New chiral ebselen analogues with antioxidant and cytotoxic potential

Article abstract of DOI:10.3390/molecules22030492

New chiral camphane-derived benzisoselenazol-3(2H)-ones and corresponding diselenides have been synthetized using a convenient one-pot procedure. Se-N bond was efficiently converted to an Se-Se bond, which could also be easily re-oxidized to the initial benzisoselenazolone moiety. The antioxidant activity of camphor derivatives was evaluated and compared to the reactivity of a series of N-amino acid benzisoselenazol-3(2H)-ones obtained by a modified procedure involving the improved synthesis and isolation of the diseleno bis(dibenzoic) acid. The most efficient peroxide scavengers, N-bornyl and N-leucine methyl ester benzisoselenazol-3(2H)-ones, were further evaluated as cytotoxic agents on four cancer cell lines (MCF-7, HEP G2, HL 6, and DU 145) and normal cell line PNT1A. The highest antiproliferative potential was evaluated for two compounds bearing a 3-methylbutyl carbon chain, N-leucine methyl ester and N-3-methylbutyl benzisoselenazol-3(2H)-ones.

Full text of DOI:10.3390/molecules22030492

molecules
Article
New Chiral Ebselen Analogues with Antioxidant and
Cytotoxic Potential
Agata J. Pacuła , Katarzyna B. Kaczor , J e˛ drzej Antosiewicz 2,5, Anna Janecka 3,
1
2
3
4
1
´
1,
Angelika Długosz , Tomasz Janecki , Andrzej Wojtczak and Jacek Scianowski *
1
Department of Organic Chemistry, Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7,
8
7-100 Torun, Poland; pacula@doktorant.umk.pl (A.J.P.); awojt@umk.pl (A.W.)
Department of Bioenergetics and Physiology of Exercise, Medical University of Gdansk, Debinki 1,
0-211 Gdansk, Poland; katarzyna.kaczor@gumed.edu.pl (K.B.K.); jant@gumed.edu.pl (J.A.)
Department of Biomolecular Chemistry, Faculty of Medicine, Medical University of Lodz, Mazowiecka 6/8,
2-215 Lodz, Poland; anna.janecka@umed.lodz.pl (A.J.); angelika.dlugosz@stud.umed.lodz.pl (A.D.)
2
3
4
5
8
9
Institute of Organic Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland;
tomasz.janecki@p.lodz.pl
Department of Biochemistry, Gdansk University of Physical Education and Sport, Kazimierza Gorskiego 1,
8
0-336 Gdansk, Poland
*
Correspondence: jsch@chem.umk.pl; Tel.: +48-566-114-532
Academic Editors: Claudio Santi, Luana Bagnoli and Roman Dembinski
Received: 30 January 2017; Accepted: 17 March 2017; Published: 20 March 2017
Abstract: New chiral camphane-derived benzisoselenazol-3(2H)-ones and corresponding diselenides
have been synthetized using a convenient one-pot procedure. Se-N bond was efficiently converted
to an Se-Se bond, which could also be easily re-oxidized to the initial benzisoselenazolone moiety.
The antioxidant activity of camphor derivatives was evaluated and compared to the reactivity
of a series of N-amino acid benzisoselenazol-3(2H)-ones obtained by a modified procedure
involving the improved synthesis and isolation of the diseleno bis(dibenzoic) acid. The most
efficient peroxide scavengers, N-bornyl and N-leucine methyl ester benzisoselenazol-3(2H)-ones,
were further evaluated as cytotoxic agents on four cancer cell lines (MCF-7, HEP G2, HL 6,
and DU 145) and normal cell line PNT1A. The highest antiproliferative potential was evaluated for
two compounds bearing a 3-methylbutyl carbon chain, N-leucine methyl ester and N-3-methylbutyl
benzisoselenazol-3(2H)-ones.
Keywords: organoselenium compounds; terpenes; antioxidant activity; biological activity
1
. Introduction
Since it was proven that selenium is an essential micronutrient mainly associated with the antioxidant
defense system of the living cell, the discovery of ebselen (N-phenylbenzisoselenazol-3(2H)-one)
1 has
been considered a milestone in this constantly developing field of research [ ]. This organoselenium
1
compound is able to act as selenocysteine (Sec), thus mimicking the activity of glutathione
peroxidase (GPx). Sec is the key element of the enzymes active site that provides GPx with the unique
ability to reduce hydrogen peroxide and other reactive oxygen and nitrogen species [
property makes ebselen a potent bioactive molecule which is effective in the treatment of various
diseases including diabetes [ ], Alzheimer’s disease [ ], stroke [ ], hearing loss [ ], and cancer [10].
It is also non-toxic, as the selenium atom incorporated in its structure is not bioavailable [11].
The mechanism of peroxides reduction by ebselen is controversial and several activity pathways
have been proposed. It was postulated that the elimination of peroxide is performed by a specific active
species, formed from ebselen . According to different assays, can be transformed into selenol 12
2]. This unique
3
–6
7
8
9
1
1
1
2
[
]
Molecules 2017, 22, 492; doi:10.3390/molecules22030492
www.mdpi.com/journal/molecules

