Phenylpropanoid-based sulfonamide promotes cyclin D1 and cyclin E down-regulation and induces cell cycle arrest at G1/S transition in estrogen positive MCF-7 cell line

Article abstract of DOI:10.1016/j.tiv.2019.04.023

Cancer is one of the most critical problems of public health in the world and one of the main challenges for medicine. Different biological effects have been reported for sulfonamide-based compounds including antibacterial, antifungal, and antitumor activ

Full text of DOI:10.1016/j.tiv.2019.04.023

Accepted Manuscript
Phenylpropanoid-based sulfonamide promotes cyclin D1 and
cyclin E down-regulation and induces cell cycle arrest at G1/S
transition in estrogen positive MCF-7 cell line
Helloana Azevedo-Barbosa, Guilherme Álvaro Ferreira-Silva,
Carolina Faria Silva, Thiago Belarmino de Souza, Danielle
Ferreira Dias, Ana Claudia Chagas de Paula, Marisa Ionta, Diogo
Teixeira Carvalho
PII:
DOI:
Reference:
S0887-2333(18)30529-0
https://doi.org/10.1016/j.tiv.2019.04.023
TIV 4532
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
4 September 2018
3 April 2019
18 April 2019
Please cite this article as: H. Azevedo-Barbosa, G.Á. Ferreira-Silva, C.F. Silva, et al.,
Phenylpropanoid-based sulfonamide promotes cyclin D1 and cyclin E down-regulation
and induces cell cycle arrest at G1/S transition in estrogen positive MCF-7 cell line,
Toxicology in Vitro, https://doi.org/10.1016/j.tiv.2019.04.023
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Phenylpropanoid-based sulfonamide promotes cyclin D1 and cyclin
E down-regulation and induces cell cycle arrest at G1/S transition in
estrogen positive MCF-7 cell line
Helloana Azevedo-Barbosaa, Guilherme Álvaro Ferreira-Silvab, Carolina Faria Silvaa,
Thiago Belarmino de Souzac, Danielle Ferreira Diasd, Ana Claudia Chagas de Paulae,
Marisa Iontab,* marisa.ionta@unifal-mg.edu.br, Diogo Teixeira Carvalhoa,* diogo.carvalho@unifal-
mg.edu.br
aLQFar - Laboratory of Research in Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences,
Federal University of Alfenas, zip code 37130-001, Alfenas, MG, Brazil
bLabaInt - Laboratory of Integrative Animal Biology, Institute of Biomedical Sciences, Federal University of
Alfenas, zip code 37130-001, Alfenas, MG, Brazil
cSchool of Pharmacy, Federal University of Ouro Preto, zip code 35400-000, Ouro Preto, MG, Brazil
dLFQM - Laboratory of Phytochemistry and Medicinal Chemistry, Institute of Chemistry, Federal University of
Alfenas, zip code 37130-001, Alfenas, MG, Brazil
eDepartment of Pharmaceutical Sciences, College of Pharmacy, Federal University of Juiz de Fora, zip code
36036-900, Juiz de Fora, MG, Brazil
*Corresponding authors at: 700, Gabriel Monteiro da Silva Street, Zip code: 37130-001, Alfenas, MG, Brazil
Abstract
Cancer is one of the most critical problems of public health in the world and one of the
main challenges for medicine. Different biological effects have been reported for
sulfonamide-based compounds including antibacterial, antifungal, and antitumor activities.
Herein, a series of phenylpropanoid-based sulfonamides (4a, 4a’, 4b, 4b’, 5a, 5a’, 5b and
5b’) were synthesized and their cytotoxic activity was evaluated against four cell lines
derived from human tumours (A549 – lung, MCF-7 – breast, Hep G2 - hepatocellular
carcinoma, and HT-144-melanoma). Cell viability was significantly reduced in the MCF-7
cell line when compounds 4b, 4b’ and 5a were used; IC50 values were lower than those
found for their precursors (eugenol and dihydroeugenol) and sulfanilamide. We observed
that 4b induced cell cycle arrest at G1/S transition. This is probably due to its ability to
reduce cyclin D1 and cyclin E expression. Moreover, 4b also induced apoptosis in MCF-7
cells as demonstrated by an increase in the cell population positive for annexin V in
treated cultures in comparison to the control group. Taken together, the data showed that
4b is a promising antitumor agent and it should be considered for further in vivo studies.
Keywords:

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Phenylpropanoids; Sulfonamides; Molecular hybridization; Antiproliferative activity; Breast
cancer.
1. Introduction
Cancer is the second major cause of death in the world population and breast
cancer is the most frequent cancer in women worldwide (Torre et al., 2016). In 2012, about
1.67 million new cases of breast cancer were diagnosed, representing 25% of all cancers
reported in women (Ferlay et al., 2015). Breast cancer is a heterogeneous disease
characterized by a wide spectrum of clinical, pathological, and molecular features (Perou
et al., 2000; Sorlie et al., 2003). Histologically, breast cancer has been classified into in
situ carcinoma (cancer cells are confined to the pre-existing normal lobules or ducts) and
invasive carcinoma (cancer cells invade the surrounding stroma) with their respective
subdivisions (Malhotra et al., 2010). According to the gene expression profile, breast
cancer has been classified as luminal A/B (estrogen and progesterone receptors positive);
HER2-enriched (HER+) (amplification and/or overexpressing of human epidermal growth
factor type 2 gene; basal-like (also known as triple negative once the majority of these
tumours do not express ER, PR, or HER2); claudin-low (emerged from basal-like subtype,
but display expression of claudins 3/4/7); and normal breast-like (Perou et al., 2000; Sorlie
et al., 2003). Invasive carcinoma is the most common subtype (70-80%) diagnosed, and
luminal A/B tumour subtypes are ER-positive corresponding to 40% of cases (Prat and
Perou, 2011; Prat et al., 2015). The recognition of the tumour subtype at histological and
molecular levels is essential for the choice of a more adequate therapy (Ross et al., 2008;
Maughan et al., 2010).
Various anticancer drugs have been introduced in chemotherapeutic protocols,
however many patients are refractory to currently available chemotherapy (Weir et al.,
2011). Therefore, it is relevant to identify new substances with anticancer activity to
overcome chemotherapy resistance.
Sulfonamides are classically known for being the central core in the structures of
many antibacterial synthetic drugs such as sulfanilamide (4-aminobenzenesulfonamide,
Scheme 1), a precursor substance of the class of sulfonamidic antibacterials (Lesch, 2007;
Wainwright and Kristiansen, 2011). On the other hand, various bioactive molecules
possessing the sulfonamide group exhibit potential antifungal (Wang et al., 2010), anti-
inflammatory (Chen et al., 2005), antiviral (Supuran et al., 2004), antihypertensive (Kanda
et al., 2001), diuretic (Neff and Nawarskas, 2010), hypoglycemic (Boyd, 1988),
anticonvulsive (Parker et al., 2009), and hypolipemic (Takahashi et al., 2010) activity. In

