Thiosemicarbazones and 4-thiazolidinones indole-based derivatives: Synthesis, evaluation of antiproliferative activity, cell death mechanisms and topoisomerase inhibition assay

Article abstract of DOI:10.1016/j.ejmech.2017.05.023

In this study, we report the synthesis and structural characterization of a series of thiosemicarbazone and 4-thiazolidinones derivatives, as well as their in?vitro antiproliferative activity against eight human tumor cell lines. For the most potent compo

Full text of DOI:10.1016/j.ejmech.2017.05.023

Accepted Manuscript
Thiosemicarbazones and 4-thiazolidinones indole-based derivatives: Synthesis,
evaluation of antiproliferative activity, cell death mechanisms and topoisomerase
inhibition assay
Jamerson Ferreira de Oliveira, Talitha Santos Lima, Débora Barbosa Vendramini-
Costa, Sybelle Christianne Batista de Lacerda Pedrosa, Elizabeth Almeida Lafayette,
Rosali Maria Ferreira da Silva, Sinara Monica Vitalino de Almeida, Ricardo Olímpio
de Moura, Ana Lúcia Tasca Gois Ruiz, João Ernesto de Carvalho, Maria do Carmo
Alves de Lima
PII:
S0223-5234(17)30384-7
10.1016/j.ejmech.2017.05.023
EJMECH 9453
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 January 2017
Revised Date: 4 May 2017
Accepted Date: 6 May 2017
Please cite this article as: J.F. de Oliveira, T.S. Lima, Dé.Barbosa. Vendramini-Costa, S.C.B. de
Lacerda Pedrosa, E.A. Lafayette, R.M.F. da Silva, S.M.V. de Almeida, Ricardo.Olí. de Moura,
Ana.Lú.Tasca.Gois. Ruiz, Joã.Ernesto. de Carvalho, M.d.C.A. de Lima, Thiosemicarbazones and 4-
thiazolidinones indole-based derivatives: Synthesis, evaluation of antiproliferative activity, cell death
mechanisms and topoisomerase inhibition assay, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.05.023.
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Graphical Abstract

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Thiosemicarbazones and 4-Thiazolidinones Indole-based derivatives:
synthesis, evaluation of antiproliferative activity, cell death
mechanisms and topoisomerase inhibition assay
Jamerson Ferreira de Oliveiraa; Talitha Santos Limaa; Débora Barbosa Vendramini-
Costab,c; Sybelle Christianne Batista de Lacerda Pedrosad; Elizabeth Almeida
Lafayettee; Rosali Maria Ferreira da Silvaf; Sinara Monica Vitalino de Almeidag;
Ricardo Olímpio de Mourah; Ana Lúcia Tasca Gois Ruizb; João Ernesto de Carvalhob,i;
Maria do Carmo Alves de Limaa,*
aUniversidade Federal de Pernambuco (UFPE), Departamento de Antibióticos
(DANTI), 50670-901, Recife, PE (Brazil)
bUniversidade Estadual de Campinas (Unicamp), Centro Pluridisciplinar de Pesquisas
Químicas, Biológicas e Agrícolas (CPQBA) 13083-970, Campinas, SP (Brazil)
c Cancer Biology, Fox Chase Cancer Center, 19111, Philadelphia, PA (USA)
dUniversidade Federal do Vale do São Francisco (UNIVASF), Colegiado de Ciências
Farmacêuticas (CFARM), 56304-205, Petrolina, PE (Brazil)
eUniversidade Federal da Paraíba (UFPB), Departamento de Química e Física (DQF),
58397-000, Areia, PB (Brazil)
fUniversidade Federal de Pernambuco (UFPE), Departamento de Ciências
Farmacêuticas (DCFAR), 50670-901, Recife-PE, Brazil
gUniversidade de Pernambuco (UPE), Faculdade de Ciências, Educação e Tecnologia
de Garanhuns (FACETEG), 55290-000, Garanhuns-PE, Brazil
hUniversidade Estadual da Paraíba (UEPB), Departamento de Farmácia, 58429-500,
Campina Grande, PB (Brazil)
iUniversidade Estadual de Campinas (Unicamp), Faculdade de Ciências Farmacêuticas
(FCF), 13083-970, Campinas, SP (Brazil)
To whom correspondence should be addressed: Maria do Carmo Alves de Lima
*Email: nenalima.mariadocarmo@gmail.com
Phone number: +55 8121268347
Fax: +55 8121268346

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Abstract
In this study, we report the synthesis and structural characterization of a series of
thiosemicarbazone and 4-thiazolidinones derivatives, as well as their in vitro
antiproliferative activity against eight human tumor cell lines. For the most potent
compound further studies were performed evaluating cell death induction, cell cycle
profile, ctDNA interaction and topoisomerase IIα inhibition. A synthetic three-step
route was established for compounds (2a-e and 3a-d) with yields ranging from 32-95%.
Regarding antiproliferative activity, compounds 2a-e and 3a-d showed mean GI50
values ranging between 1.1 µM (2b) - 84.65 µM (3d). Compound 2b was the most
promising especially against colorectal adenocarcinoma (HT-29) and leukemia (K562)
cells (GI50 = 0.01 µM for both cell lines). Mechanism studies demonstrated that 24h-
treatment with compound 2b (5 µM) induced phosphatidylserine residues exposition
and G2/M arrest on HT-29 cells. Moreover, 2b (50 µM) was able to interact with
ctDNA and inhibited topoisomerase IIα activity. These results demonstrate the
importance of thiosemicarbazone, especially the derivative 2b, as a promising candidate
for anticancer therapy.
Keywords: Medicinal chemistry; anticancer therapy; cellular death; DNA interaction;
topoisomerase inhibition