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or diselenide
3
[13], which can reduce hydrogen peroxide forming selenic acid
4
. Elimination of water
from acid 4 regenerates ebselen 1 (Scheme 1).
Scheme 1. Probable catalytic pathways for the reduction of H O by ebselen.
2
2
In both cases, the rate determining the step of the cycle is the cleavage of the Se-S bond, in the
selenenylsulfide and the formation of the active organoselenium species or . Our recent studies
5
2
3
confirmed this assumption, showing that ebselen derivatives undergo facile Se-N bond cleavage and
rapidly form the corresponding diselenides, exhibiting high antioxidant potential [14]. To further
examine this issue, we synthesized a series of new chiral ebselen derivatives, transformed them to
corresponding diselenides, and compared their antioxidant activity. It seems probable that the direct
application of a diselenide could increase the rate of peroxide reduction.
So far, the synthesis of N-phenylbenzisoselenazol-3(2H)-one
a multi-step synthesis based on the formation of 2,2-diselenobis(benzoic acid)
conversion to corresponding dichloride with thionyl chloride and further reaction with aniline
Method A) [15] or o-metalation of benzamide , selenium insertion, and oxidative cyclization promoted
by copper bromide (Method B) [16]. Several methods in which N-phenyl-o-iodobenzamide is used
as a starting material are described. Ebselen was obtained by treatment with a copper catalyst and
1
has been accomplished by
6, followed by the
7
(
8
9
1
selenium in the presence of potassium carbonate as a base (Method C) [17], a nucleophilic reagent
formed from selenium and potassium tert-butoxide (Method D) [18] or by the methodology presented
in our previous paper, in which lithium diselenide, formed in the reaction of lithium hydroxide and
elemental selenium in the presence of hydrazine hydrate, is applied (Method E) [19] (Scheme 2).
Scheme 2. Methodologies to synthesize ebselen 1.

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Although various benzisoselenazolones have been synthetized and tested as bio-molecules,
there are only few examples of chiral ebselen derivatives [18 20 22]. The aim of our research
,
–
was to fill this gap and combine the benzisoselenazolone structure with a chiral terpene scaffold.
Terpenes possess several significant properties. As natural products abundant in plants, they provide
a broad spectrum of structurally diverse motifs and were used not only as fragrance or taste carriers
but also as pharmacologically potent agents [23]. In our research group, we efficiently used terpenes to
obtain a broad spectrum of various chiral organoselenium derivatives including selenols, selenides,
and diselenides [24–31].
Herein, we report the synthesis of benzisoselenazolones and corresponding diselenides substituted
on the nitrogen atom with monoterpene skeletons from camphane group. We also tested the obtained
compounds for their antioxidant and anticancer activity. Obtained results were additionally compared
to the activity of a series of other chiral, naturally derived benzisoselenazolones bearing amino
acid substituents.
2
. Results and Discussion
In the conducted research, we planned to use and compare the efficiency of two metal-free
procedures (method D and E) in the synthesis of new camphane-derived benzisoselenazolones and
corresponding diselenides. The first step included the synthesis of N-terpenyl-o-iodobenzamides 15
and 16 used as substrates. R(
procedure consisting of formation of corresponding oxime 10, its conversion to oxime methyl ether 11
−
)-Isobornylamine 12 was obtained from camphor by a three-step
,
and reduction of 11 with lithium aluminium hydride to amine 12. During the reduction of oxime methyl
ether, we observed the formation of isobornyl amine 12 and bornyl amine 13 (64:36). Isobornyl amine
1
1
2
6
was separated by column chromatography (silica gel, dichloromethane). Iodobenzamides 15 and
were obtained from the reaction of acid chloride 14 with previously synthetized amine 12 and
commercially available R(+)-bornylamnie 13 (Scheme 3).
Scheme 3. Synthesis of N-isobornyl and N-bornyl-o-iodobenzamides 15 and 16.
In the next step, we decided to obtain benzisoselenazolones by the reaction of amides 15 and 16 with
nucleophilic reagent formed in situ from elemental selenium and potassium tert-butoxide (Method D).
Unfortunately, corresponding diselenides 17 and 18 were obtained in low yields (Scheme 4).

