Combined Acylselenourea-Diselenide Structures: New Potent and Selective Antitumoral Agents as Autophagy Activators

Article abstract of DOI:10.1021/acsmedchemlett.7b00482

A series of 16 new diselenide-acylselenourea conjugates have been designed following the fragment-based drug strategy. Compound in vitro cytotoxic potential was evaluated against six human cancer cell lines and two nonmalignant derived cell lines with the aim of determining their potency and selectivity. Nine derivatives exhibited GI₅₀ values under 10 μM in at least four cancer cell lines. A clear gap situated phenyl substitution over heterocyclic moieties in terms of selectivity. Among carbocyclic compounds, derivatives 2 and 7 significantly inhibited cell growth of breast adenocarcinoma cells with GI₅₀ values of 1.30 and 0.15 nM, respectively, with selectivity indexes 12 and 121 times higher than those obtained for doxorubicin. Preliminary mechanistic studies indicated that compounds 2 and 7 induce cell cycle arrest and autophagy-dependent cell death evidenced by the blockage of cell death with pretreatment with wortmannin or chloroquine and confirmed by the upregulation of the markers Beclin1 and LC3B in MCF-7 cells.

Full text of DOI:10.1021/acsmedchemlett.7b00482

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Combined acylselenourea-diselenide structures: new potent
and selective antitumoral agents as autophagy activators
Pablo Garnica, Ignacio José Encío, Daniel Plano, Juan Antonio Palop, and Carmen Sanmartin
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00482 • Publication Date (Web): 13 Mar 2018
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ACS Medicinal Chemistry Letters
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Combined acylselenourea-diselenide structures: new potent
and selective antitumoral agents as autophagy activators
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Pablo Garnica , Ignacio Encío , Daniel Plano , Juan A. Palop , Carmen Sanmartín
1
University of Navarra, Faculty of Pharmacy and Nutrition, Department of Organic and Pharmaceutical Chemistry, Irun-
2
larrea 1, E-31008 Pamplona, Spain. Instituto de Investigación Sanitaria de Navarra (IdiSNA), Irunlarrea 3, E-31008 Pam-
plona, Spain. Department of Health Sciences, Public University of Navarra, Avda. Barañain s/n, E-31008 Pamplona, Spain
3
KEYWORDS: selenium, acylselenoureas, diselenides, autophagy.
ABSTRACT: A series of 16 new diselenide-acylselenourea conjugates have been designed following the fragment-based drug
strategy. Compound in vitro cytotoxic potential was evaluated against six human cancer cell lines and two non-malignant derived
cell lines with the aim of determining their potency and selectivity. Nine derivatives exhibited GI50 values under 10 µM in at least
four cancer cell lines. A clear gap situated phenyl substitution over heterocyclic moieties in terms of selectivity. Among carbocyclic
compounds, derivatives 2 and 7 significantly inhibited cell growth of breast adenocarcinoma cells with GI50 values of 1.30 and 0.15
nM, respectively with selectivity indexes 12 and 121 times higher than those obtained for doxorubicin. Preliminary mechanistic
studies indicated that compounds 2 and 7 induce cell cycle arrest and autophagy-dependent cell death evidenced by the blockage of
cell death with pre-treatment with wortmannin or chloroquine and confirmed by the upregulation of the markers Beclin1 and LC3B
in MCF-7 cells.
Cancer can be categorized as a serious clinical problem as it
affects millions of patients worldwide. Only in the United
States of America statistics show death rate by cancer will
of investigation, our research group has explored this chemical
entity and the most potent antitumoral structures synthesized
in our group containing the diphenyldiselenide moiety corre-
1
17
reach the alarming ratio of 1,600 deaths per day. Even
sponds to bis(4-aminophenyl)diselenide structure. Therefore,
though big progress and investment in the field has been
made, selectivity and side effects are still issues for current
treatments. Moreover, both acquired and de novo resistances
we selected this fragment as a starting point for our structure
design with the aim of enhancing its potency and reducing its
toxicity.