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1 Introduction
Cancer remains a major health problem with 14.1 million new cases and 8.2
million deaths by cancer in 2012 worldwide [1]. Although chemotherapy is one of the
main therapeutic options for cancer, this form of treatment has limitations such as the
possibility of severe adverse effects and the development of resistance. Moreover, as
cancer is a group of diseases, there is no specific treatment for some kinds of tumors. In
this context, the continuous research and development of new chemical entities is of
great relevance for cancer therapy [2].
Among the available cancer therapeutic targets, DNA remains as an attractive
target for the development of new drugs that can bind to its structure and artificially
alter and/or inhibit its function [3]. Extensive chemical and biochemical studies have
characterized a variety of molecules that can react with DNA, conferring various
pharmacological effects, such as antibiotic, antitumor, antiviral or antiprotozoal [4].
Once drugs bind to DNA and protein interfaces, the activity of enzymes that metabolize
the DNA, such as topoisomerase can be inhibited by the induction of cell death [5].
Topoisomerases are ubiquitous enzymes that alter DNA topology by relieving
supercoiling-associated tension in double stranded DNA. These enzymes can perform
their function by transiently cutting one strand (type I topoisomerases, Top1) or both
DNA strands (type II topoisomerases, Top2) [6]. The ubiquitous presence and crucial
biological roles of topoisomerases explain their prevalence as therapeutic target [5].
Moreover, it is widely known that cancer cells possess high levels of topoisomerase
activity and show remarkable sensitivity to DNA-targeted drugs [7].
Most clinical drugs that target Top1 or Top2 convert the topoisomerase-
associated DNA breaks (known as topoisomerase cleavage complexes) into irreversible
DNA damage that initiates the apoptotic program [8]. These topoisomerase-targeted
drugs, referred to as topoisomerase poisons, stabilize the topoisomerase cleavage
complexes by preventing DNA religation [6]. In addition to their role in the initiation of
apoptosis, a growing number of studies show that Top1 and Top2 also participate
directly in the execution of the apoptotic program [9]. Since topoisomerase inhibitors
can trigger cell death by apoptosis, the elucidation of the relationship between these
mechanisms provide opportunities to develop novel therapeutic strategies.
In this context, thiosemicarbazones arise as a promising class of molecules, due
to their reported antiproliferative activity in human tumor cell lines [10-12] and
associated mechanisms of action, including the ability of interacting with DNA and

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topoisomerase inhibition [13-16]. Recent studies in our research group have shown that
thiophene-thiosemicarbazone and acridine-thiosemicarbazone derivatives are promising
for antitumor activity, presenting the ability to interfere in the cell cycle and to interact
with DNA [12,14]. Additionally, the indole nucleus has also been disclosed as a
privileged structure in the design of new molecules [17-19].
Continuing our studies with thiosemicarbazone derivatives, this study aimed to
synthesize a new series of thiosemicarbazone and 4-thiazolidinones exploring the indole
heterocycle. The antiproliferative activity of the new series was evaluated in a panel of
eight human tumor cell lines and the most active derivative (compound 2b) was selected
for evaluation of cell death induction, cell cycle profile, DNA interaction and
topoisomerase inhibition.
2 Results and Discussion
2.1 Synthesis of thiosemicarbazone and 4-thiazolidinones derivatives
The synthesis of thiosemicarbazone (2a-e) and 4-thiazolidinones (3a-d)
derivatives was performed in three steps (Scheme 1). First, a nucleophilic addition
reaction between hydrazine and substituted isothiocyanates afforded
a
thiosemicarbazide. Then, the synthesized thiosemicarbazides reacted with indole-3-
carboxaldehyde, in the presence of a catalytic amount of AcOH, via condensation
reaction, leading the thiosemicarbazones [12]. Afterwards, the obtained
thiosemicarbazone derivatives were treated with ethyl-2-chloroacetate and sodium
acetate affording the respective N-3-substituted-4-thiazolidinones [20, 21]. The yields
ranged from 32 to 95%
PLEASE INSERT SCHEME 1 HERE
After crystallization, the thiosemicarbazone (2a-e) and 4-thiazolidinones (3a-d)
structures were determined by 1H and 13C NMR, IR and high-resolution mass.
In general, the IR spectrum of thiosemicarbazones 2a-e displayed stretching of
C=N and C=S bonds around 1537-1547 and 1203-1243cm−1, respectively. Thus, the
predominance of the thione form of the synthesized thiosemicarbazones was confirmed
through the absence of absorption in the 2500-2600 cm-1 region [22, 23]. For the 4-
thiazolidinones 3a-d, the IR spectra showed bands at 1398–1362 cm−1 characteristic of

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NCS bending vibration, providing evidence of ring closure [24, 25]. Finally, the IR
spectra of 3a-d displayed C=O absorbance in the 1701-1722 cm−1 region [26].
1
All the compounds synthesized showed in H NMR signals at δ 7.88-7.17 ppm
corresponding to protons related to a phenyl group besides a singlet between δ 7.32 and
8.47 ppm attributed to azomethine group (H–C=N), which is in accordance with the
reports involving thiosemicarbazones [27, 28]. Moreover, the protons of 4-
thiazolidinone ring (S-CH2) occurred between δ 4.07 and 4.41 ppm [29]. The N–H
aromatic (N4) proton signals appeared at δ 9.50-9.99 ppm region, except for 2d and 2e.
The presence of a spacer group in N4 of thiosemicarbazone 2d and 2e promoted an
electron-releasing inductive effect shifting to downfield region (δ 7.79 and 7.87 ppm,
respectively) the N–H aromatic (N4) proton signals. Finally, the other signals related to
thiosemicarbazone, such as N–H hydrazine proton presented chemical shifts at δ 11.49-
11.60 ppm region and N–H indole ring showed a singlet peak in the range of δ 11.22
and 11.83 ppm.
13
Similarly, in the C NMR spectra, the chemical shift of azomethine group (H–
C=N) appeared at region δ 138.0-153.8 ppm while C=S group occurred in the range δ
174.6-179.8 ppm, both comparable to the literature [30, 31]. The carbon of carbonyl
group of 4-thiazolidinone ring occurred between 171.9 and 172.3 δ ppm beside signals
in δ 20.64 - 44.9 ppm region attributed to methylene, methyl and spacer groups. In
addition, HRMS analysis confirmed the identity of all the synthesized compounds.
The relative configuration (E or Z) of thiosemicarbazone depends on the
aldehyde substituent [32]. For the 2a-e and 3a-d series, indole-carboxaldehyde was
used. In the 1H NMR spectrum analysis, it was possible to detect a single peak, referring
to a chemical shift of 7.32 to 8.47 δ ppm for the singlet azomethine hydrogen.
Furthermore, it was also observed in our experiments the appearance of a single spot in
thin layer chromatography (TLC), indicating the presence of a single isomeric form.
In this study, compound 2b, the most promising compound in the series, was
chosen for elucidation of isomerism of the derivatives synthetized. Thus, in 2D NMR
NOESY spectrum, it was observed the spatial correlation between the hydrogen of =N-
NH- at δ 11.68 ppm and azomethine (-CH=N-) at δ 8.49 ppm, as well as no spatial
interaction between the hydrogen in position 2 of the indole (-CH=C-) at δ 7.92 ppm
and the hydrogen azomethine (-CH=N-) at δ 8.47 ppm m (See supplementary material).
These results suggested the E configuration, which corroborates the crystallographic

