Synthesis of thiophene-thiosemicarbazone derivatives and evaluation of their in vitro and in vivo antitumor activities

Article abstract of DOI:10.1016/j.ejmech.2015.09.036

A series of thiophene-2-thiosemicarbazones derivatives (5-14) was synthesized, characterized and evaluated for their antitumor activity. They were tested in vitro against human tumor cell lines through the colorimetric method. The results revealed that compounds 7 and 9 were the most effective in inhibiting 50% of the cell growth after 48 h of treatment. As compound 7 showed a potent antiproliferative profile, it has been chosen for further studies in 786-0 cell line by flow cytometry. Treatments with compound 7 (50 μM) induced early phosphatidylserine exposure after 18 h of exposure and this process progressed phosphatidylserine exposure with loss of cell membrane integrity after 24 h of treatment, suggesting a time-dependent cell death process. Regarding the cell cycle profile, no changes were observed after treatment with compound 7 (25 μM), suggesting a mechanism of cell death independent on the cell cycle. The in vivo studies show that compound 7 possess low acute toxicity, being the doses of 30-300 mgKg^-1 chosen for studies in Ehrlich solid tumor model in mice. All doses were able to inhibit tumor development being the lowest one the most effective. Our findings highlight thiophene-2-thiosemicarbazones as a promising class of compounds for further studies concerning new anticancer therapies.

Full text of DOI:10.1016/j.ejmech.2015.09.036

Accepted Manuscript
Synthesis of thiophene-thiosemicarbazone derivatives and evaluation of their in vitro
and in vivo antitumor activities
Jamerson Ferreira de Oliveira, Anekécia Lauro da Silva, Débora Barbosa
Vendramini-Costa, Cezar Augusto da Cruz Amorim, Júlia Furtado Campos, Amélia
Galdino Ribeiro, Ricardo Olímpio de Moura, Jorge Luiz Neves, Ana Lúcia Tasca Gois
Ruiz, João Ernesto de Carvalho, Maria do Carmo Alves de Lima
PII:
S0223-5234(15)30280-4
10.1016/j.ejmech.2015.09.036
EJMECH 8133
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 August 2015
Revised Date: 26 September 2015
Accepted Date: 29 September 2015
Please cite this article as: J.F. de Oliveira, A.L. da Silva, D.B. Vendramini-Costa, C.A. da Cruz Amorim,
J.F. Campos, A.G. Ribeiro, R. Olímpio de Moura, J.L. Neves, A.L. Tasca Gois Ruiz, J. Ernesto de
Carvalho, M. do Carmo Alves de Lima, Synthesis of thiophene-thiosemicarbazone derivatives and
evaluation of their in vitro and in vivo antitumor activities, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.09.036.
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Graphical abstract

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Synthesis of thiophene-thiosemicarbazone derivatives and evaluation
of their in vitro and in vivo antitumor activities
Jamerson Ferreira de Oliveiraa, Anekécia Lauro da Silvaa, Débora Barbosa Vendramini-
Costab, Cezar Augusto da Cruz Amorima, Júlia Furtado Camposc, Amélia Galdino
Ribeiroa, Ricardo Olímpio de Mourad, Jorge Luiz Nevese, Ana Lúcia Tasca Gois Ruizb,
João Ernesto de Carvalhob, Maria do Carmo Alves de Limaa,*
aUniversidade Federal de Pernambuco (UFPE), Departamento de Antibióticos, 50670-
901, Recife, PE (Brazil)
bUniversidade Estadual de Campinas (Unicamp), Divisão de Farmacologia e
Toxicologia - Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas
(CPQBA) 13083-970, Campinas, SP (Brazil)
cInstituto Nacional de Tecnologia Nordeste/MCT, Centro de Tecnologias Estratégicas
do Nordeste, 50740-540 - Recife, PE (Brazil)
dUniversidade Estadual da Paraíba (UEPB), Departamento de Farmácia, 58429-500,
Paraíba, PB (Brazil)
eUniversidade Federal de Pernambuco (UFPE), Departamento de Química
Fundamental, 50670-901, Recife, PE (Brazil)
To whom correspondence should be addressed: Maria do Carmo Alves de Lima
*Email: nenalima.mariadocarmo@gmail.com
Phone number: +558121268347
Fax: +558121268346
Keywords: antitumor, cytotoxicity, medicinal chemistry, thiosemicarbazone, thiophene.

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Abstract
A series of thiophene-2-thiosemicarbazones derivatives (5-14) was synthesized,
characterized and evaluated for their antitumor activity. They were tested in vitro
against human tumor cell lines through the colorimetric method. The results revealed
that compounds 7 and 9 were the most effective in inhibiting 50% of the cell growth
after 48 hours of treatment. As compound 7 showed a potent antiproliferative profile, it
has been chosen for further studies in 786-0 cell line by flow cytometry. Treatments
with compound 7 (50 µM) induced early phosphatidylserine exposure after 18 hours of
exposure and this process progressed phosphatidylserine exposure with loss of cell
membrane integrity after 24 hours of treatment, suggesting a time-dependent cell death
process. Regarding the cell cycle profile, no changes were observed after treatment with
compound 7 (25 µM), suggesting a mechanism of cell death independent on the cell
cycle. The in vivo studies show that compound 7 possess low acute toxicity, being the
doses of 30-300 mgKg-1 chosen for studies in Ehrlich solid tumor model in mice. All
doses were able to inhibit tumor development being the lowest one the most effective.
Our findings highlight thiophene-2-thiosemicarbazones as a promising class of
compounds for further studies concerning new anticancer therapies.

