Organoseleno cytostatic derivatives: Autophagic cell death with AMPK and JNK activation

Article abstract of DOI:10.1016/j.ejmech.2019.04.074

Selenocyanates and diselenides are potential antitumor agents. Here we report two series of selenium derivatives related to selenocyanates and diselenides containing carboxylic, amide and imide moieties. These compounds were screened for their potency and selectivity against seven tumor cell lines and two non-malignant cell lines. Results showed that MCF-7 cells were especially sensitive to the treatment, with seven compounds presenting GI₅₀ values below 10 μM. Notably, the carboxylic selenocyanate 8b and the cyclic imide 10a also displayed high selectivity for tumor cells. Treatment of MCF-7 cells with these compounds resulted in cell cycle arrest at S phase, increased levels of pJNK and pAMPK and caspase independent cell death. Autophagy inhibitors wortmannin and chloroquine partially prevented 8b and 10a induced cell death. Consistent with autophagy, increased Beclin1 and LC3-IIB and reduced SQSTM1/p62 levels were detected. Our results point to 8b and 10a as autophagic cell death inducers.

Full text of DOI:10.1016/j.ejmech.2019.04.074

European Journal of Medicinal Chemistry 175 (2019) 234e246
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Organoseleno cytostatic derivatives: Autophagic cell death with AMPK
and JNK activation
,
,
,
, b
Pablo Garnica a b, Ignacio Encío b c, Daniel Plano a b, Juan A. Palop a
,
Carmen Sanmartín a
, b, *
a
ꢀ
ꢀ
Universidad de Navarra, Facultad de Farmacia y Nutricion, Departamento de Tecnología y Química Farmaceuticas, Campus Universitario, 31080,
Pamplona, Spain
b
ꢀ
Instituto de Investigacion Sanitaria de Navarra (IdiSNA), Irunlarrea 3, E-31008, Pamplona, Spain
c
~
Department of Health Sciences, Public University of Navarra, Avda. Baranain s/n, E-31008, Pamplona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Selenocyanates and diselenides are potential antitumor agents. Here we report two series of selenium
derivatives related to selenocyanates and diselenides containing carboxylic, amide and imide moieties.
These compounds were screened for their potency and selectivity against seven tumor cell lines and two
non-malignant cell lines. Results showed that MCF-7 cells were especially sensitive to the treatment,
Received 18 February 2019
Received in revised form
17 April 2019
Accepted 29 April 2019
Available online 4 May 2019
with seven compounds presenting GI50 values below 10 mM. Notably, the carboxylic selenocyanate 8b
and the cyclic imide 10a also displayed high selectivity for tumor cells. Treatment of MCF-7 cells with
these compounds resulted in cell cycle arrest at S phase, increased levels of pJNK and pAMPK and caspase
independent cell death. Autophagy inhibitors wortmannin and chloroquine partially prevented 8b and
10a induced cell death. Consistent with autophagy, increased Beclin1 and LC3-IIB and reduced SQSTM1/
p62 levels were detected. Our results point to 8b and 10a as autophagic cell death inducers.
Published by Elsevier Masson SAS.
Keywords:
Autophagy
Cancer
Cyclic imide
Diselenide
Selenocyanate
1. Introduction
During the past decade, extensive studies of selenium com-
pounds have demonstrated their antitumor and chemopreventive
Despite recent advances in development of anticancer agents,
this illness remains a leading cause of disease-related death
worldwide [1]. Due to their effect on several survival or death
signaling pathways that may decide the fate of cancer cells [2,3],
therapies based on autophagy targeted agents are now in the focus
of a wide range of researchers. Among the signaling pathways
implicated in these processes, JNK activation has been proven to
participate in multiple autophagic events such as Beclin1 expres-
sion and autophagic-mediated cell death [4,5]. Energetic stress has
also been described to be a trigger for autophagy [6]; in this
context, AMPK has been proven to have an essential role promoting
autophagy by inhibiting the mTORs regulatory cascade [7]. The
phosphorylation of AMPK and JNK in autophagy-mediated cell
death has been previously described in breast adenocarcinoma
[8,9] and several other cancer types such as myeloma [10] and
leukemia [11].
activities in a vast array of experimental models [12]. These de-
rivatives interfere with the redox homeostasis and signaling of
cancer cells. The mechanism by which they cause their effect
include alterations in cell cycle checkpoints, proliferation, senes-
cence, and death pathways [13]. In addition, some selenium de-
rivatives such as selenite, selenocysteine and Se-allylselenocysteine
play an effective role in cancer treatment as autophagy inducers
and modulators of the JNK signaling pathway [14e17].
Many chemical entities containing selenium, with potent anti-
tumor activity, have been explored by the scientific community.
Among them, selenocyanate [18] and diselenide [19] moieties have
been highlighted due to their interesting antitumor properties. In
this line of investigation two effective derivatives, the diselenide
analog bis(4-aminophenyl)diselenide (0a) and the corresponding
selenocyanate (0b), were recently identified in our laboratory [20].
In order to obtain a second generation of selenium structures with
improved activity, selectivity and water solubility these compounds
have been used as a starting point to continue with their modula-
tion [21]. It is remarkable that one of the limitations of the selenium
derivatives is their poor water solubility which is detrimental for
* Corresponding author. Department of Pharmaceutical Technology and Chem-
istry, University of Navarra, Irunlarrea, 1, E-31008, Pamplona, Spain.
E-mail address: sanmartin@unav.es (C. Sanmartín).
https://doi.org/10.1016/j.ejmech.2019.04.074
0223-5234/Published by Elsevier Masson SAS.

P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
235
their bioavailability and drug development. To obtain compounds
with improved pharmacokinetic properties, modifications in the
hydrophobic scaffold that improve water solubility are usually
assayed. As an example of this strategy, introduction of a hydroxyl
group in the phenyl ring of the natural product camptothecin has
been shown to counteract efficiently this drawback [22]. Thus, a
useful option when using this approach is to incorporate polar
functional groups, such as acidic or basic groups. These fragments
enable the possibility of salt formation and therefore might
enhance water solubility [23,24]. In this study, substantial efforts
had been directed towards finding different chemical scaffolds that,
while maintaining the cytotoxic activity, should increase the hy-
drophilicity further contributing to the solubility optimization.
Among the structural features incorporated, we surmised that the
introduction of the carboxylic core could be a logical approach for
improving its aqueous solubility. This moiety is present in widely
described organoselenium compounds such as 3,30-dis-
elenodipropionic acid [25]. In addition, the dicarboxylic acids are of
special importance because of their versatility in the preparation of
the corresponding cyclic imide homologs which are widely
described in the literature as potential antitumor agents [26,27].
Taking into consideration the facts stated above and our previ-
ous work in the field of new selenium compounds as antitumor
agents [21,28e33], the present study aimed to synthesize seleno-
cyanates and diselenides containing carboxylic, amide and imide
moieties. The general outline of this series of compounds is pre-
sented in Fig. 1. Variations were made in the group linked to the
carboxy feature through selection of different cyclic symmetric
anhydrides commercially available such as maleic, succinic,
phthalic … Finally, with the objective of widening the structural
variations, a new anhydride was synthesized through the Diels-
Alder cycloaddition. The rationality behind this proposal is the
synthesis of a structural analog of norcantharidin, a well-known
active antitumor autophagy inducer [34].
work can be categorized into two different subseries according to
their selenium moiety:
- Diselenide derivatives containing carboxylic and amide or imide
moieties (1a-3a, 5a, 7a-11a).
- Selenocyanate derivatives containing carboxylic and amide
moieties (1b-8b).
The resulting compounds were numbered according to the
corresponding anhydride used as starting material.
Derivatives were synthesized following the synthetic path
depicted in Fig. 2. The corresponding anhydrides were reacted with
either
bis(4-aminophenyl)diselenide
(0a)
or
4-
aminophenylselenocyanate (0b) in acetone at room temperature
for 8 h up to 24 h. The formed precipitate was filtered and washed
with n-hexane or ethyl ether to yield the final compounds. The
mechanism proposed for this reaction is a nucleophilic acyl sub-
stitution illustrated in Fig. 3A. Moreover, all our attempts to
generate derivatives 4a and 6a were unsuccessful since the reaction
of the corresponding anhydrides with 0a in different conditions
(temperature, solvents and catalyst) failed to yield the desired
compound. Unfortunately, the alternative strategy of reducing their
selenocyanate analogs (4b and 6b) to obtain derivatives 4a and 6a
under different conditions only resulted in the degradation of the
start-up derivatives. To obtain the cyclic imides (9a-11a), the cor-
responding amidic acids (1a, 2a, 5a) were heated in presence of
acetic anhydride and sodium acetate. This reaction probably starts
by a deprotonation of the carboxylic group, followed by the
nucleophilic attack of the oxygen to the carbonyl group on the
acetic anhydride followed by a subsequent intramolecular cycliza-
tion to yield the final cyclic imides. The reaction was quenched with
water causing the prompt precipitation of the desired compound.
The proposed mechanism of reaction to yield the cyclic imides is
exemplified in Fig. 3B.
2. Results and discussion
2.2. Biology
2.1. Chemistry
2.2.1. Cytotoxicity and antiproliferative activity
The cytotoxic potential of the seventeen synthesized com-
pounds was evaluated against a panel of cell lines including seven
The seventeen compounds synthesized and presented in this
Fig. 1. Structures of novel selenium containing compounds.