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Scheme 4. Synthesis of diselenides 17 and 18.
These results prompted us to modify the procedure and design a new methodology in which
the nucleophile was changed to lithium diselenide, formed from lithium hydroxide and elemental
selenium in the presence of hydrazine hydrate. To our delight, this modification gave expected
benzisoselenazolones 19 and 20 in high yields. The structure of compound 20 was additionally
confirmed by an X-ray analysis (Scheme 5).
Scheme 5. Synthesis of N-isobornyl and N-bornyl benzisoselenazol-3(2H)-ones 19 and 20.
The absolute configuration of compound 20, determined by the Flack method [32] is (1R,2S,4R).
The bulk of the N-bornyl moiety seems to be the largest among the chiral benzisoselenazolones reported
so far [13,15]. Two bonds formed by Se1 are almost identical, with the Se1-N1 and Se1-C17 distances
being 1.883(2) and 1.887(2) Å, respectively and the N1-Se1-C17 angle is 85.89(10) deg. These values
are similar to those found for other chiral derivatives. However, a search in the Cambridge Structural
Database [33] revealed that N-Se-C angle values range from 77.05 to 90.39 deg, with the smaller
angles for compounds having the additional substituent at Se atom. Comparison revealed that Se-N
and Se-C bond distances reported here were significantly longer than those Se-C 1.868(4) and Se-N
1
.870(5) Å reported for the analog containing two ebselen moieties linked by ethylene bridge [34]. In that
structure, the C-Se-N angle of 84.97(18) is significantly smaller than that in the structure reported here.
In selenoxide, analog of ebselen [35], the Se-N and Se-C bonds of 1.899(3) and 1.928(3) Å, respectively,
are longer than those found in compound 20 reported here. Also, the N-Se-C angle 84.68(13) deg
was significantly smaller than that in 20. In the structure of ebselen with no substituent at N [36],

Molecules 2017, 22, 492
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the significant difference up to 0.039 Å was reported for Se-N and Se-C distances, and the C-Se-N angle
was significantly smaller than in the structure reported here. However, geometry of N-Se-C fragment
in compound containing N-Met [37] was almost identical with that reported here. That might result
from the steric and electronic effect of Se=O bond in selenoxide. Contrary to this, the values of Se-C
1
.856(2), Se1-N 1.916(2) and C-Se-N 84.33(10) reported for of ebselen [38] differed significantly from
those in the structure reported here. The comparison given above indicates that the electronic effect
and not only the bulk or nature of N-substituent affect the ebselen geometry in a significant manner.
Geometry of the bornyl moiety is typical for such systems. Conformation of the molecule is described
with Se1-N1-C2-C1 and Se1-N1-C2-C3 torsion angles being 93.9(2) and
−
27.7(3) deg, respectively.
See detailed information about NMR spectra and crystallographic data in Supplementary Materials.
Benzisoselenazolones 19 and 20 were further quantitatively converted to corresponding
diselenides 17 and 18 using sodium borohydride. On the other hand, the Se-N bond could be efficiently
regenerated in the reaction with potassium iodate used as an oxidant (Scheme 6).
Scheme 6. Cleavage and regeneration of Se-N bond.
To compare the biological activity of the synthetized compounds to other chiral ebselen analogues,
a series of amino acid derivatives was obtained and similarly evaluated. To achieve this goal, several
amino acid methyl esters were converted into N-substituted-o-iodobenzamides. Conversion to the
final organoselenium derivatives using the methodology developed in our research group (Method E)
was unsuccessful due to the hydrolysis of the ester moiety. As procedures involving cyclization
of iodobenzamides using nucleophilic selenium reagents could not be used, we focused on the
commonly used multistep Method A. During this investigation, we developed a revised version of
this method, i.e., a convenient procedure to obtain the 2,2-diselenobis(benzoic acid)
6
. Synthesis of
6
significantly limits the overall yield of the reaction, thus improving the efficiency of this step
highly upgrades the methodology. Acid 6 is usually prepared from the diazonium salt of anthranilic
acid followed by substitution with sodium diselenide. The yield of the reaction depends on the
procedure by which sodium diselenide is formed and by the efficiency of the final product isolation.
Sodium diselenide can be prepared using different reagents, elemental selenium and sodium hydroxide
in the presence of hydrazine in methanol [39], with addition of rongalite in water [40] or from elemental

