Water-dependent synthesis of biologically active diaryl diselenides

Article abstract of DOI:10.24820/ark.5550190.p010.311

A new one-step method for the synthesis of diaryl diselenides has been developed. The reaction of o-iodobenzamides with dilithium diselenide can be controlled by the presence of water providing a simple and efficient protocol to obtain benzisoselenazolones or diaryl diselenides. A series of N-Aryl ebselen derivatives and the corresponding diselenides was obtained. All synthesized compounds were tested in vitro as antioxidants and cytotoxic agents. N-(2,3,4-Trimethoxyphenyl)benzisoselenazol-3(2H)-one was the best in vitro antioxidant and the corresponding diselenide the most potent cytotoxic agent against prostate cancer cell line DU145, being inactive towards healthy prostate cell line PNT1A. Formula parented.

Full text of DOI:10.24820/ark.5550190.p010.311

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Organic Chemistry
Arkivoc 2018, part iii, 153-164
Water-dependent synthesis of biologically active diaryl diselenides
Agata J. Pacułaa, Magdalena Obieziurska a, Jacek Ścianowski*a, Katarzyna B. Kaczorb,
and Jędrzej Antosiewiczb,c
aDepartment of Organic Chemistry, Faculty of Chemistry, Nicolaus Copernicus University in Torun,
7 Gagarin Street, 87-100 Torun, Poland
bDepartment of Bioenergetics and Physiology of Exercise, Medical University of Gdansk,
1 Debinki Street, 80-211 Gdansk, Poland; cDepartment of Biochemistry, Gdansk University of Physical Education
and Sport, 1 Kazimierza Gorskiego Street,, 80-336 Gdansk, Poland
E-mail: jsch@chem.umk.pl
Received 08-23-2017
Accepted 11-30-2017
Published on line 02-18-2018
Abstract
A new one-step method for the synthesis of diaryl diselenides has been developed. The reaction of o-
iodobenzamides with dilithium diselenide can be controlled by the presence of water providing a simple and
efficient protocol to obtain benzisoselenazolones or diaryl diselenides. A series of N-aryl ebselen derivatives
and the corresponding diselenides was obtained. All synthesized compounds were tested in vitro as
antioxidants and cytotoxic agents. N-(2,3,4-trimethoxyphenyl)benzisoselenazol-3(2H)-one was the best in vitro
antioxidant and the corresponding diselenide the most potent cytotoxic agent against prostate cancer cell line
DU145, being inactive towards healthy prostate cell line PNT1A.
Keywords: Benzisoselenazolones, diselenides, selenium, cytotoxicity, antioxidants, biological activity
DOI: https://doi.org/10.24820/ark.5550190.p010.311
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Introduction
Selenium-selenium bond is a unique structural motif that makes organic molecules highly applicable as
reagents and catalysts in various organic transformations1-3 and pharmacologically potent compounds in
biological systems.4-7 Several research papers and reviews reveal this undeniable potential and prove that the
search for simple protocols to synthesize diselenides is a highly desirable. Considering their usage in organic
synthesis diselenides possess several advantages - they are stable and can be easily converted to electrophilic,
nucleophilic and radical reagents.8-24
The biological potential of diaryl diselenides is mainly associated with their GPx-like activity. Glutathione
peroxidase (GPx) is a selenoenzyme that, based on the presence of catalytically active aminoacid
selenocysteine (Sec, U), eliminates reactive oxygen and nitrogen species enabling to maintain the redox
homeostasis inside the cell.25,26 It is assumed that the most-studied GPx mimic ebselen (N-
phenylbenzisoselenazo-3(2H)-one, 1), through the reaction with thiols, can be transformed to the
corresponding diaryl diselenide with amido function 2 and in this form act as a bio-active intermediate that
eliminates peroxides.27-29
It has been proven that Se-N bond is not necessary to observe an enhanced GPx-like activity. Non-bonding
selenium-heteroatom interactions can increase the antioxidant capacity what was established in the case of
several diaryl diselenides possessing an additional heteroatom. Examples of highly reactive molecules 3-7 are
presented in Scheme 1.30-32
Scheme 1. Examples of diaryl diselenides with enhanced GPx-like activity.