2
to chemotherapy have been documented. In this context, over
On the other hand, selenourea moiety is emerging as privi-
leged structural fragment in some antiproliferative studies.
Those resulting molecules behaved as reactive oxygen species
(ROS) scavengers as well as glutathione peroxidase mimetics
the last decade, selenium compounds have demonstrated im-
portant effects on cancer progression distressing cell growth
and proliferation, this activity is both form and dose depend-
3,4
1
8
ent. Many mechanisms of action have been identified, in-
and proved to have potent cytotoxic effects.
N,N’-
cluding the induction of apoptosis either caspase dependent or
disubstitued selenourea derivatives have been reported as non-
toxic polyfunctional antioxidants with potency over sulphur or
5,6
independent.
Furthermore, modulation of some kinases
19
activity, antioxidant effects through selenoproteins involved in
the modulation of oxidative stress, mitotic catastrophe, au-
tophagy or a combination of the previously described mecha-
nisms that have been proven for selenium-containing mole-
oxygen analogs. We have recently confirmed that selenourea
derivatives have more potent in vitro activities against several
20
cancer cell lines than their sulphur analogs. Since combina-
tion of two or more active structural moieties can possibly
enhance bioactivity and reduce adverse effects, we combined
7,8
cules.
Autophagy has been deeply studied as a factor of cancer
regulation. Drug-induced autophagy in breast cancer cell lines
(PhSe) nucleus and selenourea fragment with the intention of
2
9
achieving this synergic effect. In addition, and considering that
a variety of drugs are activated by biotransformation, the sele-
noureas were functionalized with different acyl substituents in
order to facilitate or to modulate the release of the active
groups. These modifications enclose:
has already been described in the past and has been correlated
1
0
with apoptosis promotion. Beyond the interest of the on-
cosuppressive effect, autophagy assures anticancer inmuno-
11
surveillance. Some autophagy activators have been approved
1
2
by FDA for cancer treatment such as everolimus.
a)
Systematic variations of substituents at different di-
versification points in phenyl rings with electron-
withdrawing or electron-donor groups in order to ob-
tain the maximal 3D structural enrichment as well as
information about the effects of electronic properties
in the antitumoral activity (compounds 1–7). In addi-
tion, compounds 7 and 8 were included with the aim
Among the chemical entities containing selenium that have
been identified, the chemical form diselenide stands out as an
appealing skeleton for development of new anticancer
13
agents. In particular diphenyl diselenide (PhSe) has been
2
widely studied and has proven activity as oxidative stress
14,15
16
protector,
presenting also cytotoxic activity. In this line
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of evaluating the effect of polymethoxy substitution
Page 2 of 7
The reaction of benzoyl chlorides (1–8) or hetero aroyl
chlorides (9–16) with potassium selenocyanate in a molar ratio
1:1 respectively, in dry acetone yielded the corresponding acyl
isoselenocyanates. These intermediates were used without
purific tion and the reaction in situ with bis (4-
aminophenyl)diselenide in acetone at room temperature gener-
ated the final compounds.
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2
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5
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7
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and biomimeticing combretastatin. This agent has
proven ability to induce cell death thought various
mechanisms including mitotic catastrophe and au-
tophagy induction in various adenocarcinoma-
2
1
derived cell lines.
b)
c)
Replacing the lateral phenyl group by heterocyclic
system (compounds 9–16) possessing a functional
group able to form hydrogen bonding as well as anti-
tumor activity against different tumor cell lines as fu-
2
2
23
24
ran, thiophene, benzothiophene, isoxazole, ben-
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27
zodioxole, quinoline and phenylquinoline deriv-
atives.
Carbonyl group was included as a cleavable linker
largely used in targeted drug strategies with intention
of modulating metabolism.
Scheme 1. Mechanism proposed for the synthesis of bis(4-
aminophenyl)diselenide.