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studies of X-ray and NOESY spectrum of E-thiosemicarbazone presented in the
literature [12, 33].
2.1 Evaluation of the antiproliferative activity in human tumor cell lines
Considering that different cell lines display different sensitivities toward the
same cytotoxic compound, the antiproliferative activity of thiosemicarbazone (2a-e) and
4-thiazolidinones (3a-d) were evaluated in vitro against eight different human cancer
cell lines [U251 (glioma), MCF-7 (breast), NCI-ADR/RES (multidrug resistant ovary
carcinoma), 786-0 (renal), NCI-H460 (non-small cell lung cancer), OVCAR-3 (ovary),
HT-29 (colon), and K-562 (leukemia)] according to the National Cancer Institute of
United States protocols (NCI-US) [34]. The antiproliferative activity of each compound
was also evaluated in vitro against spontaneously transformed keratinocytes from
histologically normal skin (HaCat cells). Doxorubicin was employed as the positive
control [35].
The antiproliferative activity was represented as the GI50 (concentration of
compound that inhibits 50% of cell growth, Table 1) and the mean values ranged
between 1.1 µM (compound 2b) and 84.65 µM (compound 3d). Based on this mean
values expressed as logarithm, Fouche et al. (2008) [36] described four levels of activity
named inactive (mean log GI50 > 1.50), weak activity (1.50 ≥ mean log GI50 > 1.10),
moderate activity (1.10 > mean log GI50 > 0) and potent activity (mean log GI50 < 0).
Considering these criteria, compounds 2e and 3a-d were considered inactive while 2a,
2c and 2d showed a moderate activity besides 2b that presented a potent
antiproliferative activity (Table 1).
These results suggest that transforming thiosemicarbazone 2a-d into N-3-
substituted-4-thiazolidinones 3a-d by a non-classical bioisosteric modification resulted
in lack of antiproliferative effects (Table 1). Between thiosemicarbazone derivatives, the
absence of bromine in the indole ring associated to the 1-naphthyl group connected to
N4 (2b) resulted in better antiproliferative activity (mean log GI50 = -0.69, potent
activity) with selectivity against colorectal adenocarcinoma (HT-29) and leukemia
(K562) cell lines (GI50 = 0.01 µM, for both cell lines). The substitution of the naphthyl
group (2b) for the p-toluyl (2c) or 2-phenethyl (2e) resulted in moderate (mean log GI50
= 0.39) to inactive (mean log GI50 > 1.47) results, respectively (Table 1).
The strategy for the design of derivatives in this study was based on previous
studies by our research group [12,14]. The best compounds evaluated by Oliveira et al.

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(2015) and Almeida et al. (2015) presented mean log GI50 of 0.75 and 0.88,
respectively, characterizing these compounds as being of moderate activity. Our
proposal for molecular modification of thiophene and acridine heterocycles by the
heteroaromatic nucleus of indole increased substantially antiproliferative activity (mean
log GI50 = -0.69).
The introduction of an aliphatic spacer group between N4-thiosemicarbazone and
the phenyl ring, as found in 2d and 2e, did not exert a positive effect on the cytostatic
activity. Oliveira et al. (2015) [12] described that this kind of substitution did not
improve the cytostatic activity of 2-thiophene-thiosemicarbazone derivatives. Moreover,
the presence of a bromine in the indole ring (2d) improved the antiproliferative activity
of the 2-phenethyl thiosemicarbazone derivatives. Thus, while 2e was inactive, 2d
showed a moderate antiproliferative activity (Table 1).
Therefore, the presence of an aromatic group at N4 seemed to be the main factor
for the biological activity displayed by this series of compounds (2a-e) while the
substitution in the indole nucleus of thiosemicarbazones afforded a secondary
contribution.
In order to simulate the behavior of compounds against normal cells, the HaCat
cell line (immortalized human keratinocyte) was inserted into the evaluation panel.
Inactive compounds (2e, 3b, 3c and 3d) were shown to be non-cytotoxic. On the other
hand, the most potent compound of the series (2b), also presented cytostatic effect in
this lineage (GI50 = 0.50 µM). However, the results presented in vitro cytotoxicity do not
necessarily have repercussions on in vivo toxicity assays. Oliveira et al. (2015) [12]
chose, in their antiproliferative in vitro study, a potent compound against tumor cells
that was also cytotoxic in the HaCat lineage. Subsequently, the acute toxicity of this
compound was evaluated, in which no severe clinical side effects were observed.
PLEASE INSERT TABLE 1 HERE
2.2 Cell Cycle and Cell Death Analyses of Compound 2b
Based on antiproliferative activity (Table 1), compound 2b was chosen for
analyses of cell cycle and cell death on colorectal adenocarcinoma cell line (HT-29,
GI50 = 0.01 µM, Table 1). In the first set of experiments, the induction of
phosphatidylserine (PS) exposure was evaluated by the ability of annexin-V to bind PS

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residues translocated to the outer face of the cell membrane during the early stage of
cell death, while 7-AAD binds to the DNA only after loss of cell membrane integrity.
After 24h, compound 2b reduced HT29 cells viability (unstained cells) in a
concentration-dependent manner and increased double stained cell subpopulations (PS
residues exposition along with lack of cell membrane integrity) and 7-AAD stained
(only lack of cell membrane integrity) in a similar profile. Only at the lowest
concentration (5 µM), 2b increased (12.6 ± 1.6 %) the cell subpopulation with PS
residues exposition without lack of membrane integrity (Figure 1). These data suggested
that compound 2b induces cell death in a concentration-dependent way, possibly
through externalizing phosphatidylserine residues prior to membrane integrity lack.
PLEASE INSERT FIGURE 1 HERE
Furthermore, 2b (5, 10 and 30 µM) induced G2/M arrest, more pronouncedly in
smaller concentrations, followed by a decrease in G1- and S-phase populations (Table
2). These results were similar to those obtained for colchicine (1.25 nM, positive
control), a natural product that binds to tubulin leading microtubule destabilization and
prevents mitosis progress [37]. Moreover, the induction of G2/M arrest by microtubule-
targeting agents can be followed by induction of programmed cell death process [38].
Consequently, the promotion of cell cycle arrest in G2/M phase by 2b (Table 2) can
induce programmed cell death process initialized by PS residues externalization (Figure
1).
PLEASE INSERT TABLE 2 HERE
One of the features of cancer cells is presenting sustained proliferative capacity,
which favors the propagation of mutations and contributes to genetic instability, one of
the characteristics of tumorigenesis [39]. Thus, therapies that interfere and limit this
capacity are desirable, to which compound 2b has proved to be a promising alternative.
2.3 DNA binding properties of compound 2b
Indole derivative biological activities have been associated to DNA binding
power and correlated to apoptosis of cancer cells since this type of death can be

