Synthesis and antiproliferative activity of novel selenoester derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.11.034

A series of 31 new selenoesters were synthesized and their cytotoxic activity was evaluated against a prostate cancer cell line (PC-3). The most active compounds were also tested against three tumoural cell lines (MCF-7, A-549 and HT-29) and one non-tumou

Full text of DOI:10.1016/j.ejmech.2013.11.034

European Journal of Medicinal Chemistry 73 (2014) 153e166
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of novel selenoester
derivatives
,
Enrique Domínguez-Álvarez a d, Daniel Plano a, María Font b, Alfonso Calvo c, Celia Prior c,
,
a
Claus Jacob d, Juan Antonio Palop a , Carmen Sanmartín
*
a Synthesis Section, Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain
b Molecular Modeling Section, Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea 1, E-31008 Pamplona, Spain
c Oncology Division, Center for Applied Medical Research, CIMA, University of Navarra, Pío XII 53, E-31008 Pamplona, Spain
d Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, Campus, 66123 Saarbruecken, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 31 new selenoesters were synthesized and their cytotoxic activity was evaluated against a
prostate cancer cell line (PC-3). The most active compounds were also tested against three tumoural cell
lines (MCF-7, A-549 and HT-29) and one non-tumour prostate cell line (RWPE-1). Thirteen compounds
showed significant activity towards all tumour cells investigated, and some of them were even more
potent than etoposide and cisplatin, which were used as reference drugs. Because of their pronounced
potency and/or selectivity, four analogues (5, 21, 28 and 30), were selected in order to assess their redox
properties related to a possible redox modulating activity. The glutathione peroxidase (GPx) assay
showed slight activity for compound 30 and the 2,2-diphenyl-1-picrylhydrazyl-(DPPH) assay showed a
weak activity for compounds 5 and 28. The present results revealed that analogues 5, 21, 28 and 30 might
serve as a useful starting point for the design of improved anti-tumour agents.
Received 27 March 2013
Received in revised form
8 November 2013
Accepted 25 November 2013
Available online 12 December 2013
Keywords:
Selenium
Antiproliferatives
Cytotoxicity
Cancer
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
selenium moiety have attracted reasonable attention [4e9]. The
role of selenium as part of potential redox-modulating, chemo-
Cancer represents one of the most serious clinical problems in
the world and has become the second major cause of death in
developed countries. Over 1.5 million cases of cancer occur in the
United States annually and cancer-related deaths are estimated to
reach 12 million worldwide in 2015 [1]. Unlimited replication, self-
sufficiency in growth signals, insensitivity to antigrowth signals,
sustained angiogenesis, metastasis, and evasion of apoptosis are
the hallmarks of cancer cells [2]. Most of the anticancer agents
currently used in clinical practice exhibit undesirable side effects
such as reduced bioavailability, toxicity and drug-resistance [3].
Therefore, cancer treatment has been a major endeavour of
research and development in academia and pharmaceutical in-
dustry for the last decades. For this reason, the discovery and
development of novel molecules that possess ideally both, a che-
moprotective effect and an anticancer chemotherapeutic effect, are
pertinent.
therapeutic and chemopreventive agents has been supported by
epidemiological, preclinical and clinical studies, having shown
many authors associations among them [10e13]. Several mecha-
nisms have been suggested to explain the anticancer effects of
selenium derivatives. The most accepted ones are reduction of DNA
damage, oxidative stress, inflammation, enhancement of the im-
mune response, incorporation into selenoproteins, tumour sup-
pressor gene silencing by alteration of their DNA methylation
status, cell cycle arrest, inhibition of angiogenesis and induction of
apoptosis [14,15]; depending on the chemical form of selenium
involved, the type of cancer and its dosage [16,17]. Related to che-
mopreventive activity, different mechanisms of action have been
reported. Among them, the action of certain selenoderivatives as
interleukin-18 expression suppressors [18] and angiogenesis in-
hibitors [19] can be highlighted; being the control of the level of
oxidative stress the point of convergence of both activities [20e27].
This control can be exerted by modulating the expression of sele-
noproteins [20,23,25], regulating the intracellular redox state
[21,22,24] or scavenging the reactive oxygen species [26].
Amidst the wide range of compounds tested as potential anti-
cancer agents, derivatives comprising functionalities based on a
Furthermore, it is well known that the selenium metabolism is
critical for the anticancer activity of selenocompounds. Amidst the
active metabolites, a-keto acids as well as methylselenol have been
* Corresponding author. Tel.: þ34 948 425 600; fax: þ34 948 425 649.
E-mail address: jpalop@unav.es (J.A. Palop).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.034

154
E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
hypothesized as responsible of the anticancer and chemo-
preventive effects [28e30]. Recently, glutaredoxin proteins, which
exert a critical role in the maintenance of the cell redox homeo-
stasis, have been proposed to be implicated in a novel pathway for
selenium metabolism [31].
In the past few years, we have been involved in the develop-
ment, design as well as the synthesis of structurally modified
selenium derivatives, some of which have exhibited significant
anticancer activity as cytotoxic and antiproliferative agents acting
through the induction of apoptosis, as antioxidants or even as
kinase modulators [32e40].
Among these various selenium compounds, our research group
described in 2009 the synthesis and antiproliferative activity of
particular selanylacetic acid derivatives [33]. Prompted by the
abovementioned results and by the promising activity profile
associated with several selanylacetic derivatives, and as an exten-
sion of our previous studies, the aim of this study is to design and
synthesis of a range of novel selenoester-substituted derivatives
using the structural derivatization approach. It is expected that the
potential of these compounds as anticancer and chemopreventive
agents should be higher than the one of the original compounds.
The rationale behind this strategy is to retain the selenoester
group as scaffold, for considering that its possible hydrolysis rep-
resents a crucial “redox activation” step which could be modulated
by the inclusion of different functional groups in the moiety bound
to the selenium atom of the selenoester. The hydrolysis subse-
quently generates ionic species of selenium (such as selenols)
which can readily participate in redox processes. These charged
selenocompounds may possess inherent biological activity, and it
would be beneficial if they could enhance the cytotoxic impact on
cancer cells.
Furthermore, it seems reasonable to bear in mind the biological
activity of related compounds which contain heterocyclic rings
instead of carbocycles, hence certain representative pyridyl and
thienyl derivatives have been also synthesized. Finally, we have
investigated whether or not the length of the aliphatic side chain is
a key feature for in vitro activity.
Herein, we report the synthesis of thirty-one new compounds
and their in vitro cytotoxic activity against prostate human tumour
cell line (PC-3). The most active compounds of the preliminary
screening have been evaluated further against the immortalized
non-tumour cell line RWPE-1 in order to assess the selectivity of
the compounds for cancer cells (compared to non-cancer cells). In
addition, some of these active compounds have been tested against
other tumour cell lines (i.e. breast, lung and colon) with the aim of
ascertain whether there are differences of susceptibility amidst the
cancer cell lines derived from different tissues. Furthermore, the
involvement of the compounds in redox-modulating processes is
determined through the evaluation of their electrochemical prop-
erties, their glutathione peroxidase (GPx)-like activity as well as the
assessment of their capability to interact with a stable free radical
as 2,2-diphenyl-1-picrylhydrazyl (DPPH).
2. Results and discussion
2.1. Synthetic chemistry
The key step in the synthetic strategy for the majority of the
compounds synthesized (1e4 and 14e31) was the reaction be-
tween sodium hydrogen selenide, produced by the reaction of
powdered grey selenium with sodium borohydride in an adequate
medium, and the aroyl or heteroaroyl chloride, followed by the
treatment of the intermediate sodium aroylselenide generated
The strategy to optimize the activity of these new selenoester
derivatives (Fig. 1) involved the modification of the lead molecules
previously reported [33], taking into account the synthetic
accessibility.
with the corresponding a-halo derivative according to Scheme 1.
This synthesis was carried out following our own protocol [33],
which was based on a published procedure [41]. Derivative 5 was
isolated with a 94% yield, whereby the selenating agent was
generated by the reaction of lithium aluminium hydride with
selenium in anhydrous THF, the subsequent reaction with
2-benzofuran-1,3-dione or phthaloyl chloride and the final dehy-
dration with sulphuric acid. Compound 5 could also be obtained
following the sodium borohydride based-procedure, although in
this case the yield is lower (18%). In general, the yields are moderate
with the exception of compounds 25e29, for which yields up to 70%
could be achieved. The derivatives 6e13 were synthesized from the
corresponding selanylacetic acids [33] by Fischer esterification with
yields ranging from 18 to 63%.
All the newly synthesized compounds were pure and chemically
stable on air, according to the spectroscopic (IR, 1H and 13C NMR,
MS) and the elemental analysis carried out to confirm the struc-
tures of the different derivatives. The patterns observed in the IR
spectra were similar for all derivatives, including the carbonyl
stretch of the selenoester as the most remarkable band observed in
the IR spectra. On the other hand, no general features were found in
1H and 13C NMR spectra due to the high structural variations amidst
the different compounds. Multidimensional COSY, HMQC and
HMBC NMR experiments were performed for selected compounds
to confirm their respective structures.
Therefore, all the compounds share as common feature a sele-
noester group whose carbonylic carbon is bounded to an aromatic or
heteroaromatic ring with or without substituents through a linker of
variable length (0e2 carbons), whereas the selenium atom is linked
to different functionalities through an alkyl bridge of the same
length as the aforementioned linker; the functionalities being
considered include amides, ketones, methylgroupsand methyl, tert-
butyl and phenyl esters. These functional groups have been selected
in order to obtain compounds with different physicochemical
characteristics related to hydrophobic or hydrophilic properties and
hydrogen bond donors or acceptors, with the purpose of maintain-
ing a gradient in their polarityand to modify their ability to establish
electrostatic interactions. The placement of different substituents in
the aryl ring leads to variations of the electronic, hydrophobic and
steric properties of the molecule, thus exerting a noteworthy influ-
ence over the magnitude of the hydrolysis process as well as over the
compounds’ expected biological activity.
The selection of the substituents is mainly based on the results
obtained for the previously reported selanylacetic acids [33].
In the mass spectra of methylselenoesters (derivatives 24e31),
the molecular ion peak is observed, although with a low abun-
dance; whereas the most intense peak in the mass spectra of the
majority of the selenocompounds is either the carbonyl-containing
cation generated after the selenoester bond breakdown or the
fragment resulting when carbon dioxide is liberated from the
aforesaid carbonyl ion.
Fig. 1. General scheme of structural modulation.

