Design, synthesis and antiproliferative activity evaluation of fluorine-containing chalcone derivatives

Article abstract of DOI:10.1080/07391102.2020.1848627

A series of new chalcones containing fluoro atom at B ring have been designed, synthesized, and evaluated to be antiproliferative activity against a panel of human tumor cell lines. Some of the analogs (8, 9, 12, 45, 46 and 48) displayed powerful antiproliferative effects to certain human tumor cells, but all of them were devoid of any cytotoxicity towards the normal HEK 293. Acridine orange staining data supported that the cytotoxic and antiproliferative effects of the synthesized analogs on tumor cells are mediated through apoptosis. The compounds 12 and 46 manifested concentration-dependent antiproliferative activity in human hepatocellular carcinoma cell lines using an xCELLigence assay. The structures and antiproliferative activity relationship were further supported by in silico molecular docking study of the compounds against tubulin protein which suggests our compounds interference to cell division. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1848627

Journal of Biomolecular Structure and Dynamics
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Design, synthesis and antiproliferative activity
evaluation of fluorine-containing chalcone
derivatives
Serdar Burmaoglu , Derya Aktas Anil , Arzu Gobek , Deryanur Kilic , Derya
Yetkin , Nizami Duran & Oztekin Algul
To cite this article: Serdar Burmaoglu , Derya Aktas Anil , Arzu Gobek , Deryanur Kilic , Derya
Yetkin , Nizami Duran & Oztekin Algul (2020): Design, synthesis and antiproliferative activity
evaluation of fluorine-containing chalcone derivatives, Journal of Biomolecular Structure and
Dynamics, DOI: 10.1080/07391102.2020.1848627
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1848627
Design, synthesis and antiproliferative activity evaluation of fluorine-containing
chalcone derivatives
Serdar Burmaoglua, Derya Aktas Anilb, Arzu Gobeka, Deryanur Kilica, Derya Yetkinc, Nizami Durand and Oztekin
Algule
a
b
€
€
Department of Chemistry, Faculty of Science, Ataturk University, Erzurum, Turkey; Department of Chemistry and Chemical Process
c
Technologies, Erzurum Vocational High School, Ataturk University, Erzurum, Turkey; Advanced Technology Education Research and
Application Center, Mersin University, Mersin, Turkey; Department of Medical Microbiology, Medical Faculty, Mustafa Kemal University,
Antakya-Hatay, Turkey; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mersin University, Mersin, Turkey
d
e
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 13 April 2020
Accepted 4 November 2020
A series of new chalcones containing fluoro atom at B ring have been designed, synthesized, and eval-
uated to be antiproliferative activity against a panel of human tumor cell lines. Some of the analogs
(8, 9, 12, 45, 46 and 48) displayed powerful antiproliferative effects to certain human tumor cells, but
all of them were devoid of any cytotoxicity towards the normal HEK 293. Acridine orange staining
data supported that the cytotoxic and antiproliferative effects of the synthesized analogs on tumor
cells are mediated through apoptosis. The compounds 12 and 46 manifested concentration-depend-
ent antiproliferative activity in human hepatocellular carcinoma cell lines using an xCELLigence assay.
The structures and antiproliferative activity relationship were further supported by in silico molecular
docking study of the compounds against tubulin protein which suggests our compounds interference
to cell division.
KEYWORDS
Synthesis; Chalcone; MTT;
antiproliferative activity;
flour effect
Introduction
potent and more selective anti-cancer agents. Presence of
chalcone-compounds as main components or as a substitu-
ent in biologically active molecules encourages medicinal
chemists to synthesize chalcone-containing derivatives. Also
known as a,b-unsaturated ketones, chalcones are not only
good precursors for synthetic derivatization, but also an
important component of natural products (Singh et al.,
2014). Chalcones and their synthetic analogs have consider-
able number of biological activities (Karthikeyan et al., 2014).
Cancer disease is one of the leading causes of death world-
wide. Prevalence of various cancer types has nearly doubled
within recent years. With this increased rate in mind, if new
drugs and therapeutic methods cannot be developed, this
rate is expected to increase even further within the coming
years (Singh et al., 2014). Although there are currently many
treatment methods, the rapidly increasing rates of the dis-
ease prevalence urge researchers for developing safer, more Although in literature, there are many known methods to
CONTACT Serdar Burmaoglu
sboglu@atauni.edu.tr; Oztekin Algul
oztekinalgul@mersin.edu.tr
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1848627.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
S. BURMAOGLU ET AL.
Figure 1. Selected examples of fluorinated drugs.
synthesize chalcones, in general, chalcones can be readily (Burmaoglu et al., 2016). In particular, it has been determined
that the molecules whose literature data are designed will
have high efficiency in five cancer cells.
synthesized with a Claisen-Schmidt condensation reaction in
the presence of an acid or a base (Matos et al., 2015).
Molecules that contain multiple pharmacophores are known
to be much more beneficial in treatment of cancer (Abbot
et al., 2017).
Based upon these observations, we designed and synthe-
sized a series of chalcone derivatives containing fluorine
atom at different positions of a series of B-ring and their
antiproliferative properties against various cancer cell lines
were investigated.
On the other hand, fluorine is the 13th most common
element found on Earth. Despite this fact, it is quite rare in
natural organic products, with currently only 13 natural
organic products known to include fluorine atom within their
structures (Menaa et al., 2013). Also, it is becoming increas-
ingly common in the synthetic derivatives, in parallel with
the increasing number of medicinal chemistry studies on
pharmaceutical compounds including fluorine atom. For
instance, the rate of fluorine-containing pharmaceutical com-
pounds was about 2% back in 1970s, whereas now it is
around 18%. In USA, 9 out of 31 new drugs that were
licensed in 2002 contain fluorine in their structure (Figure 1).
Thus, it can be said that the proportion of drugs that contain
at least one fluorine atom is 25% in the whole world. When
we consider that organofluorine compounds are rare in nat-
Finally, the antiproliferative activities of thirty-four new syn-
thesized chalcone and dihydrochalcone derivatives (11, 13,
17, 19, 22-33, 42, 44, 45, 48, 50, 51, 61-66, 68-73) were eval-
uated. In addition, the previously synthesized twelve chal-
cones/dihydrochalcones (8-10, 12, 14-16, 18, 43, 46, 49 and
52) were included only for comparison purposes. 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). Acridine orange staining and MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay
were conducted on different cancer cell types in the presence
of our derivatives in order to measure their apoptotic as well
as antiproliferative activities, respectively. The concentration-
dependent antiproliferative activities of the highest active
compounds against HepG2 cell line were also revealed using
an xCELLigence assay. The structure activity properties were
further supported by in silico molecular docking study of their
interaction with the tubulin protein.
ꢀ
ꢀ
ural products, this 25% is obviously a quite high rate (Begue
& Bonnet-Delpon, 2008).
Due to its unique properties, fluorine atom is increasingly
being incorporated to the structure of small bioactive mole-
cules. However, despite the supportive findings related to
the structure-activity relationship, biological effects of fluor-
ine atom are still not exactly clear. Surely, many effects of
the fluorine atom indicate that if fluorine is strategically
bound to appropriate site of the molecule, it results in very
important changes in the biological properties of the mol-
ecule compared to the non-fluorine derivatives of the same
molecule. Nonetheless, for certain situations, even all this
knowledge fails to aid in predicting the effect of fluorine
atom on the entire pharmacokinetics. Therefore, determin-
ation of its effect still continues as a trial and error process
(Isanbor & O’Hagan, 2006). Thus, organofluorine compounds
in medicinal chemistry still have significance for the purpose
of understanding the enigmatic effect of introduction of
fluorine atom to the molecular structure.
Results and discussion
Chemistry
As shown in Scheme 1, the target compounds 8-13 and 14-
19 were synthesized. The condensation of trimethoxyaceto-
phenone (1) with benzaldehyde (2) and fluoro-substituted
benzaldehydes (3-7) in an aqueous solution of KOH afforded
related chalcones 8-13 in 70-92% yields through the adop-
tion of a general synthesis protocol (Burmaoglu et al., 2016).
In this series, the reaction between 2,4,5-trifluoro benzalde-
hyde (7) and trimethoxy acetophenone (1) resulted in com-
pound 13. The fluorine atom at para position of compound
7 is very suitable for nucleophilic aromatic substitution. Since
this reaction is carried out in methanol and in strong basic
condition, the methoxide ion readily reacts with the fluorine
atom at the para position to give compound 13.
Thus, we have recently reported a preliminary account on
a new and facile route to chalcones containing fluoro atom
at B ring. The results of preliminary in vitro antiproliferative
activity confirmed that the fluoro-substituted chalcones at B
ring inhibited the growth of certain human cancer cell lines Hydrogenation of trimethoxychalcones 8-13 on Pd/C

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Scheme 1. Preparation of fluoro-substituted trimethoxy chalcones (8-13) and dihydrochalcones (14-19). Reagents and conditions: (i) KOH, MeOH, room tempera-
ture, 15 h; (ii) H2, Pd-C, EtOH, room temperature, 16 h.
Scheme 2. Preparation of fluoro-substituted mono/di-methoxychalcones (22-27) and dihydrochalcones (28-33). Reagents and conditions: (i) KOH, MeOH, room
temperature, 15 h; (ii) H2, Pd-C, EtOH, room temperature, 16 h.
afforded related dihydrochalcones 14-19 in 67-87% yields
(Scheme 1).
Condensation of trihydroxyacephenone (34) with benzal-
dehyde (2) and fluoro-substituted benzaldehydes (3-7) did
To synthesize fluoro-substituted mono/di-methoxy chal- not afford the related chalcones and only the starting materi-
cones and their dihydrochalcone derivatives (22-27 and als were recovered. Because of this setback, we decided to
28-33) we used the same method above. For this, the use a protective group for the hydroxyl groups. For this,
condensation of di or mono-methoxyacetophenone (20 or chloromethyl methyl ether (MOMCl), which is stable under
21) with fluoro-substituted benzaldehydes (3, 5, and 6) in an basic conditions and can be deprotected easily under mild
aqueous solution of KOH afforded related chalcones 22-27 in acidic conditions, was used. After the base-catalysed reaction
51-79% yields. Hydrogenation of mono/di-methoxychalcones of compound 35 with related benzaldehydes 2-7, the related
22-27 on Pd/C afforded related dihydrochalcones 28-33 in chalcones, except compound 47, were gotten by removal of
44-78% yields (Scheme 2).
MOM with HCl in MeOH. As a result of the reaction between

4
S. BURMAOGLU ET AL.
Scheme 3. Preparation of fluoro-substituted tri-hydroxychalcones (42-47) and dihydrochalcones (48-52 and 55). Reagents and conditions: (i) MOMCl, DIPEA, DCM,
0 ꢀC, 3 h; (ii) KOH, MeOH, room temperature, 15 h; (iii) HCl, room temperature, 12 h; (iv) H2, Pd-C, EtOH, room temperature, 16 h; (v) BnBr, K2CO3, DMF, room tem-
perature, 12 h, under N2; (vi) KOH, MeOH, room temperature, 15 h; (vii) H2, Pd-C, EtOH, room temperature, 16 h.
compound 35 and 7 a mixture of product was obtained and chalcones 61-63 in 42-76% yields. Hydrogenation of com-
could not be separated in any way by chromatographic
methods for compound 47. Therefore, we decided to use
benzyl chloride instead of MOMCl as a protecting group.
Condensation of 53 with compound 7 pleasingly afforded
related benzyl-protected chalcone 54. Hydrogenation of tri-
hydroxychalcones 42-46 on Pd-C afforded related dihydro-
chalcones 48-52 in 64-95% yields. Hydrogenation of 54 on
Pd-C did not afford the related dihydrochalcone 55. We got
a mixture of products and this mixture could not be sepa-
rated in any way by chromatographic methods for com-
pound 55 (Scheme 3).
To synthesize fluoro-substituted dihydroxy chalcones, we
used the same synthetic route of the trihydroxy chalcones.
For this, the reaction between 56 and fluoro-substituted ben-
zaldehydes (3-6) did not afford the related chalcones and
only the starting materials were recovered. Then we used
MOM-protected acetophenone 57 instead of compound 56
and condensation of 57 with fluoro-substituted benzalde-
hydes 3-6 gave related MOM-protected chalcones 58-60.
pounds 61-63 in the presence of Pd-C gave related dihydro-
chalcones 64-66 in 60-69% yields (Scheme 4).
Finally, preparation of fluoro-substituted monohydroxy
chalcones was summarized in Scheme 5. For this, we did not
need to protect the hydroxyl group with any protecting
group. Condensation of 67 with related benzaldehydes 3-6
gave related chalcones 68-70 in 54-83% yields and hydro-
genation of 68-70 in the presence of Pd-C gave desired
products 71-73 in 50-54% yields.
Consequently, five class of chalcone and dihydrochalcone
derivatives were synthesized according to functional group
differences in ring A as shown in Figures 2 and 3.
Biological evaluation
After successful completion of the synthesis studies, all syn-
thesized compounds were evaluated for their in vitro antipro-
liferative activities against a panel of five human malignant
After the removal of MOM with HCl in MeOH gave related cell lines, including human lung adenocarcinoma epithelial

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
Scheme 4. Preparation of fluoro-substituted dihydroxychalcones (61-63) and dihydrochalcones (64-66). Reagents and conditions: (i) MOMCl, DIPEA, DCM, 0 ꢀC, 3 h;
(ii) KOH, MeOH, room temperature, 15 h; (iii) HCl, room temperature, 12 h; (iv) H2, Pd-C, EtOH, room temperature, 16 h.
cell line (A549), human renal cancer cell line (A498), human
cervical cancer cell line (HeLa), human skin malignant melan-
oma cell line (A375), human hepatocellular cell line (HepG2)
and normal cell lines; human embryonic kidney cell line (HEK
293) and compared with those of methotrexate (MTX).
Then, in order to further strengthen our MTT data, we cal-
culated the apoptotic indexes of chalcone derivatives at 8
and 24 h time points by acridine orange staining test (Figure
4a-e). Compounds with the highest activities were further
examined by using acridine orange dye that stains the cyto-
plasmic changes during the apoptosis as well as by using
ethidium bromide that would indicate the chromosomal
changes through its binding to DNA. The samples were ana-
lyzed by immuno-flurosence microscope. Our compounds
effects were compared to that of the methotrexate, MTX.
Apoptotic indexes were determined by measuring the levels
Inhibitory studies in vitro antiproliferative activities and
structure-activity relationship (SAR)
To assess the specificity of the compounds, their toxicity was
tested against normal cells using HEK. The specificity of the
compounds was calculated as their IC50 values for normal cells
divided by their IC50 values for the specific cancer cells, accord-
ing to the specificity equation (Specificity ¼ IC50 values of com-
pound against normal cells/IC50 values of compound against
cell line of interest). The specificity of the compounds appeared
to be relatively moderate specificity toward all cancer cells.
The non-toxic concentrations of all compounds were
determined on the HEK-293 cell line. Then, antiproliferative
activity studies were performed at these non-toxic concentra-
tions. The compounds with the best selectivity values for
each cancer cell line are shown in Table 2.
The synthesized compounds were mono/di/tri methoxy or
of cell and nucleus shrinkage, chromatin condensation, and hydroxy substituted analogs of chalcone with varying com-
accumulation of the nuclear envelope at the periphery. Both ponents: fluorine atoms, ketone groups, or alkyl chains.
the floating and adherent cells were analyzed after 8th and Accordingly, a structure–activity relationship (SAR) was estab-
24th hours of incubation, 100 cells were counted in the same lished by comparing the antiproliferative activities of the dif-
square area to calculate the apoptotic index of each sample.
Cell growth inhibition was evaluated by using the stand-
ferent chalcone groups.
According to the resulting IC50 values of the cytotoxic
ard MTT colorimetric assay after exposure of cells to the test assay, potency and specificity (Table 1) the A549, A498, HeLa,
compounds for 96 h. The commercial antitumor agent MTX A375, and HepG2 cell lines were medium and sensitive to all
was used as positive control (Table 1).
of the synthesized analogues. The highest potency in the

