New glutathione peroxidase mimetics—Insights into antioxidant and cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2016.10.018

A series of N-alkyl benzisoselenazol-3(2H)-ones has been obtained and transformed to corresponding diselenides by the reduction with sodium borohydride. Additionally, efficient methodology for the oxidative Se–N bond formation by potassium iodate has been presented, new conversion of diselenide to benzisoselenazolone was observed. The GPx-like activity of all synthetized derivatives has been evaluated by NMR. N-Allyl diselenide was up to five times better antioxidant than ebselen. Anticancer capacity towards MCF7 and DU145 cancer cells has been also tested. The highest antiproliferative activity was obtained for N-cyclohexyl benzisoselenazolone.

Full text of DOI:10.1016/j.bmc.2016.10.018

Accepted Manuscript
New glutathione peroxidase mimetics – insights into antioxidant and cytotoxic
activity
Agata J. Pacuła, Katarzyna B. Kaczor, Angelika Wojtowicz, Jędrzej
Antosiewicz, Anna Janecka, Angelika Długosz, Tomasz Janecki, Jacek
Ścianowski
PII:
S0968-0896(16)30995-6
DOI:
Reference:
http://dx.doi.org/10.1016/j.bmc.2016.10.018
BMC 13345
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 July 2016
11 October 2016
15 October 2016
Please cite this article as: Pacuła, A.J., Kaczor, K.B., Wojtowicz, A., Antosiewicz, J., Janecka, A., Długosz, A.,
Janecki, T., Ścianowski, J., New glutathione peroxidase mimetics – insights into antioxidant and cytotoxic activity,
Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.10.018
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New glutathione peroxidase mimetics – insights into antioxidant
and cytotoxic activity
Agata J. Pacułaa, Katarzyna B. Kaczorb, Angelika Wojtowiczb, Jędrzej Antosiewiczb, Anna
Janeckac, Angelika Długoszc, Tomasz Janeckid and Jacek Ścianowskia*
aDepartment of Organic Chemistry, Faculty of Chemistry, Nicolaus Copernicus University, 7 Gagarin Street,
87-100 Torun, Poland; bDepartment of Bioenergetics and Physiology of Exercise, Medical University of Gdansk,
c
1 Debinki Street, 80-211 Gdansk, Poland; Department of Biomolecular Chemistry, Faculty of Medicine,
d
Medical University of Lodz, Mazowiecka 6/8, 92-215 Lodz, Poland; Institute of Organic Chemistry, Lodz
University of Technology, Zeromskiego 116, 90-924 Lodz, Poland; *e-mail: jsch@chem.umk.pl
Abstract: A series of N-alkyl benzisoselenazol-3(2H)-ones has been obtained and
transformed to corresponding diselenides by the reduction with sodium borohydride.
Additionally, efficient methodology for the oxidative Se-N bond formation by potassium
iodate has been presented, new conversion of diselenide to benzisoselenazolone was
observed. The GPx-like activity of all synthetized derivatives has been evaluated by NMR. N-
allyl diselenide was up to five times better antioxidant than ebselen. Anticancer capacity
towards MCF7 and DU145 cancer cells has been also tested. The highest antiproliferative
activity was obtained for N-cyclohexyl benzisoselenazolone.
Key words: benzisoselenazolones/ diselenides / antioxidant activity / cytotoxicity
1. Introduction
Reactive oxygen species (ROS) are important factors both in health and disease. At
physiological concentrations ROS act as modulators of the cells signaling pathways and
immune system responses.1 When the formation of ROS increases, exceeding the
compensatory antioxidant processes, oxidative stress is generated. This state can be explained
as the imbalance between the formation and elimination of ROS leading to the disruption of
the redox homeostasis of cells. This can induce various pathological processes leading to
cancer, diabetes, neurodegenerative diseases and cardiovascular system dysfunctions.2
Conversely, ROS can stimulate proliferation and activate pro survival signaling pathways in
cancer cells. 3,4 The redox homeostasis in the cell can be maintained by a natural antioxidant
defense system in which several enzymes take part including the selenoenzyme glutathione
peroxidase (GPx). The presence of selenocysteine in the active site of GPx is crucial for its

