Exploitation of new chalcones and 4H-chromenes as agents for cancer treatment

Article abstract of DOI:10.1016/j.ejmech.2018.07.058

Chalcone and chromene derivatives were synthesized in good yield through simple and effective reactions using innocuous solvents such as water and ethanol and high yielding aldol condensations. Generally, the reactions were performed at room temperature, leading to the isolation of highly pure compounds. These compounds were tested on breast cancer cells (MCF-7 and Hs578T) and breast non-neoplastic cells (MCF-10A). After determination of IC₅₀ value, specific assays were performed to analyze the potential of these compounds, and those bearing halogenated substituents presented enhanced activity comparing to methoxyl or methyl groups. More specifically, the bromine atom was often present in the bioactive molecules that proceeded to the final assays and showed to be promising candidates for further studies. The selected chromene acted as a cell migration inhibitory agent and triggered regulated cell death associated with G₂/M cell-arrest and microtubule destabilization. For chalcones, the results suggest an anti-proliferative activity. Also, results for combination-therapy potentiated the antitumor effect of doxorubicin and reduced cytotoxicity in MCF-10A cells.

Full text of DOI:10.1016/j.ejmech.2018.07.058

Accepted Manuscript
Exploitation of new chalcones and 4H-chromenes as agents for cancer treatment
Olívia Pontes, Marta Costa, Filipa Santos, Belém Sampaio-Marques, Tatiana Dias,
Paula Ludovico, Fátima Baltazar, Fernanda Proença
PII:
S0223-5234(18)30623-8
10.1016/j.ejmech.2018.07.058
EJMECH 10593
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 April 2018
Revised Date: 20 July 2018
Accepted Date: 25 July 2018
Please cite this article as: Olí. Pontes, M. Costa, F. Santos, Belé. Sampaio-Marques, T. Dias, P.
Ludovico, Fá. Baltazar, F. Proença, Exploitation of new chalcones and 4H-chromenes as agents for
cancer treatment, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.058.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Exploitation of new chalcones and 4H-chromenes as agents for
cancer treatment
Olívia Pontes a,b,$, Marta Costa a,b,*,$, Filipa Santos a,b, Belém Sampaio-Marques a,b,
Tatiana Dias c, Paula Ludovico a,b, Fátima Baltazar a,b, # and Fernanda Proença c,*,#
aLife and Health Sciences Research Institute (ICVS), University of Minho, Campus of Gualtar,
Braga, Portugal; bICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães,
Portugal; cDepartment of Chemistry, University of Minho, Campus of Gualtar, Braga, Portugal
* Corresponding authors.
$ and #These authors contributed equally to this work.
E-mail address: fproenca@quimica.uminho.pt; martasilviacosta@gmail.com
Keywords:
4H-Chromenes
Chalcones
Michael addition
Anticancer activity
Breast cancer
Abstract
Chalcone and chromene derivatives were synthesized in good yield through simple and
effective reactions using innocuous solvents such as water and ethanol and high
yielding aldol condensations. Generally, the reactions were performed at room
temperature, leading to the isolation of highly pure compounds. These compounds were
tested on breast cancer cells (MCF-7 and Hs578T) and breast non-neoplastic cells
(MCF-10A). After determination of IC50 value, specific assays were performed to
analyze the potential of these compounds, and those bearing halogenated substituents
presented enhanced activity comparing to methoxyl or methyl groups. More
specifically, the bromine atom was often present in the bioactive molecules that
proceeded to the final assays and showed to be promising candidates for further studies.
The selected chromene acted as a cell migration inhibitory agent and triggered regulated
cell death associated with G2/M cell-arrest and microtubule destabilization. For
chalcones, the results suggest an anti-proliferative activity. Also, results for
combination-therapy potentiated the antitumor effect of doxorubicin and reduced
cytotoxicity in MCF-10A cells.

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1. Introduction
Cancer is a rather heterogeneous pathology related to defects in terms of the regulatory
circuits that control cellular homeostasis, including proliferation, differentiation, cell
migration and death and, due to this dynamic system, there is an explicit difficulty to
reach an effective treatment [1, 2]. Breast cancer is the most frequent type of cancer in
women worldwide and the fifth cause of death from cancer. Furthermore, with an
estimated 1.67 million new cases, it is considered the second most common cancer in
the world [3]. The treatment for breast cancer depends on the stage and grade of cancer
and may include a combination of surgery and radiation, chemical, immunological and
targeted therapies [4]. However, not all the cases of breast cancer have good prognosis
leading to the importance of understanding the tumor heterogeneity and survival for the
specific subtypes [5].
Chalcones and chromene derivatives have been studied for many years and several
therapeutic applications were found including anti-HIV [6], antidiabetic [7],
antibacterial [8], antifungal [9], anti-inflammatory [10, 11], anti-coagulant [12],
antioxidant [10, 13], antidepressant [14, 15] and antileishmanial activity [16]. A general
overview of recent works reveals an infinite search for naturally occurring compounds
with anticancer activity [17, 18]. Additionally, newly synthetized molecules have
proven their potential for cancer treatment. Crolibulin, a 4H-chromene, is currently in
phase I/II of clinical trials on anaplastic thyroid cancer patients [19, 20]. Described as a
tubulin inhibitor, its activity includes induction of apoptosis and disruption of
neovascular endothelial cells with interruption of blood flow to the tumor.
Further, many other chalcones and chromenes with promising anticancer effectiveness
against a diversity of breast cancer cell lines have been identified and led to many
synthetically inspired compounds with enhanced antitumor properties [21]. The
mechanisms of action include triggering of apoptosis either via death receptor-mediated
or mitochondrial-mediated pathways [22-24]. Other compounds are effective through
cell-cycle arrest [25], estrogen receptor agonist and antagonist [26-28] degradation of
Hsp90 client proteins [29], generation of reactive oxygen species (ROS) [30], inhibiting
HIF-1 activation [31] or monoamine oxidase (MAO) A [32].

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Since cancer is a disease that affects the population worldwide and chalcones and
chromenes have already proven anticancer activity via a diversity of modes of action,
this became an interesting research area [33]. Inspired by the important biological
activity of these compounds, a series of chalcones and chromenes were synthesized
using an eco-friendly methodology and a range of compounds were selected to assess
their anticancer activity on breast cell lines.
2. Results and Discussion
2.1. Chemistry
Chalcone and chromene derivatives were synthesized as illustrated in Scheme 1. The
synthesis of α,β-unsaturated carbonyl compounds 3 was performed under base-
catalyzed aldol condensation conditions. The reaction of aromatic benzaldehydes 1 with
acetophenones 2 occurred in EtOH and aqueous NaOH (3M), at room temperature or
with gentle heating, leading to the isolation of the expected compounds 3 with yields
between 46 and 100%.
Scheme 1. Synthetic pathway for the synthesis of chalcones 3 and 4H-chromenes 4 and 5.
In order to synthetize 4H-chromenes 4 and 5, α,β-unsaturated carbonyl
compounds 3 were combined with active methylene compounds (methyl cyanoacetate,
R7=CO2Me or malononitrile, R7=CN) (Scheme 1). Reactions were carried out in

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methanol or ethanol and in the presence of triethylamine, leading to the expected
products 4 and 5 by simple filtration in 12 to 94% yields.
2.2.Biological evaluation
2.2.1. In vitro cytotoxic activity
The 34 newly synthetized compounds 3, 4 and 5 with good purity level were
evaluated in an initial screening for their anticancer properties by the SRB assay, after
72 hours treatment. The first screening was performed on the Luminal-A subtype breast
cancer cell line- MCF-7 - using two different concentrations (10 and 30 µM) for each
compound (Tables 1 and 2 Supporting Information). By analysis of structure-activity,
compounds with halogenated substituents presented better anticancer activity when
compared to molecules substituted with methoxyl (-OCH3) and methyl (-CH3) groups.
However, within the group of halogens, bromine (-Br) presented enhanced activity
comparing with chlorine (-Cl) and, even more, for fluorine (-F) [34].
From this first screening a range of 20 compounds were selected due to the low
cell survival rates induced in MCF-7 cells. Treatment of Hs578T cells (basal-like breast
cancer subtype) was also performed to assess if the subtype of breast malignancy could
lead to a different response to the treatment, given to the differential receptor expression
[35, 36]. Cytotoxicity was also evaluated for the non-neoplastic mammary epithelial
MCF-10A cell line aiming to determine if the promising molecules would be harmful
for non-tumoral cells [37]. Since chemotherapy presents side effects, screening for
cytotoxicity, could allow to manage the molecular scaffold attending to the reduction of
these body-responses. In general, Hs578T cell’s sensitivity to the compounds was
inferior to MCF-7 cells, which could be explained by the different subtype of cancer,
including differential hormone receptor expression. For MCF-10A cells, almost every
compound assessed led to a 50% (or more) cell survival for the 30 µM concentration,
which compared to the effect on breast cancer cells, corresponds to lower cytotoxicity.
After careful analysis of the results from this first screening, a range of
chalcones 3 (b, e, g and i) and chromenes 5 (c, e and i) (Figure 1) were selected to
further assess the survival curves and determine the respective IC50 values for viability
(Table 1 and Figure 40 Supporting Information).

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Figure 1. Selected compounds for IC50 determination in MCF-7, Hs578T and MCF-10A cell lines.
Table 1. Anticancer activity (IC50, µM) of the selected compounds 3 and 5 against MCF-7, Hs578T and
MCF-10A cell lines. Each compound was tested at least in triplicate and the data are presented as mean
values±SD.
Selectivity Index [38]
IC50 (µM)
Compound
(MCF-10A
vs MCF-7)
0.17
(MCF-10A
vs Hs578T)
1.96
MCF-7
Hs578T
MCF-10A
1.32 ± 0.042
5.15 ± 0.007
5.07 ± 0.009
3.98 ± 0.071
3.82 ± 0.022
2.00 ± 0.033
3.65 ± 0.021
5.94 ± 0.015
7.24 ± 0.017
0.52 ± 0.006
5.33 ± 0.010
7.27 ± 0.016
4.80 ± 0.011
5.88 ± 0.012
1.60 ± 0.023
4.52 ± 0.019
10.26 ± 0.017
16.48 ± 0.027
1.54 ± 0.078
12.11 ± 0.028
40.64 ± 0.319
24.25 ± 0.031
29.26 ± 0.026
6.52 ± 0.017
18.87 ± 0.020
18.93 ± 0.031
14.66 ± 0.027
Doxorubicin
1.35
7.02
5.09
6.66
2.26
4.17
2.19
1.02
1.27
4.59
4.05
3.98
3.08
3.08
0.85
-0.11
3b
3e
3g
3i
3k
5c
5e
5i
Doxorubicin was used as reference compound helping to validate the in vitro
results of IC50 determination. In general, the newly synthetized compounds presented
lower cytotoxic effect than doxorubicin for malignant cells however with enhanced
selectivity index. Chalcones 3 showed in general lower IC50 values than chromenes 5, in
both cancer cell lines. In 2013, a series of new chalcones and flavonol derivatives were
synthetized and tested against HCT116 cells (colorectal carcinoma cell line) in our
research group [34]. Comparing with compounds 3 and taking into account the different
cell lines, these new molecules seem to present enhanced anticancer activity with lower
IC50 values for malignant cells. This improvement in activity may be due to the 2-
hydroxy group in ring B of the chalcone instead of, the previously described, ring A.