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1. Introduction
Cancer is a leading cause of death worldwide, responsible for 8.2 million deaths
in 2012. These numbers can be linked to population growth, the process of
industrialization and changes in habit life [1-2]. In general, cancer treatment is based on
radiotherapy, surgical removal, hormone therapy, immunotherapy and chemotherapy,
alone or combined, aiming to increase the patients survival [3].
The design of new compounds prototypes based on medicinal chemistry
followed by the biological approach has been a good strategy for the discovery of new
therapies [4]. Molecular hybridization is a strategy for drug design widely used in
medicinal chemistry and aims at the junction of two chemical moieties into a single
compound, targeting an increase in biological activity [5]. The presence of heterocyclic
systems prototype molecules is of great interest in design of the new drugs. Features as
small molecular weight, presence of heteroatoms and aromatic structures can mimic
endogenous substrates, which may lead to the development of a biological activity [6].
Raltitrexed (Tomudex®) (Figure 1) is a quinazoline folate analogue that has in its
structure the thiophene heterocycle, which is also found in many promising antitumor
compounds [6-9]. The mechanism of action of this drug is the selective inhibition of the
enzyme thymidylate synthase, which leads to DNA fragmentation and subsequent cell
death [10]. Raltitrexed is indicated for the treatment of colorectal cancer (CRC) been
considered a promising alternative therapy when combined with 5-fluorouracil (5-FU)
and capecitabine (Figure 1). This treatment schedule is better acceptance by patients
since it results in lower clinical complications, such as thrombosis and cardiotoxicity,
than the use of 5-FU and capecitabine alone [11-13].
PLEASE, INSERT FIGURE 1 HERE
Recent studies have been aimed to the discovery of new drugs for cancer
treatment. The thiosemicarbazones comprise a class of molecules known for their
diverse biological activities such as antimicrobial [14], antitumor [15-17],
antileishmanial [18] and antiplasmodic [19] properties. Among the thiosemicarbazones,
triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone, 3-AP) is the most
promising molecule undergoing clinical phase II studies [20]. The probable mechanism

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of action of this drug is the inhibition of the enzyme ribonucleotide reductase [21], but
there is also evidence of topoisomerase II inhibition [22].
Thus, this study aimed to describe the production of novel thiophene-2-
thiosemicarbazones through molecular hybridization strategy between the thiophene
core of raltitrexed and the thiosemicarbazone of triapine properly synthesized and
characterized. The derivatives were evaluated for their antiproliferative activity in a
panel of human tumor cell lines. The most promising molecule (compound 7) was
chosen for further studies concerning cell death and cell cycle profile by flow
cytometry, focusing on the human renal adenocarcinoma cell line 786-0. Finally, we
also describe the in vivo antitumor activity of 7 in the murine model of Ehrlich solid
tumor in mice.

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2. Results and Discussion
2.1 Chemistry
2-thiophene-thiosemicarbazone derivatives (5-14) were prepared in two steps.
Thiosemicarbazide was obtained via nucleophilic addition reaction between hydrazine
and substituted isothiocyanates. The synthesized thiosemicarbazides reacted with 2-
thiophene-carboxaldehyde, in presence of a catalytic amount of AcOH, via
condensation reaction, leading to the formation of compounds of interest [23]. Good
reaction yields were observed using this methodology (40-87%) (Scheme 1). Some
compounds evaluated in this study were previously synthesized (compounds 5-8) [24].
However, such compounds have not been investigated against its antitumor potential.
Thus, both compounds previously synthesized (5-8) as the novel compounds (9-14)
were assessed for their pharmacological potential.
PLEASE, INSERT SCHEME1 HERE
1
The structures were determined by H and 13C NMR, DEPT, IR and high-
resolution mass. Analyzes of these data were compatible with the proposed compounds.
The IR spectra showed characteristic stretches of connections C=S, C=N and NH.
Additionally, the absence of bands between 2700 cm-1 and 2500 cm-1 excluded the
possibility of a thione-thiol tautomerism (e.g., H-N-C=S, -N=C-SH) [25].
As exemplified with the 1H NMR analysis of N-(p-tolyl)-2-(thiophen-2-
ylmethylene)-thiosemicarbazone (9), the singlet peak δ 2.50 corresponded to the para
methyl group at phenyl ring. The phenyl protons occurred as doublets peak at 7.15 and
7.42 ppm. The protons of thiophene group occurred at δ 7.13, 7.52 and 7.69. Singlet
peak at δ 8.33 confirms the success of the reaction between N-(p-tolyl)-
thiosemicarbazone and 2-thiophene-carboxaldehyde, relative to iminic proton (H-C=N).
In addition, signals related to N-H the presented chemical shifts δ 9.70 and 11.76 ppm.
The IR spectrum showed stretches on the C=N and C=S at 1593 and 1557 cm-1,
respectively, and the absorption band of N-H at 3297 cm-1. The predominance of the
thione form of the synthesized thiosemicarbazones may be observed in the chemical
13
shift of carbon of the thiocarbonyl C NMR at δ 175.5. Quaternary carbon peaks were
confirmed by DEPT experiments to appear at δ 134.3, 136.3, 138.3 and 175.5. In a

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manner complementary, HRMS confirmed the identity of all the synthesized
compounds.
All target compounds 5-14 could exist in either the E or Z isomeric form due to
the imino bond (-CH=N-). However, in 1H NMR spectra, the –N=CH- signal was seen
as a single peak for each compound indicating that only one isomer was generated. The
1
hydrogen attached to carbon of imine presented chemical shift in H NMR spectra δ
8.24-8.40 for all target compounds. X-ray crystallography and NOESY studies confirm
the preference of the E diastereoisomer [26,27].
Compound 10 was chosen for elucidation of isomerism of the derivatives
obtained in this study. Many NOESY signals were observed for this compound. By on
side, NOESY signals of the studied molecule were employed to assign and corroborate
the obtained structure. By the other side, portion of the NOESY spectra reveals the
interactions between H-C=N (at 8.33 ppm), N-H (at 9.70 ppm) and H-C=C (at 7.53
ppm) moieties. The close proximity between this group of protons suggests (E)-isomer
as main product. Additionally, no signal relative to (Z)-isomer of the analyzed structure
was detected in the NOESY spectrum (See supplementary material).
As reported in the literature, chemical shift values around 11 ppm (9-12 ppm)
were correlated to the E isomers, whereas higher ppm values were reported for the Z
forms (over 12 ppm) [28]. The most important signal in the process of describing the
1
correct stereoisomer is that connected with NH group (CS)NH-proton [29]. The H
NMR of all synthesized compounds shows a singular peak around 11.50-11.97 ppm
relative to the NH signal which indicates E isomer was generated.
2.2 Biological activities
2.2.1 In vitro studies
The antiproliferative effects of the thiophene-2-thiosemicarbazones derivatives
on nine human tumor cell lines and one non-tumor cell line are summarized in Table 1,
using doxorubicin as an positive control. The results were expressed as concentration
required to inhibit in 50% the cell growth, named as GI50 in µM (Table 1). Moreover,
the average activity was expressed as mean GI50 that was the arithmetical average of all
GI50 values obtained for the human tumor cell lines. All ten derivatives evaluated have

