Design, synthesis and QSAR study of 2′-hydroxy-4′-alkoxy chalcone derivatives that exert cytotoxic activity by the mitochondrial apoptotic pathway

Article abstract of DOI:10.1016/j.bmc.2018.10.045

Eleven 4′-alkoxy chalcones were synthesized and biologically evaluated for their antiproliferative activity against four human tumor cell lines (PC-3, MCF-7, HF-6, and CaSki). Compounds 3a-3d and 3f were selective against PC-3, with IC₅₀ values ranging from 8.08 to 13.75 μM. In addition, chalcones 3a-3c did not affect the normal fibroblasts BJ cells. The most active and selective compounds were further evaluated for their effect on the progression of cell cycle in PC-3 cells, and chalcones 3a and 3c induced a G2/M phase arrest. Furthermore, it was found that these three chalcones induced the mitochondrial apoptotic pathway by regulating Bax and Bcl-2 transcripts and by increasing caspase 3/7 activation. Otherwise, the QSAR model indicates that the double bond of the α,β-unsaturated carbonyl, as well as the planar structure geometry, are important to the biological activity of the synthetized chalcones. Based on these studies, it was concluded that withdrawing substituents in ring A, decrease the antiproliferative activity. This is related to the possible mechanism of action of these compounds, where a Michael addition needs to take place in order to be a potent anticancer agent.

Full text of DOI:10.1016/j.bmc.2018.10.045

Accepted Manuscript
Design, synthesis and QSAR study of 2′-hydroxy-4′-alkoxy chalcone deriva-
tives that exert cytotoxic activity by the mitochondrial apoptotic pathway
Silvia Marquina, Maritza Maldonado-Santiago, Jessica Nayelli Sánchez-
Carranza, Mayra Antúnez-Mojica, Leticia González-Maya, Rodrigo Said Razo-
Hernández, Laura Alvarez
PII:
S0968-0896(18)31437-8
https://doi.org/10.1016/j.bmc.2018.10.045
BMC 14604
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 August 2018
17 October 2018
30 October 2018
Please cite this article as: Marquina, S., Maldonado-Santiago, M., Nayelli Sánchez-Carranza, J., Antúnez-Mojica,
M., González-Maya, L., Said Razo-Hernández, R., Alvarez, L., Design, synthesis and QSAR study of 2′-
hydroxy-4′-alkoxy chalcone derivatives that exert cytotoxic activity by the mitochondrial apoptotic pathway,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.045
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Design, synthesis and QSAR study of 2-hydroxy-4-alkoxy chalcone
derivatives that exert cytotoxic activity by the mitochondrial apoptotic
pathway
Silvia Marquina1, Maritza Maldonado-Santiago1, Jessica Nayelli Sánchez-Carranza2, Mayra
Antúnez-Mojica1, Leticia González-Maya2, Rodrigo Said Razo-Hernández3, Laura
Alvarez1*
1Centro de Investigaciones Químicas-IICBA, 2Facultad de Farmacia, 3Centro de
Investigación en Dinámica Celular-IICBA. Universidad Autónoma del Estado de Morelos,
Avenida Universidad 1001, Chamilpa, Cuernavaca, Morelos 62209, México.
smarquina@uaem.mx; maritza.maldonados@uaem.edu.mx; jes_chazaq@hotmail.com;
myam@uaem.mx; letymaya@uaem.mx; rodrigo.razo@uaem.mx; lalvarez@uaem.mx
Corresponding author
Dra. Laura Alvarez
Centro de Investigaciones Químicas-IICBA
Universidad Autónoma del Estado de Morelos (UAEM)
Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos 62209, México
Tel: +52 777 3297997
lalvarez@uaem.mx

Abstract
Eleven 4-alkoxy chalcones were synthesized and biologically evaluated for their
antiproliferative activity against four human tumor cell lines (PC-3, MCF-7, HF-6, and
CaSki). Compounds 3a-3d and 3f were selective against PC-3, with IC50 values ranging
from 8.08 to 13.75 M. In addition, chalcones 3a-3c did not affect the normal fibroblasts
BJ cells. The most active and selective compounds were further evaluated for their effect
on the progression of cell cycle in PC-3 cells, and chalcones 3a and 3c induced a G2/M
phase arrest. Furthermore, it was found that these three chalcones induced the
mitochondrial apoptotic pathway by regulating Bax and Bcl-2 transcripts and by increasing
caspase 3/7 activation. Otherwise, the QSAR model indicates that the double bond of the
α,β-unsaturated carbonyl, as well as the planar structure geometry, are important to the
biological activity of the synthetized chalcones. Based on these studies, it was concluded
that withdrawing substituents in ring A, decrease the antiproliferative activity. This is
related to the possible mechanism of action of these compounds, where a Michael addition
needs to take place in order to be a potent anticancer agent.
Keywords: Chalcones, antiproliferative activity, QSAR study, cell cycle, apoptosis,
Bax/Bcl-2, caspase 3/7, cancer.

1. Introduction
Chalcones (1,3-diaryl-2-propen-1-ones) are natural open chain flavonoids widely
biosynthesized in plants.1 Structurally, they consist of two aryl groups (A- and B-rings)
connected by an , -unsaturated ketone moiety that typically assumes the
thermodynamically more stable E configuration. Scientific research has demonstrated that
chalcones display a variety of interesting biological activities such as antioxidant,
cytotoxic, anticancer, antimicrobial, antiviral, antiprotozoal, antiulcer, antihistaminic and
anti-inflammatory.2,3 As for antitumor activity, chalcones exhibit various effects including
anti-initiation, apoptosis induction, antiproliferation, antimetastasis, antiangiogenesis, DNA
and mitochondrial damage, tubulin inhibition and so forth.4,5
Because of their structural simplicity and the associated ease of synthesis, chalcones
continue to enjoy considerable attention from medicinal chemists exploring new molecular
scaffolds for the novel therapeutics design.6,7
Among the naturally occurring hydroxy chalcones, 2,4-dihydroxychalcone
8,9
(2,4DHC; 3a), which is a flavonoid isolated from various medicinal plants,
displays
antibacterial and antifungal properties.10 Also, this compound has been proved to be
antigenotoxic on HepG2 cells.11 Experimental studies have shown the antiproliferative
activity of 2,4DHC on cancer cells.12,13 Further, it has been demonstrated that induce
apoptosis on human gastric cancer cells,14 and damage to the DNA which results in a Rad3-
dependent and Chk1-dependent cell cycle and block at the G2/M transition.15
Inspired in the chemical structure of 2,4DHC (3a), and in an effort to discover more
effective compounds, here, we synthesized 2-hydroxy, 4-alkoxy-chalcone derivatives (3a-
k) and were evaluated for their antiproliferative activity against a panel of four human
cancer cell lines including MCF-7 (breast), HF-6 (colon), CaSki (cervical), and PC-3
(prostate). Among all the compounds, chalcones 3a-c displayed the most potent antitumor
activity against the PC-3 cancer cell line and were choose for determine their capacity to
cause cell cycle arrest and to induce apoptosis. In addition, the antiproliferative properties
of these molecules were evaluated by a quantitative structure activity relationship study
(QSAR), employing global and local chemical reactivity molecular descriptors such as:

chemical hardness ( ), softness ( ), chemical potential ( ), Electrophilicity ( ) and Fukui
function.
2. Results and discussion
2.1. Synthesis
In this study we synthesized eleven derivatives of 2,4-dihydroxychalcone (3a), to
try to potentiate its cytotoxic effects. In comparison with the parttern structure 3a, we
inserted different substituents in C-4, such as methyl, ethyl, propyl, benzyl and acetate, as
well as halogens in C-3 and C-5, leaving intact B ring. It is important to mention that there
are few reports about this type of chalcones.
Alcoxyacetophenones (2b-e) were obtained by alkylation of 2,4-dihydroxy
acetophenone with methyl iodine, bromoethane, bromopropane, and benzyl bromide,
respectively, in the presence of K2CO3. The 2-hydroxy-4-acetylacetophenone (2f) was
prepared by acylation of 2a with acetic anhydride and pyridine. Otherwise, chalcones were
prepared by Claisen-Schmidt condensation of the appropriate 2-hydroxy-4-alkoxy-
acetophenone (2b-2f) with benzaldehyde (1) in the presence of KOH. 2,4-dihydroxy
chalcone (3a) was prepared through demethylation with BBr3 of the corresponding 4-
methoxy derivative chalcone (3e). Substitution pattern on A-ring was changed adding Br
and I atoms at C-3’ and C-5’ (derivatives 3g-j). Finally, catalytic hydrogenation of 3b
afforded the dihydroderivative 3k. Chalcone derivatives were characterized by 1D NMR
and mass spectra analysis. The general procedures for the synthesis of chalcone derivatives
are described in Scheme 1.

Scheme 1. Synthesis of chalcones 3a-3k.
2.2. Biological Activity
The synthesized chalcones (3a-3k) with purity values over 95% were evaluated by
the MTT assay for their capacity to inhibit the in vitro growth of breast (MCF-7), colon
(HF-6), cervical (CaSki), and prostate (PC-3) human cancer cell lines. We also included
human fibroblasts cell line (BJ) as a control of non-cancerous cells. Results are summarized
in Table 1 which indicate that chalcones displayed selectivity against PC-3 cell line, where
the derivatives 2,4-dihydroxychalcone (3a), 2-hydroxy-4-methoxychalcone (3b), 4-
ethoxy-2-hydroxy-chalcone (3c), 2-hydroxy-4-propoxychalcone (3d) and 4-acetoxy-2-
hydroxy-chalcone (3f) were the most potent with IC50 values of 13.75, 11.81, 8.2, 8.08, and
10.035 M, respectively. In addition, compounds 3a, 3b and 3c were selective to cancer
cells, unlike 3d and 3f that were cytotoxic also against human fibroblast cells.
Table 1. Antiproliferative activity (IC50 values) of synthetic chalcones 3a-k against four
human tumor cell lines and one no tumoral (BJ).
Compound
IC50 (Mean ± S.D., M)
BJ
PC-3
MCF-7
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
HF-6
ND
> 50
ND
ND
ND
> 50
> 50
> 50
> 50
38.90±0.02
CaSki
ND
ND
262.5±8
239.17±7
207.87±7
15.95±2.5
ND
21.27±3.5
ND
ND
13.75±0.35
11.81±0.25
8.2±0.15
8.08±0.032
34.66±0.18
10.035±0.21
34.037±0.5
> 50
3a
3b
3c
3d
3e
3f
3g
3h
3i
ND
> 50
> 50
> 50
> 50
> 50
> 50
> 50
ND
ND
47.78±0.45
> 50
3j

ND
> 50>
> 50
> 50
> 50
3k
5.0±0.35
0.12±0.027
0.28±0.026
0.0369±0.014
0.3±0.003
PDX
ND = no determined
PDX = Podophyllotoxin (positive control)
Similar studies reported that the structurally related 2,2,-dihydroxychalcone caused
growth inhibition in four cell lines, showing to be more effective in the prostate cancer cell
line (IC50 = 10.26 ꢀμM).16
By the other hand, a series of 2,4-dihydroxychalcones possessing both halogen and
methoxyl groups on B ring and hydroxyl and alkyl groups on A ring were investigated for
their ability to inhibit in vitro growth of three human tumor cell lines, MCF-7 (breast
adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma). The
results showed that 3,4,5-trimethoxy-2,4-dihydroxychalcone and 4-fluoro-2,4-
dihydroxychalcone were the most active against MCF-7 cell line with IC50 values of 7.8 ±
0.5 and 7.3 ± 0.6 M respectively.17
2.3. Cell Cycle analysis
Several studies have shown that the antiproliferative activity of chalcones is due to
their interference with different cell cycle phases. Previous studies with chalcone
derivatives have demonstrated that many of these compounds cause arrest in G2/M
phase.16,18,19 Some chalcones may bind to tubulin, preventing polymerization of the
microtubule and therefore causing blocking in the G2/M phase of the cell cycle of K562
and Jurkat cells.20-22
This information agrees with our results, we examined the effect of the synthesized
chalcones (3a-3c) in the cell cycle progression of PC3 cancer cells, using the IC50 values
previously determined. Figure 1 displays DNA histograms of PC3 cells treated in the
presence of 3a-3c and controls. One of the changes more frequently observed in the cell
cycle of PC3 cancer cells is the G2/M phase arrest. For instance, compound 3a (13.75 µM)
induced a G2/M phase arrest in PC3 cells by increasing from 24.9 % to 40.8 % with respect
to the negative control (Figure 1B), while 3c (8.2 µM) to 50.1% (Figure 1D) and chalcone
3b at 11.81 µM the increase in G2/M was not significant (27.1 %) (Figure 1C).
In addition, in this study, chalcones 3a-3c significantly increased the subG1 phase
population, this suggests DNA damage, cell death or cellular senescence (Figures 1B-1D).

Fig. 1. Effect of chalcones on cell cycle in PC3 cell line of prostate cancer. A) Negative Control; B) 3a; C)
3b; D) 3c; E) Podophyllotoxin (positive control).
2.4. Cell death
Several studies have reported that chalcones can block the cell cycle in the G2/M
phase,21 while others show blocking in the G0/G1 phase.23,24 However, the effect of
chalcones and their derivatives in the transduction of signals which control the cell cycle
and induce apoptosis of cells are still controversial. To elucidate the type of cell death
induced by compounds 3a-3c in PC3 cells, we investigated whether these compounds could
induce apoptosis. PC3 cells treated with compound 3a, 3b and 3c, according to their IC50,
were observed by fluorescence microscopy of orange-ethidium acridine bromide clear
condensation of chromatin, plasma membrane blistering and apoptotic bodies formation
were observed; similar morphological changes were observed when apoptosis was induced
by the positive controls H2O2 (Figure 2F) and podophyllotoxin (Figure 2G).