6
S. BURMAOGLU ET AL.
Scheme 5. Preparation of fluoro-substituted mono-hydroxychalcones (68-70) and dihydrochalcones (71-73). Reagents and conditions: (i) KOH, MeOH, room tem-
perature, 15 h; (ii) H2, Pd-C, EtOH, room temperature, 16 h.
Figure 2. The structures of fluoro-substituted methoxy chalcones/dihydrochalcones.
culture of A549, A498 was recorded after treatment with tri-
hydroxy/trimethoxy in A ring and non/2-F/and 2,5-diF substi- the following compounds: trimethoxy (A ring) and nonsubsti-
tuted in B ring on chalcone derivatives 46 and 8, 9 and 12 tuted or 2,5-diF (B ring) chalcone derivatives (IC50:
The highest active molecules against A498 cell lines were
8
(IC50: 0.067 ꢁ 0.137 mM), which demonstrated about approxi- 0.067 mM), and 12 (IC50: 0.030 mM), and trihydroxy (A ring)
mately 6-fold lower potency than the antitumor agents MTX. and nonsubstituted (B ring) saturated chalcone derivative 48
A
potent submicromolar antiproliferative activity was (IC50: 0.15 mM), respectively, which exhibited 4 and 1.8 or 9-
recorded after treatment of A549 cells with trihydroxy and fold lower potency than MTX.
difluoro substituted saturated chalcone derivative 52 (IC50:
Several compounds exhibited submicromolar activities
0.136 mM), and dihydroxy and 2,5-difluouro substituted satu- toward the HeLa cells. These are 12 and 16 (IC50: 0.060 and
rated chalcone derivative 66 (IC50: 0.150 mM) and monohy- 0.066 mM), trimethoxy (A ring) and 2,5-difluoro (B ring) chal-
droxy and 4-fluouro substituted chalcone derivative 69 cone and trimethoxy (A ring) 3-F (B ring) saturated chalcone,
(IC50: 0.160 mM).
respectively. Also, trihydroxy (A ring) and 2,5-difluoro (B ring)

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 3. The structures of hydroxy chalcones/dihydrochalcones.
unsaturated and saturated chalcone derivatives 46, and 52 established by comparing the IC50 values of analogues with
(IC50: 0.068 mM) had significantly active against cancer cell those recorded for the corresponding linker group should
line, respectively.
be saturated.
The most potent compound in the culture of A375 cell
Remarkably, all of the synthesized chalcones and satu-
was analogue trihydroxy (A ring) 2,5-difluoro (B ring) chal- rated chalcones including mono and dimethoxy, mono and
cone 52 (IC50: 0.068 mM), which demonstrated about 3.5-fold dihydroxy completely inactive toward all cell lines, in con-
lower potency than MTX, in contrast to mono/di methoxy trast to trihydroxy and trimethoxy group of compounds and
and mono/di hydroxy chalcone and saturated chalcone MTX, which exhibited a potency cytotoxic activities (IC50:
derivatives, which was completely inactive against this cell 0.03-9.91 mM and 0. 022 mM respectively) against this cell
line. But, trimethoxy (A ring) and nonsubstituted or 2,5-diF (B lines. These results do suggest that the synthesized chal-
ring) unsaturated chalcone derivatives (8 and 12) were the cones represent selective antitumor agents, but this assump-
most active compounds against to A375 cancer cell lines, tion should be verified by additional in vitro experiments
0.067 and 0.060 mM, respectively.
with additional normal cell lines.
The highest potency in the HepG2 cell line was recorded
Based on the initial survey that we showed in Table 2,
after treatment with 12 (IC50: 0.060 mM), trimethoxy (A ring) indicating the selectivity of the compounds with non-toxic
2,5-diF (B ring); 43 (IC50: 0.029 mM) trihydroxy (A ring) 2-F concentrations of each compound on each cancer cell type.
(B ring), and 46 (IC50: 0.068 mM) trihydroxy (A ring) 2,5-diF In our further investigations, we focused on the compounds
(B ring) which exhibited approximately 1 and 2-fold lower with the highest selectivity at the lowest concentration on
potency than MTX.
each cell line.
In order to correlate the structure of synthesized chalcone
The selected compounds and MTX were exposed for a
derivatives with their cytotoxic activities, we first considered period of 0–96 h for each cancer cell line. At the end of the
the effect of flour atoms at B ring. None substituted com- incubation, all cells were evaluated and subjected to an
pounds; 8, 14, 42 and 48 have been used as controls in this assessment of their cytotoxic responses using an MTT assay
SAR analysis. As the data in Table 1 reveal the introduction (Figures 5–9) (McGahon et al., 1995; Mosmann, 1983).
of fluoro atom caused the increased antitumor potency ori-
As shown in 5-9, the proliferation of cancer cells treated
ginally displayed by control compounds, especially 2,5-diF with a non-toxic and in lower concentration dose of com-
compounds. As the data in Table 1 indicate the removal of pounds and MTX was inhibited in a time-dependent manner.
fluoro atom decreased antiproliferative activity, against After a short treatment (24 h), MTX was more effective and
majority of tumor cell lines under evaluation.
the amount of live cells was lower than that observed with
The next round of modifications undertaken to improve the compounds. The antiproliferative activity of all the tested
the antiproliferative activities have been performed by chal- compounds increased as the exposure time was extended, as
cone structure to saturated chalcone structure of linker proven by the decline in the number of surviving cells. After
group of two phenyl rings. The relationships between these a longer exposure, all tested compounds had an activity
structural changes and antiproliferative potencies were equal to that of MTX.

8
S. BURMAOGLU ET AL.
Figure 4. a. Apoptotic cell index of compound “12” in A549 cell culture; b. Apoptotic cell index of compound “48” in A489 cell culture; c. Apoptotic cell index of
compound “12” in HeLa cell culture; d. Apoptotic cell index of compound “12” in A375 cell culture; e. Apoptotic cell index of compound “12” in HepG2 cell culture.
control reagent had comparable effects on the cancer cells.
In order to further strengthen our MTT data, we did acridine Overall, this data supports high efficiency of the newly gener-
Detection of apoptosis by acridine orange staining test
orange staining on the cancer cell lines treated with the chal-
cone derivatives with the highest specificity as well as activity
against them. After their careful evaluation at 8 and 24 h time
points, we calculated their apoptotic indexes as shown in
Figure 4a-e. In line with MTT assay data appropriate compounds
exerted anticancer effect on each cancer cell line in a dose and
time dependent manner by inducing the apoptosis. These
results hint the possible intracellular pathway of antiprolifera-
tive effect of our compounds on each cancer cell type.
Apoptosis was the main route of their anticancer activities
ated chalcone derivatives on versatile cancer cell types. Their
activities and specificities against the cancer cell types ranged
based on the differences in their structures.
xCELLigence cytotoxicity protocol
The use of the xCELLigence system for label-free and real-
time monitoring of cell viability was described here. The
monitoring of cell viability by the xCELLigence system was
made it possible to distinguish between different perturba-
based on acridine orange staining. Our compounds and MTX tions of cell viability. We have used the cell index

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Table 1. In vitro cytotoxicity of fluoro-substituted chalcones analogues and MTX.
ꢂ
HEK293
A549
A498
Hela
A375
HepG2
Specificity
Hela
Structure
Substituent
Trimethoxy
IC50 (lM)a
A549
A498
A375
HepG2
Comp. No
8
9
–
2-F
3-F
4-F
0.335
0.632
0.632
3.161
0.598
9.91
1.665
1.643
0.657
8.21
0.134
0.126
0.253
0.253
0.120
4.95
0.533
0.526
0.263
1.03
0.067
0.126
0.126
0.253
0.030
9.91
0.533
0.526
0.526
1.03
0.134
0.126
0.506
4.047
0.060
2.48
0.533
0.263
0.066
1.03
0.067
0.126
0.506
1.265
0.060
1.24
0.533
0.526
0.263
2.05
0.134
0.126
0.253
1.265
0.060
9.91
0.533
0.263
0.263
2.05
2.5
5.0
2.5
12.5
5.0
2.00
3.125
3.125
2.5
5.0
5.0
5.0
12.5
20.0
1.00
3.1
3.1
1.3
7.97
6.3
2.5
5.0
1.25
0.8
10.0
4.00
3.125
6.25
10.0
1.00
6.25
3.99
5.0
5.0
1.25
2.5
10.0
7.99
3.125
3.125
2.5
0.50
3.125
3.99
2.5
5.0
2.5
10
11
12
13
14
15
16
17
18
19
2.5
2,5-diF
2,5-DiF-4-OMe
–
2-F
3-F
4-F
2,5-diF
2,5-DiF-4-OMe
Dimethoxy
2-F
4-F
2,5-diF
2-F
10.0
1.00
3.125
6.25
2.5
0.50
3.125
2.00
7.97
6.3
3.99
1.551
7.35
0.248
1.84
0.248
7.35
0.248
1.84
0.496
1.84
0.496
3.67
1.00
22
23
24
28
29
30
1.09
2.18
4.11
1.22
4.88
2.28
0.27
2.18
1.03
9.76
4.88
2.28
0.27
2.18
1.03
2.44
9.76
4.56
0.54
1.09
1.03
4.88
4.88
9.12
0.27
1.09
1.03
9.76
2.44
2.28
0.54
1.09
1.03
4.88
2.44
1.14
4.04
1.00
3.99
0.13
1.00
1.00
4.04
1.00
3.99
0.50
0.50
0.50
2.02
2.00
3.99
0.25
1.00
0.25
4.04
2.00
3.99
0.13
2.00
1.00
2.02
2.00
3.99
0.25
2.00
2.00
4-F
2,5-diF
Monomethoxy
2-F
4-F
2,5-diF
2-F
25
26
27
31
32
33
2.28
2.28
2.14
2.56
0.64
1.19
0.28
1.14
0.53
1.28
0.32
0.59
0.57
1.14
0.53
1.28
0.64
0.30
0.57
2.28
0.53
0.64
0.32
0.30
0.57
2.28
1.07
1.28
1.28
0.59
0.28
1.14
0.53
1.28
0.16
0.59
8.14
2.00
4.04
2.00
2.00
2.02
4.00
2.00
4.04
2.00
1.00
3.97
4.00
1.00
4.04
4.00
2.00
3.97
4.00
1.00
2.00
2.00
0.50
2.02
8.14
2.00
4.04
2.00
4.00
2.02
4-F
2,5-diF
Trihydroxy
–
2-F
3-F
4-F
2,5-diF
–
2-F
3-F
42
43
44
45
46
48
49
50
51
52
2.44
0.365
4.56
2.28
1.711
2.42
1.810
1.19
1.19
0.340
0.61
0.146
1.14
0.57
0.137
0.15
0.145
0.15
0.30
0.136
0.61
0.729
1.14
0.57
0.274
0.15
0.145
0.30
0.15
0.136
0.61
2.334
2.28
0.28
0.068
0.30
0.579
0.30
1.19
0.068
0.61
0.146
1.14
0.14
0.274
0.30
0.579
0.59
1.19
0.068
0.61
0.029
1.14
0.14
0.068
0.30
0.290
0.59
0.30
0.136
4.00
2.5
4.00
0.5
4.00
4.00
6.3
16.13
12.5
3.97
7.93
2.5
4.00
0.156
2.00
8.14
25.0
8.07
3.125
3.97
1.00
5.0
4.00
2.5
4.00
12.5
4.00
16.29
25.0
8.07
6.25
2.02
3.97
2.5
4.00
4.00
12.5
16.13
12.5
7.93
3.97
2.5
4.00
16.29
6.25
8.07
3.125
2.02
1.00
5.0
4-F
2,5-diF
Dihydroxy
2-F
4-F
2.5-diF
2-F
61
62
63
64
65
66
1.20
1.21
0.56
2.54
2.40
1.18
0.30
0.30
1.13
5.08
0.30
0.15
0.30
0.30
1.13
2.54
0.30
0.15
0.60
0.15
1.13
5.08
0.60
0.59
0.30
0.60
2.26
5.08
1.20
1.18
0.30
0.60
2.26
2.54
0.60
0.15
4.00
4.03
0.50
0.50
8.00
7.87
4.00
4.03
0.50
1.00
8.00
7.87
2.00
8.07
0.50
0.50
4.00
2.00
4.00
2.02
0.25
0.50
2.00
1.00
4.00
2.02
0.25
1.00
4.00
7.87
4-F
2.5-diF
Monohydroxy
2-F
4-F
2.5-diF
2-F
4-F
2.5-diF
MTX
68
69
70
71
72
73
2.58
1.29
2.40
2.71
2.60
5.12
0.028
0.32
0.16
1.20
0.34
0.65
0.32
0.025
0.32
0.64
1.20
0.17
1.30
0.64
0.016
0.64
0.64
1.20
0.34
0.16
0.32
0.022
1.29
0.64
1.20
2.71
1.30
0.64
0.020
0.32
0.32
1.20
0.34
1.30
0.32
0.041
8.06
8.06
2.00
7.97
4.00
16.00
1.12
8.06
2.02
2.00
15.94
2.00
8.00
1.75
4.03
2.02
2.00
2.00
2.02
2.00
1.00
2.00
8.00
1.40
8.06
4.03
2.00
7.97
2.00
16.00
0.68
7.97
16.25
16.00
1.27
aIC50 is the concentration of compound required to inhibit the cell growth by 50% compound to an untreated control. Values are means of three independent
experiments. Coefficients of variation were less than 10%. HEK293, human embryonic kidney cells; A549, human lung adenocarcinoma epithelial cells; A498, human
ꢂ
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.
the significance of time resolution by xCELLigence readout
was exemplified by its ability to pinpoint the optimal time
points for conducting end point viability and apoptosis
assays. The specificity of the compounds 12 and 46 deter-
mined to be cell-dependent with good specificity toward
HepG2 cells. Therefore, these compounds were selected for
xCELLigence assay. In order to evaluate the cytotoxicity
response of BJ-5ta homo sapiens foreskin normal cell line
Table 2. The compounds with the best selectivity values and non-toxic con-
centrations for each cancer cell line (human nontoxic concentration).
Cancer
The highest
Non-toxic
cell line
selective compound
concentration (lM)
A549
A498
HeLa
A375
HepG2
12 and 48
12 and 48
12 and 46
12 and 45
12 and 46
0.12 and 0.15
0.03 and 0.15
0.06 and 0.068
0.06 and 0.14
0.029 and 0.068
determination on monitoring of viability by xCELLigence to and the liver cancer cells to the compound 12 and 46 appli-
systematically examine cytotoxic effects triggered with differ- cations, the time-dependent graph of the proliferation curves
ent cell-killing kinetics. Good correlation was observed and obtained from the real-time cell analyzer was plotted.