Currently, we can specify few methodologies to synthesize N-substituted diselenides 8. Direct reduction of
benzisoselenazolones with different reducing agents – sodium borohydride, hydrazine, ascorbate or
phosphoric acid (h),33-36 nucleophilic substitution of o-iodobenzamides with lithium diselenide (i),37 under
radical conditions from the reaction of 2-benzylselenobenzamides with triphenyltin hydride and further
treatment with benzoyl peroxide (j)38 and few protocols starting from 2,2-diselenobis(benzoic acid) - through
the formation of the corresponding dichloride and further reaction with amine (k)39 or by EDC (l)40 and DCC-
mediated (m)41 coupling reactions (Scheme 2).
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Scheme 2. Approaches to diselenides with amido function 8.
The aim of this study was the synthesis of diaryl diselenides with an amido function substituted on the
nitrogen atom by a phenyl ring carrying electron withdrawing or electron donating groups and comparison of
their antioxidant activity to the GPx-capacity of the corresponding benzisoselenazolones and ebselen.
Results and Discussion
First, a series of benzisoselenazolones 1, 9-14 was prepared by two procedures, from 2,2-diselenobis(benzoic
acid) (method A)39 and by our method B,42-44 involving the treatment of N-substituted o-iodobenzamide with
dilithium diselenide, providing significantly better yields (Scheme 3).
Scheme 3. Synthesized benzisoselenazolones 1, 9-14.
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Compounds 1, 9-14 were further transformed into the corresponding diselenides 2, 15-20 using sodium
borohydride as the reducing agent followed by air oxidation (method C), however, low yields required further
studies. During our investigations on the synthesis of ebselen derivatives, based on the reaction of N-
substituted o-iodobenzamide with dilithium diselenide, we observed that water influences the reaction course
leading to the formation of diselenides as by-products. Consequently, a new one-step synthesis of compounds
2, 15-20 was developed (method D). According to our previously proposed mechanism for the formation of
ebselen derivatives, in the first reaction step corresponding diselenides are formed, which are then oxidized to
benzisoselenazolones.42 Apparently, the addition of a small amount of water (5%) decreases the solubility of
diselenides what prevents further oxidation reactions. Increasing the amount of added water completely
inhibited the product formation and the unreacted substrate was isolated after 24 h. Although the newly
presented procedure is based on the known reaction of o-iodobenzamides with LiSeSeLi (method i) significant
improvements have been achieved. Lithium diselenide is obtained by a modified protocol from selenium and
lithium hydroxide in the presence of hydrazine hydrate, and no catalyst is needed for the reaction to
proceed.42 Formation of monoselenides in the reaction mixture was not observed.37 Addition of water (5%) to
the reaction medium enables to obtain diaryl diselenides as the only products directly from o-iodobenzamides
(Scheme 4).
Scheme 4. Water-controlled synthesis of diaryl diselenides.
Results for the synthesis of diaryl diselenides 2, 15-20 by both methods C and D are collected in Table 1.
Reducing the procedure to one step increased the overall yields of the reactions. The newly developed water-
dependent methodology was significantly more efficient than the synthesis of benzisoselenazolone and its
further reduction and air oxidation.
All of the obtained benzisoselenazolones and diselenides were evaluated as in vitro antioxidants using the
NMR assay proposed by Iwaoka.45 Time of conversion of dithiol (DTTred) to a disulphide (DTTox) in the presence
of H2O2 and 10% of selenocatalyst was measured in selected time intervals (Table 2). The highest activity was
obtained for benzisoselenazolone 14 with an additional methoxy moiety. In this case no substrate was
observed after 5 min of the reaction time.
