Antioxidant-prooxidant properties of a new organoselenium compound library

Article abstract of DOI:10.3390/molecules15107292

The present study describes the biological evaluation of a library of 59 organoselenium compounds as superoxide (O₂^-) generators and cytotoxic agents in human prostate cancer cells (PC-3) and in breast adenocarcinoma (MCF-7). In orde

Full text of DOI:10.3390/molecules15107292

Molecules 2010, 15, 7292-7312; doi:10.3390/molecules15107292
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antioxidant-Prooxidant Properties of a New Organoselenium
Compound Library
Daniel Plano 1, Ylenia Baquedano 1, Elena Ibáñez 1, Iosu Jiménez 1, Juan Antonio Palop 1,
Julian E. Spallholz 2 and Carmen Sanmartín 1,*
1
Department of Organic and Pharmaceutical Chemistry, University of Navarra, Irunlarrea, 1,
E-31008 Pamplona, Spain
2
Texas Tech University, Department of Nutrition, Lubbock, TX 79430, USA
* Author to whom correspondence should be addressed; E-Mail: sanmartin@unav.es.
Received: 21 September 2010; in revised form: 18 October 2010 / Accepted: 20 October 2010 /
Published: 21 October 2010
Abstract: The present study describes the biological evaluation of a library of 59 organo-
─
selenium compounds as superoxide (O2 ) generators and cytotoxic agents in human prostate
cancer cells (PC-3) and in breast adenocarcinoma (MCF-7). In order to corroborate that the
biological activity for selenium compounds depends on the chemical form, a broad structural
variety is presented. These structures include selenocyanates, diselenides, selenoalkyl
functional moieties and eight newly synthesized symmetrically substituted dithioselenites and
selenylureas. Eleven of the derivatives tested showed high levels of superoxide generation in
vitro via oxidation of reduced glutathione (GSH) and nine of them were more catalytic than the
reference compound, diselenodipropionic acid. Eighteen of the library compounds inhibited
cell growth more than or similar to reference chemotherapeutic drugs in PC-3 and eleven were
more potent cytotoxic agents than etoposide in the MCF-7 cell line. Considering both
parameters (superoxide generation and cell cytotoxicity) compounds B1, C6 and C9 displayed
the best therapeutic profiles. Considering that many diselenide compounds can generate
─
superoxide (O2 ) in vitro via oxidation of GSH and other thiols, the analogue B1, that contains
a diselenide moiety, was selected for a preliminary mechanistic investigation, which . revealed
that B1 has apoptogenic effects similar to camptothecin mediated by reactive oxygen species
(ROS) in lymphocytic leukemia cells (CCRF-CEM) and affected the MCF-7 cell-cycle in
G2/M and S-phases.
Keywords: selenium; superoxide; diselenodipropionic acid; cytotoxicity

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1. Introduction
Selenium (Se) is an important nutritional trace element involved in different physiological functions
with antioxidative, antitumoral and chemopreventive properties [1]. There is increasing evidence that
dietary Se intakes are sub-optimal in the populations of many countries and that human cancer
mortalities would significantly decline with supplementation [2], although various authors [3,4] have
considered some potential limitations to this fact. The essential trace element Se is the catalytic
cofactor of important endogenous antioxidative systems of the human body, attracting more and more
attention of laypersons and research groups [5]. Se is present in 25 human selenoproteins, many of
them involved in anti-oxidant defense systems and in cancer prevention [6]. Selenoproteins include
enzymes, such as the glutathione peroxidases (GPx), thioredoxin reductases (TrxR) and iodothyronine
deiodinases (ID) [7]. It is well established [8] that these Se enzymes play a key role in redox regulation
as modulators of reactive oxygen species (ROS). Reactive oxygen species are constantly generated and
eliminated in all biological systems, and they play important roles in a variety of normal biochemical
─
functions and abnormal pathological processes. These species, including superoxide (O2 ), hydrogen
peroxide (H2O2) and hydroxyl radicals (OH·) are known to mediate apoptosis acting as inducers of
cancer chemoprevention and as therapeutic agents derived from catalytic selenium compounds. Excess
intracellular ROS may attack cellular membrane lipids, proteins, and DNA, inhibiting their normal
functions by causing oxidative damage. This is one of the proposed anticancer mechanisms of
selenium compounds [9]. In spite of these facts, the effectiveness of the incorporation of Se into
anticarcinogenic compounds can be conceptualized as a multi-tiered process whereby
chemical/biochemical actions of its metabolites, such as methylselenol (CH3SeH/CH3Se-), hydrogen
selenide (H2Se, HSe−) or other selenide anions (RSe−) arise (Figure 1) [9-12].
Figure 1. A multi-tiered model of Se anticarcinogenesis.