10
S. BURMAOGLU ET AL.
Figure 5. MTT assay on Human Lung Adenocarcinoma Epithelial Cells (A549 cell line) after 24, 48, 72 and 96 h incubation periods with methotrexate and com-
pounds. The absorbance values were measured at 570 nm for the MTT assay. Control cells were untreated and MTX treated cells were used as positive controls.
Cell viability was calculated based on the ratio of the absorbance of the sample groups over those of the untreated group. P values were calculated by student t
test by using at least three independent experimental values.
Note: p values: Concentration: 0.039 ¼ Control-12: p>.05; Control-48: p>.05; Concentration: 0.078 ¼ Control-12: p<.001; Control-48: p<.001; Concentration: 0.156 ¼ Control-12: p<.0001;
Control-48: p<.0001; Control-MTX: p < 0.001
Figure 6. MTT assay on A498 Cell (Human Renal Cancer Cells) after 24, 48, 72 and 96 h incubation periods with methotrexate and compounds. The absorbance val-
ues were measured at 570 nm for the MTT assay. Control cells were untreated and MTX treated cells were used as positive controls. Cell viability was calculated
based on the ratio of the absorbance of the sample groups over those of the untreated group. P values were calculated by student t test by using at least three
independent experimental values.
Note: p values: Concentration: 0.039 ¼ Control-12: p>.05; Control-48: p>.05; Concentration: 0.078 ¼ Control-12: p<.001; Control-48: p<.001; Concentration: 0.156 ¼ Control-12: p<.001;
Control-48: p<.001; Control-MTX: p < 0.001
Figure 7. MTT assay on HeLa after 24, 48, 72 and 96 h incubation periods with methotrexate and compounds. The absorbance values were measured at 570 nm
for the MTT assay. Control cells were untreated and MTX treated cells were used as positive controls. Cell viability was calculated based on the ratio of the absorb-
ance of the sample groups over those of the untreated group. P values were calculated by student t test by using at least three independent experimental values.
Note: p values: Concentration: 0.039 ¼ Control-12: p>.05; Control-46: p>.05; Concentration: 0.078 ¼ Control-12: p<.001; Control-46: p>.05; Concentration: 0.156 ¼ Control-12: p<.001;
Control-46: p<.001; Control-MTX: p < 0.01
compound 12, it is not significant differences between the
same correlation (Figures 13, 14, and 15). The statistical
accuracy of the data is shown in Table 3. The decrease in
cell proliferation of all concentrations 24, 48 and 72 h after
the application of 12 and 46 compounds in BJ-5ta cell line
was not significant compared to the control group
(p ¼ 0,059). As a result, the values indicate that all 12 and 46
concentrations had a no significant antiproliferative effect on
BJ-5ta cell line.
Compounds 12 and 46 effects the proliferation of BJ-5ta
cells. BJ-5ta homo sapiens foreskin normal cell line was used
to evaluate the effect of compounds 12 and 46 on normal
cells. The cell proliferation of all concentrations (39 nM,
78 nM, and 158 nM) and 24, 48 and 72 h after the application
of 12 (Figures 10, 11 and 12) and 46 (Figures 13, 14 and 15)
(Table 3) compounds in BJ-5ta (ATCC CRL-4001TM) cell line
R
V
was compared to the control group.
As shown in Figures 10, 11, and 12, a correlation is not
significantly observed between the concentration of com-
pound 12 and cytotoxic response kinetics, cell index and cell
viability. It was determined that compound 46 such as
Compound 12 and 46 effects the proliferation of HepG2
cells. In order to evaluate the cytotoxicity response of the

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 8. MTT assay on A375 Cell (Human Skin Malignant Melanoma Cells) after 24, 48, 72 and 96 h incubation periods with methotrexate and compounds. The
absorbance values were measured at 570 nm for the MTT assay. Control cells were untreated and MTX treated cells were used as positive controls. Cell viability
was calculated based on the ratio of the absorbance of the sample groups over those of the untreated group. P values were calculated by student t test by using
at least three independent experimental values.
Note: p values: Concentration: 0.039 ¼ Control-12: p>.05; Control-45: p<.001; Concentration: 0.078 ¼ Control-12: p<.001; Control-45: p<.001; Concentration: 0.156 ¼ Control-12: p<.001;
Control-45: p<.001; Control-MTX: p < 0.01
Figure 9. MTT assay on HepG2 Cell (Human Hepatocellular Carcinoma Cells) after 24, 48, 72 and 96 h incubation periods with methotrexate and compounds. The
absorbance values were measured at 570 nm for the MTT assay. Control cells were untreated and MTX treated cells were used as positive controls. Cell viability
was calculated based on the ratio of the absorbance of the sample groups over those of the untreated group. P values were calculated by student t test by using
at least three independent experimental values.
Note: p values: Concentration: 0.039 ¼ Control-12: p>.05; Control-46: p>.05; Concentration: 0.078 ¼ Control-12: p<.001; Control-46: p>.05; Concentration: 0.156 ¼ Control-12: p<.001;
Control-46: p<.001; Control-MTX: p < 0.01
liver cancer cells to the 12 and 46 substance applications, et al., 2017). Many of the clinically used natural product anticancer
the time-dependent graph of the proliferation curves
obtained from the real-time cell analyser was plotted. The
data obtained were digitized in terms of cell index by the
software of the device (Figures 16–20).
agents such as colchicines, MDL-27048, taxanes, combretastatin
A-4, podophyllotoxin and vinca alkaloids are tubulin-binding
agents. Colchicine, combretastatin A-4 and podophyllotoxin are
targets for the colchicine domain, while vinblastine targets the
vinca alkaloid site, which are known as microtubule destabilizing
agents. On the other hand, agents such as paclitaxel that bind to
the paclitaxel site are called microtubule-stabilizing agents (Banu
et al., 2017). Huang and co-workers have synthesized novel chal-
cone derivatives (74, 75) and compound 74 has inhibited tubulin
polymerization by in vitro tubulin polymerization experiments. It
has also been shown by molecular docking study that 74 can be
binding to the colchicine site of tubulin (Huang et al., 2017). Anti-
tubulin polymerization activities and anticancer activities of chal-
cone derivatives containing the diaryl ether moiety were eval-
uated by Wang et al. In addition thanks to the molecular docking
studies, the binding mechanism of 75 in the colchicine binding
site of tubulin has been demonstrated (Wang et al., 2020). Based
on large number of such literatures results, it has been reported
that chalcone derivatives have been as tubulin inhibitors (Kamal
et al., 2010; Mirzaei et al., 2020; Sabina et al., 2017).
As a result of the experiment (Figures 16–20), the difference
between the mean values of the control group and the other
group before administration of the substance (p > 0.05), the
difference between the mean measurements taken at 24, 48
and 72 h after the 12 and 46 applications was statistically sig-
nificant (p < 0.001). After 12 administration 24 h (39 nM
(p < 0.001), 78 nM (p ¼ 0.001) and 158 nM (p ¼ 0.031)) and 48 h
(39 nM (p ¼ 0.002), 78 nM (p ¼ 0.011). 158 nM (p ¼ 0.021))
decrease in cell proliferation such as 46 was significant com-
pared to the control group, but there was no significant differ-
ence between the groups at 72 h (p ¼ 0.084).
Molecular docking studies
Microtubules composed of a and b-tubulin heterodimers are one
of the most important structural components of the cell skeleton
in eukaryotic cells. These microtubules are involved in many
important cellular functions such as cytoplasmic organelle motil-
ity, formation of mitotic spindles, and protection of the integrity
of the cell shape, intracellular transport and cell replication. A
large number of anticancer ligands interfere with the microtubule
polymerization/depolymerisation process resulting in the arrest
Thus, in our current study, molecular docking was per-
of the cell cycle in the G2/M phase (Choudhury et al., 2013; Qiu formed by inserting the compounds into the colchicine

12
S. BURMAOGLU ET AL.
Figure 10. BJ-5ta cell line was treated with various cytotoxic compounds and monitored in real time for cytotoxic response kinetics. Representative compounds 12
generated distinct response profiles. Pink: Control, red: 39 nM, green: 78 nM, navy blye: 158 nM, turkois: blank.
Figure 11. Cell index values of BJ-5ta cells treated with 39,78,158 nM of 12.
Figure 12. Determination of the concentration of 12 that is required for cell proliferation and cell viability. BJ-5ta cells were incubated with 39, 78 and 158 nM
were followed for 72 h using xCELLigence cell analysis system. UT column corresponds to untreated cells and 39,78,158 nM concentrations. This allows to follow in
real time proliferation of the cells and to image the cells at different times. Representative images after 72 h of treatment are shown for each condition.
binding site of tubulin in order to recognize possible binding the combretastatin formed a hydrogen bond interaction with
mode and key active site interactions. Combretastatin is a the Gly A:144 residue and pi-cation interaction with Lys
natural phenol that has antimitotic activity that specifically B:254. The compound 45 formed a hydrogen bond with the
binds to the colchicine binding domain of tubulin (Banu Asn B:249, the compound 46 formed a hydrogen bond with
et al., 2017). For this reason, combretastatin was used as a the Thr A:179. Lastly the compound 48 formed a hydrogen
reference molecule in the docking study. The compounds bond with the Val B:238.
The residues Asn B:258, Thr A:179, Ser A:178, Thr B:353,
Asn A:101, Thr A:145, Gln B: 247 and Gln A:11 made polar
interactions with combretastatin, while the residues Ala
and the reference molecule were docked with tubulin pro-
tein to identify possible binding mode and key active
site residues.
When the docking results of combretastatin, 12, 45, 46 A:180, Val A:181, Ala B:354, Ala A:99, Ala A:12 and Leu B:248
and 48 molecules were examined, it was determined that made hydrophobic interactions. Also, in the combretastatin

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
Figure 13. BJ-5ta cell line was treated with various cytotoxic compounds and monitored in real time for cytotoxic response kinetics. Representative compounds 46
generated distinct response profiles. Pink: Control, red: 39 nM, green: 78 nM, navy blue: 158 nM, turkois: blank.
Figure 14. Cell index values of BJ-5ta cells treated with 39,78,158 nM of 46.
Figure 15. Determination of the concentration of 46 that is required for cell proliferation and cell viability. (A) BJ-5ta cells were incubated with 39, 78 and 158 nM
were followed for 72 h using xCELLigence cell analysis system. UT column corresponds to untreated cells and 39,78,158 nM concentrations. This allows to follow in
real time proliferation of the cells and to image the cells at different times. Representative images after 72 h of treatment are shown for each condition.

14
S. BURMAOGLU ET AL.
Table 3. Average of measurements between groups 24, 48 and 72 h after 12 and 46 administration.
H0 H24
H48
H72
Mean SD
Min-Max
Mean SD
Min-Max
Mean SD
Min-Max
Mean SD
Min-Max
Control
1.21 0.019
1.179 0.145
1.096 0.063
1.096 0.207
1.059 0.04
1.075 0.082
0.946 0.073
1.191-1.229
1.087-1.396
1.044-1.175
0.809-1.261
1.015-1.107
0.984-1.151
0.848-1.015
1.97 0.023
1.856 0.301
1.851 0.057
1.82 0.333
1.638 0.092
1.72 0.169
1.496 0.068
1.951-1.995
1.648-2.302
1.781-1.907
1.419-2.232
1.501-1.702
1.574-1.879
1.406-1.556
2.584 0.042
2.373 0.333
2.427 0.112
2.474 0.496
2.117 0.151
2.232 0.272
1.962 0.075
2.541-2.626
2.077-2.849
2.272-2.535
1.951-3.145
1.908-2.269
2.013-2.581
1.856-2.032
3.035 0.066
2.682 0.265
2.835 0.182
2.936 0.639
2.405 0.196
2.562 0.312
2.315 0.078
2.969-3.101
2.421-3.04
2.61-3.006
2.402-3.863
2.139-2.598
2.298-3.009
2.212-2.402
12-39 nM
12-78 nM
12-158 nM
46-39 nM
46-78 nM
46-158 nM
P
0,059
Figure 16. HepG2 cell line was treated with various cytotoxic compounds and monitored in real time for cytotoxic response kinetics. Representative compound 46
generated distinct response profiles. Pink: Control, red: 39 nM, green: 78 nM, navy blue: 158 nM, turkois: blank.
Figure 17. Cell index values of HepG2 cells treated with 39,78,158 nM of 12.
binding site, positive and negative charge interactions were positive and negative charge interactions were found with
found with Lys B:352- Lys B:254 and Glu A:71- Asp A:69 resi- Lys B:352- Lys B:254 and Asp B:251 residues, respectively.
dues, respectively. The docking score of combretastatin was The docking score of the compound 45 was determined as
determined as ꢁ4.82 kcal/molꢁ1
.
ꢁ7.26 kcal/molꢁ1
.
The residues Asn B:258, Thr A:179, Thr B:353, Asn A:101
The residues Asn B:258, Thr A:179, Thr B:353 and Asn
and Asn B:249 made polar interactions with compound 12, B:249 made polar interactions with compound 46, while the
while the residues Ala A:180, Val A:181, Ala B:354, Leu B:255, residues Ala A:180, Ala B:354, Leu B:255, Ala B:250, Cys B:241,
Ala B:250, Met B:259, Cys B:241, Ile B:378, Ala B:316, Ala Ile B:378, Ala B:316, Ala B:317, Val B:318 and Leu B:248 made
B:317, Val B:318 and Leu B:248 made hydrophobic interac- hydrophobic interactions. Also, in the binding site, positive
tions. Also, in the binding site, positive and negative charge and negative charge interactions were found with Lys B:352-
interactions were found with Lys B:352- Lys B:254 and Asp Lys B:254 and Asp B:251 residues, respectively. The docking
B:251 residues, respectively. The docking score of the com- score of the compound 46 was determined as
pound 12 was determined as ꢁ5.78 kcal/molꢁ1
.
ꢁ6.04 kcal/molꢁ1
.
The residues Asn B:258, Thr A:179, Thr B:314, Asn A:101,
The residues Asn B:258, Thr A:179 and Thr B:239 made
Gln A:11 and Asn B:249 made polar interactions with com- polar interactions with compound 48, while the residues Ala
pound 45, while the residues Ala A:180, Val A:181, Leu B:255, A:180, Val A:181, Met B:259, Leu B:255, Ala B:354, Ala B:250,
Ala B:316, Leu B:248, Ala B:250, Val B:315 and Met B:259 Cys B:241, Ile B:378, Ala B:316, Ala B:317, Val B:318, Val B:238,
made hydrophobic interactions. Also, in the binding site, Leu B:242 and Leu B:248 made hydrophobic interactions.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
15
Figure 18. Cell index values of HepG2 cells treated with 39,78,158 nM of 46.
Figure 19. Determination of the concentration of 12 that is required for cell proliferation and cell viability. (A) HepG2 cells were incubated with 39, 78 and 158 nM
were followed for 72 h using xCELLigence cell analysis system. UT column corresponds to untreated cells and 39,78,158 nM concentrations. This allows to follow in
real time proliferation of the cells and to image the cells at different times. Representative images after 72 h of treatment are shown for each condition.
Also, in the binding site, positive charge interaction was
In addition, pharmacokinetic properties such as absorp-
found with Lys B:352. The docking score of the compound tion, distribution, metabolism and excretion (ADME) are
48 was determined as ꢁ8.48 kcal/molꢁ1
.
important for the usability of the compounds in human ther-
These docking results revealed that the four compounds apy (Kalani et al., 2012). ADME calculations of the chalcone
(12, 45, 46 and 48) interacted with several common residues derivatives (12, 45, 46 and 48) were obtained using QikProp
(Asn B:258, Thr A:179, Ala A:180, Leu B:255 and Lys B:352) (details are provided in the Supplementary content in Table
when compared to the reference molecule. When the glide S1). In the Lipinski rules, which also consider the numbers of
score values of the molecular dockings were examined, it hydrogen bond donor-hydrogen bond acceptor atoms,
was determined that compound 48 had the best interaction molecular mass and octanol - water distribution coefficients,
energy (8.48 kcal molꢁ1) in all the docked compounds. In no deviation was observed for all these compounds (12, 45,
addition, one of the most important findings in this study is 46 and 48) (Lipinski et al., 1997). In addition, the number of
that the docking results of the compounds with the best deviations from the Jorgensen rule for these compounds (12,
anticancer effect are better than the reference molecule. 45, 46 and 48) was determined as 0. This result says that the
Figures 21 and 22 represent the docked poses of combretas- compounds are more likely to be obtained orally (Duffy &
tatin, 12, 45, 46 and 48 compounds in the tubulin as two Jorgensen, 2000). The compounds (12, 45, 46 and 48) have
and three dimension, respectively.
QikProp parameters within the acceptable range, except for