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Compounds 3e, 3g, 3i and 5c were concluded to be the most promising due to
their less aggressive effect on the non-malignant cell line MCF-10A and better
selectivity index. These compounds proceeded to more specific tests such as cell
migration capacity (wound-healing assay), proliferation (BrdU incorporation), protein
expression using Western Blotting and Annexin/PI and cell cycle arresting assays by
flow cytometry.
2.2.2. Effect of selected compounds on cell migration
Migration and invasion capacity of malignant cells are two of the main features
involved in cancer progression and metastasis [39]. To assess if cell migration was
being affected, compounds 3e, 3g, 3i and 5c were tested by treating MCF-7 cells with
respective ½ IC50 and IC50 during 48 hours for each compound and performing the
wound-healing assay (Figure 41 Supporting Information). At the beginning and regular
timepoints (12, 24 and 48 hours), images were captured (Figure 41 Supporting
Information) and the progression of the scratch closure for each compound was
analyzed by be_Wound_1.7.1 and summarized in Figure 2.
Figure 2. Effect of chalcones 3e (A), 3g (B) and 3i (C) and chromene 5c (D) on MCF-7 cell migration
(12, 24 and 48 hours of treatment) by the wound-healing assay. Results are presented as mean ± SD of
three independent experiments. *p=0.01, **p=0.0022, ***p=0.0001, ****p<0.0001 compared to control
(DMSO).

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As shown in Figure 41A of Supporting Information, MCF-7 cells presented a
satisfactory migratory capacity since the scratch reduced significantly after 48 hours
when cells were not treated. In general, chromene 5c presented a much more promising
inhibitory ability than chalcones (Figure 2 and Figure 41E Supporting Information).
Treatment with compound 5c, for 48 hours, was able to significantly decrease the
percentage of cell migration relative to control (around 50%) as shown in Figure 2.
Several chromenes were already reported as being good anti-migratory agents against
breast cancer cell lines tested through wound-healing assay [40, 41]. Both apigenin and
luteolin are naturally occurring chromenes with similar and simple structures as
compound 5c. In the case of apigenin, the inhibitory effect was reported as being caused
by the downstream signaling blockage of PI3K/Akt pathway necessary for motility
induced by HGF (Hepatocyte growth factor) as well as adherence junction disassembly
[41]. However, as it can be seen in Figure 41E of Supporting Information, the majority
of the cells were non-adherent indicating that they died by the treatment not allowing to
determine if the inhibitory power was due to loss of migratory ability or by cell death
induction.
Cell migration was not significantly affected by chalcones (Figure 2 and Figure
41B-D Supporting Information). In the literature, not many articles describe chalcones
as inhibitors of breast cancer cell migration. In 2014, researchers found, through the
wound-healing assay, a chalcone able to significantly inhibit cell migration of highly
metastatic AGS cells (human gastric carcinoma) [42]. According to a quantitative
assessment, treatment with 1 and 2 µg/ml of chalcone inhibited 30.1 and 50.6% of cell
migration at 48 hours, respectively.
2.2.3. Effect of chalcones and chromenes on cell proliferation
The inhibitory influence on cell proliferation of the four best chalcones and
chromenes was evaluated through the ability of BrdU incorporation during DNA
synthesis. MCF-7 cells were treated with IC50 and ½ IC50 values, for 24 and 48 hours
(Figure 3).

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Figure 3. Effect of chalcones 3e (A), 3g (B) and 3i (C) and chromene 5c (D) on MCF-7 cell proliferation
after 24 and 48 hours of treatment. Results are presented as mean ± SD of at least three independent
experiments. **p<0.005, ****p<0.0001 compared to control (0.5% DMSO).
As shown by the analysis of Figure 3A and 3B, chalcones induced a significant
decrease in cell proliferation, in a time- and concentration-dependent manner, for MCF-
7 cells. The highest effect was observed for compounds 3e and 3g with more than 50%
of reduction in cell proliferation at 48 hours, with the IC50 treatment. On the other hand,
chalcone 3i did not affect cell proliferation maintaining similar rates comparing to the
control (0.5% DMSO) (Figure 3C) and evidencing the differences in activity of the two
halogen atoms (-Br and -Cl) present in the aromatic moiety.
In 2015, a series of synthesized chalcones, very similar to those tested in this
assay, were shown to present good anti-proliferative activity against several cell lines,
including MCF-7 [43]. One chalcone of this range, substituted with a methoxy group in
the aromatic moiety, was described to inhibit significantly BrdU incorporation against
NCI-H460 cells (human lung cancer cell line) and, consequently, cell proliferation
around 33% after 48 hours of treatment when compared to control.
A slightly weaker effect was observed for compound 5c (Figure 3D) that
reduced cell proliferation in about 20% with the IC50 treatment at 48 hours.

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2.2.4. Effect of chalcones and chromenes on cell death
Annexin V is commonly combined with Propidium iodide (PI) to understand if
cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity
and permeability [44]. To better understand the ability of compounds 3e, 3g, 3i and 5c
to induce cell death, the AnnexinV/PI assay was performed. MCF-7 cells were treated
with IC50 of each previously described compound for 48h and then harvested and
stained with annexin V and PI. Analysis by flow cytometry showed that MCF-7 cells
are affected by treatment with compounds 3e and 5c as revealed by the elevated
percentage of early and late apoptotic cells when compared to untreated control cells
(0.5% DMSO) (Figure 4). Treatment with the compounds 3g and 3i did not show major
alterations on cell viability in comparison with control cells (Figure 4).
A
B
****
****
****
****
****
****
Figure 4. Flow cytometry analysis of MCF-7 cell’s viability assessed by annexin V/PI assay. (A) Representative dot
plots of MCF-7 cells untreated (control) or treated for 48 h with IC50 concentrations for chalcones (3e, 3g or 3i) or the
chromene 5c. (B) Graphical representation after quantification of the percentage of cells in each quadrant of the dotplot.
The results were obtained using the non-treated cells as control (100 %) and mean±SEM of two independent
experiments. Annexin V/PI data was analyzed bytwo-way ANOVA and Bonferronipost hoctest. ****p<0.0001.
Many other chromenes were previously described as apoptosis-inducers both by
receptor-mediated extrinsic or mitochondrial-mediated intrinsic pathway. For example,
the newly synthetized Gen-27 was demonstrated to be a bioactive molecule increasing
apoptotic cell rates in human breast cancer cells, including MCF-7 cells [22].

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Researchers found that the release of cytochrome c from mitochondria to cytosol
activates caspases and induces PARP cleavage. Another study showed that the new
thiadiazole hybrid (IPTBC) inhibits cell growth of human breast adenocarcinoma cells
in vitro mainly due to apoptosis rather than necrosis, as showed by multiple apoptosis
assays [23]. Therefore, chalcones and chromenes are recognized as anticancer agents
through pro-apoptotic mechanisms. Since tumor cells are highly adaptable to different
conditions due to their ability of proliferate and escape apoptosis, further studies to
better understand the nature of the apoptotic behavior would be important. Thus, the
levels of several proteins involved in cell death pathways were determined by Western-
blot.
As represented in Figure 5, regulated cell death seems to be mainly induced by
compound 5c. The level of cleaved PARP (c-PARP) after 24 hours of treatment is
considerably enhanced with this chromene comparing with compounds 3. Nevertheless,
molecule 3e seems also to induce a discreet increase in the levels of c-PARP. This data
is in agreement with the data above described related to the evaluation of cell survival
by AnnexinV/PI.
Regarding Bim, a BH-3 only protein from Bcl-2 family, an evident increase of
its levels is noted (Figure 5). Moreover, biophysical studies have indicated that BH3-
domain proteins, of which Bim is a member, were described to interact with tubulin that
is also overexpressed upon treatment with chalcones and chromene [45]. In an initial
phase, microtubules are described to sequester Bim by binding to the dynein light chain,
thereby preventing initiation of apoptotic signaling. When released from the
microtubules, Bim migrates to the mitochondria, interacting with several proteins (e.g.
Bax, Bcl-2 and Bcl-xL) and, finally, promoting apoptosis [45]. Finally, levels of Bcl-xL
(Figure 5), an anti-apoptotic protein from the Bcl-2 family, seem to considerably
decrease upon treatment at 48 hours thereby promoting apoptosis [46]. Together, this
information suggests that an early apoptosis signal is released from cells affecting then
the levels of pro- and anti-apoptotic proteins involved in mitochondrial-induced
apoptosis.

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Figure 5. Representative immunoblots MCF-7 cells treated for 24h (A) and 48h (B) with compounds 3e,
3g, 3i and 5c. Similar blots were obtained from at least two independent experiments.
2.2.5. Effect on cell cycle arrest
Inhibition of cell cycle progression, allied to apoptosis, is an important factor to control
cancer cell growth. To investigate whether the four selected compounds affect the cell cycle of
MCF-7 cells, the cell cycle distribution of the cells stained with PI were analyzed using flow
cytometry for determination of the DNA content. As shown in Figure 6, compared to the control
group, cells treated with compound 5c showed a statistically significant accumulation of cells in
G2/M cell cycle phase accompanied by a notorious decrease of cells in G0/G1 phase of the cell
cycle. Furthermore, treatment of MCF-7 cells with the compound 3e resulted in an extensive
fragmentation of DNA and accumulation of cells with a sub-G0/G1 content of DNA (Figure 6A).
On the contrary, the other chalcones did not presented such marked effect revealing a very similar
pattern to control cells.

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In 2014, a newly synthetized flavonoid (LL-202) revealed anticancer effects against human
breast cells in vitro inhibiting growth and proliferation as well as an apoptosis-inducing behavior
both by mitochondrial- and death-receptor-mediated pathways [24]. By flow cytometry, it was
reported that LL-202 induced cell cycle arrest at G2/M phase in MCF-7 cells. Finally, the newly
synthetized flavonoid significantly inhibited the growth of MCF-7 breast cancer xenograft tumors
in a dose-dependent manner, with low systemic toxicity.
Figure 6. Flow cytometry analysis of the DNA content of MCF-7 cells. (A) Cell cycle profile of MCF-7
cells untreated (control) or treated for 48 hours with the IC50 concentrations for chalcones (3e, 3g or 3i) or
the chromene 5c. (B) Quantification of the cells in different phases of cell cycle. Results are presented as
mean ± SD of 2 independent experiments. Data was analyzed by two-way ANOVA and Bonferroni post
hoc test. **p<0.005, ****p<0.0001 compared to control.
2.2.6. Effects of chalcones and chromene on microtubule dynamics
It is well-known that the tubulin binding agents are divided into two types: microtubule
stabilizer agents (MSAs) as paclitaxel and microtubule destabilizer agents (MDAs) as colchicine
[47]. To evaluate to which type compounds 3e, 3g, 3i and 5c are related to, newly synthetized
compounds were employed at their IC50 value. Also, paclitaxel (at 2 µM) was administrated to
malignant cells to assess its effect and visualize the main differences in comparison with our
compounds.