E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
155
Scheme 1. Synthesis of compounds 1e31.
2.2. Biological evaluation
as GI50, TGI and LC50, are determined after an incubation of the cells
with the compounds for 72 h. The LC50 indicates the cytotoxic
potency of a derivative, whereas the TGI and the GI50 measure the
antiproliferative properties as they represent the concentrations at
which the cell growth of treated cells is suppressed completely or
reduced by 50%, respectively, in comparison to the untreated
control.
2.2.1. Cytotoxicity
Many of the studies addressing a prospective anticancer activity
of selenium compounds have been performed in prostate cancer
cell lines [42e46]. Thus, the 31 derivatives synthesized have been
tested in vitro against cultured prostate cancer cells (PC-3). To
evaluate the antiproliferative potential of the aforementioned
compounds, the values of cytostatic and cytotoxic parameters, such
Cytotoxicity assays were based on the reactivity of MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] as

156
E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
described by Denizot and Lang [47], measured over a range of
concentrations of each substance between 0.01 and 100 M. To
contrast, steric hindrance can explain that the R0-oxicarbonylsele-
noesters whose R0 is a bulky substituent, such as a tert-butyl group
(compounds 6, 7, 9 and 12), exhibit a lower activity compared to
similar compounds in which R0 is a methyl group. According to a
previous work of our group the number of methylene groups of the
n1 chain that maximizes the activity were 0 and 1, as further ex-
tensions of the chain inactivates the compounds [33]. For this
reason only these values of n1 were considered, finding that, in
compounds which contain an amide or a tert-butyl ester group, the
molecule with a methylene group in n1 has a slightly higher activity
than the comparable arylselenoester; whereas the effect is the
opposite one in the derivatives with a methyl ester group in the Se-
bounded alkyl chain. Regarding the length of this second alkyl
chain (n2), it is observed that its extension (from 1 to 2 methylene
groups) entails a higher increase of the toxicity in non-tumoural
prostate cells than the observed increase of the activity in PC-3
cell line.
m
place the data in perspective, cisplatin and etoposide were selected
as a positive control. The analyses were all performed with a
minimum of three independent experiments. As shown in Table 1,
20 of the 31 derivatives assayed (i.e. compounds 3, 5, 6, 7, 8, 9, 10, 11,
12, 13, 18, 19, 21, 22, 23, 25, 28, 29, 30 and 31) exhibited potent
antiproliferative activity against PC-3 cells, with GI50 values lower
than 10
28, 29, 30 and 31) were also active in improving the GI50 of cisplatin
(5 M). Furthermore, the three most cytostatic compounds (29, 30
mM. 13 of them (i.e. compounds 6, 8, 9, 11, 12, 13, 21, 22, 23,
m
and 31) show GI50 values in nanomolar range, i.e. between 8.4 and
57 nM. Amazingly, it appears that these compounds are 11e70-fold
more active than etoposide. Interestingly, the derivatives with the
selenium-moiety showing higher polarity used to have a lower
activity. In other words, the replacement of the original acid group
of selanylacetic acids by an ester or ketone group might improve
the cytostatic efficiency against this cancer cell line. In fact, this
hypothesis is confirmed through the comparison of the activity of
derivatives 1 versus 6 and 18; and 2 versus 7, 19, 21 and 22. In
Finally, several methylselenoesters (compounds 24e31) have
been synthesized and evaluated since methylselenol has been hy-
pothesized to be
a selenium metabolite with its very own
Table 1
Average GI50, TGI and LC50 values (mM) for compounds.
Comp
Ring
n1
n2
Z
PC-3
RWPE-1
GI50
SIe
GI50
TGIb
97.55
>100
70.6
>100
79.66
8.76
73.99
9.06
LC50
TGI
LC50
a
c
1
2
3
4
5
6
7
8
Phenyl
4-Chlorophenyl
3,5-Dimethoxyphenyl
Phenyl
Anhydride
0
0
0
1
d
0
0
0
0
0
0
1
1
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
d
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
CONH2
CONH2
CONH2
CONH2
d
46.25
40.96
6.36
35.68
7.91
2.94
9.10
4.18
4.29
5.61
4.87
4.81
2.22
45.22
63.72
27.0
43.56
6.38
6.86
36.64
1.58
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NDd
ND
5.76
ND
28.9
4.97
8.60
3.84
6.10
10.4
5.62
3.06
0.88
ND
ND
ND
36.0
ND
>100
ND
ND
>100
ND
>100
88.3
>100
91.2
89.4
>100
76.6
99.2
9.55
ND
ND
0.9
ND
3.7
1.7
0.9
0.9
1.4
1.9
1.2
0.6
0.4
ND
ND
ND
ND
1.1
1.1
ND
1.3
1.0
1.3
ND
3.2
ND
ND
6.4
0.4
3.1
1.0
1.4
0.4
Phenyl
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOCH3
COOC(CH3)3
COOC(CH3)3
COOC(CH3)3
COOC(CH3)3
COOC6H5
COOC6H5
COOC6H5
COCH3
COC(CH3)3
COC(CH3)3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
16.3
4-Chlorophenyl
2-Chlorophenyl
3,5-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
2-Thienyl
Phenyl
Phenyl
Phenyl
55.8
15.8
16.3
57.1
14.11
35.7
4.85
9
9.07
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
57.31
18.38
10
19.71
>100
>100
77.7
>100
79.41
82.68
99.19
3.14
28.3
6.91
>100
71.31
77.36
71.28
18.41
23.69
1.69
ND
ND
4-Chlorophenyl
3,5-Dimethoxyphenyl
Phenyl
ND
ND
ND
ND
ND
ND
46.3
>100
ND
ND
ND
>100
>100
ND
Phenyl
7.16
7.54
ND
2.03
2.29
3.34
ND
27.8
ND
ND
5.24
0.0244
0.0264
0.0091
0.9
4-Chlorophenyl
3,5-Dimethoxyphenyl
4-Chlorophenyl
4-Chlorophenyl
3,5-Dimethoxyphenyl
Phenyl
4-Chlorophenyl
4-Cyanophenyl
3,5-Dimethoxyphenyl
4-Methylselenoacylphenyl
3-Methylselenoacylphenyl
6-Methylselenoacylpyridyl
5-Methylselenoacylthienyl
Etoposide
ND
4.71
5.32
5.60
6.30
8.60
8.91
9.25
2.19
2.66
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
13.03
8.58
36.30
14.41
0.82
0.0570
0.0084
0.0089
0.63
ND
>100
ND
ND
39.4
0.975
ND
>100
ND
ND
>100
65.0
67.2
>100
>100
>100
3.01
0.729
4.8
CH3
0.81
3.98
50.12
79.43
>100
Cisplatin
5.01
2.4
5.87
a
GI50: growth inhibition 50%.
TGI: total growth inhibition.
LC50: lethal concentration 50%.
ND: not determined.
SI: selectivity index (GI50 RWPE-1/GI50 PC-3).
b
c
d
e

E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
157
anticancer properties [48e52]. Five of the compounds considered
(i.e. 25, 28, 29, 30 and 31) exhibited GI50 values below 10 M.
cancer (A-549) and colon carcinoma (HT-29) cell lines (Table 2).
Here, having a GI50 value <5 M in the PC-3 cell line was the
m
m
Remarkably derivatives 28e31, which possess a molecular sym-
metry in their structure, are the most potent amidst all compounds
tested. Molecular symmetry is a structural property frequently
presented in anticancer drugs [53,54] used in prostate cancer
treatment [55,56]. Additionally, the effect of substituents on the
phenyl ring of the tail as well as the length of the alkyl chains be-
tween the carbonyl groups has also been investigated (Fig. 1);
although no apparent correlations were found between these pa-
rameters and the biological activity.
Compounds are classified as cytostatic or cytotoxic depending of
the separation between the different cell survival/proliferation pa-
rameters obtained by the MTT assay (LC50, TGI, GI50). In this way,
compounds whose three parameters reside more or less in the same
orderare considered as cytotoxic derivatives; whereas the ones with
a noteworthy gap between the cytotoxic and the two-antiprolifer-
ative markers are denoted as cytostatic agents. For example, com-
arbitrary and demanding criterion considered for selecting the
aforesaid derivatives. With the exception of compounds 8 and 11,
that exerted a modest antiproliferative activity in MCF-7, the
remaining compounds tested demonstrated a potent cytostatic
effect on all the cell lines tested in a dose-dependent manner
(Fig. 2). Interestingly, all the selenoesters tested in these cell lines
exhibited a higher activity than cisplatin in HT-29 cells, whereas
nine and three of them improved the action of this reference drug
in A-549 and MCF-7 cell lines, respectively. In addition, three de-
rivatives (29, 30 and 31) had nanomolar GI50 values in all the cell
lines studied. It is quite remarkable that compounds 28 and 29
exhibited rather different activity profiles, with a marked cytostatic
activity found for 29 e but not for 28 e in A-549 and HT-29 cells, in
spite of their structural similarity.
In terms of cell line sensitivity, the higher activities generally were
observed for A-549 > PC-3 > HT-29 > MCF-7 cell lines.
pounds 6 (GI50 ¼ 2.94
(GI50 ¼ 4.18 M, TGI ¼ 9.06
TGI ¼ 9.07 M and LC50 >100
and LC50 > 100
M), 30 (GI50 ¼ 0.0084
and LC50 > 100
M) and 31 (GI50 ¼ 0.0089
LC50 > 100
m
M, TGI ¼ 8.76
m
M and LC50 > 100
m
M), 8
m
m
m
M and LC50 >100
m
M), 9 (GI50 ¼ 4.29
m
M,
2.2.2. Redox modulating activity
mM), 23 (GI50 ¼ 2.66
m
M, TGI ¼ 6.91
mM
Nowadays, there is an increased interest of using antioxidants
for medical purposes. Thus, drugs possessing redox modulating
activities, such as free radical scavenging and antioxidant proper-
ties, are considered for prevention and/or treatment of diseases
directly related to a disturbed redox homeostasis present in the
organisms. Moreover, recent literature reported the possible role of
selenium in the etiology of carcinogenesis by modulating oxidative
stress [25,57e59]. The aim of developing hybrid compounds
through the combination of the antitumour and antioxidant ac-
tivities in the same molecule may have a synergistic effect, result-
ing in a higher activity than the sum of the different components.
Systems-oriented drug design, which considers network crosstalk
and regulation instead of an individual target, has thus become a
promising alternative approach in overcoming the problems of
m
m
M, TGI ¼ 1.69
mM
m
mM, TGI ¼ 0.81
mM and
m
M) exhibited a very interesting pattern of selective
cytostatic potency against the PC-3 cell line. In contrast, compound
M) may be
21 (GI50 ¼ 1.58
m
M, TGI ¼ 3.14
m
M and LC50 ¼ 4.71
m
classified as cytotoxic agent for showing the highest overall potency
in all three parameters, including the LC50 value.
The activity of the compounds that presented a good inhibitory
activity (GI50 < 10 mM) in PC-3 cells has been determined in a non-
tumour prostate cell line (RWPE-1), following the same experi-
mental procedure, to obtain a rough measure of their selectivity
between cancer cells and normal cells. In order to achieve this, the
selectivity index (SI) was calculated as the ratio of the GI50 values
determined for the non-tumour and the malignant cells. Com-
pounds 5, 25, 28 and 30 were the most selective ones for exhibiting
SI values of 3.6, 3.4, 6.4 and 3.1, respectively. In contrast, the
remaining derivatives showed similar values in cancer and non-
tumour cells, pointing towards a more general, unspecific cyto-
toxic/cytostatic activity.
targeting
a single preselected target. The results previously
described prompted us to perform further biological studies in
order to gain a preliminary insight into the mechanisms of action of
selected compounds. Derivatives 28 and 30 were chosen because
they were the most selective ones among the four compounds with
nanomolar GI50 values in PC-3 cells. Compound 21 was selected for
being the only one that exerted a cytotoxic action in prostate cancer
cells. And finally, derivative 5 was chosen for being a selenoanhy-
dride instead a selenoester: here, selenium anion formation should
be favoured upon hydrolysis due to its higher reactivity.
Finally, taking into account that each cancer cell line presents a
distinctive pattern of sensitivity, thirteen compounds (6, 8, 9, 11, 12,
13, 21, 22, 23, 28, 29, 30 and 31) were selected for additional
cytotoxicity assays in breast adenocarcinoma (MCF-7), human lung
Table 2
Average GI50, TGI and LC50 values (
mM) for selected compounds.
Compound
MCF-7
A-549
GI50
HT-29
GI50
TGIb
LC50
TGI
LC50
38.3
38.6
44.8
23.7
82.0
51.8
87.0
>100
92.5
>100
>100
>100
>100
>100
>100
GI50
TGI
37.0
LC50
a
c
6
8
9
11
12
13
21
22
23
28
29
30
31
Etoposide
Cisplatin
8.1
10.9
8.9
16.3
9.5
7.8
4.5
5.3
6.3
>100
>100
>100
>100
74.9
>100
9.3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
2.7
4.1
4.2
3.7
2.2
1.3
1.7
1.2
6.5
7.1
7.2
6.9
6.9
5.8
7.1
5.6
5.5
3.6
4.7
3.4
3.7
3.3
2.6
3.6
5.0
5.5
>100
>100
96.5
9.2
9.5
25.7
24.7
7.3
>100
>100
>100
5.8
6.8
8.9
9.1
>100
94.2
6.01
2.9
27.5
42.7
8.9
60.8
7.9
3.5
7.99
>100
>100
>100
>100
>100
>100
>100
>100
0.52
0.29
0.32
19.95
3.16
0.061
0.062
0.079
4.46
3.63
0.680
4.2
0.710
0.331
0.047
0.295
31.62
7.95
>100
39.9
a
GI50: growth inhibition 50%.
TGI: total growth inhibition.
LC50: lethal concentration 50%.
b
c