16
S. BURMAOGLU ET AL.
Figure 20. Determination of the concentration of 46 that is required for cell proliferation and cell viability. (A) HepG2 cells were incubated with 39, 78 and 158 nM
were followed for 72 h using xCELLigence cell analysis system. UT column corresponds to untreated cells and 39,78,158 nM concentrations. This allows to follow in
real time proliferation of the cells and to image the cells at different times. Representative images after 72 h of treatment are shown for each condition.
(QPlogBB,
QPlogKp,
and
QPlogKhsa)
parameters.
The preliminary SAR analysis suggested that the following
structural criteria was required for the antiproliferative action
of the synthesized compounds: (a) the removal of the ketone
or double bond in the linker group of two phenyl ring in
chalcone structures may increase antiproliferative activity
depending on the substitution of A rings, methoxy or
hydroxyl groups; (b) the presence of fluoro substituent at the
B ring increases the antitumor potency originally displayed
by lead 8 or 42, but at least two fluoro substituents at 2, 5
Consequently, the estimated pharmacological properties
obtained for these compounds would help design and
develop more effective drug candidates in the bio-
logical system.
Conclusion
In this study, we designed and synthesized a series of novel positions of the B ring; (c) generally fluoro substituent at the
ortho position of the B ring compounds was more potent
against cell lines, 2,5-diF substituted compounds more
potent against A549 and A498 cell lines. The methoxy substi-
tuted chalcone compounds without any substituents in the B
ring were generally striking.
It is noteworthy that, the replacement of either the enone
or ethylenic bridge of the chalcone with the rigid heterocyc-
lic moieties resulted in ligands with superior antitumor
potential. This information would pave the way towards the
design and synthesis of chalcone derivatives with efficient
anticancer activities. We have also tested the effect of our
compounds on regular cell types such as unmodified breast
cells and hepatic cells. Our results suggest that the doses
that we used in our experiments were relatively safer against
these cell types compared to the cancer cells.
34 compounds. These compounds were further compared to
12 previously synthesized compounds with known biological
activities as antiproliferative agents. These pharmacophore
chalcones were tagged with fluoro atom(s) in B rings to fur-
ther strengthen their antiproliferative activities. These com-
pounds were synthesized by using an efficient reaction
method. Their biological activities were examined in vitro
against a panel of human cancer cell lines. Furthermore, the
structure-activity relationship for each of the compound was
determined. These compounds exerted their antiproliferative
effect partially through apoptosis, the monitoring of cell via-
bility by the xCELLigence system and also possibly by inhibit-
ing
the
cell
division
through
inhibition
of
microtubule formation.
Among the synthesized compounds, 12 showed the most
promising anticancer activity against the majority of the cell
lines. Compounds numbered with 8, 43, 46, 48, 52, 55 and
73 were active against all of the tested cancer cell types
A549, A498, HeLa, A375, and HepG2. Each of the compounds
is listed with the cancer type that it was the most effective
as in the following: 8, 9, 12, 43, 46, 48-55, 66, 69 (0.12-
0.16 mM against A549). 8, 12, 48-55, 66 and 71 (0.30-0.17 mM
against A498), 12, 16, 46 and 52 (0.060-0.068 mM against
HeLa), 8, 12 and 52 (0.060-0.068 mM against A375), and 12,
43 and 46 (0.029-0.068 mM against HepG2).
Moreover, acridine orange staining studies of the com-
pounds with the highest specificities and activities suggested
that these compounds had their anticancer effects partly due
to induction of apoptosis. Our compounds were compared
to widely use chemotherapeutic MTX and they exerted simi-
lar anticancer effects while sparing the normal tissue based
cell lines. Based on the in vitro data we can conclude that
our reagents were relatively safer than MTX while having
similar anticancer efficiencies. The compounds 12 and 46
determined to good specificity toward HepG2 cells.
Therefore, these compounds were selected for xCELLigence

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
17
Figure 21. Molecular modeling (2 D and 3 D) of 12, 45, 46 in colchicine binding site of tubulin. Illustrated are the proposed binding mode and interaction between
tubulin and selected compounds. (A) compound 12, (B) compound 45, (C) compound 46. The compound 12, 45 and 46 are represented in ball and stick model
with yellow, orange and dark blue carbon atoms, respectively. In the ligands, the hydrogen atoms are white, the oxygen atoms are red, and the fluorine atoms are
bright green, Mg2þ atom is represented as pink ball. Important amino acids in the binding pockets are shown in stick model with black carbon atoms, white hydro-
gen atoms, blue nitrogen atoms and red oxygen atoms, whereas tubulin is depicted in the ribbon model as red (A-chain) and yellow (B-chain).
Figure 22. Molecular modeling (2 D and 3 D) of 48 and combretastatin-A4 in colchicine binding site of tubulin. Illustrated are the proposed binding mode and
interaction between tubulin and selected compounds (D) compound 48, (E) combretastatin-A4. The compound 48 and combretastatin-A4 are represented in ball
and stick model with pink and green carbon atoms, respectively. In the ligands, the hydrogen atoms are white, the oxygen atoms are red, and the fluorine atoms
are bright green, Mg2þ atom is represented as pink ball. Important amino acids in the binding pockets are shown in stick model with black carbon atoms, white
hydrogen atoms, blue nitrogen atoms and red oxygen atoms, whereas tubulin is depicted in the ribbon model as red (A-chain) and yellow (B-chain).
assay. In order to evaluate the cytotoxicity response of BJ-5ta significantly observed between the concentration of com-
homo sapiens foreskin normal cell line and the liver cancer pounds (12 and 46) and cytotoxic response kinetics, cell
cells to the compound 12 and 46 applications, the time- index and cell viability. As a result of the xCELLigence cell
dependent graph of the proliferation curves obtained from analysis experiment, the difference between the mean values
the real-time cell analyzer was plotted. The correlation is of the control group and the other group before

18
S. BURMAOGLU ET AL.
administration of the substance, the difference between the
mean measurements taken at 24, 48 and 72 h after the com-
pound 12 and 46 applications was statistically significant.
For compound 12, all concentrations except 158 mM were
statistically decreased compared to control group. It has
been interpreted as the development of resistance to com-
pound 12 developed at high concentrations (158 nM).
In summary, these conclusions were in line with the
results obtained from the MTT assay which supported these
derivatives antiproliferative effects on different cancer cell
types. In order to determine their intracellular effect, we
used simulations deciphering their molecular docking on the
cellular proteins. Molecular docking studies revealed that our
compounds have great potential to bind tubulin dimers.
Tubulins are the structural proteins that form the microtu-
bules and microtubule formation is required for the separ-
ation of the chromosomes and other organelles at anaphase
of the cell cycle. Therefore, our compounds’ antiproliferative
effect stems from their ability to bind tubulin and possibly
by interfering the formation of the mitotic spindles consist-
ing of microtubules.
Overall, our results suggest that these unique fluoro sub-
stituted chalcone derivatives specially 12 and 46 require fur-
ther characterization to draw their pharmacokinetic effects as
anticancer agents for HepG2 cancer cell lines. Also, these
chalcone derivatives could provide an effective solution to
improve the bioavailability and cell growth inhibitory activity
against specially HepG2 cancer cell lines. We are currently
working on the synthesis and mechanism of actions and
in vivo studies of new derivatives of chalcones. These results
will be published in due course.
(E)-3-phenyl-1-(2,4,6-trimethoxyphenyl)prop-2-en-1-one (8)
Yellow solid; yield 98%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.27;
m.p. 85-90 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.53 ꢁ 7.51 (m,
2H). 7.38 ꢁ 7.34 (m, 4H). 6.96 (d, 1H, A part of AB system,
J ¼ 16.1 Hz), 6.16 (s, 2H), 3.86 (s, 3H), 3.77 (s, 6H); 13C NMR
(100 MHz, CDCl3) d 194.3, 162.5, 158.9, 144.1, 135.1, 130.2,
129.1, 128.8, 128.4, 113.9, 90.7, 55.9, 55.5; IR (KBr, cmꢁ1
)
Vmax 2941, 1606, 1457, 1269, 1206, 1129; Anal. calcd for
C18H18O4: C: 72.47; H: 6.08; Found: C: 72.10; H: 6.18.
(E)-3-(2-fluorophenyl)-1-(2,4,6-trimethoxyphenyl)prop-2-en-
1-one (9)
Yellow solid; yield 92%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.23;
m.p. 103-106 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.59 ꢁ 7.50 (m,
2H), 7.36 ꢁ 7.30 (m, 1H), 7.17 ꢁ 7.13 (m, 1H), 7.09-7.01 (m,
2H), 6.16 (s, 2H), 3.86 (s, 3H), 3.78 (s, 6H); 13C NMR (100 MHz,
CDCl3) d 194.2, 161.1 (d, C-16, JCF ¼ 252.1 Hz), 162.6, 159.1,
136.2 (d, C-10, JCF ¼ 3.3 Hz), 131.5 (d, C-14, JCF ¼ 8.7 Hz),
131.1 (d, C-12, JCF ¼ 5.7 Hz), 128.9 (d, C-8, JCF ¼ 2.9 Hz), 124.4
(d, C-13, JCF ¼ 3.6 Hz), 123.2 (d, C-11, JCF ¼ 11.8 Hz), 116.1 (d,
C-15, JCF ¼ 21.6 Hz), 111.6, 90.7, 55.9, 55.5; IR (KBr, cmꢁ1
)
Vmax 2941, 1607, 1458, 1269, 1228; Anal. calcd for
C18H17FO4: C: 68.35; H: 5.42; Found: C: 68.09; H: 5.51.
(E)-3-(3-fluorophenyl)-1-(2,4,6-trimethoxyphenyl)prop-2-en-
1-one (10)
Yellow solid; yield 91%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.3;
m.p. 86-90 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.35 ꢁ 7.19 (m,
4H), 7.11 ꢁ 7.02 (m, 1H), 6.94 (d, 1H, A part of AB system,
J ¼ 16.0 Hz), 6.16 (s, 2H), 3.86 (s, 3H), 3.78 (s, 6H); 13C NMR
(100 MHz, CDCl3) d 193.7, 162.9 (d, C-15, JCF ¼ 241.5 Hz) ,
162.7, 158.9, 142.2, 137.4 (d, C-11, JCF ¼ 7.5 Hz), 130.3 (d, C-
13, JCF ¼ 8.4 Hz), 130.1, 124.3 (d, C-12, JCF ¼ 2.7 Hz), 116.9 (d,
C-14, JCF ¼ 21.2 Hz), 114.6 (d, C-16, JCF ¼ 21.6 Hz), 111.6, 90.8,
55.9, 55.5; IR (KBr, cmꢁ1) Vmax 2942, 1652, 1607, 1457, 1228,
1157, 1129; Anal. calcd for C18H17FO4: C: 68.35; H: 5.42;
Found: C: 68.16; H: 5.39.
Experimental
Chemistry
General: All reagents used were commercially available unless
otherwise specified and all solvents were distilled before use.
Melting points were measured with Gallenkamp melting
point devices. IR Spektra: Perkin Elmer 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.
(E)-3-(4-fluorophenyl)-1-(2,4,6-trimethoxyphenyl)prop-2-en-
1-one (11)
Yellow solid; yield 80%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.27;
m.p. 122-123 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.52–7.48 (m,
2H). 7.32 (d, 1H, B part of AB system, J ¼ 16 Hz), 7.07–7.01
(m, 2H), 6.87 (d, 1H, A part of AB system, J ¼ 16 Hz), 6.15 (s,
2H), 3.84 (s, 3H), 3.76 (s, 6H); 13C NMR (100 MHz, CDCl3) d
194.1, 164.0 (d, C-20, JCF ¼ 249.8 Hz), 162.7, 159.1, 142.8,
131.5, 130.4 (d, C-18, JCF ¼ 8.4 Hz), 129.0, 116.1 (d, C-19, JCF
¼ 21.7 Hz), 111.9, 90.9, 56.1, 55.7; IR (KBr, cmꢁ1) Vmax 3502,
2941, 2841, 1651, 1599; Anal. calcd for C18H18O4: C: 68.35; H:
5.42; Found: C: 68.16; H: 5.38.
General procedure for preparation of compounds 8-13,
22-27 and 68-70
To a solution of related acetophenone (1, 20, 21 or 67) (1
eq.) in MeOH (2.5 mL/1 mmol of substrate). benzaldehyde
derivatives (2-7) (1.6 eq.) and %50 KOH solution (1.5 mL/
1 mmol of substrate) were added sequentially and stirred for
15 h at room temperature. After 15 h solvent was evaporated.
Crude material extracted with 2 M HCl solution (2 mL/1 mmol
of substrate) and DCM (2 mL/1 mmol of substrate x 3). The
combined extracts were dried over Na2SO4. The solvent was
removed in vacuo and the remaining residue purified via
(E)-3-(2,5-difluorophenyl)-1-(2,4,6-trimethoxyphenyl)prop-
2-en-1-one (12)
flash chromatography over silica gel using gradient elution Yellow solid; yield 80%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.4;
with EtOAc and Hexanes to yield compounds 8-13, 22-27 m.p. 118-120 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.48 (d, 1H, B
and 68-70.
part of AB system, J ¼ 16.0 Hz), 7.28 ꢁ 7.23 (m, 2H),