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the thiophene core in their structure, with modifications present in the distal portion of
the compounds.
Considering the average activity (mean GI50), three compounds (6, 7 and 9)
exhibited values lower than 50 µM, while six compounds (5, 8, 10, 12, 13 and 14)
showed mean GI50 between 50 - 100 µM, been 11 inactive (mean GI50 >100 µM).
Compound 5 represents the basic skeleton of this series of derivatives, with no
replacements in the phenyl portion. This compound did not exert antiproliferative effect
in most tumor cell lines evaluated been effective against K562 (leukemia cell line, GI50
= 16.7 µM) (Table 1).
The introduction of a halogen into phenyl group (6 and 7, mean GI50 < 50 µM)
resulted in an important increase in cytostatic activity. The derivative with a p-bromo-
phenyl moiety (7) stood out among the other compounds, being active against all cell
lines excepting leukemia (K-562), with GI50 values ranging from 0.74 to 79.4 µM.
Besides, the p-chlorophenyl analogue (6) was active against K-562, NCI-ADR/RES
(multiresitant ovarian cells), 786-0 (renal cell adenocarcinoma) and U251 (glioma) with
GI50 values of 2.0, 2.8, 7.4 and 8.3 µM, respectively (Table 1).
Moreover, the results observed for 9 and 10 seems to suggest that the size of the
alkyl group introduced in the aromatic group affect greatly the cytostatic effective of a
thiophene-2-thiosemicarbazones derivatives. This way, when a C2 chain (p-ethylphenyl,
10) was attached to the base molecule, this resulted in lower inhibitory effect than that
observed for 5 for almost cell lines, while a p-methylphenyl derivative (9) afforded an
increment in cytostatic activity (Table 1). Also, it was interesting to notice that both 9
(p-methylphenyl) and 10 (p-ethylphenyl) showed a high selectivity to K-562 (leukemia,
GI50 <0.9 and 1.6. µM, respectively), suggesting that the presence of an alkyl group
introduced in the aromatic group affect increase the cytostatic effective against
leukemia (Table 1).
Besides, when an ethyl group was introduced as a spacer group (compound 11,
2-phenyl-ethyl derivative, a structural isomer of 10 (p-phenyl-ethyl) even the cytostatic
activity against leukemia was not observed resulting in an inactive thiosemicarbazone
derivative (mean GI50 >100 µM). These results suggest that large aliphatic carbon
chains do not favor the antiproliferative activity. Also, the introduction of a
hydroxyalkyl chain in the aromatic group (8, p-methoxyphenyl) might be considered
unfavorable, since 8 was inactive for most of cell lines (mean GI50 < 89 µM) except for
leukemia (GI50 = 1.7 µM, Table 1).

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The substitution of phenyl ring for a 1-naphthyl ring (12) or for a 3-pyridyl
group (13) resulted in an increased antiproliferative effect (mean GI50 > 59.5 and > 65.9
µM, respectively) compared to 6 (mean GI50 > 81.8 µM) mainly due the increment on
activity against U251 (glioma), 786-0 (renal adenocarcinoma) for both 12 and 13
besides against NCI-ADR/RES (multidrug resistant ovarian adenocarcinoma) and K562
(leukemia) for 12 and NCI-H460 (no-small lung adenocarcinoma) for 13. Finally, the
structural modification presented by compound 14 (allyl), certainly did not favor an
increase in the antiproliferative activity. Removing the phenyl ring and the insertion of
an unsaturated aliphatic chain in this class of compounds significantly reduced their
activity in the tumor cell lines used in this experiment (Table 1).
The non-tumor cell line HaCaT (immortalized human keratinocyte) was inserted
into the panel in order to simulate normal human cells since immortalized cells share
with tumor cells the characteristic of rapid mitosis of tumor cells without the presence
of oncogenes. Considering the obtained results, the most potent derivatives 2-thiophene-
thiosemicarbazones 6, 7 and 9, also showed cytostatic effect against non-tumor cell line
(Table 1).
The thiosemicarbazones are known for their antiproliferative activity, and our
results corroborate with previous studies that show a cytostatic profile for the cell line
K-562 (leukemia) [30]. The N-acylhydrazones are analogous of thiosemicarbazone
compounds. One of the common points between these two classes of compounds is the
presence of the C=N bond. Recent studies show the importance of N- acylhydrazones
for the antiproliferative activity against cell lines, as we can observe in our study for the
compounds that possess the substitution p-methylphenyl and p-bromophenyl [31].
In summary, it can be suggested that the inclusion of aromatic rings in the
structure of thiosemicarbazones improves the antiproliferative activity of this class of
compounds. Furthermore, the presence of halogens directly connected to this core also
has a large contribution to the increase in this activity. However, the presence of alkyl
groups either directly connected to the thiosemicarbazone or attached to an aromatic
ring skeleton does not appear to increase the effect on cell growth, unless the alkyl
moiety is a short chain. With these preliminary results, it was possible to assume that
these thiosemicarbazones derivatives show a remarkable antiproliferative activity.
PLEASE, INSERT TABLE 1 HERE