peroxide scavenging capacity. In recent years organoselenium compounds had emerged as
promising antioxidants able to act as an artificial selenocysteine and mimic the activity of
GPx.5 Ebselen (N-phenylbenzisoselenazol-3(2H)-one) 1 is currently one of the most
thoroughly studied organoselenium molecules that exhibit high therapeutic potential.6 It was
9
proven to be active in the treatment of Alzheimer’s disease,7 retinopathies,8 diabetes and
inflammation 10 and is also at clinical trial for bipolar disorder 11 and hearing loss.12
Recent mechanistic investigations performed by Mugesh and co-workers resulted in
new conclusions concerning the catalytic cycle for the antioxidant activity of ebselen.
Compound 1 reacts with a thiol to produce an unstable selenenyl sulfide 2 which, through a
disproportionation reaction, forms the diselenide 3. Compound 3 reduces hydrogen peroxide,
producing seleninic 4 and selenenic acid 5. In the presence of an excess of thiol, compound 4
and 5 react with RSH to form selenenyl sulfide 2. Ebselen is regenerated by the elimination of
a water molecule from selenenic acid 5 (Scheme 1).13
Scheme 1. Catalytic cycle for the elimination of hydrogen peroxide by ebselen
According to Mugesh the rate limiting step of the cycle is the disproportionation
reaction to diselenide 3, which depends not on the structure of the reduced peroxide but on the

type of the thiol used as cofactor. In the assay mixture with the excess of thiol and peroxide
the predominant form is the seleninic acid 4 and the selenenyl sulfide 2. The presence of
seleninic acid was established using 77Se NMR spectroscopy and the structure was
additionally confirmed by X-ray analyses14,15 However, current investigations, applying DFT
techniques, indicate that after the formation of selenenyl sulfide 2, the preferred path is the
reaction with a second molecule of RSH to form a selenol which is able to reduce hydrogen
peroxide with the lowest activation barrier giving the selenenic acid 5.16
As suggested in the known ebselen catalytic cycle the benzisoselenazolone reduces
hydrogen peroxide after being converted to a diselenide. To investigate if this conversion
takes place, and formation of the diselenide is the rate determining step of the cycle, we
decided to test in an NMR antioxidant activity assay both the benzisoselenazoles and
diselenides to evaluate if applying directly the diselenide would increase the rate of the
reaction proving this activity-structure relation.
In vivo both ebselen and diselenides can be easily metabolized. After intravenous
administration the extracellular pool of ebselen is bounded to the cysteine residue of albumin
and transported to cells. Inside the cell the favored target organelle are the endoplasmic
reticulum and mitochondria.17 The metabolic pathway includes isoselenazolone ring opening
with Se-N bond cleavage, further methylation to 2-methyl selenobenzanilide or
glucuronidation to 2-glucuronyl selenobenzanilide released with bile.6 For the diaryl
diselenides a convenient way of excretion includes a reaction with GSH to form a selenol-
GSH adduct, further transformation to mercapturic acid derivatives which can be released
with urine.18
Selenium atom incorporated in the structure of ebselen is not bioavailable, thus not
toxic. The ability to form Se-S bonds with the thiol group of cysteine residues in proteins is
associated with significant pharmacological functions, including the cytotoxic activity.19
Several research groups had modified the structure of ebselen to increase its antitumor
activity. Various substitutions on the nitrogen atom, with a saccharide moiety 5,20 a triazole
scaffold 6,21 conversion to a diselenide 722 or combining with the structure of resveratrol 823
enabled to obtain promising results revealing the potential of this field of research in the
search for new organoselenium chemotherapeutics (Scheme 2).

Scheme 2. Examples of ebselen derivatives with antiproliferative potential
During our previous investigations we have observed a significant decrease in
solubility when an aryl moiety was introduced on the nitrogen atom. In contrary, N-alkyl
derivatives were easily dissolved in the media used for the performed antioxidant activity
assays. As good solubility is an important feature when choosing the right candidate for
screening and preliminary results were promising we have decided to evaluate further the
biological potential of the N-alkyl benzisoselenazolones. Moderate GPx-like activity of
ebselen is also associated with its low solubility. Derivatives presented in this paper bear an
N-alkyl moiety which improves solubility while maintaining the stable arylseleno core. This
arylseleno structure is less susceptible to converse to more toxic inorganic forms than
alkylseleno compounds. The aim of this work was to synthesize a series of N-substituted
benzisoselenazolones, transform them to corresponding diselenides and compare their
antioxidant and cytotoxic activity in vitro.