It was also observed the chromatin condensation and yellow-green nuclei (white
arrows) in the presence of all compounds (Figures 2C-2E), while membrane invaginations
are clearly seen in the effect caused by compound 3c; Figure 2E presents the morphological
changes such as chromatin condensation, plasma membrane blebbing and formation of
apoptotic bodies. Our results in the increase of SubG1 phase were correlated with the
morphological changes observed which are characteristic of apoptosis. These results are in
agreement with the increase of the subG1 population previously observed in the cell cycle
histograms showed in Figure 1.
Fig. 2. Effect of chalcones on cell death by epifluorescence microscopy in PC3 cell line of prostate cancer. A)
andB) Negative Controls; C) 3a; D) 3b; E) 3c; F) H2O2 apoptosis positive control; G) Podophyllotoxin 0.005
µM apoptosis positive control; H) Necrosis control.
2.4.1. Apoptosis characterization: Evaluation of the effect of chalcones 3a-c on the
expression of Bax, Bcl-2 and caspases 3/7 activation in PC-3 cell line
Apoptotic pathway has been described as an important signaling of cell death for
mammalian cells.25 It is well documented that activating caspase-9, cause the activation of
effector caspases (-3, -6 and -7) in the apoptosis of intrinsic pathway; also an increase of
the pro-apoptotic Bax expression and down-regulation of the anti-apoptotic Bcl-226,27 is
observed. The ratio of Bax/Bcl-2 is critical for the apoptosis induction by the mitochondrial
pathway.28,29 It has been described in the literature some studies showing that chalcones

induce cell death via apoptotic pathways, for instance, the K562 cells treated with a
synthetic chalcone induced apoptosis through the mitochondrial pathway and activated
caspase-3, while Jurkat cells can reduce the expression of the anti-apoptotic gene Bcl-2 and
increase the expression of the pro-apoptotic gene.20,30
This motivated us to look at the effect of the 3a-3c chalcones on the levels of
Bax/Bcl-2 and caspase3/7 activation on PC3 cells. The results showed that compounds, 3a-
3c clearly inhibited Bcl-2 and increased Bax transcripts in PC3 cells (Figure 3) and showed
a clear increase in caspase 3/7 activation statistically significant as compared to the control
(Figure 4) suggesting that 3a-3c cause cell death by mitochondrial apoptotic pathway.
2.6. Computational results
2.6.1. Geometry optimization and Molecular Graphics
From the conformational analysis of all the chalcone derivatives (3a-3k) and their
subsequent optimization geometry, the minimum energy structure of all the chalcones was
obtained, since all their frequencies were positive. The optimized structure of all the
chalcone derivatives was obtained, and the LUMO and ionization potential maps, are
displayed in Figures 5 and 6 respectively.

Fig. 5. LUMO map of all the chalcone derivatives (3a-3k). Blue, green, yellow and red colors indicate the
most, medium, poor and none populated region of the LUMO orbital, respectively.
It can be noted that almost all the chalcones share a similar plane structure
geometry, regardless molecule 3k. This fact is related to its lack of double bound between
the phenyl ring and the carbonyl moiety. From the LUMO map analysis we can see how
this lack of double bond in the chalcone skeleton affects its electronic distribution (Figure
5). The rest of the chalcones had their LUMO map with similar characteristics; LUMO map
indicated the regions of a molecule that are most sensitive to nucleophilic attack (blue color
regions). One region is on the carbonyl carbon, and the other region is on the β carbon; like
in a simple α,β-unsaturated carbonyl compound. This fact may explain why molecule 3k
has not anticancer activity. Nevertheless, molecules 3j and 3h possess the same LUMO
map characteristics of the biological active chalcones and they are inactive. That is why we
employed the potential ionization map to identify the molecule zones that are more prone to
suffer an electrophilic attack (red zones, Figure 6).

Fig. 6. Local ionization potential map of all the chalcone derivatives (3a-3k). Red, yellow, green and blue
colors indicate zones from which electrons are most easily, medium, less and unlikely ionized, respectively.
From Figure 6 we can observe that the substitution of an halogen atom in the
benzene ring affects its local ionization potential, like in 3g, 3h, 3i and 3j. In addition, a
decrease in the ionization potential occurs in the region of the double bond where the Cα
and Cβ are located. This may explain the low and lack of biological activity of the
molecules that possess one halogen atom in their benzene ring (3g-3j). From these results,
we can deduce that electron withdrawing substituents in ring A affects the biological
activity of the chalcones, specially substitutions in the C-5 of ring A, since they losses their
anticancer biological activity.
Analyzing the values of the global reactivity molecular descriptors (Table S1) it can be
observed that 3k has the greater chemical hardness value, this can be caused by its lack in
the double bond between the ring B and the carbonyl group and this fact may explain its
biological inactivity. Contrary to 3h and 3j that have the lowest values of chemical
hardness, therefore 3h and 3j are the softest molecules. These molecules for our study were
biologically inactive, we cannot attribute this to the presence of halogen atoms in their

structure (Br and I) because 3g and 3i also have them. Therefore, the location of halogen
atoms in ring A is crucial for their anticancer activity, this can be related to the resonance
and inductive effects related to the position were the substitution occurred. It is worthy of
mention the works of Fortuna et al, who used the Volsurf approach to design and
synthesized several new halogenated heterocyclic compounds with potent antiproliferative
activity against different cell lines, especially MCF-7.31, 32
2.6.4. QSAR study
On the other hand, from the molecular graphics results we can observe that the
carbonyl and substituted benzene of the chalcones are key for their biological activity.
Nevertheless, we still cannot explain the differences in between the active chalcones.
For this reason and considering the small number of molecules, we performed a QSAR
study employing global and local reactivity chemical quantum descriptors of the chalcones;
QSAR models of a small number of compounds have been successfully used to help
explaining their biological activity. The best mathematical model according to all the
statistical parameters is shown below.
Because of the small number of compounds in this study, the number of descriptors
allowed in the QSAR model is very low. Therefore, to ensure a non-collinearity between
descriptors the QUIK rule had to be approved ( ). In the same manner to avoid a
QSAR model with an excess of good or bad descriptors the redundancy ( ) and
overfitting ( ) rules were checked. Additionally, to have a greater guarantee of
the QSAR model predictive ability we used the Asymptotic rule ( ) as a
determinant for the model selection. QSAR model indicates that the double bond of the α,β-
unsaturated carbonyl is directly correlated to the biological activity of the chalcones. The
of the Cα and Cβ are the molecular descriptors that helps us to describe how the
chemical reactivity of the chalcones affects their anticancer activity. This equation agrees
with the LUMO and potential ionization maps described before. According to the QSAR