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In view of the potency of 7 towards almost all cell lines (Table 1), this
compound was selected for further studies against the human kidney tumor cell line
786-0, which is an epithelial adenocarcinoma that produces the parathyroid hormone
(PTH) like peptide as breast and lung tumors [32]. First, the proliferation of 786-0 cells
were determined after short time exposition (24 h) and a narrow concentration range
(25, 50, 100 and 200 µM) of compound 8 in order to stablish better experimental
condition for flow cytometric evaluations (phosphatidylserine exposition and cell
cycle). This way, after 24h, the necessary concentration to promote total growth
inhibition and 50% of cell death were 28 and 91.8 µM, respectively, thus an
intermediary concentration (50 µM) was selected for further experiments.
One feature of some cell death subroutines, like apoptosis, parthantos and
netosis [33], is the early exposure of phosphatidylserine (PS) on external cell membrane
surface. This PS externalization signalizes for recognition and engulfment of dying
cells, as viable cell membranes exhibits substantial phospholipid asymmetry, with most
of the PS residing on the inner leaflet of the plasma membrane [34]. For assessment of
PS exposure, we used the double staining with Annexin V and 7-amino-actinomycin D
(7-AAD). Annexin V binds to PS translocated to the outer face of the cell membrane
during the initial process of cell death while 7-AAD binds to the DNA only after loss of
cell membrane integrity. This way, cells stained only with annexin-V-PE could be
considered as in early stage of cell death subroutine.
After 24h of treatment, compound 7 (50 µM) increased 786-0 cell population
with PS externalization (21.9 ± 2.4 %) and with PS externalization and loss of cell
membrane integrity (35.7 ± 2.4%) in comparison to vehicle-treated cells (3.8 ± 0.5%
and 3.6 ± 0.8%, respectively; p<0.001) (Figure 2). These results are in accordance with
the antiproliferative activity assay, where after 24 hours of treatment, lethal
concentration for 50% of the cells was 91.8 µM, thus cell death rate of around 35% of
the cells was expected after a treatment with 50 µM of the compound. Moreover, the PS
externalization induced by compound 7 (50 µM) seemed to be a dynamic, time-
dependent process such as 18 hours of exposition resulted 15.5 ± 3.5% of 786-0 cells
with PS externalization without damage to cell membrane in comparison to 7.2 ± 1.4%
of vehicle-treated 786-0 cells with the same markup (p<0.05) (Figure 2).
PLEASE, INSERT FIGURE 2 HERE

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For flow cytometer cell cycle evaluation, 786-0 cells were treated for 24 hours
with a lower concentration of 7 (25 µM) in order to minimize a sub-G1 phase arrest.
Colchicine (1.25 nM) was employed a positive control, as it promotes a cell cycle arrest
on G2/M phase. The cell cycle distribution of treated 780-6 cells is presented in Table 2.
As expected, colchicine (1.25 nM) increased the population of 786-0 cells in
G2/M phase (57.5 ± 3.3%) followed by a decreasing on G1 phase cell population (21.5
± 2.2%) in comparison to vehicle-treated cells (Table 2). At these experimental
conditions, no interference on cell cycle profile was observed for compound 7 at 25 ꢀM
(Table 2).
PLEASE, INSERT TABLE 2 HERE
Several studies have been conducted in order to understand the mechanism by
which thiosemicarbazones act on tumor cells [35,36]. For example, a copper complex of
thiosemicarbazone was evaluated for antitumor activity and it was able to induce PS
exposition without loss of cell membrane integrity in a concentration-dependent way in
the HepG2 (human hepatocellular carcinoma) cell line [37]. Another study showed that
anthraquinone-thiosemicarbazone derivatives (AQ-thiosemicarbazone) were able to
induce apoptosis in HeLa cell line (human cervix adenocarcinoma) in a caspase-
dependent pathway, mainly through the mitochondrial pathway activation of caspase 8
[38]. These data are consistent with our findings, as compound 7 also led to PS
exposition without loss of cell membrane integrity in a time-dependent way.
The complex network of cell death subroutines and cell cycle are closely linked,
as many of cancer cells treatment with anticancer agents usually result in cell cycle
arrest, which subsequently leads the cells to initiate a cell death subroutine [39]. The
cell cycle analysis by flow cytometry enables to quantify the amount of cells in the
different cell cycle phases and thereby infer a possible interference of the compounds
evaluated on DNA replication [40]. Although compound 7 promotes cell death in 786-0
as evidenced by PS exposition, no interference on cell cycle was observed in our study.
There are several studies showing molecules that are also cytotoxic for non-
tumor cells, by inducing cell death through a mechanism other then inhibition of DNA
duplication, synthesis, cell cycle check points or inhibition of mitosis, as would be
observed in the cell cycle experiment by flow cytometry. An example is Goniothalamin,

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a styryl lactone with well-known antitumor activity in vitro and in vivo that also does
not promote cell cycle arrest [41].
2.2.2 In vivo studies
Owing to the promising in vitro antiproliferative activity of 7, we evaluated its in
vivo antitumor activity in the Ehrlich solid tumor model in mice. In order to choose the
properly doses to perform the antitumor studies in mice, the acute toxicity of compound
7 was evaluated.
2.2.2.1 Oral Acute Toxicity
Animals orally treated with compound 7 (200 and 400 mgKg-1) showed no
severe clinical side effects with no mortality (Table 3). Moreover, the single treatment
with 600 mgKg-1 of compound 7 promoted adhesion of organs. Based on these results,
the in vivo anticancer assay was conducted with 30, 100 and 300 mgKg-1of compound
7.
PLEASE, INSERT TABLE 3 HERE
2.2.2.2 In vivo antitumor activity
In this experiment, Ehrlich tumor cells were injected in the paw subcutaneous of
the mice and treatments began 3 days after the cells inoculation. The tumor
development was accompanied by measures of paw volume using a pletismometer at
time 0 (basal volume) and each three days until the end of the experiment (Table 4)
[43], Treatment with compound 7, at 30 mgKg-1, was effective since the 6th day of
experiment, when inhibited tumor development in 30% in comparison to negative
control group. At the same day, positive controls doxorubicin (3 mgKg-1) and piroxicam
(40 mgKg-1) showed an inhibition of 35.8 and 34% respectively (Table 4). At the 9th
day, all treatments presented smaller tumor volumes than negative control group being
compound 7 (30 mgKg-1) and piroxicam (40 mgKg-1) the most potent treatments. From
the 12th day until the 15th day (end of experiment), all treatments presented statistical
differences with negative control, being treatments with 30 mgKg-1 of compound 7 the