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
19
7.05 ꢁ 6.98 (m, 2H), 6.16 (s, 2H), 3.86 (s, 3H), 3.78 (s, 6H); 13C
(E)-3-(2,5-difluorophenyl)-1-(2,4-dimethoxyphenyl)prop-2-
en-1-one (24)
NMR (100 MHz, CDCl3) d 193.5, 162.7, 158.6 (d, C-12, JCF
¼
Light yellow solid; yield 71%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
244.1 Hz), 159.1, 157.3 (d, C-15, JCF ¼ 248.1 Hz), 134.4, 131.9
1
0.56; m.p. 107-109 ꢀC; H NMR (400 MHz, CDCl3) d 7.77 (d, 1H,
(d, C-9, JCF ¼ 5.0 Hz), 124.5 (dd, C- 10, JCF ¼ 13.9, 7.5 Hz),
J ¼ 8.8 Hz), 7.71 (d, 1H, J ¼ 16 Hz), 7.58 (d, 1H, J ¼ 16.0),
7.29 ꢁ 7.24 (m, 1H), 7.06 ꢁ 6.96 (m, 2H), 6.54 (dd, 1H, J ¼ 8.8,
2.4 Hz), 6.47 (d, 1H, J ¼ 2.4 Hz), 3.89 (s, 3H), 3.84 (s, 3H); 13C
NMR (100 MHz, CDCl3) d 189.9, 164.7, 160.9, 158.8 (dd, C-12,
JCF ¼ 239.2, 2.1 Hz), 157.8 (dd, C-14, JCF ¼ 248.1, 2.3 Hz),
133.3, 132.9, 130.7, 125.1 (dd, C-10, JCF ¼ 14.4, 7.9 Hz), 121.9,
117.8 (dd, C-14, JCF ¼ 24.2, 8.8 Hz), 117.2 (dd, C-13, JCF
¼
16.3, 8.6 Hz), 114.5 (dd, C-11, JCF ¼ 24.3, 4.0), 111.5, 90.8,
55.9, 55.4; IR (KBr, cmꢁ1) Vmax 2942, 1655, 1606, 1489, 1272,
1228, 1158, 1129; Anal. calcd for C18H16F2O4: C: 64.67; H:
4.82; Found: C: 64.66; H: 4.81.
117.8 (dd, C-11, JCF ¼ 23.2, 8.9 Hz), 117.5 (dd, C-13, JCF
¼
24.9, 8.5 Hz), 114.9 (dd, C-15, JCF ¼ 24.2, 3.5 Hz), 105.7, 98.7,
55.9, 55.8; IR (KBr, cmꢁ1) Vmax 2952, 1653, 1596, 1485, 1332,
1256; Anal. calcd for C17H14F2O3: C: 67.10; H: 4.64; Found: C:
67.06; H: 4.68.
(E)-3-(2,5-difluoro-4-methoxyphenyl)-1-(2,4,6-trimethoxy-
phenyl)prop-2-en-1-one (13)
Yellow solid; yield 70%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.33;
m.p. 176-178 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.43 (d, 1H,
J ¼ 16.0 Hz), 7.29 ꢁ 7.24 (m, 1H), 6.86 (d, 1H, J ¼ 16.4 Hz),
6.69 ꢁ 6.65 (m, 1H), 6.15 (s, 2H), 3.89 (s, 3H), 3.85 (s, 3H), 3.77
(s, 3H); 13C NMR (100 MHz, CDCl3) d 193.8, 162.8, 159.2 (C-4/
C-6), 157.9 (dd, C-16, JCF ¼ 256.2, 2.2 Hz), 148.9 (dd, C-13, JCF
(E)-3-(2-fluorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-
one (25)
Light yellow solid; yield 51%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
1
¼ 242.1, 2.8 Hz), 134.9, 131.8 (dd, C-14, JCF ¼ 5.8, 1.5 Hz), 0.5; m.p. 108-110 ꢀC; H NMR (400 MHz, CDCl3) d 8.03 (d, 2H,
J ¼ 8.8 Hz), 7.88 (d, 1H, J ¼ 16.0 Hz) 7.67 ꢁ 7.61 (m, 2H),
7.38 ꢁ 7.34 (m, 1H), 7.20 ꢁ 7.09 (m, 2H), 6.97 (d, 2H,
J ¼ 8.8 Hz), 3.88 (s, 3H); 13C NMR (100 MHz, CDCl3) d 188.8,
163.8, 161.9 (d, C-14, JCF ¼ 252.7 Hz), 136.8, 131.8 (d, C-15,
JCF ¼ 8.8 Hz), 131.2, 131.1, 129.9 (d, C-8, JCF ¼ 2.9 Hz), 124.7
(C-11/C-12) , 123.4 (d, C-10, JCF ¼ 11.5 Hz), 116.5 (d, C-13, JCF
¼ 22.0 Hz), 114.1, 55.7; IR (KBr, cmꢁ1) Vmax 2935, 1655, 1599,
1456, 1229; Anal. calcd for C16H13FO2: C: 74.99; H: 5.11;
Found: C: 74.92; H: 5.32.
129.9, 114.5 (dd, C-12, JCF ¼ 20.5, 4.9 Hz), 111.9, 106.5 (dd, C-
11, JCF ¼ 24.4, 2.8 Hz), 101.7 (dd, C-15, JCF ¼ 28.4, 1.8 Hz),
91.0 (C-1/C-3), 56.7, 56.1, 55.7; IR (KBr, cmꢁ1) Vmax 2941,
1604, 1516, 1457, 1227, 1128; Anal. calcd for C19H18F2O5: C:
62.64; H: 4.98; Found: C: 62.50; H: 4.62.
(E)-1-(2,4-dimethoxyphenyl)-3-(2-fluorophenyl)prop-2-en-1-
one (22)
Light yellow solid; yield 79%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
1
0.5; m.p. 55-57 ꢀC; H NMR (400 MHz, CDCl3) d 7.81 ꢁ 7.76 (m,
(E)-3-(4-fluorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-
one (26)
1H), 7.64 ꢁ 7.57 (m, 2H), 7.34 ꢁ 7.29 (m, 2H), 7.16 ꢁ 7.05 (m,
2H), 6.55 (dd, 1H, J ¼ 8.4, 2.0 Hz), 6.48 (d, 1H, J ¼ 2.0 Hz), 3.89
(s, 3H), 3.85 (s, 3H); 13C NMR (100 MHz, CDCl3) d 190.4, 164.5,
161.8 (d, C-14, JCF ¼ 256.2 Hz), 160.8, 134.6, 133.2, 131.4 (d,
C-15, JCF ¼ 8.6 Hz), 129.8 (d, C-11, JCF ¼ 6.6 Hz), 129.6 (d, C-
10, JCF ¼ 3.1 Hz), 124.6 (d, C-12, JCF ¼ 3.6 Hz), 122.2, 120.5,
White solid; yield 59%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.5;
m.p. 119-121 ꢀC; 1H NMR (400 MHz, CDCl3) d 8.01 (d, 2H,
J ¼ 8.8 Hz), 7.75 (d, 1H, J ¼ 16.0 Hz), 7.63 ꢁ 7.59 (m, 2H), 7.46
(d, 1H, J ¼ 15.6 Hz), 7.13 ꢁ 7.04 (m, 2H). 7.1 (d, 2H, J ¼ 8.8 Hz),
3.87 (s, 3H); 13C NMR (100 MHz, CDCl3) d 188.7, 164.1 (d, C-
116.4 (d, C-13, JCF ¼ 21.9 Hz), 105.5, 98.8, 55.9, 55.8; IR (KBr, 15, JCF ¼ 250.1 Hz), 163.6, 142.8, 131.5 (d, C-10, JCF ¼ 5.3 Hz),
cmꢁ1) Vmax 2942, 1657, 1607, 1457, 1327, 1259, 1212; Anal.
131.2, 131.0 (C-4/C-6), 130.5 (d, C-11/C-14, JCF ¼ 8.5 Hz),
121.8, 116.2 (d, C-12/C-13, JCF ¼ 21.8 Hz), 114.1 (C-1/C-3),
calcd for C17H15FO3: C: 71.32; H: 5.28; Found: C: 71.10;
H: 5.31.
55.7; IR (KBr, cmꢁ1) Vmax 2936, 1654, 1598, 1506, 1227; Anal.
calcd for C16H13FO2: C: 74.99; H: 5.11; Found: C: 74.84;
H: 5.22.
(E)-1-(2,4-dimethoxyphenyl)-3-(4-fluorophenyl)prop-2-en-1-
one (23)
(E)-3-(2,5-difluorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-
one (27)
Light yellow solid; yield 54%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
0.5; m.p. 97-99 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.75 (d, 1H,
J ¼ 8.8 Hz), 7.65 ꢁ 7.54 (m, 3H), 7.44 (d, 1H, J ¼ 16.0 Hz),
7.08 ꢁ 7.04 (m, 2H), 6.55 (dd, 1H, J ¼ 8.4, 2.0 Hz), 6.48 (d, 1H,
J ¼ 2.4 Hz), 3.88 (s, 3H), 3.84 (s, 3H); 13C NMR (100 MHz,
CDCl3) d 190.4, 164.5, 163.9 (d, C-15, JCF ¼ 249.4 Hz), 160.7,
140.6, 133.1, 131.9 (d, C-10, JCF ¼ 3.4 Hz), 130.3 (d, C-14/C-11,
JCF ¼ 8.4 Hz), 127.2, 122.3, 116.1 (d, C-13/C-12, JCF ¼ 21.7 Hz),
105.5, 98.9, 56.0, 55.8; IR (KBr. cmꢁ1) Vmax 2941, 1655, 1599,
1508, 1418, 1327, 1213; Anal. calcd for C17H15FO3: C: 71.32;
H: 5.28; Found: C: 71.52; H: 5.54.
Light yellow solid; yield 51%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
1
0.53; m.p. 105-107 ꢀC; H NMR (400 MHz, CDCl3) d 8.08 (d, 2H,
J ¼ 8.8 Hz), 7.82 (d, 1H, J ¼ 15.6 Hz), 7.62 (d, 1H, J ¼ 16.0 Hz),
7.35 ꢁ 7.31 (m, 2H), 7.13 ꢁ 7.05 (m, 1H), 6.99 (d, 2H,
J ¼ 8.8 Hz), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3) d 188.3,
163.9, 158.9 (dd, C-14, JCF ¼ 2.4, 241.7 Hz), 157.8 (dd, C-12,
JCF ¼ 2.4, 248.6 Hz), 135.4, 131.1, 130.9, 125.5, 124.7 (dd, C-
10, JCF ¼ 7.9, 14.1 Hz), 118.2 (dd, C-13, JCF ¼ 9.0, 24.2 Hz),
117.6 (dd, C-11, JCF ¼ 8.5, 25.0 Hz), 115.3 (dd, C-15, JCF ¼ 3.4,
24.3 Hz), 114.2, 55.7; IR (KBr, cmꢁ1) Vmax 2929, 1659, 1598,

20
S. BURMAOGLU ET AL.
1489, 1338, 1247; Anal. calcd for C16H12F2O2: C: 70.07; H:
4.41; Found: C: 69.93; H: 4.56.
3-Phenyl-1-(2,4,6-trimethoxyphenyl)propan-1-one (14)
Colourless liquid; yield 84%; Rf ¼ (EtOAc/Hexanes 40:60) ¼
1
0.27; H NMR (400 MHz, CDCl3) d 7.43 ꢁ 7.09 (m, 5H). 6.10 (s,
2H), 3.81 (s, 3H), 3.75 (s, 6H), 3.13 ꢁ 2.98 (m, 4H); 13C NMR
(100 MHz, CDCl3) d 204.0, 162.2, 158.4, 141.7, 128.7, 128.5,
126.1, 113.5, 90.8, 56.3, 55.7, 46.2, 29.9; IR (KBr, cmꢁ1) Vmax
3420, 1608, 1455, 1340, 1206, 1125; Anal. calcd for C18H20O4:
C: 71.98; H: 6.71; Found: C: 71.89; H: 6.59.
(E)-3-(2-fluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-
one (68)
Yellow solid; yield 56%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.46;
1
m.p. 167-169 ꢀC; H NMR (400 MHz, CD3OD) d 8.04 ꢁ 7.99 (m,
2H), 7.89 ꢁ 7.81 (m, 3H), 7.47 ꢁ 7.42 (m, 1H), 7.27 ꢁ 7.16 (m,
2H). 6.89 (d, 2H, J ¼ 8.4 Hz); 13C NMR (100 MHz, CD3OD) d
189.1, 163.1, 161.8 (d, C-15, JCF ¼ 250.7 Hz). 135.5, 132.0 (d,
2-(3-(2-Fluorophenyl)propyl)-1,3,5-trimethoxybenzene (15)
Colourless liquid; yield 67%; Rf ¼ (EtOAc/Hexanes 40:60) ¼
0.36; 1H NMR (400 MHz, CDCl3) d 7.23 ꢁ 7.19 (m, 1H),
7.15 ꢁ 7.09 (m, 1H), 7.04 ꢁ 6.94 (m, 2H), 6.12 (s, 2H), 3.80 (s,
3H), 3.77 (s, 6H), 2.68 ꢁ 2.61 (m, 4H), 1.81 ꢁ 1.73 (m, 2H); 13C
NMR (100 MHz, CDCl3) d 161.2 (d, C-15, JCF ¼ 242.6 Hz),
159.2, 158.9, 130.4 (d, C-11, JCF ¼ 5.2 Hz), 129.9 (d, C-10, JCF
C-17, JCF ¼ 8.8 Hz), 131.3, 129.4, 129.0, 124.7 (d, C-13, JCF
¼
3.6 Hz), 124.0 (d, C-12, JCF ¼ 5.3 Hz), 123.1 (d, C-11, JCF ¼
11.4 Hz), 115.8 (d, C-14, JCF ¼ 22.1 Hz), 115.4; IR (KBr, cmꢁ1
)
Vmax 3184, 2818, 1649, 1606, 1485, 1338, 1283; Anal. calcd
for C15H11FO2: C: 74.37; H: 4.58; Found: C: 74.16; H: 4.59.
¼ 15.8 Hz), 127.9 (d, C-13, JCF ¼ 3.8 Hz), 123.7 (d, C-12, JCF
¼
3.6 Hz), 115.9 (d, C-14, JCF ¼ 22.2 Hz), 111.3, 90.5, 55.6, 55.3,
29.5, 28.7, 22.4; IR (KBr, cmꢁ1) Vmax 2938, 1610, 1596, 1455,
1205, 1151, 1124; Anal. calcd for C18H21FO3: C: 71.03; H: 6.95;
Found: C: 70.78; H: 7.09.
(E)-3-(4-fluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-
one (69)
Yellow solid. yield 54%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.43;
1
m.p. 176-178 ꢀC; H NMR (400 MHz, CD3OD) d 8.03 ꢁ 7.99 (m,
2H), 7.79 ꢁ 7.70 (m, 4H), 7.18 ꢁ 7.14 (m, 2H), 6.89 (d, 2H,
J ¼ 8.8 Hz), 3.83 (bs, 1H); 13C NMR (100 MHz, CD3OD) d 189.3,
164.2 (d, C-17, JCF ¼ 248.6 Hz), 162.9, 142.5, 131.8 (d, C-11,
JCF ¼ 3.1 Hz), 131.2, 130.6 (d, C-12/C-15, JCF ¼ 8.5 Hz), 129.6,
121.6, 115.7 (d, C-13/C-14, JCF ¼ 21.9 Hz), 115.3; IR (KBr,
cmꢁ1) Vmax 3181, 2961, 1654, 1600, 1508, 1338, 1220; Anal.
calcd for C15H11FO2: C: 74.37; H: 4.58; Found: C: 74.56;
H: 4.91.
2-(3-(3-Fluorophenyl)propyl)-1,3,5-trimethoxybenzene (16)
White solid; yield 74%; Rf ¼ (EtOAc/Hexanes 10:90) ¼ 0.5;
m.p. 40-44 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.21 ꢁ 7.16 (m,
1H), 6.97 ꢁ 6.90 (m, 1H), 6.85 ꢁ 6.80 (m, 2H), 6.12 (s, 2H), 3.79
(s, 3H), 3.77 (s, 6H), 2.64 ꢁ 2.59 (m, 4H), 1.82 ꢁ 1.73 (m, 2H);
13C NMR (100 MHz, CDCl3) d 162.9 (d, C-14, JCF ¼ 243.0 Hz),
159.3, 158.9, 145.9, 129.4 (d, C-12, JCF ¼ 8.3 Hz), 124.1 (d, C-
11, JCF ¼ 2.7 Hz), 115.2 (d, C-15, JCF ¼ 20.6 Hz), 112.2 (d, C-13,
JCF ¼ 20.9 Hz), 111.2, 90.5, 55.6, 55.3, 35.5, 30.6, 22.4; IR (KBr,
cmꢁ1) Vmax 2939, 1611, 1595, 1455, 1205, 1152, 1118; Anal.
calcd for C18H21FO3: C: 71.03; H: 6.95; Found: C: 70.89;
H: 6.97.
(E)-3-(2,5-difluorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-
one (70)
Light yellow solid. yield 83%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
0.66; m.p. 190-192 ꢀC; 1H NMR (400 MHz, CD3OD) d 8.03 (d,
2H, J ¼ 8.8 Hz), 7.87 ꢁ 7.79 (m, 2H), 7.70 ꢁ 7.64 (m, 1H),
7.24 ꢁ 7.17 (m, 2H), 6.90 (d, 2H, J ¼ 8.8 Hz); 13C NMR
2-(3-(4-Fluorophenyl)propyl)-1,3,5-trimethoxybenzene (17)
White solid; yield 83%; Rf ¼ (EtOAc/Hexanes 10:90) ¼ 0.66;
m.p. 57-58 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.16 ꢁ 7.13 (m,
2H), 6.96 ꢁ 6.91 (m, 2H), 6.13 (s, 2H), 3.81 (s, 3H), 3.78 (s, 6H),
2.62 ꢁ 2.59 (m, 4H), 1.75 (tt, 2H, J ¼ 15.2, 7.6 Hz); 13C NMR
(100 MHz, CDCl3) d 160.1 (d, C-13, JCF ¼ 20.8 Hz), 159.4, 159.0
(C-6/C-4), 138.9, 129.8 (d, C-15/C-11, JCF ¼ 7.7 Hz), 114.8 (d,
C-14/C-12, JCF ¼ 20.8 Hz), 111.5, 90.7 (C-1/C-3), 55.9, 55.5,
35.2, 31.3, 22.5; IR (KBr, cmꢁ1) Vmax 3445, 2937, 1609, 1597,
1508, 1455, 1204, 1150; Anal. calcd for C18H21FO3: C: 71.03;
H: 6.95; Found: C: 71.04; H: 6.85.
(100 MHz, CD3OD) d 188.7, 163.1, 159.2 (d, C-13, JCF
¼
240.2 Hz), 157.8 (d, C-15, JCF ¼ 244.3 Hz), 134.1, 131.4 (C-4/C-
6), 129.3, 129.1, 125.1 (d, C-11, JCF ¼ 4.8 Hz), 118.3 (dd, C-14,
JCF ¼ 24.4, 8.0 Hz), 117.3 (dd, C-17, JCF ¼ 25.7, 8.9 Hz), 115.4
(C-1/C-3), 114.4 (dd, C-12, JCF ¼ 25.1, 2.8 Hz); IR (KBr, cmꢁ1
)
Vmax 3318, 2956, 1604, 1487, 1338, 1220; Anal. calcd for
C15H10F2O2: C: 69.23; H: 3.87; Found: C: 68.97; H: 4.09.
General procedure for preparation of compounds 14-19,
28-33 and 71-73
To a solution of tri/di/mono-methoxy-chalcones (8-13, 22-27
and 68-70) substrates in EtOH (10 mL/1 mmol of substrate)
Pd/C (20%) was added. The reaction flask was purged with
hydrogen gas three times before being allowed to stir under
a hydrogen balloon for 16 h at room temperature. Then, the
reaction mixture was filtered and concentrated in vacuo and
the remaining residue purified via flash chromatography over
silica gel using gradient elution with EtOAc and Hexanes to
yield compounds 14-19, 28-33 and 71-73.
2-(3-(2,5-Difluorophenyl)propyl)-1,3,5-trimethoxyben-
zene (18)
Colourless liquid; yield 87%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
0.53; 1H NMR (400 MHz, CDCl3) d 6.97 ꢁ 6.88 (m, 2H),
6.81 ꢁ 6.76 (m, 1H), 6.12 (s, 2H), 3.79 (s, 3H), 3.77 (s, 6H),
2.65 ꢁ 2.61 (m, 4H), 1.81 ꢁ 1.73 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 158.6 (d, C-18, JCF ¼ 241.0 Hz), 159.3, 158.8, 157.1
(d, C-15, JCF ¼ 250.0 Hz), 131.6 (dd, C-13, JCF ¼ 18.7, 7.5 Hz),
116.6 (dd, C-14, JCF ¼ 23.6, 5.5 Hz), 115.6 (dd, C-16, JCF
¼