158
E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
the kind of redox modulating activity e and subsequent cytotoxic/
cytostatic effects e observed in cell culture. Secondly, the redox
couple at the heart of this activity seems to involve selenol(ate) and
diselenide species, confirming that the compounds can become
hydrolyzed and, as expected, liberate a redox active, anionic sele-
nolate species (there are additional signals which may represent
the non-hydrolyzed precursors and/or further decomposition
products). Thirdly, whilst it is not always possible to calculate exact
redox potentials (the redox couple in question is sometimes quasi-
reversible and hence calculating E1/2 is complicated), both, the
generally observed reversibility and position of the potentials
bodes well for a pronounced biological redox activity which,
because of reversibility, may also include aspects of catalysis (see
below).
The GPx-like catalytic activity for the four selected compounds
(5, 21, 28 and 30) was investigated using a thiophenole(PhSH)
based assay [61], which measures the ability of the compounds to
catalyse the reaction of hydrogen peroxide with a selected thiol
in vitro (since this activity resembles the one of the selenium
enzyme GPx, the assay is sometimes also referred to as a
“GPx_assay”). The results are expressed as the ratio between the
initial rates of the catalyzed reaction by the tested compound (Vcat)
and the spontaneous reaction (Vsp). Diphenyldiselenide was used
as positive control and all the compounds were studied at a con-
Fig. 2. Cytotoxicity of compounds, cisplatin and etoposide (GI50) against MCF-7
(breast), HT-29 (colon), A-549 (lung) and PC-3 (prostate) human cancer cell lines,
being the z axis logarithmic and cut by the basal xy plane at 1 mM to highlight the
potency of the most cytostatic compounds.
Therefore, measurements of the electrochemical behaviour, the
capacity to interact with hydrogen peroxide (GPx-like activity) and
the capability to interact with a stable free radical (DPPH) may
provide useful information about the redox modulating and/or
antioxidant profile of the four selected active compounds.
centration of 100 mM. The results obtained show that the activity of
compound 30 was comparable to the one of the reference, whilst
derivatives 5, 21 and 28 presented a clearly lower activity, as shown
in Table 4. It is therefore unlikely that these compounds per se are
good antioxidants.
The DPPH radical has been widely used to test the ability of
compounds to behave as free radical scavengers or hydrogen do-
nors. Briefly, the assay measures the decrease in absorbance of the
DPPH radicals at a characteristic wavelength after a half-hour in-
cubation of the DPPH radical with the different concentrations (5,
In a first step we have employed cyclic voltammetry in order to
evaluate whether the compounds are involved in redox reactions,
using a mercury-drop or carbon electrode and aqueous, buffered
electrolyte solutions (to represent certain aspects of biological
conditions) [60]. Voltammograms of compounds 5, 21 and 28 could
be recorded, whilst it has been impossible to analyze compound 30
due to its lower solubility in aqueous media. The compounds
examined showed peaks in mercury electrode, and two of them (5
and 21), also showed signals related to electrochemical processes at
the carbon electrode (at compound concentrations of 5 mM). These
signals can be interpreted as follows: in general terms, selenoesters
showed a cathodic reduction peak Epc in the potential interval
between ꢀ550 and ꢀ700 mV and a corresponding anodic oxidation
peak Epa generally a couple of tens of millivolts at more positive
potentials (see Table 3). Not unexpectedly, these oxidation and
reduction potentials point to the presence of a readily reductable
diselenide (ReSeSeeR0) and a readily oxidizable anionic selenium
species, probably the corresponding selenolate (RSeꢀ). In contrast,
the potentials of the selenoanhydride (5) are in the range
between ꢀ250 and ꢀ400 mV, indicating a very different redox
behaviour due to the distinct structural features of this compound.
Fig. 3 includes a representative voltammogram for the mercury
electrode. Whilst it is fairly difficult to derive at a more detailed
interpretation of the various oxidation (and reduction) potentials
measured due to complications with adsorption, a few interesting
observations can be made: first of all, the results confirm that the
compounds studied are redox active within the physiologically
relevant potential range. In turn, this activity may form the basis for
15, 50, 150 and 500 mM) of the antioxidant compound. Hence we
carried out experiments to explore the free radical scavenging
ability of the novel selenoesters through their capacity for scav-
enging DPPH. The values obtained were then compared with the
ones measured for the positive control, i.e. the ascorbic acid [62].
Results are expressed as the percentage of the DPPH free radical
scavenging at five concentrations, as shown in Table 5 and Fig. 4.
Each value is expressed as the average of three experiments per
concentration ꢁ SEM.
Table 3
Electrochemical parameters obtained for selected selenoesters. Mercury-drop
electrode. v ¼ 200 mV/s; MeOH:H2O (phosphate buffer pH 7.4) 1:4; t ¼ 5 min.
Please see text for further details.
Compound
Epc
Epa
DEp
E1/2
5
21
28
ꢀ393 mV
ꢀ588 mV
ꢀ696 mV
ꢀ274 mV
ꢀ518 mV
ꢀ639 mV
119 mV
70 mV
57 mV
ꢀ334 mV
ꢀ553 mV
ꢀ668 mV
Fig. 3. Voltammogram corresponding to compound 5 in mercury electrode.

E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
159
Table 4
The GPx-like catalytic activity determined for the four selected
compounds (using diphenyldiselenide as benchmark).
Compound
Ratio Vcat/Vsp
5
21
4.0
2.4
28
4.9
30
11.5
13.3
Diphenyldiselenide
The tested compounds showed a weak to moderate capacity for
scavenging the radical DPPH in comparison to the positive control
(ascorbic acid). It was not possible to calculate an IC50 value for
most of these compounds as only compound 5, which was the one
with the most promising antioxidant profile, has a DPPH scav-
enging over 50% at least one of the concentrations assayed; the IC50
Fig. 4. Variation of DPPH radical scavenging activity vs concentration of test
compounds.
of this compound is 74.1 mM. As for the thiophenol assay, the DPPH
assay counts against a pronounced antioxidant activity of the
compounds tested. Whilst these compounds are clearly redox
active (see electrochemical results), their redox activity may result
in a more pro-oxidant, redox modulating activity, rather than in a
classical antioxidant activity. Despite being speculative at this time,
such a pro-oxidant activity may indeed explain why these com-
pounds do not protect and hence enhance the growth of cancer
cells, but actually hinder proliferation and on occasion even exhibit
outright cytotoxic properties.
derivatives included in this work. It is noteworthy to remark that
the most active compounds presented in this work (5, 25, 28 and
30) have higher selectivity indexes than the reference drugs
employed. Therefore, this fact justifies the realization of more in-
depth studies to ascertain their mechanism of action; as well as
they become a promising starting point to future modulations of
their activity through the design, synthesis and evaluation of
structural variants of these selenoesters. Due to the better activity
and/or selectivity as cytostatic or cytotoxic agents, compounds 5,
21, 28 and 30 have been selected for subsequent screening pro-
cedures in order to assess their redox properties relevant to a
biological, possibly redox modulating activity. Here, our results
indicate that the compounds studied are all redox active within the
physiological potential range, and that the key parameters of this
redox activity point towards the presence of a possibly catalytically
active selenolate/diselenide redox pair. While the mechanism of
the aforesaid cytostatic/cytotoxic activity is therefore neither due to
a significant peroxidase activity or free radical scavenging antiox-
idant properties, it is possible that these redox compounds
modulate the intracellular redox state of the cancer cells affected
and hence prevent proliferation and/or induce apoptosis. Indeed,
such redox modulating, often pro-oxidant and pro-apoptotic
properties have been observed by us and others for a wide range
of selenium and tellurium compounds attacking the cellular thi-
olstat in the past. Additional experiments are obviously required in
the future to investigate the precise mechanism or mechanisms
responsible of the pronounced biological activity apparently asso-
ciated with these selenocompounds. In any case, it can be stated
that the compounds investigated show considerable anti-
proliferative and cytotoxic properties, which places them at the
centre for future anticancer drug research.
3. Conclusion
In summary, we describe the synthesis and the cytotoxic and
cytostatic evaluation of 31 new selenoesters against the prostate
cancer cell line PC-3, for the most active compounds, a more
detailed investigation of the biological activity against three further
cancer cell lines (breast ¼ MCF-7, lung ¼ A-549 and colon ¼ HT-29)
and one non-tumour prostate cell line (RWPE-1).
The majority of the compounds exerted a remarkable cytostatic
activity in PC-3 cancer cells. Compounds 29, 30 and 31, with GI50
values in the nanomolar order, were even more potent than eto-
poside, while 13 of them (6, 8, 9, 11, 12, 13, 21, 22, 23, 28, 29, 30 and
31) were more active than cisplatin. The same compounds, with the
exception of derivatives 8 and 11 in MCF-7, exhibited GI50 values
below 10 mM against the other cancer cell lines tested. It was found
that biological activity is often selective for the cancer cells
(compared to the normal cells) and tends to increase when the
polarity of the alkyl moiety bound to the selenium atom of the
selenoester group is diminished; whereas the existence of sym-
metric elements in the molecular structure seems to enhance the
biological activity. On the other hand, results enable us to confirm
that the presence of methyl-seleno moiety substantially increase
the antiproliferative activity of the compounds previously pub-
lished by our group [33,36], being noteworthy the activity
improvement observed as regards carboxymethylarylselenoates
described [33] and used as starting compounds in the design of the
4. Experimental protocols
4.1. Chemistry
Melting points were determined with a Mettler FP82 þ FP80
apparatus (Greifense, Switzerland) and have not been corrected.
The 1H and 13C NMR spectra were recorded on a Bruker 400
UltrashieldÔ spectrometer (Rheinstetten, Germany) using TMS as
the internal standard. The IR spectra were obtained on a Thermo
Nicolet FT-IR Nexus spectrophotometer with KBr pellets. Elemental
microanalyses were carried out on vacuum-dried samples using a
LECO CHN-900 Elemental Analyzer. Silica gel 60 (0.040e0.063 mm)
1.09385.2500 (Merck KGaA, 64271 Darmstadt, Germany) was used
for Column Chromatography and AlugramÒ SIL G/UV254 (Layer:
Table 5
Percentages of free radical scavenging activity (DPPH radical) obtained for the
various compounds.
Compound
5
m
M
15
m
M
50
m
M
150
m
M
500 mM
5
14.1 ꢁ 5.3 21.9 ꢁ 0.9 48.0 ꢁ 2.3 56.4 ꢁ 2.4 46.4 ꢁ 1.6
21
28
30
4.1 ꢁ 0.7
4.1 ꢁ 1.9
6.5 ꢁ 2.6
5.7 ꢁ 0.4
3.8 ꢁ 1.6
4.3 ꢁ 3.4
8.9 ꢁ 2.0 15.9 ꢁ 1.6 43.1 ꢁ 2.9
9.7 ꢁ 4.9 21.5 ꢁ 3.5
5.2 ꢁ 3.4
8.8 ꢁ 1.2
4.9 ꢁ 6.7 10.5 ꢁ 5.3
Ascorbic acid 29.1 ꢁ 2.1 87.5 ꢁ 0.9 98.5 ꢁ 1.2 98.5 ꢁ 1.2 98.9 ꢁ 1.6