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Figure 7. Effect of 3e, 3g, 3i and 5c on the microtubule network of MCF-7 cells. Untreated (control),
paclitaxel (2 µM) and cells treated with compounds 3e, 3g, 3i and 5c at the IC50 value for 12h were
stained with β-tubulin and counterstained with 4,6-diamidino-2-phenylindole (DAPI). Microtubules and
unassembled tubulin are shown in green. DNA, stained with DAPI, is shown in blue.
As shown in Figure 7, chalcones treatment did not significantly affect cells maintaining
morphological similarities to the control. Also, no tubulin network disturbance seems to happen
when MCF-7 cells are treated with compounds3e, 3g and 3i.
On the other hand, compound 5c induced significant morphological alterations compatible
with a disruption of the microtubules dynamics after 12 hours treatment. Images suggest that
compound 5c promotes cell detachment, shrinkage and round up in contrast with the elongated
shape of the chalcone treated cells that are similar to the control. Also, nuclei seem to be
contracted and present condensed chromatin (Figure 7). These cell characteristics point to a
regulated cell death phenotype in accordance with previous results.
In fact, crolibulin, a chromene with similar scaffold to compound 5c, has proved its activity
as a MDA binding to the colchicine-binding site on β-tubulin and being on clinical trials for
anaplastic thyroid cancer treatment [48]. Moreover, this small molecule activity leads to the cell
cycle arrest, induction of apoptosis, and inhibition of tumor cell proliferation, being found a
comparable mode of action for this compound [20]. Our results demonstrate that compound 5c

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seems to inhibit tubulin dynamics and might be a promising novel tubulin polymerization
inhibitor agent. The increased levels ofβ-tubulin assessed by immunoblot (Figure 5) togetherwith
the analysis of Bim levels, further support the activation of the intrinsic apoptosis signal pathway.
2.2.7. Effect of the compounds in combination withdoxorubicin
Anthracyclines are antitumor antibiotics that alter the cancer cell DNA, preventing
uncontrolled cell proliferation and replication, byDNA intercalation or formation of free radicals.
Doxorubicin (Figure 8) has demonstrated a great treatment potency, being considered one of the
most potent chemotherapeutic drugs approved by FDA [49]. Slow disease progression by
interfering with dividing cells has been broadly acknowledged for several decades. Doxorubicin is
used as therapy for hematological malignancies and solid tumors, including breast cancer. On the
other hand, its high cytotoxicity on noncancerous cells in the human body complicates cancer
treatment [50]. The literature reports that doxorubicin toxicity affects several organs including the
heart, brain, kidneyand liver.
Figure8. Doxorubicin.
Aiming to enhance doxorubicin anticancer potential and, possibly, diminish its side effects,
a combinatory study with compounds 3e, 3g, 3i and 5c and doxorubicin was performed on MCF-
7 cancer cells and MCF-10A non-malignant cells (Table 2 and Figure 42 Supporting
Information). The combined treatment was performed using a combination of half the IC50 of
doxorubicin (0.66 and 0.77 µM for MCF-7 and MCF-10Acell lines, respectively) and varying the
concentration (from 30 to 0.005 µM) of each selected compound (3e, 3i, 3g and 5c) (Figure 43
Supporting Information). Comparing to the IC50 of doxorubicin (Table 1), the combination with
compounds 3e, 3g, 3i and 5c improves the anticancer effect by decreasing the minimal effective
concentration need to reduce cell viability in 50% of the malignant cells. Furthermore, IC50 for
MCF-10A cells is greatly improved by the combined treatment, increasing the selectivity index of

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the newly synthetized compounds. These results suggest that this combined-therapy may decrease
the toxicityof non-neoplastic cells, anticipatinga potential reduction of side effects.
Table 2. Antiproliferative activity (IC50, µM) of the selected compounds 3 and 5 against MCF-7 and MCF-10A cell
lines in combination with doxorubicin after 72h treatment.
IC50 (µM)
Compound
Doxo + 3e
Doxo + 3g
Doxo + 3i
Doxo + 5c
Selectivity index
MCF-7
MCF-10A
>60
0.42 ± 0.050
0.27 ± 0.025
0.28 ± 0.034
0.11 ± 0.009
>141.9
3.07
52.96
13.36
1.10 ± 0.063
15.11 ± 0.872
1.58 ± 0.091
3. Conclusions
The search for novel and effective breast cancer treatments is increasing with the
particular concern to reduce side effects. These advances improve the patient’s quality
of life and lead to higher survival rates.
Good yields through clean reactions were obtained for chalcone and chromene
derivative synthesis. Innocuous solvents such as water and ethanol were used and aldol
condensations were highly effective. Generally, the reactions were performed at room
temperature, leading to the isolation of many pure compounds.
Synthetic chalcones and chromenes demonstrated to be powerful anticancer agents
through all the assays performed. Halogenated substituents presented more interesting
capacities than methoxyl or methyl groups when they were the only substituents in the
molecular scaffold. The bromine atom was the most present halogen in the final
molecules that proceeded to the final assays and showed to be more promising
candidates for further studies.
Specifically, for chalcones, the results suggest an anti-proliferative activity. On the other
hand, chromene 5c demonstrated to be involved in the inhibition of cell migration and
induction of apoptosis, apparently triggering cell death associated with a G2/M phase
cell cycle arrest. The increased levels of β-tubulin together with the analysis of PARP
and Bim levels, support the activation of the intrinsic apoptosis signal pathway. Also,
our results demonstrate that compound 5c seems to alter tubulin polymerization and
might be a promising novel MDA.

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Finally, when combining these novel compounds with the commercially available drug
doxorubicin, results lead to a decrease in the IC50 value of MCF-7 malignant cells and,
more importantly, increase in the selectivity index.
4. Experimental
4.1. Chemistry
All chemicals, reagents and solvents for compounds synthesis were analytical grade, purchased
from commercial sources and used without further purification, unless otherwise specified.
Reactions were monitored by thin layer chromatography (TLC) using silica gel 60 plates
purchased from Macherey-Nagel with a 0.2 mm and a fluorescence indicator. An UV chamber
(CN-6 Vilber Lourmat) with a 254 nm lamp was used for revelation. Dry flash chromatography
was performed with silica gel (particle size <0.063 mm) from MN Kieselgel 60 (230 ASTM).
For reactions at high temperatures, a hot plate stirrer IKAMAG RCT was used, with magnetic
stirring at 40 rpm and at different temperatures according to the specific procedure. For solvent
evaporation, a Buchi RE 11 rotary evaporator was used with vacuum and variable bath
1
temperature. NMR spectra were obtained in Bruker Avance III (at 400 MHz for H NMR and
100 MHz for 13C NMR), at 25ºC, with deuterated dimethylsulfoxide (DMSO-d6) as solvent.
Chemical shifts were recorded in parts per million (ppm) using the residual solvent peak as an
internal standard. IR spectra were recorded in FT-IR Bomem MB 104 using nujol mulls and
NaCl cells. Melting points were determined in a Stuart SMP3 melting point apparatus and are
uncorrected. Elemental analysis was performed on a LECO CHNS-932 instrument. 3,5-
Difluoro-2-hydroxybenzaldehyde was synthesized as described before [51].
4.1.1. General procedure for the synthesis of chalcone derivatives 3
Aqueous 3M NaOH was added to a solution of substituted salicylaldehyde 1 (1.1 equiv.) and
acetophenone 2 in ethanol. The solution was stirred at room temperature (4-31 hours) or heated
at 40-60 ºC (for 3a, b, q and u; 18-27 hours). The solution was neutralized with concentrated
HCl (37%) and a suspension was obtained, cooled at 4 ºC for 1-16 hours. The solid was filtered
and washed with ethanol and water leading to the respective α,β-unsaturated carbonyl
compound 3. This procedure was adapted from reference [34].
4.1.1.1. 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-1-phenylprop-2-en-1-one (3a). Yellow solid,
yield 100%, mp 175-177 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.85 (s, 3H, -OCH3), 7.17 (d, J
= 2.0 Hz, 1H, H4), 7.65 (tt, J = 1.3 Hz, 1H, H4’), 7.77 (d, J = 2.4 Hz, 1H, H6), 7.88 (d, J = 15.6
Hz), 8.03 (d, J = 15.6 Hz), 8.13 (d, J = 6.8Hz, 1H, H6’), 9.74 (s, 1H, -OH). 13C NMR (100
MHz, DMSO-d6):
δ 56.36 (-OCH3), 110.50 (C5), 116.14 (C4), 121.49 (C6), 122.09, 123.20