equation, if we increase the negative value of of Cα and Cβ atoms, the biological
activity of the chalcones will increment (Table 2).
Table 2. Values of the molecular descriptors present in the QSAR model and the
biological activity (IC50) of chalcones.
Molecule
-0.025 -0.025
-0.021 -0.025
-0.018 -0.027
-0.019 -0.025
-0.017 -0.023
-0.027 -0.023
-0.019 -0.018
13.75
11.81
8.2
3a
3b
3c
3d
3e
3f
3g
3h
3i
8.84
34.67
10.04
47.79
˃ 50
34.04
˃ 50
˃ 50
-0.01
-0.023
-0.023 -0.019
-0.008 -0.019
3j
3k
0.016
0.013
Since we do not possess an exact quantitative IC50 value for the 3f, 3h, and 3j
molecules, they were not considered for the construction of the QSAR model. All the
experimental biological activity ( ), the calculated and predicted biological activities
( and ) by the QSAR model and leverage values ( ) of chalcones are presented
in Table 3. Also, the Calculation Error and Prediction Error values which states for the
differences between and both and , are presented by the and the
terms respectively.
Table 3. Values of the anticancer experimental , calculated and predicted
activities, and and values are shown.
Molecule
13.75
11.81
8.2
6.69
13.05
8.99
16.24
28.26
12.34
47.17
-
2.7
13.32
9.59
18.32
25.26
14.79
46.32
-
0.361
0.18
0.431
0.22
0.40
0.51
0.58
-
-7.06
1.24
0.79
7.4
-6.41
2.31
-0.62
-
-11.05
1.51
1.39
9.48
-9.41
4.75
-1.47
-
3a
3b
3c
3d
3e
3f
3g
3h
3i
8.84
34.67
10.04
47.79
˃ 50
34.04
˃ 50
36.39
-
37.94
-
0.31
-
2.35
-
3.9
-
3j
˃ 50
-
-
-
-
-
3k

The linear relation of versus plot is shown in Figure 7A, values are
obtained using the QSAR model. The squared correlation coefficient ( ) of the QSAR,
which explain the variance of the description model, is shown. From this figure it can be
noted that the mathematical model possesses a respectable descriptive ability taking into
account the small number of molecules, even the low values of the biological activity are
well approximated. Also, linear relation of the versus
plot is shown in Figure 7B,
values are obtained employing the leave one out technique. The squared correlation
coefficient ( ) corresponds to the value of the explained variance in prediction of the
QSAR model
.
Fig. 7. A) Linear relation of calculated activity versus experimental activity. B) Linear relation of predicted
activity versus experimental activity.
William plot based on the prediction residuals and the leverage values was used to
define the applicability domain of the chalcones anticancer activity prediction model
(Figure 8). From William plot, both structural outside compounds ( ) and response
outliers (
) can be detected.

Fig. 8. Williams plot of prediction residuals versus leverage values of chalcones. The horizontal line shows
the warning leverage ( , n is the number of chalcones and p is the number of descriptors in the
model plus one), the two vertical lines indicate the bounds within which all of residuals should lie
(3SDEC=13.38).
Compounds outside the area formed by the three black lines are identified as
outliers. These lines represent the warning leverage (h*, horizontal line) and three times the
standard deviation in calculation error (SDCE, vertical lines). All the chalcones fell within
the applicability domain of the model, suggesting the application of this model for the
prediction of the anticancer activity of new chalcones with high structure similarity.
From these Figures it can be shown that the QSAR model calculates in a good
proportion the IC50 experimental values, with an exception for 3a and 3e. Where their
calculation is complicated since 3a has the same value of for Cα and Cβ, and 3e
possesses a low value of for Cα.
QSAR equation shows that is the descriptor with major importance for the
biological activity of chalcones (higher coefficient value). This can be explained by the
chemical mechanism that these compounds (α,β-unsaturated carbonyl) may suffer. Since

represents the Fukui function in terms of the removal of an electron from the molecule,
and more specific, the effect of removing one electron from atom A. If we have values of
that are higher than those of , this will indicate that a Michael addition is taking
place rather than a carbonyl addition. Implying that chalcones undergoing a Michael
addition will possess higher anticancer activity that those undergoing a carbonyl addition.
Analyzing the molecular graphics and QSAR results it can be deduced that the
incorporation of electron withdrawing groups in ring A, reduces the anticancer activity of
chalcones over the PC-3 cell line. These electron withdrawing groups trigger a nucleophilic
attack over the carbon of the carbonyl group; facilitating that a carbonyl addition takes
place. This is related to the possible mechanism of action of these compounds, where a
Michael addition needs to take place so that the chalcones can have a potent anticancer
activity. Additionally, this hypothesis is supported by the fact that molecule 3k, which
lacks a double bond between ring B and the carbonyl moiety, did not display anticancer
activity. Corroborating the importance of the double bond for the activity of these
molecules, which fits perfectly with our QSAR model. Where these chalcones undergoes a
covalent mechanism to be active as anticancer compounds, reacting with a protein (with
their serine and threonine residues) or nucleic acid (phosphate groups) important for the
cell stability.
3. Conclusions
In this study, derivatives of 2-hydroxy-4-alkoxy chalcones (3a-k) were selected
and synthesized from different side chains at position 4 of ring A and leaving ring B
without substituents. Antiproliferative assay revealed that the synthetized compounds
displayed selective cytotoxicity against PC-3 cell line, and three compounds 3a-3c showed
the highest activity. Further studies on the mechanism demonstrated that these three
chalcones induced the mitochondrial apoptotic pathway by regulating Bax and Bcl-2
transcripts and by increasing caspases 3/7 activation. In addition, chalcones 3a and 3c
induced a G2/M phase arrest. Finally, QSAR study suggested the importance of the α,β-
unsaturated ketone and the planar structure geometry for the biological activity, in which,
electron withdrawing substituents in ring A decrease the anticancer activity of chalcones,
by switching its reactivity so that a carbonyl addition occurs rather than a Michael addition.