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attributed to the alteration of nucleic acid structure, function or stability [40]. Moreover,
interaction between indole compounds and specific regions of nucleic acid can
modulate several key cell processes as DNA replication [41].
Aiming to understand the means by which 2b exerts G2/M arrest on HT-29
cells, the mode of interaction of 2b with DNA was evaluated. Generally, small organic
compounds can interact to DNA by intercalation, DNA-groove binding or by attractive
electrostatic interactions [42]. Monitoring UV-Visible absorption in Tris-HCl buffer (10
mM, pH 7.6), 2b showed maximum absorption peak at 334 nm and calf thymus DNA
(ctDNA) did not demonstrate absorption at the same λ, when they were analyzed
separately. Titration of increasing amounts of ctDNA into 2b solution led to increased
absorption (hyperchromic effect), presenting hyperchromicity of 13 % at 100 µM of
ctDNA. Both hyperchromic and hypochromic effects are characteristic of
conformational alterations of the DNA double helix after compound binding [43].
Generally, hypochromism combined with a redshift is considered an indicative of
intercalation of small molecules to the DNA due to the stacking of the chromophore
pairs [3, 14, 44]. Besides, the hyperchromic effect suggests changes on DNA
conformation and structure (denaturation) [4, 45]. Therefore, the UV-Visible absorption
result suggested that 2b promoted changes on DNA conformation.
Using the model proposed by McGhee and Von Hippel (1974) [46] the binding
constant value (Kb) of 2b was estimated in 4.3 x 104 M-1 (Table 3). Binding constant
values in the range of 104 are typical of complexes formed between DNA and organic
dyes [42], indicating moderate binding to DNA [47].
PLEASE INSERT TABLE 3 HERE
The positive influence of hydrogen donor groups on ctDNA binding affinity is
observed in several studies [48-50]. Here, the presence of NH groups in the hydrazine-
carbothioamide portion of 2b could contribute to ctDNA binding ability. Janardan et al.
(2014) [51] have synthesized hydrazine and semicarbazide derivatives with DNA
binding properties confirmed by UV-Vis and IR techniques. For both intercalation and
groove bind, DNA and organic compound interactions occur by hydrogens and Van der
Waals bonds [52]. Compounds bearing typical aromatic rings such as pyrrole, furan or
benzene as lateral chain with torsional freedom, can bind to the DNA groove producing
little or no structural rearrangement of the DNA helix, unlike intercalators [4, 53].

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Almeida et al (2015) [14] showed that derivative 2-(acridin-9-ylmethylene)-N-
(naphtalen-1-yl)-hydrazinecarbothioamide intercalates DNA with a binding constant
value of 8.47 x 105 M-1. The presence of the naphthlen ring and hydrazine-
carbothioamide group strengthens their interaction with DNA. In addition, acridine ring
is the structural difference compared to derivative 2b and confirms the importance of
planarity of the aromatic ring in the increased affinity to DNA [54].
Moreover, fluorescence spectroscopy was used to confirm the interaction
between 2b and ctDNA, since fluorescence quenching studies can give some
information on the binding of small molecules to biomacromolecules on the molecular
level [55, 56]. Fluorescence spectra of 2b (15 µM; λexcitation 335 nm; λemission 435 nm)
were monitored in the absence and presence of increasing amounts of ctDNA (0-100
µM) demonstrating that increases in ctDNA concentration promoted decrease on 2b
fluorescence emission (Figure 2). Suppression in fluorescence intensity in the presence
of DNA can be observed when groove binding agents [57, 58], electrostatic [59],
hydrogen bonding [60] or hydrophobic interactions are involved [61].
Quantitative analysis of fluorescence quenching was determined from the plot of
the relative intensity of fluorescence versus the concentration of ctDNA, and the
fluorescence suppression constant (Ksv) was obtained using the Stern-Volmer equation
[62]. Derivative 2b presented a Ksv value of 1.19 x 103 M-1, indicating the interaction
with ctDNA [3]. Emission-quenching phenomena reflect the interaction between the
derivative and ctDNA, consistent with the electronic absorption spectroscopy results
[63]. Both Ksv and Kb constants of the derivative 2b indicate static quenching due to
complex formation between the new derivative and ctDNA. Moreover, the reduction of
fluorescence emission by derivative 2b was accompanied by an apparent change in the
maximum wavelength of fluorescence emission, with a shift of 8 nm that is indicative of
a physical interaction with ctDNA [64]. These DNA-binding results could explain the
cell cycle arrest and the topoisomerase-DNA interface interaction that were evaluated
by topo IIα inhibition assay and that can be related to apoptosis pathway.
PLEASE INSERT FIGURE 2 HERE
2.4 Topoisomerase IIα assay
Topoisomerase (Topo) inhibitors are molecules that impair the enzymatic
activity by forming of a ternary complex (DNA-Topo-compound) or by catalytic

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inhibition, resulting in cell death by apoptosis [9, 65, 66]. Some antitumor drugs already
used in clinical practice have topoisomerase as biological target, such as doxorubicin
and daunorubicin (anthracycline group), etoposide and teniposide (epipodophyllotoxins
group), amsacrine (acridines group) and some thiosemicarbazones [67, 68]. However,
these drugs present many side effects, which prompts the development of new
topoisomerase inhibitors [69].
Among the mammalian topoisomerases, Topo IIα is preferentially expressed in
proliferating cells consisting in an important target for the discovery of new antitumor
drugs [8]. Compound 2b was evaluated in the DNA plasmid relaxation assay. This
assay consisted in the evaluation of topo IIα enzymatic activity in the absence and
presence of the compound followed by electrophoresis analysis [70]. As demonstrated
in Figure 3, the circular plasmid pUC19 can exist in three different topological
conformations: supercoiled circular DNA, nicked circular DNA and linear DNA, all of
which have identical sequences, but migrate at different rates through agarose gel.
Owing to their compact nature, supercoiled DNA topoisomers migrate faster through
agarose in comparison to linear DNA, nicked circular DNA, or relaxed DNA. When
topo IIα is active, the plasmid migrates as a single band and much more slowly. On the
other hand, inhibition of topo IIα results in a similar band pattern compared to plasmid
DNA (first lane). The activity of 2b as topo II inhibitor was visualized by the formation
of topoisomers for both tested concentration, but the inhibition was partial when
compared to the positive control used (m-AMSA).
PLEASE INSERT FIGURE 3 HERE
The topo II inhibition probably corroborates other mechanisms of action
identified in this work, such as cell cycle arrest and phosphatidylserine residues
exposition to the external plasma membrane surface. The formation of DNA
topoisomers indicates that the cleavage complex was stabilized which could be due to
alkylation of the thiol residues in the DNA-topo II complex by the thiosemicarbazone
moiety [71].
Indole derivatives are also known for their inhibitory activity against topo II.
Some indenoindolone-thiosemicarbazones were synthesized and evaluated for their
potential in inhibiting topo II. Kashyap et al. (2013) in their study evaluated the capacity
of inhibition of topoisomerase of indeneindolones. It was found that two derivatives of

