Novel merosesquiterpene exerts a potent antitumor activity against breast cancer cells in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2014.03.071

This article describes the antitumor properties of a new family of merosesquiterpenes, which were synthesized by Diels-Alder cycloaddition of the labdane diene trans-communic acid, highly abundant in Cupressus sempervirens, or its methyl ester, with the appropriate dienophile. These compounds demonstrated potent cytotoxic activity in vitro against human breast, colon, and lung tumor cells. We highlight the elevated activity (IC₅₀: 0.35 ± 0.10 μM) and specificity (TI: 9) of compound 13 against the MCF-7 line, which corresponds to the most prevalent breast cancer cell subtype, luminal A. It was found that compound 13 exerts its anti-tumor action by inducing oxidative stress, arresting the cell cycle in stages G₀-G_1, and activating apoptosis, which are all associated with low cyclin D1 regulation, pRb hypophosphorylation, increased expression of p27 and p53, and poly (ADP-ribose) polymerase (PARP) fractioning. Epithelial-mesenchymal transition, a phenomenon associated with metastasis promotion and a worsened prognosis also appeared to be inhibited by compound 13. In addition, it markedly reduced tumor development in immunocompetent C57BL/6 mice with allografts of E0771 mouse breast tumor cells (luminal A subtype). According to these findings, this new family of compounds, especially compound 13, may be highly useful in the treatment of human breast cancer.

Full text of DOI:10.1016/j.ejmech.2014.03.071

European Journal of Medicinal Chemistry 79 (2014) 1e12
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel merosesquiterpene exerts a potent antitumor activity against
breast cancer cells in vitro and in vivo
,
Esther Carrasco a b, Pablo Juan Álvarez a, Consolación Melguizo a, José Prados a,
Enrique Álvarez-Manzaneda c, Rachid Chahboun c, Ibtissam Messouri c,
,
María Isabel Vázquez-Vázquez a, Antonia Aránega a, Fernando Rodríguez-Serrano a
*
a Institute of Biopathology and Regenerative Medicine (IBIMER), University of Granada, 18071 Granada, Spain
b PhD Program in Biomedicine, University of Granada, 18071 Granada, Spain
c Department of Organic Chemistry, Institute of Biotechnology, University of Granada, 18071 Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
This article describes the antitumor properties of a new family of merosesquiterpenes, which were
synthesized by DielseAlder cycloaddition of the labdane diene trans-communic acid, highly abundant in
Cupressus sempervirens, or its methyl ester, with the appropriate dienophile. These compounds
demonstrated potent cytotoxic activity in vitro against human breast, colon, and lung tumor cells. We
Received 23 January 2014
Received in revised form
19 March 2014
Accepted 25 March 2014
Available online xxx
highlight the elevated activity (IC50: 0.35 ꢀ 0.10
mM) and specificity (TI: 9) of compound 13 against the
MCF-7 line, which corresponds to the most prevalent breast cancer cell subtype, luminal A. It was found
that compound 13 exerts its anti-tumor action by inducing oxidative stress, arresting the cell cycle in
stages G0eG1, and activating apoptosis, which are all associated with low cyclin D1 regulation, pRb
hypophosphorylation, increased expression of p27 and p53, and poly (ADP-ribose) polymerase (PARP)
fractioning. Epithelialemesenchymal transition, a phenomenon associated with metastasis promotion
and a worsened prognosis also appeared to be inhibited by compound 13. In addition, it markedly
reduced tumor development in immunocompetent C57BL/6 mice with allografts of E0771 mouse breast
tumor cells (luminal A subtype). According to these findings, this new family of compounds, especially
compound 13, may be highly useful in the treatment of human breast cancer.
Keywords:
Allografts
Apoptosis
Cancer
Cell cycle arrest
Merosesquiterpene
Oxidative stress
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
Breast cancer is the most frequent cancer among women
worldwide, with 1.38 million new cases being diagnosed in 2008.
The term cancer defines a group of diseases characterized by the
uncontrolled growth of cells and their dissemination to other body
tissues through blood and lymphatic vascular systems. Cancer is
one of the main causes of death worldwide, with 12.7 million new
cases and 7.6 million deaths in 2008 according to the World Health
Organization [1,2]. Generally, cancers of the breast, lung, colorectal,
and prostate cancer have been the most frequent types in devel-
oped countries and cancers of the stomach, liver, oral cavity, and
cervix cancer the most frequent in developing countries, although
these patterns are changing, especially due to population aging and
life style changes [3].
Although the mortality rate is much lower than the incidence
(around 6e19 per 100,000), it remains the first cause of death by
cancer in females [1]. Breast cancer is currently treated with sur-
gical resection, chemotherapy, radiotherapy, and/or hormone
therapy, but there is an elevated rate of recurrence and develop-
ment of metastatic disease, largely attributable to the onset of
resistance to some chemotherapies or other treatments. There is a
need to develop more effective and selective treatments that pro-
duce lesser systemic toxicity and do not generate resistance [4,5].
Medicinal plants have been used as a source of therapeutic
agents against all types of disease for thousands of years. For
instance, natural compounds and their derivatives have been used
as anticancer agents, including many widely administered “stan-
dard” chemotherapeutics such as paclitaxel, vinblastine, or
vincristine, among others [6]. Attention first centered on terrestrial
organisms, given their accessibility, and then turned to marine
* Corresponding author. Institute of Biopathology and Regenerative Medicine,
Biomedical Research Centre, Avd. del Conocimiento s/n., 18100 Armilla, Granada,
Spain.
E-mail address: fernrs@ugr.es (F. Rodríguez-Serrano).
http://dx.doi.org/10.1016/j.ejmech.2014.03.071
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

2
E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
organisms and microorganisms, aided by advances in analysis
techniques [7].
Natural products of mixed biosynthetic origin (polyketidee
terpenoid) containing a sesquiterpene unit joined to a phenolic or
quinone moiety are generally named “merosesquiterpenes” [8]. The
most important metabolites of this family are the compounds
bearing a bicyclic terpene (drimane) moiety, mainly due to their
potent biological activities. Drimenyl phenols include the choles-
teryl ester transfer protein (CETP) inhibitors wiedendiol A (1) and
wiedendiol B (2), isolated from the marine sponge Xestospongia
wiedenmayeri [9]. Drimenyl quinones include: the antitumor taur-
anin (3), isolated from Phyllosticta spinarum, a fungal strain endo-
phytic in Platycladus orientalis [10], which also inhibits cholesterol
biosynthesis; the anti-HIV (þ)-hyatellaquinone (4), found in the
sponge Hyatella intestinalis [11]; and the recently reported dis-
comycete metabolite (ꢁ)-F-12509 A (5), which exhibits sphingo-
sine kinase inhibitory activity [12] (Fig. 1).
More recently, merosesquiterpenoids with different function-
alities on the C-4 of the drimane moiety were reported. These
include: the 15-human lipoxygenase (15-HLO) inhibitor jaspic acid
(6), found in the Papua New Guinea marine sponge Jaspis cf. john-
stoni [13]; the antileishmanial disulfated meroterpenoid ilhabrene
(7), isolated from the Brazilian marine sponge Callyspongia sp. [14];
and the fungitoxic terpenoid-quinone pycnanthuquinone C (8),
isolated from the stem bark of Pycnanthus angolensis (Welw.) [15].
The two-synthon strategy is the most frequently applied
method for synthesizing this type of compound and usually in-
volves the reaction of a drimane electrophile with a nucleophilic
phenol derivative [16]. However, this strategy has some drawbacks:
a nucleophilic aromatic synthon must be utilized, preventing the
straightforward generation of compounds bearing electrophilic
groups, such as COOR, COR, and CN in the phenolic moiety, i.e., the
immediate precursors of metabolites 3 and 6. In addition, the two-
synthon strategy involves the use of a drimane electrophile, which
is usually prepared in a multistep synthetic sequence from a lab-
dane diterpene.