2. Results and discussion
2.1. Chemistry
We have used our previously published methodology in which lithium diselenide,
obtained in situ from elemental selenium and lithium hydroxide in the presence of hydrazine
hydrate, reacts with N-substituted o-iodobenzamides to form N-substituted
9
benzisoselenazolones 9a-d (Scheme 3).24 N-methyl 9a, N-butyl 9b, N-3-methylbutyl 9c and
N-cyclohexyl 9d derivatives were further converted to corresponding diselenides 10b-d by the
reduction with sodium borohydride and oxidation with air.
Scheme 3. Synthesis of N-substituted benzisoselenazolones 9a-d and diselenides 10b-d
We also noticed that obtained diselenides 10b-d can be transformed into starting
benzisoselenazolones 9b-d by the oxidative formation of the Se-N bond using potassium
iodate. Cyclization of the diselenides proceeded in good yields (Scheme 4).
Scheme 4. Synthesis of N-substituted benzisoselenazolones 9b-d

In turn, N-allyl benzisoselenazolone 9e was obtained by transformation of anthranilic acid 11
into 2-chloroselenobenzoyl chloride 12, followed by the reaction with allyl amine 1325
(Scheme 5). Procedure previously described for the synthesis of 9a-d did not work in this
case. Benzisoselenazolone 9e was also reduced to corresponding diselenide 10e. Interestingly,
the formation of Se-N bond by treatment with KIO3 did not occur leading to the assumption
that due to the electronic effect of the allylic moiety, the Se-N bond can be rapidly cleaved to
form a more stable diselenide 10e (Scheme 4).
Scheme 5. Synthesis of N-allyl derivatives 9e and 10e
2.2. Antioxidant activity
Second part of our research involved evaluation of the antioxidant activity of the
synthesized benzisoselenazolones and diselenides using the methodology presented by
Iwaoka and co-workers. Dithiothreitol (DTTred) was oxidized to dithiane (DTTox) by
hydrogen peroxide in the presence of 10% of the Se-catalyst. Rate of the reaction was
evaluated on the basis of 1H NMR spectra. Decay of the substrate and increase of the product
concentration were observed (Table 1, Fig. 1).26
The best peroxide scavenging capacity was obtained for N-allyl diselenide 10e, a total
conversion of the substrate was assessed after 30 min of the reaction.
All N-alkyl benzisoselenazolones showed better antioxidant activity than ebselen. In case of
diselenides 10 b-d we did not observe any increase of antioxidant activity. Only diselenide

10e demonstrated a satisfying result. Probably, the ease of formation of ebselen derivatives
from diselenides influences the speed of this reaction. When the diselenide is not converted to
ebselen in the reaction conditions (see scheme 1) the antioxidant properties are better (see also
reaction with KIO3, only diselenide 10e did not form the ebselen 9e). In our opinion the
stability of Se-N bond in ebselen derivatives influences the speed of this reaction. Strong Se-
N bond gives lower antioxidant activity.
Remaining Dithiothreitol [%]
Catalyst
3 min
5 min
15 min 30 min 60 min
[0.1 equiv.]
9a
9b
9c
9d
9e
78
81
77
75
76
84
93
65
92
59
65
59
58
69
69
75
88
49
85
36
35
41
42
62
58
64
82
38
75
12
11
32
28
55
53
58
77
31
65
0
0
29
13
44
41
52
73
21
54
0
Ebselen
10b
10c
10d
10e
Table 1. Antioxidant activity of the tested Se-catalysts
Fig 1. Graphical presentation of antioxidant activities

2.3. Cytotoxic activity
Cytotoxic activity of the obtained derivatives was evaluated by the MTT assay using
breast carcinoma MCF-7 cell line. SRB viability assay was performed on prostate cancer cell
line DU-145 and noncancerous epithelial cell line PNT1A. The IC50 values are presented in
Table 2.
Table 2. Cytotoxic activity evaluated in vitro [IC50]
Entry
Compound
MCF-7 (MTT assay) DU-145 (SRB assay) PNT1A (SRB assay)
IC50, µM
32.67 ± 1.15
13.77 ± 0.29
29.0 ± 1.90
7.31 ± 0.14
25.70 ± 3.40
> 500
30.18±0.09
20.76±0.36
30.39± 0.13
5.71±0.39
10.30 ±0.15
>40
>40
>40
>40
>40
>40
-
1
2
3
4
5
7
9a
9b
9c
9d
9e
10b
> 500
>40
-
8
10c
90 ± 0.0
125 ± 15
>40
20.41±0.12
-
-
9
10
10d
10e
The cytotoxic activity was correlated with the structure of the tested compounds. For both
cancer cell lines the N-cyclohexyl benzisoselenazolone 9d was the most active derivative. N-
allylic diselenide 10e exhibited a moderate antiproliferative activity in comparison to the high
antioxidant activity obtained for the oxidation of DTTred. When we compare the antioxidant
and cytotoxic properties of N-alkyl ebselen derivatives we can see that the best cytotoxicity
was observed for ebselen 9d with the lowest antioxidant activity. Stable S-N bond gives better
cytotoxic properties. In normal prostate cell line all ebselen derivatives did not reveal high
cytotoxivity and antiproliferative activity. The IC50 values were above 40 µM in all of the
tested compunds.
Moreover, to get insight to the mechanism of antiproliferative activity of the studied
compounds, active form of Akt kinase (p-Akt) has been measured. Akt is a serine threonine
27,28
kinase which is often up regulated in many cancer types including breast and prostate
It
has been shown to stimulate proliferation and survival of cancer cells and its activity can be
increased by ROS.3 Additionally, most of anticancer drugs efficacy depends on their ability to
target Akt kinase.29 Interestingly, both compounds 9d and 9c significantly reduced p-Akt
level in DU145 cell line (Figure 1). Moreover, compound 9d had no effect on Akt activity in
normal epithelial cell line (PNT1A).