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P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
Fig. 2. General procedure of synthesis.
Fig. 3. Mechanism proposed for the synthesis diselenide derivatives. Mechanism proposed for the synthesis of compounds 1a-3a, 5a and 7a-8a (A). Mechanism proposed for the
synthesis of cyclic imide derivatives 9a-11a (B).
different cancer cell lines and two other cell lines derived from non-
malignant tissue. Evaluation was performed at 48 h treatment
the figure, MCF-7 cells were the most sensitive cells toward the
tested derivatives. In fact, seven compounds (1b, 2b, 4b, 8a, 8b, 9a
and 10a) reduced cell growth to less than 50% when assayed at
following
the
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) methodology as previously
described [33]. The cancer cell lines included in the panel were PC-3
(prostatic adenocarcinoma); HTB-54 (lung carcinoma), and HT-29
(colon carcinoma); MOLT-4 and CCRF-CEM (acute lymphoblastic
leukemia); K-562 (chronic myelogenous leukemia) and MCF-7
(breast adenocarcinoma). The selected cell lines derived from
non-malignant tissue were 184B5 and BEAS-2B. Cisplatin was used
as positive control. In addition, the parent compounds bis(4-
aminophenyl)diselenide (0a) and 4-aminophenylselenocyanate
(0b) were tested as a reference to identify whether the second
generation compounds accomplished the objective of improving
potency and selectivity.
10 mM in these cells. Some compounds also matched this threshold
in PC-3, CCRF-CEM, HTB-54, MOLT-4 and HT-29 cells. However,
none of the derivatives was able to reduce the cell growth effec-
tively in K-562, the most resistant cell line to the treatments.
Interestingly, compounds 1b, 2b, 4b, 8a, 8b, 9a and 10a did not
significantly affect cell growth in 184B5 cells, thus suggesting a
potential selectivity of the compounds for breast cancer cells.
Consequently, those seven compounds were further analysed in full
dose-response curves in every cell line. GI50, TGI and LC50 values
were calculated form the curves and are shown in Table 1. Selec-
tivity for tumor cells was estimated according to the formulas GI50
(184B5)/GI50 (MCF-7) and GI50 (BEAS-2B)/GI50(HTB-54) (Table 2).
As shown in Tables 1 and 2, derivatives 8a, 9a and 10a exhibited
To narrow down the number of derivatives moving on to the full
dose-response cytotoxic profiling assay, a two-dose concentration
GI50 values under 10 mM in three of the tested cancer cell lines.
(100
mM and 10
mM) screening was first performed. The results
Besides, compounds 1b, 2b, 4b, 8b and 10a were highly selective for
obtained for the 10
mM treatment are shown in Fig. 4. As shown in
tumor cells in the breast model. Remarkably, though highly

P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
237
Fig. 4. Heat map representing average percentages of cell growth for every tested structure at a 10 mM concentration for 48 h
* NCI data (http://dtp.nci.nih.gov).
cytotoxic, parent compounds 0a and 0b showed low selectivity for
breast cancer cells (SI < 9). Therefore, we decided to focus on the
effects of these compounds in the breast cancer cell line. When
ranked in terms of potency and selectivity for breast cancer cells, a
clear gap established 10a, a compound with a nanomolar GI50 value
and a staggering SI, as the leader structure. Despite less cytotoxic,
1b and 8b were also highly selective. Among them, the analog
derivative of norcantharidin (8b) was selected in order to evaluate
whether this structure was able to mimic norcantharidin's effect
and induce autophagy. As a result, derivatives 8b and 10a were
selected to further analyse their mechanism of action in MCF-
7 cells. As only the highest concentration tested led to negative
growth values as exemplified on Fig. 5, these results uncover a
mainly cytostatic profile.

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P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
Table 2
Average values of GI50, TGI and LD50 (mM) for 48 h treatment and calculated SI.
Code 184B5
SIa
BEAS-2B
GI50
SIb
GI50
TGI
GI50
TGI
LD50
1b
2b
4b
8a
8b
9a
10a
0a
0b
>100
>100
>100
8.93
>100
>100
>100
>100
>100
>100
>51.73
>12.98
15.46
10.45 42.82
4.43 24.63
52.93 >100
1.05 4.43
15.82 51.02
80.15 0.22
>100
>100
<0.39
<0.16
11.25 15.67 1.19
23.19
15.53 0.04
77.25 0.33
73.77 0.24
61.91 0.15
77.25 91.19 >100
40.51 63.07 86.29 6.74
60.78 >100
6.29
0.88
1.97
43.62
20.86
0.88
>100
6.3 ꢀ 104 0.75
14.03 30.44 8.39
1.06 1.27 0.88
0.73
7.92
1.09
3.44
14.69
27.51 9.06
a
Selectivity index (SI) calculated as GI50 (184B5)/GI50 (MCF-7).
SI calculated as GI50 (BEAS-2B)/GI50(HTB-54).
b
Besides, comparison between 10a and 0a clearly showed a great
enhancement in selectivity for the second generation compound. In
fact, SI was 7,000 times higher for compound 10a than for 0a, its
parent compound. This effect was less notorious when compound
8b was compared with 0b. However, both 10a and 8b succeeded in
increasing the selectivity towards the cancer cells, thus meeting
one of our goals.
The importance of estrogen receptors (ER) in cell cycle and cell
proliferation in breast cancer cells, as well as the crucial role of
estradiol synthesis pathways has been widely described. In fact,
antiestrogens have been found to repress transcription of several
ERa target genes in MCF-7 cells, specifically in S phase [35]. Besides,
when MCF-7 cell cultures were exposed to a genotoxic agent higher
levels of DNA damage in S- and G2/M-enriched cultures correlated
with higher levels of CYP1A1 y CYP1B1 [36]. MCF-7 is an ER
a
expressing cell line. Therefore, to compare we decided to test 8b
and 10a in MDA-MB-231, a breast cancer cell line non-expressing
ERa. Obtained results for MDA-MB-231 cells are shown in Fig. 5.
As shown in the figure, 8a and 10a dose-response curves in MDA-
MB-231 differ from those obtained in MCF-7 cells. Moreover,
lying in the micromolar range GI50, TGI and LD50 values for 8b
(GI50 ¼ 33.61
m
M, TGI ¼ 61.92
m
m
M and LD50 ¼ 83.92 M) and 10a
m
(GI50 ¼ 13.65
mM, TGI ¼ 51.49
M
and LD50 > 100 mM) are also
higher than in MCF-7 cells. These data suggest that ER signalling
and/or estradiol metabolism play a relevant role in cytostatic effect
displayed by 8b and 10a in MCF-7 cells.
In terms of structure-activity relationship, most diselenide
structures containing carboxylic moieties were discarded in the
screening process. For instance, when comparing 8b with its dis-
elenide homolog a complete loss of selectivity could be observed.
On the other hand, if we stablish a comparison between carboxylic
derivatives and their cyclic imide homologs data suggests that this
modification was crucial for both potency and selectivity.
2.2.2. Compounds 8b and 10a induce cell cycle arrest in S phase
and cell death
Many selenium containing compounds involve cell cycle regu-
lation among their therapeutic effects [13]. Therefore, as a first
approach to the mechanism of action we studied the effect of 8b
and 10a on cell cycle. With this purpose, the cell cycle status of
MCF-7 cell cultures treated with different concentrations of 8b and
10a and for different time points was determined by flow cytom-
etry. Camptothecin was used as positive control. As shown in the
Fig. 6, both a reduction in the number of G0/G1 cells and a signifi-
cant increase in the percentage of cells in S phase were detected for
both compounds even at the lowest concentration (10 mM) and the
shortest time tested (24 h). This result, indicative of S phase arrest
was both dose (Fig. 6A) and time (Fig. 6B and C) dependent.
To study the role of apoptosis in the induction of cell death by 8b

