rf (EtOAc/Hexanes 60:40) = 0,53; mp = 139-140 °C; 1H NMR
(400 MHz, CDCl3) δ 14.27 (s, 1H), 7.74 (d, 2H, J = 16.0 Hz), 7.59 (t, 2H, J = 7.6 Hz), 7.54 (d, 2H,
J = 16.0 Hz), 7.35 (dd, 2H, J = 13.6, 5.6 Hz), 7.17 (t, 2H, J = 7.6 Hz), 7.12 – 7.05 (m, 2H), 6.05 (s,
1H), 3.96 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 193.1, 164.9, 164.1, 163.1, 160.5, 136.3, 131.9,
131.8, 130.5, 130.5, 129.8, 129.7, 124.7, 124.6, 123.5, 123.4, 116.5, 116.3, 108.8, 86.8, 56.2; Anal.
calcd for C26H20F2O5: C, 69.33; H, 4.48; Found: C,69.42; H, 4.75.
4.1.5. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(3-fluorophenyl)prop-2-en-
1-one) (4c)
The above general procedure was followed with 3-fluorobenzaldehyde (3c) to yield 4c as a
1
dark yellow solid (99% yield). Rf (EtOAc/Hexanes 60:40) = 0,56; mp = 175-176 °C; H NMR
(400 MHz, CDCl3) δ 14.29 (s,1H), 7.56 (d, 2H, J = 15.8 Hz), 7.40 (d, 2H, J = 15.8 Hz), 7.36 – 7.29
(m, 4H), 7.27 – 7.21 (m, 2H), 7.10 – 7.01 (m, 2H), 6.05 (s, 1H), 3.96 (s,6H); 13C NMR (100 MHz,
CDCl3) δ 192.8, 164.9, 164.4, 164.1, 161.9, 142.2, 142.2, 137.6, 137.6, 130.7, 130.6, 129.2, 124.8,
124.7, 117.4, 117.2, 114.8, 114.6, 108.7, 86.9, 86.8, 56.3, 56.3; Anal. calcd for C26H20F2O5: C,
69.33; H, 4.48; Found: C, 69.37; H, 4.68.
4.1.6. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(4-fluorophenyl)prop-
2-en-1-one) (4d)
The above general procedure was followed with 4-fluorobenzaldehyde (3d) to yield 4d as
a dark yellow solid (78% yield). Rf (EtOAc/Hexanes 60:40) = 0,53; mp = 177-178 °C; 1H NMR
(400 MHz, CDCl3) δ 14.33 (s, 1H), 7.61 – 7.50 (m, 6H), 7.35 (d, 2H, J = 15.8 Hz), 7.07 (t, 4H, J =
13
8.6 Hz), 6.05 (s, 1H), 3.95 (s, 6H); C NMR (100 MHz, CDCl3) δ 192.9, 165.4, 164.8, 163.9,
162.9, 142.6, 131.6, 131.5, 130.6, 130.5, 127.8, 116.3, 116.1, 108.8, 86.9, 86.8, 56.3, 56.2; Anal.
calcd for C26H20F2O5: C, 69.33; H, 4.48; Found: C, 69.42; H, 4.69.

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4.1.7. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(2,3-
difluorophenyl)prop-2-en-1-one) (4e)
The above general procedure was followed with 2,3-difluorobenzaldehyde (3e) to yield 4e
as a dark yellow solid (90% yield). Rf (EtOAc/Hexanes 60:40) = 0,33; mp = 215-216 °C; 1H NMR
(400 MHz, CDCl3) δ 14.22 (s, 1H), 7.69 (d, 2H, J = 16.0 Hz), 7.55 (d, 2H, J = 16.1 Hz), 7.34 (s,
2H), 7.24 – 7.03 (m, 4H), 6.06 (s, 1H), 3.97 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 192.7, 165.1,
164.3, 134.9, 131.7, 131.6, 125.8, 124.6, 124.5, 124.5, 118.7, 118.5, 108.7, 86.8, 56.2; Anal. calcd
for C26H18F4O5: C, 64.20; H, 3.73; Found: C, 64.57; H, 4.11.