Molecules 2010, 15
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These species can each undergo oxidation/reduction, according to Figures 1 and 2. For example
GSSe− and HSe- may affect the redox cycle, helping to deplete GSH and produce ROS as superoxide,
hydrogen peroxide and the hydroxyl radical. Other selenium compounds have been reported to
generate superoxide and H2O2 via oxidation of GSH following diselenide reduction and methyl
selenide (CH3Se−) formation (Figure 2). Numerous research studies have shown that these redox
effects depend upon the dose and the chemical form of the selenium compound [13–15].
Figure 2. Redox cycling of methylselenide and other selenides (RSe−).
In the last few years, a number of novel synthetic organoselenium compounds have been
synthesized for their use as antioxidants in medicinal chemistry. In addition, many of these derivatives
have exhibited antitumoral activities. Evidence indicates several different mechanisms for this Se
activity. The properties of diselenides have received significant attention, particularly with the
discovery that these compounds possess a mode of antioxidant action similar to that of glutathione
peroxidase. Such antioxidant diselenides include diphenyl diselenide [16,17], 3,3´-diselenodipropionic
acid [18,19], binaphthyl diselenide [20] and 3,3´-ditrifluoromethyldiphenyl diselenide [21]. Other
chemical forms of Se that are also able to protect against oxidative damage are selenomethionine,
methylseleninic acid, dimethyldiselenide [22] and Se-methylselenocysteine. These methylated
selenium derivatives [23] can undergo the metabolic generation of the monomethylated selenium
species, methylselenide, which can undergo oxidation/reduction processes. Selenocyanate derivatives
also represent an important class of agents with antioxidative properties; as 1,4-phenylenebis-
(methylene)selenocyanate (p-XSC) [24] or monoselenocyanate [25]. Redox activity can be correlated
with inhibition of tumor formation. Drake [26], contrary to the prevailing thought, presented evidence
that Se in the +4 oxidation state is more effective as an anticarcinogenic agent than Se in the +2
oxidation state. Examples of biologically active antioxidant selenium compounds are illustrated in
Figure 3.
Much experimental evidence suggests that modification of organoselenium compounds can have a
profound effect on their chemical and therefore on their biological activity. From this viewpoint, and
taking into account that during the last several years, the principal interest in our research group has
been the study, syntheses and biological evaluation of a variety of compounds containing Se as
candidates for new antineoplastic agents [27–31], the objectives of the present study were twofold.
Firstly, an evaluation was planned in order to choose compounds that present a dual activity:
superoxide generation complemented with cytotoxicity. Several studies have found a good correlation
between the redox potential of selenium compounds and their cytotoxicity [9,10,13].

Molecules 2010, 15
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Figure 3. Chemical structures of some representative selenium compounds with
antioxidant properties.
Secondly, the synthesis and biological evaluation of an original series consisting of a small group of
compounds which possess Se in the +4 states oxidation with the aim of establishing that these Se +4
derivatives can catalyze the oxidation of thiol groups generating superoxide [26]. Therefore the
biological evaluation as potential antioxidant and cytotoxic agents of a library of 59 selenium
compounds is presented, eight of them newly synthesized selenylurea and dithioselenite derivatives.
2. Results and Discussion
2.1. Chemistry
The compounds reported in this paper correspond to four different chemical series (A-D) as
illustrated in Figure 4.
Figure 4. General chemical structures for the series of compounds studied A-D.
The preparation of the series A compounds began with the reaction and addition of selenium dioxide
to malononitrile in dimethyl sulfoxide (DMSO) at room temperature, followed by reaction with the
corresponding aminoaryl derivatives or by the reaction of haloarenes with potassium selenocyanate in
acetone under reflux conditions, according to a literature procedure [32]. Conversion of the derivatives in

Molecules 2010, 15
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series A to the corresponding series B compounds was carried out by reduction with sodium borohydride
in ethanol, according to the synthetic method previously described [32].
The imidoselenocarbamate derivatives in series C were synthesized following previously published
methods [27,33], starting from the appropriate Se-alkyl imidoselenocarbamate hydroiodide and the
corresponding acyl chloride.
To explore the effect of substitution of Se +2 with Se in the +4 oxidation new compounds related to
dithioselenites and selenylureas (compounds within series D) were synthesized. The synthetic routes
for the preparation of these series D compounds are presented in Scheme 1.
Scheme 1. Synthetic routes for compounds related to dithioselenites and selenylureas
(series D).
Dithioselenites D1-D5 were prepared from the corresponding functionalized benzylmercaptan and
selenium oxychloride (SeOCl2) in dichloromethane in the presence of potassium bicarbonate [34].
When the mercaptans were not commercially available they were obtained from the corresponding
benzyl halide and sodium hydrosulfide by refluxing in ethanol [35]. For the preparation of selenylureas
D6-D8 the key benzylamines were subjected to reaction with SeOCl2 under a nitrogen atmosphere.
This methodology was tentatively performed in order to obtain derivatives with strong electron
withdrawing substituents. Despite numerous efforts, all attempts at introducing these strong electron
withdrawing moieties using various reaction conditions failed. A possible explanation to this fact could
be that the amine function in the presence of SeOCl2 generates a chloroamine group that can progress
to an aldehyde group through an imine intermediate which interferes with the main desired addition
reaction. This effect is even more marked when the substituent in the aryl ring are strongly electron
withdrawing. For this reason the yield for the derivative D8 in the D series was very poor (8%) and for
other attempted benzylamines (NO2, CN) the yields were negligible and the products could not be
isolated. The physical constants of compounds D1-D8 are listed in Table 1. The IR, NMR and MS
spectral data obtained for the newly synthesized compounds all correspond to the theoretical chemical
structures.