Scheme 1. Reaction protocol for synthesis of the new merosesquiterpenes.
Recently, Alvarez-Manzaneda et al. reported a new procedure
for producing this type of compound. The merosesquiterpene
skeleton of these compounds is constructed by DielseAlder cyclo-
addition of the labdane diene trans-communic acid (9a), highly
abundant in Cupressus sempervirens, or its methyl ester (9b), with
the appropriate dienophile. The DielseAlder adduct was converted
into the corresponding merosesquiterpene derivative after
aromatization (Scheme 1) [17].
Fig. 2 shows some DielseAlder adducts, aromatization products
(merosesquiterpene derivatives), and other compounds obtained
after functionalization of the aromatic compounds.
When the methyl trans-communate (9b) was treated with
2-chloroprop-2-enenitrile in refluxing toluene for 48 h, the
a
-chloronitrile 10 was obtained (88% yield). The treatment of ester
9b with methyl prop-2-ynoate in xylene under reflux for 12 h and
the subsequent treatment of the resulting crude product with DDQ
(1.1 eq) in refluxing dioxane rendered a 1:1 mixture of regioisomers
11a and 11b (94% yield). Compound 11a was isolated unreacted
after treating the mixture with MeMgBr. When ester 9b was heated
with dimethyl but-2-ynedioate in xylene under reflux for 6 h and
then aromatized by treating with DDQ (1.1 eq) in refluxing dioxane,
the result was diester 12 (95% yield). The heating of ester 9b with
cyclohexa-2,5-diene-1,4-dione or 2-methoxycyclohexa-2,5-diene-
Fig. 1. Chemical structures of compounds 1e8.

E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
3
Table 1
Antiproliferative activity of merosesquiterpenes against MCF-7, A-549, and T-84
cells.
Compound
IC50
(
m
M)a
MCF-7
A-549
T-84
11a
12
13
14
15
16
18
40.41 ꢀ 9.75
33.01 ꢀ 2.79
0.35 ꢀ 0.10
16.02 ꢀ 0.40
38.37 ꢀ 5.30
39.63 ꢀ 6.00
45.06 ꢀ 6.5
28.14 ꢀ 0.45
19.89 ꢀ 0.22
1.38 ꢀ 0.83
17.51 ꢀ 0.75
41.17 ꢀ 2.70
20.17 ꢀ 0.21
26.96 ꢀ 0.14
29.02 ꢀ 0.48
25.04 ꢀ 0.24
0.56 ꢀ 0.13
16.74 ꢀ 0.62
30.01 ꢀ 3.92
23.66 ꢀ 4.40
40.02 ꢀ 0.23
a
Data are means ꢀ SD of four determinations.
compound is poorly understood, the active compound usually
possesses a quinone moiety. It has been postulated that the activity
of this group may be attributable to the Michael acceptor (elec-
trophile) character of the unsaturated carbonyl system present in
this type of compound; this proposal was supported by our
observation of the activity of quinones 13, 14, and 15. Thus, com-
pounds 14 and 15, which have methoxy and hydroxyl groups,
respectively, in the double bond, thereby diminishing the electro-
philic character of the quinone system, exerted a markedly lower
Fig. 2. Chemical structure of novel synthetic merosesquiterpenes.
activity in comparison to compound 13 (IC50 ¼ of 38.37
mM for 15
1,4-dione in toluene, under appropriate reaction conditions, and
further treatment with DDQ (2.2 eq) in dioxane under reflux gave a
good yield of naphthoquinones 13 and 14, respectively. Methox-
yquinone 14 was efficiently converted into hydroxyquinone 15 after
treatment with KOH in MeOH at room temperature.
versus 0.35 M for 13 in MCF-7 cells).
m
Lipophilicity (log P) is a key parameter of the biological effect
developed by these compounds, because it can determine their
capacity to penetrate biological membranes and therefore influ-
ence intracellular bioavailability [20]. The log P of the studied
molecules was determined by using Marvin 6.0.5 software
(ChemAxon Ltd., 2013). Table 2 shows the resulting theoretical
values. A very strong and inversely proportional relationship was
found between the IC50 in the three tumor lines treated with
compounds 13, 14, and 15 and the log P values of the lines
(Spearman’s correlation coefficient ꢁ1, p ¼ 0), although no major
differences in these values were observed. This increase in com-
pound activity with higher log P was not observed in the remaining
compounds.
Cycloadduct 10 was transformed into aromatic compounds 16
and 17. The
a-chloronitrile 10 was readily transformed into the
aromatic nitrile 16 after dehydrohalogenation, by treating with
DBU in refluxing benzene and subsequent aromatization with DDQ
in dioxane under reflux. Alternatively, chloronitrile 10 was con-
verted into the corresponding
refluxing with KOH in t-BuOH. This ketone was transformed into
salicylate 18 after methoxycarbonylation and further
aromatization.
a,b-unsaturated ketone 17 by
This study investigated the antitumor properties of this new
family of synthetic merosesquiterpenes, whose synthesis and
antitumor effects are protected under international patents WO/
2009/112622 [18] and WO/2010/076358 [19], respectively. After an
initial screening in breast, lung, and colon cancer cells, the com-
pound showing the highest activity and therapeutic index, com-
pound 13, was selected for study of its mechanism of action against
breast tumor cells, which were found to be the most susceptible
cells to this compound. We studied its effects on oxidative stress,
the cell cycle, and apoptosis, given their high involvement in the
action of chemotherapeutics. Finally, we examined the effect of this
compound on the development of in vivo tumors in a murine model
of breast cancer allograft.
Based on the initial screening results, we elected to study the
effect of 13 on other breast and colon cell lines, comparing the
results with those obtained under the same experimental condi-
tions with 5-fluorouracil (5-Fu), a commercial pharmaceutical
widely administered to treat colon and breast cancer, among other
solid tumors [21,22]. The IC50 values of these two compounds were
determined in human breast tumor lines MCF-7, MDA-MB-231, and
T-47D, mouse breast tumor line E0771, and human colon tumor
lines T-84, HT-29, RKO, and SW-480. Their IC50 was also obtained in
normal human breast (MCF-10A) and colon (CCD18Co) cell lines
(Table 3) in order to calculate their in vitro therapeutic index (TI).
The IC50 of compound 13 was <1
exception of HT-29 (1.97 M). In general, the activity of 13 was
highly superior to that of 5-Fu. Thus, the mean IC50 was 0.48 M for
13 versus 10.51 M for 5-Fu in human breast tumor lines and
1.04 M for 13 versus 3.44 M for 5-Fu in human colon tumor lines.
mM in all lines with the
m
m
m
2. Results and discussion
m
m
Exceptionally, the activity of 5-Fu was almost 30% higher than that
of 13 in the mouse E0771 line, which has an elevated proliferative
rate, with a three-fold shorter duplication time in comparison to
MCF-7 cells. Hence, E0771 cells would require high rates of
2.1. In vitro cytotoxicity assay
In a first phase, we assessed the cytotoxic action of the com-
pounds listed in Fig. 2 against human breast (MCF-7), lung (A-549),
and colon (T-84) tumor lines. In general, activity was observed by
all of these molecules, with inhibitory concentration 50 (IC50
values < 50 M in all cases (Table 1). The optimal results were
observed for compounds 13 and 14. The highest activity was evi-
denced by 13, with an IC50 < 1 M in both MCF-7 and T-84 cells.
Although the structureeactivity relationship for this type of
)
Table 2
m
Lipophilicity values of synthetic merosesquiterpenes.