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Table 1. Results of diselenides synthesis according to method C and D
R=
-Ph
Compound
Method C [%]
Method D [%]
2
34
53
22
24
17
26
22
42
70
45
69
76
43
29
-C6H4-4-OCH3
-C6H4-4-NO2
-C6H4-4-Cl
-C6H4-4-Br
-C6H4-4-I
15
16
17
18
19
20
-C6H2-2,3,4-(OCH3)3
Table 2. Results of the in vitro antioxidant activity measurement for benzisoselenazolones and diselenides
Remaining dithiothreitol [%]
Catayst
1
3 min
84
64
25
78
86
42
5
89
84
79
61
81
89
96
5 min
75
41
7
68
83
0
15 min
64
5
30 min
58
0
60 min
52
0
9
10
11
12
13
14
2
15
16
17
18
19
20
5
0
0
50
79
0
38
75
0
36
71
0
0
0
0
0
83
73
58
53
72
83
82
74
63
10
26
55
81
41
68
57
0
19
41
78
6
63
52
0
12
23
74
0
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All derivatives were also tested as cytotoxic agents towards prostate cancer cell line DU145. Results are
collected in Table 3. The highest cytotoxic activity against cancer cells was observed for diselenides bearing
additional methoxy substituents in the N-phenyl ring 15 and 20. Compounds exhibiting cytotoxic potential
were additionally tested on normal cell line PNT1A. The data showed that both compounds demonstrated low
toxicity towards PNT1A cells. They were also more active than commercially used cisplatin. Conversely,
compounds 9 and 2 exhibited the same cytotoxic activity against noncancerous and cancer cells.
Table 3. Cytotoxic activity of benzisoselenazolones and diselenides expressed as IC50. Values (µM) are means
± SD
Entry Compound
DU-145
21.16 ± 0.60
19.80 ± 13
>40
PNT1A
30.57 ± 0.12
20.35 ± 0.08
1
2
1
9
3
10
-
4
11
>40
-
5
12
>40
-
6
13
>40
-
7
8
9
10
11
12
13
14
15
14
2
15
16
17
18
19
20
20.84 ± 0.37
20.70 ± 0.28
11.24 ± 0.97
>40
>40
19.76 ± 0.14
>40
-
-
-
-
>40
-
>40
>40
>40
11.12 ± 0.99
24.14 ± 0.79
Cisplatin
Conclusions
Herein, we have established that by adding 5 % of water we can initiate the formation of a Se-Se instead of Se-
N bond. Simple modification of the previously presented methodology to synthesize benzisoselenazolones led
to the development of an efficient protocol to obtain diaryl diselenides with amido function. A series of
diselenides was synthesized by two methods: from the reduction and further air oxidation of previously
obtained benzisoselenazolones and by the newly developed methodology which was more efficient. An
additional advantage of the new methodology is the lack of monoselenides as the side products. All
compounds were tested as in vitro antioxidants and cytotoxic agents. The highest in vitro antioxidant activity
was obtained for benzisoselenazolone 14 for which no substrate was observed after 5 min. The cytotoxic
activity assay performed on prostate cancer cell line DU145 showed that the presence of the methoxy
substituent, also attached to the N-phenyl ring of compound 14, influences the biological activity of
derivatives 15 and 20 with the highest cytotoxic potential. It is important to note that both compounds 15 and
20 showed specific activity against cancer cells higher than cisplatin but not against normal epithelial cell line
PNT1A. Thus, it can be concluded that both compounds are worth of further study as potential drugs.
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Experimental Section
1
General. H 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 100.6 MHz and chemical shifts were
recorded relative to solvent resonance (CDCl3 77.25). NMR spectra were carried out using ACD/NMR
Processor Academic Edition. 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.