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the series could inhibit the enzyme activity, without DNA intercalation [72]. In this
way, this pharmacophore may also have contributed to the biological action of
derivative 2b.
It is worthwhile to highlight that there is a relationship between inhibition of
topo II and cell cycle arrest. Recent studies show that the cell cycle arrest observed in
cancer cells treated with topo II poisons is primarily in the G2/M phase [73]. These data
corroborate our results, where the compound 2b could cause arrest at the G2/M phase of
the cell cycle. This suggests indole-thiosemicarbazone derivative, in particular
compound 2b, as candidate for anticancer therapy.
3 Conclusions
This study showed the antiproliferative activity of thiosemicarbazones and 4-
thiazolidinones indole-based derivatives, with emphasis on derivative 2b that presents
important chemical characteristics such as the presence of the indole, thiosemicarbazone
and naphthalene moieties. As far as our results have suggest, 2b can interact with DNA
and inhibit topo IIα that probably could explain PS residues externalization and G2/M
arrest in HT-29 cells highlighting as a promising candidate for use in anticancer therapy.
4 Experimental Section
4.1 Chemistry
All reagents used in this study are commercially available (Sigma-Aldrich,
Acros Organics, Vetec). The melting points were determined on Quimis 340 (Quimis,
Brazil) apparatus and are uncorrected. IR spectra were measured on Bruker IFS-66 IR
spectrophotometer (Bruker, Germany) using KBr pellets. NMR spectra were recorded
on Varian UnityPlus spectrometer 400 MHz (400 MHz for 1H and 100 MHz for 13C) or
a Bruker AMX-300 MHz (300 MHz for 1H and 75.5 MHz for 13C) instruments by using
tetramethylsilane as an internal standard. DMSO-d6 was purchased from Sigma-Aldrich.
HRMS were performed on a MALDI-TOF Autoflex III (Bruker Daltonics, Billerica,
MA, USA). The chemical shifts were reported in δ units, and coupling constants (J)
were reported in Hertz (Hz). The multiplicities were given as s (singlet), d (doublet), t
(triplet), m (multiplet), dd (double doublet). TLC development was conducted on 0.25
mm silica gel plates (Merck, silica gel 60 F254 in aluminium foil).
4.1.2 General procedure for compounds 2a-e

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Substituted thiosemicarbazide (1 mmol) and 3-5 drops of acetic acid were added
to a solution of indole-3-carboxaldehyde (1 mmol) in ethanol (10 mL). The reaction was
processed under magnetic stirring for 2h at room temperature. The precipitate was
filtered off, washed with ethanol then dried in desiccator under vacuum. Additional
amount of desired compound could be recovered from the filtrate after cooling. After
drying, the product was recrystallized from ethanol [12].
4.1.2.1 (E)-2-(5-bromo-1H-indol-3-ylmethylene)-N-(naphthalen-1yl)hydrazinecarbothi-
oamide (2a)
White powder; MP: 205-207°C; Yield: 77%; Rf: 0.54 (CHCl3/MeOH 9.5:0.5) NMR 1H
(300 MHz, DMSO) δ: 7.33 (m, 1H, indole), 7.41 (s, 1H, indole), 8.46 (s, 2H, indole),
8.00 (s, 1H, =CH-), 8.00-7.43 (m, 7H, naphtyl), 9.99 (s, 1H, NH), 11.69 (s, 1H, NH),
13
11.88 (s, 1H, NH). NMR C (75 MHz, DMSO) δ: 110.73, 113.38, 113.86, 122.69,
123.88, 125.31, 125.31, 125.68, 125.68, 125.99, 126.26, 126.42, 128.09, 130.13,
132.53, 133.65, 135.42, 135.80, 140.98, 176.21. IR (KBr, cm−1): 3320.50 (NH),
3200.00 (NH), 3164.90 (NH), 750.00 (CS). HRMS m/z [M + H]+ calcd for
C20H15BrN4S: 422.0201; found: 423.3289.
4.1.2.2
(2b)
(E)-2-(1H-indol-3ylmethylene)-N-(naphthalen-1-yl)hydrazinecarbothioamide
1
White powder; MP: 185-187°C; Yield: 95%; Rf: 0.44 (CHCl3/MeOH 9:1). NMR H
(300 MHz, DMSO) δ: 7.09 (t, 1H, J= 7.5Hz, indole), 7.20 (t, 1H, J= 7.5Hz, indole), 7.46
(d, 1H, J= 8.1Hz, naphtyl), 7.63-7.51 (m, 3H, naphtyl), 7.69 (d, 1H, J= 7.2Hz, indole),
7.89 (d, 1H, J= 8.2Hz, napthyl), 7.93 (d, 1H, J= 2.7Hz, indole), 8.03-7.95 (m, 2H,
naphtyl), 8.37 (d, 1H, J= 7.9 Hz, indole), 8.51 (s, 1H, =CH-), 9.87 (s, 1H, NH), 11.69 (s,
2H, NH). NMR 13C (75 MHz, DMSO) δ: 111.07, 111.75, 120.55, 122.17, 122.64,
122.92, 124.01, 125.40, 125.88, 125.97, 126.08, 126.51, 128.06, 130.38, 131.38,
133.68, 135.70, 137.06, 141.34, 176.22. IR (KBr, cm−1): 3319.51 (NH), 3158.5 (NH),
773.75 (CS) HRMS m/z [M + H]+ calcd for C20H16N4S: 344.1096; found: 344.4328.
4.1.2.3 (E)-2-(1H-indol-3-ylmethylene)-N-(p-toluyl)hydrazinecarbothioamide (2c)
1
White powder; MP: 195-197°C; Yield: 32%; Rf: 0.55 (CHCl3/MeOH 9:1). NMR H
(300 MHz, DMSO) δ: 3.43 (s, 3H, CH3), 7.17 (d, 2H, J= 8.1Hz, fenil), 7.48 (d, 2H, J=
8.4Hz, phenyl), 7.23-7.11 (m, 2H, indole), 7.44 (d, 1H, J= 7.5Hz, indole), 7.88 (d, 1H,