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
21
25.3, 8.8 Hz), 113.1 (dd, C-17, JCF ¼ 24.0, 8.6 Hz), 110.9, 90.3, 5.3 Hz), 116.7 (dd, C-13, JCF ¼ 25.2, 8.6 Hz), 113.6 (dd, C-15,
55.6, 55.3, 28.9, 28.5, 22.3; IR (KBr, cmꢁ1) Vmax 2939, 1611,
1596, 1496, 1457, 1205, 1153, 1121; Anal. calcd for
JCF ¼ 23.9, 8.5 Hz), 103.9, 98.7, 55.5, 55.4, 30.1, 29.5, 28.8; IR
(KBr, cmꢁ1) Vmax 2936, 1613, 1495, 1464, 1208; Anal. calcd
C18H20F2O3: C. 67.07; H: 6.25; Found: C: 66.72; H: 6.22.
for C17H18F2O2: C: 69.85; H: 6.21; Found: C: 69.59; H: 6.29.
2-(3-(2,5-Difluoro-4-methoxyphenyl)propyl)-1,3,5-trime-
thoxybenzene (19)
1-Fluoro-2-(3-(4-methoxyphenyl)propyl)benzene (31)
Colorless liquid; yield 47%; Rf ¼ (EtOAc/Hexanes 20:80) ¼
0.93; 1H NMR (400 MHz, CDCl3) d 7.22 ꢁ 7.13 (m, 4H),
7.11 ꢁ 7.02 (m, 2H), 6.89 ꢁ 6.86 (m, 2H), 3.82 (s, 3H), 2.72 (t,
2H, J ¼ 7.6 Hz), 2.66 (t, 2H, J ¼ 8 Hz), 1.96 (tt, 2H, J ¼ 15.2,
White solid; yield 80%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.83;
m.p. 80-82 ꢀC; 1H NMR (400 MHz, CDCl3) d 6.98 ꢁ 6.93 (m,
1H). 6.66 ꢁ 6.61 (m, 1H), 6.13 (s, 2H), 3.84 (s, 3H), 3.80 (s, 3H),
3.79 (s, 6H), 2.63 ꢁ 2.55 (m, 4H), 1.74 (tt, 2H, J ¼ 15.6, 8.0 Hz);
13C NMR (100 MHz, CDCl3) d 159.4, 159.0 (C-4/C-6), 156.8 (dd,
C-15, JCF ¼ 239.2, 2.3 Hz), 148.6 (dd, C-12, JCF ¼ 239.1,
8.8 Hz); 13C NMR (100 MHz, CDCl3) d 161.4 (d, C-14, JCF
¼
243.3 Hz), 158.0, 134.4, 130.6 (d, C-11, JCF ¼ 5.3 Hz), 129.6 (C-
4/C-6), 129.4 (d, C-10, JCF ¼ 15.7 Hz), 127.7 (d, C-15, JCF
7.9 Hz), 124.1 (d, C-12, JCF ¼ 3.6 Hz), 115.4 (d, C-13, JCF
¼
¼
2.7 Hz), 145.9 (d, C-13, JCF ¼ 22.6 Hz), 121.6 (dd, C-10, JCF
¼
21.9 Hz), 113.9 (C-1/C-3), 55.5, 34.9, 32.2, 28.9; IR (KBr, cmꢁ1
)
18.7, 6.0 Hz), 116.9 (dd, C-11, JCF ¼ 20.1, 7.0 Hz), 111.3, 101.6
(d, C-14, JCF ¼ 28.9 Hz), 90.8 (C-1/C-3), 56.7, 55.8, 55.5, 29.4,
27.9, 22.3; IR (KBr, cmꢁ1) Vmax 2939, 1609, 1596, 1519, 1455,
1332, 1151; Anal. calcd for C19H22F2O4: C. 64.76; H. 6.29;
Found: C: 64.65; H: 6.00.
Vmax 2934, 2860, 2061, 1879, 1612, 1512; Anal. calcd for
C16H17FO: C: 78.66; H: 7.01; Found: C: 78.53; H: 7.10.
3-(4-Fluorophenyl)-1-(4-methoxyphenyl)propan-1-ol (32)
Light yellow liquid; yield 44%; Rf ¼ (EtOAc/Hexanes 20:80) ¼
0.33; 1H NMR (400 MHz, CDCl3) d 7.26 (d, 2H, J ¼ 8.8 Hz),
7.14 ꢁ 7.11 (m, 2H), 6.99 ꢁ 6.94 (m, 2H), 6.89 (d, 2H,
J ¼ 8.8 Hz), 4.62 ꢁ 4.58 (m, 1H), 3.80 (s, 3H), 2.73 ꢁ 2.57 (m,
2H), 2.14 ꢁ 2.10 (m, 1H), 2.03 (s, 1H, C-OH), 2.0 ꢁ 1.91 (m, 1H);
13C NMR (100 MHz, CDCl3) d 161.5 (d, C-15, JCF ¼ 242.2 Hz),
159.3, 137.6 (d, C-10, JCF ¼ 3.3 Hz), 136.8, 129.9 (d, C-11/C-14,
1-(3-(2-Fluorophenyl)propyl)-2,4-dimethoxybenzene (28)
Light yellow liquid; yield 45%; Rf ¼ (EtOAc/Hexanes 10:90) ¼
0.93; 1H NMR (400 MHz, CDCl3) d 7.26 ꢁ 7.15 (m, 2H).
7.10 ꢁ 7.00 (m, 3H), 6.48 (d, 1H, J ¼ 2.4 Hz), 6.45 (dd, 1H,
J ¼ 8.0, 2.4 Hz), 3.82 (s, 6H), 2.77 (t, 2H, J ¼ 8.0 Hz), 2.65 (t, 2H,
J ¼ 7.6 Hz), 1.92 (tt, 2H, J ¼ 15.6, 8.0 Hz); 13C NMR (100 MHz,
CDCl3) d 161.4 (d, C-14, JCF ¼ 243.2 Hz), 159.3, 158.6, 130.8
(d, C-11, JCF ¼ 5.1 Hz), 130.15, 129.7 (d, C-10, JCF ¼ 15.7 Hz),
127.5 (d, C-15, JCF ¼ 8.1 Hz), 124.0 (d, C-12, JCF ¼ 3.4 Hz),
123.2, 115.3 (d, C-13, JCF ¼ 22.3 Hz), 103.9, 98.7, 55.6, 55.5,
30.5, 29.6, 29.0; IR (KBr, cmꢁ1) Vmax 2936, 2861, 2071, 1613,
1587, 1505; Anal. calcd for C17H19FO2: C: 74.43; H: 6.98;
Found: C: 74.14; H: 7.24.
JCF ¼ 7.6 Hz), 127.4 (C-4/C-6), 115.2 (d, C-13/C-12, JCF
¼
20.8 Hz), 114.2 (C-1/C-3), 73.6, 55.5, 40.7, 31.4; IR (KBr, cmꢁ1
)
Vmax 3387, 2933, 1709, 1610, 1509; Anal. calcd for
C16H17FO2: C: 73.83; H: 6.58; Found: C: 73.67; H: 6.71.
1,4-Difluoro-2-(3-(4-methoxyphenyl)propyl)benzene (33)
Colorless liquid; yield 73%; Rf ¼ (EtOAc/Hexanes 20:80) ¼
0.93; 1H NMR (400 MHz, CDCl3) d 7.13 (d, 2H, J ¼ 8.4 Hz),
6.99 ꢁ 6.93 (m, 1H), 6.91 ꢁ 6.82 (m, 4H), 3.81 (s, 3H),
2.68 ꢁ 2.61 (m, 4H), 1.93 (tt, 2H, J ¼ 15.2, 7.6 Hz); 13C NMR
(100 MHz, CDCl3) d 158. 8 (dd, C-12, JCF ¼ 242.3, 2.3 Hz),
158.1, 157.3 (dd, C-14, JCF ¼ 239.1, 2.5 Hz), 134.1, 131.1 (dd,
C-10, JCF ¼ 18.7, 7.7 Hz), 129.6 (C-4/C-6), 116.9 (dd, C-11, JCF
¼ 23.3, 5.3 Hz), 116.2 (dd, C-13, JCF ¼ 25.4, 8.9 Hz), 114.0 (C-
1/C-3), 113.8 (dd, C-15, JCF ¼ 24.1, 8.7 Hz), 55.5, 34.7, 31.8,
28.8; IR (KBr, cmꢁ1) Vmax 2935, 2861, 1612, 1513, 1495, 1246;
Anal. calcd for C16H16F2O: C: 73.27; H: 6.15; Found: C: 73.31;
H: 6.28.
1-(3-(4-Fluorophenyl)propyl)-2,4-dimethoxybenzene (29)
Yellow liquid; yield 57%; Rf ¼ (EtOAc/Hexanes 10:90) ¼ 0.83;
1H NMR (400 MHz, CDCl3) d 7.18 ꢁ 7.15 (m, 2H), 7.05 ꢁ 6.96
(m, 3H), 6.47 ꢁ 6.44 (m, 2H), 3.81 (m, 6H), 2.67 ꢁ 2.59 (m, 4H),
1.89 (tt, 2H, J ¼ 15.6, 8.0. Hz); 13C NMR (100 MHz, CDCl3) d
161.4 (d, C-15, JCF ¼ 241.3 Hz), 159.4, 158.6, 138.5 (d, C-10,
JCF ¼ 3.0 Hz), 130.2, 129.9 (d, C-11/C-14, JCF ¼ 7.7 Hz), 123.2,
115.0 (d, C-13/C-12, JCF ¼ 20.9 Hz), 103.9, 98.7, 55.6, 55.5,
35.1, 31.9, 29.5; IR (KBr, cmꢁ1) Vmax 2935, 2858, 1613, 1508,
1208; Anal. calcd for C17H19FO2: C: 74.43; H: 6.98; Found: C:
74.25; H: 6.92.
4-(3-(2-Fluorophenyl)propyl)phenol (71)
1-(3-(2,5-Difluorophenyl)propyl)-2,4-dimethoxyben-
zene (30)
Light yellow liquid; yield 50%; Rf ¼ (EtOAc/Hexanes 20:80) ¼
0.56; 1H NMR (400 MHz, CDCl3) d 7.20 ꢁ 7.15 (m, 2H),
7.08 ꢁ 6.99 (m, 4H), 6.77 (d, 2H, J ¼ 8.4 Hz), 4.96 (bs, 1H), 2.68
(t, 2H, J ¼ 7.6 Hz), 2.61 (t, 2H, J ¼ 7.6 Hz), 1.92 (tt, 2H, J ¼ 15.6,
Colorless liquid; yield 78%; Rf ¼ (EtOAc/Hexanes 20:80) ¼
0.83; 1H NMR (400 MHz, CDCl3) d 7.03 (d, 1H, J ¼ 8.4 Hz),
6.98 ꢁ 6.89 (m, 2H), 6.86 ꢁ 6.80 (m, 1H), 6.46 (d, 1H,
J ¼ 2.4 Hz), 6.43 (dd, 1H, J ¼ 8.0, 2.4 Hz), 3.80 (s, 6H),
2.68 ꢁ 2.59 (m, 4H), 1.88 (tt, 2H, J ¼ 15.6, 8.0 Hz); 13C NMR
(100 MHz, CDCl3) d 159.4, 158.5, 158.8 (dd, C-12, JCF ¼ 237.7,
2.3 Hz), 157.3 (dd, C-14, JCF ¼ 239.2, 2.4 Hz), 131.4 (dd, C-10,
8.0 Hz); 13C NMR (100 MHz, CDCl3) d 161.4 (d, C-16, JCF
¼
243 Hz), 153.7, 134.6, 130.8 (d, C-12, JCF ¼ 20.8 Hz), 129.7 (C-
4/C-6), 129.3 (d, C-11, JCF ¼ 15.8 Hz), 127.6 (d, C-14, JCF
8 Hz), 124.1 (d, C-13, JCF ¼ 3.5 Hz), 115.4 (d, C-15, JCF
¼
¼
JCF ¼ 18.7, 7.5 Hz), 130.2, 122.9, 116.8 (dd, C-11, JCF ¼ 23.3, 22.1 Hz), 115.3 (C-3/C-1), 34.8, 32.2, 28.8; IR (KBr, cmꢁ1) Vmax