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most effective, inhibiting in 71.1%, in comparison to negative control group, in the 12th
day, the tumor development (Table 4). However, the compound 7 at doses of 100 and
300 mg/Kg showed reductions of paw volume 39.6 and 49%, respectively, showing
good activity of the compound in the model adopted. The measurement of variation in
paw volume on Ehrlich tumor is a good indication of antitumor activity being well
correlated with the antiinflammatory effect of compound 7.
PLEASE, INSERT TABLE 4 HERE
The Ehrlich tumor is a murine breast adenocarcinoma, which presents an
aggressive fast-growing behavior [44]. Ehrlich tumor cells generate an inflammatory
response that leads to increase in vascular permeability, edema formation and cell
migration [45]. In fact, the paw volume increased in the range of 25 to 40% (in
comparison basal volume) in all experimental groups. Studies show that in the Ehrlich
tumor occurs upregulation of cyclooxygenase-2 (COX-2) and activation of nuclear
factor-kappa B (NF-κB), a transcription factor that regulates the expression of many
pro-inflammatory and pro-proliferative genes [46]. Thus, the use of non-steroidal anti-
inflammatory drugs (NSAID) as adjuvants on cancer therapy is a good approach, as
they target COX-2 and downregulate NF-κB pathway, leading to inhibition of tumor
proliferation [47]. Our results obtained for Piroxicam (NSAID) corroborate with these
facts as this NSAID was inhibited effectively the Ehrlich tumor development (Table 4).
It is well-known that inflammation promotes all steps of carcinogenesis [48].
The inflammatory process establishes a microenvironment which is favorable to tumor
growth and maintenance [49,50]. In fact, epidemiological data confirm the close
relationship between cancer and inflammation, up to 20% of all cancer types are
preceded by chronic inflammation [51].
Molecules presenting the thiophene moiety have antiinflammatory and
antitumoral properties. 3-[4-(methylsulfonyl)phenyl]thiophene-2-carbaldehyde O-
arylmethyl oximes derivatives are able to inhibit COX-2 activity [52] and as this moiety
is present on compound 7, it could be involved on the antiproliferative activity of this
molecule, suggesting also an anti-inflammatory activity.
In our study, treatments with compound 7 were conducted by oral route and cell
inoculation and tumor development were in the paw subcutaneous. The treatments
conducted every two days (7 treatments in total) did not promote clinical side effects.

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All these results highlight compound 7 as a promising molecule for further studies for
cancer chemotherapy, as the toxicity promoted by most of the drugs used on
chemotherapy is still a challenge to overcome.
3. Conclusions
The novel thiophene-2-thiosemicarbazone derivatives have been prepared and
shown cytostatic activity against several cancer cell lines. It can be suggested that the
inclusion of aromatic rings in the structure of thiosemicarbazones improves the
antiproliferative activity of this class of compounds and that the presence of halogens
directly connected to this core also has a large contribution to the increase in this
activity.
Compound 7 promotes PS externalization as a cell death signaling mechanism
on the human renal adenocarcinoma cell line 786-0 without changes on cell cycle
profile. Moreover, compound 7 possesses low acute toxicity, being able to inhibit
Ehrlich solid tumor development in low doses (30 mgKg-1) through orally
administration without evidence of toxic effects. Our findings highlight thiophene-2-
thiosemicarbazones as a promising class of compounds for further studies concerning
new anticancer therapies.
4. Experimental Section
4.1 Chemistry
All reagents used in this study are commercially available (Sigma-Aldrich,
Acros Organics, Vetec). Progress of the reactions was followed by thin-layer
chromatography (TLC) analysis (Merck, silica gel 60 F254 in aluminium foil). Melting
points were determined on a Quimis 340 (Quimis, Brazil) capillary melting point
apparatus and were uncorrected. IR spectra were recorded with a Bruker model IFS66
FT-IR spectrophotometer (Bruker, Germany) using KBr pellets. NMR spectra were
1
measured on either a Varian UnityPlus 400 MHz (400 MHz for H and 100 MHz for
13C) or a Bruker AMX-300 MHz (300 MHz for 1H and 75.5 MHz for 13C) instruments.
DMSO-d6 was purchased from Sigma-Aldrich. Chemical shifts are reported in ppm and
multiplicities are given as s (singlet), d (doublet), t (triplet), m (multiplet), dd (double

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doublet), and coupling constants (J) in hertz. Mass spectrometry experiments were
performed on a MALDI-TOF Autoflex III (Bruker Daltonics, Billerica, MA, USA).
4.1.1 General procedure for the synthesis of thiosemicarbazides (3)
80% hydrazine hydrate (1) (311 µL, 10 mmol) was added slowly to a solution of
different isothiocyanates (2) (5 mmol) in dichloromethane (10 mL). The reaction was
processed under magnetic stirring for 2 h at room temperature. The precipitate was
filtered off, washed with dichloromethane and 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 n-hexane.
4.1.2 General procedure for the synthesis of thiosemicarbazones (5-14)
Substituted thiosemicarbazide (3) (1 mmol) and few drops of acetic acid were
added to a solution of 2-thiophene-carboxaldehyde (4) (93 µL, 1 mmol) in ethanol (10
mL). The reaction was processed under magnetic stirring for 2 h 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 toluene.
4.1.2.1 (E)-N-(p-tolyl)-2-(thiophen-2-ylmethylene)-thiosemicarbazone (9)
Compound 9 was obtained as white powder (119 mg, 40%): Rf= 0.45 (n-Hex/AcOEt
1
8:2); mp: 193-194°C; H NMR (400 MHz, DMSO-d6): δ= 2.50 (s, 3H), 7.13 (t, J= 4.8
Hz, 1H), 7.15 (d, J= 8 Hz, 2H), 7.42 (d, J= 8.4 Hz, 2H), 7.52 (d, J= 3.6 Hz, 1H), 7.69
(d, J= 4.8 Hz, 1H), 8.33 (s, 1H), 9.70 (s, 1H), 11.76 ppm (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ= 20.5 (CH3), 125.3 (CH Ar), 127.9 (CH Ar), 128.5 (CH Ar), 129.1 (CH
Ar), 130.8 (CH Ar), 134.3 (Cq Ar), 136.3 (Cq Ar), 137.8 (C=N), 138.3 (Cq Ar), 175.5
(C=S) ppm; IR (KBr): v~= 1557 (C=S), 1593 (C=N), 3297 (N-H) cm-1; HRMS m/z
[M+H]+ calcd for C13H13N3S2: 276.058; found: 276.089.
4.1.2.2 (E)-N-(4-ethylphenyl)-2-(thiophen-2-ylmethylene)-thiosemicarbazone (10)
Compound 10 was obtained as white powder (162 mg, 55%): Rf= 0.46 (n-Hex/AcOEt
7:3); mp: 186-188°C; 1H NMR (400 MHz, DMSO-d6): δ= 1.19 (t, J= 7.6 Hz, 3H), 2.60