P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
239
Fig. 5. Dose response curves obtained for 8b (A) and 10a (B) in MCF-7 and MDA-MB-231 cell lines.
Fig. 6. Cell cycle phase distribution of MCF-7 cell cultures after treatment with compounds 8b and 10a. (A) Dose-dependent induction of cell cycle arrest after 48 h treatment
with compounds 8b and 10a. Time-course analysis of cell cycle distribution at 10 M (B) and 40 M (C) of 8b and 10a. Camptothecin (6 M) was employed as a positive control.
Results are expressed as a mean SEM of at least three independent experiments performed in duplicate. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control cells.
m
m
m
and 10a, MCF-7 cells were incubated in the presence of increasing
concentrations of 8b and 10a for 48 h. Then, the apoptotic status of
the cells was studied by TUNEL. As shown in Fig. 7A, when tested at
treatment of the cultures with either an autophagy inhibitor
(wortmannin, chloroquine) [37e39] or a pan-caspase inhibitor (Z-
VAD-FMK) on the induction of cell death by these compounds. As
shown in Fig. 8, pre-treatment of the cells with the PI3K inhibitor
wortmannin or the lysosomal inhibitor chloroquine led to a sig-
nificant reduction in the number of dead cells in the cultures after
exposure to compounds 8b and 10a. However, pre-incubation of
the cultures with Z-VAD-FMK could not prevent 8b and 10a-
induced cell death. These results suggest that autophagy is the way
by which 8b and 10a cause their effect.
concentrations higher than 40
significant increase in the number of death cells (subdiploid cells).
Fig. 7B shows that at 40 M concentration the induction of cell
mM, both compounds induced a
m
death could be detected as soon as 24 h.
2.2.3. Compounds 8b and 10a induce autophagy-mediated cell
death and AMPK/JNK pathway activation
To further analyse the molecular mechanism by which 8b and
10a reduced MCF-7 cell viability, we explored the effect of pre-
To further confirm the involvement of autophagy in 8b and 10a
induced cell death the levels of expression of the autophagy

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P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
Fig. 7. Compounds 8b and 10a induced cell death in a dose- and time-dependent manner in MCF-7 cell cultures. Cells were treated with increasing concentrations of com-
pounds 8b and 10a for 48 h (A) or at 40 M concentration for different periods of time (B). Camptothecin was used as positive control. Results are expressed as a mean SEM of at
m
least three independent experiments performed in duplicate. *p < 0.05 and **p < 0.01 compared to control cells.
Fig. 8. Cell death induced by compounds 8b and 10a is partially blocked by wortmannin or chloroquine but not by caspase inhibitor Z-VAD-FMK. Cell death determination in
MCF-7 cell cultures pre-incubated with (A) 100 nM wortmannin, 10
mM chloroquine or (B) 50 mM Z-VAD-FMK before treatment with 80
mM 8b, 80 mM 10a or 30 mM rapamycin for
48 h. Rapamycin was used as reference autophagy control at 30
mM treatment. Results are expressed as a mean SEM of at least three independent experiments performed in
duplicate. *p < 0.05 and **p < 0.01 compared to the control.
markers Beclin-1 and LC3B were determined. Autophagic flux was
also assessed by testing SQSTM1/p62 [40]. As shown in Fig. 9, when
MCF-7 cells were treated with 80 mM of either compound for 48 h,
However, in the specific context of breast adenocarcinoma cells
PI3K activation does not necessarily lead to autophagy suppression
[45]. Moreover, specific activation of the isoform 1 of AKT in breast,
neck and head carcinomas has been shown to interfere with their
metastatic progression [46,47]. Whether AKT-mediated repression
of metastasis would represent an additional beneficial effect of the
treatment with compounds 8b and 10a merits further research.
Beclin-1, LC3BeI and LC3B-II were augmented while SQSTM1/p62
was downregulated thus confirming autophagy. Since the activa-
tion of AMPK and JNK have been shown to play a role in autophagy-
mediated cell death [6,41], AMPK and JNK phosphorylation were
also studied. As shown in Fig. 9, both 8b and 10a induced AMPK and
JNK phosphorylation.
Inhibition of mTORC1 after AMPK activation is a main step in
AMPK-mediated autophagy. The PI3K/AKT pathway also has a
regulatory effect on mTOR and therefore in autophagy. This
pathway is commonly deregulated in cancer cells [42]. Aimed to
analyze the effect of 8b and 10a on PI3K and AKT signaling, we
determined the phosphorylation status of both, the PI3K catalytic
3. Conclusion
To sum up, nine diselenide (1a-3a, 5a, 7a-11a) and eight sele-
nocyanate monoamidic acids (1b-8b) were synthesized with high
yields. A screening in a panel of cancer cell lines revealed that MCF-
7 was the most sensitive among the tested ones to treatment with
these compounds. Due to their high potency and stunning selec-
tivity towards MCF-7 cells, derivatives 8b and 10a emerged as the
most promising structures. Full dose response curves in MCF-7 cells
showed up a cytostatic effect for these compounds. Further analysis
uncovered their ability to induce both S phase arrest and a caspase-
independent cell death program in these cells. Besides,
subunit p110
phospho-p110
a
a
and AKT (Ser473). As shown in Fig. 10, increased
and phospho-AKT (Ser473) were detected indi-
cating activation of the pathway. PI3K activation is usually related
to tumor migration enhancement [43] and has been reported to be
associated with inhibition of autophagy and tumorgenesis [44].