3. Conclusions
We have obtained a series of N-alkyl benzisoselenazolones and developed an efficient
methodology for their conversion to diselenides with sodium borohydride and the reverse
reaction to form the Se-N bond with the use of potassium iodate as an oxidant. All prepared
compounds were evaluated as antioxidants and anticancer agents. N-allyl diselenide 10e was
the most reactive antioxidant. The highest antiproliferative activity was obtained for N-
cyclohexyl benzisoselenazolone 9d. We have shown that antioxidant and cytotoxic ativity
strongly depends on the stability of the Se-N bond present in the ‘ebselen-like’ structures. The
highest cytotoxic activity was observed for compounds with the lowest antioxidant activity.
In addition, our preliminary data indicate that compounds with antiproliferative activity 9d

and 9c target Akt kinase in cancer cells but not in PNT1A normal human prostate epithelial
cells. This suggests that they can have potential anticancer activity in in vivo conditions.
The newly obtained compounds were selected based on their antiproliferative activity
and are currently under further investigation. They are evaluated for the molecular mechanism
of action on cancer cell lines with different genetic background.
4. Experimental
1H NMR spectra were obtained at 400 or 700 MHz and chemical shifts were recorded relative
to SiMe4 ( 0.00) or solvent resonance (CDCl3 7.26, CD3OD 3.31). Multiplicities were
given as: s (singlet), d (doublet), dd (double doublet), ddd (double double doublet), t (triplet),
td (triple doublet), dt (double triplet) and m (multiplet). The number of protons (n) for a given
resonance was indicated by nH. Coupling constants were reported as a J value in Hz. 13C
NMR spectra were acquired at 100.6Hz and chemical shifts were recorded relative to solvent
77
resonance (CDCl3 77.25). Se NMR spectra were recorded on Bruker Avance III / 400 or
Bruker Avance III / 700 with diphenyl diselenide as an external standard. Commercially
available solvents DMF, DCM and MeOH (Aldrich) and chemicals were used without further
purification. Column chromatography was performed using Merck 40-63D 60Å silica gel.
4.1. Synthesis of compound 9e.25
4.1.1. N-allyl-1,2-benzisoselenazol-3(2H)-one 9e
1
Yield: 91%, mp 119-121oC, H NMR (400 MHz, CDCl3) δ = 4.49 (dt, J=1.2, 6.0 Hz, 2H),
5.32 (ddd, J=1.2, 2.4, 10.0 Hz, 1H), 5.38 (ddd, J=1.2, 2.4, 16.8 Hz, 1H), ), 5.93-6.03 (m, 1H),
7.43 (ddd, J=1.2, 7.2, 8.0 Hz, 1Har), 7.57-7.67 (m, 2Har), 8.07 (ddd, J=0.4, 1.2, 7.6 Hz, 1Har)
13
ppm; C NMR (100.6 Hz, CDCl3) δ = 47.1 (CH2), 119.2 (CH2), 124.0 (CH), 126.1 (CHar),
127.6 (Car), 128.8 (CHar), 131.9 (CHar), 133.5 (CHar), 137.9 (Car), 166.9 (C=O) ppm; 77Se
(76.3 MHz, CDCl3), δ = 887.32 ppm, IR 3270, 2956, 2925, 2865, 1634, 1601, 1585, 1257 cm-
1. Elemental Anal. Calcd for C10H9NOSe (238.14): C, 50.43; H, 3.81. Found: C, 50.12; H,
3.89.
4.2. Synthesis of diselenides
General procedure for the synthesis of compounds 10b-10d: To a solution of
benzisoselenazolone 9b-9d (1.0 mmol) in methanol (10 ml) cooled to 0oC, sodium