Molecules 2010, 15
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Table 1. Physical constants for compounds D1-D8.
Yield
(%)
M.p.
(ºC)
Recryst.
Solvent
CHN (%)
Molecular
formula
Ref.
D1
Ar
X
Calcd/Found
C, 40.77/40.54
H, 3.39/3.48
N, 0.00/0.00
p-chlorophenyl
S
9
126–128 Methanol C14H14Cl2OS2Se
C, 47.88/48.10
H, 4.49/4.76
N, 0.00/0.00
D2
p-methoxyphenyl
S
S
14
11
18
7
96–98
Methanol
C16H18O3S2Se
C16H12F6OS2Se
C, 40.25/40.51
H, 2.52/2.68
N, 0.00/0.00
D3 p-trifluoromethylphenyl
131–133 Methanol
C, 49.10/49.37
H, 3.07/3.35
N, 0.00/0.00
D4
D5
D6
D7
p-cyanophenyl
p-nitrophenyl
S
170–172 Methanol C16H12N2OS2Se
124–126 Methanol C14H12N2O5S2Se
C, 38.98/39.10
H, 2.78/2.60
N, 0.00/0.00
S
C, 44.68/44.49
H, 3.72/3.40
N, 7.44/7.56
p-chlorophenyl
p-methoxyphenyl
NH
NH
22
39
8
245–247 Ethanol
176–178 Ethanol
159–161 Ethanol
C14H14Cl2N2OSe
C16H20N2O3Se
C16H14F6N2OSe
C, 52.32/52.40
H, 5.45/5.40
N, 7.62/7.37
C, 43.34/43.49
H, 3.16/3.10
N, 6.32/6.30
D8 p-trifluoromethylphenyl NH
2.2. Biological Evaluation
All of the selenium compounds described in this paper (A1-A13, B1-B13, C1-C25 and D1-D8)
were screened for their ability to generate superoxide by the oxidation of GSH by a lucigenin
dependent chemiluminescent (CL) assay [36]. Their ex vivo cytotoxic activity against the human
prostate cancer (PC-3) and breast adenocarcinoma (MCF-7) cell lines using the MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) protocol was followed [37].

Molecules 2010, 15
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2.2.1. Superoxide anion scavenging activity
The superoxide anion is considered to be biologically important since it can dismutate to form
stronger oxidative species, H2O2, singlet oxygen and hydroxyl radicals. In the PMS/NADH-NBT
(phenazine methosulphate/nicotinamide adenine dinucleotide-nitroblue tetrazolium) system,
superoxide anions are derived from the dissolved oxygen by a PMS/NADH coupling reaction, which
can specifically reduce NBT. The decrease of NBT absorbance at 560 nm indicates the consumption of
superoxide anions in the reaction mixture [36]. Experiments were carried out using a concentration of
4 µg/µL for all the compounds and the results are shown in Tables 2 (compounds of series A and B), 3
(compounds of series C) and 4 (compounds of series D). Diselenodipropionic acid, known to affect the
redox cycle generating superoxide, was used as the reference compound.
2.2.2. Cytotoxic activity in PC-3 and MCF-7
Initially, the compounds were evaluated for their ex vivo cytotoxic activity against human prostate
cancer (PC-3, ATCC, Manassas, VA) and breast adenocarcinoma (MCF-7, ATCC, Manassas, VA) cell
lines. These cell lines were selected as there are many clinical trials in the literature showing the
activity of selenium compounds in the reduction of hormone dependent cancer [38,39]. Cytotoxicity
results were tabulated as IC50 values. All experiments were independently performed at least three
times and the values calculated after 72 hours of selenium compound exposure (concentrations of 2, 5,
7 and 10 μM were employed throughout). The results are shown in Tables 2 (compounds of series A
and B), 3 (compounds of series C) and 4 (compounds of series D). Methylseleninic acid (MSA) in PC-
3 and etoposide in PC-3 and MCF-7 were used as controls in the cytotoxic assays [40–42].
All compounds of the selenium library that presented chemiluminescence units (CLU) values more
than tenfold the control have been considered as potent superoxide generators. The results presented in
Table 2 indicate that three compounds (B1, B6, and A10 in this decreasing CLU order) were potent
superoxide generators. Comparison of the results with the values for the standard diselenodipropionic
acid showed that compound B1 was 1.5 times more active. It was observed that six compounds (A1, A2;
A7, A12, B1 and B13) possessed cytotoxic activity against PC-3 cells, of which four (A2, A12, B1 and
B13) were more active than methylseleninic acid and etoposide. Compound B1 was the most potent (IC50
= 1.7 μM) and was five times more active than standard methylseleninic acid (IC50 = 8.4 μM) and eight
times more active than etoposide (IC50 = 13.6 μM), an agent used in the treatment of prostate cancer.
Moreover, two derivatives (A12 and B1) were more potent cytotoxic agents than standard etoposide
(IC50 = 17.5 μM) in MCF-7.
The results obtained for imidoselenocarbamate derivatives (Table 3) showed that three compounds,
which presented a furyl (C6 and C16) and thienyl (C9) heteroaromatic rings, were potent superoxide
generators with CLU values higher than the control reference standard. Furthermore, 13 compounds
(C1-C10 and C12-C14) possessed cytotoxic activity and 11 (C2, C4-C10, C12-C14) of them
presented IC50 values lower than MSA or etoposide in PC-3. In addition, considering MCF-7 cells
eight of them (C1-C5, C8-C10) were more active than etoposide, highlighting C3 and C10 which
showed IC50 values below 1 μM. It was also observed that selenomethyl derivatives (C1-C15) were
more active as cytotoxic agents than selenobenzyl analogues (C16-C25). These results reinforced the
earlier hypothesis concerning the determinant role of the selenomethyl group as a scaffold for the