P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
241
Fig. 9. Beclin-1, p62, LC3BeI, LC3B-II, p-AMPK and p-JNK proteins were determined by western blot. (A) A representative experiment is exemplified. (B) Aggregate results
(mean SEM; n ¼ 3) expressed as fold induction relative to control cells. *p < 0.05, **p < 0.01 and ***p < 0.001.
Fig. 10. PI3K and p-AKT proteins were determined by western blot. (A) A representative experiment is exemplified. (B) Aggregate results (mean SEM; n ¼ 3) expressed as fold
induction relative to control cells. *p < 0.05, **p < 0.01 and ***p < 0.001.
wortmannin and chloroquine partially prevented induction of cell
death, thus suggesting autophagy. Increased levels of Beclin1 and
LC3-IIB and reduced levels of SQSTM1/p62 in MCF-7 cells after
exposure to 8b or 10a also supported autophagy. Since pJNK
upregulation and AMPK phosphorylation were also detected after
the treatments, the modulation of the AMPK and JNK signaling
pathways seems to be involved in the induction of autophagy by 8b
and 10a. Finally, the phosphorylation of both, AKT and the PI3K
all final compounds was 95% or higher. Chemicals were purchased
from E. Merck (Darmstadt, Germany), Panreac Química S.A. (Mon-
tcada i Reixac, Barcelona, Spain), Sigma-Aldrich Quimica, S.A.
(Alcobendas, Madrid, Spain) and Acros Organics (Janssen Pharma-
ceuticalaan, Geel, Belgium).
4.1.2. General procedure for the synthesis of compounds 1a-3a, 5a
and 7a-8a
catalytic subunit p110a were also detected. Whether the activation
Bis(4-aminophenyl)diselenide (1 mmol) was dissolved in of dry
acetone (10 mL) and the corresponding anhydride (2.1 mmol) then
added. The reaction was then stirred for a variable time of 8 h up to
24 h at room temperature. Then reaction was quenched with water,
compound was filtered and purified by stirring or washing with
ethyl ether.
of the PI3K/AKT pathway by 8b and 10a in MCF-7 cells restricts
their invasive capacity and represents an extra beneficial effect of
these compounds for cancer treatment deserves to be studied
profoundly.
In order to assign the chemical shifts in NMR spectroscopy the
following assignment has been done: central rings A and A0,
external fragments B and B’ (Fig. 11).
4. Experimental
4.1. Chemistry
4.1.1. Material and methods
4.1.2.1. (2Z,20Z)-4,4’-[diselenodiylbis(benzene-4,1-diylimino)]bis(4-
oxobut-2-enoic acid) (1a). From maleic anhydride. Conditions:
8 h at room temperature. The product was kept under stirring with
water (25 mL) for 2 h, filtered and then washed with ethyl ether
(2 ꢀ 25 mL). A yellow powder was obtained. Yield: 68.9%. Mp:
186e186.5 ꢁC. IR (KBr) cmꢂ1: 3305, 3193 (NeH), 1723 (C¼O car-
boxylic acid), 1623 (C¼O, amide), 818 (SeeSe). 1H NMR (400 MHz,
Proton (1H) and carbon (13C) NMR spectra of every compound
and selenium (77Se) NMR spectra of representative derivatives
were recorded on a Bruker Advance Neo 400 Ultrashield™ spec-
trometer (Rheinstetten, Germany) using DMSO‑d6 as solvent. IR
spectra were recorded on a Thermo Nicolet FT- IR Nexus spectro-
photometer using KBr pellets for solid samples. Elemental analysis
was performed on a LECO CHN-900 Elemental Analyzer. Purity of
DMSO‑d6) d: 13.01 (bs, 2H, COOH), 10.54 (s, 2H, NH), 7.60 (d, 4H,