160
E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
0.2 mm) (MachereyeNagel GmbH & Co. KG. Postfach 101352, D-
52313 Düren, Germany) was used for Thin Layer Chromatography.
Chemicals were purchased from E. Merck (Darmstadt, Germany),
Scharlau (F.E.R.O.S.A., Barcelona, Spain), Panreac Química S.A.
(Montcada i Reixac, Barcelona, Spain), SigmaeAldrich Química, S.A.
(Alcobendas, Madrid, Spain), Acros Organics (Janssen Pharmaceu-
ticalaan 3a, 2440 Geel, Belgium) and Lancaster (Bischheim-Stras-
bourg, France).
(OCH3); 105.2 (C2 þ C6); 106.6 (C4); 140.9 (C1); 161.7 (C3 þ C5); 170.7
(CONH2); 194.3 (COSeCH2). MS (m/z% abundance): 303 (Mþꢄ, 2), 165
(100). Elemental Analysis for C11H13NO4Se, calcd/found (%): C:
43.72/43.53; H: 4.34/4.44; N: 4.64/4.86.
4.1.1.4. Carbamoylmethyl phenylethaneselenoate (4). From phenyl-
acetyl chloride (1.96 g, 12.7 mmol), grey selenium (1.00 g,
12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and chlor-
oacetamide (1.18 g, 12.7 mmol). The solid was solved in ethyl ether/
methanol (9:1) and extracted with hexane (3 ꢃ 25 mL). The organic
layer was dried and evaporated for obtained a white-grey solid
(1.685 g, 52%); mp: 107e109 ꢂC. IR (KBr) cmꢀ1: 3345e3173 (m, Ne
H), 3061e2798 (w, CeH), 1697 (s, C]O ester), 1659 (s, C]O amide).
4.1.1. General procedure for compounds 1e4
A solution of sodium borohydride (1.00 g, 26.4 mmol) in 12.5 mL
of distilled water was added to a stirred suspension of grey sele-
nium (1.00 g, 12.7 mmol) in 12.5 mL of distilled water at room
temperature. The reaction mixture was stirred until an almost
colourless solution of NaHSe was formed. The corresponding acyl
chloride (12.7 mmol) was added in small portions and the reaction
mixture was magnetically stirred at 50 ꢂC for 1 h. The solid was
filtered and after that, chloroacetamide (1.18 g, 12.7 mmol) was
added to the filtrate. The mixture was heated at 50 ꢂC for 2 h. The
solid formed was solved in diethyl ether:methanol (9:1), to remove
metallic impurities by filtration. Final compound is precipitated by
addition of hexane to the filtrate.
1H NMR (400 MHz, DMSO-d6)
3.99 (s, 2H, ArCH2COSe); 7.03e7.49 (br s, 2H, NH2); 7.33e7.36 (m,
5H, HAr). 13C NMR (100 MHz, DMSO-d6)
: 27.4 (SeCH2); 53.9
d: 3.45 (s, 2H, CH2COSeCH2CONH2);
d
(ArCH2COSe); 128.6 (C4); 129.2 (C3 þ C5); 130.6 (C2 þ C6); 132.4
(C1); 172.8 (CONH2); 201.9 (COSe). MS (m/z% abundance): 253/254/
255/257/259 (Mþ, 1/1/2/4/2), 91 (100). Elemental Analysis for
C10H11NO2Se, calcd/found (%): C: 46.89/46.78; H: 4.33/4.51; N: 5.47/
5.43.
4.1.2. Benzo[c]selenophen-1,3-dione (5)
4.1.1.1. Carbamoylmethyl benzoselenoate (1). From benzoyl chloride
(1.78 g, 12.7 mmol), grey selenium (1.00 g, 12.7 mmol), sodium
borohydride (1.00 g, 26.4 mmol) and chloroacetamide (1.18ꢀ1g,
A 1 M solution of lithium aluminium hydride (7.00 mL,
7.00 mmol) was added to a stirred suspension of grey selenium
(1.10 g, 13.9 mmol) in 15 mL of THF at room temperature. The re-
action mixture was stirred until the end of the gas liberation.
Phthaloyl chloride (1.41 g, 6.9 mmol) solved in dichloromethane
(20 mL) was added in small portions and the reaction mixture was
magnetically stirred at 50 ꢂC for 1 h. After this time sulphuric acid
(10 mL) was added during five minutes. The solid was filtered and
was washed with chloroform (4 ꢃ 15 mL). The organic layer was
dried and evaporated for obtaining a brown solid that was recrys-
tallized from hexane to isolate an orange solid. Yield 95% (1.378 g);
mp: 122e124 ꢂC. IR (KBr) cmꢀ1: 1749 (C]O), 1685 (C]O). 1H NMR
12.7 mmol). Yield 37% (1.444 g); mp: 114e115 ꢂC. IR (KBr) cm
:
3383e3191 (m, NeH), 3055e2930 (w, CeH), 1654 (s, C]O ester),
1625 (s, C]O amide). 1H NMR (400 MHz, DMSO-d6)
d: 3.73 (s, 2H,
SeCH2CONH2); 7.15e7.65 (br s, 2H, CONH2); 7.59 (t, 2H, H3 þ H5, J3-
¼ 7.9 Hz, J3-4,5-4 ¼ 7.6 Hz); 7.74 (t, 1H, H4); 7.91 (dd, 2H, H2 þ H6,
2,5-6
J2-4,6-4 ¼ 1.0 Hz). 13C NMR (100 MHz, DMSO-d6)
d: 29.0 (SeCH2);
127.7 (C2 þ C6); 130.3 (C3 þ C5); 135.3 (C4); 138.8 (C1); 170.8
(CONH2); 194.4 (COSe). MS (m/z% abundance): 243 (Mþ, 1), 105
(100). Elemental Analysis for C9H9NO2Se, calcd/found (%): C: 44.64/
44.73; H: 3.75/3.88; N: 5.78/5.77.
(400 MHz, CDCl3)
d
: 7.95 (dd, 2H, H4 þ H5, J4-6,5-3 ¼ 3.2 Hz, J4-3,5-
¼ 5.7 Hz); 7.98 (dd, 2H, H3 þ H6). 13C NMR (100 MHz, CDCl3)
d:
6
4.1.1.2. Carbamoylmethyl 4-chlorobenzoselenoate (2). From 4-
chlorobenzoyl chloride (2.22 g, 12.7 mmol), grey selenium (1.00 g,
12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and chlor-
oacetamide (1.18 g, 12.7 mmol). The solid was solved in ethyl ether
(50 mL), washed with water (3 ꢃ 40 mL) and finally extracted with
hexane (3 ꢃ 25 mL). The organic layer was dried and evaporated for
obtaining a white solid (0.193 g, 6%); mp: 135e136 ꢂC. IR (KBr)
cmꢀ1: 3384e3192 (m, NeH), 3096e2923 (w, CeH), 1677 (s, C]O
124.0 (C3 þ C6); 135.3 (C1 þ C2), 142.2 (C4 þ C5), 194.5 (COSeCO). MS
(m/z% abundance): 212 (Mþꢄ, 11), 76 (100). Elemental Analysis for
C8H4O2Se, calcd/found (%): C: 45.48/45.33; H: 1.89/1.91.
4.1.3. General procedure for compounds 6e13
0.50 g of the corresponding selanylacetic acid is solved in
methanol (20 mL) in presence of hydrochloric acid (0.2 mL). The
mixture was heated by refluxing during 4 h. After this period, water
(25 mL) was added and extracted with chloroform (2 ꢃ 30 mL). The
organic phase was washed with water (3 ꢃ 20 mL), dried over
Na2SO4 and concentrated to dryness. The resultant residue was
purified by the appropriate treatment.
ester), 1657 (s, C]O amide). 1H NMR (400 MHz, DMSO-d6)
d: 3.74
(s, 2H, SeCH2CONH2); 7.16 (br s, 2H, NH2); 7.66 (d, 2H, H3 þ H5, J3-2,5-
¼ 8.6 Hz); 7.91 (d, 2H, H2 þ H6). 13C NMR (100 MHz, DMSO-d6)
d:
6
28.6 (SeCH2); 129.0 (C2 þ C6); 130.5 (C3 þ C5); 137.4 (C1); 140.0 (C4);
170.6 (CONH2); 193.4 (COSeCH2). MS (m/z% abundance): 277 (Mþ,
1), 139/141 (100/34). Elemental Analysis for C9H8ClNO2Se, calcd/
found (%): C: 39.08/39.03; H: 2.92/2.96; N: 5.06/5.02.
4.1.3.1. Methoxycarbonylmethyl benzoselenoate (6). From benzoyl-
selanylacetic acid (0.50 g, 2.06 mmol), methanol (20 mL) and hy-
drochloric acid (0.2 mL). The residue was stirred during 48 h with
ethyl ether (100 mL), silica gel and activated carbon. The organic
phase was filtered, dried and evaporated to dryness. A yellowish
liquid was obtained. Yield 35% (0.184 g). IR (KBr) cmꢀ1: 3061e2850
(m, CeH), 1738 (s, C]O ester), 1678 (s, C]O selenoester). 1H NMR
4.1.1.3. Carbamoylmethyl-3,5-dimethoxybenzoselenoate
(3).
From 3,5-dimethoxybenzoyl chloride (1.27 g, 6.3 mmol), grey se-
lenium (0.50 g, 6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol)
and chloroacetamide (0.59 g, 6.33 mmol). The solid was solved in
ethyl ether/methanol (1:5, 50 mL), washed with water (3 ꢃ 40 mL)
and finally extracted with hexane (3 ꢃ 25 mL). The organic layer
was dried and evaporated for obtaining a white solid (0.380 g, 20%);
mp: 121e124 ꢂC. IR (KBr) cmꢀ1: 3374e3187 (s, NeH), 3096e2931
(w, CeH), 1686 (m, C]O ester), 1658 (s, C]O amide). 1H NMR
(400 MHz, CDCl3) d: 3.77 (s, 3H, COOCH3); 3.87 (s, 2H, SeCH2
-
COOCH3); 7.49 (dt, 2H, H3 þ H5, J3-2,5-6 ¼ 8.1 Hz, J3-4,5-4 ¼ 7.6 Hz);
7.64 (m, 1H, H4, J4-2,4-6 ¼ 1.3 Hz); 7.92 (dd, 2H, H2 þ H6). 13C NMR
(100 MHz, CDCl3)
d
: 25.5 (SeCH2); 53.2 (COOCH3); 127.8 (C2 þ C6);
129.4 (C3 þ C5); 134.5 (C4); 138.8 (C1); 171.1 (COO); 192.7 (COSe).
MS (m/z% abundance): 258 (Mþ, 1), 225/227 (1/2), 105 (100).
Elemental Analysis for C10H10O3Se, calcd/found (%): C: 46.71/46.98;
H: 3.92/4.08.
(400 MHz, CDCl3) d: 3.67 (s, 2H, SeCH2CONH2); 3.86 (s, 6H, OCH3);
5.47e6.29 (br s, 2H, NH2); 6.73 (t, 1H, H4, J4-2,4-6 ¼ 2.3 Hz); 7.04 (d,
2H, H2 þ H6). 13C NMR (100 MHz, DMSO-d6)
d: 29.3 (SeCH2); 56.5