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addition, recent reports have demonstrated the antitumor potential of sulfonamide
compounds. Despite the variety of structural patterns of these compounds, it has been
demonstrated that the sulfonamide group is critical for their cytotoxic activity against
cancer cell lines (Kamal et al., 2011; Fortin et al., 2011; Lal et al., 2013; Facchinetti et al.,
2014; Kwon et al., 2015; Ghorab et al., 2015).
Eugenol (4-allyl-2-methoxyphenol, 1, Scheme 1) is a natural allylphenol found in
essential oils of many aromatic plants, such as cloves (Syzygium aromaticum), sassafras
(Ocotea odorifera), and cinnamon (Cinnamomum zeylanicum) (Baskaran et al., 2010).
Pharmacological activity of eugenol has been reported, especially relating to antibacterial
and antifungal activities (Ahmad et al., 2010; Devi et al., 2010; Carrasco et al., 2012;
Cortés-Rojas et al., 2014; Li et al., 2015). We have previously demonstrated that synthetic
compounds obtained from natural phenylpropanoids have important antimicrobial activity
(Souza et al., 2014; Souza et al., 2015; Abrão et al., 2015; Souza et al., 2016). There are
several reports of anticancer properties of eugenol due to its ability to induce apoptosis in
breast cancer cells (Al-Sharif et al., 2013; Júnior et al., 2016), leukaemia cells (Yoo et al.,
2005), melanoma cells (Ghosh et al., 2005; Kim et al., 2006; Pisano et al., 2007),
osteosarcoma cells (Shin et al., 2007), and prostate cancer cells (Ghosh et al., 2009).
Furthermore, Manikandan et al. (2011) demonstrated the antitumor potential of eugenol
against gastric carcinoma due to its ability to inhibit the angiogenesis process.
Molecular hybridization is a fairly new concept in drug design and its development is
based on the combination of pharmacophoric moieties of different natural bioactive or
synthetic substances to produce a new hybrid compound with improved affinity and
efficacy when compared to the parent drugs (Viegas-Junior et al., 2007; Abbot et al.,
2017). In the present study, we describe the synthesis of new sulfonamides prepared from
eugenol (4a, 4a’, 5a, 5a’) or dihydroeugenol (4b, 4b’, 5b, 5b’) and the investigation of their
cytotoxic potential against different cancer cell lines (A549, HepG2, HT-144 and MCF-7).
The structural pattern of compounds was designed by molecular hybridization between the
phenylpropanoid and sulfonamide subunit from sulfanilamide (Scheme 1). Furthermore,
we evaluated the molecular mechanism underlying the cytotoxic activity of the compound
4b on MCF-7 cells.

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Scheme 1. Design of the new phenylpropanoid-based sulfonamides derivatives (4a, 4a’, 4b, 4b’, 5a, 5a’, 5b
and 5b’).
2. Results and Discussion
2.1 Synthesis and characterization of sulfonamide derivatives
The synthetic route used to obtain the molecular hybrids is depicted in Scheme 2.

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Scheme 2. Synthetic route to phenylpropanoid-based sulfonamide derivatives: i) NaNO3, KHSO4, SiO2/H2O
(1:1), CH2Cl2, r.t.; ii) Bi(NO3)3.5H2O, SiO2, CHCl3, 70 C; iii) SnCl2.2H2O, EtOH, 80 C; iv) 4-
acetamidobenzenesulfonyl chloride, pyridine, 100 C; v) SOCl2, MeOH, r.t.
The synthesis of the desired compounds was achieved initially by nitration of
eugenol (1) and dihydroeugenol (1’) under mild reaction conditions as described by
Zolfigol et al. (2001) and Canales et al. (2011), who obtained ortho-nitrophenols and meta-
nitrophenols, respectively, with high regioselectivity. The next step consisted of reducing
these nitrated intermediates 2a, 2b, 2a’ and 2b’ with stannous chloride in ethanol (Belamy
and Ou, 1984). The amphoteric aminophenol derivatives 3a, 3b, 3a’ and 3b’ were
obtained as solids after careful acid-base extractions. The spectrometric data of nitro and
amino intermediates showed perfect agreement with the expected ones. Subsequently,
these aminophenols were converted to the respective 4-acetamidobenzenesulfonamides
4a, 4b, 4a’ and 4b’ by reaction with 4-acetamidobenzenesulfonyl chloride in refluxing
pyridine (Silva et al., 2015). The identities of these compounds were confirmed by some
key signals in their NMR spectra such as the sulfonamide NH in the range 8.95-8.74 ppm,
the presence of a para-disubstituted aromatic ring-pattern, and the acetamide CH3 at 2.05
ppm. Finally, the final sulfonamides (5a, 5b, 5a’ and 5b’) were obtained by deacetylation
of 4-acetamidobenzenesulfonamides with thionyl chloride in methanol (Wang et al., 2012).

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These products were obtained after preparative TLC for structural characterization and
desired biological evaluations. NMR and IV spectra did not show the characteristic signals
of the acetamidic group. In addition, it was possible to detect the presence of the typical
aniline amino groups in the region of 8.6 and 8.4 ppm. The eight sulfonamides are
undescribed substances and were fully characterized by IR, NMR, and HRMS analysis.
2.2 Biological assays
The synthetized compounds (4a, 4b, 4a’, 4b’, 5a, 5b, 5a’ and 5b’) were tested at
125 µM against four tumour cell lines derived from human tumours (A549, HT-144, HepG2
and MCF-7). The concentration used in pre-screening was determined in a previous pilot
study (data not known). We observed a significant reduction in viability only in MCF-7
cultures treated with 4b, 4b’ and 5a compounds (Fig. 1A). Thus, MCF-7 cells were
selected to determine IC50 values of these molecules (Table 1 and Fig.1B). We found that
4b, 4b’ and 5a were more cytotoxic than their precursors (eugenol and dihydroeugenol) or
sulfanilamide. IC50 values of eugenol and dihydroeugenol were upward to 1,500 µM, while
IC50 values of 4b, 4b’ and 5a were, respectively,133.80 ± 2.36 µM, 116.60 ± 1.83 µM and
164.90 ± 6.13 µM. Therefore 4b, 4b’, and 5a were potentially more efficient in reducing the
viability of MCF-7 cells (more than 10-fold) in relation to their precursors.
The cytotoxic potential of 4b, 4b’, and 5a on MCF-7 cells may be related to the
propyl side chain and acetamide group, because 4b and 4b’ were more active than their
allylic analogues 4a and 4a’ or even those with the free arylamine group, 5b and 5b’.The
ortho position of the sulfonamide group in relation to the phenolic hydroxyl seems to also
contribute to the observed activity profile since the analogues 4a’, 4b’, 5a’, and 5b’, that
present the sulfonamide group meta in relation to the hydroxyl phenol, were less active.
We also sought to investigate the cytotoxic profile of 4b, 4b’, and 5a on normal
cells, an important aspect for the development of therapeutic drugs. For this purpose, we
performed dose-response curves using fibroblasts derived from human normal skin (CCD-
1059Sk), and non-transformed breast epithelial cells (MCF 10A). We could observe a
lower cytotoxicity of 4b, 4b’, and 5a on normal cells when compared to tumour cells (Fig.
1C). Thus, these compounds seem to have selectivity toward MCF-7 cells.

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Fig.1. Cell viability determined by rezasurin assay. (A) Cells were treated with different molecules at 125 µM
for 48 h. (B) Dose response curves determined after 48 h of treatment with 4b, 4b’, 5a, and their precursors
(sulfanilamide, eugenol and dihydroeugenol) in MCF-7 cells. (C) Dose response curves determined after 48
h of treatment with 4b, 4b’, and 5a in normal cells. Significant differences from control groups were
determined according to ANOVA followed by Tukey’s post-test from three independent experiments.*** p 
0.001, ** p  0.01, and * p  0.05.