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P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
NMR (400 MHz, DMSO‑d6) d: 12.76 (bs, 2H, COOH), 10.09 (s, 2H,
NH), 7.62 (d, 4H, A þ A0, J2-3 ¼ J6-5 ¼ 8.5 Hz, H2þH6), 7.55 (d, 4H,
A þ A0, J3-2 ¼ J5-6 ¼ 8.5 Hz, H3þH5), 4.19 (s, 4H, B þ B0, H1), 4.17 (s,
4H, B þ B0, H2). 13C NMR (100 MHz, DMSO‑d6)
d: 172.68 (COOH),
168.99 (C¼O), 139.56 (A þ A0, C4), 133.94 (A þ A0, C2þC6), 125.01
(A þ A0, C1), 121.22 (A þ A0, C3þC5), 71.38 þ 69.11 (B þ B’, C2þC1). MS
[m/z (% abundance)]: 93 (95), 172 (100). Elemental analysis calcu-
lated (%) for C20H20N2O8Se2: C: 41.83, H: 3.51, N: 4.88; found: C:
41.83, H: 3.82, N: 5.19.
4.1.2.5. 2,2’-[(Diselenodiyldibenzene-4,1-diyl)dicarbamoyl]bis(cyclo-
hexanecarboxylic acid) (7a). From cis-1,2-cyclohexanecarboxylic
anhydride. Conditions: 24 h at room temperature. The product
was kept under stirring with water (25 mL) for 2 h, filtered, then
stirred with ethyl ether (100 mL) for 24 h and then filtered. A light
brown powder was obtained. Yield: 97.7%. Mp: 150e151 ꢁC. IR (KBr)
cmꢂ1: 3307 (NH), 1698 (C¼O carboxylic acid), 1665 (C¼O, amide),
Fig. 11. General NMR assignation for compounds of series a.
A þ A0, J2-3 ¼ J6-5 ¼ 8.8 Hz, H2þH6), 7.57 (d, 4H, A þ A0, J3-2 ¼ J5-
¼ 8.8 Hz, H3þH5), 6.46 (d, 2H, B þ B0, J1-2 ¼ 12.0 Hz, H1), 6.31 (d,
6
2H, B þ B0, J2-1 ¼12.0 Hz, H2). 13C NMR (100 MHz, DMSO‑d6)
d:
166.76 (COOH), 163.17 (C¼O), 138.71 (A þ A0, C4), 132.88 (A þ A0,
C2þC6), 131.33 þ 130.23 (B þ B0, C1þC2), 124.19 (A þ A0, C1), 120.02
(A þ A’, C3þC5). MS [m/z (% abundance)]: 172 (100), 344 (25).
Elemental analysis calculated (%) for C20H16N2O6Se2 ∙ 2H2O: C:
41.83, H: 3.51, N: 4.88; found: C: 41.54, H: 3.53, N: 4.80.
820 (SeeSe). 1H NMR (400 MHz, DMSO‑d6)
d: 11.88 (bs, 2H, COOH),
9.87 (s, 2H, NH), 7.55 (d, 4H, A þ A0, J2-3 ¼ J6-5 ¼ 8.8 Hz, H2þH6), 7.50
(d, 4H, A þ A0, J3-2 ¼ J5-6 ¼ 8.8 Hz, H3þH5), 2.93 (d, 2H, B þ B0, J1-
¼ 5.4 Hz, H1), 2.70e2.56 (m, 2H, B þ B0, Hchex), 2.09 (d, 2H,
CHchex
B þ B0, JCHchex-1 ¼5.4 Hz, Hchex), 1.98 (d, 2H, B þ B0, J ¼ 8.9 Hz, Hchex),
1.83e1.57 (m, 6H, B þ B0, 3Hchex), 1.48e1.24 (m, 6H, B þ B0, 3Hchex).
13C NMR (100 MHz, DMSO‑d6)
d
: 175.56 (COOH), 173.36 (C¼O),
4.1.2.2. 2,2’-[(Diselenodiyldibenzene-4,1-diyl)dicarbamoyl]bis(ben-
zoic acid) (2a). From phthalic anhydride. Conditions: 12 h at room
temperature. The product was kept under stirring with water
(25 mL) for 1 h, filtered and then washed with ethyl ether
(2 ꢀ 25 mL). A yellow powder was obtained. Yield: 26.7%. Mp:
154e155 ꢁC. IR (KBr) cmꢂ1: 3282 (NeH),1708 (C¼O carboxylic acid),
140.42 (A þ A0, C4), 133.78 (A þ A0, C2þC6), 123.66 (A þ A0, C1),
120.26 (A
þ
A0, C3þC5), 43.00
þ
42.44 (B
þ
B0, C1þC2),
28.13 þ 25.62þ24.47 þ 22.78 (B þ B’, C3þC4þC5þC6). MS [m/z (%
abundance)]: 81 (70), 172 (100), 344 (25). Elemental analysis
calculated (%) for C28H32N2O6Se2: C: 51.70, H: 4.96, N: 4.31; found:
C: 52.06, H: 5.09, N: 4.71.
1657 (C¼O, amide), 819 (SeeSe). 1H NMR (400 MHz, DMSO‑d6)
d:
11.02 (s, 2H, NH), 7.85 (d, 2H, B þ B0, J3-4 ¼ 8.8 Hz, H3), 7.72e7.66 (m,
4H, B þ B0, H4þH6), 7.63e7.52 (m, 10H, A þ A0, H2þH3þH4þH5,
4.1.2.6. 3,3'-[(Diselenodiyldibenzene-4,1-diyl)dicarbamoyl]bis(7-
oxabicyclo[2.2.1]hept-5-ene-2-carboxylic acid) (8a). From 3,6-
epoxy-1,2,3,6-tetrahydrophthalic anhydride obtained by the
classic procedure described for a Diels-Alder reaction using furan
and maleic anhydride as reagents to yield the Diels-Alder adduct.
Conditions: 24 h at room temperature. The product was kept under
stirring with water (25 mL) for 2 h, filtered, then stirred with ethyl
ether (100 mL) for 24 h and then filtered. A yellow powder was
obtained. Yield: 22.5%. Mp: 126e127 ꢁC IR (KBr) cmꢂ1: 3299 (NH),
1706 (C¼O carboxylic acid), 1669 (C¼O, amide), 819 (SeeSe). 1H
B þ B0, H5), 3.39 (bs, H2Oþ2COOH). 13C NMR (100 MHz, DMSO‑d6)
d:
171.22 (COOH), 168.50 (C¼O), 140.48 (A þ A0, C4), 134.06 þ 132.02
(A
þ
A0, C2þC6þC1), 130.40
þ
130.27 (B
þ
B0, C5þC4),
128.65 þ 126.48 (B þ B0, C1þC2), 124.60 (A þ A0, C3þC5),
121.03 þ 120.51 (B þ B’, C3þC6). 77Se NMR (76 MHz, DMSO‑d6)
d:
482.83 (SeeSe). MS [m/z (% abundance)]: 104 (100), 172 (93), 344
(25). Elemental analysis calculated (%) for C28H20N2O6Se2 ∙ 2H2O:
C: 49.87, H: 3.59, N: 4.15; found: C: 49.73, H: 3.53, N: 4.35.
4.1.2.3. 4,4’-[Diselenodiylbis(benzene-4,1-diylimino)]bis(4-
oxobutanoic acid) (3a). From succinic anhydride. Conditions:
24 h at room temperature. The product was kept under stirring
with water (25 mL) for 2 h, filtered, then stirred with ethyl ether
(100 mL) for 24 h and then filtered. A yellow powder was obtained.
Yield: 72.27%. Mp: 179e180 ꢁC. IR (KBr) cmꢂ1: 3318 (NH), 1696
(C¼O carboxylic acid), 1666 (C¼O, amide), 818 (SeeSe). 1H NMR
NMR (400 MHz, DMSO‑d6) d: 12.19 (bs, 2H, COOH), 10.03 (s, 2H,
NH), 7.65 (d, 4H, A þ A0, J2-3 ¼ J6-5 ¼ 9.0 Hz, H2þH6), 7.62 (d, 4H,
A þ A0, J3-2 ¼ J5-6 ¼ 9.0 Hz, H3þH5), 6.50 (s, 4H, B þ B0, H3þH5), 5.14
(s, 2H, B þ B0, H5), 5.06 (s, 2H, B þ B0, H2), 2.82 (d, 2H, B þ B0, J1-
¼ 9.1 Hz, H1), 2.71 (d, 2H, B þ B0, J6-1 ¼9.1 Hz, H6). 13C NMR
6
(100 MHz, DMSO‑d6)
d
: 173.08 (COOH), 170.48 (C¼O), 141.07
(A þ A0, C4), 137.49 þ 137.08 (B þ B0, C4þC5), 135.29 (A þ A0, C2þC6),
120.79 (A þ A0, C1), 116.51 (A þ A0, C3þC5), 80.82 þ 79.61 (B þ B0,
C3þC6), 47.96 þ 47.36 (B þ B’, C1þC2). MS [m/z (% abundance)]: 172
(100), 344 (25). Elemental analysis calculated (%) for
(400 MHz, DMSO‑d6) d: 12.21 (bs, 2H, COOH), 10.11 (s, 2H, NH), 7.56
(d, 4H, A þ A0, J2-3 ¼ J6-5 ¼ 8.4 Hz, H2þH6), 7.51 (d, 4H, A þ A0, J3-
¼ J5-6 ¼ 8.4 Hz, H3þH5), 2.60e2.46 (m, 8H, B þ B0, H1þH2). 13C
2
NMR (100 MHz, DMSO‑d6)
d
: 174.27 (COOH), 170.80 (C¼O), 140.09
C28H24N2O8Se2 ∙ 2H2O: C: 47.34, H: 3.97, N: 3.94; found: C: 47.66, H:
(A þ A0, C4), 133.76 (A þ A0, C2þC6), 123.85 (A þ A0, C1), 120.12
(A þ A0, C3þC5), 31.57 þ 29.23 (B þ B’, C2þC1). MS [m/z (% abun-
dance)]: 172 (100), 344 (15), 424 (10). Elemental analysis calculated
(%) for C20H20N2O6Se2 ∙ 2H2O: C: 41.51, H: 4.18, N: 4.84; found: C:
41.11, H: 3.79, N: 4.77.
4.13, N: 4.23.
4.1.3. General procedure for the synthesis of compounds 9ae11a
A reaction mixture containing 1.3 mmol of the corresponding
carboxylic derivatives (1a, 2a or 5a) in 15 mL of acetic anhydride
and 200 mg of sodium acetate was heated for 3 h under reflux, then
quenched with water (50 mL) and kept under stirring for 3 h. The
aqueous solution was extracted with CH2Cl2 (2 ꢀ 25 mL), dried with
sodium sulphate anhydrous and the solvent was evaporated under
vacuum.
4.1.2.4. 2,2’-[(Diselenodiylbis(benzene-4,1-diylimino)]bis(2-
oxoethane-2,1-diyloxy)diacetic acid (5a). From diglycolic anhydride.
Conditions: 24 h at room temperature. The product was kept under
stirring with water (25 mL) for 2 h, filtered, then stirred with ethyl
ether (100 mL) for 24 h and then filtered. A yellow powder was
obtained. Yield: 69.7%. Mp: 140e141 ꢁC. IR (KBr) cmꢂ1: 3337 (NH),
1709 (C¼O carboxylic acid), 1683 (C¼O, amide), 818 (SeeSe). 1H
4.1.3.1. 1,1'-(Diselenediyldibenzene-4,1-diyl)bis(1H-pyrrole-2,5-
dione) (9a). From compound 1a. The product was then washed