E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
161
4.1.3.2. Methoxycarbonylmethyl 4-chlorobenzoselenoate (7). From 4-
O selenoester). 1H NMR (400 MHz, CDCl3)
d: 3.76 (s, 3H, COOCH3);
chlorobenzoylselanylacetic acid (0.55 g, 1.26 mmol), methanol
(20 mL) and hydrochloric acid (0.2 mL). The residue was dried in
vacuum to give a white solid. Yield 61% (0.230 g); mp: 46e47 ꢂC. IR
(KBr) cmꢀ1: 3001e2945 (w, CeH), 1737 (s, C]O ester); 1677 (s, C]O
3.86 (s, 2H, SeCH2); 7.17 (dd, 1H, H4, J4-5 ¼ 3.9 Hz, J4-3 ¼ 4.9 Hz); 7.73
(dd, 1H, H3, J3-4 ¼ 4.9 Hz, J3-5 ¼ 1.1 Hz); 7.83 (dd, 1H, H5, J5-4 ¼ 3.9, J5-
¼ 1.1 Hz). 13C NMR (100 MHz, CDCl3)
d: 25.6 (SeCH2COOCH3); 53.2
3
(COOCH3); 128.5 (C4); 132.6 (C5); 134.4 (C3); 142.9 (C2); 170.9
(CH2COOCH3); 182.8 (COSeCH2). MS (m/z% abundance): 264 (Mþ,
2), 111 (100). Elemental Analysis for C8H8O3SSe, calcd/found (%): C:
36.51/36.87; H: 3.06/3.10.
selenoester). 1H NMR (400 MHz, CDCl3)
d: 3.77 (s, 3H, COOCH3); 3.87
(s, 2H, SeCH2COOCH3); 7.48 (d, 2H, H3 þ H5, J3-2,5-6 ¼ 8.7 Hz); 7.86 (d,
2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d: 25.7 (SeCH2); 53.2
(COOCH3); 56.1 (ArOCH3),105.4 (C2 þ C6); 106.8 (C4); 140.3 (C1); 161.4
(C3 þ C5); 171.1 (COO); 192.7 (COSe). MS (m/z% abundance): 139/141
(100/34), 111/113 (46/15). Elemental Analysis for C10H9ClO3Se, calcd/
found (%): C: 41.19/41.49; H: 3.11/3.16.
4.1.3.7. Methoxycarbonylmethyl
phenylethaneselenoate
(12).
From phenylacetoylselanylacetic acid (0.50 g,1.94 mmol), methanol
(20 mL) and hydrochloric acid (0.2 mL). The residue was stirred
during 48 h with ethyl ether (100 mL), silica gel and activated
carbon. The organic phase was filtered, dried and evaporated to
dryness. A yellowish liquid was obtained. Yield 25% (0.132 g); mp:
88e89 ꢂC. IR (KBr) cmꢀ1: 3062e2846 (w, CeH), 1736 (m, C]O
4.1.3.3. Methoxycarbonylmethyl
2-chlorobenzoselenoate
(8).
From 2-chlorobenzoylselanylacetic acid (0.46 g, 1.66 mmol),
methanol (20 mL) and hydrochloric acid (0.2 mL). The residue was
stirred during 48 h with ethyl ether (100 mL), silica gel and acti-
vated carbon. The organic phase was filtered, dried and evaporated
to dryness. A yellowish liquid was obtained. Yield 48% (0.230 g). IR
(KBr) cmꢀ1: 3064e2846 (m, CeH), 1738 (s, C]O ester); 1692 (s, C]
ester), 1704 (m, C]O selenoester). 1H NMR (400 Hz, CDCl3)
d: 3.62
(s, 2H, SeCH2COO); 3.70 (s, 3H, COOCH3); 3.89 (s, 2H, ArCH2COSe);
7.30e7.34 (m, 2H, H2 þ H6); 7.35e7.40 (m, 3H, H3 þ H4 þ H5). 13
C
NMR (100 MHz, CDCl3)
d: 25.8 (SeCH2); 41.6 (ArCH2CO); 53.7
O selenoester). 1H NMR (400 MHz, CDCl3)
d: 3.77 (s, 3H, COOCH3);
(COOCH3); 128.5 (C4); 129.2 (C3 þ C5); 130.6 (C2 þ C6); 132.6 (C1);
171.0 (COO); 198.8 (COSe). MS (m/z% abundance): 272 (Mþꢄ, 1), 91
(100). Elemental Analysis for C11H12O3Se, calcd/found (%): C: 48.72/
48.90; H: 4.46/4.28.
3.89 (s, 2H, SeCH2COO); 7.35e7.39 (m, 1H, H5); 7.46e7.48 (m, 2H,
H4 þ H3); 7.75 (dd, 1H, H6, J6-5 ¼ 7.7 Hz, J6-4 ¼ 2.0 Hz). 13C NMR
(100 MHz, CDCl3) d: 26.9 (SeCH2); 53.2 (OCH3); 127.4 (C5); 129.8
(C3); 130.8 (C2); 131.7 (C6); 133.4 (C4); 138.0 (C1); 170.8 (COO); 192.1
(COSe). MS (m/z% abundance): 139/141 (100/34), 111/113 (38/11),
85/87 (5/1). Elemental Analysis for C10H9ClO3Se, calcd/found (%): C:
41.19/40.83; H: 3.11/2.86.
4.1.3.8. Methoxycarbonylethyl
phenylethaneselenoate
(13).
From 3-phenylacetoylselanyl propionic acid (0.50 g, 1.84 mmol),
methanol (20 mL) and hydrochloric acid (0.2 mL). The propionic
acid used as reagent was prepared according the following proto-
col. A solution of sodium borohydride (1.00 g, 26.4 mmol) in
12.5 mL of distilled water was added to a stirred suspension of grey
selenium (1.00 g, 12.7 mmol) in 12.5 mmol of distilled water at
room temperature. The reaction mixture was stirred until an almost
colourless solution of NaHSe was formed. The 2-phenylacetyl
chloride (1.96 g, 12.7 mmol) was added in small portions and the
reaction mixture was magnetically stirred for 1 h. A yellow solution
was formed and the 3-bromo propionic acid (1.94 g, 12.7 mmol)
was added. Within 60 min at 40 ꢂC a solid was formed. The product
was filtered and purified by column chromatography (ethyl acetate)
and recrystallized from carbon tetrachloride for obtaining a white
solid. Yield 17% (0.569 g). IR (KBr) cmꢀ1: 3064e2565 (s, CeH); 1709
(s, C]O acid); 1689 (s, C]O selenoester). 1H NMR (400 MHz, CDCl3)
4.1.3.4. Methoxycarbonylmethyl 3,5-dimethoxybenzoselenoate (9).
From 3,5-dimethoxybenzoylselanylacetic acid (1.0 g, 3.30 mmol),
methanol (20 mL) and hydrochloric acid (0.2 mL). The residue was
stirred during 48 h with ethyl ether (100 mL), silica gel and acti-
vated carbon. The organic phase was filtered, dried and evaporated
to dryness. An orangish liquid was obtained which was purified by
silica gel column chromatography (hexane/acetate 4:1) to give 9.
Yield 35% (0.371 g). IR (KBr) cmꢀ1: 3091e2840 (m, CeH), 1738 (s,
C]O ester),1668 (s, C]O selenoester). 1H NMR (400 MHz, CDCl3)
d:
3.77 (s, 3H, COOCH3); 3.85 (s, 2H, SeCH2COOCH3); 3.85 (s, 6H,
ArOCH3); 6.71 (t,1H, H4, J4-2,4-6 ¼ 2.3 Hz); 7.05 (d, H2 þ H6). 13C NMR
(100 MHz, CDCl3) d: 25.7 (SeCH2); 53.2 (COOCH3); 56.1 (ArOCH3),
105.4 (C2 þ C6); 106.8 (C4); 140.3 (C1); 161.4 (C3 þ C5); 171.1 (COO);
192.7 (COSe). MS (m/z% abundance): 165 (100). Elemental Analysis
for C12H14O5Se, calcd/found (%): C: 45.44/45.39; H: 4.45/4.42.
d
: 2.80 (t, 2H, SeCH2CH2CO, JCH(CO)eCH(Se) ¼ 6.9 Hz); 3.07 (t, 2H,
SeCH2CH2CO); 3.87 (s, 2H, ArCH2CO); 7.30 (dd, 2H, H2 þ H6, J2-3,6-
¼ 7.6 Hz, J2-4,6-4 ¼ 1.8 Hz); 7.34e7.38 (m, 3H, H3 þ H4 þ H5). 13
C
5
4.1.3.5. Methoxycarbonylmethyl 3,4,5-trimethoxybenzoselenoate
NMR (100 MHz, CDCl3) d: 19.6 (SeCH2CH2); 35.4 (CH2CH2CO); 54.5
(10). From
3,4,5-trimetoxybenzoylselanylacetic
(0.40
g,
(ArCH2); 128.2 (C4); 129.1 (C3 þ C5); 130.5 (C2 þ C6); 133.7 (C1);
178.8 (COOH); 200.7 (COSe). MS (m/z% abundance): 268/269/270/
272/274 (Mþꢄ, 1/1/2/4/1, Se), 91 (100). Elemental Analysis for
1.20 mmol), methanol (20 mL) and hydrochloric acid (0.2 mL). The
residue was dried in vacuum to give a white solid. Yield 49%
(0.203 g); mp: 36e38 ꢂC. IR (KBr) cmꢀ1: 3000e2837 (m, CeH), 1738
(m, C]O ester), 1680 (m, C]O selenoester). 1H NMR (400 MHz,
C11H12O3Se, calcd/found (%):C: 48.72/48.62; H: 4.46/4.28.
The residue was stirred during 48 h with ethyl ether (100 mL),
CDCl3)
d
: 3.66 (s, 3H, COOCH3); 3.77 (s, 3H, 4-OCH3); 3.87 (s, 6H,
silica gel and activated carbon. The organic phase was filtered, dried
and evaporated to dryness. A yellowish liquid was obtained which
was purified by silica gel column chromatography (dichloro-
methane, 100) to give 13. Yield 26% (0.137 g). IR (KBr) cmꢀ1: 3062e
2846 (m, CeH), 1737 (s, C]O ester); 1697 (s, C]O, selenoester). 1H
3,5-diOCH3); 3.89 (s, 2H, SeCH2COOH); 7.13 (2H, H2 þ H6). 13C NMR
(100 MHz, CDCl3) d: 25.7 (SeCH2); 53.2 (COOCH3); 56.1 (ArOCH3),
105.4 (C2 þ C6); 106.8 (C4); 140.3 (C1); 161.4 (C3 þ C5); 171.1 (COO);
192.7 (COSe). MS (m/z% abundance): 195 (100). Elemental Analysis
for C13H16O6Se, calcd/found (%): C: 44.97/44.60; H: 4.64/4.63.
NMR (400 MHz, CDCl3)
d: 2.75 (t, 2H, SeCH2CH2COOCH3, JCH(CO)e
¼ 7.0 Hz); 3.08 (t, 2H, COSeCH2CH2COOCH3); 3.69 (s, 3H,
CH(Se)
4.1.3.6. Methoxycarbonylmethyl 2-thiophenecarboselenoate (11).
From thieno-2-ylselanylacetic acid (0.50 g, 2.01 mmol), methanol
(20 mL) and hydrochloric acid (0.2 mL). The residue was stirred
during 48 h with ethyl ether (100 mL), silica gel and activated
carbon. The organic phase was filtered, dried and evaporated to
dryness. A yellowish liquid was obtained. Yield 31% (0.164 g). IR
(KBr) cmꢀ1: 3103e2847 (m, CeH), 1738 (s, C]O ester); 1668 (s, C]
COOCH3); 3.86 (s, 2H, ArCH2COSe); 7.30 (dd, 2H, H2 þ H6, J2-3,6-
¼ 7.7 Hz, J2-4,6-4 ¼ 1.8 Hz); 7.34e7.39 (m, 3H, H3 þ H4 þ H5). 13
C
5
NMR (100 MHz, CDCl3) d: 20.1 (SeCH2CH2CO); 35.4 (SeCH2CH2CO);
52.2 (OCH3); 54.5 (ArCH2COSe); 128.2 (C4); 129.0 (C3 þ C5); 130.5
(C2 þ C6); 133.2 (C1); 173.0 (COOCH3); 200.6 (COSe). MS (m/z%
abundance): 284 (Mþ ꢀ 2, 3), 91 (100). Elemental Analysis for
ꢄ
C
12H14O3Se, calcd/found (%): C: 50.54/50.70; H: 4.95/4.86.