Founded on these facts, the experience and results of our re-
search team in the field of diselenide and selenourea synthe-
17,20,28,29
sis
a general structure was designed, included in Fig-
Reaction times range from 1 h up 12 h and was then
quenched with cold water where potassium chloride from the
formation of isoselenocyanate is dissolved. At this point some
compounds precipitate along with some starting reagents.
Purification is performed by various methodologies such as
washing with ethyl ether or extraction with dichloromethane.
After purification yields range from 16% to 95%, this variabil-
ity in yield values may be due to acyl chloride reactivity or to
other physical properties of the reagents such as solubility in
the solvent used.
ure 1. In this entity the fragments incorporated are bis(4-
aminophenyl)diselenide coloured in green, selenourea moiety
coloured in red and lateral cyclic fragment coloured in blue.
All the synthesized compounds were screened for their cyto-
toxic activity against a panel of cancer cell lines and a prelim-
inary approach to the undergoing mechanism of action was
performed.
Given the absence of commercially available acyl chlorides
such as those needed for the synthesis of compounds 12, 13,
4 and 15, they were obtained by chlorination of their corre-
sponding carboxylic acids with thionyl chloride under reflux
conditions according to the literature.
1
We propose a two-phase reaction to obtain the novel
diselenides. With regard to the proposed mechanism (Scheme
), the first reaction could be a nucleophilic attack of the de-
scribed negative charge over the nitrogen atom over the car-
bonylic carbon following an acyl substitution mechanism to
yield the isoselenocyanate. Secondly the lone pair of electrons
of the nitrogen atom attached to the phenyl ring could perform
a nucleophilic attack over the isoselenocyanate carbon atom
leading to the formation of the final structure.
2
Figure 1. Structures of novel selenium containing compounds.
Sixteen novel compounds matching the designed scaffold
described in the introduction were synthesized. They can be
classified according to their lateral rings, carbocyclic (1–8)
and heterocyclic (9–16) and their structures are summarized in
Figure 1. The structures were confirmed by nuclear magnetic
1
13
resonance ( H NMR and C NMR), infrared spectroscopy
IR) and mass spectrometry (MS) and purity was confirmed by
(
Scheme 2. Proposed mechanisms for the two-phase reac-
tion to form the novel diselenides.
elemental microanalysis.
The novel organoseleno derivatives were synthesized start-
ing form bis(4-aminophenyl)diselenide which has been syn-
The sixteen novel compounds were screened for their effect
on cancer cell viability against a panel of six human cancer
cell lines: breast adenocarcinoma (MCF-7), colon carcinoma
HT-29), lymphocytic leukaemia (K-562), prostate cancer
PC-3), lymphoblastic leukaemia (CCRF-CEM) and lung
29
thesized according to previously published procedure. First
step of the proposed mechanism (Scheme 1) is an electrophilic
aromatic substitution that curses through an intermediate stabi-
lized by the four possible resonance structures leading to 4-
aminophenylselenocyante by deprotonation. Secondly, treat-
ment with NaBH4 to cause dimerization of the 4-
(
(
carcinoma (HTB-54). The cytotoxic potency was evaluated by
the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay according to the method-
aminophenylselenocyanate
aminophenyl)diselenide.
to
yield
bis(4-
28
ology previously described.
Initially each compound
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ACS Medicinal Chemistry Letters
Table 1. Average GI50 values (µM) and selectivity indexes.
1
2
3
4
5
6
7
8
9
Cell line
K-562
Code
R/Compound
a
b
MCF-7
9.76 × 10
1.30 × 10
3.24 × 10
5.60 × 10
4.95 × 10
1.51 × 10
9.10
PC-3
3.96 × 10
5.87 × 10
0.27
HTB-54
24.78
7.74
HT-29
9.12
6.61
9.02
5.27
8.95
17.09
21.97
3.21
6.66
6.58
5.90
3.78
6.82
7.71
11.45
CCRF-CEM
0.26
184B5
9.79
SI
BEAS-2B
SI
-
3
-2
-2
1
2
3
4
5
7
8
9
phenyl
25.47
25.56
32.25
>100
25.38
>100
9.24
1,003.20
7,005.30
253.18
2,264.07
1,660.78
69,673.53
0.70
20.01
4.17
4.01
7.44
2.51
9.72
9.49
5.45
0.80
7.64
7.13
13.59
>100
3.48
>100
0.81
0.54
0.14
0.24
0.19
0.48
0.59
0.86
0.14
0.13
0.15
0.60
n.d.c
0.38
n.d.