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Table 1. IC50 values (µM) determined after 48 of treatment with the compounds 4b, 4b’, 5a, and their
precursors.
IC50 ± Standard Deviation
Compounds
MCF-7
CCD-1059Sk
MCF10A
˃ 300
4b
4b’
133.80 ± 2.36
282.80 ± 5.11
˃ 300
116.60 ± 1.83
164.90 ± 6.13
> 1500.00
185.90 ± 5.04
˃ 300
5a
215.80 ± 6.79
-
-
-
Eugenol
Dihydroeugenol
Sulfanilamide
-
-
-
> 1500.00
> 1000.00
We selected 4b for further studies to understand better the effect of this substance
on MCF-7 cells. We investigated the proliferative behaviour of MCF-7 cells treated with 4b
through different methodological approaches. The morphological features of the MCF-7
cells after treatment with 4b are shown in Fig. 2A. Large cells were observed in cultures
treated with 4b at 50 µM. While small and rounded cells were frequently observed in
cultures treated with 4b at 100 µM or 200 µM. This indicates cell detachment from the
substrate, similar to the process observed in cell death (Fig. 2A). In addition, it was
possible to observe cell density reduced in all treated samples compared to untreated
cultures (Fig. 2A). The colonies number was also reduced in samples treated with 4b at 50
or 100 µM in comparison to control samples, and 4b at 200 µM had high cytotoxic activity
on MCF-7 cells inhibiting completely the colony formation (Fig. 2B).
We observed a drastic reduction of viable cells in samples treated for 48 h and 72 h
when compared to control groups (Fig. 2C). Furthermore, we demonstrated that 4b
reduced the frequency of mitosis and the clonogenic capacity of MFC-7 cells (Figs. 2B and
2D). The quantification of the different phases of mitosis showed that, with exception of
prophase, all other sub-phases of the mitosis were drastically reduced in samples treated
with 4b at 100 µM (Fig. 2E). Therefore, taken together, the data showed that 4b effectively
inhibited the proliferation of MCF-7 cells.
DNA quantification was performed by flow cytometry to investigate the possible
influence of 4b compounds on cell cycle progression of MCF-7 cells. The dynamic of
progression of the cell cycle was altered after 48 h treatment (Fig. 3A and 3B). Cultures
treated with 4b exhibited a G0/G1 population increase (p  0.001), while S- and G2/M-
populations were decreased (p  0.05) when compared to control groups, indicating G1/S
arrest. This finding is very promising considering that human cancers commonly show a
deregulated control of G1 phase progression (Kumari et al., 2016).

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Unfortunately, we could not compare our results concerning cell cycle progression
with the literature, considering that 4b represents an innovative chemical structural pattern.
However, it has been reported that eugenol and isoeugenol modulated the expression
profile of important regulators of cell cycle at G1/S transition, including RB protein
(retinoblastoma protein), CDK6 (cyclin-dependent kinase 6), and p27(KIP1) in normal
keratinocytes (Kalmes and Blömeke, 2012). In addition, the sulfonamide compounds
synthesized by Kamal et al. (2011) induced G0/G1 cell cycle arrest in the K562 leukaemia
cell line.
Cell cycle regulation at G1 progression and G1/S transition involve activation of
protein complexes such as cyclin D-CDK4/6 and cyclin E-CDK2 (Matsuura et al., 2004).
These complexes are responsible for RB phosphorylating, a tumour suppressor protein.
Hyperphosphorylated RB dissociates from the E2F (transcription factor) that promotes the
transcription of important genes for S phase (Foster et al., 2010).
In the present study, we investigated the influence of 4b on cyclin D1 and cyclin E
expression. The results showed that 4b reduced expression levels of cyclin D1 in samples
treated with 100 µM and cyclin E in samples treated with both 50 and 100 µM (Fig. 3C).
Cyclin E is a critical regulator of G1/S transition and we associated the reduction in cyclin
E expression levels induced by 4b to the increase in the G1-cell population. Our findings
are promising because cyclins D and E are commonly overexpressed in breast cancer and
they are closed related to breast tumour progression (Kim and Diehl, 2009; Davis et al.,
2014).
Finally, we verified whether the increased sub-G1 population observed by flow
cytometry was related to apoptosis induction. We frequently observed in cytological
preparation cells with condensed chromatin (Fig. 4A) similarly to cells in early stage of
apoptosis (Yang et al., 2015). Thus, we performed an Annexin V assay and assessed
mitochondria integrity using MitoTracker Red fluorescent dye. There was a significant
increase in cell population positive for Annexin V in samples with 50 and 100 µM,
demonstrating pro-apoptotic activity of 4b on MCF-7 cells (Fig. 4B). Interestingly, we found
a cell population less labelled to MitoTracker Red in samples treated with 4b at 100 µM
(Fig. 4C) reinforcing pro-apoptotic activity of this substance (Cottet-Rousselle et al., 2011).
The resistance to apoptosis is a hallmark of cancer cells (Hanahan and Weinberg, 2011),
and apoptosis evasion drastically contributes to resistance both to cancer treatments and
tumour progression.

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Fig. 2. Effect of 4b on proliferative behaviour of MCF7. (A) Representative images obtained by phase
contrast microscopy (60× magnification) showing morphological features of MCF-7 cells treated with 4b or
vehicle (DMSO) for 48 h. (B) Illustrative images and quantitative analysis of clonogenic capacity assay. (C)
Cell proliferation determined by trypan blue exclusion assay after 24, 48, and 72 h of treatment with 4b. (D)

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Mitotic indexes determined from cytological preparations stained with DAPI and anti--tubulin. The pattern of
staining is shown in (E) that also shows quantitative analysis of the different stages of mitosis after 48 h of
treatment. Cells in anaphase and telophase were not observed in treated samples. Significant differences
from control groups were determined using ANOVA followed by Tukey’s post-test from three independent
experiments. *p  0.05, **p  0.01, and ***p  0.001
Fig. 3. Cell cycle analyses. (A) Illustrative histograms showing cell populations distributed in different phases
of the cell cycle after 48 h of treatment with 4b. Tamoxifen was used as a positive control at 25 µM. (B) Cell
cycle analysis. Brown, pink, green, and blue bars represent, respectively, Sub-G1, G0/G1, S, and G2/M
phases. (C) Cyclin D1 and cyclin E expression profiles were determined by western blot. -tubulin was used
as a loading control. Significant differences from control groups were determined using ANOVA followed by
Tukey’s post-test from three independent experiments. *p < 0.05, **p  0.01, and ***p  0.001

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Fig.4. Pro-apoptotic analyses. (A) Fluorescent images from cell cultures treated at 100 µM with 4b showing
cells with condensed chromatin (green: microtubule network and blue: nuclei). (B) Annexin V-PE/7-AAD
assay. Viable cells: lower left quadrants; early apoptotic cells: lower right quadrants; late apoptotic cells:
upper right quadrants; necrotic cells: upper left quadrants. (C) Viable cells with intact mitochondrial