Synthesis of compounds 1, 9-14
Method A.44 Preparation of 2-(chloroseleno)benzoyl chloride. To a suspension of selenium (12.7 mmol) in
water (7 mL) sodium borohydride (25.3 mmol) was added under argon atmosphere. Mixture was stirred for
o
0.5h at 0 C, selenium (12.7 mmol) was added, and stirring was continued at the same temperature for 1h.
Mixture was warmed to room temperature and stirred for 18 h. After adding 40% NaOH (5 mL) the reaction
o
was cooled to 5 C and the diazonium salt of anthranilic acid was added dropwise. (The diazonium salt was
prepared z in advance — to a solution of anthranilic acid (26.0 mmol) and concentrated hydrochloric acid (5
o
mL) in water (15 mL) cooled to 5 C a cooled solution of sodium nitrite (27.5 mmol) in water (15 mL) was
o
o
added dropwise and the reaction was stirred for 15 min at 5 C). The mixture was stirred for 3 h at 60 C and
for 18 h at room temperature. The formed precipitate was filtered off and the solution was acidified to pH = 1
by 36% HCl. The formed precipitate was filtrated. The crude product was purified by washing with boiling
water and dried in air. Acid (10.0 mmol) was further converted to 2-(chloroseleno)benzoyl chloride by heating
o
with thionyl chloride (20 mL) at 85 C for 3 h. Thionyl chloride was distilled off, and the crude product was
used without further purification.
Preparation of benzisoselenazlones. To a solution of amine (2.0 mmol) and triethylamine (4.0 mmol) in
dichloromethane 2-(chloroseleno)benzoyl chloride (1.0 mmol) was added. The mixture was stirred for 24h at
room temperature, poured on water and extracted with DCM. The combined organic layers were dried over
anhydrous magnesium sulfate and evaporated. Yields: 54% 1, 49% 9, 32% 10, 52% 11, 45% 12, 19% 13, 55%
14.
Method B.42 Hydrazine hydrate (8.0 mmol) was added dropwise to the mixture of selenium powder (1.2
o
mmol) and lithium hydroxide (3.6 mmol) in DMF (3 mL). Reaction was heated to 120 C and stirred for 15 min
under argon atmosphere. After cooling to room temperature the amide (1.0 mmol) in DMF (2 mL) was added.
o
The reaction mixture was heated to 120 C and stirred for 20 h under argon atmosphere. The solution was
cooled, 25 mL of brine was added and the mixture was stirred for additional 20h. Precipitate was filtered
under vacuum, washed with water and dried in air. The crude product was purified by column
chromatography (silica gel, DCM:MeOH 99.2 : 0.8). Yields: 91% 1, 86% 9, 60% 10, 67% 11, 72% 12, 82% 13,
75% 14.
N-(4-Chlorophenyl)-1,2-benzisoselenazol-3(2H)-one (11).33 Yield: Method A: 52%, Method B: 67%, mp 179-
o
o
1
181 C (mp 181-181.5 C), H NMR (700 MHz, DMSO) δ = 7.51 (t, J 7.0 Hz, 1Har), 7.54 (d, J=8.4 Hz, 2Har), 7.71-
7.73 (m, 3Har), 7.93 (d, J 6.3 Hz, 1Har), 8.11 (d, J 8.4 Hz, 1Har) ppm; 13C NMR (100.6 MHz, CDCl3) δ = 124.73
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(CHar), 127.53 (2xCHar), 127.72 (CHar), 128.24 (Car), 130.42 (2xCHar), 130.49 (CHar), 133.15 (Car), 133.77 (CHar),
138.33 (Car), 138.70 (Car), 166.72 (C=O) ppm; 77Se (76.3 MHz, CDCl3), δ = 967.61 ppm, IR 1649, 1631, 1600,
1584, 1527, 1505, 1487, 1448, 1432, 1409, 1381, 1348, 1309, 1274, 1232, 1198, 1128, 1069, 1025 cm-1.