-3
4-methylphenyl
6.79
9.09
-2
4-nitrophenyl
28.61
30.84
13.16
20.22
16.03
6.35
0.96
8.21
-3
-2
4-methylthiophenyl
4-chlorophenyl
4.46
6.29 × 10
12.68
8.20
-3
-2
4.11
2.59 × 10
-4
-2
-2
3,5-dimethoxyphenyl
3,4,5-trimethoxyphenyl
furan-2-yl
7.85 × 10
8.16
9.35 × 10
10.49
6.41
6.64
0.62
2.33
6.40
26.71
8.71
6.39
4.25
12.09
7.93
5.51
84.87
9.02
4.58
0.58
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
isoxazol-2-yl
3.81
4.33
5.86
2.80
0.73
11
12
thiophene-2-yl
15.85
73.92
9.74
60.75
48.23
22.58
60.62
9.11
10.20
15.13
25.13
26.81
25.36
17.88
6.96
0.44
3-chloro-thiophene-2-yl
benzo[b]thiophene-2-yl
1,3-benzodioxole-5-yl
quinoline-3-yl
13.60
8.05
0.59
1
1
1
1
3
4
5
6
27.85
17.64
66.03
36.42
10.65
70.83
20.44
9.66
2.54
25.33
0.81
15.10
0.64
2-phenylquinoline-4-yl
14.72
>100
8.64
0.59
Reference drugs
-3
-2
-2
-2
-2
doxorubicin
cisplatin
etoposide
MSA
2.00 × 10
3.16
1.00 × 10
5.01
<1.00 × 10
9.64
0.10
7.94
31.62
n.d.
2.00 × 10
5.01
3.30 × 10
1.15
n.d.
n.d.
1.79
574
n.d.
n.d.
1.40
0.13
n.d.
n.d.
3.66
>13
n.d.
n.d.
1.03
1
19.95
1.28
0.63
n.d.
12.59
n.d.
1.26
1.08
2.45
3.54
a
b
SI :
calculated
as
GI50
(184B5)/GI50
(MCF-7)
SI :
calculated
as
GI50
(BEAS-2B)/GI (HTB-54)
50
was tested at five different concentrations from 0.01 µM to
00 µM after 72 h treatment; for some compounds further
fulfill the 10 µM GI value condition in at least five out of six
50
1
tested cell lines. Besides, GI50 values for compound 10 are in
the same order of magnitude as those exhibited by cisplatin
and MSA in those cell lines were data is available. However,
although compound 10 was cytotoxic showing LD50 values
under 10 µM in CCRF-CEM and MCF-7, this activity was not
accompanied by the required selectivity for breast adenocarci-
noma cells. If we draw the focus to MCF-7, PC-3 and CCRF-
CEM cell lines, all seven carbocyclic derivative compounds
dilutions were needed due to their potency. Doxorubicin,
etoposide and cisplatin were used as reference drugs. Me-
thylseleninic acid (MSA) was introduced as a selenium con-
3
0
taining molecule with promising cytotoxic profile.
Compound 6 was not screened due to its lack of solubility
under the assay conditions. As an approximation, compound 4
will be considered as an analogue in terms of electronic and
steric modulations.
presented GI values under 10 µM in all three cell lines.
50
In CCRF-CEM cells, compounds 4, 5 and 7 present GI50
As Table 1 illustrates, CCRF-CEM, PC-3 and MCF-7 were
generally the most sensitive cell lines. On the other hand, K-
values at least ten-fold lower than etoposide, cisplatin and
MSA. Comparison of those reference drugs in PC-3 cells
reveals that compounds 1, 2 and 3 satisfy these same criteria.