Compound
Log P
11a
6.21
12
6.22
13
5.73
14
5.32
15
5.21
16
6.07
18
6.56
m

4
E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
Table 3
accumulation can damage the DNA, producing the instability and
the mutation of genes (e.g., tumor suppressor gene p53) associated
with cancer development, and it can also alter proteins involved in
signaling pathways related to cell proliferation. Conversely, when
ROS levels are regulated and oxidative balance is maintained, they
can increase p53 expression and even induce apoptosis, inhibiting
the tumor process [27]. ROS levels are higher in cancer cells than in
normal cells due to the elevated metabolic activity of the former,
which leads to a chronic state of oxidative stress. It has been sug-
gested that the action mechanism of numerous anticancer drugs is
based on the promotion of ROS production, contributing to oxida-
tive imbalance and the death of tumor cells. The first known anti-
cancer drug to act by ROS induction and oxidative stress was
elesclomol, whose antitumor activity is strongly reduced when
administered with the antioxidant N-acetylcysteine (NAC), as in the
case of paclitaxel [27,28]. Moreover, some drugs, e.g., endostatin,
only act when used with other conventional chemotherapeutic
drugs capable of generating ROS [29].
IC50 and TI of 13 and 5-fluorouracil against breast and colon cancer cells.
Cell line
13
5-Fu
IC50
IC50
(
m
M)a
TI
(
m
M)a
TI
2.82
Breast
Colon
MCF-7
MDA-MB-231
T-47D
E0771
MCF-10A
T-84
HT-29
RKO
SW-480
CCD18Co
0.35 ꢀ 0.10
0.46 ꢀ 0.18
0.63 ꢀ 0.08
0.39 ꢀ 0.04
3.15 ꢀ 0.08
0.56 ꢀ 0.13
1.97 ꢀ 0.22
0.59 ꢀ 0.24
0.92 ꢀ 0.03
0.33 ꢀ 0.24
9.00
6.85
5.00
8.08
1.00
0.59
0.17
0.56
0.36
1.00
2.86 ꢀ 0.21
18.12 ꢀ 1.41
10.56 ꢀ 0.92
0.28 ꢀ 0.01
8.06 ꢀ 0.18
3.35 ꢀ 0.58
2.58 ꢀ 0.68
4.39 ꢀ 0.22
3.69 ꢀ 0.49
9.26 ꢀ 1.28
0.44
0.76
28.79
1.00
2.76
3.60
2.11
2.51
1.00
a
Data are means ꢀ SD of four determinations.
nucleoside and nucleotide synthesis to include them in their
nucleic acids and metabolic mediators. 5-Fu inhibits thymidilate
synthase enzyme, responsible for the de novo synthesis of thymi-
dylate, a nucleotide required for DNA synthesis and repair. 5-Fu can
also introduce errors during DNA and RNA synthesis through its
inclusion in the chain instead of thymidine or uracil [22,23]. These
effects of the drug on nucleic acid metabolism may explain why its
effect on the highly proliferative E0771 cells was greater than that
of 13.
In order to determine whether the mechanism of action of 13 on
line MCF-7 is mediated by oxidative stress induction, MCF-7 cells
were induced with this compound in the presence or absence of
NAC at 20 mM. After 3 days, the cultures were processed for calo-
rimetric quantification with sulforhodamine B, following the pro-
cedure reported in Material and Methods. A dose-dependent
decrease in cell viability was observed in the cultures induced at
The TI values of 13 were ꢂ5 in the breast cell lines and were
markedly higher than those of 5-Fu with the exception again of the
E0771 line, in which the value was 28.79. In the colon cell lines,
however, the TI was <0.6 for 13 versus >2 for 5-Fu. According to
these findings, 13 is more selective against human breast tumor
lines and 5-Fu is more selective against colon tumor lines. Taking
the IC50 and TI values together, the best results were obtained using
concentrations of 1, 2, and 3 mM, with reductions of 90.21%, 46.09%,
and 23.26%, respectively, versus controls (Fig. 3). In the presence of
NAC, however, the compound showed no cytotoxicity, and no sig-
nificant differences were found with controls, indicating that the
action of 13 is based on oxidative stress induction.
These findings were confirmed by measuring the intracellular
ROS production in cultures induced with 2 mM 13 for 12 h in the
13 against the MCF-7 line, with values of 0.35
respectively. We therefore selected this line for in-depth study of
the action mechanism of compound 13.
mM and 9.00,
presence or absence of NAC. Fig. 4 shows that the intracellular ROS
concentration was increased by 13 in the absence but not in the
presence of NAC, when the fluorescence intensity was very similar
to that of the control group.
These results allow us to become involved in the continuing
debate on the benefits of antioxidants for cancer patients. Evidence
has been published since the 1990s on the chemopreventive effect
of antioxidants consumed as food supplements or as part of the
diet, and many of them have been attributed with action against
some types of cancer, e.g., resveratrol, folic acid, vitamins A and E,
among many others. However, there has been an increase in studies
reporting a negative effect of antioxidants on cancer therapy,
The MCF-7 cell line was the first human breast cancer line to be
isolated and permanently maintained in culture (in 1970), and it
has been one of the most widely used for the study of breast cancer
biology and treatment. Its phenotypic and genotypic properties and
characteristics are well documented. It is known to be wild-type for
p53 and to express estrogen and progesterone receptors, making it
a good model for studying the most prevalent breast cancer sub-
type, luminal A. These features made the MCF-7 line ideal for our
study [24,25].
The assays described below were conducted under conditions of
high cell density, 5 ꢃ 104 cells/cm2, to obtain sufficient biomass.
Hence, for the induction of the cultures, the IC50 calculated under
these conditions was taken as the reference value, i.e.,
2.09 ꢀ 0.25
mM for compound 13 and 18.21 ꢀ 1.66
mM for 5-Fu.
2.2. Study of oxidative stress induction
Reactive oxygen species (ROS) intervene in signal transduction
and gene transcription, activating different pathways involved in
biological processes, including cell growth, proliferation, differ-
entiation, and apoptosis. They appear as metabolic by-products of
oxygen and present unpaired electrons; therefore, ROS can accept
electrons from other biological molecules, such as lipids, proteins,
and nucleic acids, altering their function. Antioxidants act to
avoid this, being responsible for neutralizing ROS by electron
donation and establishing a dynamic balance (oxidative balance).
Oxidative stress develops when this balance is altered in favor of
ROS [26].
Fig. 3. Effect of 13 on the viability of MCF-7 cells in the presence (þ) or absence (ꢁ) of
These free radicals have a dual role in cancer and can either
favor or inhibit its development. Because of their cell toxicity, their
the antioxidant NAC for
determinations.
3
days. The histogram depicts means
ꢀ
SD of four

E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
5
Fig. 4. ROS production in MCF-7 cultures induced with 2 mM of compound 13 for 12 h in the presence or absence of NAC. (a) Percentage of DCF-positive cells and mean fluorescent
intensity. The histogram depicts means ꢀ SD of four determinations. (b) Comparative cytogram of intracellular ROS production (c) Bright field micrographs (c1 and c3) and DCF-
fluorescence (c2 and c4) of control (left) and 13-treated (right) MCF-7 cells.
mainly in initial stages. It has been suggested that foods with
antioxidant capacity should not be consumed during treatment
with certain drugs, but there is no clinical evidence to endorse this
measure, and published clinical trials have been controversial [27e
30]. It is clear that oxidative stress and ROS levels affect cancer
development and treatments, and this issue warrants continued
research to reach a consensus on the optimal recommendations for
patients. In the present case, the effect of 13, a highly active com-
pound with a promising TI value, was completely annulled in the
presence of an antioxidant, as in the aforementioned cases of ele-
sclomol and paclitaxel.
respectively (Fig. 6), suggesting that the effect of compound 13 is
mediated by apoptosis induction.