borohydride (1.0 mmol) was added and the mixture was stirred for 1h. Water (15ml) was
added and the mixture was oxidized with air for 1h. Formed precipitate was filtered and dried
in air.
4.2.1. 2,2‘-Diselenobis(N-butylbenzamide) 10b
1
Yield: 78%, mp 175-177oC (lit.30 mp 181-183oC), H NMR (400 MHz, CDCl3) δ = 0.99 (t,
J=7.2 Hz, 3H, CH3), 1.40-1.51 (m, 2H), 1.60-1.70 (m, 2H), 3.53 (dt, J=6.0, 7.2 Hz, 2H), 6.81
(bs, 1H, NH), 7.23 (td, J=1.2, 7.2 Hz, 1Har), 7.28-7.32 (m, 1Har), 7.49 (dd, J=1.6, 7.6 Hz,
1Har), 7.91 (dd, J=1.2, 8.0 Hz, 1Har) ppm; 13C NMR (100.6 Hz, CDCl3) δ = 13.7 (CH3), 20.18
(CH2), 31.7 (CH2), 40.0 (CH2), 126.1 (CHar), 126.5 (CHar), 131.4 (CHar), 131.6 (CHar), 132.9
(Car), 133.4 (Car), 168.2 (C=O) ppm; 77Se (76.3 MHz, CDCl3), δ = 451.98 ppm, IR 3301,
2954, 1609, 1584, 1541, 1433, 1317, 1026 cm-1.
4.2.2. 2,2‘-Diselenobis[N-(3-methylbutyl)benzamide) 10c
Yield: 60%, mp 160-162oC, 1H NMR (700 MHz, CDCl3) δ = 0.98 (d, J=0.7 Hz, 6H, 2xCH3),
1.53-1.57 (m, 2H), 1.70-1.75 (m, 1H), 3.50-3.54 (m, 2H), 6.12 (bs, 1H, NH), 7.22 (td, J=1.4,
7.7 Hz, 1Har), 7.28 (td, J=1.4, 7.7 Hz, 1Har), 7.45 (dd, J=1.4, 7.7 Hz, 1Har), 7.89 (dd, J=0.7,
7.7 Hz, 1Har) ppm; 13C NMR (100.6 Hz, CDCl3) δ = 22.1 (2xCH3), 25.6 (CH), 38.06 (CH2),
38.2 (CH2), 125.6 (CHar), 126.1 (CHar), 131.0 (CHar), 131.2 (CHar), 132.5 (Car), 132.9 (Car),
167.7 (C=O) ppm; 77Se (76.3 MHz, CDCl3), δ = 451.67 ppm, IR 3292, 2953, 2925, 1620,
1541, 1450, 1309, 1282 cm-1. Elemental Anal. Calcd for C24H32N2O2Se2 (538.44): C, 53.54;
H, 5.99. Found: C, 53.42; H, 5.93.
4.2.3. 2,2‘-Diselenobis(N-cyclohexylbenzamide) 10d
1
Yield: 76%, mp 261-263oC, H NMR (400 MHz, CDCl3) δ = 1.18-1.36 (m, 4H), 1.40-1.45
(m, 1H), 1.66-1.75 (m, 1H), 1.77-1.85 (m, 2H), 2.10 (dd, J=4.0, 12.8 Hz, 2H), ), 4.00-4.09 (m,
1H), 6.03 (d, J=6.8 Hz, 1H, NH), 7.23-7.26 (m, 1Har), 7.48 (dd, J=1.6, 7.6 Hz, 1Har), 7.61-
7.65 (m, 1Har), 7.9 (d, J=8.0 Hz, 1Har) ppm; 13C NMR (100.6 Hz, DMSO) δ = 25.3 (2xCH2),
25.6 (CH2), 32.7 (2xCH2), 49.1 (CH), 126.5 (CHar), 128.4 (CHar), 129.8 (Car), 131.8 (CHar),
132.3 (CHar), 134.0 (Car), 166.9 (C=O) ppm; 77Se (76.3 MHz, DMSO), δ = 443.47 ppm, IR
3275, 2924, 2851, 1614, 1583, 1536, 1450, 1432, 1372, 1281, 1149, 1080, 1026 cm-1.
4.2.4. 2,2‘-Diselenobis(N-allylbenzamide) 10e