5
62 cell line was the less sensitive in the general picture. TGI
and LC50 values for every cell line are also provided in the
supplementary material (Tables S1 and S2).
When antiproliferative activities for structures with carbo-
cyclic core ending were analyzed in MCF-7 cells, compounds
–5 and 7 showed GI50 values at least two orders of magnitude
lower than those for cisplatin, etoposide and MSA. Due to this
data, a more potent reference drug is needed for comparison
such as doxorubicin. Besides, doxorubicin was highly selec-
Interestingly compounds with carbocyclic lateral rings (1–5,
1
7
and 8) and those with heterocyclic rings (9–16) displayed
different cytotoxic profiles. Whereas compounds with carbo-
cyclic substituted endings exhibited GI50 values in the nano-
molar range in MCF-7, PC-3, CCRF-CEM, no heterocyclic
derivatives matched this condition.
tive for the breast adenocarcinoma cell line (SI184B5/MCF-7
=
574), which makes it an ideal reference for compounds 1–5
and 7. Taking into account this benchmark, five out of the six
compounds (1, 2, 4, 5 and 7) equal the nanomolar range for
GI values. Moreover, selectivity index for mammary adeno-
carcinoma exceeded the one calculated for doxorubicin in up
The assay was also performed on two cell lines derived
from non-malignant cells, one mammary gland derived
(
(
184B5) and the other derived from bronchial epithelium
BEAS-2B). Selectivity index (SI) for breast and cancer cells
50
was calculated as the ratio of the GI50 values obtained for the
non-malign derived and the homolog cancer cell line.
to 120 folds (compound 7).
Taking into account that one of the goals of fragment-based
design strategy is to improve potency and reduce the toxicity,
we compared those results with the ones obtained for bis(4-
aminophenyl)diselenide itself. GI50 values (µM) for each cell
line were 0.75 (MCF-7), 0.80 (PC-3), 0.21 (HTB-54), <0.01
HT-29), 0.95 (K-562) and 5.28 (CCRF-CEM). Selectivity
wise the GI50 value obtained when the assay is performed in
84B5 cell cultures was of 6.29 µM leading to a SI of 8.39.
Moreover, there are also clear differences in SI values for
breast cell lines. Table 1 shows a clear range gap correlated
with ending core nature, with the exception of compound 8
that did not pair the level of selectivity shown by the rest of
the carbocyclic lateral ring compounds (1–5, 7 and 8).
(
In spite of this, GI50 values of compounds 9–15 are under
the 10 µM threshold for HT-29 treatment and in the same
order of magnitude as cisplatin. Furthermore, among the dif-
ferent heterocyclic substituents, furan and isoxazole (9–10)
1
Our target when SI was calculated as GI50 (BEAS-2B)/ GI50
(HTB-54) was a value of 3.44 as the GI50 for BEAS-2B was
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Page 4 of 7
0
.73 µM. In general terms, this compound could be considered
significance at a 1 µM concentration for derivative 7 (Figure
3B) and at 5 µM treatment for compound 2 (Figure 3A). Con-
comitant with this, a 10 µM treatment for 6 h with either com-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
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3
3
4
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4
4
4
4
4
4
4
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5
5
5
5
6
potent as it induces fifty percent cell growth inhibition under
10 µM concentrations in all six cell cancer cell lines. Although
HT-29 was the most sensitive cell line while CCRF-CEM was
the most resistant, when we have a look at SI values we can
state that the selectivity for this compound is in general scarce.
On the other hand, when we compare these values with the
results obtained for the novel diselenide derivatives we can
observe that compounds 1–5 and 7 have a striking selectivity
for MCF-7 cell line over the rest of the cancer cell lines and
over 184B5 cell line. From the data, we can conclude that the
introduction of the acylselenourea group plays a determinant
role in terms of selectivity when compared to the core struc-
ture, bis(4-aminophenyl)diselenide alone.
pound 2 or 7 augmented the number of cells in subG phase
1
(Figures 3C and 3D). Furthermore, both compounds 2 and 7
induced arrest in G /M in a time- and dose-dependent manner.