P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
243
with n-hexane (100 mL). A yellow solid was obtained. Yield: 55.4%.
Mp: 91.5e92.5 ꢁC. IR (KBr) cmꢂ1: 1710 (C¼O), 818 (SeeSe). 1H NMR
H2þH3þH5þH6), 6.47 (d, 1H, B, J1-2 ¼ 12.0 Hz, H1), 6.33 (d, 1H, B, J2-
¼12.0 Hz, H2). 13C NMR (100 MHz, DMSO‑d6)
d: 167.37 (COOH),
1
(400 MHz, DMSO‑d6)
d
: 7.77 (d, 4H, A þ A0, J2-3 ¼ J6-5 ¼ 8.6 Hz,
164.02 (C¼O), 140.42 (A, C4), 135.24 (A, C2þC6), 132.04 þ 130.75 (B,
H2þH6), 7.33 (d, 4H, A þ A0, J3-2 ¼ J5-6 ¼ 8.6 Hz, H3þH5), 7.19 (s, 4H,
C1þC2), 121.16 (A, C3þC5), 117.57 (A, C1), 105.77 (CN). 77Se NMR
B þ B0, H1þH2). 13C NMR (100 MHz, DMSO‑d6)
d
: 169.69 (C¼O),
(76 MHz, DMSO‑d6) d: 322.45 (SeCN). MS [m/z (% abundance)]: 118
134.75 (A þ A0, C4), 131.37 þ 131.27 (A þ A0, C2þC6; B þ B0, C1þC2),
129.15 (A þ A0, C1), 127.51 (A þ A’, C3þC5). MS [m/z (% abundance)]:
57 (75), 252 (100), 311 (65). Elemental analysis calculated (%) for
(100), 198 (25), 278 (10), 296 (7). Elemental analysis calculated (%)
for C11H8N2O3Se: C: 44.74, H: 2.73, N: 9.49; found: C: 44.35, H: 3.08,
N: 9.10.
C
20H12N2O4Se2 ∙ H2O: C: 46.17, H: 2.71, N: 5.38; found: C: 46.10, H:
3.03, N: 4.94.
4.1.4.2. 2-[(4-Selenocyanatophenyl)carbamoyl]benzoic acid (2b).
From phthalic anhydride. Conditions: 14 h at room temperature.
The product was kept under stirring with water (25 mL) for 2 h,
filtered and then washed with n-hexane (25 mL) and ethyl ether
4.1.3.2. 1,1'-(Diselenodiyldibenzene-4,1-diyl)bis(1H-isoindole-
1,3(2H)-dione) (10a). From compound 2a. The product was then
washed with n-hexane (100 mL). A yellow solid was obtained.
Yield: 90.6%. Mp: 248e249 ꢁC. IR (KBr) cmꢂ1: 1709 (C¼O), 815
(25 mL).
A white powder was obtained. Yield: 89.5%. Mp:
162e164 ꢁC. IR (KBr) cmꢂ1: 3317, 3122 (NeH), 2149 (CN), 1718 (C¼O
carboxylic acid), 1647 (C¼O, amide). 1H NMR (400 MHz, DMSO‑d6)
(SeeSe). 1H NMR (400 MHz, DMSO‑d6)
d
: 8.15e7.79 (m, 12H, A þ A0,
H2þH6; B þ B0, H2þH3þH4þH5), 7.55 (d, 4H, A þ A0, J3-2 ¼ J5-
d
: 13.14 (s, 1H, COOH), 10.60 (s, 1H, NH), 7.92 (d, 1H, B, J3-4 ¼ 7.5 Hz,
¼ 8.4 Hz, H3þH5). 13C NMR (100 MHz, DMSO‑d6)
: 172.73 (C¼O),
d
H3), 7.78 (d, 2H, A, J2-3 ¼ J6-5 ¼ 8.3 Hz, H2þH6), 7.73e7.64 (m, 3H, A,
6
H3þH5, B, H4), 7.62e7.54 (m, 2H, B, H5þH6). 13C NMR (100 MHz,
140.18 þ 140.09 (A þ A0, C4; B þ B0, C1þC6), 133.49 (B þ B0, C4þC5),
131.99 þ 131.73 (A þ A0, C1þC2þC6), 128.65 (B þ B0, C2þC5), 120.26
DMSO‑d6)
d: 168.23 (COOH),167.80 (C¼O),141.32 þ 139.03 (A, C4; B,
(A þ A’, C3þC5). 77Se NMR (76 MHz, DMSO‑d6)
d: 481.88 (SeeSe).
C1), 135.29 (A, C2þC6), 132.31 (B, C5), 130.30 þ 130.09þ130.05 (B,
C2þC3þC4), 128.25 (B, C6), 121.16 (A, C1), 116.99 (A, C3þC5), 105.91
(CN). MS [m/z (% abundance)]: 76 (50), 104 (55), 118 (100), 198 (20).
Elemental analysis calculated (%) for C15H10N2O3Se: C: 52.19, H:
2.92, N: 8.11; found: C: 52.06, H: 3.24, N: 8.06.
MS [m/z (% abundance)]: 93 (65), 172 (100), 302 (15), 604 (5).
Elemental analysis calculated (%) for C28H16N2O4Se2 ∙ 2H2O: C:
52.68, H: 3.16, N: 4.39; found: C: 52.82, H: 3.20, N: 4.77.
4.1.3.3. 1,1'-(Diselenodiyldibenzene-4,1-diyl)bis(morpholine-3,5-
dione) (11a). From compound 5a. The product was then washed
with ethyl ether (3 ꢀ 10 mL). A yellow solid was obtained. Yield:
72.6%. MP: 150e152. IR (KBr) cmꢂ1: 1708 (C¼O), 819 (SeeSe). 1H
4.1.4.3. 4-Oxo-4-[(4-selenocyanatophenyl)amino]butanoic acid (3b).
From succinic anhydride. Conditions: 24 h at room temperature.
The product was kept under stirring with water (25 mL) for 2 h,
filtered, then stirred with ethyl ether (100 mL) for 24 h and then
NMR (400 MHz, DMSO‑d6)
d
: 7.76 (d, 4H, A þ A0, J2-3 ¼ J6-5 ¼ 7.8 Hz,
H2þH6), 7.23 (d, 4H, A þ A0, J3-2 ¼ J5-6 ¼ 7.8 Hz, H3þH5), 4.54 (s, 8H,
filtered.
A brown powder was obtained. Yield: 36.7%. Mp:
154e156 ꢁC. IR (KBr) cmꢂ1: 3340 (NH), 2158 (CN), 1693 (C¼O car-
B þ B0, H2þH3). 13C NMR (100 MHz, DMSO‑d6)
: 170.19 (C¼O),
d
boxylic acid), 1636 (C¼O, amide). 1H NMR (400 MHz, DMSO‑d6)
d:
133.32 (A þ A0, C4),131.42 (A þ A0, C2þC6),130.71 (A þ A0, C1),130.30
(A þ A0, C3þC5), 67.74 (B þ B’, C1þC2). MS [m/z (% abundance)]: 184
(100), 271 (25), 538 (15). Elemental analysis calculated (%) for
12.18 (bs, 1H, COOH), 10.21 (s, 1H, NH), 7.66 (bs, 4H, A,
H2þH6þH3þH5), 2.58 (d, 2H, A, J2-1 ¼6.0 Hz, H2), 2.54 (d, 2H, A, J1-
¼ 6.0 Hz, H1). 13C NMR (100 MHz, DMSO‑d6)
d: 174.27 (COOH),
C
20H16N2O6Se2 ∙ H2O: C: 43.18, H: 3.26, N: 5.04; found: C: 43.45, H:
2
171.03 (C¼O), 141.06 (A, C4), 135.36 (A, C2þC6), 120.60 (A, C3þC5),
116.47 (A, C1), 105.90 (CN), 31.54 (B, C2), 29.10 (B, C1). MS [m/z (%
abundance)]: 101 (25), 118 (100), 198 (40), 298 (28). Elemental
analysis calculated (%) for C11H10N2O3Se2 ∙ H2O: C: 41.92, H: 3.84,
N: 8.89; found: C: 41.59, H: 3.58, N: 8.69.
3.52, N: 5.36.
4.1.4. General procedure for the synthesis of compounds 1be8b
4-Aminophenyl selenocyanate (2 mmol) was dissolved in dry
acetone (15 mL) and the corresponding anhydride (2 mmol) then
added. The reaction was then stirred for a variable time of 12 h up to
48 h at room temperature. Reaction was quenched with water,
compound was then filtered and purified by stirring or washing
with solvents such as n-hexane and ethyl ether. The chemical shifts
assignment in NMR spectroscopy for these compounds is exem-
plified in Fig. 12.
4.1.4.4. 3-[(4-Selenocyanatophenyl)carbamoyl]pyrazine-2-carboxylic
acid (4b). From 2,3-pyrazinedicarboxylic anhydride. Conditions:
24 h at room temperature. The product was kept under stirring
with water (25 mL) for 3 h, filtered, then stirred with ethyl ether
(100 mL) for 24 h and then filtered. A yellow powder was obtained.
Yield: 49.3%. Mp: 164e165 ꢁC. IR (KBr) cmꢂ1: 3280 (NH), 2153 (CN),
1765 (C¼O carboxylic acid), 1671 (C¼O, amide). 1H NMR (400 MHz,
4.1.4.1. (2Z)-4-oxo-4-[(4-selenocyanatophenyl)amino]but-2-enoic
acid (1b). From maleic anhydride. Conditions: 14 h at room tem-
perature. The product was kept under stirring with water (25 mL)
for 2 h, filtered and then washed with n-hexane (25 mL) and ethyl
ether (25 mL). A yellow powder was obtained. Yield: 51.8%. Mp:
161e162 ꢁC. IR (KBr) cmꢂ1:3299, 3196 (NeH), 2157 (CN), 1722 (C¼O
carboxylic acid), 1624 (C¼O, amide). 1H NMR (400 MHz, DMSO‑d6)
DMSO‑d6) d: 13.82 (bs, 1H, COOH), 11.03 (s, 1H, NH), 8.92 (s, 2H, B,
H3þH4), 7.87 (d, 2H, A, J2-3 ¼ J6-5 ¼ 8.6 Hz, H2þH6), 7.74 (d, 2H, A, J3-
¼ J5-6 ¼ 8.6 Hz, H3þH5). 13C NMR (100 MHz, DMSO‑d6)
d: 167.05
2
(COOH), 163.64 (C¼O), 146.99 þ 146.75þ146.15 þ 145.49 (B,
C1þC2þC3þC4), 140.49 (A, C4), 135 (A, C2þC6), 122.19 (A, C3þC5), 118
(A, C1), 106.37 (CN). MS [m/z (% abundance)]: 79 (100), 107 (95), 118
(30), 304 (75). Elemental analysis calculated (%) for C13H8N4O3Se:
C: 44.97, H: 2.32, N: 16.14; found: C: 44.73, H: 2.72, N: 15.82.
d: 12.96 (s, 1H, COOH), 10.58 (s, 1H, NH), 7.69 (bs, 4H, A,
4.1.4.5. {2-Oxo-2-[(4-selenocyanatophenyl)amino]ethoxy}acetic acid
(5b). From diglycolic anhydride. Conditions: 24 h at room temper-
ature. The product was kept under stirring with water (25 mL) for
1 h, filtered and then washed with ethyl ether (2 ꢀ 25 mL). A white
powder was obtained. Yield: 51.1%. Mp: 141e142 ꢁC. IR (KBr) cmꢂ1
:
3305 (NH), 2151 (CN), 1716 (C¼O carboxylic acid), 1660 (C¼O,
Fig. 12. NMR assignation rules followed for series b.
amide). 1H NMR (400 MHz, DMSO‑d6)
d
: 12.91 (bs, 1H, COOH), 10.14