1
Yield: 82%, mp 201-203oC, H NMR (700 MHz, CDCl3) δ = 4.14 (tt, J=1.4, 5.6 Hz, 2H),
5.23 (ddd, J=1.4, 2.8, 10.5 Hz, 1H), 5.32 (ddd, J=1.4, 2.8, 10.5 Hz, 1H), ), 5.90-6.01 (m, 1H),
6.23 (s, 1H, NH), 7.23 (td, J=1.4, 7.7 Hz, 1Har), 7.30 (td, J=1.4, 7.7 Hz, 1Har), 7.51 (dd,
13
J=1.4, 7.7 Hz, 1Har), 7.91 (dd, J=1.4, 7.7 Hz, 1Har) ppm; C NMR (100.6 Hz, CDCl3) δ =
42.6 (CH2), 117.2 (CH2), 126.1 (CH), 126.6 (CHar), 131.5 (CHar), 131.8 (CHar), 133.0 (Car),
133.2 (Car), 133.8 (CHar), 168.0 (C=O) ppm; 77Se (76.3 MHz, CDCl3), δ = 453.62 ppm, IR
3301, 2954, 2924, 1609, 1539, 1317, 1260, 1163 cm-1. Elemental Anal. Calcd for
C20H20N2O2Se2 (478.30): C, 50.22; H, 4.21. Found: C, 49.90; H, 4.27.
4.3. Conversion of diselenides to benzisoselenazolones.
To a solution of diselenide 10b-10d (1.0 mmol) in DMF (3 ml), under argon atmosphere,
potassium iodate (1.0 mmol) was added and the mixture was stirred at 120oC for 24h. Mixture
was cooled to ambient temperature and brine (5ml) was added. Mixture was stirred for 3h and
the formed precipitate was filtered and dried in air. Yield: 70% (9b), 86% (9c), 71% (9d).
4.4. Evaluation of antioxidant activity26.
To a solution of compounds 9a-9e and 10b-10e (0.015mmol) and dithiothreitole DTTred
1
(0.15mmol) in 1.1mL of CD3OD 30% H2O2 (0.15mmol) was added. H NMR spectra were
measuared right after addition of hydrogen peroxide and then in specific time intervals. The
concentarion of the substrate was determined according to the changes in the integration on
the 1H NMR spectra.
4.5. Cell viability assays
4.5.1. MTT viability assay
Cell culture. The MCF-7 cell line was purchased from the European Collection of
Cell Cultures (ECACC). The MCF-7 cells were cultured in Minimum Essential Medium
Eagle (MEME, Sigma-Aldrich) with glutamine (2 mM) (Sigma-Aldrich), MEM Non-essential
amino acid solution 100x (Sigma-Aldrich), gentamycin (5 lg/mL) and 10% heatinactivated
fetal bovine serum (FBS) (both from Biological Industries, Beit-Haemek, Israel). Cells were
maintained at 37°C in a 5% CO2 atmosphere and were grown until 80% confluent.
MTT assay. The MTT assay was performed according to the known procedure.31 The
cells were grown to sub-confluent levels in the culture medium and then plated onto 24-well
plates (104 cells/well) in the final volume of 1 ml of the culture medium. After 24 h, vehicle