Molecules 2010, 15
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biological activity of these types of compounds. The likely explanation for this finding is that
compounds with a selenomethyl moiety act as methylselenol precursors which generate superoxide by
redox cycling. It has been suggested that methylselenol is the critical Se metabolite for anticancer
activity of the dietary selenoamino acids. [43–45] so as the selenoketo acids [12] generated from
metylselenocysteine and selenomethionine through amino acid oxidases and aminotransferases.
Table 2. Chemiluminescence (CLU) and cytotoxic activities (IC50) of the compounds A1-
A13 and B1-B13.
PC-3 cell line
MCF-7 cell line
Compound
Ar
n
CLUa
IC50 (μM)
10.0
2.0
IC50 (μM)
A1
4-aminophenyl
4-(N,N-dimethylamino)phenyl
4-amino-3-carboxyphenyl
4-acetamido-3-carboxyphenyl
4-bromophenyl
0
0
0
0
1
1
1
1
1
1
1
1
2
0
0
0
0
1
1
1
1
1
1
1
1
2
0.242
1.095
0.075
0.301
0.372
0.814
0.091
0.798
0.810
1.490
0.580
0.141
0.232
3.928
0.281
0.129
0.085
0.315
1.543
0.141
0.735
1.089
0.570
0.119
0.101
0.176
0.103
2.526
-
>10
>10
>10
>10
>10
>10
>10
>10
NE
A2
A3
NEb
> 10
> 10
NE
A4
A5
A6
phenyl
A7
4-nitrophenyl
8.9
A8
4-trifluoromethylphenyl
4-methylthiophenyl
4-methylphenyl
> 10
NE
A9
A10
NE
NE
A11
4-cyanophenyl
> 10
6.0
NE
A12
naphthyl
9.4
A13
4-nitrophenyl
NE
NE
B1
4-aminophenyl
1.7
4.3
B2
4-(N,N-dimethylamino) phenyl
4-amino-3-carboxyphenyl
4-acetamido-3-carboxyphenyl
4-bromophenyl
> 10
NE
>10
>10
NE
B3
B4
NE
B5
NE
NE
B6
phenyl
NE
NE
B7
4-nitrophenyl
NE
NE
B8
4-trifluoromethylphenyl
4-methylthiophenyl
4-methylphenyl
> 10
> 10
NE
>10
>10
NE
B9
B10
B11
4-cyanophenyl
NE
NE
B12
B13
naphthyl
NE
NE
4-nitrophenyl
5.0
>10
-
Control
-
Diselenodipropionic acid
MSAc
-
-
8.4 [40]
13.6 ± 2.2 [41]
-
Etoposide
-
17.5 ± 1.2 [42]
a CLU are average CLU/30 sec integrals (N = 20); b No effect; c Methylseleninic acid.

Molecules 2010, 15
Table 3. Chemiluminescence (CLU) and cytotoxic activities (IC50) of the compounds C1-C25.
7300
PC-3 cell line MCF-7 cell line
Aromatic or
heteroaromatic
3-pyridyl
R
CLUa
Comp.
IC50 (μM)
IC50 (μM)
C1
methyl 0.110
methyl 0.157
methyl 0.085
methyl 0.040
methyl 0.221
>10
1.8
6.2
C2
4-methylphenyl
4-cyanophenyl
3,5-dimethoxyphenyl
2-chloro-3-pyridyl
2-furyl
3.0
C3
>10
6.5
0.1
C4
2.8
C5
9.1
1.3
C6
methyl
2353
7.4
>10
C7
2-thienyl
methyl 0.165
methyl 0.517
6.1
>10
C8
3-chloro-2-thienyl
5-nitro-3-thienyl
2-isoxazolyl
6.3
8.3
C9
methyl
methyl 0.408
methyl
NDb
2760
1.2
6.0
C10
7.6
0.6
C11
2-benzothienyl
> 10
8.1
>10
C12
3,4-methylenedioxybenzyl methyl 0.043
>10
C13
3-quinolinyl
2-phenyl-4-quinolinyl
9-acridinyl
methyl 0.100
methyl 0.054
methyl 0.185
7.6
>10
C14
7.7
NE
C15
> 10
> 10
NEc
NE
NE
C16
2-furyl
benzyl
508
>10
C17
2-thienyl
benzyl 0.073
benzyl 0.052
benzyl 0.063
benzyl 0.216
benzyl 0.076
benzyl 0.046
benzyl 0.051
benzyl 0.061
benzyl 0.051
0.060
NE
C18
3-chloro-2-thienyl
5-nitro-3-thienyl
2-isoxazolyl
>10
C19
> 10
NE
>10
C20
>10
C21
2-benzothienyl
3,4-methylenedioxybenzyl
3-quinolinyl
> 10
NE
>10
C22
NE
C23
> 10
> 10
> 10
-
NE
C24
C25
2-phenyl-4-quinolinyl
9-acridinyl
>10
NE
Control
-
Diselenodipropionic acid
MSAd
2.526
-
-
-
8.4 [40]
13.6 ± 2.2 [41]
-
Etoposide
-
17.5 ± 1.2 [42]
a CLU are average CLU/30 sec integrals (N = 20); b Not determined; c No effect; d Methylseleninic acid.
The compounds of series D were the most potent superoxide anion generators with five active
compounds (D2-D5 and D8), especially compound D5, with an average value of 4,743 CLU/30
seconds. Compounds therefore with Se in the +4 oxidation state, like selenite, seemed to be more
active as superoxide generators than Se compounds in the +2 oxidation state. These results are