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J= 2.7Hz, indole), 8.20 (d, 1H, J= 7.5Hz, indole), 8.39 (s, 1H, =CH-), 9.50 (s, 1H, NH),
13
11.49 (s, 1H, NH), 11.66 (s, 1H, NH). NMR C (DMSO, 75MHz) δ: 20.64, 111.03,
111.98, 120.79, 121.83, 122.77, 124.13, 125.19, 125.19, 128.70, 128.70, 131.29,
134.26, 136.74, 137.11, 141.23, 174.62. IR (KBr, cm−1): 3409.4 (NH), 3314.1 (NH),
3164.9 (NH), 744.1 (CS). HRMS m/z [M + H]+ calcd for C17H16N4S: 308.1096; found:
308.4007.
4.1.2.4
(E)-2-((5-bromo-1H-indol-3-yl)methylene)-N-phenethylhydrazinecarbothio-
amide (2d)
1
White powder; MP: 220-222°C; Yield: 86%; Rf: 0.46 (CHCl3/MeOH 9:1). NMR H
(300 MHz, DMSO) δ: 2.38 (t, 2H, J= 6.9Hz, CH2), 3.82 (m, 2H, J= 5.7Hz, CH2), 7.21-
7.17 (m, 1H, indole), 7.43-7.40 (m, 1H, indole), 7.98 (d, 1H, J= 2.4Hz, indole), 8.27 (d,
1H, J= 3.9Hz, indole), 7.35-7.21(m, 5H, phenyl), 7.87 (d, 1H, J= 5.4Hz, NH), 8.25 (s,
1H, =CH-), 11.25 (d, 1H, J= 3Hz, NH), 11.81 (s, 1H, NH). NMR 13C (75 MHz, DMSO)
δ: 34.82, 44.98, 110.75, 113.34, 113.89, 123.67, 125.31, 125.57, 126.18, 128.44,
128.44, 128.56, 128.56, 132.07, 135.76, 139.26, 140.13, 176.09 (CS). IR (KBr, cm−1):
3420.25 (NH), 3365.13 (NH), 3200.00 (NH), 1737.84 (CS). HRMS m/z [M + H]+ calcd
for C18H17BrN4S: 400.0357; found: 401.3234.
4.1.2.5 (E)-2-((1H-indol-3-yl)methylene)-N-phenethylhydrazinecarbothioamide (2e)
1
White powder; MP: 197-199°C; Yield: 73%; Rf: 0.51 (CHCl3/MeOH 9:1). NMR H
(300 MHz, DMSO) δ: 2.95 (t, 2H, J= 7.8Hz, CH2), 3.85 (quart., 2H, J= 7.2Hz, CH2),
7.15-7.10 (m, 1H, indole), 7.42 (d, 1H, J= 8.1Hz, indole), 7.39-7.18 (m, 5H, phenyl),
7.79 (d, 1H, J= 2.7Hz, NH), 7.84 (d, 1H, indole, J=7.8Hz), 7.98 (d, 1H, indole,
J=5.4Hz), 8.28 (s, 1H, =CH), 11.22 (s, 1H, NH), 11.60 (s, 1H, NH). NMR 13C (75 MHz,
DMSO) δ: 34.88, 44.71, 111.03, 111.76, 120.57, 121.83, 122.60, 123.82, 126.20,
128.47, 128.47, 128.57, 128.57, 131.06, 137.04, 139.27, 140.68, 175.84 (CS). IR (KBr,
cm−1): 3365.00 (NH), 3256.37 (NH), 3200.00 (NH), 746.08 (CS). HRMS m/z [M + H]+
calcd for C18H18N4S: 322.1252; found: 322.4273.
4.1.3 General procedure for compounds 3a-d
A solution of 1 mmol of thiosemicarbazones, 1.1 mmol of ethyl-2-chloroacetate,
and 4 mmol of sodium acetate anhydrous in 20 mL of ethanol was stirred until reflux to
the completion of the reaction (16-24 h). After, the solution was cooled to 0º C, the

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precipitate was collected with filter under vacuum and washed with hot methanol and
water [20,21].
4.1.3.1 (E)-2-(E)-(5-bromo-1H-indol-3ylmethylene)hydrazono)-3-(naphthalen-1
yl)thiazolidin-4-one (3a)
1
White powder; MP: 280-282°C; Yield: 35%; Rf: 0.58 (CHCl3/MeOH 9:1). NMR H
(300 MHz, DMSO) δ: 4.41-4.33 (m, 2H, CH2), 7.32 (m, 1H, indole), 7.35 (m, 1H,
indole), 8.24 (m, 1H, indole), 8.40 (m, 1H, indole), 7.42 (m, 1H, naphtyl), 7.76-7.57 (m,
5H, naphtyl), 8.07 (m, 1H, naphtyl), 8.07 (s, 1H, =CH-), 11.79 (s, 1H, NH). IR (KBr,
cm−1): 3345.7 (NH), 3053.8 (NH), 2977.5 (NH), 1707.48 (CO). HRMS m/z [M + H]+
calcd for C22H15BrN4OS: 462.0150; found: 463.3497.
4.1.3.2 (E)-2-(E)-(1H-indol-3-ylmethylene)hydrazono)-3-(naphthalen-1-yl)thiazolidin-
4-one (3b)
White powder; MP: 265-267°C; Yield: 45%; Rf: 0.59 (CHCl3/MeOH 9.5:0.5). NMR 1H
(300 MHz, DMSO) δ: 4.35 (d, 1H, J= 17.1Hz, CH2), 4.19 (d, 1H, J= 16.2Hz, CH2), 7.20
(m, 2H, indole), 8.08 (d, 1H, J= 8.1Hz, indole), 8.23 (d, 1H, J= 8.4Hz, indole), 7.44 (m,
1H, naphtyl), 7.69-7.58 (m, 5H, naphtyl), 7.79-7.76 (m, 1H, naphtyl), 8.27 (s, 1H,
=CH), 11.62 (s, 1H, NH). NMR 13C (75 MHz, DMSO) δ: 32.40, 111.75, 111.98,
120.79, 121.95, 122.46, 122.69, 124.42, 125.83, 126.52, 127.13, 127.13, 128.34,
129.37, 129.37, 132.01, 132.01, 133.91, 137.10, 153.89, 161.51, 172.32 (CO). IR (KBr,
cm−1): 3394.93 (NH), 1613.64 (CO). HRMS m/z [M + H]+ calcd for C22H16N4OS:
384.1045; found: 384.4536.
4.1.3.3 (E)-2-(E)-(5-bromo-1H-indol-3-ylmethylene)hydrazono)-3-(p-toluyl)thiazolidin-
4-one (3c)
1
White powder; MP: 295°C; Yield: 32%; Rf: 0.56 (CHCl3/MeOH 9.5:0.5). NMR H
(300 MHz, DMSO) δ: 2.37 (s, 3H, CH3), 4.11 (s, 2H, CH2), 7.28 (d, 2H, J= 8.7Hz,
phenyl), 7.43 (d, 2H, J= 8.7Hz, phenyl), 7.35-7.24 (d, 2H, indole), 8.40 (m, 2H, indole),
7.82 (s, 1H, =CH-), 11.83 (s, 1H, NH). NMR 13C (75 MHz, DMSO) δ: 111.42, 113.45,
114.01, 125.17, 127.93, 127.93, 127.93, 127.93, 129.49, 129.49, 129.49, 129.49,
132.58, 133.10, 135.78, 135.78, 138.05, 153.22, 171.98 (CO). IR (KBr, cm−1): 3255.75
(NH), 1608.29 (CO). HRMS m/z [M + H]+ calcd for C19H15BrN4OS: 426.0150; found:
427.3176.