244
P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
(s, 1H, NH), 7.74 (d, 2H, A, J2-3 ¼ J6-5 ¼ 8.8 Hz, H2þH6), 7.68 (d, 2H, A,
116.51 (A, C1), 105.93 (CN), 80.82 þ 79.61 (B, C3þC6), 47.96 þ 47.36
J3-2 ¼ J5-6 ¼ 8.8 Hz, H3þH5), 4.21 (s, 2H, B, H1), 4.20 (s, 2H, B, H2). 13
C
(B, C1þC2). 77Se NMR (76 MHz, DMSO‑d6)
d: 320.95 (SeCN). MS [m/z
NMR (100 MHz, DMSO‑d6)
d: 172.25 (COOH), 168.78 (C¼O), 140.14
(% abundance)]: 68 (100), 118 (100), 198 (25), 278 (10). Elemental
analysis calculated (%) for C15H12N2O4Se ∙ ½ H2O: C: 48.35, H: 3.49,
N: 7.52; found: C: 48.30, H: 3.70, N: 7.53.
(A, C4), 135.27 (A, C2þC6), 121.30 (A, C3þC5), 117.43 (A, C1), 105.92
(CN), 70.83 (B, C1), 68.51 (B, C2). MS [m/z (% abundance)]: 118 (85),
198 (40), 211 (30), 314 (100). Elemental analysis calculated (%) for
C
8.82.
11H10N2O4Se: C: 42.19, H: 3.22, N: 8.95; found: C: 41.92, H: 3.53, N:
4.2. Biological evaluation
4.2.1. Cell cultures
4.1.4.6. 2’-[(4-Selenocyanatophenyl)carbamoyl]-[1,10-biphenyl]-2-
carboxylic acid (6b). From diphenic anhydride. Conditions: 48 h at
room temperature. The product was kept under stirring with water
(25 mL) for 3 h, filtered and then washed with ethyl ether
(2 ꢀ 25 mL). A white powder was obtained. Yield: 21.8%. Mp:
146e147 ꢁC. IR (KBr) cmꢂ1: 3296 (NH), 2153 (CN), 1726 (C¼O, car-
Cell lines were purchased from the American Type Culture
Collection (ATCC). PC-3, HTB-54, HT-29, MOLT-4, CCRF-CEM, K-562
and MCF-7 cell lines were grown in RPMI 1640 medium (Gibco)
supplemented with 10% fetal bovine serum (FBS; Gibco), 100 units/
mL penicillin and 100 mg/mL streptomycin (Gibco). BEAS-2B cell
line (normal epithelial lung) was cultured in DMEM (Gibco), 10%
boxylic acid), 1631 (C¼O, amide). 1H NMR (400 MHz, DMSO‑d6)
d:
FBS, 100 units/mL penicillin and 100 mg/mL streptomycin. 184B5
12.80 (bs, 1H, COOH), 10.24 (s, 1H, NH), 7.83 (d, 1H, B, J9-10 ¼ 7.6 Hz,
H9), 7.67e7.58 (m, 3H, B, H2þH5þH12), 7.58e7.48 (m, 5H, A,
H2þH3þH5þH6, B, H11),7.41 (t, 1H, B, J4-3 ¼ J4-5 ¼ 7.4 Hz, H4), 7.24 (t,
2H, B, J3-2 ¼ J3-4 ¼ J10-9 ¼ J10-11 ¼5.9 Hz, H3þH10). 13C NMR
cells were grown in DMEM/F12 medium supplemented with 5%
FBS, 1 ꢀ ITS (Lonza), 100 nM hydrocortisone (Aldich), 2 mM sodium
pyruvate (Lonza), 20 ng/mL EGF (Sigma- Aldrich), 0.3 nM trans-
retinoic acid (Sigma-Aldrich), 100 units/mL penicillin and 100 mg/
mL streptomycin. Cells were maintained at 37 ꢁC and 5% CO2.
(100 MHz, DMSO‑d6)
d
: 169.20 (COOH), 167.55 (C¼O), 141.41 (A, C4),
140.79 þ 140.76 (B, C6þC7), 136.14 (B, C8), 135.19 (A, C2þC6), 131.67
(B, C1), 131.42 130.11þ129.84 (B,
131.07þ130.45
C2þC3þC4þC9þC11), 127.88 127.78þ127.55 (B, C5þC10þC12),
þ
þ
þ
4.2.2. Cytotoxic and antiproliferative activities
Cell viability was determined using the MTT 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) method
121.06 (A, C3þC5), 117.14 (A, C1), 105.89 (CN). MS [m/z (% abun-
dance)]: 152 (70), 181 (100), 225 (30), 422 (10). Elemental analysis
calculated (%) for C21H14N2O3Se ∙ 2H2O: C: 55.15, H: 3.97, N: 6.13;
found: C: 55.39, H: 3.59, N: 6.22.
at 10 and 100
dose-response curves five different doses ranging from 0.01 to
100 M, for some compounds lower doses where needed in order
mM to perform the screening. In order to build full
m
4.1. 4. 7. 2-[(4-Selenocyanatophenyl)carbamoyl]cyclo-
hexanecarboxylic acid (7b). From cis-1,2-cyclohexanecarboxylic
anhydride. Conditions: 24 h at room temperature. The product
was kept under stirring with water (25 mL) for 2 h, filtered and then
washed with ethyl ether (2 ꢀ 25 mL). A white powder was ob-
tained. Yield: 47.0%. Mp: 150e151 ꢁC. IR (KBr) cmꢂ1: 3335 (NH),
2152 (CN), 1702 (C¼O, carboxilic acid), 1677 (C¼O, amide) 1H NMR
to reach 50% cell growth. Depending on cell size, 8,000 to
40,000 cells were seeded per well in 96-well plates and incubated
overnight. Then treated with the compounds for 48 h, cells were
then incubated with 50
was removed by aspiration and formazan crystals dissolved in
150 L of DMSO. The absorbance was measured at 550 nm in a
mL of MTT (2 mg/mL stock) for 4 h, medium
m
microplate reader (Sunrise reader, Tecan). At least three indepen-
dent experiments performed in quadruplicate were analysed. Re-
sults are expressed as GI50, the concentration that reduces by 50%
the growth of treated cells with respect to untreated controls, TGI,
(400 MHz, DMSO‑d6) d: 11.99 (bs, 1H, COOH), 9.95 (s, 1H, NH), 7.66
(d, 2H, A, J2-3 ¼ J6-5 ¼ 8.5 Hz, H2þH6), 7.62 (d, 2H, A, J3-2 ¼ J5-
¼ 8.5 Hz, H3þH5), 2.94 (d, 1H, B, J1-Hchex ¼ 4.0 Hz, H1), 2.60 (d, 1H,
6
B, Jchex-1 ¼4.0 Hz, Hchex), 2.09 (d, 1H, B, J ¼ 9.7 Hz, Hchex), 1.99 (d, 1H,
B, J ¼ 8.8 Hz, Hchex), 1.82e1.57 (m, 3H, B, 3Hchex), 1.48e1.24 (m, 3H,
the concentration that completely inhibits cell growth, and LC50
,
the concentration that kills 50% of the cells.
B, 3Hchex). 13C NMR (100 MHz, DMSO‑d6)
d: 175.55 (COOH), 173.56
(C¼O), 141.39 (A, C4), 135.24 (A, C2þC6), 120.76 (A, C3þC5), 116.18 (A,
C1), 105.87 (CN), 43.05 42.42 (B,
þ
C1þC2),
4.2.3. Evaluation of cell cycle progression and cell death
28.06 þ 25.64þ24.44 þ 22.78 (B, C3þC4þC5þC6). MS [m/z (%
abundance)]: 67 (90), 81 (93), 118 (100), 198 (60), 334 (100).
Elemental analysis calculated (%) for C15H16N2O3Se ∙ H2O: C: 48.79,
H: 4.91, N: 7.59; found: C: 48.56, H: 4.72, N: 7.66.
A fixed population of MCF-7 cells per flask were seeded in
25 cm2 flasks then incubated overnight. Cultures were treated with
the corresponding amount of compounds 10a, 8b, DMSO (control)
or 6 mM camptothecin (positive control). Seeded population was
dependent on studied time point: 3 ꢀ 106 cells/flask for 24 h or
shorter treatment, 2 ꢀ 106 cells/flasks for 48 h treatment and finally
1 ꢀ 106 cells/flask for 72 h experiments. Apo-Direct kit (BD Phar-
migen) was used to determine cell cycle distribution and cell death
percentage. Cells were fixed in a 1% paraformaldehyde solution in
PBS for 30e40 min at 0 ꢁC, washed with PBS twice and incubated
for 30 min with 70% ethanol on ice. Staining was performed
following manufacturer's protocol and samples were analysed by
flow cytometry using a Counter Epics XL cytometer (Beckman
Counter).
4.1.4.8. 3-[(4-Selenocyanatophenyl)carbamoyl]-7-oxabicyclo[2.2.1]
hept-5-ene-2-carboxylic
acid
(8b). From
3,6-epoxy-1,2,3,6-
tetrahydrophthalic anhydride obtained by the classic procedure
described for a Diels-Alder reaction using furan and maleic anhy-
dride as reagents to yield the Diels-Alder adduct. Conditions:
24 h at room temperature. The product was kept under stirring
with water (25 mL) for 4 h, filtered and then washed with ethyl
ether (2 ꢀ 25 mL). A light-yellow powder was obtained. Yield:
20.3%. Mp: 155e156 ꢁC. IR (KBr) cmꢂ1: 3267 (NH), 1711 (C¼O car-
boxylic acid), 1689 (C¼O, amide). 1H NMR (400 MHz, DMSO‑d6)
d
:
Inhibition assays cells were pre-treated with 50
mM of the pan-
12.19 (s, 1H, COOH), 10.03 (s, 1H, NH), 7.65 (d, 2H, A, J2-3 ¼ J6-
caspase inhibitor Z-VAD-FMK (BD Pharmigen) or 100 nM of the
¼ 9.0 Hz, H2þH6), 7.62 (d, 2H, A, J3-2 ¼ J5-6 ¼ 9.0 Hz, H3þH5), 6.50
autophagy inhibitor wortmannin (Santa Cruz) for 1 h or 10
chloroquine (Sigma Aldrich). The cells were treated with 80
m
m
M of
M of
5
(s, 2H, B, H3þH5), 5.14 (s, 1H, B, H5), 5.06 (s, 1H, B, H2), 2.82 (d, 1H, B,
J1-6 ¼ 9.1 Hz, H1), 2.71 (d,1H, B, J6-1 ¼9.1 Hz, H6). 13C NMR (100 MHz,
8b or 10a, DMSO was added to the control cells. Samples were
processed following the same methodology stated above. At least
three independent experiments were performed in duplicate.
DMSO‑d6)
d:
173.08 (COOH), 170.48 (C¼O), 141.07 (A, C4),
137.49 þ 137.08 (B, C4þC5), 135.29 (A, C2þC6), 120.79 (A, C3þC5),