4.1.8. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(2,4-
difluorophenyl)prop-2-en-1-one) (4f)
The above general procedure was followed with 2,4-difluorobenzaldehyde (3f) to yield 4f
as a dark yellow solid (74% yield). Rf (EtOAc/Hexanes 60:40) = 0,46; mp = 177-178 °C; 1H NMR
(400 MHz, CDCl3) δ 14.28 (s, 1H), 7.67 (d, 2H, J = 16.0 Hz, A part of AB system), 7.58 (dd, 2H,
J = 15.0, 8.4 Hz), 7.47 (d, 2H, J = 16.1 Hz, B part of AB), 6.87 (ddd, 4H, J = 21.8, 13.8, 5.3 Hz),
6.04 (s, 1H), 3.95 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 192.8, 165.4, 165.3, 164.9, 164.1, 163.4,
163.3, 162.9, 162.8, 160.8, 160.7, 135.2, 130.9, 130.9, 130.8, 130.8, 130.0, 129.9, 120.0, 119.9,
112.4, 112.4, 112.2, 112.1, 108.7, 105.1, 104.8, 104.6, 86.8, 56.2; Anal. calcd for C26H18F4O5: C,
64.20; H, 3.73; Found: C, 64.45; H, 3.95.
4.1.9. (2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(2,5-
difluorophenyl)prop-2-en-1-one) (4g)
The above general procedure was followed with 2,5-difluorobenzaldehyde (3g) to yield 4g
as a dark yellow solid (99% yield). Rf (EtOAc/Hexanes 60:40) = 0,60; mp = 166-167 °C; 1H NMR
(400 MHz, CDCl3) δ 14.24 (s,1H), 7.66 (d, 2H, J = 16.0 Hz), 7.47 (d, 2H, J = 16.0 Hz), 7.32 – 7.16
13
(m, 2H), 7.10 – 6.89 (m, 4H), 6.04 (s,1H), 3.96 (s, 6H); C NMR (100 MHz, CDCl3) δ 192.5,
176.0, 165.1, 164.3, 160.1, 159.0, 157.7, 156.5, 134.7, 131.3, 131.2, 124.8, 124.7, 124.7, 124.6,
118.4, 118.3, 118.1, 118.1, 117.7, 117.6, 117.4, 117.4, 115.2, 115.2, 114.9, 108.6, 86.9, 86.9, 56.3,
56.2; Anal. calcd for C26H18F4O5: C, 64.20; H, 3.73; Found: C, 64.21; H, 4.03.

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4.1.10.(2E,2'E)-1,1'-(2-hydroxy-4,6-dimethoxy-1,3-phenylene)bis(3-(3,4-
difluorophenyl)prop-2-en-1-one) (4h)
The above general procedure was followed with 3,4-difluorobenzaldehyde (3h) to yield 4h
as a dark yellow solid (80% yield). Rf (EtOAc/Hexanes 60:40) = 0,43; mp = 211-212 °C; 1H NMR
(400 MHz, CDCl3) δ 14.26 (s, 1H), 7.52 (d, 2H, J = 15.8 Hz), 7.44 – 7.37 (m, 2H), 7.33 (d, 2H, J
= 15.8 Hz), 7.28 (d, 2H, J = 2.2 Hz), 7.17 (dd, 2H, J = 18.0, 8.3 Hz), 6.05 (s, 1H), 3.97 (s, 6H); 13C
NMR (100 MHz, CDCl3) δ 192.4, 164.9, 164.1, 153.0, 152.9, 152.1, 151.9, 150.5, 150.4, 149.6,
149.5, 141.2, 132.7, 132.6, 132.6, 128.9, 128.9, 125.5, 125.5, 125.4, 125.4, 118.1, 117.9, 116.7,
116.6, 108.7, 86.8, 56.3; Anal. calcd for C26H18F4O5: C, 64.20; H, 3.73; Found: C, 64.26; H, 3.90.
4.2. Cytotoxicity Studies
The cytotoxicity of the compounds was evaluated by MTT (3- (4,5-Dimethylthiazol-
2-yl) -2,5-Diphenyltetrazolium Bromide) test. MCF-7 (human breast adenocarcinoma) and
Caco-2 (human colorectal adenocarcinoma) cell lines were used in the study. First, cells
o
were incubated with DMEM medium at 37 C under 5% CO2 until being 80% confluent.