ACCEPTED MANUSCRIPT
(C1), 128.51 (C2’+C6’), 128.72 (C3’+C5’), 137.34, 133.08, (C4’), 137.60 (C1’), 145.93 (C3),
149.04 (C2), 189.17. Anal. Calcd for C16H15O3Br: C, 57.33; H, 4.52. Found: C, 57.02; H, 4.77.
4.1.1.2. 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-1-(4-chlorophenyl)prop-2-en-1-one (3b).
Light brown solid, yield 66%, mp 163-165 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.85 (s, 3H, -
OCH3), 7.17 (d, J = 2.4 Hz, 1H, H4), 7.63 (dt, J = 8.4Hz, 1H, H5’), 7.77 (d, J = 2.0 Hz, 1H,
H6), 7.91 (d, J = 16.0 Hz), 8.00 (d, J = 16.0 Hz), 8.18 (d, J = 6.0Hz, 1H, H6’), 9.77 (s, 1H, -
OH). 13C NMR (100 MHz, DMSO-d6):
δ 56.37 (-OCH3), 110.59 (C5), 116.26 (C4), 121.47
(C6), 121.67, 123.09 (C1), 128.84 (C2’+C6’), 130.45 (C3’+C5’), 136.24 (C4’), 137.75, 138.06
(C1’), 146.03 (C2), 149.05 (C3), 188.01. Anal. Calcd for C16H15O3BrCl: C, 51.99; H, 4.10.
Found: C, 52.19; H, 4.04.
4.1.1.3. 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (3c).
1
Yellow solid, yield 100%, mp 148-150 ºC. H NMR (400 MHz, DMSO-d6):
δ 3.84 (s, 3H, -
OCH3), 7.17 (d, J = 2.4Hz, 1H, H4), 7.23 (dd, J = 8.4, 2.8 Hz, 1H, H4’), 7.47 (t, J = 8.0 Hz,
1H, H5’), 7.59 (dd, J = 2.8, 1.6 Hz, 1H, H2’), 7.75 (d, J = 1.2Hz, 1H, H6’), 7.77 (d, J = 2.4 Hz,
1H, H5), 7.88 (d, J = 15.6Hz), 7.98 (d, J = 15.6Hz), 9.75 (s, 1H, -OH). 13C NMR (100 MHz,
DMSO-d6):
δ 55.36 (-OCH3), 56.36 (-OCH3), 110.57 (C5), 113.06 (C2’), 116.16 (C4), 119.06
(C6’), 121.07 (C4’), 121.51 (C6), 122.12, 123.19 (C1), 129.87 (C5’), 137.43, 139.05 (C1’),
145.94 (C2), 149.03 (C3), 159.52 (C3’), 188.92. Anal. Calcd for C17H17O4Br: C, 55.91; H, 4.70.
Found: C, 56.06; H, 4.69.
4.1.1.4. 3-(5-chloro-2-hydroxyphenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (3d). Yellow solid,
yield 86%, mp 158-160 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.84 (s, 3H, -OCH3), 6.95 (d, J =
8.8 Hz, 1H, H3), 7.24 (dd, J = 5.6, 2.8 Hz, 1H, H6’), 7.30 (dd, J = 8.8, 2.4 Hz, 1H, H4), 7.37 (d,
J = 7.6Hz, 1H, H4’), 7.48 (t, J = 8.0Hz, 1H, H5’), 7.60 (dd, J = 1.6, 1.2Hz, 1H, H2’), 7.92 (d, J
13
= 16.0 Hz), 7.96 (d, J = 16.0 Hz), 8.03 (d, J = 2.8Hz, 1H, H5), 10.55 (s, 1H, -OH). C NMR
(100 MHz, DMSO-d6):
δ 55.28 (-OCH3), 113.05 (C2’), 117.85 (C3), 119.05 (C6’), 121.04
(C4’), 122.05, 123.05 (C1), 123.23 (C5), 127.47 (C6), 129.88 (C5’), 131.40 (C4), 137.67,
139.08 (C1’), 155.95 (C2), 159.53 (C3’), 188.95. Anal. Calcd for C16H15O3Cl: C, 66.24; H,
1
13
5.22. Found: C, 65.98; H, 4.95. The H- and C-NMR spectra were identical to those reported
for the compound synthesized through a previously published procedure [52].
4.1.1.5. 3-(5-bromo-2-hydroxyphenyl)-1-(3-methoxyphenyl)prop-2-en-1-one (3e). Yellow solid,
yield 82%, mp 162-164 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.84 (s, 3H, -OCH3), 6.90 (d, J =
8.8Hz, 1H, H3), 7.23 (dd, J = 5.6, 2.8Hz, 1H, H6’), 7.37 (dt, J = 7.6, 1.6Hz, 1H, H4’), 7.42 (dd,
J = 8.8, 2.4Hz, 1H, H4), 7.48 (t, J = 8.0 Hz, 1H, H5’), 7.60 (q, J = 1.6 Hz, 1H, H2’), 7.92 (d, J =
15.6 Hz), 7.96 (d, J = 15.6Hz), 8.13 (d, J = 2.4Hz, 1H, H5), 10.60 (s, 1H, -OH). 13C NMR (100
MHz, DMSO-d6):
δ 55.37 (-OCH3), 113.06 (C2’), 117.86 (C3), 119.05 (C6’), 121.07 (C4’),
122.03, 123.13 (C1), 123.19 (C5), 127.40 (C6), 129.89 (C5’), 131.24 (C4), 137.60, 139.09

ACCEPTED MANUSCRIPT
(C1’), 155.92 (C2), 159.54 (C3’), 188.95. Anal. Calcd for C16H15O3Br: C, 57.33; H, 4.52.
Found: C, 57.44; H, 4.75.
4.1.1.6. 3-(3,5-difluoro-2-hydroxyphenyl)-1-phenylprop-2-en-1-one (3f). Yellow solid, yield
75%, mp 180-182 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 7.33 (m, 1H, H4), 7.57 (t, J = 7.2 Hz,
2H, H3’+H5’), 7.65 (m, 1H, H4’), 7.97 (d, J = 15.6 Hz), 7.77 (m, 1H, H6), 8.03 (dd, J = 15.6,
1.2 Hz), 8.14 (d, J = 7.2 Hz, 2H, H2’+H6’), 10.33 (s, 1H, -OH). 13C NMR (100 MHz, DMSO-
d6):
δ 109.08 (dd, J=23.0, 4.0Hz, C6), 106.28 (dd, J=23.0, 28.0Hz, C4), 123.29, 136.67 (t, J =
3.0 Hz), 124.94 (dd, J = 9.0, 4.0 Hz, C1), 128.54 (C2’+C6’), 128.80 (C3’+C5’), 133.34 (C4’),
137.44 (C1’), 141.19 (dd, J = 11.0, 3.0 Hz, C2), 151.59 (dd, J = 241.0, 13.0 Hz, C3), 154.33
(dd, J = 235.7, 11.6 Hz, C5), 188.17. Anal. Calcd for C15H14O2F2: C, 68.73; H, 5.39. Found: C,
68.58; H, 5.18.
4.1.1.7. 3-(5-chloro-2-hydroxyphenyl)-1-(p-tolyl)prop-2-en-1-one (3g). Yellow solid, yield
1
87%, mp 179-181 ºC. H NMR (400 MHz, DMSO-d6):
δ 2.39 (s, 3H, -CH3), 6.97 (d, J = 8.8
Hz, 1H, H3), 7.28 (dd, J = 8.8, 2.8 Hz, 1H, H4), 7.37 (d, J = 8.0 Hz, 2H, H3’+H5’), 7.93 (d, J =
16.0 Hz), 7.94 (d, J = 16.0 Hz), 8.06 (q, J = 6.4, 1.6 Hz, 2H, H2’+H6’), 10.58 (s, 1H, -OH). 13C
NMR (100 MHz, DMSO-d6):
δ 21.17 (-CH3), 117.86 (C3), 122.04, 123.13 (C1), 123.19 (C5),
127.40 (C6), 128.65 (C2’+C6’), 129.31 (C3’+C5’), 131.24 (C4), 135.12 (C1’), 137.24, 143.50
(C4’), 155.92 (C2), 188.63. Anal. Calcd for C16H15O2Cl: C, 69.94; H, 5.51. Found: C, 69.88; H,
1
5.36. The H- and 13C-NMR spectra were identical to those reported for the compound
synthesized through a previously published procedure [52].
4.1.1.8. 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-1-(p-tolyl)prop-2-en-1-one (3h). Yellow
1
solid, yield 77%, mp 193-195 ºC. H NMR (400 MHz, DMSO-d6):
δ 2.39 (s, 3H, -CH3), 3.85
(s, 3H, -OCH3), 7.17 (d, J = 2.4Hz, 1H, H4), 7.37 (d, J = 8.0Hz, 2H, H3’+H5’), 7.76 (d, J = 2.0
Hz, 1H, H6), 7.90 (d, 16.0 Hz), 7.97 (d, J = 16.0 Hz), 8.07 (d, J = 8.0 Hz, 2H, H2’+H6’), 9.71
(s, 1H, -OH). 13C NMR (100 MHz, DMSO-d6):
δ 21.18 (-CH3), 53.36 (-OCH3), 110.57 (C5),
116.06 (C4), 121.45 (C6), 122.11, 123.28 (C1), 128.67 (C2’+C6’), 129.30 (C3’+C5’), 135.08
(C1’), 136.96, 143.52 (C4’), 145.85 (C2), 149.03 (C3), 188.58. Anal. Calcd for C17H17O3Br: C,
58.47; H, 4.92. Found: C, 58.27; H, 4.99.
4.1.1.9. 3-(5-bromo-2-hydroxyphenyl)-1-(4-bromophenyl)prop-2-en-1-one (3i). Yellow solid,
1
yield 70%, mp 171-173 ºC. H NMR (400 MHz, DMSO-d6):
δ 6.79 (d, J = 8.8 Hz, 1H, H3),
7.31 (dd, J = 8.8, 2.4 Hz, 1H, H4), 7.67 (d, J = 6.8 Hz, 2H, H3’+H5’), 7.83 (d, J = 15.6 Hz),
7.86 (d, J = 15.6 Hz), 7.98 (d, J = 6.4 Hz, 2H, H2’+H6’), 8.04 (d, J = 2.4 Hz, 1H, H6), 10.49 (s,
13
1H, -OH). C NMR (100 MHz, DMSO-d6):
δ 110.85 (C5), 118.33 (C3), 121.56, 123.56 (C1),
127.25 (C4’), 130.34 (C6), 130.55 (C2’+C6’), 131.80 (C3’+C5’), 134.38 (C4), 136.59 (C1’),
137.94, 156.43 (C2), 188.24. Anal. Calcd for C15H12O2Br2: C, 46.90; H, 3.16. Found: C, 47.12;
H, 3.10.