Synthesis of diselenides 2, 15-20
Method C. To a solution of benzisoselenazolone 1, 9-14 (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 (15 mL) was added and the
mixture was oxidized with air for 1 h. Formed precipitate was filtered and dried in air.
Method D. Selenium powder (1.2 mmol) and lithium hydroxide (3.6 mmol) were weight into a single neck flask
under argon atmosphere and dissolved in a mixture of DMF (2.85 mL) and water (0.15 mL). Hydrazine hydrate
o
(0.8 mmol) was added dropwise and the mixture was heated to 120 C and stirred for 15 minutes. After
cooling to room temperature the amide (1.0 mmol) dissolved in DMF (2 mL) was added. The reaction mixture
o
was heated to 120 C and stirred for 20 h under argon atmosphere. The solution was cooled to room
temperature, 25mL of brine were added, and acidified with hydrochloric acid to pH=5. The mixture was stirred
for 1h at room temperature. The obtained precipitate was filtered under vacuum, washed with water and
dried in air. The crude product was purified by multiple rinsing with boiling DCM.
o
2,2‘-Diselenobis(N-phenylbenzamide) (2)42 Yield: Method C: 34%, Method D: 42%, mp 259-261 C (lit.46 mp
o
1
256-257 C) , H NMR (700 MHz, DMSO) δ = 7.15 (t, J 7.0 Hz, 2Har), 7.36-7.43 (m, 6Har), 7.45 (td, J 1.4, 7.0 Hz,
13
2Har), 7.76 (d, J 7.7 Hz, 4Har), 7.78 (dd, J 1.4, 8.4 Hz, 2Har), 7.96 (d, J 7.7 Hz, 2Har), 10.55 (s, 2H, 2xNH) ppm; C
NMR (100.6 MHz, DMSO) δ 120.66 (4xCHar), 124.24 (2xCHar), 126.52 (2xCHar), 128.73 (2xCar), 128.82 (4xCHar),
130.27 (2xCar), 132.06 (2xCHar), 132.13 (2xCar), 133.88 (2xCHar), 138.73 (2xCHar), 166.43 (2xC=O) ppm; 77Se
(76.3 MHz, DMSO), δ = 443.67 ppm, IR 3291, 1666, 1636, 1596, 1581, 1559, 1520, 1499, 1457, 1434, 1322,
1290, 1255, 1176, 1154, 1133, 1107, 1075, 1044, 1026 cm-1.
2,2-Diselenobis[N-(4-metoxyphenyl)benzamide] (15)47 Yield: Method C: 53%, Method D: 70%, mp 287-289 oC
o
1
(mp 290-292 C), H NMR (700 MHz, DMSO) δ 3.75 (s, 6H, 2xOCH3), 6.96 (d, J 9.1 Hz, 4Har), 7.39 (t, J 7.0 Hz,
2Har), 7.44 (t, J 7.0 Hz, 2Har), 7.67 (d, J 9.1 Hz, 4Har), 7.77 (d, J 8.4 Hz, 2Har), 7.93 (d, J 7.7 Hz, 2Har), 10.42 (s, 2H,
2xNH) ppm; 13C NMR (100.6 MHz, DMSO-d6) δ 55.70 (2xOCH3), 114.33 (4xCHar), 122.66 (4xCHar), 126.84
(2xCHar), 128.92 (2xCHar), 130.62 (2xCHar), 132.15 (2xCHar), 132.35 (2xCar), 132.43 (2xCar), 134.30 (2xCar),
156.37 (2xCar), 166.41 (2xC=O) ppm; 77Se (76.3 MHz, CDCl3), δ 443.49 ppm, IR 1631, 1510, 1410, 1247, 1027
cm-1.