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membrane (to the right from red line), and cells with loss of the mitochondrial integrity (to the left from red
line). Significant differences from control groups were determined using ANOVA followed by Tukey’s post-
test from three independent experiments. **p  0.01 and ***p  0.001
3. Conclusion
A phenylpropanoid-sulfonamide hybrid (4b) has an innovative structural scaffold
and displays promising antitumor activity against MCF-7 cells. We demonstrated that this
substance reduced cyclin D1 and cyclin E expression in MCF-7 cells, leading to cell cycle
arrest in G1/S transition, and subsequently, apoptosis. Taken together, these findings
demonstrate that 4b should be considered for further in vivo studies to validate its
antitumor activity against positive estrogen receptor breast cancer.
4. Material and methods
4.1 Materials and instrumentation
Eugenol and dihydroeugenol were purchased from Sigma (São Paulo, Brazil) and
were used as such. Thin-layer chromatography (TLC) on silica gel-G plates (Macherey-
Nagel, DC-Fertigfolien ALUGRAM® Xtra Sil G/UV254) was used to monitor reactions
courses. For column chromatography, column grade silica gel (Sorbiline; 0.040-0.063mm
mesh size) was employed. Melting points of the compounds were obtained on a Bücher
535 melting-point apparatus and are uncorrected. IR spectra were recorded on a
Shimadzu FTIR-Affinity-1. NMR spectra were recorded on a Bruker AC-300 spectrometer
1
(Rheinstetten, Germany) (300 MHz for H-NMR and 75 MHz for 13C-NMR spectra) in
deuterated chloroform or dimethylsulfoxide. Chemical shifts () were reported in parts per
million (ppm) with reference to tetramethylsilane (TMS) as internal standard and coupling
constants (J) were reported in Hertz (Hz). High resolution mass (HRMS) spectra were
obtained on a quadrupole time-of-flight instrument (Waters Xevo G2-S QTOF,
Massachusetts, U.S.A), equipped with an ESI positive and negative ion source.
4.2 Synthesis of phenylpropanoid-based sulfonamide derivatives
4.2.1 Synthesis of nitro derivatives 2a and 2a’

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To a solution of eugenol (1, 32.53 mmol) or dihydroeugenol (1’, 31.26 mmol) in
dichloromethane (50 mL) were added equimolar amounts of sodium bisulfate and
potassium nitrate, followed by addition of wet silica gel (1:1 w/w; 6.5 g for eugenol reaction
and 6.3 g for dihydroeugenol reaction). The reaction was kept under vigorous magnetic
stirring at room temperature for 48 h and monitored by TLC (chloroform/methanol, 9.5:0.5,
v/v). After completion of the reaction, the mixture was filtered off, the filtrate was dried over
anhydrous sodium sulfate and concentrated under reduced pressure. The nitro compound
was purified by column chromatography (silica gel, hexane/ethyl acetate, 9/1, v/v).
4-allyl-2-methoxy-6-nitrophenol (2a) Yellow oil; 47% yield; IR (ῡ máx, in cm-1): 3520 (O-H),
1
1600, 1550 (C=C), 1515, 1440 (NO2), 1270 (C-O-C); H NMR (δ ppm; CDCl3; 300 MHz):
10.6 (s, 1H, O-H), 7.4 (d, 1H, H5, J = 1.8 Hz), 6.9 (d, 1H, H3, J = 1.8 Hz), 5.9-5.8 (m, 1H,
CH2=CH-CH2), 5.1-5.0 (m, 2H, CH2=CH-CH2), 3.9 (s, 3H, OCH3), 3.3 (d, 2H, CH2=CH-
CH2, J = 6.6 Hz); 13C NMR (δ ppm; CDCl3; 75 MHz): 149.8 (C6), 144.8 (C1), 135.9
(CH2=CH-CH2), 133.5 (C2), 131.2 (C4), 118.5 (C5), 117.1 (CH2=CH-CH2), 115.0 (C3),
56.6 (OCH3), 39.3 (CH2=CH-CH2).
2-methoxy-6-nitro-4-propylphenol (2a’) Yellow oil; 67% yield; IR (ῡ máx, in cm-1): 3250 (O-
H), 1591 (C=C), 1541, 1448 (NO2), 1260 (C-O-C); 1H NMR (δ ppm; CDCl3; 300 MHz): 10.6
(s, 1H, O-H), 7.4 (d, 1H, H5, J = 1.8 Hz), 6.9 (d, 1H, H3, J = 1.8 Hz), 3.9 (s, 3H, OCH3), 2.5
(t, 2H, CH3-CH2-CH2, J = 7.2 Hz), 1.7-1.5 (m, 2H, CH3-CH2-CH2), 0.9 (t, 3H, CH3-CH2-CH2,
J = 7,2 Hz); 13C NMR (δ ppm; CDCl3; 75 MHz): 149.7 (C6), 144.5 (C1), 133.8 (C4), 133.6
(C2), 118.7 (C5), 114.8 (C3), 56.7 (OCH3), 37.3 (CH3-CH2-CH2), 24.1 (CH3-CH2-CH2),
13.5 (CH3-CH2-CH2).
4.2.2 Synthesis of nitro derivatives 2b and 2b’
To a suspension of bismuth nitrate pentahydrate (1 eq.) in chloroform (50 mL) were
added eugenol (1, 32.53 mmol) or dihydroeugenol (1’, 31.26 mmol) and silica gel (8 eq.).
The mixture was refluxed under stirring for 30 minutes and was monitored by TLC
(chloroform/methanol, 9.5/0.5, v/v). The mixture was then filtered off, the filtrate was dried
over anhydrous sodium sulfate and concentrated under reduced pressure. The nitro
compound was purified by column chromatography (silica gel, hexane/ethyl acetate, 9/1,
v/v).