P. Garnica et al. / European Journal of Medicinal Chemistry 175 (2019) 234e246
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0929867325666171129220544.
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Statistical data represent the mean SEM of at least three in-
dependent experiments performed in duplicate. Mann-Whitney U
test was used to stablish statistical significance of differences be-
tween control and treatment groups. GraphPad Prism version 7 was
used, significant differences were considered at p < 0.05.
4.2.5. Protein analysis
Proteins were detected by western blot. Specific antibodies for
LC3B, Beclin-1 (D40C5), SQSTM1/p62, AMPK, JNK, pAKT (Se473)
and the PI3K catalytic subunit p110a were obtained from Cell Sig-
nalling. Anti-actin (H-300) was from Santa Cruz Biotechnology.
Anti-rabbit IgG conjugated with peroxidase (Cell Signaling) was
used as secondary antibody.
Acknowledgments
The research leading to these results has received funding from
“la Caixa” Banking Foundation. P. Garnica wishes to express his
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halides: their antiproliferative effects against human breast cancer MCF-7
cells, Arch Pharm. Res. (Seoul) 38 (2014) 659e665. https://doi.org/10.1007/
s12272-014-0407-4.
ꢀ
gratitude to the Asociacion de Amigos de la Universidad de Navarra
for the pre-doctoral fellowship. Furthermore, the authors wish to
express their gratitude to the Plan de Investigacion de la Uni-
ꢀ
[20] D. Plano, Y. Baquedano, E. Ibanez, I. Jimenez, J.A. Palop, J.E. Spallholz,
C. Sanmartin, Antioxidant-prooxidant properties of a new organoselenium
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versidad de Navarra, PIUNA (Ref 2014e26) as well as Calgary
Foundation for financial support for the project.
[21] P. Garnica, I. Encio, D. Plano, J.A. Palop, C. Sanmartin, Combined
acylselenourea-diselenide structures: new potent and selective antitumoral
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2019.04.074.
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