2.4. Apoptosis assays with annexin-V
Cultures of MCF-7 cells were induced with compound 13 for
48 h, and flow cytometry was used in a viability study with annexin
V-FITC to determine whether the increased fraction of cells in Sub-
G1 was due to apoptosis promotion. Treatment with compound 13
reduced the cell viability from 93.0% in the control group to 80.5,
58.4, and 32.6% in the cultures treated with 1, 2, and 3
mM,
respectively. This viability reduction was correlated with an in-
crease in the fraction of cells in both early and late apoptosis, with
total apoptosis increasing from 3.44% in the control group to 11.07,
2.3. Cell cycle analysis
29.13, and 57.8% in the cultures treated with 1, 2, and 3
respectively (Fig. 7). These results indicate that the compound ex-
erts its action by apoptosis induction.
mM,
The cell cycle is one of the most important chemotherapeutic
targets. In order to determine the effect of compound 13 on this
process, we first synchronized MCF-7 cell cultures, maintaining
them in a serum-free medium for 24 h. At that time, as shown in
Fig. 3 (control 0), most of the cells were in phase G0eG1 (81.00%),
while 12.86% were in phase S, and 6.15% in phase G2-M. After 24 h in
the presence of serum, culture cells begin to enter the cell cycle,
with 42.34% of the cells in phase G0eG1, 49.46% in phase S, and
8.20% in phase G2-M (control 24 h). However, the induction of the
Taken together, the above findings indicate that the action of
compound 13 on MCF-7 cells is mediated by the induction of
oxidative stress, cell cycle arrest in G0eG1, and induction of
apoptosis. An increasing number of studies report the involvement
of oxidative stress in the action mechanism of a wide range of
antitumor compounds. The manner in which compound 13 exerts
its action is similar to that reported for various other compounds,
including: natural extracts of Garcina epunctata against promyelo-
cytic leukemia [31], chromenopyrazolediones against hormone-
sensitive prostate tumor cells [32], acetylsalicylic acid against he-
patocellular carcinoma cells [33], and the synergic action of quer-
cetin and 2-methoxyestradiol against human hepatocellular
carcinoma lines [34].
synchronized cultures with compound 13 at 1, 2, and 3 mM for 24 h
produced dose-dependent cell cycle arrest in phase G0eG1, with an
increase of cells in this stage to 51.40%, 66.61%, and 75.67%,
respectively, in comparison to 42.34% in the control group. This
increase in G0eG1 is associated with a corresponding reduction in
the proportion of cells in phases S and G2-M (Fig. 5).
At 24 h after induction with compound 13, no major differences
were found in the fraction of cells in sub-G1; therefore, the cyto-
grams in Fig. 3 only represent the cells found in any cycle phase.
However, at 48 h post-induction, there was a dose-dependent in-
crease in the sub-G1 population from 8.02% in the control group, to
2.5. Western blot analysis
After confirmation that the antitumor effect of compound 13 is
15.56%, 31.09%, and 39.91% after induction with 1, 2, and 3
mM of 13,
mediated by cell cycle arrest and apoptosis activation, the

6
E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
Fig. 5. Effect of compound 13 on MCF-7 cell cycle. Cultures were synchronized for 24 h with serum-free medium (control 0), and were subsequently incubated with complete
medium for 24 h without (control 24 h) or with compound 13 at 1, 2, and 3 M. The experiment was repeated independently three times yielding similar results.
m
expression of proteins involved in these processes was studied by
western blot and band densitometry (Fig. 8).
involved in the transition between phases G1 and S, remained low
during phase G0 and then gradually increased during G1 and was
maintained during the remainder of the cycle [37]. Researchers
have demonstrated that the degradation of cyclin D1 is sufficient
for cycle arrest in G0eG1 [43]; therefore, its decreased expression is
evidence of cycle arrest in phase G0eG1. In the present study, 4 h of
treatment of MCF-7 cells with compound 13 produced an 80%
decrease in its expression in comparison to baseline values.
pRb is the product of the retinoblastoma tumor suppressor gene,
an essential protein for the transition between phases G1 and S. It
forms a complex with transcription factor E2F, inhibiting its activity
and progression throughout the cell cycle. When there are
Cell cycle is mainly regulated by cyclin-dependent kinase (CDK)
proteins. Their activation depends on their binding to cyclins,
which are expressed throughout the cycle and regulate the tran-
sition between phases [35]. The expression of cyclin B1, which is
essential for the passage from phase G2 to M [36], was markedly
reduced after treatment with compound 13 and was almost absent
(6%) at 16 h. This result is in agreement with the cell cycle study
findings, given that the induction by 13 of arrest in phase G0eG1
means that the proteins involved in subsequent phases are no
longer expressed. Likewise, the expression of cyclin D1, which is

E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
7
Fig. 6. Effect of compound 13 on the fraction of MCF-7 cells in Sub-G1. Cultures were induced in complete medium for 48 h without (control) or with compound 13 at 1, 2, and 3
mM.
The experiment was repeated independently three times yielding similar results.
appropriate mitogenic signals, pRb is phosphorylated by the cyclin
D1/CDK4 complex, permitting the release of E2F and consequent
activation of the genes required for progression to phase S [38].
Total and phosphorylated forms of pRb expression both decreased
in the 13-treated cells, reaching a reduction of around 90% at 16 h in
both cases (Fig. 8). One of the target genes of E2F is proto-oncogene
c-myc, a transcription factor that participates in numerous cell
processes, including the transition from G0eG1 to S. Overexpression
and amplification of this gene is frequent in many tumors,
including breast cancer, and novel strategies are under develop-
ment to achieve its inhibition [39]. In the present study, treatment
with compound 13 reduced c-myc expression by 50% at 16 h and by
80% at 24 h (Fig. 8), supporting the results reported above for cell
cycle arrest markers.
Besides activators such as cyclins, Cip/Kip protein family mem-
bers are also of major importance in CDK expression and therefore
in cell cycle regulation, leading to inhibition of the kinase activity of
CDK/cyclin complexes and cell cycle arrest [40]. One of the family
members, p27, is used as a prognostic marker in breast cancer,
because its decreased expression is associated with tumor devel-
opment and progression [41]. Treatment of the MCF-7 line with
compound 13 produced a significant increase in p27 expression
(Fig. 8). In previous studies, arrest in G0eG1 induced by lapatinib
and euphol compounds was also associated with the over-
expression of p27 in breast tumor cells; euphol compounds also
produced reductions in cyclins B1 and D1 and in pRb hypo-
phosphorylation [42,43].
apoptosis. Tumor suppressor gene p53 is one of the main genes
involved in regulating this response. The baseline expression of p53
under normal conditions increases in response to stress and un-
dergoes a series of posttranslational modifications, including
phosphorylation, which lead to its stabilization and activation. This
permits its action as a transcription factor at nuclear level, regu-
lating the expression of genes involved in the cell cycle and
apoptosis, among others [44e46]. Given that the MCF-7 line is
wild-type for p53, we assessed the effect of 13 on its expression,
finding that it increases the levels of total p53 and its active
phosphorylated form (Fig. 8). This may indicate that 13-induced
apoptosis is dependent on p53. It may also support the effect of 13
on the cell cycle, because it has been reported that there may be an
increase in p53 expression in phase G1 arrest as well as a decrease
in cyclin D1 [47].
Poly (ADP-ribose) Polymerase (PARP) is the protein responsible
for the synthesis of poly (ADP-ribose) chains, which are essential
for DNA repair, among other processes. However, PARP cleavage by
caspases is an unequivocal sign of apoptosis [48,49]. In the present
study, we found an increase in the cleaved fraction of the protein,
confirming the induction of apoptosis (Fig. 8).
Finally, the expression of phospho-p44/42 mitogen-activated
protein kinases (MAPKs) was analyzed, because these proteins are
involved in cell proliferation and survival signaling pathways [50].