ACCEPTED MANUSCRIPT
4.1.1.10. 1-(4-chlorophenyl)-3-(2-hydroxy-5-methoxyphenyl)prop-2-en-1-one (3j). Orange solid,
yield 84%, mp 142-134 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.83 (s, 3H, -OCH3), 6.83 (t, J =
8.0 Hz, 1H, H6), 6.89 (d, J = 8.8 Hz, 1H, H4), 7.05 (dd, J = 8.0, 0.8 Hz, 1H, H5), 7.63 (d, J =
8.8 Hz, 2H, H2’+H6’), 7.81 (d, J = 16.0 Hz), 8.09 (d, J = 16.0 Hz), 8.12 (d, J = 6.4 Hz, 2H,
H3’+H5’), 9.47 (s, 1H, -OH). 13C NMR (100 MHz, DMSO-d6):
δ 55.95 (-OCH3), 113.76 (C4),
119.05 (C5), 119.82 (C6), 120.73, 121.51 (C1), 128.85 (C2’+C6’), 130.25 (C3’+C5’), 136.49
(C4’), 137.82 (C1’), 139.70, 146.72 (C2), 148.01 (C3), 188.28. Anal. Calcd for C16H15O3Cl: C,
66.10; H, 5.21. Found: C, 66.00; H, 5.27.
4.1.1.11. 3-(5-bromo-2-hydroxyphenyl)-1-(p-tolyl)prop-2-en-1-one (3k). Yellow solid, yield
1
87%, mp 191-193 ºC. H NMR (400 MHz, DMSO-d6):
δ 2.40 (s, 3H, -CH3), 6.89 (d, J = 8.8
Hz, 1H, H3), 7.37 (d, J = 8.0 Hz, 2H, H3’+H5’), 7.40 (dd, J =8.8, 2.4 Hz, 1H, H4), 7.93 (d, J =
16.0Hz), 7.94 (d, J = 16.0 Hz), 8.06 (d, J = 8.0 Hz, 2H, H2’+H6’), 8.13 (d, J = 2.4Hz, 1H, H6),
10.55 (s, 1H, -OH). 13C NMR (100 MHz, DMSO-d6):
δ 21.17 (-CH3), 110.83 (C5), 118.28
(C3), 122.01, 123.75 (C1), 128.66 (C2’+C6’), 129.30 (C3’+C5’), 130.26 (C6), 134.09 (C1’),
134.09 (C4), 137.11, 143.51 (C4’), 156.28 (C2), 188.59. Anal. Calcd for C16H15O2Br: C, 60.20;
H, 4.75. Found: C, 59.93; H, 4.77.
4.1.1.12. 3-(3,5-difluoro-2-hydroxyphenyl)-1-(p-tolyl)prop-2-en-1-one (3l). Yellow solid, yield
79%, mp 202-204 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 2.40 (s, 3H, -CH3), 7.32 (m, 1H, H4),
7.38 (d, J = 8.0 Hz, 2H, H3’+H5’), 7.75 (m, 1H, H6), 7.95 (d, J = 15.6 Hz), 8.01 (dd, J = 15.6,
1.6 Hz), 8.07 (d, J = 8.4 Hz, 2H, H2’+H6’), 10.30 (s, 1H, -OH). 13C NMR (100 MHz, DMSO-
d6):
δ 21.17 (-CH3), 106.16 (dd, J = 23.0, 28.0 Hz, C4), 109.02 (dd, J = 23.0, 4.0 Hz, C6),
123.30, 125.02 (dd, J = 10.0, 4.0 Hz, C1), 128.69 (C2’+C6’), 129.36 (C3’+C5’), 134.93 (C1’),
136.27 (t, J = 4.0 Hz), 141.10 (dd, J = 15.0, 3.0 Hz, C 2), 143.72 (C4’), 151.53 (dd, J = 240.0,
13.0 Hz, C3), 154.34 (dd, J = 236.0, 12.0 Hz, C5), 188.55. Anal. Calcd for C16H14O2F2: C,
69.55; H, 5.12. Found: C, 69.80; H, 4.93.
4.1.1.13. 3-(5-bromo-2-hydroxy-3-methoxyphenyl)-1-(4-bromophenyl)prop-2-en-1-one (3m).
1
Yellow solid, yield 80%, mp 189-191 ºC. H NMR (400 MHz, DMSO-d6):
δ 3.86 (s, 3H, -
OCH3), 7.19 (d, J = 2.4 Hz, 1H, H4), 7.78 (d, J = 3.2 Hz, 1H, H6), 7.91 (d, J = 16.0 Hz), 7.79
(d, J = 8.8 Hz, 2H, H3’+H5’), 8.05 (d, J = 16.0 Hz), 8.11 (d, J = 8.4 Hz, 2H, H2’+H6’), 9.79 (s,
1H, -OH). 13C NMR (100 MHz, DMSO-d6):
δ 56.37 (-OCH3), 110.59 (C5), 116.27 (C4),
121.48 (C6), 121.65, 123.09 (C1), 127.27 (C4’), 130.56 (C2’+C6’), 131.80 (C3’+C5’), 136.57
(C1’), 137.74149.06 (C2), 146.04 (C3), 188.23. Anal. Calcd for C16H14O3Br2: C, 46.40; H, 3.41.
Found: C, 46.55; H, 3.47.
4.1.1.14. 3-(3-bromophenyl)-1-(p-tolyl)prop-2-en-1-one (3n). Yellow solid, yield 80%, mp 127-
1
129 ºC. H NMR (400 MHz, DMSO-d6):
δ 2.40 (s, 3H, -CH3), 7.38 (d, J = 7.2 Hz, 2H,
H3’+H5’), 7.42 (d, J = 8.0 Hz, 1H, H3), 7.63 (m, 1H, H4), 7.69 (d, J = 15.6 Hz), 7.85 (d, J = 7.6

ACCEPTED MANUSCRIPT
Hz, 1H, H2), 8.00 (d, J = 15.6 Hz), 8.04 (t, J = 1.6Hz, 1H, H6), 8.10 (d, J = 8.4 Hz, 2H,
H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 21.18 (-CH3), 122.36 (C5), 123.51, 128.78
(C2’+C6’), 128.22 (C2), 129.34 (C3’+C5’), 130.76 (C6), 130.89 (C3), 132.93 (C4), 134.82
(C1’), 137.23 (C1), 141.83, 143.79 (C4’), 188.40. Anal. Calcd for C16H15OBr: C, 54.71; H,
4.31. Found: C, 54.59; H, 4.33.
4.1.1.15. 3-(2-hydroxyphenyl)-1-phenylprop-2-en-1-one (3o). Yellow solid, yield 48%, mp 169-
1
171 ºC. H NMR (400 MHz, DMSO-d6):
δ 6.87 (t, J = 7.6 Hz, 1H, H5), 6.95 (dd, J = 8.0, 0.8
Hz, 1H, H3), 7.27 (td, J = 7.2, 1.6, 1H, H4), 7.56 (t, J = 7.2 Hz, 2H, H2’+H6’), 7.79 (d, J = 8.8
Hz, 2H, H3’+H5’), 7.84 (dd, J = 8.0, 1.6 Hz, 1H, H6), 7.86 (d, J = 16.0 Hz), 8.02 (d, J = 16.0
Hz), 10.29 (s, 1H, -OH). 13C NMR (100 MHz, DMSO-d6):
δ 116.22 (C3), 119.38 (C5), 120.92,
121.33 (C1), 128.31 (C2’+C6’), 128.70 (C6), 128.75 (C3’+C5’), 132.03 (C4), 132.86 (C4’),
137.90 (C1’), 139.54, 157.25 (C2), 189.50. Anal. Calcd for C15H14O2: C, 79.61; H, 6.25. Found:
1
C, 79.64; H, 6.22. The H- and 13C-NMR spectra were identical to those reported for the
compound synthesized through a previously published procedure [53].
1
4.1.1.16. 3-(5-bromo-2-hydroxyphenyl)-1-phenylprop-2-en-1-one (3p). Yellow solid. H NMR
(400 MHz, DMSO-d6):
δ 6.90 (d, J = 8.8 Hz, 1H, H3), 6.40 (dd, J = 8.8, 2.4 Hz, 1H, H4’), 7.42
(dd, J = 8.8, 2.4 Hz, 1H, H4), 7.56 (t, J = 6.4Hz, 1H, H5), 7.92 (d, J = 8.0 Hz, 2H, H3’+H5’),
7.95 (d, J = 16.0 Hz), 7.97 (d, J = 16.0 Hz), 8.13 (d, J = 4.8 Hz, 2H, H2’+H6’), 8.15 (d, J =
4.8Hz, 1H, H6), 10.56 (s, 1H, -OH). The product was contaminated with aldehyde in a 1:0.13
1
molar ratio according to H NMR data and was used as starting material in the next step,
1
13
without further purification. The H- and C-NMR spectra were identical to those reported for
the compound synthesized through a previously published procedure [53].
4.1.1.17. 3-(5-chloro-2-hydroxyphenyl)-1-(4-chlorophenyl)prop-2-en-1-one (3q). Yellow solid.
1H NMR (400 MHz, DMSO-d6):
δ 6.98 (d, J = 8.8Hz, 1H, H3), 7.31 (dd, J = 8.8, 2.4, 1H, H4),
7.63 (d, J = 8.4Hz, 1H, H6), 7.57 (d, J = 8.4Hz, 2H, H3’+H5’), 8.17 (d, J = 8.8Hz, 2H, H2’+H6’),
10.64 (s, 1H, -OH). The product was contaminated with aldehyde in a 1:0.25 molar ratio
1
according to H NMR data and was used as starting material in the next step, without further
purification.
4.1.1.18. 3-(5-bromo-2-hydroxyphenyl)-1-(4-chlorophenyl)prop-2-en-1-one (3r). Yellow solid.
1H NMR (400 MHz, DMSO-d6):
δ 6.84 (d, J = 8.4 Hz, 1H, H3), 7.27 (dd, J = 8.4, 2.0, 1H, H4),
7.63 (dd, J = 8.4, 2.0Hz, 1H, H6), 7.93 (d, J = 15.6 Hz), 7.95 (d, J = 7.6 Hz, 2H, H2’+H6’), 7.97
(d, J = 15.6 Hz), 8.18 (d, J = 8.8 Hz, 2H, H3’+H5’), 10.05 (s, 1H, -OH). The product was
1
contaminated with aldehyde in a 1:0.6 molar ratio according to H NMR data and was used as
starting material in the next step, without further purification.
1
4.1.1.19. 3-(5-chloro-2-hydroxyphenyl)-1-phenylprop-2-en-1-one (3s). Yellow solid. H NMR
(400 MHz, DMSO-d6):
δ 6.96 (d, J = 8.0 Hz, 1H, H3), 7.30 (dd, J = 8.8, 2.8 Hz, 1H, H6), 7.56

ACCEPTED MANUSCRIPT
(t, J = 7.6 Hz, 2H, H2’+H6’), 7.65 (t, J = 7.2 Hz 1H, H4), 7.94 (d, J = 16.0 Hz), 7.97 (d, J =
16.0 Hz), 8.14 (d, J = 8.8 Hz, 2H, H3’+H5’), 10.57 (s, 1H, -OH). The product was contaminated
with traces of aldehyde according to 1H NMR data and was used as starting material in the next
step, without further purification.
4.1.1.20. 1-(4-chlorophenyl)-3-(2-hydroxy-4-methoxyphenyl)prop-2-en-1-one (3t). Isolates as
1
sodium salt, neutralization with HCl was not performed. Red solid, mp 156-159 ºC. H NMR
(400 MHz, DMSO-d6): δ 3.75 (s, 3H, -OCH3), 6.48 (d, J = 2.4 Hz, 1H, H5), 6.50 (d, J = 2.4 Hz,
1H, H4), 7.62 (dd, J = 8.8, 2.0 Hz, 2H, H3’+H5’), 7.72 (d, J = 15.6 Hz), 7.82 (d, J = 9.6 Hz, 1H,
H6), 8.02 (d, J = 15.6 Hz), 8.09 (dd, J = 8.8, 2.0 Hz, 2H, H2’+H6’), 10.43 (s, 1H, -OH). The
1
product was contaminated with traces of aldehyde according to H NMR data and was used as
starting material in the next step, without further purification.
4.1.2. General procedure for the synthesis of 4H-chromene-3-carboxylate derivatives 4
Triethylamine (2-6 equiv.) was added to a solution of chalcone 3 and methyl 2-cyanoacetate
(1.1-2 equiv.) in methanol. The mixture was stirred for 4-24 hours at room temperature, cooled
and then filtered, leading to the expected chromene derivative 4.
4.1.2.1. Methyl 2-amino-6-bromo-4-(2-(4-chlorophenyl)-2-oxoethyl)-8-methoxy-4H-chromene-
1
3-carboxylate (4b). Pink solid, yield 69%, mp 183-186 ºC. H NMR (400 MHz, DMSO-d6):
δ
3.13 (m, 2H), 3.49 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3), 4.24 (dd, J = 4.8, 2.0 Hz, 1H), 7.03 (d,
J = 2.0, 1H, H5), 7.08 (d, J = 2.0 Hz, 1H, H7), 7.53 (dd, J = 8.4, 1.6 Hz, 2H, H3’+ H5’), 7.62 (s,
2H), 7.86 (dd, J = 6.8, 2.0 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.74, 47.45,
50.38, 56.19 (-OCH3), 73.84 (C3), 113.72 (C7), 115.44 (C6), 122.08 (C5), 128.67 (C3’+C5’),
128.67 (C4a), 129.87 (C2’+C6’), 135.38 (C4’), 137.98 (C1’), 138.28 (C8a), 147.77 (C8),
161.56 (C2), 168.07, 197.46. Anal. Calcd for C20H18NO5Br: C, 55.66; H, 4.21; N, 3.24. Found:
C, 55.39; H, 4.37; N, 3.40.
4.1.2.2.
Methyl
2-amino-6-bromo-8-methoxy-4-(2-(3-methoxyphenyl)-2-oxoethyl)-4H-
1
chromene-3-carboxylate (4c). White solid, yield 62%, mp 159-161 ºC. H NMR (400 MHz,
DMSO-d6):
δ 3.18 (m, 2H), 3.52 (s, 3H, -OCH3), 3.79 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3),
4.25 (dd, J = 6.8, 4.8 Hz, 1H), 7.00 (d, J = 2.0, 1H, H5), 7.08 (d, J = 2.0 Hz, 1H, H7), 7.16 (dd,
J = 8.0, 2.0 Hz, 1H, H4’), 7.37 (t, J = 1.6 Hz, 1H, H2’), 7.38 (t, J = 8.0Hz, 1H, H5’), 7.44 (d, J
= 7.6 Hz, 2H, H6’), 7.63 (s, 2H). 13C NMR (100 MHz, DMSO-d6):
δ 30.60, 47.94, 50.41, 55.25
(-OCH3), 56.10 (-OCH3), 73.98 (C3), 112.25 (C2’), 113.67 (C7), 115.36 (C6), 119.27 (C4’),
120.46 (C6’), 122.06 (C5), 128.71 (C4a), 129.73 (C5’), 138.14 (C1’), 138.29 (C8a), 147.76
(C8), 161.55 (C2), 168.12, 198.21. Anal. Calcd for C21H20NO6Br: C, 54.56; H, 4.37; N, 3.03.
Found: C, 54.46; H, 4.33; N, 3.28.