Then, the cells were removed from the surface by trypsin and counted with Thoma counting
chamber and seeded in 96 well plates as 7x103 cells/well. Next, plates were incubated under
the same conditions for 24 h and the medium was replaced with compound solutions
prepared with media (including less than 1% DMSO) at the certain concentrations (3.125,
6.25, 12.5, 25, 50 and 100 µM for each cell type). After incubating the plates 24 h under the
same conditions, the compound solutions were then removed from the plates and 90 µL of
DMEM and 10 µL of MTT (5 mg/mL in PBS, pH 7.4) were added and incubated for 4 h
under the same conditions. At the end of the period, the MTT solution was removed
followed by adding 100 µL of DMSO and the absorbance at 550 nm with a microplate
reader was measured. Control wells were considered as 100% viable and IC50 of each
compound was calculated by the linear curve of concentration vs % inhibiton graphics [16].
4.3. In vitro Assay of Xanthine Oxidase (XO) Inhibitory Activity
In vitro bovine XO inhibitory activity was performed spectrophotometrically by
measuring the uric acid formation at 295 nm at 37 oC. In order to determinate IC50 value for
XO inhibition, the compounds at different concentrations were added to the reaction

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mixture and the absorbance was measured at 295 nm. The assayed method was based on
the procedure reported by Sweeney a. P. et al. [17]. For enzyme assay, 50 mM of phosphate
buffer (pH = 7.5), 1 mM of xanthine, and 0.2 U of XO enzyme were used. The enzymes
were pre-incubated for 10 min, with tested compounds, then the reaction was started by
addition xanthine to the reaction mixture. The tested compounds were dissolved in DMSO,
then diluted with phosphate buffer, the final concentration of DMSO in the reaction mixture
was less than 0.01% v/v which is not an interference value with enzyme assay [18]. All the
experiments were performed in triplicates, and values were expressed as means of three
experiments. Allopurinol was used as a positive control. The IC50 values of compounds
were determined by using the percent inhibition of XO. The percent inhibition of XO
activity was calculated as the following formula:
% Inhibition = (A-B)/A×100
where A = the absorbance at 295 nm without the test compound,
B = the absorbance at 295 nm with the test compound.
4.4. Molecular Modelling
The three dimensional (3D) structures of ligands were drawn by using the chemical
modeling software Avogadro [19]. Geometry optimization tool embedded in Avogadro was
used for structural refinement and small molecules were minimized using MMF94x force
field [20].
In order to theoretically investigate the potency and binding pose of these eight
molecules, Autodock molecular docking software was used [21]. The coordinates of the
protein were obtained from PDB databank, 3NVY [22]. This structure of XO at 2 Å
resolution contains quercetin molecule in its binding site. First quercetin and water
molecules were removed and the catalytic part of the protein was prepared with the
molybdenum metal ions (Mo) and cofactor MTE (phosphoric acid mono 2-amino-5,6-
dimercapto-4-oxo-3,7,8a,9,10,10a-hexahydro-4H-8-oxa-1,3,9,10-tetraaza-anthracen-7-yl
methyl).
To create coordinate, grid box and docking parameter files, AutoDockTool was used
[21]. Gasteiger partial charges were assigned to each atom. Autodock 4.2 was used for grid

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mapping and docking [21]. Energy Grid box was arranged as 50 × 50 × 50 × Å and its center
was taken according to its native ligand center. To search for all conformations, Lamarckian
genetic algorithm was applied [23]. For each ligand 1000 runs were performed using the
population size of 300, 10.000.000 million energy evaluations, and the maximum of 27000
generations to span all the conformational space. From resulting 1000 docked positions, the
poses are ranked according to the binding energy obtained from AutoDock 4.2. For the eight
bis-chalcone derivatives, three best scored docking clusters are displayed in the results
section. Docked conformers are clustered according to their Root Mean Square Deviation
(RMSD). All molecular graphics material was prepared using VMD [13].
For an initial validation study of the binding site, quercetin and salicylic molecules
were re-docked to the prepared binding site. Best-scoring docked pose of the molecules
obtained from the software was checked against X-ray crystal orientation and conformation
of the ligand. This is often referred to as the “bound” docking problem [24]. Additionally,
allopurinol and oxypurinol known inhibitors of XO, were also docked to the prepared
binding site for calculating reference binding energy values.
Acknowledgements
We thank the Scientific and Technological Research Council of Turkey (TUBITAK.
Grant Number: 114Z554).
Appendix A. Supplementary material
Supplementary data to this article can be found online at
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