Molecules 2010, 15
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according to those reported in the literature [26]. However, only one of these derivatives (D6)
presented a significant cytotoxic effect in PC-3 and MCF-7 cell lines.
Table 4. Chemiluminescence (CLU) and cytotoxic activities (IC50) of the compounds D1-D8.
PC-3 cell line
MCF-7 cell line
Comp.
Ar
X
CLUa
IC50 (μM)
IC50 (μM)
D1
D2
D3
D4
p-chlorophenyl
p-methoxyphenyl
p-trifluoromethylphenyl
p-cyanophenyl
S
S
S
S
S
0.160
890
1451
701
NEb
NE
NE
>10
NE
NE
NE
NE
NE
D5
p-nitrophenyl
4743
NE
D6
D7
p-chlorophenyl
p-methoxyphenyl
NH 0.072
NH 0.562
3.9
NE
8.4
>10
D8
Control
p-trifluoromethylphenyl NH
1973
0.137
2.526
-
NE
-
-
NE
-
-
-
Diselenodipropionic acid
MSAc
8.4 [40]
13.6 ± 2.2 [41]
Etoposide
-
17.5 ± 1.2 [42]
a CLU are average CLU/30 sec integrals (N = 20); b No effect; c Methylseleninic acid.
Taking together all the superoxide generating and cytotoxicity results, the evaluated selenium
compounds presented four types of behaviour: a) compounds both inactive as superoxide generators
and non-toxic xenobiotics to cancer cells, (A3-A6, A8, A9, A11, A13, B2-B5, B7-B12, C15, C17-
C25, D1 and D7) b) superoxide generating compounds but non-toxic xenobiotics to cancer cells,
(A10, B6, C16, D2-D5 and D8) c) compounds inactive as superoxide generators but cytotoxic
xenobiotics to cancer cells (A1, A2, A7, A12, B13, C1-C5, C7, C8, C10, C12-C14 and D6) and d)
both superoxide generators and cytotoxic xenobiotics to cancer cells (B1, C6, C9). Taking into
account that one of the objectives of the present study was the selection of organoselenium compounds
with dual biological activity, the ability to induce superoxide generation and show cytotoxic activity,
B1, C6 and C9 were selected for further study as the most interesting selenium derivatives. To gain
further insight into the mechanisms of action of these active compounds, compound B1 was also
selected for an extensive study because it is a diselenide derivative and this moiety has been postulated
as one of the important species in redox cycling (Figure 2). In addition, this compound, bis(4-amino-
phenyl)diselenide exhibited cytotoxic activity in other cell lines (CCRF-CEM, IC50 = 9.0 μM; HT-29,
IC50 = 9.8 μM). Various in vivo studies [18,20,21] have reported the diselenide moiety as a protective
and antigenotoxic agent. Therefore, based on the chemical analogy between compound B1 and the
diselenides above mentioned, B1 emerge as a potential candidate for in vivo studies.

Molecules 2010, 15
Figure 5. Apoptotic effects of compound B1 and camptothecin at 10 or 25 μM on CCRF-
7302
CEM and MCF-7 cells for 24 and 48 h, respectively. (A) Apoptotic effects of compound
B1 on CCRF-CEM were assessed by Flow Cytometry analysis after staining with Annexin
V-FITC. Annexin V staining is represented on the x-axis and PI staining is represented on
the y-axis. The representative results of three independent experiments are shown; (B)
Apoptotic effects of compound B1 were assessed by Flow Cytometry analysis using the
Apo-Direct kit, based on the TUNEL assay; (C) Percentage of apoptotic and or necrotic
cells after compound B1 or camptothecin treatment were calculated from the flow
cytometry results. Each bar represents the mean of three independent experiments and the
error bars indicate standard deviation. ** p < 0.01 with respect to the control.

Molecules 2010, 15
7303
2.2.3. Apoptosis
Recent evidence indicates that ROS may function as intracellular messengers to modulate apoptosis
and apoptotic responses have been demonstrated in many types of cancer cells treated with selenium
compounds [9,11,46,47]. For this reason it was decided to assess whether this superoxide generating
activity was related to their ability to induce apoptosis. B1, one of the most active compounds as a
superoxide generator and cytotoxic agent, was further analyzed for apoptosis in MCF-7 cells. The
apoptotic status of the cells after 48 hours of treatment with 25 μM of the compound B1 was
determined using the Apo-Direct kit (BD Pharmingen) based on the TUNEL assay as described by the
manufacturer. Camptothecin was used as a positive control. The results obtained are shown in Figure 5.
As can be seen, B1 exerted apoptogenesis similar to camptothecin. The apoptogenic action of B1 was
confirmed to occur in lymphocytic leukemia cells (CCRF-CEM) (Figure 5). The apoptotic status of
CCRF-CEM cells was confirmed by measuring the exposure of phosphatidylserine on the cell
membranes using the Annexin V-FITC Kit (BD Pharmingen) [48]. After 24 hours of treatment with 10
μM of B1 both apoptopic and necrotic effects were observed in the CCRF-CEM cells.
2.2.4. Effects on cell cycle progression
Cell cycle arrest is one consequence of treatment with many anticancer drugs, including
camptothecin. In an effort to ascertain whether compound B1 could affect the cell cycle progression in
MCF-7 cells, these cells were treated with 25 M of B1 for 48 h and cell cycle progression was
determined by flow cytometry analysis.
Figure 6. Cell cycle profile of MCF-7 cells after 48 h treatment with compound B1 and
camptothecin at 25 μM. Each bar represents the mean of three independent experiments
and error bars indicate the standard deviation. ** p < 0.01 with respect to the control.
As shown in Figure 6, DNA flow cytometric analysis indicated that treatment of the cells with
compound B1 triggered a marked change in the MCF-7 cell cycle distribution. This compound induced