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4.1.3.4 (E)-2-(E)-(1H-indol-3ylmethylene)hydrazono)-3-(p-toluyl)thiazolidin-4-one (3d)
White powder; MP: 274-276°C; Yield: 72%; Rf: 0.57 (CHCl3/MeOH 9.5:0.5). NMR 1H
(300 MHz, DMSO) δ: 2.38 (s, 3H, CH3), 4.07 (s, 2H, CH2), 7.25 (d, 2H, J= 8.4Hz,
phenyl), 7.32 (d, 2H, J= 8.4Hz, phenyl), 7.21-7.14 (m, 1H, indole), 7.45 (d, 1H, J=
6.6Hz, indole), 7.77 (d, 1H, J= 2.4Hz, indole), 8.22 (d, 1H, J= 6.6Hz, indole), 8.42 (s,
13
1H, =CH-), 11.64 (s, 1H, NH). NMR C (DMSO, 75MHz) δ: 32.14, 120.77, 122.69,
124.47, 127.96, 127.96, 127.96, 127.96, 129.49, 129.49, 129.49, 129.49, 132.01,
132.64, 137.11, 138.02, 153.70, 154.49, 172.00 (CO). IR (KBr, cm−1): 3405.71 (NH),
1722.24 (CO). HRMS m/z [M + H]+ calcd for C19H16N4OS: 348.1045; found: 348.4215.
4.2 Biological Assay
4.2.1 Cell culture conditions
Eight different human cancer cell lines [U251 (glioma), MCF-7 (breast), NCI-
ADR/RES (multidrug resistant ovary carcinoma), 786-0 (renal), NCI-H460 (non-small
cell lung cancer), OVCAR-3 (ovary), HT-29 (colon), and K-562 (leukemia)] were
kindly provided by National Cancer Institute at Frederick (NCI-USA). The
immortalized human keratinocyte (HaCaT) cell line was kindly provided by prof. Dr.
Ricardo Della Coletta (University of Campinas). All cell lines were maintained in RPMI
1640 (Gibco, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco) and
1% (v/v) penicillin:streptomycin (Nutricell, 1000 U/mL:1000 g/mL) in a humidified
atmosphere with 5% CO2, at 37°C. For the experiments, HaCaT and CHO-K1 were
used between passages 5 to 12.
4.2.2 Samples preparations
Compounds 2a-e and 3a-d (5 mg) were initially diluted in DMSO (50 µl)
followed by the addition of 950 µL of RPMI 1640/FBS 5% (working solution). Final
concentrations (0.25, 2.5, 25 and 250 µg/mL, for antiproliferative assay; 5, 10, 20, 30
and 50 µM, for flow cytometry experiments of 2b) were obtained by serial dilution in
RPMI 1640/FBS 5%. Doxorubicine (0.025, 0.25, 2.5 and 25 µg/mL, final
concentration) and Colchicine (1.25nM, final concentration) were used as positive
controls on antiproliferative and cell cycle assays, respectively.
4.2.3 Antiproliferative assay

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The in vitro antiproliferative activity assay was performed as described by
Monks et al. (1991) [74]. Each cell line was plated in 96-well plates (100 µL cells/well)
for 24h and then exposed to the derivatives 2a-e and 3a-d in different concentrations at
37º C, 5% of CO2 in air for 48 hours. Final DMSO concentration (< 0,25%) did not
affect cell viability. Before (T0 plate) and after (T1 plates) sample addition, cells were
fixed with 50% trichloroacetic acid and cell proliferation determined by
spectrophotometric quantification (540 nm) of cellular protein content using
sulforhodamine B assay. The GI50 (concentration expressed in µM inhibiting 50% of
cell growth or cytostatic effect) were determined through non-linear regression analysis
using the concentration-response curve for each cell line in software ORIGIN 8.0®
(OriginLab Corporation) [74, 75].
4.2.4 Cell treatments for Cytometry assays:
For the flow cytometry experiments, HT-29 (human colorectal adenocarcinoma,
epithelial, adherent) cells were seeded in 6 well plates (2 x 105 cells/well) and incubated
for 24 h prior to treatments.
4.2.5 Measurement of phosphatidylserine externalization
Phosphatidylserine externalization was analysed with Guava Nexin Assay Kit
(Guava Technologies, Hayward, CA) in accordance with manufacturer’s instructions.
After 24 h, HT-29 cells were treated with 2b (5, 20, 30 and 50 µM) for 24 h, harvested
(Trypsin-EDTA 0.25%) and resuspended at a density of 1 x 105 cells in 100 µL of
phosphate buffered saline (PBS). The binding buffer containing annexin-V-PE and 7-
AAD was added to the cells (100 µL/sample) and the suspension was incubated in the
dark for 20 min at room temperature. After that, cells were analyzed (5000
events/replicate) by flow cytometry (Guava Easycyte Mini – Guava Technologies,
Hayward, CA).
4.2.6 Cell Cycle analyses
Cell cycle analyses were performed with the Guava Cell Cycle reagent (Guava
Technologies, Hayward, CA) in accordance with manufacturer’s instructions. After the
first 24h, HT-29 cells were deprived of FBS for 24 h (cell cycle synchronization). After
that, cells were treated with 2b (5, 10 and 30 µM) and Colchicine (1.25 nM) for 24 h,
harvested (Trypsin-EDTA 0.25%) and resuspended at a density of 1 x 105 cells in 100

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µL of PBS. The binding buffer containing propidium iodide (PI) was added to the cells
(100 µL) and suspension was incubated in the dark for 20 min at room temperature.
Finally, cells were analyzed (5000 events/replicate) by flow cytometry (Guava Easycyte
Mini).
4.2.7 DNA interaction
4.2.7.1 Preparation of the ct-DNA solution
Calf thymus DNA (ctDNA) was purchased from Sigma and was used without
further purification. The solution of ctDNA in Tris-HCl (0,01 M, pH 7.6) buffer was
sonicated for 5 min and the DNA concentration was determined using the molar
extinction coefficients 6600 M-1 cm-1 at 260 nm [76]. The purity of DNA was
determined by monitoring the rate value of A260/A280. DNA concentration was
expressed as micromolar equivalents of the base pairs.
4.2.7.2 UV-vis absorption measurements
The derivative 2b was dissolved in DMSO at a concentration of 1 mM (stock
solution) from which the working solutions were prepared by dilution using Tris-HCl
(0.01 M, pH 7.6) buffer at concentrations ranging from 10 to 80 µM.
Under optimized concentration, the compounds were titrated with increasing
concentrations of ctDNA. Before the measurements, the system was stirred and
incubated at room temperature for 10 minutes. The measurements were performed in a
rectangular quartz cuvette with a path length of 1cm.
The intrinsic binding constant (Kb) was obtained by fitting the data to equation 1
[46]:
[DNA]/(εa – εf) = [DNA]/(εb – εf) + 1 / Kb (εb – εf)
(1)
where, εa; εb and εf are the apparent, bound, and free extinction coefficients,
respectively. Plot fitting of [DNA]/(εa – εf) vs. [DNA] used Kb obtained from the ratio
of the slope to the Y intercept. The binding data were fitted using a SigmaPlot 10.0
software.
4.2.7.3 Fluorescence Measurements
Fluorescence measurements of non-bound derivative were performed with 15
µM of compound 2b in 0.01 M Tris buffer, pH 7.6. Emission spectra were recorded in
the region 370-700 nm using an excitation wavelength of 220-400 nm. All