ACCEPTED MANUSCRIPT
4.1.2.3. Methyl 2-amino-6-bromo-4-(2-(4-bromophenyl)-2-oxoethyl)-8-methoxy-4H-chromene-
3-carboxylate (4d). White solid, yield 57%, mp 164-166 ºC. 1H NMR (400 MHz, DMSO-d6):
δ
3.15 (m, 2H), 3.49 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3), 4.24 (dd, J = 6.4, 4.8 Hz, 1H), 7.03 (d,
J = 2.0 Hz, 1H, H5), 7.08 (d, J = 2.0 Hz, 1H, H7), 7.63 (s, 2H) 7.67 (dd, J = 8.4, 1.6 Hz, 2H,
H3’+ H5’), 7.77 (dd, J = 5.2, 1.6 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.71,
47.43, 50.36, 56.11 (-OCH3), 73.82 (C3), 113.71 (C7), 115.43 (C6), 122.06 (C5), 127.16 (C4’),
128.62 (C4a), 129.97 (C2’+C6’), 131.61 (C3’+C5’), 135.69 (C1’), 138.27 (C8a), 147.76 (C8),
161.54 (C2), 168.05, 197.65. Anal. Calcd for C20H17NO5Br2: C, 46.99; H, 3.36; N, 2.74. Found:
C, 46.72; H, 3.28; N, 2.69.
4.1.2.4.
Methyl
2-amino-6-bromo-4-(2-(4-bromophenyl)-2-oxoethyl)-4H-chromene-3-
1
carboxylate (4i). White solid, yield 94%, mp 140-141 ºC. H NMR (400 MHz, DMSO-d6):
δ
3.16 (m, 2H), 3.50 (s, 3H, -OCH3), 4.27 (dd, J = 6.8, 5.2 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H, H8),
7.36 (dd, J = 8.8 2.4 Hz, 1H, H7), 7.48 (d, J = 2.4, 1H, H5), 7.56 (s, 2H), 7.68 (dd, J = 6.8, 2.0
Hz, 2H, H3’+ H5’), 7.79 (dd, J = 6.4, 2.0 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ
30.62, 50.37, 47.33, 73.80 (C3), 115.63 (C6), 117.79 (C8), 127.22 (C4’), 128.52 (C4a),
130.45 (C7), 130.97 (C5), 130.00 (C2’+C6’), 131.65 (C3’+C5’), 135.66 (C1’), 148.92
(C8a)161.61 (C2), 168.03, 197.68. Anal. Calcd for C19H15NO4Br2: C, 47.43; H, 3.15; N, 2.91.
Found: C, 47.52; H, 3.26; N, 2.86.
4.1.2.5.
Methyl
2-amino-4-(2-(4-chlorophenyl)-2-oxoethyl)-8-methoxy-4H-chromene-3-
1
carboxylate (4j). Beige solid, yield 72%, mp 141-143 ºC. H NMR (400 MHz, DMSO-d6):
δ
3.13 (m, 2H), 3.50 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 4.27 (dd, J = 4.8, 2.4 Hz, 1H), 6.80 (dd,
J = 7.6, 1.2 Hz, 1H, H5), 6.90 (dd, J = 8.0, 1.6 Hz, 1H, H7), 6.97 (t, J = 8.0 Hz, 1H, H6), 7.53
(dd, J = 6.4, 2.0 Hz, 2H, H2’+H6’), 7.59 (m, 1H, H4’), 7.62 (s, 2H), 7.86 (dd, J = 6.8, 2.0Hz,
2H, H3’+ H5’). 13C NMR (100 MHz, DMSO-d6):
δ 38.18, 48.09, 50.31, 55.59 (-OCH3), 74.30
(C3), 110.61 (C7), 119.57 (C5), 124.04 (C6), 126.88 (C4a), 128.88 (C3’+C5’), 129.88
(C2’+C6’), 135.34 (C4’), 137.89 (C1’), 138.78 (C8a), 146.88 (C8), 161.94 (C2), 168.23,
197.45. Anal. Calcd for C20H18NO5Cl: C, 61.94; H, 4.69; N, 3.61. Found: C, 61.78; H, 4.68; N,
3.57.
4.1.2.6. Methyl 2-amino-6-bromo-4-(2-oxo-2-phenylethyl)-4H-chromene-3-carboxylate (4p).
Yellow solid, yield 72%, mp 145-147 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.17 (m, 2H), 3.51
(s, 3H, -OCH3), 4.29 (dd, J = 4.8, 2.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H, H8), 7.35 (dd, J = 8.8,
2.4 Hz, 1H, H7), 7.45-7.49 (m, 3H), 7.48 (dd, J = 6.4, 2.0 Hz, 2H, H3’+ H5’), 7.88 (dd, J = 8.0,
0.8Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.51, 47.34, 50.37, 73.94 (C3),
115.58 (C6), 117.77 (C8), 127.94 (C2’+C6’), 128.60 (C3’+C5’), 128.67 (C4a), 130.39 (C7),
130.95 (C5), 133.10 (C4’), 136.67 (C1’), 148.93 (C8a), 161.62 (C2), 168.08, 198.40. Anal.

ACCEPTED MANUSCRIPT
Calcd for C19H16NO4Br: C, 56.74; H, 4.02; N, 3.48 Found: C, 56.48; H, 4.04; N, 3.50.
4.1.2.7.
Methyl
2-amino-6-bromo-4-(2-(4-chlorophenyl)-2-oxoethyl)-4H-chromene-3-
1
carboxylate (4r). Yellow solid, yield 57%, mp 115-117 ºC. H NMR (400 MHz, DMSO-d6):
δ
3.16 (m, 2H), 3.50 (s, 3H, -OCH3), 4.27 (dd, J = 4.8, 2.4 Hz, 1H), 7.36 (dd, J = 8.8, 2.4 Hz, 1H,
H7), 7.35 (d, J = 8.4Hz, 1H, H8), 7.49 (d, J = 2.4 Hz, 1H, H5), 7.54 (dd, J = 6.4, 1.6 Hz, 2H,
H3’+ H5’), 7.59 (s, 2H), 7.88 (dd, J = 11.2, 2.4 Hz, 2H, H2’+H6’). 13C NMR (100 MHz,
DMSO-d6):
δ 30.65, 47.36, 50.38, 73.82 (C3), 117.80 (C8), 128.70 (C3’+C5’), 128.75 (C4a),
128.75 (C6), 129.90 (C2’+C6’), 130.46 (C7), 130.98 (C5), 135.36 (C1’), 138.03 (C4’), 148.93
(C8a), 161.63 (C2), 168.05, 197.48. Anal. Calcd for C19H15NO4ClBr: C, 52.25; H, 3.47; N, 3.21.
Found: C, 52.34; H, 3.47; N, 2.93.
4.1.2.8. Methyl 2-amino-6-chloro-4-(2-oxo-2-phenylethyl)-4H-chromene-3-carboxylate (4s).
1
White solid, yield 56%, mp 116-118 ºC. H NMR (400 MHz, DMSO-d6):
δ 3.20 (m, 2H), 3.51
(s, 3H, -OCH3), 4.29 (dd, J = 6.8, 4.4 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H, H8), 7.23 (dd, J = 8.8,
2.4 Hz, 1H, H7), 7.35 (d, J = 2.8 Hz, 1H, H5), 7.57 (s, 2H), 7.59 (m, 1H, H4’), 7.48 (t, J = 7.2
Hz, 2H, H3’+ H5’), 7.88 (dd, J = 8.0, 0.8 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ
30.62, 47.33, 50.39, 73.92 (C3), 117.40 (C8), 127.53 (C6), 127.65 (C7), 127.96 (C2’+C6’),
128.07 (C5), 128.24 (C4a), 128.63 (C3’+C5’), 133.13 (C4’), 136.68 (C1’), 148.49 (C8a),
161.69 (C2), 168.12, 198.43. Anal. Calcd for C19H16NO4Cl: C, 63.78; H, 4.52; N, 3.92. Found:
C, 63.79; H, 4.61; N, 3.82.
4.1.3. General procedure for the synthesis of 4H-chromene-3-carbonitrile 5
Triethylamine (3-5 equiv) was added to a solution of chalcone 3 and malononitrile (2 equiv.)
in ethanol. The solution was stirred for 2-23 hours, at room temperature and filtered or first
cooled to -20 ºC for 1 hour to 7 days and then filtered and identified as the respective product 5.
4.1.3.1.
2-Amino-6-bromo-8-methoxy-4-(2-oxo-2-phenylethyl)-4H-chromene-3-carbonitrile
1
(5a). White solid, yield 42%, mp 177-179 ºC. H NMR (400 MHz, DMSO-d6):
δ 3.39 (m, 2H),
3.81 (s, 3H, -OCH3), 4.06 (t, J = 5.2 Hz, 1H), 6.90 (s, 2H), 7.05 (d, J = 1.6 Hz, 1H, H7), 7.10 (d,
J = 2.0 Hz, 1H, H5), 7.61 (tt, J = 7.6, 1.2 Hz, 1H, H4’), 7.48 (t, J =8.0 Hz, 2H, H3’+H5’), 7.91
(d, J = 4.8 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.88, 46.27, 53.82 (C3),
56.15 (-OCH3), 113.89 (C5), 115.62 (C6), 120.26, 121.62 (C7), 126.57 (C4a), 127.99
(C2’+C6’), 128.65 (C3’+C5’), 133.26 (C4’), 136.61 (C1’), 138.18 (C8a), 147.88 (C8), 161.19
(C2), 197.65. Anal. Calcd for C19H15N2O3Br: C, 57.16; H, 3.80; N, 7.02. Found: C, 57.35; H,
3.86; N, 6.92.
4.1.3.2.
2-Amino-6-bromo-4-(2-(4-chlorophenyl)-2-oxoethyl)-8-methoxy-4H-chromene-3-
1
carbonitrile (5b). White solid, yield 68%, mp 169-171 ºC. H NMR (400 MHz, DMSO-d6):
δ