o
2,2‘-Diselenobis[N-(4-nitrophenyl)benzamide] (16)47 Yield: Method C: 22%, Method D: 45%, mp 254-256 C,
1H NMR (700 MHz, DMSO) δ 7.44-7.52 (m, 4Har), 7.82 (d, J 7.2 Hz, 2Har), 8.02 (d, J 7.2 Hz, 2Har), 8.06 (d, J 9.6
Hz, 4Har), 8.31 (d, J 9.6 Hz, 4Har), 11.09 (s, 2H, 2xNH) ppm; 13C NMR (100.6 MHz, DMSO) δ 120.7 (4xCHar), 125.3
(4xCHar), 127.1 (2xCHar), 129.6 (2xCHar), 130.9 (2xCar), 132.8 (2xCar), 133.1 (2xCHar), 133.6 (2xCar), 143.3
(2xCHar), 145.4 (2xCar), 167.4 (2xC=O) ppm; 77Se (76.3 MHz, DMSO), δ 447.41 ppm, IR 3392, 1660, 1610, 1595,
1583, 1535, 1493, 1430, 1403, 1328, 1302, 1280, 1237, 1177, 1138, 1110, 1094, 1055, 1026 cm-1.
1
2,2‘-Diselenobis[N-(4-chlorophenyl)benzamide] (17)33 Yield: Method C: 24%, Method D: 69%, H NMR (700
MHz, DMSO) δ = 7.45 (dt, J 0.7, 7.0 Hz, 2Har), 7.47-7.51 (m, 6Har), 7.81 (dd, J 1.4, 8.4 Hz, 2Har), ), 7.84 (d, J 9.1
Hz, 4Har), 7.98 (dd, J 1.4, 7.7 Hz, 2Har), 10.69 (s, 2H, 2xNH) ppm; 13C NMR (100.6 MHz, DMSO) δ 123.6 (4xCHar),
127.6 (2xCar), 128.0 (2xCHar), 129.3 (2xCar), 130.1 (4xCHar), 130.5 (2xCHar), 131.7 (2xCar), 133.5 (2xCHar), 135.0
(2xCar), 139.0 (2xCHar), 167.9 (2xC=O) ppm; 77Se (76.3 MHz, DMSO), δ 444.45 ppm, IR 3283, 1641, 1593, 1512,
1396, 1317, 1252, 1096, 1015 cm-1.
2,2-Diselenobis[N-(4-bromophenyl)benzamide] (18)48 Yield: Method C: 17%, Method D: 76%, mp 248-250 oC,
1
1H NMR (700 MHz, DMSO) H NMR (700 MHz, DMSO-d6) δ 7.42 (dt, J 0.7, 7.0 Hz, 2Har), 7.46 (dt, J 1.4, 7.7 Hz,
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2Har), 7.58 (d, J 8.4 Hz, 4Har), 7.75 (d, J 9.1 Hz, 4Har), 7.78 (dd, J 0.7, 8.4 Hz, 2Har), 7.96 (dd, J 0.7, 7.0 Hz, 2Har),
10.67 (s, 2H, 2xNH) ppm; 13C NMR (100.6 MHz, DMSO) δ 116.36 (2xCHar), 122.92 (4xCHar), 126.96 (2xCHar),
129.20 (2xCar), 130.74 (2xCar), 132.05 (4xCHar), 132.52 (2xCar), 132.69 (2xCar), 134.00 (2xCHar), 138.54 (2xCHar),
166.88 (2xC=O) ppm; 77Se (76.3 MHz, DMSO), δ 445.26 ppm, IR 1643, 1587, 1509, 1489, 1471, 1455, 1425,
1391, 1314, 1286, 1251, 1237, 1097, 1073, 1045, 1025, 1011 cm-1.