162
E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
4.1.4. General procedure for compounds 14e17
(0.50 g, 6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol) and
tert-butyl bromoacetate (1.235 g, 6.3 mmol). Purified by column
chromatography (toluene, 100%) to obtain 17 as a clear yellowish
liquid. Yield 24% (0.472 g). IR (KBr) cmꢀ1: 3064e2932 (w, CeH);
1728 (s, C]O ester), 1714 (s, C]O selenoester). 1H NMR (400 MHz,
A solution of sodium borohydride (1.00 g, 26.4 mmol) in 12.5 mL
of distilled water was added to a stirred suspension of grey sele-
nium (1.00 g, 12.7 mmol) in 12.5 mmol of distilled water at room
temperature. The reaction mixture was stirred until an almost
colourless solution of NaHSe was formed. The corresponding acyl
chloride (12.7 mmol) was added in small portions and the reaction
mixture was magnetically stirred at 50 ꢂC for 1 h. The solid was
filtered and tert-butyl bromoacetate (2.47 g, 12.7 mmol) was added
upon the solution. The mixture was heated during 2 h at 50 ꢂC. The
mixture of the reaction was extracted with chloroform (3 ꢃ 40 mL),
washed with water (3 ꢃ 40 mL), dried over Na2SO4 and concen-
trated to dryness. The residue was stirred with ethyl ether (100 mL),
silica gel and active carbon during 24 h. The organic phase was
filtered, dried and evaporated to dryness and the residue was pu-
rified by column chromatography.
CDCl3)
d
: 1.43 (s, 3H, C(CH3)3); 3.55 (s, 2H, SeCH2); 3.89 (s, 2H,
ArCH2); 7.33e7.36 (m, 5H, HAr). 13C NMR (100 MHz, CDCl3)
d: 27.8
(SeCH2); 28.3 (C(CH3)3); 53.9 (ArCH2); 82.2 (C(CH3)3); 128.4 (C4);
129.2 (C3 þ C5); 130.5 (C2 þ C6); 132.8 (C1); 169.6 (COO); 198.9
(COSe). MS (m/z% abundance): 316 (Mþ þ 2, 1), 91 (100). Elemental
ꢄ
Analysis for C14H18O3Se, calcd/found (%): C: 53.68/53.49; H: 5.79/
6.02.
4.1.5. General procedure for compounds 18e20
A solution of sodium borohydride (0.50 g, 13.2 mmol) in 12.5 mL
of distilled water was added to a stirred suspension of grey sele-
nium (0.50 g, 6.3 mmol) in 12.5 mmol of distilled water at room
temperature. The reaction mixture was stirred until an almost
colourless solution of NaHSe was formed. The corresponding acyl
chloride (6.3 mmol) was added in small portions and the reaction
mixture was magnetically stirred at 50 ꢂC for 1 h. The solid was
filtered and phenyl bromoacetate (6.3 mmol) was added upon the
solution. The mixture was heated during 2 h at 50 ꢂC. After this
period, the reaction was filtered and the solid extracted with
dichloromethane (3 ꢃ 40 mL), washed with water (3 ꢃ 40 mL),
dried over Na2SO4 and concentrated to dryness. The residue was
purified.
4.1.4.1. tert-Butoxycarbonylmethyl benzoselenoate (14). From ben-
zoyl chloride (1.78 g,12.7 mmol), grey selenium (1.00 g,12.7 mmol),
sodium borohydride (1.00 g, 26.4 mmol) and tert-butyl bromoa-
cetate (2.47 g, 12.7 mmol). Purified by column chromatography
(toluene, 100%) to obtain 14 as a yellowish liquid. Yield 26%
(0.987 g). IR (KBr) cmꢀ1: 3062e2932 (w, CeH), 1731 (s, C]O ester),
1677 (s, C]O selenoester). 1H NMR (400 MHz, CDCl3)
d: 1.49 (s, 9H,
O(CH3)3); 3.79 (s, 2H, SeCH2CO); 7.49 (t, 2H, H3 þ H5, J3-2,5-
¼ 8.1 Hz, J3-4,5-4 ¼ 7.9 Hz); 7.63 (m, 1H, H4, J4-2,4-6 ¼ 1.3 Hz); 7.92
6
(dd, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d: 27.6 (SeCH2CO);
29.0 (OC(CH3)3); 82.3 (OC(CH3)3); 127.7 (C2 þ C6); 129.3 (C3 þ C5);
134.4 (C4); 138.7 (C1); 169.7 (COO); 193.1 (COSe). MS (m/z% abun-
dance): 240/241/242/244/246 (1/1/2/4/1), 223/224/225/227/229
(1/1/2/4/1).
4.1.5.1. Phenoxycarbonylmethyl benzoselenoate (18). From benzoyl
chloride (1.245 g, 8.9 mmol), grey selenium (0.70 g, 8.5 mmol),
sodium borohydride (0.70 g, 18.5 mmol) and phenyl bromoacetate
(1.36 g, 6.3 mmol). The residue was purified by stirring in
dichloromethane (100 mL), and filtered; mp: 40e41 ꢂC. Yield 53%
(1.070 g). IR (KBr) cmꢀ1: 3062e2940 (w, CeH), 1739 (s, C]O ester),
4.1.4.2. tert-Butoxycarbonylmethyl 4-chlorobenzoselenoate (15).
From 4-chlorobenzoyl chloride (2.22 g, 12.7 mmol), grey selenium
(1.00 g, 12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and
tert-butyl bromoacetate (2.47 g, 12.7 mmol). Purified by column
chromatography (toluene, 100%) to obtain 15 as a yellowish liquid.
Yield 8% (0.328 g). IR (KBr) cmꢀ1: 3090e2932 (s, CeH), 1731 (s, C]
1679 (s, C]O selenoester). 1H NMR (400 MHz, CDCl3)
d
0
: 4.08 (s, 2H,
0
0
0
0
0
0
0
0
0
SeCH2); 7.15 (dd, 2H, H2 þ H6 , J2 -3 ,6 -5 ¼ 8.3 Hz, J2 -4 ,6 -4 ¼ 1.2 Hz);
0
0
0
0
0
0
0
7.25 (m, 1H, H4 , J4 -3 ,4 -5 ¼ 7.7 Hz); 7.40 (dt, 2H, H3 þ H5 ); 7.52 (dt,
O ester), 1678 (s, C]O selenoester). 1H NMR (400 MHz, CDCl3)
d:
2H, H3 þ H5, J3-4,5-4 ¼ 8.2 Hz, J3-2,5-6 ¼ 8.0 Hz); 7.66 (m, 1H, H4, J4-2,4-
¼ 1.0 Hz); 7.95 (dd, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d: 25.7
6
1.49 (s, 9H, C(CH3)3); 3.79 (s, 2H, SeCH2); 7.47 (d, 2H, H3 þ H5, J3-
0
0
0
¼ 8.7 Hz); 7.86 (d, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d
:
(SeCH2); 121.8 (C2 þ C6 ); 126.4 (C4 ); 127.8 (C2 þ C6); 129.5
2,5-6
0
0
0
(C3 þ C5); 129.8 (C3 þ C5 ); 134.6 (C4); 138.3 (C1); 151.2 (C1 ); 169.3
(COO); 192.4 (COSe). MS (m/z% abundance): 223/224/225/227/229
(3/3/8/16/4, Se), 195/197/199/201 (1/1/2/2, Se), 105 (100).
Elemental Analysis for C15H12O3Se, calcd/found (%): C: 56.44/56.15;
H: 3.79/3.86.
27.8 (SeCH2); 29.2 (C(CH3)3); 82.5 (C(CH3)3); 129.0 (C2 þ C6); 130.2
(C3 þ C5); 136.7 (C4); 140.6 (C1); 169.4 (COO); 191.9 (COSe). MS (m/
z% abundance): 276 (3), 139/141 (20/7, Cl), 111/113 (13/4, Cl), 57
(100). Elemental Analysis for C13H15ClO3Se, calcd/found (%): C:
46.80/46.52; H: 4.53/4.67.
4.1.4.3. tert-Butoxycarbonylmethyl 3,5-dimethoxybenzoselenoate
(16). From 3,5-dimethoxybenzoyl chloride (2.54 g, 12.7 mmol),
grey selenium (1.00 g, 12.7 mmol), sodium borohydride (1.00 g,
26.4 mmol) and tert-butyl bromoacetate (2.47 g, 12.7 mmol). Pu-
rified by column chromatography (hexane/acetate, 4:1) to obtainꢀ116
4.1.5.2. Phenoxycarbonylmethyl
4-chlorobenzoselenoate
(19).
From 4-chlorobenzoyl chloride (1.11 g, 6.33 mmol), grey selenium
(0.50 g, 6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol) and
phenyl bromoacetate (1.36 g, 6.3 mmol). The residue was purified
by column chromatography (hexane/toluene, 11:9) to obtain 19 as a
as a white solid; mp: 65e66 ꢂC. Yield 21% (0.952 g). IR (KBr) cm
:
white solid; mp: 67e68 ꢂC. Yield 24% (0.547 g). IR (KBr) cmꢀ1
:
3004e2844 (m, CeH), 1735 (s, C]O ester), 1671 (s, C]O sele-
3067e2854 (s, CeH), 1752 (s, C]O ester), 1676 (s, C]O sele-
noester). 1H NMR (400 MHz, CDCl3)
d
: 1.49 (s, 9H, C(CH3)3); 3.77 (s,
noester). 1H NMR (400 MHz, CDCl3)
d
: 4.08 (s, 2H, SeCH2COOPh);
0
0
0
0
0
0
0
0
0
0
2H, SeCH2); 3.85 (s, 6H, OCH3); 6.70 (t, 1H, H4, J4-2,4-6 ¼ 2.2 Hz); 7.05
7.14 (dd, 2H, H2 þ H6 , J2 -3 ,6 -5 ¼ 7.6 Hz, J2 -4 ,6 -4 ¼ 1.0 Hz); 7.26 (dt,
(d, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d: 27.8 (C(CH3)3); 29.2
1H, H4 , J4 -3 ,4 -5 ¼ 7.7 Hz); 7.40 (t, 2H, H3 þ H5 ); 7.49 (dd, 2H,
0
0
0
0
0
0
0
(SeCH2); 56.1 (OCH3); 82.4 (C(CH3)3); 104.5 (C4); 106.0 (C2 þ C6);
140.6 (C1); 1614 (C3 þ C5); 169.6 (COO); 193.0 (COSe). MS (m/z%
abundance): 274/275/276/278/280 (2/1/3/3/1, Se), 57 (100).
Elemental Analysis for C15H20O5Se, calcd/found (%): C: 50.15/50.00;
H: 5.61/5.48.
H3 þ H5, J3-2,5-6 ¼ 8.7 Hz); 7.89 (dd, 2H, H2 þ H6). 13C NMR
0
0
(100 MHz, CDCl3)
d
: 26.0 (SeCH2COOPh); 121.7 (C2 þ C6 ); 126.5
0
0
0
(C4 ); 129.1 (C2 þ C6); 129.8 (C3 þ C5); 129.9 (C3 þ C5 ); 136.6 (C4);
0
141.1 (C1); 151.1 (C1 ); 169.1 (COSeCH2COOPh); 191.3 (COSeCH2
-
COOPh). MS (m/z% abundance): 300/302/303/304/306/308 (3/7/4/
11/12/4), 255/257/258/259/260/261/263/265 (1/3/2/6/1/12/5/1),
156/158 (5/3), 139/141 (100/34), 111/113 (28/10). Elemental Anal-
ysis for C15H11ClO3Se, calcd/found (%): C: 50.94/51.26; H: 3.14/3.27.
4.1.4.4. tert-Butoxycarbonylmethyl
phenylethaneselenoate
(17).
From phenylacetyl chloride (0.978 g, 6.3 mmol), grey selenium