The combined in vitro effects suggest that chalcones 3a-3c are compounds with bioactive
potential that might be used as lead compounds for the development of antitumoral drugs in
the coming future.
4. Experimental section
4.1. General procedure: Chemistry
All commercial regents: 2,4-dihydroxyacetophenone (99%), benzaldehyde (99.5%),
iodomethane (99%), bromoethane (98%), bromopropane (99%) and benzyl bromide (98%),
were obtained from Sigma-Aldrich and were used without further purification. Melting
points where determined in a Fisher Johns and are uncorrected. NMR spectra were recorder
1
with a Varian System instrument (400 MHz for H, and 100 MHz for 13C), employing
CDCl3 as solvent and TMS as internal reference; chemical shifts (δ) are expressed as ppm
and coupling constants (J) in Hertz. Multiplicities are indicated as singlet (s), doublet (d),
triplet (t), quartet (q), double of double (dd), multiplet (m) and broad singlet (bs). Open
column chromatographies were carried out on silica gel 60 (70–230 and 230–400 mesh),
different solvent systems as mobile phase was used for the purification (n-hexane and
EtOAc). Mass spectra were obtained in a Joel M-station JEOL JMX-AX 505 HA mass
spectrometer.
4.2. Synthesis of 2'-hydroxy-4'-alkoxy-acetophenones 2a-2f
2'-hydroxy-4'-alkoxy-acetophenones 2b-2e were obtained separately by alkylation
with 2,4-dihydroxy acetophenone (2a) (1 g, 6.58 mmol) in presence of each halide;
iodomethane (1.23 mL, 19.74 mmol), bromoethane (1.47 mL, 19.74 mmol), bromopropane
(1.69 mL, 19.74 mmol) and benzyl bromide (0.39 mL, 3.28 mmol) respectively, using
K2CO3 (0.91 g, 6.58 mmol) as a base, in acetone (10 mL). Each reaction mixture was
heated at reflux for 13 h. The crude reactions were purified separately by column
chromatography on silica gel eluting with 98:02 n-hexane-EtOAc This procedure afforded
2b (720 mg, 65.9%), 2c (870 mg, 73.5%), 2d (950 mg, 74%), and 2e (370 mg, 23.2%). 2f
was prepared by acetylation of 2a (1g, 6.58 mmol) with acetic anhydride/pyridine 2:1. The
crude reaction was ended with 20 mL Na2CO3. Then was neutralized with 10% HCl
solution (x3). The organic phase was purified by column chromatography on silica gel with
96:04, n-hexane-EtOAc, to afford 2f (810 mg, 63.43 %). The structure of these compounds
was established by NMR and mass data.

4.3. Synthesis of chalcones
The general procedure of the synthesis of chalcones is described in Scheme 1.
Chalcones were prepared by Claisen-Schmidt condensation of benzaldehyde (1) and the
alcoxyacetophenones (2b-f) in presence of KOH at reflux for 4 h. The reactions were
monitoring by TLC and purified by column chromatography (n-hexane/EtOAc, 98:2) to
give the respectively chalcones as yellow crystals (11.6 to 86.8 % yield). The halogenated
chalcones 3g-j were synthesized by allylic-halogenation using NBS and NIS with hν light
radiation. 3k was obtained by catalytic hydrogenation with Pd/C in CH2Cl2 at room
1
13
temperature for 16 h. The new chalcone derivatives (3h-3j) were characterized by H, C
NMR and mass spectra analysis.
4.3.1. 2-hydroxy-4-methoxy-chalcone (3b)
A solution of 2-hydroxy-4-methoxy acetophenone (2b) (600 mg, 3.6 mmol),
benzaldehyde (0.44 mL mL, 4.32 mmol) and KOH (304.21.mg, 5.4 mmol) in MeOH, was
heated at reflux for 4 h, it was monitoring by TLC and purified by column chromatography
(n-hexane/EtOAc, 98:2) to afford 3b as yellow crystals (790 mg, 86.8 % yield) M.p. 95-97
°C. EIMS, m/z: 254.09 [M]+. NMR data were in good accordance with the literature.33
4.3.2. 2-hydroxy-4-ethoxy-chalcone (3c)
Chalcone 3c was prepared from 2c in a similar manner to the described for compound 3b.
EIMS, m/z: 268 [M]+. NMR data were in good accordance with the literature.34
4.3.3 2-hydroxy-4-propoxy-chalcone (3d)
Chalcone 3d was prepared from 2d in a similar manner to the described for compound 3b.
EIMS, m/z: 282 [M]+. NMR data were in good accordance with the literature.35
4.3.4. 2-hydroxy-4-benzyloxy-chalcone (3e)
Chalcone 3e was prepared from 2e in a similar manner to the described for compound 3b.
EIMS, m/z: 330 [M]+. NMR data were in good accordance with the literature.36
4.3.5. 2-hydroxy-4-acetoxy-chalcone (3f)
Chalcone 3f was prepared from 2f in a similar manner to the described for compound 3b.
EIMS, m/z: 282 [M]+. NMR data were in good accordance with the literature.37

4.3.6. 2-hydroxy-3-iodine-4-methoxy chalcone (3g) and 2-hydroxy-4-methoxy-5-iodine-
chalcone (3h)
A mixture of 2-hydroxy-4-methoxy chalcone (3b) (100 mg, 0.4 mmol) and NIS (88.5 mg,
0.4 mmol) in chloroform was stirred and exposed to hν light for 60 min; affording a
mixture of two compounds which were purified by column chromatography (n-
hexane/EtOAc, 98:2→ 70:30), to obtain chalcones 3g and 3h.
4.3.6.1. 2-hydroxy-3-iodine-4-methoxy chalcone (3g). Yellow crystals (52.3 mg, 22.9 %);
mp 140-142 ºC; 1H NMR (400 MHz, CDCl3) δ: 14.26 (s, OH-2), 7.93 (1H, d, J = 9.6 Hz,
H-6), 7.92 (1H, d, J = 15.6 Hz, H-β), 7.65-7.43 (5H, m, H-2-H-6), 7.58 (1H, d, J = 16 Hz,
H-α), 6.55 (1H, d, J = 9.2 Hz, H-5), 3.99 (3H, s, H-1); 13C NMR (100 MHz, CDCl3) δ:
191.80 (C-7), 164.83 (C-4), 164.78 (C-2), 145.70 (C-β), 134.75 (C-6), 131.95 (C- 1),
131.16 (C-4), 129.26 (C-3, 5), 128.86 (C-2, 6), 119.77 (C-α), 115.13 (C-1), 102.61 (C-3,
C-5), 57.01 (C-1); EIMS, m/z: 380 [M]+.Anal. calcd for C16H13IO3: C, 50.55; H, 3.45; I,
33.38. Found: C, 49.80; H, 3.40; I, 32.36.
4.3.6.2. 2-hydroxy-4-methoxy-5-iodine-chalcone (3h). Yellow crystals (73.5 mg, 32.2%);
1
mp 130-132°C; H NMR (400 MHz, CDCl3) δ: 13.41 (1H, s, OH-2), 8.25 (1H, s, H-6),
7.90 (1H, d, J = 15.2 Hz, H-β), 7.68-7.44 (5H, m, H-2-H-6), 7.51 (1H, d, J = 15.2 Hz, H-α),
6.46 (1H, s, H-3), 3.92 (3H, s, H-1); 13C NMR (100 MHz, CDCl3) δ: 191.22 (C-7), 166.95
(C-4), 164.07 (C-2), 145.59 (C-β), 140.24 (C-6), 134.73 (C-1), 131.18 (C-4), 129.24 (C-
3, C-5), 128.94 (C-2, C-6), 119.88 (C-α), 116.38 (C-1), 100.48 (C-3), 73.55 (C-5), 56.95
(C-1). EIMS, m/z: 380 [M]+. Anal. calcd for C16H13IO3: C, 50.55; H, 3.45; I, 33.38.
Found: C, 49.78; H, 3.40; I, 32.25.
4.3.7. Synthesis of 2-hydroxy-4-methoxy-3-bromo-chalcone (3i) and 2-hydroxy-4-
methoxy-5-bromo-chalcone (3j)
A mixture of 2-hydroxy-4-methoxy chalcone (3b) (100 mg, 0.4 mmol) and NBS (70.07
mg, 0.4 mmol) in chloroform was stirred and exposed to hν light for 60 min; afforded a
mixture of two compounds and were purified by column chromatography (n-
hexane/EtOAc, 98:2→ 70:30), to obtain a mixture of 3i and 3j (74.9 mg, 37.45%) m.p.
170-172°C. 3j spectroscopy data was in good accordance with litarature.38 The structure of
3i was established by NMR and mass data.