Molecules 2010, 15
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cell accumulation in S and G2/M phases, accompanied by a diminution of cell population in the G0/G1
phase and a significant increase in SubG1 (which is representative of cells with fragmented DNA). It is
noteworthy that cell cycle distribution analysis for compound B1 is in total agreement with the
apoptosis results obtained in MCF-7 cells. In summary, B1 affects cell-cycle checkpoints in G2/M and
S-phases causing a reduction of cell-cycle progression along with a cellular transition into apoptosis.
Figure 7. Intracellular content of ROS in CCRF-CEM cells after 4 and 24 h treatment with
compound B1 at 25 μM. (A) Flow Cytometry analysis is shown for 4 h treatment with
compound B1 or vehicle (control) for the representative results of three independent
experiments; (B) Flow Cytometry analysis is shown for 24 h treatment with compound B1
or vehicle (control) for the representative results of three independent experiments; (C)
Each bar represents the mean of three independent experiments and the error bars indicate
the standard deviation. ** p < 0.01 with respect to the control.

Molecules 2010, 15
7305
2.2.5. Effects on reactive oxygen species generation (ROS)
It is well established [8] that Se plays a key role in redox regulation as a modulator of reactive
oxygen species (ROS) and numerous investigations have documented that oxidative stress-mediated
cellular changes are frequently induced following exposure to cytotoxic drugs, UV or γ-radiation [49,50].
Therefore, we examined whether ROS generation might be involved in the induction of apoptosis by
compound B1 in the CCRF-CEM cell line. As shown in Figure 7, compound B1 induced a significant
increase in the intracellular content of ROS in CCRF-CEM cells after 4 and 24 h treatment at 25 μM.
Moreover, the intracellular content of ROS seemed to be time-dependent, i.e. ROS increased with
time. These results suggest the possible role of oxidative stress in cell death induced by compound B1
in the CCRF-CEM cell line. Puntel et al. [51] have reported a mitochondrial dysfunction mediated by
thiol oxidation induced by diphenyl diselenide. This could be a possible explanation for the oxidative
stress induced by compound B1, but further studies need to be performed.
3. Experimental
3.1. General
Melting points for all the compounds of the selenium library were determined by differential
scanning calorimetry using a Perkin-Elmer DSC Diamond and with a Mettler FP82 + FP80
1
thermomicroscopy (Greifense, Switzerland). The H-NMR spectra were recorded on a Bruker 400
UltrashieldTM (Rheinstetten, Germany), using TMS as the internal standard (Table 6). The IR spectra
were recorded on a Thermo Nicolet FT-IR Nexus in KBr pellets (Table 6). Elemental microanalyses
were carried out on vacuum-dried samples (Table 1) using an Elemental Analyzer (LECO, CHN-900
Elemental Analyzer). Silicagel 60 (0.040–0.063 mm) 1.09385.2500 (Merck KGaA, 64271 Darmstadt,
Germany) was used for Column Chromatography and Alugram SIL G/UV254 (Layer: 0.2 mm)
(Macherey-Nagel 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),
Sigma-Aldrich Química, S.A., (Alcobendas, Madrid, Spain), Acros Organics (Janssen Biological
activity assays).
3.2. General Procedure for Preparation of 4-Mercaptomethylbenzonitrile and 4-Nitrophenylmethanethiol
To a solution of the corresponding 4-bromomethylbenzonitrile (20.13 mmol) or 4-nitrobenzyl
bromide (20.13 mmol) and ethanol (50 mL) was added sodium hydrosulfide (44.66 mmol). The
mixture was stirred at its refluxing temperature for 16 h and evaporated. The residue was triturated
with water (100 mL) and extracted with dichloromethane (3 × 50 mL). Combined organic phases were
washed with water (100 mL) and dried with anhydrous Na2SO4. Solvent was removed in vacuo and the
resulting crude product was purified by washing with ethyl ether (4 × 25 mL).
1
4-mercaptomethylbenzonitrile: Yield: 79%. IR (KBr): 2226 (s, CN); H-NMR (400 MHz, DMSO-d6,
): 2.15 (s, 1H, SH); 3.85 (d, 2H, CH2); 7.43 (d, 2H, H2, H6, J2-3 = 8 Hz); 7.56 (d, 2H, H3, H5, J3-2 = 8
Hz). Anal. Calcd. for C8H7NS2 (%): C, 64.42; H, 4.70; N, 9.40. Found: C, 64.33; H, 4.75; N, 9.38.