2
A very remarkable increment of G /M phase was evidenced
2
for the 24 h treatment even at the lowest concentration tested
(1 µM) peaking at 10 µM and 20 µM with over 40% of cells in
this phase (Figures 3A and B). Similar results were found for
time course analysis, after 10 µM treatment for 6 h the growth
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of G /M phase population was meaningful (Figures 3C and
2
D).
Some general considerations can be extracted from these re-
Though the number of synthesized compounds is little to
make solid structure-activity relationship statements, some
conclusions can be withdrawn from the results. When struc-
ture ending core is replaced by heterocyclic rings a loss of
antiproliferative activity and selectivity was observed. As for
the compounds bearing carbocyclic rings a trend might be
considered as electron-donating substituents enhance activity
in MCF-7 cells, peaking this effect with the presence of two
methoxy groups (compound 7). However, the presence of
three electron-donating groups seems to revert this effect,
presumably due to steric hindrance.
sults for derivative 2. The arrest of cells in G /M phase be-
2
comes significant at the lowest times and doses tested. This
population, even though building up seems to plateau over the
5 µM concentration for 24 h treatment (Figure 3A). If we
analyze the 10 µM treatment over the time, population of cells
in G /M phase peak for the 24 h treatment while in the 48 h
2
experiment this population seems to have reached its limit
value as cells start to then accumulate in the hypodiploid
subG phase.
1
This same phenomenon can be observed when compound’s
7
behaviour is studied, in this case, arrest in G /M phase peaks
2
At this point and considering all the previous results com-
pounds 2 and 7 were selected for further biological studies due
to their activity and selectivity specially against breast adeno-
carcinoma cells (MCF-7).
at 10 µM treatment for 24 h. Higher doses or longer times of
exposure cause cells to accumulate in subG phase and the
1
number of cells in S phase to drop below the 10% value. A
slight reversion of G /M arrest can be perceived for 20 µM
2
treatment for 24 h or 10 µM for 48 h.
Apoptosis and cell cycle arrest are targets for many anti-
cancer drugs such as doxorubicin. Influence of some selenium
containing compounds over those processes has been prov-
A
B
*
**
***
***
***
***
***
***
**
***
***
***
100
50
0
1
00
31,32
en.
As a preliminary approach to the mechanism of action,
***
***
***
*
**
*
**
G2/M
S
the cell cycle distribution and cell death status was studied in
MCF-7 cultures treated with either compounds 2 or 7. These
tests were performed by flow cytometry using Apo-Direct Kit
*
**
***
***
***
G2/M
S
***
***
***
***
***
G0/G1
SubG1
50
***
G0/G1
SubG1
*
**
*
**
***
*
(BD Pharmigen) based on the TUNEL technique under the
***
***
***
0
conditions described by the manufacturer. Treatment with
camptothecin 6 µM was used as positive control.
C
D
Figure 3. Cell cycle analysis after treatment with compounds 2 (A
and C) and 7 (B and D). Cells were treated with increasing con-
centrations of compounds 2 (A) and 7 (B) for 24 h. Figures C and
D correspond to treatments with 10 µM concentration for different
periods of time for compounds 2 (C) and 7 (D).
Figure 2. Compounds 2 and 7 induced cell death in a dose- and
time-dependent manner in MCF-7 cell cultures. Cells were treated
with increasing concentrations of compounds 2 and 7 for 24 h (A)
or with 10 µM concentration for different periods of time (B).
As shown in Figure 2, both compounds 2 and 7 increased
the percentages of subdiploid cells in the cultures in a dose-
and time-dependent manner. In fact, both derivatives induced
a significant increase in cell death when added at 5 µM con-
centration (Figure 2A). At 10 µM concentration, increase of
cell death was detected as soon as 6 h for both compounds
(Figure 2B).