The p44/42 MAPK signaling pathway is known to be activated in
human breast tumor cells in response to sexual steroid hormones
and growth factors, including estrogens, progesterone, and
epidermal growth factor (EGF) [51]. It has also been verified that a
reduction in phospho-p44/42 MAPK levels in breast cancer patients
In many cases, the response to cell stress is mediated by cell
cycle arrest, DNA repair and, when damage is not repaired,

8
E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
Fig. 7. Apoptosis assay with annexin-V by flow cytometry of MCF-7 cells induced with 1, 2, and 3
mM compound 13 for 48 h. Viable (Q4), necrotic (Q1), early (Q3), and late (Q2)
apoptotic cells can be distinguished. The experiment was repeated independently three times yielding similar results.
is associated with good clinical response and higher global survival
after treatment with neoadjuvant chemotherapy [52]. In our study,
a major increase in the expression of phospho-p44/42 MAPKs was
observed at 4 h after treatment with compound 13 (Fig. 8), likely
because the compound acts downstream from these proteins in the
mitogenic signaling pathway. From 4 h onwards, a progressive
reduction was observed in the expression of proteins downstream
from p44/42 MAPK, especially that of cyclins and pRb, as well as in
phospho-p44/42 MAPK levels, which were lower than control
levels at 24 h of treatment. This may be explained by the reduced
expression of pRb, which is responsible for the expression of ste-
roidal hormones, especially the estrogen receptor [53]. Conse-
quently, pRb reduction may downregulate the estrogen receptor
and reduce the emission of mitogenic signals towards p44/42
MAPK, which would produce a decrease in its phosphorylated
active forms.
2.6. Expression of epithelialemesenchymal transition (EMT)
markers
Epithelialemesenchymal transition (EMT) is an essential pro-
cess during development, when embryonic cells change their
characteristics and acquire a mesenchymal phenotype. However,
EMTalso plays a key role in the acquisition of metastatic capacity by
cancer cells, which involves cytoskeleton remodeling, polarity and
intercellular contact loss and the consequent acquisition of high
Fig. 8. Western blot analysis of cell cycle and apoptosis-related markers in MCF-7
human breast cancer cells induced by 2 M compound 13 for 0, 4, 8, 16, and 24 h.
m

E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
9
motility and invasiveness [54,55]. At molecular level, this process is
associated with a decrease in the expression of cell adhesion
markers, such as E-cadherin and its intracellular association
molecule,
protein that binds to the cytoskeleton of actin via
extracellular region, stabilized by Ca2þ, binds to itself and forms
homodimers that interact with those of adjacent cells [57]. -cat-
b
-catenin [56]. E-cadherin is a transmembrane glyco-
b
-catenin. Its
b
enin is also a highly important protein in the regulation of the Wnt
pathway, which is crucial for correct cell development and func-
tioning [58].
Given the importance of EMT in the development of metastasis,
flow cytometry was used to assess the effect of compound 13 on the
expression of E-cadherin and its associated protein, b-catenin, after
48 h of induction. As observed in Fig. 9, 13 promoted the expression
of both markers, suggesting that merosesquiterpene acts by
inhibiting EMT development, which is of major translational in-
terest given the importance of this process.
Fig. 10. Effect of compound 13 on tumor growth in C57BL/6 mice with E0771 mouse
breast cancer cell allografts; the tumor volume is plotted against the days elapsed since
tumor cell inoculation. Arrows indicate days on which the vehicle (control) or com-
pound 13 (5, 10, or 15 mg/kg) were orally administered. **Significant difference with
control group, p < 0.01. In the photograph, a control animal (below) can be distin-
guished from an animal treated with 15 mg/kg of compound 13 (above).
2.7. In vivo assay
The in vitro assay results prompted us to assess the effect of
compound 13 on tumor development in vivo. C57BL/6 mice were
inoculated with cells from the breast cancer tumor line E0771,
established from a C57BL/6 mouse tumor. This model of murine
breast cancer syngeneic allograft generates highly proliferative
tumors in immunocompetent animals of the same species, offering
a scenario that is closer to the clinical situation from a translational
perspective. It also allows assays to be developed in a more
convenient manner and at a lower cost in comparison to immu-
nodepressed models, enhancing the histological reproducibility
and growth rate of the tumor and simplifying the statistical analysis
for data validation [59]. Crucially, E0771 cells are of the luminal A
subtype, as are the MCF-7 cells used in the in vitro assays, allowing
the study of compound 13 in cells with the same breast cancer
subtype [60].
Forty C57BL/6 mice were subcutaneously injected with 1 million
E0771 cells. At 9 days, when the tumor volume reached 75 mm3,
four experimental groups of 10 randomly assigned animals each
were orally administered with the vehicle (1% methylcellulose so-
lution) on six occasions or with compound 13 at a concentration of
5, 10, or 15 mg/kg Fig. 10 depicts the mean tumor volumes in each
experimental group, indicating the days on which 13 was admin-
istered (arrows). The volume of tumors started to increase faster in
control versus treated animals from day 27 onwards, and the dif-
ference reached significance by day 33. At the end of the assay on
day 42, the mean volume of control tumors was 2.4- to 3.3-fold that
of treated tumors (p < 0.01), confirming the in vivo antitumor ac-
tivity of compound 13. Paradoxically, no significant differences
were found on day 42 among the animals treated with 5, 10, or
15 mg/kg Fig. 10 includes a photograph that permits differentiation
between a control animal (below) and an animal treated with
15 mg/kg 13 (above), due to the major difference in tumor volumes
(arrow).
There were no significant differences in animal survival among
the study groups. No signs of toxicity, e.g., hair loss or diarrhea,
were observed in the animals treated with 13, despite its oral
administration. These findings suggest a very good tolerance to
compound 13. The absence of significant differences among groups
treated at 5, 10, or 15 mg/kg indicates that the dosage could be
increased to optimize treatment outcomes.
3. Conclusion
We screened the antitumor activity of seven new mer-
osesquiterpenes, whose synthesis and properties are under the
Fig. 9. E-cadherin and
b-catenin expression in MCF-7 cells after treatment for 48 h with 1 and 2 mM compound 13. The figures depict the superposition of the cytograms and
express the mean fluorescence intensity (MFI) values. The experiment was repeated independently three times yielding similar results.

10
E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
protection of international patents. These compounds demon-
strated elevated activity against human breast, colon, and lung
tumor cells. Compound 13 evidenced the highest activity and
specificity against MCF-7 breast cancer cells, which offer a good
model for the study of the most prevalent molecular subtype of
breast cancer, luminal A. Compound 13 acts against the cells by
inducing oxidative stress, arresting the cell cycle in phase G0eG1,
and activating apoptosis. Epithelialemesenchymal transition,
associated with metastasis promotion and a worse disease prog-
nosis, also appears to be inhibited by compound 13. This compound
strongly inhibits tumor development in C57BL/6 immunocompe-
tent mice with allografts of E0771 mouse breast tumor cells, which
are also luminal A subtype. These findings indicate that this new
family of compounds, especially compound 13, may be highly
useful to treat breast cancer in humans. Studies are under way to
further explore their mechanism of action and to gather key data
for structure-based drug design.
IC50 obtained at high cell density as reference for induction of these
cultures.
4.4. Study of the induction of oxidative stress
MCF-7 cells were seeded at high density (5 ꢃ 104/cm2) and, at
24 h, induced with different concentrations of compound 13 for 3
days in the presence or absence of 20 mM NAC. The cells in culture
were counted by staining with sulforhodamine B (see above sec-
tion). Intracellular ROS levels were also determined in these cul-
tures, using a FACScan flow cytometer (Becton Dickinson, San José,
CA, USA) to quantify the fluorescence intensity emitted after in-
cubation with 10 m
M 20,70-dichlorofluorescein diacetate (DCF-DA,
SigmaeAldrich) for 30 min at 37 ꢄC. The percentage of DCF-positive
cells and mean fluorescent intensity were calculated by FlowJo
software (v 7.6.5, Tree Star, Inc., Ashland, OR, USA). Samples of
cultures were also analyzed by fluorescence microscopy (Leica
DM5500B).