Molecules 2017, 22, 492
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selenium and sodium borohydride in water, with or without NaOH [41
,42]. We turned our attention
to the last procedure, in which sodium diselenide is synthesized from Se and NaBH in NaOHaq
.
4
By changing the reaction conditions, i.e., lower temperature and longer reaction time, we were able to
obtain pure Na Se , eliminating the common formation of Na Se. Next, we optimized the purification
2
2
2
procedure of the final product. Synthesized 2,2-diselenobis(benzoic acid)
6 is often contaminated with
salicylic acid which is hard to separate. Multiple rinsing of the crude product with boiling water
enabled to obtain the final product as pure solid in 90% yield.
The revised procedure (Method A) was used to synthesize N-substituted benzisoselenazol-3(2H)-ones
derived from glycine 21, L-alanine 22, L-leucine 23, L-phenylalanine 24 and β-alanine 25 [22] (Scheme 7).
O
O
O
O
1
) NaNO , HCl
2
OH 2) Na2Se2
SOCl2
OH
Cl
SeCl
7, (98%)
R-NH2
N
Se
R
NH2
Se)2
6
, (90%)
O
O
R =
O
O
O
OCH3
OCH3
OCH3
OH
OCH3
4, (94%)
2
1, (71%)
22, (57%)
23, (85%)
2
25
, (81%)
Scheme 7. Synthesis of amino acid derivatives 21–25.
All obtained terpene and amino acid derivatives were tested as antioxidants using NMR activity
assay proposed by Iwaoka and co-workers. The ability to eliminate hydrogen peroxide was equal to
1
the rate of the oxidation of dithiol to disulfide. Formation of the product was observed by H-NMR
spectra performed in selected time intervals [43]. The results are presented in Table 1.
Table 1. Results of the antioxidant activity measurement.
Remaining Dithiotreitol (%)
Catalyst
3 min
5 min
15 min
30 min
60 min
1
1
1
2
2
2
2
2
2
2
7
8
9
0
1
2
3
4
5
6
59
48
50
30
99
96
45
96
94
77
84
89
36
38
17
11
99
95
0
95
93
58
75
83
12
28
0
0
25
0
0
22
0
0
0
0
98
93
0
94
92
42
64
74
97
91
0
93
92
28
58
68
95
89
0
90
91
13
52
63
Ebselen (1)
Diselenide (3)
The highest reactivity was found for N-bornyl 20 and N-leucine methyl ester 23
red
ox
benzisoselenazol-3(2H)-ones. Total conversion of DTT to DTT (DDT-Dithiotreitol) was observed