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measurements were performed at 25 °C in a rectangular quartz cuvette with a 1 cm path
length. Fluorescence intensities were expressed in arbitrary units. Fluorescence
titrations were conducted by the addition of increasing amounts of ctDNA directly into
the cell containing the derivatives.
The behavior suppression emission was analyzed by the Stern-Volmer equation
was obtained through the equation (2) [62]:
F0 / F = 1 + KSV [Q]
(2)
where, F0 and F are the fluorescence intensities in the absence and presence of indoles
derivatives. KSV is the constant linear suppression. [Q] is the concentration of
derivatives. The plot of relative emission intensity (F0/F) versus [Q] was used to obtain
the constant from the linear regression. The binding data were fitted using a SigmaPlot
10.0 software.
4.2.8 In vitro topoisomerase II inhibition assay
Human topoisomerase IIα inhibition activity was determined as previously
described by Almeida et al. (2016) [70]. Briefly, 100 ng pUC19 DNA plasmid (from
Sigma) and 4.0 unit of recombinant human (p170) topoisomerase II (Sigma-Aldrich) in
relaxation buffer without or with 2b or amsacrine (m-AMSA) were incubated for 30 min
at 37º C. The tested concentrations were 50 and 100 µM. Reactions were terminated
with 1% SDS and digested with proteinase K. Then, the reaction products were
analyzed by electrophoresis gel stained with Ethidium Bromide and photographed under
UV light.
4.2.9 Statistical analyses
The results were expressed as the mean ± standard error and by analysis of
variance (ANOVA), one-way followed by Tukey tests. P values lower than 0.05
(p<0.05) were considered as indicative of significance and represented by: *p<0.05,
**p<0.01 and ***p<0.001. The calculations were performed using the statistical
software GraphPad Prism version 7.0, San Diego California, USA.
Acknowledgements
This study was supported by Brazilian agencies Fundação de Amparo Pesquisa
do Estado de Pernambuco (FACEPE, Brazil) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq).

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Conflict of Interest
The authors declare that there was no competing interests.
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Table 1. In vitro antiproliferative activity (GI50a in µM) of thiosemicarbazones and 4-thiazolidinones based-indole derivatives
2a
2b
2c
2d
2e
3a
3b
3c
3d
Cell Lines
R = 1-naphtyl
R1 = Br
7.32
R = 1-naphtyl
R1 = H
0.92
R = p-toluyl
R1 = H
8.11
R = 2-phenethyl R = 2-phenethyl R = 1-naphtyl
R = 1-naphtyl
R1 = H
72.52
R = p-toluyl
R1 = Br
54.71
59.52
64.58
39.85
96.60
98.50
>100
R = p-toluyl
R1 = H
90.16
>100
Dox
R1 = Br
7.49
R1 = H
77.90
15.54
38.74
76.29
>100
>100
>100
0.17
R1 = Br
48.81
6.06
0.14
0.07
0.77
0.11
0.09
0.22
0.23
0.93
-0.68 P
0.20
U251
0.95
0.25
2.52
6.81
34.88
MCF 7
6.18
6.48
1.46
7.14
12.41
29.09
>100
5.12
38.57
>100
NCI-ADR/RES
786-0
6.56
0.25
3.99
7.58
62.08
79.14
>100
6.99
0.38
11.72
3.88
8.48
>100
NCI-H460
OVCAR-3
HT29
1.60
0.21
8.22
25.74
>100
6.56
0.01
1.45
7.87
27.34
>100
>1.40 I
6.23
96.86
>100
5.29
0.01
0.16
2.12
>100
0.01
>100
K562
0.63 M
6.15
-0.69 P
0.59
0.39 M
0.99
0.81 M
7.85
>1.47 I
>100
>1.77 I
62.98
>1.36 I
54.00
>1.98 I
>100
mean log GI50
HaCat
a GI50 = concentration (µM) that inhibits cell growth by 50% after 48 h exposition; GI50 was determined from nonlinear regression analyses by using ORIGIN 8.0 software
(OriginLab Corporation). Experiments conducted in triplicate. Dox: doxorubicine, positive control.
Classification criteria: inactive (I, mean log GI50 > 1.50), weak activity (W, 1.50 > log GI50 > 1.10), moderate activity (M, 1.10 > mean log GI50 > 0) and potent activity (P,
mean log GI50 < 0) (Fouche et al., 2008).

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Table 2. Influence of 2b on HT29 cells cycle after 24h-exposition
Treatments
Sub-G1
3.5 ± 0.7
3.5 ± 0.6
2.0 ± 0.5
1.6 ± 0.3
4.8 ± 0.8
G1
S
G2/M
Vehicle
46.1 ± 2.1
17.2 ± 1.2
5.9 ± 1.3***
6.8 ± 1.4***
5.5 ± 0.4**
7.3 ± 0.8***
32.5 ± 2.3
Colchicine 1.25 nM
5 µM
14.6 ± 1.8***
10.7 ± 0.8***
26.7 ± 1.3***
34.9 ± 1.4***
76.0 ± 1.7***
80.5 ± 2.4***
66.2 ± 1.2***
53.1 ± 1.3***
10 µM
30 µM
2b
Distribution of cells in the different phases of cell cycle, after 24 h of treatment with (DMSO 0.1%), Colchicine (1.25 nM) and derivative 2b (5, 10 and 30
µM). Mean (%) ± standard error of two independent experiments in triplicate. A total of 5000 events were collected per experiment. ANOVA followed by
Tukey’s Multiple Comparison Test. **p < 0.01, ***p < 0.001, statistically different from vehicle.
Table 3. UV-Vis absorption and fluorescence emission data of derivative 2b in the absence and presence of ctDNA.
λmax
Extinction
Coefficient (ε) M-1
Hyperchr.a Hyperchr.a λ excitation λ emission
13% 335 nm 435 nm
4.3 x 104
Ksv M-1
Free
Bound
334 nm
Compound
2b
334 nm
18.440
1.19 x 103
aHyperchromicity for complexes formed by compound 2b and 100 µM of ctDNA in comparison to free ligands.