(0.1% DMSO) or compounds in various concentrations were added and the plates were
incubated for 24 h. Then, the cells were incubated for 2 h at 37oC with 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide [MTT, 5 mg/ml in phosphate buffered saline (GIBCO,
Invitrogen, Carlsbad, CA, USA)]. The absorbance of the blue formazan product was
measured at 540 nm using an automated plate reader (iMark Bio-Rad, Hercules, CA, USA)
and compared with the control (untreated cells). All experiments were performed in triplicate.
4.5.2. SRB viability assay
Cell culture. The prostate cancer cell line DU-145 and noncancerous human prostate
cell line PNT1A were purchased from the American Type Culture Collection (ATTC,
Manassas, VA). DU 145 cells were cultured in MEME medium supplemented with 10 %
fetal bovine serum, 1% penicillin/streptomycin, 2 mM glutamine and 1 mM sodium pyruvate
at 37°C under 5% CO2 incubator. Stock solutions of N-substituted ebselen derivatives were
prepared in (0,1%) DMSO. The PNT1A cells were cultured in RPMI 1640 supplemented
with serum, L-glutamine and antibiotics.
SRB assay. The prostate cancer cell line DU-145 and noncancerous human prostate
epithelial cells PNT1A were used in this study. Cell viability was measured by
Sulphorhodamine B (SRB) assay. The cells were grown to sub-confluent levels at the certain
3
culture medium and then seeded into 96-well plates at 6.0 x 10 cells/ well in the final
volume of 200 µl in the culture medium for 24 h. Then, they were treated with various
concentrations (2,5, 5, 10, 20, 30, 40 µl) of N-substituted ebelsen derivatives for the next 24h.
After incubation, the cells were fixed in 20% trichloroacetic acid for an 1h. The plates were
washed with distilled water and 0,4% SRB (Sigma Aldrich ) in 1% acetic acid solution was
added to the plates for 15 minutes. The SRB solution was washed with 1% acetic acid. SRB
was then solubilized in 10 mM Trisma-base solution and the absorbance was measured at 570
nm using an automated microplate reader. The experiments were done in triplicate and the
IC50 values were calculated.
4.5.3 Western blotting
The cells were treated with 40µM N-cyclohexyl-1,2-benzisoselenazol-3(2H)-one and 40 µM
and N-(3-methyl)-butyl-1,2-benzisoselenazol-3(2H)-one at different time points. Both floating
and attached cells were collected : washed in PBS; resuspended in a lysis solution containing
50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1% Triton X-100, 0.1% SDS; and

incubated for 40 min on ice with gentle shaking. The cell lysate was cleared by the
centrifugation at16.000g for 20 min. Lysate proteins were resolved in 10–12% SDS-PAGE
and transferred onto polyvinyli-dene difluoride membrane. The membrane was incubated with
a solution containing Tris-buffered saline, 0.05% Tween 20, and 5–10% (w/v) nonfat dry milk
and then exposed to the desired primary antibody for 1 h at room temperature. Following
treatment with the appropriate secondary anti-body, the bands were visualized using enhanced
chemiluminescence method. Changes in protein level was assessed by densitometric scanning
of the bands and cor-rected for β-actin loading control.
Acknowledgments
This work was supported by the National Science Centre, Poland, grant no. UMO-2015/17 /B/
NZ7/03058.
5. References
1. Zuo, L.; Zhou, T.; Pannell, K. B.; Ziegler A. C. Best, T. M. Acta. Physiol., 2015, 214,
329–348.
2. (a) Sies, H. Redox Biol, 2015, 4, 180–183. (b) Steinbrenner, H.; Sies, H. Bio. et
Biophy. Acta., 2009, 1790, 1478–1485.
3. Borkowska, A.; Sielicka-Dudzin, A.; Herman-Antosiewicz, A.; Wozniak, M.; Fedeli,
D.; Falcioni, G.; Antosiewicz, J. Eur. J. Nutr., 2012, 51, 817–825.
4. Luo, L.; Kaur Kumar, J.; Clement, M. V. Free Radic. Biol. Med. 2012, 53, 1697–
1707.
5. (a) Pacuła, A. J.; Mangiavacchi, F.; Sancineto, L.; Lenardao, E. J.; Ścianowski J.;
Santi, C. Curr. Chem. Biol., 2015, 9, 97–112. (b) Weekley, C.M.; Harris, H. H. Chem.
Soc. Rev., 2013, 42, 8870–8894. (c) Nogueira, C. W.; Rocha, J. B. T. in Organic
Selenium and Tellurium Compounds; Rappoport, Z., Ed.; Wiley: Chichester, 2012;
Volume 3, Part II, Chapter 21. (d) Back, T. G. in "Biochalcogen Chemistry: The
Biological Chemistry of Sulfur, Selenium and Tellurium", eds. Bayse, C. and
Brumaghin, J., ACS Symposium Series, American Chemical Society, 2013, Ch. 7. (e)
Day, B. J. Biochem. Pharmacol. 2009, 77, 285–296. (f) Bhabak, K. P.; Mugesh, G.
Acc. Chem. Res. 2010, 43, 1408–1419. (g) Mugesh, G.; Singh, H. B. Chem. Soc. Rev.
2000, 29, 347–357. (h) Mugesh, G.; du Mont, W.-W.; Sies, H. Chem. Rev. 2001, 101,
2125–2179.
6. Parnham J. M.; Sies, H. Biochem. Pharm., 2013, 86, 1248–1253.
7. Martini, F.; Bruning, C. A.; Soares, S. M.; Naguiera C. W.; Zeni, G. Current
Pharmaceutical Design, 2015, 21, 920–924.
8. Tan, S. M.; Deliyanti, D.; Figget, W. A.; Talia, D. M.; de Haan, J. B.; Wilkinson-
Berka, J. L. Experimental Eye Research, 2015, 136, 1–8.
9. Park, S.; Kang, S.; Kim, D. S.; Shin, B. K.; Moon, N. R.; Daily III, J. W. Free
Radical Research, 2014, 48, 864–874.
10. Schewe, C.; Schewe, T.; Wendel, A. Biochem. Pharmacol. 1994, 48, 65–74.