4.3.7.1. 2-hydroxy-4-methoxy-3-bromo-chalcone (3i). Yellow crystals (23.3 mg, 11.6%);
m.p. 158-160°C; 1H NMR (400 MHz, CDCl3) δ: 14.01 (1H, s, OH-2), 7.93 (1H, d, J = 15.2
Hz, H-β), 7.91 (1H, d, J = 9.2 Hz, H-6), 7.66-7.44 (5H, m, H-2-H-6), 7.58 (1H, d, J = 15.2
Hz, H-α), 6.55 (1H, d, J = 9.2 Hz, H-5), 4.00 (1H, s, H-1); 13C NMR (100 MHz, CDCl3) δ:
191.42 (C- 7), 166.00 (C-2), 162.09 (C-4), 145.64 (C-β), 134.70 (C-6), 133.87 (C-1),
131.19 (C-4), 129.26 (C-3, 5), 128.94 (C-2, 6), 119.85 (C-α), 115.08 (C-1), 101.45 (C- 5),
101.24 (C-3), 56.82 (C-1). EIMS, m/z: 332 [M]+. Anal. calcd for C16H13BrO3: C, 57.68;
H, 3.93; Br, 23.98. Found: C, 56.85; H, 3.97; Br, 24.02.
4.3.8. Synthesis of 2-hydroxy-4-methoxydihydro-chalcone (3k)
2-hydroxy-4-methoxydihydro-chalcone (3k) was obtained from 3b (200 mg, 0.78 mmol)
by catalytic hydrogenation with Pd/C (20 mg) in CH2Cl2 (10 mL) and stirred at room
temperature for 16 h. It was monitoring by TLC and purified by column chromatography
(n-hexane/EtOAc, 98:2→80:20) to give 3k as white crystals (156 mg, 50.6 %) M.p. 95-
97°C. EIMS, m/z: 256 [M]+. NMR data were in good accordance with the literature.39
4.3.9. Synthesis of 2, 4-dihydroxy chalcone (3a)
A mixture of 3e (50 mg, 0.15 mmol) in 3mL of anhydrous dichloromethane was stirred at -
78°C for 15 min under nitrogen, then was slowly added BBr3 (0.1 mL, 0.001 mmol) and
stirred at -78°C for 30 min. After the time, the flask was cooled for 8 h and warmed under
room temperature. Finally, the crude reaction was purified by column chromatography with
80:20 n-hexane-EtOAc. The product 3a was obtained as a crystalline yellow solid (19 mg,
52%). NMR data were in good accordance with the literature.40
4.4. Biological Activity
4.4.1. Antiproliferative Activity
Chalcone derivatives 3a-3j, were subjected to antiproliferative assays against PC-3
(prostate), MCF-7(breast), HF-6 (colon) and CaSki (cervical) human cancer cell lines, we
also included human fibroblasts cell line (BJ) as a control of non-cancerous cells, obtained
from ATCC (American Type Culture Collection, Manassas, VA, USA). PC3 and CaSKi
cells were grown in RPMI-1640 medium (Sigma Aldrich, St. Louis, MO, USA), while
MCF7 and KB in Eagle's Minimum Essential Medium, (Invitrogen, Thermo Fisher
Scientific, Inc., Waltham, MA, USA) and supplemented with fetal bovine serum 10%

(SFB, Invitrogen) and with 2 mM glutamine, all cultures were incubated at 37°C in
atmosphere of 5% CO2 atmosphere.
8000 cells per well in 96-well plate were cultured for starting the cytotoxic
evaluation. The chalcone derivatives were solubilized in DMSO, the concentrations used
were 80, 8, 0.8, 0.08, and 0.008 µg/mL for a dose/response curve and incubated at 37°C in
5% CO2 atmosphere for 48 hrs and podophyllotoxin (PDX) was used as positive control.
For determining the number of viable cells in proliferation we used CellTiter 96® AQueous
One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA), following the
manufacturer's instructions. Cell viability was determined by absorbance at 450 nm using
an automated ELISA reader. The experiments were conducted by triplicate in three
independent experiments. Data were analyzed in Prism 5.0 statistical program and the IC50
were determined by regression analysis.
4.4.2. Cell Cycle Analysis.
PC3 cancer cells lines (2.0 × 105) were plated in 6-well plates and allowed to attach
overnight at 37°C in 5% CO2. Exponential growing cells were exposed to the chalcone
derivatives in accordance with IC50 values for 48 h and compared to podophyllotoxin
(PDX) (0.12 M) which was used as the positive control of the G2/M phase arrest. Cells
from each treatment were trypsinized and collected into single cell suspensions,
centrifuged, and fixed in cold ethanol (70%) overnight at −20∘C. The cells were then
treated with RNase (0.01M, Sigma Aldrich) and stained with propidium iodide (PI) (7.5
µg/mL, Invitrogen) for 30 min in the dark, PI has the ability to bind to DNA molecules, and
then RNase was added in order to allow PI to bind directly to DNA. The percentage of cells
in G1, S, and G2 phases was analyzed with a flow cytometer (Becton, Dickinson, FACS
Calibur, San Jose, CA); the number of cells analyzed for each sample was 10,000. Data
obtained from the flow cytometer were analyzed using the FlowJo Software (Tree Star,
Inc., Ashland, OR, USA) to generate DNA content frequency histograms, and to quantify
the number of cells in the individual cell cycle phases.
4.4.3. Cell Death
PC3 were cultured, 25000 cells per well in 24-well plates, and then were treated for
48 hours according to the IC50 of each compound in each cell line. Cells were treated 30
min with H2O2 for the apoptosis control death and for necrosis control the cells were