ACCEPTED MANUSCRIPT
3.38 (m, 2H), 3.81 (s, 3H, -OCH3), 4.05 (t, J = 5.6 Hz, 1H), 6.91 (s, 2H), 7.06 (d, J = 2.4 Hz,
1H, H5), 7.10 (d, J = 2.4 Hz, 1H, H7), 7.54 (d, J = 8.4 Hz, 2H, H3’+H5’), 7.91 (d, J = 8.4 Hz,
2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.95, 46.31, 53.65 (C3), 56.13 (-OCH3),
113.89 (C7), 115.63 (C6), 120.20, 121.61 (C5), 126.40 (C4a), 128.70 (C3’+C5’), 129.92
(C2’+C6’), 135.26 (C1’), 138.15 (2C, C8a and C4’), 147.85 (C8), 161.19 (C2), 196.78. Anal.
Calcd for C19H14N2O3ClBr: C, 52.61; H, 3.26; N, 6.46. Found: C, 52.69; H, 3.15; N, 6.34.
4.1.3.3. 2-Amino-6-bromo-8-methoxy-4-(2-(3-methoxyphenyl)-2-oxoethyl)-4H-chromene -3-
1
carbonitrile (5c). White solid, yield 64%, mp 157-160 ºC. H NMR (400 MHz, DMSO-d6):
δ
3.41 (m, 2H), 3.79 (s, 3H, -OCH3), 3.81 (s, 3H, -OCH3), 4.04 (t, J = 5.2 Hz, 1H), 6.91 (s, 2H),
7.05 (d, J = 2.0 Hz, 1H, H5), 7.10 (d, J = 2.0 Hz, 1H, H7), 7.18 (dd, J = 8.4, 1.2 Hz, 1H, H6’),
7.37 (t, J = 2.4 Hz, 1H, H2’), 7.39 (d, J = 8.0 Hz, 1H, H5’), 7.49 (dt, J = 8.0, 1.2Hz, 1H, H4’).
13C NMR (100 MHz, DMSO-d6):
δ 30.92, 46.30, 53.77 (C3), 55.28 (-OCH3), 56.14 (-OCH3),
112.23 (C2’), 113.87 (C7), 115.59 (C6), 119.53 (C6’), 120.27, 120.48 (C4’), 121.61 (C5),
126.52 (C4a), 129.78 (C5’), 138.04 (C1’), 138.18 (C8a), 147.86 (C8), 159.34 (C3’), 161.18
(C2), 197.48. Anal. Calcd for C20H17N2O4Br: C, 55.95; H, 4.00; N, 6.52. Found: C, 56.09; H,
4.25; N, 6.39.
4.1.3.4.
2-Amino-6-bromo-4-(2-(4-bromophenyl)-2-oxoethyl)-8-methoxy-4H-chromene-3-
1
carbonitrile (5d). White solid, yield 57%, mp 161-163 ºC. H NMR (400 MHz, DMSO-d6):
δ
3.37 (m, 2H), 3.81 (s, 3H, -OCH3), 4.05 (t, J = 5.2 Hz, 1H), 6.91 (s, 2H), 7.06 (d, J = 2.0 Hz,
1H, H5), 7.10 (d, J = 2.4 Hz, 1H, H7), 7.70 (dd, J = 6.8, 2.0 Hz, 2H, H3’+H5’), 7.82 (dd, J =
6.8, 2.0 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.97, 46.33, 56.16 (C3), 56.16
(-OCH3), 113.91 (C7), 115.65 (C6), 120.21, 121.63 (C5), 126.43 (C4a), 127.28 (C4’), 130.04
(C2’+C6’), 131.67 (C3’+C5’), 135.60 (C1’), 138.17 (C8a), 147.87 (C8), 161.21 (C2), 197.02.
Anal. Calcd for C19H14N2O3Br2: C, 47.73; H, 2.96; N, 5.86. Found: C, 47.84; H, 2.85; N, 5.60.
4.1.3.5.
2-Amino-6-bromo-4-(2-(3-methoxyphenyl)-2-oxoethyl)-4H-chromene-3-carbonitrile
1
(5e). White solid, yield 25%, mp 134-135 ºC. H NMR (400 MHz, DMSO-d6):
δ 3.44 (m, 2H),
3.79 (s, 3H, -OCH3), 4.08 (t, J = 5.2 Hz, 1H), 6.89 (s, 2H), 6.94 (d, J = 8.8 Hz, 1H, H8), 7.18
(ddd, J = 8.0, 2.4, 0.8 Hz, 1H, H4’), 7.36 (dd, J = 7.2, 2.8 Hz, 1H, H7), 7.38 (t, J = 2.4Hz, 1H,
H2’), 7.42 (d, J = 8.0 Hz, 1H, H5’), 7.49 (m, 1H, H5), 7.50 (m, 1H, H6’). 13C NMR (100 MHz,
DMSO-d6):
δ 30.75, 46.18, 53.73 (C3), 55.31(-OCH3), 112.27 (C2’), 115.83 (C6), 118.03 (C8),
119.55 (C4’), 120.30, 120.51 (C6’), 126.41 (C4a), 129.81 (C5’), 130.58 (C5), 130.68 (C7),
138.04 (C1’), 148.71 (C8a), 159.36 (C3’), 161.25 (C2), 197.52. Anal. Calcd for C19H15N2O3Br:
C, 57.16; H, 3.80; N, 7.02. Found: C, 57.06; H, 3.68; N, 6.92.
4.1.3.6.
2-Amino-6,8-difluoro-4-(2-oxo-2-phenylethyl)-4H-chromene-3-carbonitrile
(5f).
Yellow solid, yield 21%, mp 149-151 ºC. 1H NMR (400 MHz, DMSO-d6):
δ
3.49 (m, 2H), 4.11
(t, J = 5.2 Hz, 1H), 7.03 (s, 2H), 7.07 (dt, J = 9.2, 4.4, 2.0 Hz, 1H, H5), 7.25 (m, 1H, H7), 7.48

ACCEPTED MANUSCRIPT
(t, J = 8.0 Hz, 2H, H3’+H5’), 7.60 (tt, J = 8.8, 6.8, 1.6 Hz, 1H, H4’), 7.92 (dd, J = 8.4, 1.2 Hz,
2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.96, 45.61, 53.69 (C3), 103.04 (dd, J =
27.0, 21.0 Hz, C7), 109.70 (dd, J = 24.0, 3.0 Hz, C5), 120.02, 127.63 (d, J = 9.0 Hz, C4a),
127.98 (C2’+C6’), 128.68 (C3’+C5’), 133.35 (C4’), 134.44 (dd, J = 11.0, 4.0Hz, C8a), 136.53
(C1’), 149.25 (dd, J = 248.0, 14.0 Hz, C6), 157.13 (dd, J = 240.0, 11.0 Hz, C8), 160.84 (C2),
197.58. Anal. Calcd for C18H12N2O2F2: C, 66.25; H, 3.71; N, 8.59. Found: C, 65.99; H, 3.77; N,
8.40.
4.1.3.7. 2-Amino-6-chloro-4-(2-oxo-2-(p-tolyl)ethyl)-4H-chromene-3-carbonitrile (5g). White
1
solid, yield 40%, mp 175-177 ºC. H NMR (400 MHz, DMSO-d6):
δ 2.35 (s, 3H, -CH3), 3.39
(m, 2H), 4.08 (t, J = 5.6 Hz, 1H), 6.89 (s, 2H), 7.00 (d, J = 8.8 Hz, 1H, H8), 7.24 (dd, J = 8.4,
2.4 Hz, 1H, H7), 7.30 (d, J = 8.0 Hz, 2H, H3’+H5’), 7.37 (d, J = 2.4 Hz, 1H, H5), 7.82 (d, J =
8.0 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 21.11 (-CH3), 30.81, 46.03, 53.74
(C3), 117.64 (C8), 120.25, 126.08 (C4a), 127.68 (C5), 127.87 (C6), 127.76 (C7), 128.11
(C2’+C6’), 129.18 (C3’+C5’), 134.64 (C1’), 148.20 (C8a), 143.64 (C4’), 161.26 (C2), 197.13.
Anal. Calcd for C19H15N2O2Cl: C, 67.37; H, 4.47; N, 8.27. Found: C, 67.51; H, 4.23; N, 8.08.
1
The H- and 13C-NMR spectra were identical to those reported for the compound synthesized
through a previously published procedure [52].
4.1.3.8.
2-Amino-6-bromo-8-methoxy-4-(2-oxo-2-(p-tolyl)ethyl)-4H-chromene-3-carbonitrile
2.35 (s, 3H, -
(5h). Yellow solid, yield 12%, mp 129-131 ºC. 1H NMR (400 MHz, DMSO-d6):
δ
CH3), 3.35 (m, 2H), 3.81 (s, 3H, -OCH3), 4.04 (t, J = 5.2 Hz, 1H), 6.89 (s, 2H), 7.04 (d, J =
2.0Hz, 1H, H5), 7.09 (d, J = 2.4 Hz, 1H, H7), 7.28 (d, J = 8.0 Hz, 2H, H3’+H5’), 7.81 (d, J =
8.0 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
(C3)56.15 (-OCH3), 113.87 (C7), 115.60 (C6), 120.25, 121.60 (C5), 126.65 (C4a), 128.11
(C2’+C6’), 129.18 (C3’+C5’), 134.19 (C1’), 138.15 (C8a), 143.62 (C4’), 147.88 (C8),
δ 21.12 (-CH3), 30.91, 46.19, 53.85
161.17 (C2), 197.13. Anal. Calcd for C20H17N2O3Br: C, 58.12; H, 4.15; N, 6.78. Found: C,
57.90; H, 4.29; N, 6.48.
4.1.3.9. 2-Amino-6-bromo-4-(2-(4-bromophenyl)-2-oxoethyl)-4H-chromene-3-carbonitrile (5i).
1
White solid, yield 50%, mp 186-188 ºC. H NMR (400 MHz, DMSO-d6):
δ 3.45 (m, 2H), 4.08
(t, J = 5.6 Hz, 1H), 6.90 (s, 2H), 7.38 (dd, J = 8.8, 2.4 Hz, 1H, H7), 6.92 (d, J = 8.4Hz, 1H, H8),
7.52 (d, J = 2.4 Hz, 1H, H5), 7.70 (d, J = 8.8 Hz, 2H, H3’+H5’), 7.84 (d, J = 8.8 Hz, 2H,
H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
δ 30.76, 46.20, 53.62 (C3), 115.86 (C6), 118.04
(C8), 120.22, 126.30 (C4a), 127.41 (C4’), 130.61 (C5), 130.05 (C2’+C6’), 130.72 (C7), 131.69
(C3’+C5’), 135.58 (C1’), 148.67 (C8a), 161.26 (C2), 197.01. Anal. Calcd for C18H12N2O2Br2:
C, 52.31; H, 2.93; N, 6.78. Found: C, 52.35; H, 2.69; N, 6.53.
4.1.3.10. 2-Amino-6-bromo-4-(2-oxo-2-(p-tolyl)ethyl)-4H-chromene-3-carbonitrile (5k). White
1
solid, yield 62%, mp 183-185 ºC. H NMR (400 MHz, DMSO-d6):
δ 2.35 (s, 3H, -CH3), 3.43