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4-Allyl-2-methoxy-5-nitrophenol (2b): Dark yellow oil; 42% yield; IR (ῡ máx, in cm-1): 3231
1
(O-H), 1601 (C=C), 1538, 1444 (NO2), 1261 (C-O-C). H-NMR (δ ppm; CDCl3, 300 MHz):
10.65 (s, 1H, O-H), 7.52 (s, 1H, H6), 6.97 (s, 1H, H3), 5.96-5,87 (m, 1H, CH2=CH-CH2),
5.17-5.10 (m, 2H, CH2=CH-CH2), 3.94 (s, 3H, OCH3), 3.37 (d, 2H, J = 5.0 Hz, CH2=CH-
CH2). 13C-NMR (δ ppm; CDCl3, 75 MHz): 149.7 (C5), 145.0 (C1), 136.0 (CH2=CH-CH2),
133.0 (C2), 131.1 (C4), 118.6 (C6), 117.1 (CH2=CH-CH2), 115.1 (C3), 56.8 (OCH3), 39.5
(CH2=CH-CH2).
2-methoxy-5-nitro-4-propylphenol (2b’): Yellow oil; 21% yield; IR (ῡ máx, in cm-1): 3221 (O-
1
H), 1541, 1448 (NO2), 1260 (C-O-C). H-NMR (δ ppm; CDCl3, 300 MHz): 10.62 (s, 1H, O-
H), 7.49 (d, 1H, J = 2.0 Hz, H6), 6.96 (d, 1H, J = 1.8 Hz, H3), 3.93 (s, 3H, OCH3), 2.58 (t,
2H, J = 7.5 Hz, CH3-CH2-CH2), 1.70-1.58 (m, 2H, CH3-CH2-CH2), 0.97 (t, 3H, J = 7.2 Hz,
CH3-CH2-CH2). 13C-NMR (δ ppm; CDCl3, 75 MHz): 149.8 (C5), 144.7 (C1), 133.9 (C2),
133.7 (C4), 118.8 (C6), 115.0 (C3), 56.8 (OCH3), 37.5 (CH3-CH2-CH2), 24.3 (CH3-CH2-
CH2), 13.7 (CH3-CH2-CH2).
4.2.3 Synthesis of amino derivatives 3a, 3b, 3a’ and 3b’
The nitro compound 2a, 2b, 2a’, or 2b’ (1 eq.) was solubilized in ethanol (50 mL)
and stannous chloride dihydrate (5 eq.) was added to this solution. The suspension was
refluxed under magnetic stirring for 3 h and the consumption of starting material was
followed by TLC (chloroform/methanol, 9.5/0.5, v/v). The mixture was allowed to cool down
and then poured into ice water. The pH was then adjusted to 7-8 by aqueous 5% NaHCO3.
Then, the mixture was extracted with ethyl acetate (3 x 50 mL) and the organic layer was
dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under
reduced pressure, which afforded the crude products. These were next purified by column
chromatography (silica gel, hexane/ethyl acetate, 8/2, v/v).
4-Allyl-2-amino-6-methoxyphenol (3a): Light yellow solid; 60% yield; m.p.: 95-98 ºC; IR (ῡ
1
máx, in cm-1): 3371, 3311, 3074, 1606, 1512, 1456, 1230. H-NMR (δ ppm; CDCl3, 300
MHz): 6.25 (d, 1H, J = 1.8 Hz), 6.19 (d, 1H, J = 1.8 Hz), 6.00-5.87 (m, 1H), 5.11-5.02 (m,
2H), 3.84 (s, 3H), 3.24 (d, 2H, J = 6.9 Hz). 13C-NMR (δ ppm; CDCl3, 75 MHz): 146.6 (C6),
137.9 (CH2=CH-CH2), 133.8 (C2), 131.7 (C4), 131.2 (C1), 115.3 (CH2=CH-CH2), 109.5
(C5), 102.0 (C3), 56.0 (OCH3), 40.1 (CH2=CH-CH2).

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2-amino-6-methoxy-4-propylphenol (3b): Light beige solid; 63% yield; m.p.: 63-66 ºC; IR (ῡ
1
máx, in cm-1): 3370, 3307, 3012, 1513, 1212. H-NMR (δ ppm; CDCl3, 300 MHz): 6.24 (d,
1H, J = 1.8 Hz), 6.19 (d, 1H, J = 1.8 Hz), 3.85 (s, 3H), 2.45 (t, 2H, J = 7.2 Hz), 1.66-1.53
13
(m, 2H), 0.93 (t, 3H, J = 7.5 Hz). C-NMR (δ ppm; CDCl3, 75 MHz): 146.5 (C6), 134.4
(C2), 133.8 (C4), 130.9 (C1), 109.3 (C5), 101.8 (C3), 55.9 (OCH3), 38.0 (CH3-CH2-CH2),
24.7 (CH3-CH2-CH2), 13.8 (CH3-CH2-CH2).
4-Allyl-5-amino-2-methoxyphenol (3a’): Light orange solid; 60% yield; m.p.: 105-107 ºC. IR
1
(ῡ máx, in cm-1): 3369, 3305, 3071, 1606, 1513, 1456, 1214. H-NMR (δ ppm; CDCl3, 300
MHz): 6.25 (d, 1H, J = 1.8 Hz), 6.20 (d, 1H, J = 1.7 Hz), 5.99-5.87 (m, 1H), 5.11-5.03 (m,
2H), 4.08 (s, 2H), 3.84 (s, 3H), 3.25 (d, 2H, J = 6.7 Hz). 13C-NMR (δ ppm; CDCl3, 75 MHz):
147.2 (C2), 138.5 (CH2=CH-CH2), 133.8 (C5), 131.7 (C4), 131.2 (C1), 115.9 (CH2=CH-
CH2), 110.0 (C3), 102.4 (C6), 57.0 (OCH3), 40.7 (CH2=CH-CH2).
5-amino-2-methoxy-4-propylphenol (3b’): Light beige solid; 19% yield; m.p.: 82-84 ºC. IR
(ῡ máx, in cm-1): 3370, 3307, 3016, 1514, 1214. 1H-NMR (δ ppm; CDCl3, 300 MHz): 6.24 (d,
1H, J = 1.8 Hz), 6.19 (d, 1H, J = 1.8 Hz), 3.84 (s, 3H), 2.44 (t, 2H, J = 7.7 Hz), 1.66-1.53
13
(m, 2H), 0.93 (t, 3H, J = 7.3 Hz). C-NMR (δ ppm; CDCl3, 75 MHz): 146.5 (C2), 134.4
(C5), 133.8 (C4), 130.9 (C1), 109.3 (C3), 101.8 (C6), 55.9 (OCH3), 38.0 (CH3-CH2-CH2),
24.7 (CH3-CH2-CH2), 13.8 (CH3-CH2-CH2).
4.2.4 Synthesis of 4-acetamidobenzenesulfonamides 4a, 4b, 4a’ and 4b’
The amino derivatives 3a, 3b, 3a’, or 3b’ (1 eq.) and 4-acetamidobenzenesulfonyl
chloride (1 eq.) were solubilized in pyridine (5 mL) and this mixture was refluxed for 3 h
under vigorous stirring. The progress of the reaction was monitored by TLC (hexane/ethyl
acetate, 6/4, v/v). The reaction mixture was allowed to cool down and then poured into ice
water. The pH was adjusted to 1-2 with hydrochloric acid before being extracted with ethyl
acetate (3 x 50 mL). The organic phase was dried over anhydrous sodium sulfate and
concentrated under reduced pressure to afford the crude product which was purified by
column chromatography (silica gel, hexane/ethyl acetate, 4/6, v/v).
N-(4-(N-(5-allyl-2-hydroxy-3-methoxyphenyl)sulfamoyl)phenyl)acetamide (4a): Light yellow
solid; 40% yield; m.p.: 167-168 ºC. IR (ῡ máx, in cm-1): 3307 (N-H), 1682 (C=O), 1590, 1515
1
(C=C), 1311, 1152 (S=O), 1256 (C-O-C). H-NMR (δ ppm; DMSO-d6, 300 MHz): 10.29 (s;