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(q, J= 7.6 Hz, 2H), 7.13 (t, J= 4.8 Hz, 1H), 7.18 (d, J= 8.4 Hz, 2H), 7.45 (d, J= 8 Hz,
2H), 7.53 (d, J= 4 Hz, 1H), 7.68 (d, J= 4.8 Hz, 1H), 8.33 (s, 1H), 9.70 (s, 1H), 11.76
13
ppm (s, 1H); C NMR (100 MHz, DMSO-d6): δ= 15.6 (CH3), 27.6 (CH2), 125.4 (CH
Ar), 127.3 (CH Ar), 127.9 (CH Ar), 129.1 (CH Ar), 130.8 (CH Ar), 136.5 (Cq Ar),
137.8 (C=N), 138.3 (Cq Ar), 140.7 (Cq Ar), 175.5 (C=S) ppm; IR (KBr): v~= 1521
(C=S), 1547 (C=N), 3296 (N-H) cm-1; HRMS m/z [M+H]+ calcd for C14H15N3S2:
290.074; found: 290.098.
4.1.2.3 (E)-N-phenethyl-2-(thiophen-2-ylmethylene)-thiosemicarbazone (11)
Compound 11 was obtained as white powder (240 mg, 81%): Rf= 0.56 (n-Hex/AcOEt
1
7:3); mp: 169-170°C; H NMR (400 MHz, DMSO-d6): δ= 2.90 (t, J= 8 Hz, 2H), 3.75
(q, J= 6 Hz, 2H), 7.11 (dd, J1= 3.6 Hz, J2= 4 Hz, 1H), 7.20-7.34 (m, 5H), 7.43 (d, J= 3.6
Hz, 1H), 7.66 (d, J= 5.2 Hz, 1H), 8.12 (t, J= 6 Hz, 1H), 8.24 (s, 1H), 11.50 ppm (s, 1H);
13C NMR (100 MHz, DMSO-d6): δ= 34.7 (CH2), 45.0 (CH2), 126.2, 127.9 (CH Ar),
128.4 (CH Ar), 128.5 (CH Ar), 128.7 (CH Ar), 130.5 (CH Ar), 137.3 (C=N), 138.6 (Cq
Ar), 139.2 (Cq Ar), 176.5 (C=S) ppm; IR (KBr): v~= 1523 (C=S), 1546 (C=N), 3353
(N-H) cm-1; HRMS m/z [M+H]+ calcd for C14H15N3S2: 290.074; found: 290.083.
4.1.2.4 (E)-N-(naphthalen-1-yl)-2-(thiophen-2-ylmethylene)-thiosemicarbazone (12)
Compound 12 was obtained as white powder (411 mg, 80%): Rf= 0.59 (n-Hex/AcOEt
8:2); mp: 212-214°C; 1H NMR (300 MHz, DMSO-d6): δ= 7.14 (dd, J1= 5.4 Hz, J2= 5.1
Hz, 1H), 7.51-7.55 (m, 4H), 7.57 (d, J= 5.4 Hz, 1H), 7.79 (d, J= 5.1 Hz, 1H), 7.83-7.99
(m, 3H), 8.40 (s, 1H), 10.10 (s, 1H), 11.92 ppm (s,1H); 13C NMR (75 MHz, DMSO-d6):
δ= 123.5 (CH Ar), 125.8 (CH Ar), 126.4 (CH Ar), 127.2 (CH Ar), 128.4, 129.6, 131.2,
132.5, 135.9, 138.4 (C=N), 151.5 (Cq Ar), 167.6 (Cq Ar), 177.7 (C=S) ppm; IR (KBr):
v~= 1519 (C=S), 1543 (C=N), 3328 (N-H) cm-1; HRMS m/z [M+H]+ calcd for
C16H13N3S2: 312.058; found: 312.071.
4.1.2.5 (E)-N-(pyridin-3-yl)-2-(thiophen-2-ylmethylene)-thiosemicarbazone (13)
Compound 13 was obtained as white powder (233 mg, 79%): Rf= 0.40 (n-Hex/AcOEt
7:3); mp: 186-187°C; 1H NMR (300 MHz, DMSO-d6): δ= 7.14 (t, J= 3.6 Hz, 1H), 7.39
(m, 1H), 7.54 (d, J= 3.6 Hz, 1H), 7.71 (d, J= 3.9 Hz, 1H), 7.97 (m, 1H), 8.36 (s, 1H),
8.38 (m, 1H), 8.66 (s, 1H), 9.97 (s, 1H), 11.97 ppm (s,1H); 13C NMR (75 MHz, DMSO-
d6): δ= 122.8 (CH Ar), 127.9 (CH Ar), 129.4 (CH Ar), 131.2 (CH Ar), 133.3 (CH Ar),

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135.8 (CH Ar), 138.1 (CH Ar), 138.6 (C=N), 145.9 (Cq Ar), 147.1 Cq Ar), 176.1 (C=S)
ppm; IR (KBr): v~= 1519 (C=S), 1543 (C=N), 3295 (N-H) cm-1; HRMS m/z [M+H]+
calcd for C11H10N4S2: 263.038; found: 263.061.
4.1.2.6 (E)-N-allyl-2-(thiophen-2-ylmethylene)-thiosemicarbazone (14)
Compound 14 was obtained as white powder (223 mg, 65%): Rf= 0.50 (n-Hex/AcOEt
8:2); mp: 159-160°C; 1H NMR (400 MHz, DMSO-d6): δ= 4.20 (t, J= 5.6 Hz, 2H), 5.09
(d, J= 10.4 Hz, 1H), 5.14 (d, J= 17.2 Hz, 1H), 5.95-5.86 (m, 1H), 7.11 (m, 1H), 7.44 (d,
J= 3.6 Hz, 1H), 7.64 (d, J= 5.2 Hz, 1H), 8.23 (m, 1H), 8.26 (s, 1H), 11.51 ppm (s,1H);
13C NMR (100 MHz, DMSO-d6): δ= 45.6 (CH2), 115.5 (CH2), 127.8 (CH allyl), 128.7
(CH Ar), 130.5 (CH Ar), 134.9 (CH Ar), 137.4 (C=N), 138.5 (Cq Ar), 176.8 (C=S)
ppm; IR (KBr): v~= 1533 (C=S), 1553 (C=N), 3358 (N-H) cm-1; HRMS m/z [M+H]+
calcd for C9H11N3S2: 226.043; found: 226.070.
4.2 Biological activities
4.2.1 In vitro assays
Considering that different cell lines display different sensitivities toward the
same cytotoxic compound, the antiproliferative activity of all the 2-thiophene-
thiosemicarbazone were evaluated in vitro against nine different human cancer cell lines
[U251 (glioma), UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES (multidrug
resistant ovary carcinoma), 786-0 (renal), NCI-H460 (non-small cell lung cancer), PC-3
(prostate), HT-29 (colon), and K-562 (leukemia)]. 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 [53].
Cells in 96-well plates (100 µL cells/well) were exposed to 2-thiophene-
thiosemicarbazonics derivates in concentrations 0.25, 2.5, 25 and 250 µg/mL in
DMSO/RPMI at 37º C, 5% of CO2 in air for 48 hours. Doxorubicin was used as
standard (0.025, 0.25, 2.5 and 25 µg/mL). Final DMSO concentration did not affect cell
viability (0.1%). 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