E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
163
4.1.5.3. Phenoxycarbonylmethyl 3,5-dimethoxybenzoselenoate (20).
From 3,5-dimethoxybenzoyl (1.27 g, 6.33 mmol), grey selenium
(0.50 g, 6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol) and
phenyl bromoacetate (1.36 g, 6.3 mmol). The residue was purified
by column chromatography (hexane/ethyl acetate, 4:1) to obtain 20
as a yellow liquid. Yield 26% (0.625 g). IR (KBr) cmꢀ1: 3071e2839
(m, CeH), 1752 (s, C]O ester), 1680 (s, C]O selenoester). 1H NMR
4.1.6.3. 3,3-Dimethyl-2-oxobutyl 3,5-dimethoxybenzoselenoate (23).
From 3,5-dimethoxybenzoyl chloride (1.27 g, 6.33 mmol), grey se-
lenium (0.50 g, 6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol)
and 1-chloropinacolone (0.85 g, 6.3 mmol). The solid was solved in
dichloromethane (50 mL) and washed with water (2 ꢃ 40 mL). The
organic layer was dried over Na2SO4 and concentrated to dryness
for obtaining 23 as a white solid; mp: 67e70 ꢂC. Yield 57% (1.239 g).
IR (KBr) cmꢀ1: 3091e2838 (m, CeH), 1713 (s, C]O ketone), 1668 (s,
(400 MHz, CDCl3)
d: 3.87 (s, 6H, 3,5-diOCH3); 4.06 (s, 2H, SeCH2
-
COOPh); 6.73 (t, 1H, H4, J4-2,4-6 ¼ 2.3 Hz); 7.08 (d, 2H, H2 þ H6); 7.15
C]O selenoester). 1H NMR (400 MHz, CDCl3)
SeCH2COC(CH3)3); 3.85 (s, 6H, ArOCH3); 4.16 (s, 2H, SeCH2
d: 1.29 (s, 9H,
0
0
0
0
0
0
0
0
0
0
(dd, 2H, H2 þ H6 , J2 -3 ,6 -5 ¼ 8.6 Hz, J2 -4 ,6 -4 ¼ 1.1 Hz); 7.25 (m, 1H,
-
H4 , J4 -3 ,4 -5 ¼ 7.4 Hz); 7.40 (m, 2H, H3 þ H5 ). 13C NMR (100 MHz,
COC(CH3)3); 6.70 (t, 1H, H4, J4-2,4-6 ¼ 2.3 Hz); 7.05 (d, 2H, H2 þ H6).
0
0
0
0
0
0
0
CDCl3)
d
: 25.9 (SeCH2COOPh); 56.1 (OCH3); 105.5 (C2 þ C6); 106.8
13C NMR (100 MHz, CDCl3)
d:
27.7 (SeCH2COC(CH3)3); 31.8
0
0
0
0
0
(C4); 121.8 (C2 þ C6 ); 126.4 (C4 ); 129.8 (C3 þ C5 ); 140.2 (C1); 151.1
(SeCH2COC(CH3)3); 45.2 (COC(CH3)3); 56.6 (OCH3); 104.5 (C2 þ C6);
106.0 (C4); 140.7 (C1); 161.4 (C3 þ C5); 193.9 (COSeCH2COOPh);
210.7 (COSeCH2COC(CH3)3). MS (m/z% abundance): 165 (100).
Elemental Analysis for C15H20O4Se, calcd/found (%): C: 52.48/52.37;
H: 5.87/5.92.
0
(C1 ); 161.5 (C3 þ C5); 169.2 (COSeCH2COOPh); 192.4 (COSeCH2
-
COOPh). MS (m/z% abundance): 258 (3), 165 (100). Elemental
Analysis for C17H16O5Se, calcd/found (%): C: 53.84/53.74; H: 4.25/
4.37.
4.1.7. General procedure for compounds 24e31
4.1.6. General procedure for compounds 21e23
A solution of sodium borohydride (1.00 g, 26.4 mmol) in 12.5 mL
of distilled water was added to a stirred suspension of grey sele-
nium (1.00 g, 12.7 mmol) in 12.5 mL of distilled water at room
temperature. The reaction mixture was stirred until an almost
colourless solution of NaHSe was formed. The corresponding acyl
chloride (12.7 mmol for monofunctionalized chlorides and 6.3 for
dichlorides) was added in small portions and the reaction mixture
was magnetically stirred at 50 ꢂC for 1 h. The solid was filtered and
methyl iodide (1.5 mL) was added upon the solution. The mixture
was heated during 1 h at 50 ꢂC. The final product is obtained by
filtration in the case of difunctionalized derivatives. In the case of
the monofunctionalized ones, the solid is solved in ether:-
dichloromethane (1:1) to remove metallic impurities by filtration,
being eliminated the solvent in vacuum afterwards.
A solution of sodium borohydride (0.50 g, 13.2 mmol) in 12.5 mL
of distilled water was added to a stirred suspension of grey sele-
nium (0.50 g, 6.3 mmol) in 12.5 mL of distilled water at room
temperature. The reaction mixture was stirred until an almost
colourless solution of NaHSe was formed. The corresponding acyl
chloride (6.33 mmol) was added in small portions and the reaction
mixture was magnetically stirred at 50 ꢂC for 1 h. The solid was
filtered and the adequate a-haloketone (6.3 mmol) was added upon
the solution. The mixture was heated during 2 h at 50 ꢂC. The solid
resultant was filtered, washed with appropriate solvents and
purified.
4.1.6.1. 2-Oxopropyl
4-chlorobenzoselenoate
(21). From
4-
chlorobenzoyl chloride (1.11 g, 6.33 mmol), grey selenium (0.50 g,
6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol) and 1-chloro-
2-propanone (0.59 g, 6.3 mmol). The solid was solved in dichloro-
methane (50 mL) and was washed with water (3 ꢃ 15 mL). The
organic layer was dried and evaporated and the white solid was
4.1.7.1. Methyl benzoselenoate (24). From benzoyl chloride (1.78 g,
12.7 mmol), grey selenium (1.00 g,12.7 mmol), sodium borohydride
(1.00 g, 26.4 mmol) and methyl iodide (1.5 mL, excess). Yellowish
liquid, isolated after reaction by decantation instead of by filtration.
Yield 52% (1.316 g). IR (KBr) cmꢀ1: 3060e2932 (w, CeH), 1673 (s,
identified as 21; mp: 41e42 ꢂC. Yield 54% (0.945 g). IR (KBr) cmꢀ1
:
C]O). 1H NMR (400 MHz, CDCl3)
d: 2.42 (s, 3H, SeCH3); 7.48 (m, 2H,
3078e2921 (w, CeH), 1709 (s, C]O ketone), 1660 (s, C]O sele-
noester). 1H NMR (400 MHz, CDCl3)
d
: 2.37 (s, 3H, COCH3); 3.94 (s,
H3 þ H5, J3-4,5-4 ¼ 7.7 Hz, J3-2,5-6 ¼ 7.6 Hz); 7.61 (m, 1H, H4, J4-2,4-
¼ 1.2 Hz); 7.93 (dd, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d
: 5.5
2H, SeCH2COCH3); 7.48 (d, 2H, H3 þ H5, J3-2,5-6 ¼ 8.7 Hz); 7.87 (d,
6
2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d: 29.1 (SeCH2); 35.0
(SeCH3); 127.5 (C2 þ C6); 129.2 (C3 þ C5); 133.9 (C4); 139.4 (C1);
195.3 (ArCOSeCH3). MS (m/z% abundance): 196/197/198/200 (Mþ
,
(COCH3); 129.1 (C2 þ C6); 129.7 (C3 þ C5); 136.7 (C4); 141.1 (C1);
192.0 (COSe); 203.7 (COCH3). MS (m/z% abundance): 139/141 (100/
33), 111/113 (46/15), 85/87 (4/1). Elemental Analysis for
ꢄ
1/1/1/3), 105 (100).
C
10H9ClO2Se, calcd/found (%): C: 43.58/43.30; H: 3.29/3.27.
4.1.7.2. Methyl 4-chlorobenzoselenoate (25). From 4-chlorobenzoyl
chloride (2.22 g, 12.7 mmol), grey selenium (1.00 g, 12.7 mmol),
sodium borohydride (1.00 g, 26.4 mmol) and methyl iodide (1.5 mL,
excess). Yellowish solid; mp: 46e47 ꢂC. Yield: 79% (2.341 g). IR
(KBr) cmꢀ1: 3093e2928 (w, CeH), 1668 (s, C]O). 1H NMR
4.1.6.2. 3,3-Dimethyl-2-oxobutyl 4-chlorobenzoselenoate
(22).
From 4-chlorobenzoyl chloride (1.11 g, 6.33 mmol), grey selenium
(0.50 g, 6.3 mmol), sodium borohydride (0.50 g, 13.2 mmol) and 1-
chloropinacolone (0.85 g, 6.3 mmol). The solid was solved in
dichloromethane (50 mL) and washed with water (2 ꢃ 40 mL). The
organic layer was dried over Na2SO4 and concentrated to dryness
for obtaining 22 as a white solid; mp: 119e120 ꢂC. Yield 59%
(1.184 g). IR (KBr) cmꢀ1: 3092e2871 (w, CeH), 1718 (s, C]O ke-
(400 MHz, CDCl3)
d
: 2.42 (s, 3H, SeCH3); 7.45 (d, 2H, H3 þ H5, J3-2,5-
¼ 8.6 Hz); 7.87 (d, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d: 5.8
6
(SeCH3); 128.8 (C2 þ C6); 129.5 (C3 þ C5); 137.7 (C4); 140.3 (C1);
194.1 (ArCOSeCH3). MS (m/z% abundance): 232/234/236 (Mþꢄ, 1/2/
1), 156/158 (9/3), 139/141 (100/34), 111/113 (39/13), 85 (7), 75 (12),
50 (6). Elemental Analysis for C8H7ClOSe, calcd/found (%): C: 41.14/
40.98; H: 3.02/2.92.
tone), 1665 (s, C]O selenoester). 1H NMR (400 MHz, CDCl3)
d: 1.29
(s, 9H, SeCH2COC(CH3)3); 4.19 (s, 2H, SeCH2COC(CH3)3); 7.46 (d, 2H,
H3 þ H5, J3-2,5-6 ¼ 8.5 Hz); 7.86 (d, 2H, H2 þ H6). 13C NMR (100 MHz,
CDCl3)
d: 27.1 (SeCH2COC(CH3)3); 32.0 (SeCH2COC(CH3)3); 45.2
4.1.7.3. Methyl 4-cyanobenzoselenoate (26). From 4-cyanobenzoyl
chloride (2.06 g, 12.7 mmol), grey selenium (1.00 g, 12.7 mmol),
sodium borohydride (1.00 g, 26.4 mmol) and methyl iodide (1.5 mL,
excess). Red solid; mp: 104e108 ꢂC. Yield: 71% (2.020 g). IR (KBr)
cmꢀ1: 3091e2938 (m, CeH), 2229 (s, CN), 1665 (s, C]O). 1H NMR
(COSeCH2COC(CH3)3); 128.9 (C2 þ C6); 129.6 (C3 þ C5); 137.2 (C4);
140.7 (C1); 192.8 (COSeCH2COCH3); 210.4 (COSeCH2COC(CH3)). MS
(m/z% abundance): 195 (4), 139/141 (100/31), 111/113 (40/13).
Elemental Analysis for C13H15ClO2Se, calcd/found (%): C: 49.15/
49.08; H: 4.76/4.82.
(400 MHz, CDCl3)
d
: 2.47 (s, 3H, SeCH3); 7.79 (d, 2H, H3 þ H5, J3-2,5-