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1H; OH), 8.85 (sl; 2H; NHSO2, NHCOCH3), 7.64 (s; 4H; H2’, H3’), 6.57 (d; 1H; J = 1.8 Hz;
H4), 6.53 (d; 1H; J = 1.7 Hz; H6), 5.87-5.78 (m; 1H; CH2=CH-CH2), 5.00-4.94 (m; 2H;
CH2=CH-CH2), 3.68 (s; 3H; OCH3), 3.16 (d; 2H; J = 6.5 Hz; CH2=CH-CH2), 2.05 (s; 3H;
NHCOCH3). 13C-NMR (δ ppm; DMSO-d6, 75 MHz): 169.3 (C5’), 147.8 (C3), 143.0 (C4’),
137.8 (CH2=CH-CH2), 137.5 (C1’), 134.1 (C5), 129.8 (C1), 128.1 (C2’), 124.2 (C2), 118.4
(C3’), 116.1 (C4), 115.7 (CH2=CH-CH2), 109.3 (C6), 55.9 (OCH3), 39.5 (CH2=CH-CH2),
24.2 (NHCOCH3). HRMS-ESI (m/z): calcd. for C18H20N2O5S (M+Na)+: 399.0991; found:
399.0985.
N-(4-(N-(2-hydroxy-3-methoxy-5-propylphenyl)sulfamoyl)phenyl)acetamide (4b): Beige
solid; 25% yield; m.p.: 176-180 ºC. IR (ῡ máx, in cm-1): 3537, 3364 (N-H), 3154 (O-H), 2923
(C-H), 1687 (C=O), 1589, 1512 (C=C), 1312, 1158 (S=O), 1256 (C-O-C). 1H-NMR (δ ppm;
DMSO-d6, 300 MHz): 10.26 (s; 1H; OH), 8.95 (s; 1H; NHSO2), 8.56 (s; 1H; NHCOCH3),
7.65 (s; 4H; H2’, H3’), 6.58 (d; 1H; J = 1.9 Hz; H4), 6.53 (d; 1H; J = 1.9 Hz; H6), 3.69 (s;
3H; OCH3), 2.36 (t; 2H; J = 7.4 Hz; CH3-CH2-CH2), 2.06 (s; 3H; NHCOCH3), 1.52-1.40
(sex; 2H; CH3-CH2-CH2), 0.80 (t; 3H; J = 7.3 Hz; CH3-CH2-CH2).13C-NMR (δ ppm; DMSO-
d6, 75 MHz): 169.0 (C5’), 147.6 (C3), 142.9 (C4’), 137.1 (C1’), 134.0 (C5), 132.0 (C1),
128.0 (C2’), 124.2 (C2), 118.2 (C3’), 115.8 (C4), 109.1 (C6), 55.8 (OCH3), 37,0 (CH3-CH2-
CH2), 24.1 (NHCOCH3), 24.1 (CH3-CH2-CH2), 13.4 (CH3-CH2-CH2). HRMS-ESI (m/z):
calcd. for C18H22N2O5S (M+H)+: 379.1328; found: 379.1315.
N-(4-(N-(2-allyl-5-hydroxy-4-methoxyphenyl)sulfamoyl)phenyl)acetamide (4a’): Yellow
solid; 43% yield; m.p.167-168 ºC. IR (ῡ máx, in cm-1): 3307 (N-H), 1682 (C=O), 1590, 1515
1
(C=C), 1311, 1152 (S=O), 1256 (C-O-C). H-NMR (δ ppm; DMSO-d6, 300 MHz): 10.30 (s;
1H; OH), 8.84 (sl; 1H; NHSO2), 7.64 (s; 4H; H2’, H3’), 6.57 (d; 1H; J = 1.9 Hz; H3), 6.53 (d;
1H; J = 1.8 Hz; H6), 5.86-5.75 (m; 1H; CH2=CH-CH2), 5.00-4.93 (m; 2H; CH2=CH-CH2),
3.68 (s; 3H; OCH3), 3.16 (d; 2H; J = 6.6 Hz; CH2=CH-CH2), 2.05 (s; 3H; NHCOCH3).13C-
NMR (δ ppm; DMSO-d6, 75 MHz): 169.7 (C5’), 148.1 (C4’), 143.3 (C5), 138.2 (C4), 137.8
(CH2=CH-CH2), 134.4 (C1’), 130.2 (C1), 128.4 (C2’), 124.9 (C2), 118.8 (C3’), 116.4 (C3),
116.1 (CH2=CH-CH2), 109.6 (C6), 56.2 (OCH3), 39.5 (CH2=CH-CH2), 24.5 (NHCOCH3).
HRMS-ESI (m/z): calcd. for C18H20N2O5S (M+H)+: 377.1172; found: 377.1176.
N-(4-(N-(5-hydroxy-4-methoxy-2-propylphenyl)sulfamoyl)phenyl)acetamide (4b’): Light
yellow solid; 41% yield; m.p.: 176-180 ºC. IR (ῡ máx, in cm-1): 3536, 3362 (N-), 3143 (O-H),
1
1687 (C=O), 1589, 1522 (C=C), 1313, 1158 (S=O), 1255 (C-O-C). H-NMR (δ ppm;

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DMSO-d6, 300 MHz): 10.26 (s; 1H; OH), 8.74 (sl; 2H; NHSO2, NHCOCH3), 7.65 (s; 4H;
H2’, H3’), 6.58 (d; 1H; J = 1.7 Hz; H3), 6.53 (d; 1H; J = 1.7 Hz; H6), 3.69 (s; 3H; OCH3),
2.36 (t; 2H; J = 7.4 Hz; CH3-CH2-CH2), 2.06 (s; 3H; NHCOCH3), 1.53-1.40 (sex; 2H; CH3-
13
CH2-CH2), 0.81 (t; 3H; J = 7.3 Hz; CH3-CH2-CH2). C-NMR (δ ppm; DMSO-d6, 75 MHz):
169.0 (C5’), 147.5 (C4’), 142.9 (C5), 137.0 (C4), 133.9 (C1’), 132.0 (C1), 128.0 (C2’),
124.2 (C2), 118.2 (C3’), 115.8 (C3), 109.1 (C6), 55.7 (OCH3), 36.9 (CH3-CH2-CH2), 24.1
(NHCOCH3), 24.1 (CH3-CH2-CH2), 13.4 (CH3-CH2-CH2). HRMS-ESI (m/z): calcd. for
C18H22N2O5S (M+Na)+: 401.1147; found: 401.1128.
4.2.5 Synthesis of 4-aminobenzenesulfonamides 5a, 5b, 5a’ and 5b’
Thionyl chloride (5 eq.) was added to a stirred solution of the corresponding 4-
acetamidobenzene sulfonamide (1 eq.) in dry methanol (10 mL). The solution was kept
under stirring at room temperature for 24 h. The progress of the reaction was monitored by
TLC (chloroform/methanol, 9/1, v/v). Methanol was then removed under reduced pressure.
The crude product was purified by preparative thin layer chromatography
(chloroform/methanol, 9/1, v/v).
N-(5-allyl-2-hydroxy-3-methoxyphenyl)-4-aminobenzenesulfonamide (5a): Red solid; 45%
yield. m.p.: 133-136 ºC. IR (ῡ máx, in cm-1): 3484 (N-H), 3383, 3295 (NH2), 1622, 1594,
1
1515 (C=C), 1315, 1132 (S=O), 1210 (C-O-C), 1085 (C-N). H-NMR (δ ppm; DMSO-d6,
300 MHz): 8.63 and 8.43 (sl; 2H; NH2), 7.38-7.33 (m; 2H; H2’), 6.62 (d; 1H; J = 1.9 Hz;
H4), 6.52-6.50 (m; 1H; H6), 6.9-6.47 (m; 2H; H3’), 5.91 (s; 2H; OH, NHSO2), 5.88-5.79 (m;
1H; CH2=CH-CH2), 5.02-4.96 (m; 2H; CH2=CH-CH2), 3.69 (s; 3H; OCH3), 3.17 (d; 2H; J =
6.7 Hz; CH2=CH-CH2).13C-NMR (δ ppm; DMSO-d6, 75 MHz): 152.7 (C3), 147.6 (C4’),
137.8 (CH2=CH-CH2), 136.3 (C5), 129.7 (C1), 128.8 (C2’), 125.4 (C1’), 124.9 (C2), 115.5
(CH2=CH-CH2), 114.6 (C6), 112.4 (C3’), 108.5 (C4), 55.7 (OCH3), 39.7 (CH2=CH-CH2).
HRMS-ESI (m/z): calcd. for C16H18N2O4S (M+H)+: 335.1066; found: 335.1060.
4-amino-N-(2-hydroxy-3-methoxy-5-propylphenyl)benzenesulfonamide (5b): Dark yellow
oil; 33% yield. IR (ῡ máx, in cm-1): 3472 (N-H), 3375 (NH2), 1612, 1595, 1514 (C=C), 1314,
1
1152 (S=O), 1215 (C-O-C), 1088 (C-N). H-NMR (δ ppm; DMSO-d6, 300 MHz): 8.56 and
8.39 (s; 2H; NH2), 7.36 (d; 2H; J = 8.7 Hz; H2’), 6.61 (d; 1H; J = 1.5 Hz; H4), 6.51-6.50 (m;
1H; H6), 6.49-6.48 (m; 2H; H3’), 5.91 (s; 1H; OH or NHSO2), 3.69 (s; 3H; OCH3), 2.36 (t;
2H; J = 7.4 Hz; CH2-CH2-CH3), 1.54-1.41 (m; 2H; CH2-CH2-CH3), 0.82 (t; 3H; J = 7.3 Hz;