22
S. BURMAOGLU ET AL.
3336, 2931, 1612, 1513, 1491, 1355, 1227; Anal. calcd for ofsubstrate) was added 12 M HCl solution (2 eq.) drop by
drop at room temperature. Reaction mixture stirred for 12 h.
After 12 h reaction mixture washed with H2O solution (2 mL/
1 mmol ofsubstrate) and extracted with EtOAc (2.5 mL/
1 mmol of substrate x 3). The combined extracts were dried
over Na2SO4. The solvent was removed in vacuo and the
remaining residue purified via flash chromatography over sil-
ica gel using gradient elution with EtOAc and Hexanes to
yield compounds 42-46 and 61-63.
C15H15FO: C: 78.24; H: 6.57; Found: C: 78.13; H: 6.47.
3-(4-Fluorophenyl)-1-(4-hydroxyphenyl)propan-1-one (72)
White solid; yield 50%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.6;
m.p. 133-135 ꢀC; 1H NMR (400 MHz, CD3OD) d 7.81 (d, 2H,
J ¼ 8.8 Hz), 7.20 ꢁ 7.15 (m, 2H), 6.94 ꢁ 6.89 (m, 2H), 6.81 (d,
2H, J ¼ 8.8 Hz), 3.15 (t, 2H, J ¼ 7.6 Hz), 2.90 (t, 2H, J ¼ 7.2 Hz);
13C NMR (100 MHz, CD3OD) d 199.0, 162.8, 161.6 (d, C-14, JCF
¼ 241.0 Hz), 137.4 (d, C-11, JCF ¼ 3.3 Hz), 130.7 (C-4/C-6),
129.9 (d, C-16/C-12, JCF ¼ 7.6 Hz), 128.8, 115.1 (C-1/C-3),
(E)-3-phenyl-1-(2,4,6-trihydroxyphenyl)prop-2-en-1-
one (42)
114.8 (d, C-15/C-13, JCF ¼ 21.3 Hz), 39.6, 29.5; IR (KBr, cmꢁ1
)
Orange solid; yield 49%; Rf ¼ (EtOAc/Hexanes 60:40) ¼ 0.28;
Vmax 3296, 2928, 1656, 1601, 1581, 1366, 1211; Anal. calcd
for C15H13FO2: C: 73.76; H: 5.36; Found: C.73: 58; H: 5.24.
1
m.p. 193 ꢀC; H NMR (400 MHz, CD3OD) d 8.25 ꢁ 8.20 (m, 1H),
7.80 ꢁ 7.74 (m, 1H), 7.65 ꢁ 7.58 (m, 2H), 7.45 ꢁ 7.35 (m, 3H),
4.84 (s, 2H), 3.30 (bs, 1H); 13C NMR (100 MHz, CD3OD) d
193.3, 162.3, 161.9, 142.6, 135.7, 130.1, 128.8, 128.3, 127.3,
105.7, 104.9; IR (KBr, cmꢁ1) Vmax 3159, 2324, 1624, 1591,
1437, 1401, 1338; Anal. calcd for C15H12O4: C: 70.31; H: 4.72;
Found: C: 70.29; H: 4.79.
4-(3-(2,5-Difluorophenyl)propyl)phenol (73)
White solid; yield 54%; Rf ¼ (EtOAc/Hexanes 20:80) ¼ 0.56;
m.p. 47-49 ꢀC; 1H NMR (400 MHz, CDCl3) d 7.05 (d, 2H,
J ¼ 7.6 Hz), 6.98 ꢁ 6.92 (m, 1H), 6.89 ꢁ 6.80 (m, 2H), 6.76 (d,
2H, J ¼ 8.4 Hz), 4.62 (bs, 1H), 2.65 ꢁ 2.58 (m, 4H), 1.89 (tt, 2H,
J ¼ 15.6, 8.0 Hz); 13C NMR (100 MHz, CDCl3) d 158.8 (d, C-13,
JCF ¼ 232.9 Hz), 157.3 (d, C-16, JCF ¼ 238 Hz), 153.8, 134.3,
131.1 (dd, C-11, JCF ¼ 19.0, 7.7 Hz), 129.7 (C-4/C-6), 116.9 (d,
C-12, JCF ¼ 23.5, 5.4 Hz), 116.2 (d, C-15, JCF ¼ 25.6, 9.0 Hz),
115.4 (C-1/C-3), 113.8 (d, C-14, JCF ¼ 23.9, 8.5 Hz), 34.7, 31.8,
28.8; IR (KBr, cmꢁ1) Vmax 3325, 2931, 1612, 1513, 1494, 1209;
Anal. calcd for C15H14F2O: C: 72.57; H: 5.68; Found: C: 72.81;
H: 5.89.
(E)-3-(2-fluorophenyl)-1-(2,4,6-trihydroxyphenyl)prop-2-en-
1-one (43)
Orange solid; yield 70%; Rf ¼ (EtOAc/Hexanes 50:50) ¼ 0.53;
1
m.p. 195-197 ꢀC; H NMR (400 MHz, Acetone-d6) d 8.36 (d, 1H,
J ¼ 16.0 Hz), 7.89 (d, 1H, J ¼ 15.6 Hz), 7.79 ꢁ 7.75 (m, 1H),
7.49 ꢁ 7.43 (m, 1H), 7.28 ꢁ 7.19 (m, 2H), 6.01 (s, 2H); 13C NMR
(100 MHz, Acetone-d6) d 193.0, 165.9, 165.8, 162.3 (d, C-18,
JCF ¼ 250.0 Hz), 134.5 (d, C-12, JCF ¼ 3.3 Hz), 132.7 (d, C-16,
JCF ¼ 8.8 Hz), 130.9 (d, C-14, JCF ¼ 6.0 Hz), 130.1 (d, C-11, JCF
General procedure for preparation of compounds 42-46,
61-63
¼ 3.0 Hz), 125.7 (d, C-15, JCF ¼ 3.6 Hz), 124.3 (d, C-13, JCF
¼
11.6 Hz), 116.9 (d, C-17, JCF ¼ 21.9 Hz), 105.7, 96.4; IR (KBr,
cmꢁ1) Vmax 3411, 1621, 1508, 1331, 1136, 1088; Anal. Calcd
for C15H11FO4: C. 65.69; H: 4.04; Found: C: 65.83; H. 4.04.
MOM protection part: To a solution of trihydroxy acetophe-
none (34) or dihydroxy acetophenone (56) in DCM (5 mL/
1 mmol of substrate) was added DIPEA (2.9 eq.). After stirred
for 0.5 h MOMCl (2.1 eq.) was added at 0 ꢀC, under N2 atm.
Reaction mixture stirred for 3 h. Reaction stopped with satu-
rated NH4Cl solution (2 mL/1 mmol of substrate). The com-
bined extracts were dried over Na2SO4. The solvent was
removed in vacuo and the remaining residue purified via
coloumn chromatography over silica gel using gradient elu-
tion with EtOAc and Hexanes to yield compounds 35
and 57.
Condensation part: To a solution of compounds 35 or 57
(1 eq.) in MeOH (5 mL/1 mmol of substrate) benzaldehyde
derivatives (2-7) (1.6 eq.) and %50 KOH solution (1.5 mL/
1 mmol of substrate) was added sequentially and stirred for
15 h at room temperature. After 15 h solvent was evaporated.
Crude material exracted with 2 M HCl solution (2 mL/1 mmol
of substrate) and EtOAc (2 mL/1 mmol of substrate x 3). The
combined extracts were dried over Na2SO4. The solvent was
removed in vacuo and the remaining residue purified via
flash chromatography over silica gel using gradient elution
with EtOAc and Hexanes to yield compounds 36-40 and
58-60.
(E)-3-(3-fluorophenyl)-1-(2,4,6-trihydroxyphenyl)prop-2-en-
1-one (44)
Orange solid; yield 39%; Rf ¼ (EtOAc/Hexanes 60:40) ¼ 0.5;
m.p. 190 ꢀC; 1H NMR (400 MHz, CD3OD) d 8.18 (d, 1H,
J ¼ 16.0 Hz), 7.66 (d, 1H, J ¼ 15.6 Hz), 7.42 ꢁ 7.38 (m, 1H),
7.34 ꢁ 7.30 (m, 2H), 7.14 ꢁ 7.06 (m, 1H), 4.85 (s, 2H), 3.31 (s,
1H); 13C NMR (100 MHz, CD3OD) d 192.5, 165.5, 164.9 (C-6/C-
4), 163.3 (d, C-16, JCF ¼ 243.6 Hz), 139.9, 138.4 (d, C-12, JCF
¼
7.6 Hz), 130.5 (d, C-14, JCF ¼ 8.3 Hz), 130.3 (d, C-13, JCF
¼
8.3 Hz), 129.3 (C-1/C-3), 124.2, 116.3 (d, C-15, JCF ¼ 21.6 Hz),
113.9 (d, C-17, JCF ¼ 22.0 Hz), 104.8; IR (KBr, cmꢁ1) Vmax
3203, 2917, 1626, 1585, 1448, 1344; Anal. calcd for
C15H11FO4: C: 65.69; H. 4.04; Found: C: 65.80; H. 3.75.
(E)-3-(4-fluorophenyl)-1-(2,4,6-trihydroxyphenyl)prop-2-en-
1-one (45)
Dark yellow solid; yield 55%; Rf ¼ (EtOAc/Hexanes 30:70) ¼
1
0.4; m.p. 161-163 ꢀC; H NMR (400 MHz, CD3OD) d 8.14 (d, 2H,
J ¼ 15.7 Hz), 7.71 ꢁ 7.64 (m, 3H), 7.17 ꢁ 7.12 (m, 2H), 5.85 (s,
2H), 4.89 (bs, 2H), 3.30 (bs, 1H); 13C NMR (100 MHz, CD3OD) d
Deprotection part: To a solution of MOM-protected chal-
cones (36-40 and 58-60) (1 eq.) in EtOAc (3 mL/1 mmol 192.6, 165.4, 164.9, 163.9 (d, C-17, JCF ¼ 247.8 Hz), 140.3,

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
23
132.3 (d, C-14, JCF ¼ 3.2 Hz), 130.2 (d, C-15/C-19, JCF
¼
(dd, C-12, JCF ¼ 25, 9.1 Hz), 117.3 (dd, C-18, JCF ¼ 25.4,
8.9 Hz), 114.4 (dd, C-14, JCF ¼ 27.9, 3.0 Hz), 113.4, 108.3,
102.6; IR (KBr, cmꢁ1) Vmax 3164, 2919, 1635, 1555, 1508,
1371, 1234; Anal. calcd for C15H10F2O3: C: 65.22; H: 3.65;
Found: C: 65.58; H: 3.89.
8.4 Hz), 127.6, 115.7 (d, C-16/C-18, JCF ¼ 22.0 Hz), 104.7, 94.8
(C-1/C-3); IR (KBr, cmꢁ1) Vmax 3294, 2931, 2481, 1599, 1509,
1353, 1219; Anal. calcd for C15H11FO4: C: 65.69; H: 4.04;
Found: C: 65.48; H: 4.35.
(E)-3-(2,5-difluorophenyl)-1-(2,4,6-trihydroxyphenyl) prop-
2-en-1-one (46)
General procedure for preparation of compounds 48-52,
64-66
Dark orange solid; yield 40%; Rf ¼ (EtOAc/Hexanes 50:50) ¼
0.5; m.p. 203-207 ꢀC; 1H NMR (400 MHz, Acetone-d6) d 8.32
(d, 1H, J ¼ 16.0 Hz), 7.79 (d, 1H, J ¼ 16.0 Hz), 7.52 ꢁ 7.48 (m,
1H), 7.29 ꢁ 7.19 (m, 2H), 6.00 (s, 2H); 13C NMR (100 MHz,
To a solution of compounds 42-46 or 61-63 in EtOAc
(10 mL/1 mmol of substrate) Pd/C (20%) was added. The reac-
tion flask was purged with hydrogen gas three times before
being allowed to stir under a hydrogen balloon for 18 h at
room temperature. Then, the reaction mixture was filtered
and concentrated in vacuo and the remaining residue puri-
fied via flash chromatography over silica gel using gradient
elution with EtOAc and Hexanes to yield compounds 48-52
and 64-66.
Acetone-d6) d 192.7, 166.1, 165.8, 159.7 (d, C-15, JCF
¼
239.5 Hz) , 158.3 (d, C-18, JCF ¼ 246.1 Hz), 133.1, 132.2 (d, C-
12, JCF ¼ 5.8 Hz), 125.8 (dd, C-13, JCF ¼ 14.3, 7.9 Hz), 118.8
(dd, C-14, JCF ¼ 24.6, 9.2 Hz), 118.4 (dd, C-16, JCF ¼ 25.1,
8.9 Hz). 115.5 (dd, C-17, JCF ¼ 24.7, 3.4 Hz), 105.9, 96.1; IR
(KBr, cmꢁ1) Vmax 3280, 1738, 1633, 1517, 1350, 1218; Anal.
Calcd for C15H10F2O4: C: 61.65; H: 3.45; Found: C: 61.31;
H: 3.76.
3-Phenyl-1-(2,4,6-trihydroxyphenyl)propan-1-one (48)
White solid; yield 64%; Rf ¼ (EtOAc/Hexanes 20:80) ¼ 0.2;
1
m.p. 120 ꢀC; H NMR (400 MHz, CD3OD) d 7.24 ꢁ 7.21 (m, 2H),
(E)-1-(2,4-dihydroxyphenyl)-3-(2-fluorophenyl)prop-2-en-1-
one (61)
7.17 ꢁ 7.12 (m, 2H), 7.07 ꢁ 7.03 (m, 1H), 5.81 (s, 2H), 4.84 (s,
2H), 3.83 (s, 1H), 3.32 (t, 2H, J ¼ 7.6 Hz), 2.94 (t, 2H, J ¼ 8.4 Hz);
13C NMR (100 MHz, CD3OD) d 204.9, 164.9, 164.6, 141.9,
128.3, 128.2, 127.6, 94.6, 93.7, 31.0, 27.6; IR (KBr, cmꢁ1) Vmax
3295, 2917, 1629, 1602, 1514, 1452, 1294; Anal. calcd for
C18H18O4: C. 69.76; H: 5.46; Found: C: 69.65; H: 5.61.
Yellow solid; yield 42%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.5;
1
m.p. 160-162 ꢀC; H NMR (400 MHz, CD3OD) d 7.97 ꢁ 7.93 (m,
2H), 7.88 ꢁ 7.81 (m, 2H), 7.46 ꢁ 7.41 (m, 1H), 7.26 ꢁ 7.15 (m,
2H), 6.41 (dd, 1H, J ¼ 8.8, 2.4 Hz), 6.29 (d, 1H, J ¼ 2.4 Hz); 13C
NMR (100 MHz, CD3OD) d 191.7, 166.5, 165.6, 161.8 (d, C-15,
JCF ¼ 250.9 Hz), 135.6, 132.4, 132.1 (d, C-18, JCF ¼ 8.9 Hz),
129.1, 124.7 (d, C-11, JCF ¼ 3.5 Hz), 122.9 (m, C-12/C-13),
3-(2-Fluorophenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-
one (49)
115.9 (d, C-14, JCF ¼ 22 Hz), 113.4, 108.2, 102.7; IR (KBr, cmꢁ1
)
Vmax 3300, 1635, 1569, 1455, 1366, 1229; Anal. calcd for
C15H11FO3: C: 69.76; H: 4.29; Found: C: 69.41; H: 4.52.
White solid; yield 70%; Rf ¼ (EtOAc/Hexanes 50:50) ¼ 0.5;
1
m.p. 183-186 ꢀC; H NMR (400 MHz, Acetone-d6) d 11.69 (brs,
2H), 9.25 (brs, 1H), 7.37 ꢁ 7.34 (m, 1H), 7.23 ꢁ 7.20 (m, 1H),
7.12 ꢁ 7.04 (m, 2H), 5.97 (s, 2H), 3.43 (t, 2H, J ¼ 7.2 Hz), 3.04
(t, 2H, J ¼ 6.8 Hz); 13C NMR (100 MHz, Acetone-d6) d 204.79,
165.4, 165.4, 162.1 (d, C-18, JCF ¼ 241.6 Hz), 131.8 (d, C-14,
JCF ¼ 4.7 Hz), 129.5 (d, C-13, JCF ¼ 15.5 Hz), 128.7 (d, C-16, JCF
¼ 8.1 Hz), 125.1, 115.8 (d, C-17, JCF ¼ 22.0 Hz), 105.2, 95.9,
44.5, 24.1; IR (KBr, cmꢁ1) Vmax 2925, 1698, 1602, 1457, 1166;
Anal. calcd for C15H13FO4: C: 65.21; H: 4.74; Found: C: 65.13;
H. 5.05.
(E)-1-(2,4-dihydroxyphenyl)-3-(4-fluorophenyl)prop-2-en-1-
one (62)
Yellow solid; yield 76%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.5;
m.p. 179-181 ꢀC; 1H NMR (400 MHz, CD3OD) d 7.96 (d, 1H,
J ¼ 8.8 Hz), 7.80 ꢁ 7.68 (m, 4H), 7.18 ꢁ 7.10 (m, 2H), 6.41 (dd,
1H, J ¼ 8.8, 2.4 Hz), 6.29 (d, 1H, J ¼ 2.4 Hz); 13C NMR (100 MHz,
CD3OD) d 191.9, 166.4, 165.5, 164.3 (d, C-18, JCF ¼ 248.7 Hz),
142.6, 132.4, 131.7 (d, C-11, JCF ¼ 3.1 Hz), 130.7 (d, C-12/C-15,
JCF ¼ 8.3 Hz), 120.6, 115.7 (d, C-14/C-13, JCF ¼ 22 Hz), 113.5,
108.1, 102.7; IR (KBr, cmꢁ1) Vmax 3296, 2917, 1635, 1509,
1228, 1144; Anal. calcd for C15H11FO3: C: 69.76; H: 4.29;
Found: C: 69.72; H: 3.98.
3-(3-Fluorophenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-
one (50)
White solid; yield 95%; Rf ¼ (EtOAc/Hexanes 40:60) ¼ 0.5;
1
m.p. 123 ꢀC; H NMR (400 MHz, CD3OD) d 7.27 ꢁ 7.21 (m, 1H),
7.16 ꢁ 7.13 (m, 1H), 7.03 ꢁ 6.95 (m, 1H), 6.89 ꢁ 6.84 (m, 1H),
5.82 (s, 2H), 4.85 (bs, 2H), 3.83 (bs, 1H), 3.32 (t, 2H, J ¼ 7.2 Hz),
2.95 (t, 2H, J ¼ 8.0 Hz); 13C NMR (100 MHz, CD3OD) d 204.4,
165.0, 161.7 (d, C-16, JCF ¼ 211.4 Hz), 160.6, 144.9, 142.9,
129.7 (d, C-14, JCF ¼ 7.2 Hz), 124.1 (d, C-13, JCF ¼ 2.7 Hz),
114.8 (d, C-17, JCF ¼ 21.0 Hz), 112.3 (d, C-11, JCF ¼ 27.0 Hz),
104.1, 30.6, 27.6; IR (KBr, cmꢁ1) Vmax 3295, 2917, 1630, 1522,
1450, 1248; Anal. calcd for C15H13FO4: C. 65.21; H: 4.74;
(E)-3-(2,5-difluorophenyl)-1-(2,4-dihydroxyphenyl)prop-2-
en-1-one (63)
Yellow solid; yield 56%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.56;
m.p. 201-203 ꢀC; 1H NMR (400 MHz, CD3OD) d 7.96 (d, 1H,
J ¼ 9.2 Hz), 7.89 ꢁ 7.84 (m, 2H), 7.71 ꢁ 7.66 (m, 1H),
7.23 ꢁ 7.15 (m, 2H), 6.42 (dd, 1H, J ¼ 8.8, 2.4 Hz), 6.29 (d, 1H,
J ¼ 2.4 Hz); 13C NMR (100 MHz, CD3OD) d 191.4, 166.6, 165.8,
159.2 (d, C-13, JCF ¼ 242.6 Hz), 156.8 (d, C-15, JCF ¼ 244.8 Hz),
134.1, 132.5, 124.4 (dd, C-11, JCF ¼ 13.9, 8.6 Hz), 124.2, 118.4 Found: C: 65.07; H: 4.36.