Molecules 2010, 15
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4-nitrophenylmethanethiol: Yield: 64%. IR (KBr): 1.348 (w, C-NO2); 1H-NMR (400 MHz, DMSO-d6,
): 2.14 (s, 1H, SH); 3.89 (d, 2H, CH2); 7.70 (d, 2H, H3, H5, J3-2 = 8.6 Hz); 8.17 (d, 2H, H2, H6, J2-3
=
8.6 Hz). Anal. Calcd. for C7H7NO2S2 (%): C, 49.70; H, 4.14; N, 8.28. Found: C, 49.39; H, 4.38; N,
7.95.
3.3. General Procedure for Preparation of Dithioselenites D1-D5
To a solution of the corresponding mercaptan (13.50 mmol) in dry dichloromethane (12 mL) was
added KHCO3 (0.6 g) and molecular sieves. The suspension was stirred for 1 h under nitrogen at 0°C
and then was added SeOCl2 (0.5 mL). The mixture was maintained by stirring for 5 h at room
temperature and water (50 mL) was added and extracted with dichloromethane (3 × 50 mL). Combined
organic phases were washed with water (100 mL) and dried with anhydrous Na2SO4. Solvent was
removed in vacuo and the resulting crude product was purified by recrystalization from methanol.
According to this procedure the following compounds were synthesized: N,N´-bis-(p-chlorophenyl-
methyl)-dithioselenite (D1), N,N´-bis-(p-methoxyphenylmethyl)-dithioselenite (D2), N,N´-bis-(p-
trifluoromethylphenylmethyl)-dithioselenite (D3), N,N’-bis-(p-cyanophenylmethyl)-dithioselenite (D4)
and N,N’-bis-(p-nitrophenylmethyl)-dithioselenite (D5).
3.4. General Procedure for Preparation of Selenylureas (D6-D8)
To a solution of the corresponding benzylamine (15 mmol) in dry dichloromethane (12 mL) under
nitrogen atmosphere was added SeOCl2 (0.2 mL). The mixture was stirred for 3 h at room temperature
until a black solid was formed. The residue was filtered. The organic layer was removed in vacuo and
the crude material was washed with ethyl ether (3 × 15 mL) and re-crystallized from ethanol.
According this procedure the following compounds have been synthesized: N,N´-bis-(p-
chlorophenylmethyl)-selenylurea (D6), N,N´-bis-(p-methoxyphenylmethyl)-selenylurea (D7) and
N,N´-bis-(p-trifluoromethylphenylmethyl)-selenylurea (D8).
Table 5. Spectroscopic data (IR, 1H-NMR and MS) for the new compounds.
IR (KBr;
MS 70 eV; s;
Ref.
D1
1H NMR (400 MHz, DMSO-d6, δ/ppm, J in Hz)
υ/cm−1)
(% abundance)
4.75 (d, 4H, 2 -S-CH2-), 7.32 (d, 4H, H3 + H5 + H3’ + H5’),
7.46 (d, 4H, H2 + H6 + H2’ + H6’).
3030
3003
3008
3061
3112
125 (100)
90 (100)
3.73 (s, 6H, 2OCH3), 4.84 (s, 4H, 2 -S-CH2-), 6.87 (d, 4H, H3
+ H5 + H3’ + H5’), 7.17 (d, 4H, H2 + H6 + H2’ + H6’).
D2
D3
D4
D5
5.04 (d, 4H, 2 -S-CH2-), 7.62 (d, 4H, H3 + H5 + H3’ + H5’ ),
159 (100)
116 (100)
136 (100)
7.73 (d, 4H, H2 + H6 + H2’ + H6’).
5.03 (d, 4H, 2 -S-CH2-), 7.59 (d, 4H, H3 + H5 + H3’ + H5’ ),
7.85 (d, 4H, H2 + H6 + H2 + H6’).
4.98 (d, 4H, 2 -S-CH2-), 7.75 (d, 4H, H3 + H5 + H3’ + H5’ ),
8.16 (d, 4H, H2 + H6 + H2’ + H6’).

Molecules 2010, 15
7307
Table 5. Cont.
4.21 (d, 4H, 2 –NH-CH2-), 6.54 (t, 2H, 2-NH-CH2-), 7.28 (d,
4H, H3 + H5 + H3’ + H5’ ), 7.36 (d, 4H, H2 + H6 + H2’ + H6’).
3.73 (s, 6H, 2OCH3), 4.14 (d, 4H, 2 –NH-CH2-), 6.28 (t, 2H,
2-NH-CH2-), 6.87 (d, 4H, H3 + H5 + H3’ + H5’ ), 7.17 (d, 4H,
H2 + H6 + H2’ + H6’).
D6
D7
D8
3324
140 (100)
136 (100)
174 (100)
3351-
3321
4.32 (d, 4H, 2 –NH-CH2-), 6.69 (t, 2H, 2-NH-CH2-), 7.46 (d,
4H, H3 + H5 + H3’ + H5’ ), 7.68 (d, 4H, H2 + H6 + H2’ + H6’).
3335
3.5. Biological Activity Assays
3.5.1. Superoxide anion generating activity
The control chemiluminescent (CL) assay cocktail without substrates or GSH was made using a 0.05 M
sodium phosphate buffer (pH 7.4) containing 20 μL lucigenin/mL from a stock solution of 1.0 mg/mL
lucigenin in distilled water. The assay cocktail with thiol contained 1.0 mg GSH/mL. To aliquots of thiol
(500 μL) containing assay cocktail were added each selenium compound in DMSO (20 μL). The
concentration of each compound was no more than 4 μg/uL or otherwise saturated <4 μg/μL as not all
compounds fully dissolved in the DMSO. The samples were added in one 20 μL portion from a 50 μL
Eppendorf pipette directly to the chemiluminescent tube into a Los Alamos Diagnostics Model 535
luminometer containing an attached LKB 2209 multitemp recirculating water bath. Chemiluminescent
data were recorded at 25 degrees C in 30-second integrated units over a period of up to 20 min. There
was a3-second instrumental delay between integrations. Additional details of this assay including the
quenching of chemiluminescence generated by superoxide dismutase have been previously reported [21].
This CL assay is quantitative (correlation coefficient, r = 0.99; P < 0.001) in generating CL for small
amounts of redox cycling selenium compounds [21].
3.5.2. Cytotoxic activity in PC-3 and MCF-7
PC-3 and MCF-7 cells were seeded in 96-well plates (Millipore, Eschborn, Germany) at a density
of 1.5 × 103 and 104 cells per well, respectively. The cells were incubated at 37ºC under 5% CO2
overnight prior to the addition of the compounds. All compounds were dissolved in DMSO at a
concentration of 0.01M. Sterile filtration of the compounds was achieved using 0.2 μm filter disks.
Serial dilutions with supplemented medium were prepared daily to a final concentration of 2, 5, 7 and
10 µM in cell culture. After 3 days of incubation, MTT solution (5 mg/mL in PBS, 10 μL) was added
to the cells in each well and then incubated for an additional 4 h at 37ºC. The absorbance of formazan
at λ = 570 nm was measured on a Polarstar Galaxy plate reader (BMG LabTechnologies GmbH). The
percentage of viable cells was calculated to obtain IC50-values. PC-3 and MCF-7 cells were cultured
under standard conditions (Dulbecco’s RPMI 1640 medium, with GlutamaxTM 1, Invitrogen
supplemented with 10% fetal bovine serum, Fetalclone III, SH30109.03, HYCLONE and 1%
Penicillin-Streptomycin, Invitrogen).
3.5.3. Apoptosis and cell cycle
MCF-7 cells are human breast adenocarcinoma whose apoptotic status and cell cycle analysis of the
cells were determined using the Apo-Direct kit (BD Pharmingen), based on the TUNEL assay under