ACCEPTED MANUSCRIPT
(m, 2H), 4.08 (t, J = 5.6 Hz, 1H), 6.89 (s, 2H), 6.94 (dd, J = 8.8 Hz, 1H, H8), 7.23 (dd, J = 2.4,
0.4 Hz, 1H, H5), 7.28 (d, J = 8.0 Hz, 2H, H3’+H5’), 7.38 (dd, J = 8.8, 2.4 Hz, 1H, H7), 7.81
(dd, J = 6.4, 1.6 Hz, 2H, H2’+H6’). 13C NMR (100 MHz, DMSO-d6):
46.06 53.80 (C3), 115.82 (C6), 118.03 (C8), 120.25, 126.52 (C4a), 128.11 (C2’+C6’), 129.18
δ 21.12 (-CH3), 30.74,
,
(C3’+C5’), 130.56 (C5), 130.65 (C7), 134.18 (C1’), 143.64 (C4’), 148.67 (C8a), 161.23 (C2),
197.14. Anal. Calcd for C19H15N2O2Br: C, 59.55; H, 3.95; N, 7.31. Found: C, 59.39; H, 3.96; N,
7.38.
4.1.3.11. 2-Amino-4-(2-oxo-2-phenylethyl)-4H-chromene-3-carbonitrile (5o). White solid, yield
27%, mp 136-138 ºC. 1H NMR (400 MHz, DMSO-d6):
δ 3.36 (m, 2H), 4.10 (t, J = 5.6 Hz, 1H),
6.82 (s, 2H), 6.98 (dd, J = 8.0, 0.8 Hz, 1H, H8), 7.05 (td, J = 7.2, 1.2 Hz, 1H, H7), 7.23 (m, 2H,
H5+H6), 7.47 (t, J = 8.0 Hz, 2H, H3’+H5’), 7.60 (tt, J = 14.8, 7.6, 1.2 Hz, 1H, H4’), 7.90 (dd, J
13
= 8.4, 1.2 Hz, 2H, H2’+H6’). C NMR (100 MHz, DMSO-d6):
δ 31.09, 46.90, 54.18 (C3),
115.94 (C8), 120.64, 123.92 (C4a), 124.54 (C7), 128.08 (C2’+C6’), 128.10 (C6), 128.11 (C5),
128.76 (C3’+C5’), 133.34 (C4’), 136.74 (C1’), 149.35 (C8a), 161.64 (C2), 197.94. Anal. Calcd
for C18H14N2O2: C, 74.47; H, 4.87; N, 9.65. Found: C, 74.18; H, 4.77; N, 9.91. The 1H- and 13C-
NMR spectra were identical to those reported for the compound synthesized through a
previously published procedure [52].
4.2. Biological evaluation
4.2.1 Cell lines and culture conditions
Two human breast cancer cell lines Hs578T (basal subtype) and MCF-7 (luminal-A subtype),
and a normal breast cell line MCF-10A, were obtained from ATCC (American Type Culture
Collection). The two cancer cell lines were cultured in Dulbecco’s modified Eagle medium,
4.5g/l glucose (DMEM, Gibco) supplemented with 10% heating activated Foetal Bovine Serum
(FBS, Gibco) and 1% antibiotic solution (Penicillin-Streptomycin, Gibco). The normal cell line
was cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12,
Gibco) supplemented with 5% heating activated Foetal Bovine Serum (FBS, Gibco), 1%
antibiotic solution (Penicillin-Streptomycin, Gibco), 1% steroid hormone (Hydrocortisone,
Sigma-Aldrich), 0.1% peptide hormone (Insulin, Sigma-Aldrich) and 0.01% protein complex
(Cholera Toxin, Gibco). Cells were grown in a humidified incubator at 37 ºC and 5% CO2. For
all assays, DMSO (Dimethyl Sulfoxide, Sigma-Aldrich) controls were used.
To cultivate cells for any assay, sub-confluent cells were reaped by gently washing flasks with
phosphate-buffer saline (PBS 1x) and then detaching with trypsin (TrypLE™ Express, Gibco) at
37ºC. To inactivate trypsin DMEM 10% FBS or DMEM/F12 5% FBS were added and cells
were collected and centrifuged 5 minutes at 900 rpm. Cells were suspended in new medium and
10µl of cell suspension were collected, in which 20 µl of trypan blue (Trypan Blue Solution,

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0.4%, Gibco) was added. Cells were counted in a Neubauer chamber for posterior density
calculation.
4.2.2. Cell viability by the Sulforhodamine B assay
For the first screen and to determine the compounds IC50 value for the different breast cancer
cell lines, cells were plated in 96-well plates, at a 3000 cells/100 µL density per well for all the
cell lines (Hs578T, MCF-7 and MCF-10A). Cells were allowed to adhere for 18 to 20 hours
(overnight) and then were exposed to various compound concentrations used for a total of 72
hours treatment. Controls were performed with 0.3% or 0.5% of DMSO (compound vehicle) for
the first screening and IC50 determination, respectively. The compound cytotoxic effect was
evaluated by the Sulforhodamine B assay (SRB, TOX-6, Sigma-Aldrich). Spectrophotometric
measurement of absorbance was read at 490 nm, using 690 nm as background absorbance
(Tecan Infinite M200). GraphPad Prism 5 software was used for the analysis and IC50 values
from three independent experiments (at least), each one in triplicate, applying a sigmoidal dose-
response (variable slope) non-linear regression, after logarithmic transformation.
4.2.3.Proliferation assay
MCF-10A and MCF-7 cells were plated in 96-well plates at a density of 8000cells/100µl, and
grown overnight at 37°C in a 5% CO2 humidified atmosphere. Then, adherent cells were treated
with compounds 3e, 3g, 3i and 5c at IC50 and ½ IC50 concentrations or 0.5% DMSO (controls)
for 24 and 48 hours. After incubation, cells were labelled by addition of 5µl/well BrdU labelling
solution (final concentration: 20 µM BrdU) reincubating for 6 hours to allow BrdU to
incorporate into the proliferating cell DNA, replacing thymidine. After labelling, culture
medium was removed, cells were fixed and DNA was denatured through incubation with 100 µl
of FixDenat solution at room temperature. Denaturation of DNA is essential for antibody
conjugate binding to the incorporated BrdU. After 30 minutes, FixDenat solution was removed,
and 50 µl of Anti-BrdU-POD antibody was incubated for 90 minutes at room temperature. The
Anti-BrdU-POD antibody binds to the incorporated BrdU in the newly synthesized cellular
DNA. The antibody conjugate was removed and wells were washed three times with PBS 1x.
The immune complexes were detected by the addition of Substrate solution (100 µl/well) and
the plate was incubated at room temperature until colour development was satisfactory for
photometric detection (5-10 minutes). Substrate reaction was stopped by adding 25 µl of 1M
H2SO4 to each well and softly mixed. The reaction product was quantified by measuring
absorbance at 450 nm (reference wavelength: 690 nm) in a microplate reader (Tecan Infinite
M200). A blank test was used in each experimental time point, without cells, performing all
steps described above. The results of at least three independent experiments (in triplicate) were
evaluated with GraphPad Prism 5 software.

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4.2.4.Migration assay
MCF-7 were plated in 6-well plates at a density of 9.0x105/2 ml and grown overnight at 37ºC
in a 5% CO2 humidified atmosphere. Two scratches were made with a 200 µl pipette tip in
confluent cells. Cells were gently washed with 500 µL PBS 1x and were treated with
compounds 3e, 3g, 3i and 5c at the respective IC50, ½ IC50 or 0.5% DMSO (control), for 72
hours. At 0, 12, 24 and 48 hours specific wound sites (four positions for each wound) were
photographed at 100x magnification using an Olympus IX51 inverted microscope equipped
with an Olympus DP20 Digital Camera System. Assessment of five migration distances were
performed with the MeVisLab platform and the percentage of cell migration normalized to the
control was evaluated with the GraphPad Prism 5 software. Three independent experiments
were performed for each compound.
4.2.5. Cell viability assessed by Annexin/PI assay
MCF-7 cells were seeded in 6-well culture plates and allowed to adhere overnight in DMEM.
Cells were treated with IC50 concentration of the tested compounds or 0.5% DMSO (controls)
for 48h. Triplicates were performed in each experiment. Floating and adherent cells were
collected and combined by centrifugation. Elimination of the supernatant was followed by
addition of 1 mL of Binding Buffer. To 300 mL of this solution, 8 µL FITC annexin V (BD
Pharmingen) and 30 µL of PI (50µg/mL, P1304MP, Invitrogen), were added. Samples were
incubated for 15 min at room temperature, in the dark. A further 200 mL of Binding Buffer was
added to each sample. PI signal was measured using a FACS LSRII flow cytometer (BD
Biosciences®) with a 488 nm excitation laser. The annexin V signal was collected through a
488 nm blocking filter, a 550 nm long-pass dichroic with a 525 nm band pass. Signals were
captured and FACS Diva was used as the acquisition software. The percentage of cells in each
phase was analysed using the FlowJo 7.6 (Tree Star®) software. Two independent biological
replicates were performed.
4.2.6. Protein extraction and Western blot
Cells (MCF-7) were grown in T25 flasks and, at 70-80% confluence, cells were treated with
compounds 3e, 3g, 3i and 5c at the respective IC50 or 0.5% DMSO (controls) for 24 hours. After
treatment, cells were collected by scraping and centrifuged at 2000 rpm for 5 minutes at room
temperature. The supernatant was discarded and the pellet was suspended in lysis buffer (50mM
Tris pH 7.6-8, 150mM NaCl, 5mM EDTA, 1mM Na3VO4, 10mM NaF, 1% NP-40, 1 % Triton-
X100 and 1/7 protease inhibitor cocktail (Roche Applied Sciences)) and incubated for 15
minutes on ice. Lysates were centrifuged at 13000 rpm for 15 minutes at 4 ºC and the