2,2-Diselenobis[N-(4-jodophenyl)benzamide] (19)33 Yield: Method C: 26%, Method D: 46%, mp 260-264 oC, 1H
NMR (700 MHz, DMSO) δ 7.41 (dt, J 1.4, 7.7 Hz, 2Har), 7.46 (dt, J 1.4, 7.7 Hz, 2Har), 7.62 (d, J 8.4 Hz, 4Har), 7.72
13
(d, J 8.4 Hz, 4Har), 7.78 (dd, J 0.7, 7.7 Hz, 2Har), 7.95 (dd, J 0.7, 7.7 Hz, 2Har), 10.63 (s, 2H, 2xNH) ppm; C NMR
(100.6 MHz, DMSO-d6) δ 88.44 (2xCar), 123.18 (4xCHar), 126.98 (2xCHar), 129.16 (2xCHar), 130.75 (2xCHar),
132.49 (2xCar), 132.66 (2xCHar), 134.04 (2xCar), 137.88 (4xCHar), 138.97 (2xCar), 166.89 (2xC=O) ppm; 77Se (76.3
MHz, DMSO-d6), δ 445.49 ppm, IR 3260, 1636, 1597, 1584, 1508, 1456, 1429, 1409, 1391, 1349, 1316, 1232,
1126, 1061, 1027, 1004 cm-1.
2,2-Diselenobis[N-(3,4,5-trimetoxyphenyl)benzamide] (20)49 Yield: Method C: 22%, Method D: 29%, mp 156-
160 oC, 1H NMR (700 MHz, CDCl3) δ 3.87 (s, 6H, 2xOCH3), 3.92 (s, 12H, 4xOCH3), 6.96 (s, 4Har), 7.34 (t, J 7.7 Hz,
2Har), 7.38 (t, J 7.7 Hz, 2Har), 7.67 (d, J 7.0 Hz, 2Har), 7.80 (s, 2H, 2xNH), 7.99 (d, J 7.7 Hz, 2Har) ppm; 13C NMR
(100.6 MHz, CDCl3) δ 57.26 (4xOCH3), 62.00 (2xOCH3), 99.19 (4xCHar), 104.62 (2xCar), 127.54 (2xCHar), 127.76
(2xCHar), 130.38 (2xCar), 132.95 (2xCHar), 133.12 (2xCHar), 134.51 (4xCar), 134.83 (2xCar), 136.33 (2xCar), 154.49
(2xC=O) ppm; 77Se (76.3 MHz, CDCl3), δ = 454.96 ppm, IR 3268, 2933, 2836, 1632, 1600, 1585, 1561, 1526,
1506, 1488, 1450, 1410, 1384, 1311, 1284, 1233, 1199, 1128, 1071, 1045, 1025, 1008 cm-1. Elemental Anal.
Calcd for C32H32N2O8Se2 (730.53): C, 52.61; H, 4.42. Found: C, 52.26; H, 4.38.
Evaluation of in vitro antioxidant activity45
To a solution of compounds 1, 2, 9-20 (0.015 mmol) and dithiothreitole DTTred (0.15 mmol) in 1.1mL of CD3OD
30% H2O2 (0.15 mmol) was added. 1H NMR spectra were measuared right after addition of hydrogen peroxide
and then in specific time intervals. The concentration of the substrate was determined according to the
changes in the integration on the 1H NMR spectra (Table 2).
SRB viability assay50
Cell culture: The prostate cancer cell line DU-145 was purchased from the American Type Culture Collection
(ATTC, Manassas, VA). The DU-145 cells were cultured in MEME medium supplemented with 10% fetal bovine
o
serum, 1% penicillin/streptomycin, 2 mM glutamine and 1 mM sodium pyruvate at 37 C. The PNT1A cells
were cultured in RPMI 1640 supplemented with serum, L-glutamine and antibiotics. The cells were maintained
o
at 37 C in an atmosphere containing 5% CO2. Stock solutions of N-substituted ebselen derivatives were
prepared in (0,1%) DMSO.
SRB assay: Cell viability was measured by Sulphorhodamine B (SRB) assay. The cells were grown to sub-
3
confluent levels at the certain 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 triplates and the IC50 values
were calculated.
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Pacuła, A. J. et al.
Acknowledgements
This work was supported by the National Science Centre, Poland, grant no. UMO-2015/17 /B/ NZ7/03058.
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