Molecules 2017, 22, 492
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after 5 and 3 min, respectively. Other terpene derivatives 17
–
19 exhibited much lower antioxidant
potential, and no antioxidant potential was observed for other amino acid benzisoselenazolones.
This result led us to the conclusion that the structure of leucine, bearing a 2-methylpropyl chain on
the α-carbon, could be the reason of this high reactivity. Thus, for further anticancer activity assays,
we chose to evaluate the cytotoxic potential of compounds 20 and 23, for which the best results were
obtained in the NMR activity test.
Additionally, we decided to test the anticancer activity of N-3-methylbutyl benzisoselenazol-3(2H)-one
2
6
presented in our previous paper [19], bearing a carbon chain analogous to the one present
in the structure of leucine. The cytotoxic activity was evaluated on four cancer cell lines: MCF-7
(
(
breast carcinoma), HEP G2 (liver cancer), HL60 (human promyelocytic leukemia), DU 145
prostate cancer), and non-cancerous cell line PNT1A. The results are collected in Table 2.
Table 2. Results of the cytotoxic activity assays.
MCF-7
MTT)
HEP G2
(MTT)
HL60
(MTT)
DU-145
(SRB)
PNT1A
(SRB)
Comp.
Structure
(
IC , µM
5
0
2
0
220 ± 34
232 ± 28
190 ± 20
>40
>40
>40
>40
2
3
6
18 ± 1.1
29 ± 1.9
23.5 ± 3.2
100 ± 12
21.5 ± 1.8
43.5 ± 5.0
2
30.6 ± 0.2
30.35 ± 0.06
Control
Carboplatin
0.70 ± 0.30 3.19 ± 0.46 9.70 ± 1.20
-----
-----
The highest antiproliferative activity was observed for N-leucine methyl ester derivative 23
against MCF-7, HEP G2, and HL60 cells. This compound also exhibited the best antioxidant potential.
Its activity against MCF-7, HEP G2, and HL60 was relatively good and was compared with the data
obtained for carboplatin. Structurally similar N-3-methylbutyl benzisoselenazol-3(2H)-one 26 was also
active against all three cancer cell lines and had low cytotoxicity in HEP G2 cell line, on the contrary to
data obtained on chiral N-bornyl derivative 20, revealing that the presence of α-carbon 2-methylpropyl
chain could be a structural motif providing the bio-activity.
3
. Experimental
3
.1. General
1H-NMR spectra were obtained at 400 or 700 MHz and chemical shifts were recorded relative
to SiMe ( 0.00) or solvent resonance (CDCl3 7.26, CD OD 3.31). Multiplicities were given as
δ
δ
δ
4
3
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
δ 77.25). NMR spectra were
carried out using ACD/NMR Processor Academic Edition (Advanced Chemistry Development,
Toronto, ON, Canada). Original NMR spectra and crystallographic data are included in SM. Commercially
available solvents DMF (N,N-dimethylformamide), DCM (dichloromethane), and MeOH (Sigma Aldrich,