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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)
[54-56]. To choose concentrations for flow cytometry analyses, 786-0 cells were treated
for 24 hours with 25, 50, 100 and 200 µM of compound 7. Cell growth was determined
through sulforhodamine B assay and concentration that promote total growth inhibition
(TGI) and lethal concentration 50 - LC50 (concentration that promote 50% of cell death)
were calculated in software ORIGIN 8.0® (OriginLab Corporation).
4.2.2 Measurement of phosphatidylserine externalization
Phosphatidylserine externalization was analysed by flow cytometry using
Guava® Nexin Assay Kit (Guava Technologies, Hayward, CA) in accordance with
manufacturer’s instructions. 786-0 cells were inoculated in 12 wells plate (3 x 104
cells/well) and incubated for 24 hours at 37 ºC, 5% of CO2 in air. Then, cells were
treated with compound 7 (50 µM) in DMSO/RPMI for 18 and 24 hours, harvested and
resuspended at a density of 1 x 105 cells in 100 µL of supplemented medium. One
hundred microliter of binding buffer containing annexin-V and 7-AAD were added on
the cells and incubated in the dark for 20 minutes at room temperature. After, cells were
analysed by flow cytometer (Guava Easycyte Mini - Guava Technologies, Hayward,
CA). We collected 5.000 events, as suggested by the protocols of Guava Easycyte kits.
To make it clear we added the following statement in the methods and figures legends:
"A total of 5.000 events were collected". We performed three experiments in triplicate,
as showed in the legends of figures and methodology. We performed statistical analyses
for all experiments and all the significance was showed as asterisks, according with p
value.
4.2.3 Cell cycle analyses
Cells cycle analyses were performed with the Guava®Cell Cycle reagent
(Guava Technologies, Hayward, CA) in accordance with manufacturer’s instructions.
780-0 cells were inoculated in 12 wells plate (3 x 104 cells/well) and incubated for 24
hours at 37 ºC, 5% of CO2 in air. Afterwards, cells were deprived of serum for 24 hours
for cell cycle synchronization and then treated with compound 7 (25 µM) and colchicine

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(Sigma-Aldrich, 1.25 nM) in DMSO/RPMI, for 24 hours. After treatment, cells were
harvested and resuspended at a density of 1 x 105 cells in 100 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 minutes at room temperature. After, cells were
analysed by flow cytometer (Guava Easycyte Mini - Guava Technologies, Hayward,
CA). We collected 5.000 events, as suggested by the protocols of Guava Easycyte kits.
To make it clear we added the following statement in the methods and figures legends:
"A total of 5.000 events were collected". We performed three experiments in triplicate,
as showed in the legends of figures and methodology. We performed statistical analyses
for all experiments and all the significance was showed as asterisks, according with p
value.
4.2.4 In vivo assays
4.2.4.1 Animals
Experiments were conducted with Balb/C mice (20 - 35 g) obtained from the
Multidisciplinary Center for Biological Investigation on Laboratory Animals Sciences
(CEMIB-UNICAMP). Animals were maintained at the Animal facilities of
Pharmacology and Toxicology Division, CPQBA, University of Campinas (Campinas,
Brazil), under controlled conditions (22 ± 3ºC for 12 h light/dark cycle, free access to
food and water). All mice were fasted for 16 hours prior to each experiment because
treatments were administered orally. Euthanasia was performed by cervical dislocation.
Animal care, research and animal sacrifice protocols were in accordance with the
principles and guidelines adopted by the Brazilian College of Animal Experimentation
(COBEA) and approved by the Institute of Biology/ UNICAMP - Ethical Committee
for Animal Research (number 3329-1).
4.2.4.2 Acute Oral Toxicity – Fixed Dose Procedure
A fixed dose procedure was adopted to evaluate the acute oral toxicity of
compound 7 according to Organization for Economic Cooperation and Development
(OECD) Guidelines for the Testing of Chemicals [42]. Groups of 5 mice were dosed in
a stepwise procedure using the fixed doses of 600, 400 and 200 mgKg-1. Clinical signs

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and conditions associated with pain, suffering and impending death are described in
detail in a separate OECD Guidance Document [57].
4.2.4.3 Ehrlich solid tumor in mice
The most active compound 7 was evaluated for its in vivo antiproliferative
activity in the Ehrlich solid carcinoma model in mice. The procedures were developed
according to Vendramini-Costa et al. (2010) [43] with few modifications on data
analysis.
Cell maintenance and preparations: The Ehrlich ascitic tumor (EAT), derived
from a spontaneous murine mammary adenocarcinoma, was maintained in the ascitic
form by weekly intraperitoneal transplantations of 5 x 105 tumor cells/animal, in order
to prepare cells for the following test. The ascitic fluid was removed by opening the
belly and carefully collecting all the fluid using a sterile 3 mL syringe. Ascitic tumor
cell counts were performed in a Neubauer hemocitometer, and the total number was
determined by the Trypan blue dye exclusion method, with tumor cell viability always
higher than 90%. The cells were then diluted in 0.9% phosphate buffer saline (PBS) into
final inoculation density (2.5 x 106 cells/30µL).
Ehrlich solid tumor: After cells inoculation, basal volumes of the right hind paw
were measured in a plethysmometer apparatus (7140 Ugo Basile). For the solid form
implantation, 2.5 x 106 viable tumor cells in a volume of 30 µL were injected in
subplantar site of the right hind paw of Balb-C mice (n= 8 per group) [58]. The
compound 7 was emulsified with 1% Tween 80 (SIGMA®) and dissolved in phosphate
buffered saline (PBS), pH 7,0. The test compound was administered on the 3th day after
tumor inoculation, at doses of 30, 100 and 300 mgKg-1 orally. Doxorubicin (3 mgKg-1,
i.p.) and piroxicam (40 mgKg-1, orally) were used as the standard drugs. After tumor
cell inoculation, the paw volume was measured every three days using the
plethysmometer apparatus till the 15th day, when the animals were sacrificed. The tumor
growth was measured considering the following formula:

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4.3 Statistical Analysis
The experimental results were expressed as the mean ± standard error (SE) and
by analysis of variance (ANOVA), one-way and two-way, followed by Tukey or
Bonferroni 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 5.0, San Diego
California, USA.
Acknowledgements
This study was supported by the Brazilian agencies Fundação de Amparo
Pesquisa do Estado de Pernambuco (FACEPE, Brazil) and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq).
Conflict of Interest
The authors have declared that no competing interests exist.
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[57] OECD Guidance Document on Acute Oral Toxicity Testing: 2002.
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ACCEPTED MANUSCRIPT
Table 1. In vitro antiproliferative activity (GI50a in µM) of N-substituted-2-(thiophene-2-ylmethylene)-thiosemicarbazone derivatives 5-14.
UACC-
62
NCI-
ADR/RES
NCI-
H460
Mean
GI50
Compound
U251
MCF-7
786-0
PC-3
HT-29
K-562
HaCat
74.7
>100
15.8
71.1
94.6
>100
2.8
50.4
>100
>100
4.7
>100
>100
16.7
>81.8
>48.4
>31.5
>100
5
6
7
8.3
4.2
>100
7.4
4.5
>100
79.4
>100
17.7
2.0
14.7
0.8
<0.74
1.7
>100
>100
12.6
>100
16.8
>100
38.6
>100
4.8
>100
19.5
82.6
>100
>100
80.6
>100
29.7
>100
>100
>100
>100
1.7
<0.9
1.6
>89.0
>33.7
>87.1
>100
>100
17.6
17.6
>100
8
9
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
10
11
>100
12.7
26
>100
>100
>100
>100
6.6
15.2
5.6
>100
45.9
>100
>100
>100
>100
1.8
>59.5
>65.9
82.4
45.4
12
>100
16.4
13
14
>100
>100
0,05
>100
>100
0,21
21.2
>100
>100
0,13
>100
0,30
6.0
>80.8
>100
<0,046
<0,046
<0,046
<0,046
<0,046
<0.102
<0,046
-
Dox

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aConcentration that promotes 50% of growth inhibition after 48 hours of treatment. GI50 was determined from nonlinear regression analyses by using ORIGIN 8.0 software
(OriginLab Corporation). Experiments were conducted in triplicate. Dox: doxorubicine, positive control.

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Table 2. Cell cycle profile of 786-0 cells treated with vehicle (DMSO), colchicine
(1.25 nM) and compound 7 (25 µM) for 24 hours.
Treatments
Sub-G1
3.0 ± 0.7
8.9 ± 0.8
5.4 ± 0.9
G1
S
G2/M
46.7 ± 0.8
21.5 ± 2.2***
44.9 ± 1.8
14.6 ± 1.3
12.6 ± 1.2
13.9 ± 0.7
35.7 ± 0.9
57.5 ± 3.3***
35.9 ± 0.6
Vehicle
Colchicine
Compound 7
Cell population, expressed in percentage, for each phase of cell cycle, after 24h-treatment with
vehicle, colchicine or compound 7. Results represented by mean ± standard error, in percentage of
cells, from three different experiments. A total of 5000 events were collected per experiment. ANOVA
followed by Tukey’s Multiple Comparison Test. ***p<0.001, statistically different from vehicle.
Table 3. Effects of compound 7 on acute oral toxicity assay–fixed dose procedure.
Dose (mgKg-1)
Survival rate and side effects
100% survival rate, no severe toxic effect*
100% survival rate, no severe toxic effect*
100% survival rate, adhesions of organs
200
400
600
* Clinical signs and conditions associated with pain, suffering, and impending death, are described
in detail in a separate OECD Guidance Document [42].
Table 4. Effect of 2-thiophene-thiosemicarbazone (compound 7) on Ehrlich solid tumor development in mice.
Day
Paw volume variation (%) as compared to day-0 (mean ± SD)
Doxorubicin
3 mgKg-1
Piroxicam
40 mgKg-1
Compound 7
30 mgKg-1
Compound 7
100 mgKg-1
Compound 7
300 mgKg-1
Control
40.2 ± 7.0
86.1 ± 5.7
43.7 ± 4.5
42.2 ± 3.6
26.8 ± 1.8
33.4 ± 2.4
78.1 ± 3.1
39.4 ± 3.0
75.1 ± 5.4
3
6
55.6 ± 4.3**
57.2 ± 5.0**
60.6 ± 4.6**
95.5 ± 5.2
69.8 ± 4.9*
52.3 ± 3.6***
34.9 ± 3.1***
57.1 ± 5.2***
36.3 ± 6.6***
73.7 ± 4.2
72.8 ± 5.5*
9
125.5 ± 7.5 39.6 ± 3.6***
75.8 ± 4.3***
64.0 ± 7.9***
12
15
166.5 ± 6.0 97.9 ± 12.5*** 47.3 ± 3.0*** 82.0 ± 11.6*** 118.6 ± 9.4*** 106.0 ± 17.5***
Data were expressed as the mean ± SE of 6 animals per group. Results were considered significant when *p <0.05; **p <0.01 and
***p <0.001 (in comparison to negative control group), determined by analysis of variance (ANOVA) followed by Bonferroni
posttests.

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Figure 1. Structure of Raltitrexed (1); 5-FU (2) and Capecitabine (3)
Figure 2. Percentage of 786-0 cells stained with annexin V-PE/7-AAD after 18 and 24h
of treatment.
Viable (unstained cells - viable cells), early (annexin V-PE +/7-AAD - stain), dead (annexin V-PE -/7-
AAD + stain) and late (annexin V-PE +/7-AAD + stain). Cells treated for 18 and 24 hours with vehicle
(culture medium plus DMSO 0.25%) and compound 7 (50 µM). Results represented by mean ± standard
error, in percentage of cells, from three different experiments. A total of 5000 events were collected per
experiment. ANOVA followed by Tukey’s Multiple Comparison Test. *p<0.05, ***p<0.001, *
statistically different from vehicle.