4. Materials and methods
4.5. Cell cycle analysis
4.1. Compounds
MCF-7 cells were seeded (5 ꢃ 104/cm2), and the culture medium
was replaced with a serum-free medium at 24 h to synchronize
their cell cycles. 24 h later, they were again placed in culture me-
dium with serum and induced with different concentrations of 13
for 24 or 48 h, depending on the case. After this time, cultures were
washed with PBS, fixed with 70% cold ethanol, and incubated with a
DNA extraction solution (0.2 M Na2HPO4, 0.1 M Citric Acid, pH 7.8)
for 15 min at 37 ꢄC. Cells were then centrifuged, washed with PBS,
We assessed the antitumor effect of seven merosesquiterpene
compounds, 11a, 12, 13, 14, 15, 16, and 18 (Fig. 2), which were dis-
solved in dimethyl sulfoxide (DMSO) and stored at ꢁ20 ꢄC. For each
experiment, the stock solutions were further diluted in medium to
obtain the desired concentrations. The final solvent concentration
in cell culture was <0.1% v/v DMSO.
4.2. Cell lines and culture
and resuspended in 250
ml of a solution of propidium iodide (40 mg/
ml) and RNAse (100
m
g/ml) for 30 min at 37 ꢄC in the dark. Finally,
Human breast tumor lines MCF-7, T-47D, and MDA-MB-231,
mouse breast tumor line E0771, human colon tumor lines RKO,
HT-29, SW-480, and T-84, human lung tumor line A-549, and non-
tumor lines of breast MCF-10A and colon CCD-18Co were supplied
by the Department of Cell Cultures of the Granada University Sci-
entific Instrumentation Center. All lines were cultured at 37 ꢄC in 5%
CO2 and 90% humidity and, except for MCF-10A, with Dulbecco’s
modified Eagle medium (DMEM), supplemented with 10% heat
inactivated fetal bovine serum, 10 ml/L penicillinestreptomycin
samples were analyzed in a FACScan flow cytometer, using a linear
scale for the cell cycle and a logarithmic scale to determine the sub-
G1 fraction. Results were analyzed with FlowJo software (v 7.6.5,
Tree Star, Inc.).
4.6. Apoptosis assays with annexin-V
Cell viability was determined by flow cytometry using the
Annexin V-FITC kit (Pharmingen, San Diego, CA, USA). MCF-7 cells
were seeded (5 ꢃ 104/cm2) and, after 24 h, were induced with
different concentrations of 13 for 48 h. Cells were then detached
with PBS-EDTA, washed twice with cold PBS, and collected by
centrifugation at 500 g for 10 min. Cells were stained following the
manufacturer’s protocol, and samples were then analyzed in a
FACScan flow cytometer, using FlowJo software (v 7.6.5, Tree Star,
Inc.).
100ꢃ, and 2 mM
L- glutamine. MCF-10A line was cultured in DMEM
with Ham F-12 nutrient mixture (DMEM/F-12; 1:1) supplemented
with 10 ml/L penicillinestreptomycin 100ꢃ, 20 ng/ml EGF, 100 ng/
ml cholera toxin, 10 mg/ml bovine insulin, 0.5 mg/ml hydrocortisone,
and 5% inactivated horse serum. Culture media and respective
supplements were supplied by SigmaeAldrich (St. Louis, MO).
4.3. In vitro cytotoxicity assay
4.7. Western blot analysis
In order to calculate the IC50 of compounds, 5 ꢃ 103 cells/cm2
were seeded by quadruplicate. At 24 h, cells were induced with
increasing compound concentrations for 3 days. Subsequently, cells
were fixed with 10% cold trichloroacetic acid (4 ꢄC) and stained
with 0.4% sulforhodamine in 1% acetic acid. The colorant was sol-
ubilized with 10 mM Tris-base pH 10.5, and optical density values
were determined by colorimeter at 492 nm (Multiskan EX, Thermo
Electron Corporation, Milford, MA, USA). IC50 values were calcu-
lated from the semi-logarithmic doseeresponse curve by linear
interpolation. The TI was calculated as the Normal line IC50/Tumor
line IC50, with a higher TI value indicating greater specificity of the
compound towards tumor versus normal cells.
We seeded MCF-7 cells (5 ꢃ 104/cm2) and, after 24 h, induced
them with 2 mM 13 for different time periods. The medium was
then removed, and cells were lysed with sample buffer (62.76 mM
Trise HCl pH 6.8, 5% -mercaptoethanol, 2% SDS, 10% glycerol, 0.5%
b
bromophenol blue). Proteins were separated by electrophoresis in
12% acrylamide gels in a Mini Protean Tetracell (Bio-Rad, Hercules,
CA, USA) and were then transferred to nitrocellulose membranes
(Bio-rad, 162-0115), which were blocked for 1 h at room tempera-
ture with 5% nonfat milk powder in PBS. The membranes were
incubated overnight at 4 ꢄC with primary antibodies against cyclin
B1(Cell Signaling, 4138), cyclin D1 (Cell Signaling, 2978), total pRb
(Santa Cruz Biotechnology, Inc., sc-102), phospho-pRb (Cell
Signaling, 9301), C-Myc (Santa Cruz Biotechnology, Inc., sc-70465),
p27 (Cell Signaling, 2552), total p53 (Sigma, p5813), phospho-p53
(Cell Signaling, 9284), cleaved-PARP (Cell Signaling, 9541),
The IC50 of cultures established at high cell density
(5 ꢃ104 cells/cm2) was also calculated, because the oxidative stress,
cell cycle, apoptosis, and gene expression assays were conducted
under these conditions to obtain sufficient cell biomass, taking the

E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
11
[2] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, CA: A Cancer
Journal for Clinicians 61 (2011) 69e90.
[3] C. Are, S. Rajaram, M. Are, H. Raj, B.O. Anderson, R. Chaluvarya Swamy,
M. Vijayakumar, T. Song, M. Pandey, J.A. Edney, E.L. Cazap, Journal of Surgical
Oncology 107 (2013) 221e226.
phospho-p44/22 MAPK (Cell Signaling, 4370), or
b actin (Sigma,
A3854). They were then washed and incubated for 1 h at room
temperature with the corresponding secondary antibodies (Cell
Signaling, 7074 or Sigma, A9044). Protein-antibody complexes
were detected by chemiluminescence using the ECL system
(Amersham Pharmacia Biotech, UK). The relative expression of the
proteins was semi-quantified with Quantity One software (Bio-Rad
Laboratories, Hercules, USA).
[4] X. Lü, Q. Deng, H. Li, Z. Suo, Experimental and Therapeutic Medicine 2 (2011)
821e826.
[5] A. Sheri, S. Johnston, Clinical Oncology 25 (2013) 117e126.
[6] Z. Zhong, Y. Dang, X. Yuan, W. Guo, Y. Li, W. Tan, J. Cui, J. Lu, Q. Zhang, X. Chen,
Y. Wang, Cellular Physiology and Biochemistry 30 (2012) 778e790.
[7] I. Robles-Fernández, F. Rodríguez-Serrano, P.J. Álvarez, R. Ortiz, A.R. Rama,
J. Prados, C. Melguizo, E. Álvarez-Manzaneda, A. Aránega, Recent Patents on
Anti-Cancer Drug Discovery 8 (2013) 203e215.
[8] T.J. Simpson, Topics in Current Chemistry 195 (1998).
[9] (a) S.J. Coval, M.A. Conover, R. Mierzwa, A. King, M.S. Puar, D.W. Phife, J.K. Pai,
R.E. Burrier, M.S. Ahn, G.C. Boykow, M. Patel, S.A. Pomponi, Bioorganic &
Medicinal Chemistry Letters 5 (1995) 605e610;
4.8. Expression of epithelialemesenchymal transition (EMT)
markers
MCF-7 cells (5 ꢃ 104/cm2) were seeded and, after 24 h, induced
with different concentrations of 13 for 48 h. They were subse-
quently detached with PBS-EDTA, washed with PBS, and resus-
(b) S. Chackalamannil, Y. Xia, Y. Wang, M. Tsai, M. Czarniecki, S. Nang,
A. Clemmons, H.S. Ahn, G. Boykow, Bioorganic & Medicinal Chemistry Letters
5 (1995) 2005e2010;
pended in 100
which 10 l of the corresponding antibody, E-cadherin (R&D Sys-
tems, FAB18381P) or
ml blocking buffer (PBS, 2% BSA, 0.07% EDTA) to
(c) A.R. Tall, The Journal of Lipid Research 34 (1993) 1255e1274;
(d) K.R. Marotti, C.K. Castle, T.P. Boyle, A.H. Lin, R.W. Murray, G.W. Melchior,
Nature 364 (1993) 73e75;
(e) J. Koizumi, A. Inazu, K. Yagi, I. Koizumi, Y. Uno, K. Kajinami, S. Miamoto,
P. Moulin, A.R. Tall, H. Mabuchi, R. Takeda, Atherosclerosis 90 (1991) 189e
196;
m
b-catenin (R&D Systems, IC13292F), was
added. Cells were incubated for 30 min at 4 ꢄC in the dark, washed
with PBS, and analyzed by flow cytometry (FACScan). Results were
analyzed using FlowJo software (v 7.6.5, Tree Star, Inc.).