With the aim of narrowing down the pathways implicated in
this cell death processes, MCF-7 cells were incubated with
compounds 2 and 7 in the presence or absence of the pan-
caspase inhibitor Z-VAD-FMK, the PI3K inhibitor wortman-
nin and chloroquine. As observed in Figure 4A, pre-
incubation with both wortmannin and chloroquine blocked cell
death caused by both compounds 2 and 7, reverting apoptotic
population levels to those present in the culture treated only
with wortmannin or chloroquine on their own. Rapamycin was
used as a reference autophagy inductor. This suggests that
As expected for compounds that induce cell death, when
cell cycle distribution was studied, the number of the hypodip-
loid sub G cell population was increased. This result reaches
1
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ACS Medicinal Chemistry Letters
autophagy is involved in the cell death induced by these com-
methylphenyl and 3,5-dimethoxyphenyl moieties respectively,
were selected for further biological studies due to their high
potency and selectivity against breast adenocarcinoma cells.
Cell cycle analysis through flow cytometry revealed that cell
1
2
3
4
5
6
7
8
9
pounds. On the other hand, pre-incubation with Z-VAD-FMK
could not prevent compounds 2 and 7 induced cell death (Fig-
ure 4B) which rules out caspase-dependent cell death.
death as well as G /M phase arrest are both time and dose
2
A
B
dependent. Moreover, in the presence of wortmannin or chlo-
roquine cell death was reverted to negative control levels
indicating that autophagy might be implicated in cell death
mechanism. This hypothesis is confirmed when autophagy
marker analysis is performed by western blot for LC3B and
Beclin1.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Chemical synthesis, characterization of target compounds; proto-
cols of biological assays, cytotoxicity complete data and cell
cycle, cell death and protein analysis exemplification (PDF).
Figure 4. Cell death induced by compounds 2 and 7 is blocked by
wortmannin and chloroquine but not by caspase inhibitor Z-VAD-
FMK. MCF-7 cells were pre-incubated for 1 h with 50 mM of the
pan-caspase inhibitor Z-VAD-FMK (A), 100 nM of the autophagy
inhibitor wortmannin and 10 µM of chloroquine (B) or in absence
of inhibitor prior the addition of 10 µM 2 and 7 or DMSO (con-
trol) for 24 h. 30 µM Rapamycin was used as a reference autoph-
agy control. Cells were then processed with the Apo-Direct kit
and analyzed by flow cytometry.
AUTHOR INFORMATION
Corresponding Author
* E-mail: sanmartin@unav.es
As suggested by the inhibitor assay results and with the aim
of confirming that autophagy is involved in the mechanism of
action by which compounds 2 and 7 cause cell death, we pro-
ceeded to the evaluation by western blot of autophagy markers
Funding Sources
3
3
This research was founded in part by "la Caixa" Banking Founda-
tion and Asociación de Amigos de la Universidad de Navarra (a
grant to P. Garnica), by plan de Investigación de la Universidad
de Navarra, PIUNA (Ref 2014–26) as well as Fundación Caja
Navarra -UNED.
(
Beclin1 and LC3B). MCF-7 cells were treated with 10 µM
of compounds 2 and 7 for 24 h. Formation of autophagosomes
starts with an initial nucleation step that requires the ULK1
complex that includes Beclin1 which, as shown in Figure 5 is
upregulated by treatment with both selected compounds. Con-
sistent effects are observed when LC3B-I and LC3B-II were
studied.
ACKNOWLEDGMENT
The authors wish to express their gratitude to University of Calga-
ry Chemistry Department and to Thomas G. Back for the oppor-
77
tunity to perform the Se NMR experiments.
ABREVIATIONS
GI , concentration that reduces growth by 50% compared to
50
control; TGI, concentration that completely inhibits cell growth;
LC , concentration that kills 50% of cells; SI, selectivity index;
50
N.d., not determined.
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