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CH2-CH2-CH3).13C-NMR (δ ppm; DMSO-d6, 75 MHz): 153.7 (C3), 147.9 (C4’), 136.4 (C5),
132.5 (C1), 129.2 (C2’), 125.6 (C1’), 125.3 (C1’), 114.8 (C6), 112.8 (C3’), 108.8 (C4), 56.1
(OCH3), 37.5 (CH2-CH2-CH3), 24.6 (CH2-CH2-CH3), 13.9 (CH2-CH2-CH3). HRMS-ESI
(m/z): calcd. for C16H20N2O4S (M+H)+: 337.1223; found: 337.1210.
N-(2-allyl-5-hydroxy-4-methoxyphenyl)-4-aminobenzenesulfonamide (5a’): Light yellow
solid; 40% yield; m.p.:140-143 ºC. IR (ῡ máx, in cm-1): 3486 (N-H), 3383, 3294 (NH2), 1622,
1595, 1516 (C=C), 1315, 1132 (S=O), 1210 (C-O-C), 1084 (C-N). 1H-NMR (δ ppm; DMSO-
d6, 300 MHz): 8.63 and 8.43 (sl; 2H; NH2), 7.38-7.34 (m; 2H; H2’), 6.63 (d; 1H; J = 1.9 Hz;
H3), 6.52-6.51 (m; 1H; H6), 6.50-6.47 (m; 2H; H3’), 5.91 (s; 2H; OH, NHSO2), 5.88-5.79
(m; 1H; CH2=CH-CH2), 5.02-4.96 (m; 2H; CH2=CH-CH2), 3.69 (s; 3H; OCH3), 3.17 (d; 2H;
J = 6.6 Hz; CH2=CH-CH2). 13C-NMR (δ ppm; DMSO-d6, 75 MHz): 152.8 (C4’), 147.6 (C5),
137.8 (CH2=CH-CH2), 136.3 (C4), 129.7 (C1), 128.8 (C2’), 125.4 (C1’), 124.9 (C2), 115.5
(CH2=CH-CH2), 114.6 (C3), 112.4 (C3’), 108.5 (C6), 55.7 (OCH3), 39.7 (CH2=CH-CH2).
HRMS-ESI (m/z): calcd. for C16H18N2O4S (M+Na)+: 357.0885; found: 357.0886.
4-amino-N-(5-hydroxy-4-methoxy-2-propylphenyl)benzenesulfonamide (5b’): Dark yellow
oil; 31% yield. IR (ῡ máx, in cm-1): 3462 (N-H), 3375, 3248 (NH2), 1612, 1595, 1514 (C=C),
1314, 1148 (S=O), 1217 (C-O-C), 1086 (C-N).1H-NMR (δ ppm; DMSO-d6, 300 MHz): 8.56
e 8.39 (s; 2H; NH2), 7.36 (d; 2H; J = 8.7 Hz; H2’), 6.61 (d; 1H; J = 1.7 Hz; H3), 6.51-6.50
(m; 1H; H6), 6.48-6.47 (m; 2H; H3’), 5.91 (s; 1H; OH ou NHSO2), 3.69 (s; 3H; OCH3), 2.36
(t; 2H; J = 7.4 Hz; CH2-CH2-CH3), 1.53-1.41 (m; 2H; CH2-CH2-CH3), 0.81 (t; 3H; J = 7.3
13
Hz; CH2-CH2-CH3). C-NMR (δ ppm; DMSO-d6, 75 MHz): 152.5 (C4’), 147.2 (C5), 135.7
(C4), 131.8 (C1’), 128.6 (C2’), 124.9 (C1), 124.6 (C2), 114.1 (C3), 112.1 (C3’), 108.1 (C6),
55.5 (OCH3), 36.8 (CH2-CH2-CH3), 23.9 (CH2-CH2-CH3), 13.2 (CH2-CH2-CH3). HRMS-ESI
(m/z): calcd. for C16H20N2O4S (M+H)+: 337.1223; found: 337.1210.
4.3 Biological assays
4.3.1 Cell lines and treatment schedule
Human tumour cell lines were used in the present study, A549 (lung carcinoma),
MCF-7 (breast carcinoma), HepG2 (hepatocellular carcinoma), and HT-144 (melanoma).
In addition, fibroblasts derived from human normal skin (CCD-1059Sk) and non-
transformed breast epithelial cells (MCF 10A) were also examined. The cell cultures

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(A549, MCF-7, HepG2, HT-144, and CCD-1059-Sk) were maintained in DMEM/F12
(Dulbecco’s Modified Eagle’s Minimum Essential Medium, Sigma, CA, USA)
supplemented with 10% fetal bovine serum (FBS, Cultilab, São Paulo, Brazil). MCF 10A
cells were cultivated in DMEM/F12 (Gibco) supplemented with 5% horse serum, 10 µg/mL
insulin, 0,50 µg/mL hydrocortisone, 100 ng/mL cholera toxin and 10 ng/mL EGF. Cells
were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
4.3.2 Cell viability analysis
4.3.2.1 Colorimetric assay
The cells were seeded into a 96-well plate at 5 × 104 cells/well (A549 and CCD-
1059Sk) or 1 × 104 cells/well (HT-144, HepG2, MCF-7, and MCF 10A). Resazurin
reduction assay was used according to the manufacturer’s instructions (Sigma) to assess
cell viability of A549, CCD-1059Sk, HT-144, HepG2, and MCF-7 cells. The resazurin (blue,
absorption peak at 600 nm) is reduced to resorufin (pink, absorption peak at 570nm) by
cell metabolically actives. Therefore, the amount of resorufin produced is directly
proportional to the number of viable cells. MTT (3-(4,5-dimethylthiazol)-2,5-
diphenyltetrazolium bromide) assay was used according to the manufacturer’s instructions
(Sigma) to assess cell viability of MCF 10A cells. The molecules were solubilized in DMSO
and kept at 4 °C until use. A stock solution (20 mM) was diluted in fresh medium
immediately before of the use. The substances (4a, 4b, 4a’, 4b’, 5a, 5b, 5a’, and 5b’)
were screened at 125 µM for 48 h treatment. After that, dose-response curves were
performed using the most active compounds. Thus, 4b, 4b’, 5a, and their precursors
(sulfanilamide, eugenol, and dihydroeugenol) were tested in a concentration range of 50-
400 µM for 48 h treatment on MCF-7. In these same experimental conditions, 4b, 4b’, and
5a were also tested on CCD-1059-Sk and MCF 10A. The experiments were conducted in
triplicate wells. IC50 values were determined from a non-linear regression using GraphPad
Prism® (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as the mean
± standard deviation (SD) of three independent experiments.
4.3.2.2 Trypan blue exclusion test
Cells were seeded into 35 mm plates at a density of 2 × 105. The treatment with
compound 4b was performed at 50 and 100 µM for 24, 48, or 72 h. Cells were harvested