24
S. BURMAOGLU ET AL.
3-(4-Fluorophenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-
one (51)
4-(3-(2,5-Difluorophenyl)propyl)benzene-1.3-diol (66)
White solid; yield 60%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.43;
1
m.p. 119-121 ꢀC; H NMR (400 MHz, CD3OD) d 7.02 ꢁ 6.95 (m,
White solid; yield 60%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.23;
1
m.p. 103-105 ꢀC; H NMR (400 MHz, CD3OD) d 7.23 ꢁ 7.19 (m,
2H), 6.91-6.86 (m, 1H), 6.82 (d, 1H, J ¼ 8.0 Hz), 6.26 (d, 1H,
J ¼ 2.0 Hz), 6.20 (dd, 1H, J ¼ 8.0, 2.4 Hz), 4.88 (s, 1H), 3.30 (s,
1H), 2.62 (t, 2H, J ¼ 7.6 Hz), 2.53 (t, 2H, J ¼ 7.6 Hz), 1.82 (tt, 2H,
J ¼ 15.6, 8.0 Hz); 13C NMR (100 MHz, CD3OD) d 158.9 (dd, C-
14, JCF ¼ 2.1, 238.9 Hz), 157.3 (dd, C-17, JCF ¼ 2.3, 240.1 Hz),
156.2, 155.9, 131.6 (dd, C-12, JCF ¼ 7.5, 18.6 Hz), 130.2, 119.5,
116.6 (dd, C-13, JCF ¼ 5.4, 24.9 Hz), 115.8 (dd, C-16, JCF ¼ 8.9,
25.6 Hz), 113.3 (dd, C-15, JCF ¼ 8.6, 24.2 Hz), 106.2, 102.3,
30.3, 29.3, 28.4; IR (KBr, cmꢁ1) Vmax 3400, 2931, 1620, 1525,
1489, 1305, 1236; Anal. calcd for C15H14F2O2: C: 68.17; H:
5.34; Found: C: 67.90; H: 5.59.
2H), 6.97 ꢁ 6.93 (m, 2H), 5.81 (s, 2H), 4.83 (bs, 3H), 3.30 (t, 2H,
J ¼ 7.6 Hz), 2.92 (t, 2H, J ¼ 8 Hz); 13C NMR (100 MHz, CD3OD) d
204.6, 164.9, 164.6 (C-6/C-4), 161.5 (d, C-17, JCF ¼ 240.0 Hz),
137.9 (d, C-14, JCF ¼ 3.3 Hz), 129.8 (d, C-15/C-19, JCF
¼
7.6 Hz), 114.7 (d, C-16/C-18, JCF ¼ 21 Hz), 104.1, 94.6 (C-1/C-
3), 45.7, 30.1; IR (KBr, cmꢁ1) Vmax 3342, 2924, 1630, 1509,
1453, 1299, 1218; Anal. calcd for C15H13FO4: C. 65.21; H: 4.74;
Found: C: 65.12; H: 4.53.
3-(2,5-Difluorophenyl)-1-(2,4,6-trihydroxyphenyl) propan-
1-one (52)
Biological part
White solid; yield 65%; Rf ¼ (EtOAc/Hexanes 50:50) ¼ 0.47;
1
m.p. 156-161 ꢀC; H NMR (400 MHz, Acetone-d6) d 11.67 (brs,
Cell culture studies
2H), 9.23 (brs, 1H), 7.22 ꢁ 7.07 (m, 2H), 6.99 (m, 1H), 5.94 (s,
2H), 3.43 (t, 2H, J ¼ 7.2 Hz), 3.02 (t, 2H, J ¼ 7.2 Hz); 13C NMR
(100 MHz, Acetone-d6) d 203.5, 164.5, 164.4, 158.6 (d, C-15,
JCF ¼ 240.7 Hz), 157.2 (d, C-18, JCF ¼ 239.9 Hz), 130.7 (dd, C-
13, JCF ¼ 18.2, 7.8 Hz), 116.9 (dd, C-14, JCF ¼ 24.1, 5.1 Hz),
Cytotoxicity assays against cancer cells: In this study, five dif-
ferent cancer cell lines were used [A549 Cell Line (Human
Lung Adenocarcinoma Epithelial Cells), A498 Cell Line
(Human Renal Cancer Cells), HeLa Cell Line (Human Cervical
Cancer cells), A375 Cell Line (Human Skin. Malignant
Melanoma Cells), HepG2 Cell Line (Human Hepatocellular
Carcinoma Cells)] to investigate the anti-proliferative effects
of the synthesized compounds. Other than these cancer cell
lines, HEK 293 was used as the untransformed tissue origi-
nated cell line control to determine the non-toxic concentra-
tions of the compounds on relatively healthy cells. All cell
lines were obtained from the cell culture collections of
Mustafa Kemal University. Cell incubations were done at
37 ꢀC in 5% (v/v) CO2.
116.1 (dd, C-17, JCF ¼ 25.3, 8.9 Hz), 113.8 (dd, C-16, JCF
¼
24.2, 8.6 Hz), 104.4, 94.9, 43.3, 23.7; IR (KBr, cmꢁ1) Vmax 3139,
1625, 1496, 1396, 1141; Anal. calcd for C15H12F2O4: C: 61.23;
H. 4.11; Found: C. 61.47; H: 4.39.
4-(3-(2-Fluorophenyl)propyl)benzene-1,3-diol (64)
White solid; yield 69%; Rf ¼ (EtOAc/Hexanes 30:70) ¼ 0.33;
1
m.p. 91-93 ꢀC; H NMR (400 MHz, CDCl3) d 7.26 (s, 1H), 7.21-
7.13 (m, 2H), 7.07 ꢁ 6.95 (m, 2H), 6.35 (dd, 1H, J ¼ 8.0, 4.0 Hz),
6.31 (d, 1H, J ¼ 2.4 Hz), 4.81 (bs, 2H), 2.69 (t, 2H, J ¼ 7.6 Hz),
2.60 (t, 2H, J ¼ 7.6 Hz), 1.9 (tt, 2H, J ¼ 12.8, 7.6 Hz); 13C NMR
(100 MHz, CDCl3) d 161.4 (d, C-18, JCF ¼ 243.4 Hz), 154.8,
154.5, 130.9, 130.8 (d, C-14, JCF ¼ 5.1 Hz), 129.3 (d, C-13, JCF
MTT cell viability assay
The cytotoxic activity of the compounds was determined by
using MTT assay (McGahon et al., 1995; Mosmann, 1983). The
compounds were dissolved in dimethyl sulfoxide (DMSO)
and then diluted to the appropriate concentrations in
Dulbecco’s modified Eagle’s medium (DMEM). Concentration
of DMSO was less than 1% in the culture medium.
Subsequently, the cells were seeded at 104 cells/well in
DMEM, supplemented with 10% fetal bovine serum (FBS),
100 unit/mL penicillin-G, 100 mg/mL streptomycin in each
well of 96-well microculture plates. The cells were cultured at
37 ꢀC for 24, 48, 72 and 96 h in an incubator containing
5% CO2.
¼ 16 Hz), 127.7 (d, C-16, JCF ¼ 8 Hz), 124.1 (d, C-16, JCF
¼
3.4 Hz), 120.6, 115.4 (d, C-17, JCF ¼ 22.2 Hz), 107.9, 103.1,
30.4, 29.1, 28.9; IR (KBr, cmꢁ1) Vmax 3349, 2930, 2862, 1618,
1491, 1455, 1227; Anal. calcd for C15H15FO2: C: 73.15; H: 6.14;
Found: C: 73.47; H: 6.02.
1-(2,4-Dihydroxyphenyl)-3-(4-fluorophenyl)propan-1-
one (65)
White solid; yield 60%; Rf ¼ (EtOAc/Hexanes 40:60) ¼ 0.66;
m.p. 125-127 ꢀC; 1H NMR (400 MHz, CDCl3) d 12.72 (s, 1H),
7.62 (d, 1H, J ¼ 8.0 Hz), 7.21 ꢁ 7.17 (m, 2H), 7.0 ꢁ 6.96 (m, 2H),
6.36 ꢁ 6.34 (m, 2H), 5.62 (s, 1H), 3.21 (t, 2H, J ¼ 9.2 Hz), 3.02
(t, 2H, J ¼ 7.6 Hz); 13C NMR (100 MHz, CDCl3) d 203.6, 164.0
After incubation, cells were treated with test compounds
of appropriate concentrations for 24, 48, 72 and 96 h. After
incubation period, 10 mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide) (5 mg/mL) was added to each
well and the plates were further incubated for 4 h. Then the
medium of each well was carefully removed, formazan crys-
tals were dissolved in 100 mL of DMSO. Absorbance was
determined at 570 nm for each well using microplate reader
(Bioteck) (Sabina et al., 2017).
(d, C-15, JCF ¼ 273.4 Hz), 162.9, 160.5, 136.6 (d, C-12, JCF
¼
3.3 Hz), 132.4, 130.0 (d, C-17/C-13, JCF ¼ 7.6 Hz), 115.6 (d, C-
16/C-14, JCF ¼ 20.9 Hz), 114.1, 107.9, 103.9, 39.9, 29.7; IR (KBr,
cmꢁ1) Vmax 3337, 2923, 1631, 1509, 1445, 1373, 1221; Anal.
calcd for C15H13FO3: C: 69.22; H: 5.03; Found: C: 69.16;
H: 5.24.
The cell viability was expressed as percentage of the
viable cells in each sample with respect to the control wells.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
25
Three independent experiments in triplicates were done for wells. The final volume of all wells was 190 mL. Plates were
the determination of the growth inhibition of each com-
pound. The IC50 values were calculated from concentration-
response curves using the SPSS (SPSS.Inc. Chicago) Software.
re-placed in the incubator and the cell proliferation curve
was monitored for 24 h. Different concentrations (39 nM,
78 nM and 158 nM) of 12 and 46 (200 lL) were then added
to the cells, 0.01% DMSO was used as a vehicle group and CI
was measured automatically once per hour until the end of
the experiment.
Determination of the apoptotic indexes by acridine
orange staining
Acridine orange is a dye that enters the nucleic acid (DNA or
RNA) structure in cells or can be attached to nucleic acids.
This binding is an event that occurs as a result of electro-
static interactions of the acridine molecule between nucleic
acid base pairs. Acridine orange allows cells to be examined
by fluorescence microscopy due to their metachromatic
properties. With this staining method, analysis of cellular
physiology and cell cycle status can be performed (Mirzaei
et al., 2020). The Acridine Orange dying method was per-
formed according to the method described by Mossmann
(Kalani et al., 2012). For this purpose, after preparing the cell
suspension to be 1 ꢃ 106 cells/mL sterile coverslips were
placed in culture dishes. The new synthesized chemical com-
pounds (at different concentrations) were added to the cell
cultures at the determined concentrations and the cell cul-
tures were incubated at 37 ꢀC. At the end of the incubation,
the cells were washed 3 times for 2 min with PBS solution.
After the last wash, 1 mL of p ethanol was added to the cul-
ture dishes and kept at room temperature for 5 min. After a
series of washes, the cells soaked in PBS for 2 min were
treated with acridine orange diluted with PBS. Then a mix-
ture of 200 mL of acridine orange (100 mg/mL) and ethidium
bromide (100 mg/mL) was added to the cells and incubated
at room temperature for 5 min.
Molecular docking
Molecular docking studies of the synthesized chalcone deriv-
atives were performed using the Glide XP docking protocol.
Crystal structure of ß-tubulin (PDB ID: 1SA0) (Mirzaei et al.,
2020; Sabina et al., 2017) was prepared using Protein
Preparation Wizard of Maestro. The unwanted water mole-
cules were removed from the protein and hydrogen atoms
were added. Further, the protein was optimized and mini-
mized. The receptor-binding site was defined as a Glide
enclosing box in the centroid of the co-crystallized colchicine
molecule. The structures of chalcone derivatives were built in
the Maestro program and prepared for docking with LigPrep
tool. The process was made at neutral pH and under OPLS
2005 force field (Halgren et al., 2004). The compounds were
docked into the ß-tubulin structure using Glide XP (Halgren
€
et al., 2004; Schrodinger, LLC, 2017). The best docked poses
with lowest Glide score values were documented for
each compound.
Prediction of ADME properties
€
The QikProp program of Schrodinger Maestro 9.2
€
At the end of the staining process, the cell preparations (Schrodinger, LLC, 2017) was used to obtain the ADME prop-
were examined under an immuno-fluorescent microscope
(Olympus) and evaluated. In the microscopic examination,
live cells (green stained chromatin), apoptotic cells (conden-
sate and fragmented green stained chromatin), dead cells
with normal nuclei (condensed and fragmented orange
stained chromatin) were counted in the preparade.
Subsequently, the apoptotic index calculation was made
according to the formula below.
Apoptotic index ¼ (Number of Apoptotic Cells plus
Number of Apoptotic Core Dead Cells)/Number of Live Cells
plus Number of Apoptotic Cells Number of Normal Cells with
Dead Nucleus plus Number of Apoptotic Nuclei) ꢃ 100.
erties of the chalcone derivatives. The program uses the
Jorgensen (Duffy & Jorgensen, 2000; Jorgensen & Duffy,
2000; 2002) method to calculate pharmacokinetic properties
and some identifiers. In addition, QikProp offers a range of
values to compare certain molecular properties with 95% of
known drugs.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
Scientific and Technological Research Council of Turkey10.13039/
501100004410We thank the Scientific and Technological Research
Council of Turkey (TUBITAK Grant Number: 114Z554).
xCELLigence cytotoxicity studies
In order to evaluate the cytotoxicity response of the liver
cancer cells to the 12 and 46 applications, the time-depend-
ent graph of the proliferation curves obtained from the real-
time cell analyser was plotted. The data obtained were digi-
tized in terms of cell index by the software of the device.
After the whole experimental procedure was prepared in the
system, 100 mL of doped medium was added to each well of
the e-plates and the plates were placed in the system in the
incubator. In this way, the experiment was started and blank
reading was taken. Then the cell suspension containing 90 mL
of (2 ꢃ 104 HepG2 and BJ-5ta 5 ꢃ 103) cell was added to the
ORCID
Oztekin Algul
http://orcid.org/0000-0001-5685-7511
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