(f) A.F. Barrero, E.J. Alvarez-Manzaneda, R. Chahboun, Tetrahedron Letters 38
(1997) 8101e8104;
(g) A.F. Barrero, E.J. Alvarez-Manzaneda, R. Chahboun, Tetrahedron 54 (1998)
5635e5650;
4.9. In vivo assay
(h) E.J. Alvarez-Manzaneda, R. Chahboun, I. Barranco Pérez, E. Cabrera,
E. Alvarez, R. Alvarez-Manzaneda, Organic Letters 7 (2005) 1477e1480;
(i) A. Bernet, K. Seifert, Helvetica Chimica Acta 89 (2006) 784e796;
(j) Y. Arima, M. Kinoshita, H. Akita, Tetrahedron Asymmetry 18 (2007) 1701e
1711;
We used 40 female C57BL/6 mice weighing 25e30 g (Scientific
Instrumentation Center of Granada University), which were kept in
a laminar flow cabinet in a room with a controlled environment
(37 ꢄC, 40e70% relative humidity, 12-h light/dark cycle, and
pathogen-free). The in vivo study was approved by the ethics
committee of the University of Granada. Tumors were induced by
subcutaneous injection of 1 ꢃ106 cells of the E0771 line in the right
side of the mice. At 9 days, when the tumor volume reached
75 mm3, mice were randomly distributed into four groups for oral
treatment with vehicle alone (1% methylcellulose) or with com-
pound 13 at concentrations of 5, 10, or 15 mg/kg. The compound
was administered on days 9, 12, 15, 18, 21, and 24 post-injection,
and periodical measurements were made during the in vivo assay
of the largest (a) and the next largest (b) diameter using a digital
(k) T. Laube, A. Bernet, H.M. Dahse, I.D. Jacobsen, K. Seifert, Bioorganic &
Medicinal Chemistry 17 (2009) 1422e1427.
[10] (a) K. Kawashima, K. Nakanishi, H. Nishikawa, Chemical & Pharmaceutical
Bulletin 12 (1964) 796e803;
(b) E.M. Wijeratue, P.A. Paranagama, M.T. Marrou, M.K. Gunatilaka,
A.E. Arnold, A.A.L. Gunatilaka, Journal of Natural Products 71 (2008) 218e222;
(c) S. Ishii, M. Fujii, H. Akita, Chemical & Pharmaceutical Bulletin 57 (2009)
1103e1106.
[11] (a) R. Talpir, A. Rudi, Y. Kashman, Y. Loya, A. Hizi, Tetrahedron 50 (1994)
4179e4184;
(b) S. Poigny, T. Huor, M. Guyot, M. Samadi, The Journal of Organic Chemistry
64 (1999) 9318e9320;
(c) A. Bernet, J. Schroeder, K. Seifert, Helvetica Chimica Acta 86 (2003) 2009e
2020;
(d) T. Laube, W. Beil, K. Seifert, Tetrahedron 61 (2005) 1141e1148;
(e) Y. Amano, M. Kinoshita, H. Akita, Journal of Molecular Catalysis B: Enzy-
matic 32 (2005) 141e148;
caliper. Tumor volume was calculated as V ¼ ab2
p/6.
(f) B.K. Rubio, R.W.M. Van Soest, P. Crews, Journal of Natural Products 70
(2007) 628e631;
(g) K.W.L. Yong, A. Jankam, J.N.A. Hooper, A. Suksamrarn, M.J. Garson, Tetra-
hedron 64 (2008) 6341e6348.
4.10. Statistical analysis
SPSS 14 for Windows (SPSS, Chicago, IL, USA) was used for the
statistical analysis. Results were compared with the Student’s test,
one- and two-way ANOVAs, and Spearman’s test. p < 0.05 was
considered significant. Data were graphically represented by using
Microsoft Excel 2010 software (Microsoft Corporation).
[12] (a) K. Kono, M. Tanaka, T. Ogita, T. Hosoya, T. Kohama, The Journal of Anti-
biotics 53 (2000) 459e465;
(b) K. Kono, M. Sugiura, T. Kohama, The Journal of Antibiotics 55 (2002) 99e
104;
(c) N. Maezawa, N. Furnichi, H. Tsuchikawa, S. Katsumura, Tetrahedron Letters
48 (2007) 4865e4867;
(d) E. Bonhoure, A. Lauret, D.J. Barnes, C. Martin, B. Malavaud, T. Kohama,
J.V. Melo, O. Cuvillier, Leukemia 22 (2008) 971e979.
[13] (a) L.M. Murray, A. Johnson, M.C. Diaz, P. Crews, The Journal of Organic
Chemistry 62 (1997) 5638;
Competing interests
(b) R.H. Cichewicz, V.A. Kenyon, S. Whitman, N.M. Morales, J.F. Arguello,
T.R. Holman, P. Crews, Journal of the American Chemical Society 126 (2004)
14910e14920;
(c) S.J. Robinson, E.K. Hoobler, M. Riener, S.T. Loveridge, K. Tenney,
F.A. Valeriote, T.R. Holman, P. Crews, Journal of Natural Products 72 (2009)
1857e1863.
All authors declare that they have no conflict of interest.
Acknowledgments
This study was supported by the University of Granada, the
Ministry of Science and Innovation, Spain (Project GREIB_-
PYR_2010_11) and the Regional Government of Andalusia (Project
P11-CTS-7651 and assistance to the CTS-107 and FQM-348 groups).
EC received an FPU pre-doctoral fellowship (Ministry of Education,
Culture and Sport), and this article is related to her PhD thesis.
[14] C.A. Gray, S.P. de Lira, M. Silva, E.F. Pimenta, O.H. Thiemann, G. Oliva, E. Hajdu,
R.J. Andersen, R.G.S. Berlink, The Journal of Organic Chemistry 71 (2006)
8685e8690.
[15] H.K. Wabo, S.N. Tatsimo, P. Tane, J.D. Connolly, Planta Medica 73 (2007) 187e
189.
[16] For some representative syntheses based on the two synthon strategy, see: (a)
G.L. Trammel, Tetrahedron Letters (1978) 1525e1528;
(b) A.F. Barrero, E.J. Alvarez-Manzaneda, R. Chahboun, Tetrahedron Letters 38
(1997) 2325e2328;
References
(c) O. Arjona, M. Garranzo, J. Maluego, E. Maroto, J. Plumet, B. Sáez, Tetrahe-
dron Letters 38 (1997) 7249e7252;
(d) A.F. Barrero, E.J. Alvarez-Manzaneda, R. Chahboun, M. Cortés,
V. Armstrong, Tetrahedron 55 (1999) 15181e15208;
[1] J. Ferlay, H.-R. Shin, F. Bray, D. Forman, C. Mathers, D.M. Parkin, International
Journal of Cancer 127 (2010) 2893e2917.