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with trypsin/EDTA, and trypan blue solution (0.4%) was added to the cellular suspension
(1:1). Subsequently, cells were counted using a hemocytometer chamber and a light
microscope. Non-viable (blue stained) and viable cells (unstained) were quantified from
three independent experiments. The results are presented as mean ± SD.
4.3.3 Clonogenic assay
The clonogenic assay was performed according to Franken et al. (2006). Briefly,
100 cells were seeded into 35 mm plates. Cells were treated for 48 h with compound 4b at
50, 100, and 200 µM and recovered in a drug-free medium for subsequent 15 days.
Afterward, the colonies were fixed and stained with Crystal Violet. Only colonies with > 50
cells were counted by direct visual inspection with a stereomicroscope at 20×
magnification. Assays were performed in triplicate and data were presented as mean ± SD
of three independent experiments.
4.3.4 Cell cycle analysis
To analyse cell cycle progression, cells were treated for 48 h with compound 4b at
50 and 100 µM. The cells were fixed with 75% ethanol at 4 C overnight and then rinsed
twice with cold phosphate-buffered saline (PBS). Then, the cells were resuspended in dye
solution (PBS containing 30 μg/mL propidium iodide (PI) and 3 mg/mL RNAase). DNA was
quantified 1 h after staining. The analysis was performed by flow cytometry (Guava
easyCyte 8HT, Hayward, CA, USA). Results are presented as means ± standard
deviations (SD) of three independent experiments.
4.3.5 Mitotic index determination from fluorescent cytological preparations
Cells were seeded into 35 mm Petri plates on coverslips at 2 × 105 cells/plate. After
48 h of treatment, the samples were fixed with 3.7% formaldehyde for 30 min. For α-
tubulin immunolabeling, cells were permeabilized with Triton X-100 (0.5%) for 10 min.
After blocking with 1% BSA, anti--tubulin antibody (1:100, clone DM1A, Sigma Aldrich)
was incubated overnight. On the next day, secondary anti-mouse IgG-FITC antibody
(1:100, Sigma Aldrich) was added to the sample and incubated for 2 h. Nuclei were
stained with DAPI, and the coverslips were mounted on microscope slides using Vecta-
Shield mounting medium (Vector Laboratories). Analyses were performed using a

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fluorescence microscope (Nikon). Fluorescent cytological preparations were used to
determine the frequency of mitosis (1,000 cells per sample were counted). The data are
shown as the mean ± SD of three independent experiments.
4.3.6 Immunoblot
To analyse expression protein profile, MCF-7 cells were treated for 48 h with
compound 4b at 50 and 100 µM. Cells were homogenized in RIPA lysis buffer (150 mM
NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS and 50 mM Tris pH 8.0)
containing both protease and phosphatase inhibitors (Sigma). Lysates were centrifuged
(10,000 × g) for 10 min at 4 °C. Supernatants were recovered, total proteins were
quantified (BCA kit, Pierce Biotechnology Inc., Rockford, IL, USA) and resuspended in
Laemmli sample buffer containing 62.5 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 5%
2-mercaptoethanol and 0.001% bromophenol blue. An aliquot of 50 μg protein was
separated by SDS–PAGE (12%) and transferred (100 V, 250 mA for 2h) on to a PVDF
membrane (Amersham Bioscience), blocked for 1h at 4 °C with blocking solution [5% non-
fat milk in Tris-buffered saline (TBS) + 0.1% (v/v) Tween-20] to prevent nonspecific protein
binding. The membrane was probed with primary antibodies: Cyclin D1 (Sigma – 1: 200),
Cyclin E (Santa Cruz – 1:200) and α-tubulin (Sigma– 1:1000) overnight at 4 °C. After
washing with TBS-tween (0.1%), the membrane was incubated with a secondary antibody
(anti-rabbit peroxidase conjugated) for 2h at room temperature. Immunoreactive bands
were visualized with the ECL Western Blotting Detection Kit (Amersham Pharmacia). A
reprobing protocol was followed for detecting immunoreactive bands for different
antibodies. Results were obtained from three independent experiments. The quantification
of immunoreactive bands was performed using a public program (Image J).
4.3.7 Apoptosis detection by Annexin V/7-AAD
Kit Guava Nexin® (Merk Millipore, Massachusetts, EUA) was used according to
manufacturer’s instructions. To analyse apoptosis induction, MCF-7 cells were treated for
48 h with compound 4b at 50 and 100 µM. Briefly, cells were collected by enzymatic
digestion (Trypsin/EDTA, Sigma), centrifuged at 1,000 rpm for 5 min at 4 °C, washed with
ice-cold PBS, and then 2 × 104 cells were resuspended in 100 μL of DMEM. In the next
step, 100 μL of mix solution containing buffered Annexin V-PE and 7-AAD was added. The
samples were read after 20 min of incubation at room temperature in a dark chamber. The

ACCEPTED MANUSCRIPT
analysis was performed by flow cytometry using GuavaSoft 2.7 software. The experiments
were conducted in triplicate and repeated twice. Data are presented as mean ± SD.
4.3.8 Mitochondrial integrity analysis
The integrity of mitochondria was examined by using MitoTracker Red fluorescent
dye (Invitrogen, Molecular Probes, ThermoFisher Scientific) according to manufacturer’s
instructions. MCF-7 cells were treated for 48 h with compound 4b at 50 and 100 µM. The
cells were collected by enzymatic digestion (Trypsin/EDTA), centrifuged at 1,000 rpm for 5
min at 4 °C, washed with ice-cold PBS and incubated with MitoTracker (500 nM) at room
temperature for 45 minutes in a dark chamber. The analysis was performed by flow
cytometry using GuavaSoft 2.7 software. The experiments were conducted in triplicate and
repeated twice. Data are presented as mean ± SD.
4.3.9 Statistical analysis
The results were tested for significance using one-way analysis of variance
(ANOVA) followed by a Tukey's post-test. The values were expressed as mean ± SD.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors are grateful to Brazilian Agencies FAPEMIG for financial support (APQ-
01209-13, APQ-02810-16) and CAPES for the scholarship (H.A.B and G.A.F.S).
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