164
E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
¼ 8.7 Hz); 8.01 (d, 2H, H2 þ H6). 13C NMR (100 MHz, CDCl3)
d
: 6.2
1H NMR (400 MHz, CDCl3)
d: 2.46 (s, 6H, SeCH3); 7.75 (s, 2H,
6
(SeCH3); 117.1 (CN); 118.2 (C4); 127.8 (C2 þ C6); 133.1 (C3 þ C5);
142.3 (C1); 194.4 (ArCOSeCH3). MS (m/z% abundance): 221/222/
223/225/227 (Mþꢄ, 1/1/1/2/1), 130 (100), Elemental Analysis for
C9H7NOSe, calcd/found (%): C: 48.23/47.90; H: 3.15/3.18; N: 6.25/
6.15.
H3 þ H4). 13C NMR (100 MHz, CDCl3)
d: 6.2 (COSeCH3); 130.9
(C3 þ C4); 148.9 (C2 þ C5); 186.2 (COSeCH3). MS (m/z% abundance):
319/321/323/325 (Mþ þ 1, 2/4/4/1), 255/256/257/259/261 (1/1/3/
ꢄ
5/1), 224/225/226/228/230 (2/2/4/8/2), 196/197/198/200/202 (2/2/
4/8/2), 137 (100). Elemental Analysis for C8H8O2SSe2, calcd/found
(%): C: 29.46/29.55; H: 2.47/2.79.
4.1.7.4. Methyl 3,5-dimethoxybenzoselenoate (27). From 3,5-
dimethoxybenzoyl chloride (2.54 g, 12.7 mmol), grey selenium
(1.00 g, 12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and
methyl iodide (1.5 mL, excess). White solid; mp: 54e55 ꢂC. Yield:
72% (2.374 g). IR (KBr) cmꢀ1: 3105e2836 (m, CeH), 1672 (s, C]O).
4.2. Cytotoxic and antiproliferative activities
Cell culture materials were obtained from BD Bioscience. Stock
solutions of the different compounds were prepared as follows: all
compounds were dissolved in DMSO at a concentration between
0.01 and 0.005 M. The sterile filtration of the compounds was
achieved using 0.2 mm filter disks. Serial dilutions with supple-
mented medium were prepared daily to a final concentration of less
than 2% DMSO in cell culture.
1H NMR (400 MHz, CDCl3)
d: 2.40 (s, 3H, SeCH3); 3.86 (s, 6H, 3,5-di-
OCH3); 6.69 (t, 1H, H4, J4-2,4-6 ¼ 2.3 Hz); 7.07 (d, 2H, H2 þ H6). 13
C
NMR (100 MHz, CDCl3)
d
: 5.7 (SeCH3); 56.0 (OCH3); 105.2 (C2 þ C6);
106.2 (C4); 141.4 (C1); 161.3 (C3 þ C5); 195.3 (ArCOSeCH3). MS (m/z%
abundance): 260 (Mþꢄ, 2), 165 (100). Elemental Analysis for
C
10H12O3Se, calcd/found (%): C: 46.35/46.32; H: 4.67/4.67.
The cytotoxic effect of each substance was determined accord-
ing to the protocol of Denizot and Lang [47].
4.1.7.5. Dimethyl benzene-1,4-dicarboselenoate (28). From tereph-
thaloyl chloride (1.29 g, 6.3 mmol), grey selenium (1.00 g,
12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and methyl
iodide (1.5 mL, excess). Yellow solid; mp: 111e112 ꢂC. Yield 75%
Briefly, 2 ꢃ 103 cells (PC-3, MCF-7, HT-29, A-549 or RWPE-1) in
50 mL of supplemented media, 10% fetal bovine serum (Hyclone)
and 1% penicillinestreptomycin (Lonza) were plate onto each well
of 96-well plates. After 12 h incubation, 50 L of the different
compounds at dilutions between 0.01 M and 100 M were added
and incubated for 72 h at 37 ꢂC and 5% CO2. Cells were then incu-
bated for in the presence of 10 of MTT [3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and
m
(1.511 g). IR (KBr) cmꢀ1: 3085e2928 (w, CeH), 1655 (s, C]O). 1
H
C
m
m
NMR (400 MHz, CDCl3)
NMR (100 MHz, CDCl3)
d
: 2.45 (s, 6H, SeCH3); 7.99 (s, 4H, HAr). 13
d: 6.0 (SeCH3); 127.8 (C2 þ C3 þ C5 þ C6);
4
h
ml
142.8 (C1 þ C4); 194.8 (COSeCH3). MS (m/z% abundance): 318/319/
320/322/324 (Mþꢄ, 1/1/2/2/1), 221/223/224/225/227/229 (2/20/19/
52/100/18), 213 (1), 193/195/196/197/199/201 (1/4/4/10/21/4).
Elemental Analysis for C10H10O2Se2, calcd/found (%): C: 37.52/37.36;
H: 3.15/2.95.
analyzed for their ability to generate a purple formazan dye.
Absorbance was measured at a wavelength of 590 nm and the ratio
of viable cells was calculated. Results are expressed as GI50, the
concentration that reduces by 50% the growth of treated cells with
respect to untreated controls, TGI, the concentration that
completely inhibits cell growth, and LC50, the concentration that
kills 50% of the cells. Data were obtained from at least 3 indepen-
dent experiments performed in quadruplicate.
4.1.7.6. Dimethyl benzene-1,3-dicarboselenoate (29). From iso-
phthaloyl chloride (1.29 g, 6.3 mmol), grey selenium (1.00 g,
12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and methyl
iodide (1.5 mL, excess). White solid; mp: 59e61 ꢂC. Yield: 84%
(1.710 g). IR (KBr) cmꢀ1: 3059e2932 (w, CeH); 1653 (s, C]O). 1H
4.3. Cyclic voltammetry
NMR (400 MHz, CDCl3)
d: 2.45 (s, 6H, SeCH3); 7.59 (t, 1H, H5, J5-4,5-
¼ 7.8 Hz); 8.12 (dd, 2H, H4 þ H6, J4-2,6-2 ¼ 1.8 Hz); 8.42 (t, 1H, H2).
Assays were performed with a CGME (Controlled-Growth Mer-
cury Electrode) BAS potentiostat and data was processed with an
Electrochemical Analyzer BAS 100B. Several preliminary assays
were performed to determine the most suitable conditions for the
cyclic voltammetry assays of the selenoesters presented in this
work. Optimal conditions were found to include: working elec-
trode: mercury-drop electrode, although most of the active com-
pounds give also signals at a glassy-carbon electrode. Scan rate:
200 mV/s. Concentration: 0.5 mM. Solvent: aqueous phosphate
buffer, pH 7.4, with 20% of methanol to ease solubilization of
compounds. Time: 5 min after addition compound to the voltam-
metric cell in order to allow equilibration (and, where applicable, a
sufficient degree of hydrolysis to detect the hydrolyzed (selenolate)
products).
6
13C NMR (100 MHz, CDCl3)
d: 6.0 (SeCH3); 125.9 (C5); 129.8 (C2);
131.9 (C4 þ C6); 140.0 (C1 þ C3); 194.5 (COSeCH3). MS (m/z%
abundance): 318/319/320/322/324 (Mþꢄ, 1/1/2/2/1), 221/223/224/
225/227/229 (2/20/19/52/100/18), 213 (1), 193/195/196/197/199/
201 (1/4/4/10/21/4). Elemental Analysis for C10H10O2Se2, calcd/
found (%): C: 37.52/37.16; H: 3.15/3.15.
4.1.7.7. Dimethyl pyridine-2,6-dicarboselenoate (30). From pyri-
dine-2,6-dicarbonyl dichloride (1.29 g, 6.3 mmol), grey selenium
(1.00 g, 12.7 mmol), sodium borohydride (1.00 g, 26.4 mmol) and
methyl iodide (1.5 mL, excess). Yellow solid; mp: 135e136 ꢂC. Yield
44% (0.894 g). IR (KBr) cmꢀ1: 3102e2926 (d, CeH), 1655 (s, C]O).
1H NMR (400 MHz, CDCl3)
d: 2.38 (s, 6H, SeCH3); 8.03 (ddd, 1H, H4,
J4-3,4-5 ¼ 8.4 Hz); 2.09 (dd, 2H, H3 þ H5, J3-5,5-3 ¼ 1.2 Hz). 13C NMR
(100 MHz, CDCl3)
d
: 5.6 (SeCH3); 122.8 (C3 þ C5); 139.6 (C4); 152.5
4.4. GPx-like activity
(C2 þ C6); 197.9 (COSeCH3). MS (m/z% abundance): 319/321/323/
325 (Mþ þ 1, 2/4/4/1), 255/256/257/259/261 (1/1/3/5/1), 224/225/
The evaluation of the GPx-like activity of the selenoesters is
performed following a revised procedure based in the one
ꢄ
226/228/230 (2/2/4/8/2), 196/197/198/200/202 (2/2/4/8/2), 164
(30), 137 (100). Elemental Analysis for C9H9NO2Se2, calcd/found
(%): C: 33.67/33.29; H: 2.83/2.74; N: 4.36/4.27.
described by Iwaoka and Tomoda [61]. Briefly, the initial rates of
reduction of hydrogen peroxide catalyzed by the compounds in the
presence of thiophenol are determined at 305 nm using a four-
channel Cary 50 Bio UVevis spectrophotometer. The GPx-like ac-
tivity of the compounds is expressed as the ratio of the catalyzed
over the non-catalyzed, spontaneous reaction involving thiophenol
and hydrogen peroxide. Prior to the measurements, the spectro-
4.1.7.8. Dimethyl thiophene-2,5-dicarboselenoate (31). From thio-
phene-2,5-dicarbonyl dichloride (0.97 g, 4.6 mmol), grey selenium
(0.73 g, 9.3 mmol), sodium borohydride (0.73 g, 19.3 mmol) and
methyl iodide (1.5 mL, excess). Yellow solid; mp: 99e101 ꢂC. Yield:
69% (1.042 g). IR (KBr) cmꢀ1: 3076e2928 (w, CeH), 1654 (s, C]O).
photometer is blanked with
a thiophenol solution. Then,

E. Domínguez-Álvarez et al. / European Journal of Medicinal Chemistry 73 (2014) 153e166
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percentage of the radical scavenged. The final concentrations of
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respectively.
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Acknowledgements
The authors wish to express their gratitude to the Ministerio de
Educación y Ciencia, Spain (SAF 2009-07744), the CAN Foundation
and to the University of Saarland for financial support for the
project.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.11.034.
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