boiling in water at 95°C for 10 sec. After 48 hours of treatment the cells were exposed to a
solution of acridine orange (AO) and ethidium bromide (EB) (100 µg/mL AO, 100 µg/mL
EB), according to procedures reported.41 The cells were observed using a fluorescence
microscope, AO/EB are intercalating nucleic acids specific fluorochromes and when
bounded to DNA they emitted green and orange fluorescence, respectively. It is well
known that AO can pass through cell membranes, but EB cannot. Necrotic cells stain red
but have a nuclear morphology resembling that of viable cells. Apoptotic cells appear
green, and morphological changes such as formation of apoptotic bodies are observed. The
criteria for identification are as follows: viable cells appear to have green nucleus with
intact structure; early apoptosis cells exhibit a bright green nucleus showing condensation
of chromatin; late apoptosis appears as dense orange areas of chromatin condensation; and
orange intact nucleus depicts secondary necrosis.42, 43
4.4.4. RT-PCR
1.25 × 105 PC3 cells were plated and treated with chalcones 3a-3c for 48 hrs, the RNA was
isolated. The total RNA extraction was performed employing a Quick-RNA MiniPrep Kit
(Zymo Research, Irvine, USA), following the manufacturer's instructions. RNA was
quantified using NanoDrop® ND-1000 (Thermo Scientific), and the RNA content of the
samples was normalized. The RT-PCR was performed using a One-Step RT-PCR Kit with
Thermo-Start Taq (Thermo Scientific) following the manufacturer's instructions.
The primer sequences for Bcl-2 were 5'-CCC TCC AGA TAG CTC ATT-3', and 5'-
CTAGAC AGACAA GGA AAG-3'. The Bax primer sequences were 5'-ATG GAC GGG
TCC GGG GAG-3', and 5'-TCAGAAAACATGTCAGCTGCC-3'. The GAPDH primers
were
5'-CAAGGTCATCCATGACAACTTTG-3'
and
5'-
GTCCACCACCCTGTTGCTGTAG-3'. All primers were synthesized by IDT-Integrated
DNA Technologies, the reaction products of the samples were analyzed in 1.5 % agarose
gel.
4.4.5. Caspases activity
8000 PC3 cells were plated and treated with chalcones 3a-3c, after treatment the caspase
3/7 activity was determined using The Caspase-Glo® 3/7 Assay in a luminescent assay
(Promega, cat. G811C). The manufacturer's instructions were following. The results were
represented as relative units of luminescence and represented in graphs, the statistical

analysis was performed using the Prism 5.0 statistical program and the test performed was
t-student considered significant at p < 0.05.
4.5. Computational Details
4.5.1. Conformational analysis and geometry optimization
A conformational analysis of all the chalcone derivatives by using the SYBIL force
field was performe.44 The minimum energy conformer was submitted to a geometry
optimization, employing the PM3 semi-empirical method.45 In order to obtain more reliable
values of energy and geometry, the lowest-energy structures were further optimized, within
the density functional theory (DFT) formalism. For all the calculations the B3LYP46 hybrid
functional and the 6-31G* basis set for H, C, O and Br atoms, and LACVP*
pseudopotential and basis set for Y atom were employed.47 In order to ensure that the
structure correspond to a minimum on the potential energy surface, a harmonic frequency
analysis was done. All these calculations were performed using SPARTAN'08.48
4.5.2. Molecular graphics calculation
4.5.2.1. LUMO Map
We were interested in analyzing which regions of the chalcone derivatives are most
electron deficient, and hence most subject to nucleophilic attack. For this, the lowest-
unoccupied molecular orbital (LUMO) is mapped onto an electron density isosurface of
0.002 electrons/au3. So, the LUMO map of all the chalcone structures was obtained.
4.5.2.2. Local Ionization Potential Map
Also, to identify which sites may be susceptible to electrophilic attack. We obtained the
local ionization potential was mapped onto an electron density isosurface of 0.002
electrons/au3. Therefore, local ionization potential maps of all the chalcone derivatives
were calculated. This type of surface reveals those regions from which electrons are most
easily ionized.49
4.5.2.3. Chemical reactivity molecular descriptors
As a first step, we calculated the vertical electron affinity ( ) and the vertical
ionization potential ( ) employing the finite difference approximation, as it is shown below:

Where is the total energy of the optimized structure, and are the total energy of
the anionic and cationic structures, respectively.50 With these results we obtained the global
reactivity molecular descriptors: chemical hardness ( ), softness ( ), chemical potential ( )
and Electrophilicity ( ), using the following equations:
Also, we determined the Fukui function, employing the simplification proposed by Yang et
al., where the Fukui function can be calculated in terms of the atomic charge. This is
possible by assuming that the electronic density in atom R can be expressed with its atomic
partial charge ( ). Since the derivative of the density with respect to the number of
electrons (N) is discontinue, we need to evaluate the Fukui function from the left ( ) and
from the right ( ). corresponds to a nucleophilic attack (electron wining), and
corresponds to an electrophilic attack (electron losing). The calculations were carried out
with the following equations:
Where is the partial charge of R atom in the neutral molecule, and
are the partial charge of R atom in the molecule as anion and cation
respectively.51 Furthermore, with all these results, we obtained the local reactivity
molecular descriptors of softness ( and ) and Electrophilicity ( ), as it is shown
below:

52
Where , and are the softness and Electrophilicity value of R.
4.5.3. QSAR construction and validation
All the global and local chemical reactivity molecular descriptors used in this study are
displayed in Table 4. Local chemical reactivity descriptors correspond to the atoms that are
important form the anticancer activity of chalcones, according to the molecular graphics
analysis.
Table 4. Molecular Descriptors used in this study.
Molecular Descriptors
Type
Global chemical reactivity
Local chemical reactivity
, , , , ,
, , , , , , , , ,
, , , , , , , , ,
, , , , , ,
5
8
Genetic algorithms technique (GA) was employed for the construction of the mathematical
model, with in the MobyDigs 01 software.53 Chemical reactivity molecular descriptors and
the biological activity ( ), of the chalcone derivatives, were used as the independent
variables ( ) and the dependent variable ( ), respectively. To validate our QSAR model,
we employed the coefficient of determination ( ), cross-validated ( ), standard
deviation ( ) and Fisher test ( ).54
Also, to make a more sophisticated validation we used the QUIK,
redundancy ( )
and over-fitting ( ) rules. The QUIK rule is based on the multivariate correlation index
that measures the total correlation of a set of variables. This rule is a parameter that allows
the rejection of models with high descriptor collinearity, and is defined as:
Where are the values obtained from the correlation matrix of the data set X ,
represents the number of molecules and the number of descriptors. The total correlation