11. N. Singh, N.; Sharpley, A. L.; Emir, U. E.; Masaki, C.; Herzallah, M. M.; Gluck, M.
A.; Sharp, T.; Hamer, C. J.; Vasudevan, S. R.; Coven, P. J.; Churchill, G. C.
Neuropsychopharmacology 2016, 41, 1768–1778.
12. Lynch, E. D.; Kill, J. Drug Discovery Today, 2005, 10, 1291–1298.
13. Mugesh, G. Curr. Chem. Biol., 2013, 7, 47–56.
14. Sarma, K. B.; Mugesh, G. Chem. Eur. J. 2008, 14, 10603 – 10614.
15. Bhabak, K. P.; Mugesh, G. Acc. Chem. Res., 2010, 43, 1408–1419.
16. Orian, L.; Toppo, S. Free Radic. Biol. Med., 2014, 66, 65–74.
17. Kumar Azad, G.; Tomar, R. S. Mol. Bio. Rep., 2014, 41, 4865-4879.
18. Prigol, M.; Nogueira, C. W.; Zeni, G.; Bronze, M. R.; Constantino, L. Chem. Biol.
Interact., 2012, 200, 65–72.
19. Kumar Azad, G.; Singh, V.; Mandal, P; Singh, P.; Golla, U.; Barnawal, S. Chauhan,
S.; Tomar, R. S. FEBS Open Bio., 2014, 4, 77–89.
20. Bijain, K.; Zhang, Z.; Xu, B.; Jie, S.; Chen, B.; Wan, Sh.; Wu, J.; Jiang, T.; Alaoui-
Jamali, A. M. Eur. J. Med. Chem., 2012, 48, 143–152.
21. (a) Li, B.-L.; Li, B.; Zhang, R.-L.; Zhao, J.-J.; Wang, X.-F.; Liu, Y.-M.; Shi, Y.-P.;
Liu, J.-B.; Chen, B.-Q. Bioorg. Med. Chem. Lett., 2016, 26, 1279–1281. (b) Zhao, J.;
Xuan, L; Zhao, H.; Cheng, J.; Fu, X.; Li, S.; Jing, F.; Liu, Y.; Chen, B. Chem. Res.
Chin. Univ., 2014, 30, 764–769.
22. Młochowski, J.; Kloc, K.; Syper, L.; Inglot, A. D.; Piasecki, E. Liebigs Ann. Chem.,
1993, 1239–1244.
23. Yan, J.; Guo, Y.; Wang, Y.; Mao, F.; Huang, L.; Li, X. Eur. J. Med. Chem., 2015, 95,
220–229.
24. Pacuła, A. J.; Ścianowski, J.; Aleksandrzak, K. B. RSC Adv, 2014, 4, 48959–48962.
25. Welter, A.; Christiaens, L.; Ferdinand, W.-P. Eur. Pat. Appl. EP 44453, 1982, Chem.
Abstr., 1982, 96, 199699v.
26. Kumakura, F.; Mishra, B.; Priyadarsini, K. I.; Iwaoka, M. Eur. J. Org. Chem., 2010,
440–444.
27. Altomare, D. A.; Testa, J. R. Oncogene, 2005, 24, 7455–7464.
28. Schmidt, M.; Voelker, H. U.; Kapp, M.; Krockenberger, M.; Dietl, J.; Kammerer, U.
J. Cancer. Res. Clin. Oncol., 2010, 136, 219–225.
29. Cheng, J. Q.; Lindsley, C. W.; Cheng, G. Z.; Yang, H.; Nicosia, S. V. Oncogene,
2005, 24, 7482–7492.
30. Pietka-Ottlik, M.; Potaczek, P.; Piasecki, E.; Młochowski, J. Molecules, 2010, 15,
8214–8228.
31. Mosmann, T. J. Immunol. Methods, 1983, 65, 55–63.

Graphical abstract