12
E. Carrasco et al. / European Journal of Medicinal Chemistry 79 (2014) 1e12
(e) K.I. Takao, T. Sasaki, T. Kozaki, Y. Yaganisawa, K.I. Tadano, A. Kawashima, [34] Y.F. Chang, Y.C. Hsu, H.F. Hung, H.J. Lee, W.Y. Lui, C.W. Chi, J.J. Wang, Nutrition
H. Shinonaga, Organic Letters 3 (2001) 4291;
and Cancer 61 (2009) 735e745.
(f) S. Maiti, S. Sengupta, C. Giri, B. Achari, A.K. Banerjee, Tetrahedron Letters 42
(2001) 2389e2391;
[35] W. Zhao, S.F. Zhou, Z.P. Zhang, G.P. Xu, X.B. Li, J.L. Yan, Oncology Reports 25
(2011) 1289e1295.
(g) S. Quideau, M. Lebon, A.M. Lamidey, Organic Letters 22 (2002) 3975e3978;
(h) V. Armstrong, A.F. Barrero, E.J. Alvarez-Manzaneda, M. Cortés,
B. Sepúlveda, Journal of Natural Products 66 (2003) 1382e1383.
[36] K.L. Pfaff, R.W. King, PLoS One 8 (2013) e59169.
[37] C.P. Masamha, D.M. Benbrook, Cancer Research 69 (2009) 6565e6572.
[38] S. Munro, S.M. Carr, N.B. La Thangue, Oncogene 31 (2012) 4343e4352.
[39] A. Song, J. Ye, K. Zhang, L. Sun, Y. Zhao, H. Yu, Biochemical Genetics 51 (2013)
603e617.
[40] M.K. Yoon, D.M. Mitrea, L. Ou, R.W. Kriwacki, Biochemical Society Trans-
actions 40 (2012) 981e988.
[41] W. He, X. Wang, L. Chen, X. Guan, Cancer Biotherapy & Radiopharmaceuticals
27 (2012) 399e402.
[42] L. Tang, Y. Wang, A. Strom, J.Å. Gustafsson, X. Guan, Cell Cycle 12 (2013)
2665e2674.
[43] L. Wang, G. Wang, D. Yang, X. Guo, Y. Xu, B. Feng, J. Kang, Molecular Medicine
Reports 8 (2013) 1279e1285.
[17] E. Alvarez-Manzaneda, R. Chahboun, E. Cabrera, E. Alvarez, A. Haidour,
J.M. Ramos, R. Alvarez-Manzaneda, J.L. Romera, M.A. Escobar, I. Messouri,
Synthesis (2008) 4019e4027.
[18] WO/2009/112622, Álvarez-Manzaneda Roldán E, Chahboun, 2009.
[19] A. Aránega, E. Álvarez-Manzaneda Roldán, R. Chahboun, F. Rodríguez-Serrano,
H. Boulaiz, J.A. Marchal, C. Melguizo, M. Perán, J.C. Prados, WO/2010/076358,
2010.
[20] R. Yousefi, A. Khalafi-Nezhad, M.N.S. Rad, S. Behrouz, F. Panahi, M. Esmaili,
S.M. Ghaffari, A. Niazi, A.A. Moosavi-Movahedi, Medicinal Chemistry Research
21 (2012) 1921e1928.
[21] E.T. Uluer, I. Aydemir, S. Inan, K. Ozbilgin, H.S. Vatansever, Acta Histochemica
114 (2012) 641e646.
[22] E.M. Saleh, R.A. El-Awady, N. Anis, Oncology Letters 5 (2013) 321e327.
[23] P. Álvarez, J.A. Marchal, H. Boulaiz, E. Carrillo, C. Vélez, F. Rodríguez-Serrano,
C. Melguizo, J. Prados, R. Madeddu, A. Aranega, Expert Opinion on Therapeutic
Patents 22 (2012) 107e123.
[44] K.H. Vousden, X. Lu, Nature Reviews Cancer 2 (2002) 594e604.
[45] K. Yoshida, Y. Miki, Cancer Science 101 (2010) 831e835.
[46] L.A. Carvajal, J.J. Manfredi, EMBO Reports 14 (2013) 414e421.
[47] F.C. Wong, C.C. Woo, A. Hsu, B.K. Tan, PLoS One 8 (2013) e78021.
[48] S.H. Kaufmann, S. Desnoyers, Y. Ottaviano, N.E. Davidson, G.G. Poirier, Cancer
Research 53 (1993) 3976e3985.
[24] A.S. Levenson, V.C. Jordan, Cancer Research 57 (1997) 3071e3078.
[25] J.A. Beaver, J.P. Gustin, K.H. Yi, A. Rajpurohit, M. Thomas, S.F. Gilbert,
D.M. Rosen, B.H. Park, J. Lauring, Clinical Cancer Research 19 (2013) 5413e
5422.
[26] L. Vera-Ramirez, P. Sánchez-Rovira, M.C. Ramírez-Tortosa, C.L. Ramirez-Tor-
tosa, S. Granados-Principal, J.A. Lorente, J.L. Quiles, Critical Reviews in
Oncology/Hematology 80 (2011) 347e368.
[27] S. Saeidnia, M. Abdollahi, Toxicology and Applied Pharmacology 271 (2013)
49e63.
[28] C.-Y. Yang, M. Zhang, Z. Du, J. Barsoum, J. Bertin, Molecular Cancer Thera-
peutics 7 (2008) 2319e2327.
[49] B. Singh, S.K. Guru, S. Kour, S.K. Jain, R. Sharma, P.R. Sharma, S.K. Singh,
S. Bhushan, S.B. Bharate, R.A. Vishwakarma, European Journal of Medicinal
Chemistry 70 (2013) 864e874.
[50] P.J. Roberts, C.J. Der, Oncogene 26 (2007) 3291e3310.
[51] C. Zhou, T.A. Steplowski, H.K. Dickens, K.M. Malloy, P.A. Gehrig, J.F. Boggess,
V.L. Bae-Jump, PLoS One 8 (2013) e55730.
[52] L. Huang, T. Chen, C. Chen, S. Chen, Y. Liu, J. Wu, Z. Shao, World Journal of
Surgical Oncology 11 (2013) 307.
[53] I. Caligiuri1, G. Toffoli, A. Giordano, F. Rizzolio, Oncotarget 4 (2013) 875e883.
[54] L.J. Talbot, S.D. Bhattacharya, P.C. Kuo, International Journal of Biochemistry
and Molecular Biology 3 (2012) 117e136.
[29] J. Watson, Open Biology 3 (2013) 120144.
[55] Y. Choi, H.J. Lee, M.H. Jang, J.M. Gwak, K.S. Lee, E.J. Kim, H.J. Kim, H.E. Lee,
S.Y. Park, Human Pathology 44 (2013) 2581e2589.
[56] M.A. Smit, D.S. Peeper, Aging 2 (2010) 735e741.
[57] S. Baranwal, S.K. Alahari, Biochemical and Biophysical Research 384 (2009)
6e11.
[30] R.M. Sainz, F. Lombo, J.C. Mayo, Cancers (Basel) 4 (2012) 442e474.
[31] P. Constant Anatole, S.K. Guru, N. Bathelemy, N. Jeanne, S. Bhushan,
T. Murayama, A.K. Saxena, Environmental Toxicology and Pharmacology 36
(2013) 865e874.
[32] P. Morales, D. Vara, M. Goméz-Cañas, M.C. Zúñiga, C. Olea-Azar, P. Goya,
J. Fernández-Ruiz, I. Díaz-Laviada, N. Jagerovic, European Journal of Medicinal
Chemistry 70 (2013) 111e119.
[33] H. Raza, A. John, S. Benedict, European Journal of Pharmacology 668 (2011)
15e24.
[58] M.E. Menezes, A. Mitra, L.A. Shevde, R.S. Samant, Biochemical Journal 444
(2012) 573e580.
[59] M. de Jong, T. Maina, Journal of Nuclear Medicine 51 (2010) 501e504.
[60] E. Young, L. Miele, K.B. Tucker, M. Huang, J. Wells, J.-W. Gu, Cancer Biology &
Therapy 10 (2010) 703e711.