in the set given by the model descriptors plus the response
should always be
greater than that measured only in the set of descriptors . Therefore, if
the model is rejected, where has values of 0.01 to 0.05; models with negative
differences are unacceptable.
The goal of the REDUNDANCY rule is to detect models with an excess of good molecular
descriptors and establishes that if the model is rejected. Depending on the
data, values range from 0.01 to 0.1. is defined by:
On the other hand, the purpose of the OVERFITTING rule is to detect models with an
excess of bad molecular descriptors. This rule stipulates that if the model is
rejected. The values are calculated by:
Where values range from 0.01 to 0.1 and is the number of variables in the model. is
defined by:
Where is defined by:
is the absolute value of the regression coefficient between the jth descriptors and the
response . Additionally, we evaluated the predictive ability of our model by the Leave-
.
One-Out ( method and the asymptotic squared rule
In the
method one compound is removed from the data set and the activity ( ) is
correlated using the rest of the data set. The equation to calculate
is:
Where is the predicted value of the activity (
)

establishes that a model is predictive if
The asymptotic squared rule
, and values range from -0.005 to 0.005. The equation that defines
is:
Where is the number of molecules and the number of molecular descriptors in the
model.48
Applicability domain evaluation was carried out by means of the William plot construction,
which depends in the leverage values and the standardized error in calculation. The
leverage values (h) are obtained from the leverage matrix which contains information
about the descriptors on which the model is built. The leverage matrix is defined as:
Where is the selected descriptor matrix; is the transpose matrix of ; and
is
the inverse of matrix . The leverage values are the diagonal elements of the H
matrix. The warning leverage is calculated as , where is the number of
molecules and is the number of descriptors in the model plus one. If one of the
compounds has a leverage value higher than the is will be considered an outlier, this is,
out of the applicability domain of the model.55
Acknowledgments
The authors are thankful for the financial support from CONACyT (Grant CB 240801).
RSRH thanks CONACyT for its “Retention” scholarship (C-291229-UAEMOR
MOD.ORD./54/2017). The authors thank Laboratorio Nacional de Estructura de
Macromoléculas (Conacyt 292696 and 294406) for the spectroscopic and mass analyses.
We also thank Laboratorio de Dinámica de Proteínas and the LANCAD for the computing
resources used in this work.

References
1. Veitch NC, Grayer RJ. Chalcones, Dihydrochalcones, and Aurones. In Flavonoids.
Chemistry, Biochemistry and Applications. Andersen ØM, Markham KR, eds. CRC
Press. 2005:1003–1100.
2. Yadav VR, Prasad S, Sung B, Aggarwal BB. The role of chalcones in suppression
of NF- B-mediated inflammation and cancer. Int Immunopharmacology.
2011;11:295–309.
3. Yadav JR, Elias DW, Beazely MA, Kandepu NM. Bioactivities of chalcones. Curr
Med Chem. 1999;6:1125-1149.
4. Das M, Manna K. Chalcone Scaffold in Anticancer Armamentarium: A Molecular
Insight. J Toxicol. 2016:1–4.
5. Zhang EH, Wang RF, Guo SZ, Liu B. An Update on Antitumor Activity of
Naturally Occurring Chalcones. Evid.-Based Complementary Altern. Med.
2013:81562.
6. Batovska DI, Todorova IT. Trends in Utilization of the Pharmacological Potential
of Chalcones. Curr Clin Pharmacol. 2010;5:1-29.
7. Singh P, Anand A, Kumar V. Recent developments in biological activities of
chalcones. A mini review. Eur J Med Chem. 2014:85, 758-777.
8. Chen WH, Wang R, Shi YP. Flavonoids in the Poisonous Plant Oxytropis falcate. J
Nat Prod. 2010;73:1398–1403.
9. Ghani NA, Ahmat N, Ismail NH, Zakaria I, Zawawi NK. Chemical constituents and
cytotoxic activity of Polyalthia cauliflora var. cauliflora. Res J Med Plant.
2012;6:74–82.
10. Talia JM, Tonn CE, Debattista NB, Pappano. Antibacterial efficacy of
dihydroxylated chalcones in binary and ternary combinations with nalidixic acid
and nalidixic acid-rutin against Escherichia coli ATCC 25922. Indian J Microbiol.
2012;52:638–641.
11. Zampini IC, Villarini M, Moretti M, Dominici L, Isla MI. Evaluation of genotoxic
and antigenotoxic effects of hydroalcoholic extracts of Zuccagnia punctata Cav. J
Ethnopharmacol. 2008;115:330–335.

12. Iwata S, Nishino T, Nagata N, Satomi Y, Nishino H, Shibata S. Anti-tumorigenic
activities of chalcones. I. Inhibitory effects of chalcone derivatives on Pi-
incorporation into phospholipids of HeLa cells promoted by 12-O-tetradecanoyl-
phorbol 13-acetate (TPA). Biol Pharm Bull. 1995;18:1710–1713.
13. Kupcewicz B, Jarze AA, Małecka M, Krajewska U, Rozalski M. Cytotoxic activity
of substituted chalcones in terms of molecular electronic properties. Bioorg Med
Chem Lett. 2014;24:4260–4265.
14. Lou C, Yang G, Cai H, Zou M, Xu Z, Li Y, Zhao F, Li W, Tong L, Wang M, Cai B.
2’,4’-Dihydroxychalcone-induced apoptosis of human gastric cancer MGC-803
cells via downregulation of surviving mRNA. Toxicol In Vitro. 2010;24:1333–1337.
15. Sánchez-Picó A, León-González AJ, Martín-Cordero C, Daga RR. “Screening for
natural anticancer agents using a fission yeast bioassay. Phytochem Lett.
2014;8:184–189.
16. Haddad AQ, Venkateswaran V, Viswanathan L, Teahan SJ, Fleshner NE, Klotz LH.
Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer
cell lines. Prostate Cancer Prostatic Dis 2006;9:68–76.
17. Perro MP, Cravo S, Lima RT, Vasconcelos MH, Nascimento MS, Silva AM, Pinto
S, Cidade M, Corrêa AG. Solid-phase synthesis of 20-hydroxychalcones. Effects on
cell growth inhibition, cell cycle and apoptosis of human tumor cell lines. Bioorg.
Med. Chem. 2012;20:25–33.
18. Arianingrum R, Sunarminingsih R, Meiyanto E, Mubarika S. Potential of a
Chalcone Derivate Compound as Cancer Chemoprevention in Breast Cancer. Int
Conference Chem Chemical Eng. 2012;38:41–45.
19. Wu W, Ye H, Wan L, Han X, Wang G, Hu J, Tang M, Duan X, Fan Y, He S,
Huang L, Pei H, Wang X, Li X, Xie C, Zhang R, Yuan Z, Mao Y, Wei Y, Chen L.
Millepachine, a novel chalcone, induces G2/M arrest by inhibiting CDK1 activity
and causing apoptosis via ROS-mitochondrial apoptotic pathway in human
hepatocarcinoma cells in vitro and in vivo. Carcinogenesis 2013;34:605–611.
20. Pilatova M, Varinska L, Perjesi P, Sarissky M, Mirossay L, Solar P, Ostro A,
Mojzis J. In vitro antiproliferative and antiangiogenic effects of synthetic chalcone
analogues. Toxicol. In vitro. 2010;24:1347–1355.