Molecules 2010, 15
7308
the conditions as described by the manufacturer. The apoptotic status of leukemia CCRF-CEM cells
was evaluated by measuring the exposure of phosphatidylserine on the cell membranes using the
Annexin V-FITC kit (BD Pharmingen). Briefly, 5 × 105 cells were pelleted and washed in PBS. Cells
were then stained with Annexin V-FITC and propidium iodide for 15 min at 4ºC in the dark and
analyzed using a Coulter Epics XL flow cytometer (Becton Dickinson).
3.5.4. Measurement of reactive oxygen species
The intracellular accumulation of ROS was determined using the fluorescent probes 2′,7′-
dichlorodihydrofluorescein diacetate (H2DCFDA). Briefly, CCRF-CEM cells were incubated with
compound B1 for 4 and 24 h. The cells were stained with 10 μM H2DCFDA for 60 min at 37°C and
the fluorescence was measured at the desired time intervals by flow cytometry. The ROS generation
was assessed by the dichlorofluorescein fluorescence intensity (FL-1, 530 nm) from 10,000 cells with
a Coulter Epics XL flow cytometer (Becton Dickinson).
4. Conclusions
Eight new organoselenium derivatives D1-D8 have been prepared and fifty-nine compounds related
to a series of selenium derivatives have been evaluated for their biological activities as superoxide
generators and cytotoxic agents. Three distinct cell lines (PC-3, MCF-7 and CCRF-CEM) were used in
the biological evaluation of the derivatives. Thirteen of the selenium compounds (A2, A9, A10, B1, B6,
C6, C9, C16, D2, D3, D4, D5 and D8) were of interest as superoxide generators determined by a
chemiluminescent assay. It is noteworthy that some of these were catalytically more active (B1, C6,
C9, C16, D2, D3, D4, D5 and D8) than the reference compound diselenodipropionic acid. Eighteen
compounds (A1, A2, A7, A12, B1, B13, C2, C4, C5, C6, C7, C8, C9, C10, C12, C13, C14 and D6)
and eleven compounds (A12, B1, C1, C2, C3, C4, C5, C8, C9, C10 and D6) inhibited cell growth in
PC-3 and MCF-7 cell lines, respectively, presenting values for IC50 lower than or similar to the
standard anticancer agents used (etoposide and/or methylseleninic acid). Considering the chemical and
biological data as a whole, with the aim in mind of finding new selenium compounds that fulfill within
the same molecule, both catalytic superoxide generating and cytotoxic activities, compounds B1, C6
and C9 were the most appropriate for further anticancer testing. Compound B1, a diselenide which
fulfilled both criteria, was evaluated by flow cytometric analysis for its effects on cell cycle
distribution and apoptosis induction in MCF-7 cells and its effect in ROS generation in CCRF-CEM
cells. The results indicated that B1 effectively induced cell cycle accumulation at S and G2/M phases
and a diminution in G0/G1 phase, accompanied by an increase in SubG1 phase cells. Furthermore, B1
possessed an apoptogenic effect in MCF-7 and CCRF-CEM cell lines. Finally, Se generated oxidative
stress is implicated in cell death induced by B1 in the CCRF-CEM line. In summary, considering all
the biological activities compound B1 should be considered as novel candidates for further studies to
understand the exact mechanism of action and for in vivo studies.

Molecules 2010, 15
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Acknowledgements
The authors wish to express their gratitude to the financial support from the Ministerio de
Educación y Ciencia, Spain (SAF 2009-07744). We also thank the Department of Education of the
Navarra Government for the award of three grants (E.I., I.J. and Y.B.).
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Sample Availability: Samples of the compounds A1-A13, B1-B13, C1-C25 and D1-D8 are available
from the authors.
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