Molecules 2017, 22, 492
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St. Louis, MO, USA), and chemicals were used without further purification. Column chromatography
was performed using Merck 40-63D 60 Å silica gel (Merck, Darmstadt, Germany).
3
.2. Procedures and Analysis Data
3
.2.1. (1R)-Camphor Oxime (10)
To a solution of hydroxylamine hydrochloride (0.164 mol) and sodium acetate (0.197 mol) in water
(
6
100 mL), (+)-camphor (0.131 mol) dissolved in ethanol (65 mL) was added. The mixture was stirred at
0 C for 20 h. Ethanol was evaporated and the precipitated oxime filtrated under reduce pressure.
◦
The product was used without further purification [44].
1
Yield: 88%, H-NMR (400 MHz, CDCl )
δ = 0.82 (s, 3H, CH ), 0.94 (s, 3H, CH ), 1.02 (s, 3H, CH ),
3 3 3
3
1.20–1.30 (m, 1H), 1.43–1.52 (m, 1H), 1.72 (td, J = 4.0, 12.4 Hz, 1H), 1.80–1.91 (m, 1H), 1.93 (t, J = 4, 4 Hz, 1H),
2
.07 (d, J = 18.0 Hz, 1H), 2.57 (dt, J = 4.4, 18.0 Hz, 1H) ppm.
3
.2.2. (1R)-Camphor O-methyloxime (11)
To a suspension of sodium hydride (0.183 mol) in anhydrous petroleum ether a solution of
1R)-Camphor oxime 10 (0.093 mol) in anhydrous THF (90 mL) was added portionwise. The mixture
(
was stirred for 0.5 h at room temperature and methyl iodide (0.111 mol) was added. The mixture
was stirred for additional 20 h. THF was evaporated, and the formed precipitate was washed with
water (50 mL) and extracted with petroleum ether (2
dried over anhydrous magnesium sulphate and evaporated. The product was used without further
purification [45].
×
50 mL). Combined organic layers were
1
Yield: 75%, H-NMR (700 MHz, CDCl )
δ = 0.82 (s, 3H, CH ), 0.94 (s, 3H, CH ), 1.04 (s, 3H, CH ),
3 3 3
3
1.20–1.30 (m, 1H), 1.43–1.52 (m, 1H), 1.72 (td, J = 4.0, 12.4 Hz, 1H), 1.82–1.88 (m, 1H), 1.90 (t, J = 4.2 Hz, 1H),
2
.07 (d, J = 18.2 Hz, 1H), 2.49 (dt, J = 7.7, 17.5 Hz, 1H), 3.85 (s, OCH , 3H) ppm.
3
3
.2.3. (R)-(−)-Isobornylamine (12)
To a suspension of LiAlH (0.066 mol) in anhydrous THF (50 mL) a solution of (1R)-camphor
4
O-methyloxime 11 (0.33 mol) in THF (25 mL) was slowly added. The mixture was stirred under
reflux for 24 h. After cooling to room temperature H O (2 mL), 10% NaOH (3 mL) and H O (6 mL)
2
2
were added dropwise. The formed suspension was extracted with diethyl ether. Combined organic
layers were dried and evaporated under reduce pressure. The crude product was isolated by column
chromatography (silica gel, DCM).
1
Yield: 30%, H-NMR (700 MHz, CDCl )
δ = 0.84 (s, 3H, CH ), 0.89 (s, 3H, CH ), 0.99 (s, 3H, CH ),
3 3 3
3
1
2
.00–1.05 (m, 1H), 1.08–1.12 (m, 1H), 1.52–1.59 (m, 4H), 1.67–1.72 (m, 2H), 1.77 (dd, J = 8.4, 12.6 Hz, 1H),
.74 (dd, J = 4.9, 9.1 Hz, 1H) ppm.
3
.2.4. General Procedure for the Synthesis of Compounds 15 and 16
To a solution of an amine (1.0 mmol) in DCM (2 mL), 4.4 mL of 2% NaOH was added. The mixture
◦
was cooled to 0 C and o-iodobenzoic acid chloride (1.1 mmol) dissolved in DCM (3 mL) was
added dropwise. After stirring at room temperature for 20 h, the product was extracted with DCM,
and combined organic layers were washed with 10% NaHCO and dried over magnesium sulphate.
3
The solvent was removed under reduce pressure to obtain the product as white solid.
N-Isobornyl-o-iodobenzamide (15). Yield: 92%, [α]25
δ
=
−
73.0 (c 1, CHCl ), H-NMR (400 MHz, CDCl )
1
D
3
3
= 0.83 (s, 3H, CH ), 0.90 (s, 3H, CH ), 0.95 (s, 3H, CH ), 1.14–1.22 (m, 1H), 1.31–1.38 (m, 1H),
3 3 3
1.55–1.65 (m, 1H), 1.65–1.77 (m, 3H), 1.93 (dd, J = 8.4, 12.0 Hz, 1H), 4.07 (dt, J = 4.8, 8.8 Hz, 1H),
5
.75 (d, J = 8.0 Hz, 1H, NH), 7.01–7.09 (m, 1Har), 7.30–7.35 (m, 2Har), 7.80 (d, J = 7.6 Hz, 1Har) ppm;
1
3
C-NMR (100.6 Hz, CDCl3)
δ = 12.2 (CH ), 20.2 (CH ), 20.5 (CH ), 27.0 (CH ), 36.0 (CH ), 38.9 (CH ),
3 3 3 2 2 2

Molecules 2017, 22, 492
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4
4.9 (CH), 47.2 (C), 48.8 (C), 57.4 (CH), 92.3 (Car), 128.2 (CHar), 128.3 (CHar), 130.9 (CHar), 139.9 (CHar),
1
42.6 (Car), 168.7 (C=O) ppm; Elemental Anal. Calcd for C H INO (383.267): C, 53.27; H, 5.79.
17
22
Found: C, 53.08; H, 5.81.
◦
25
D
1
N-Bornyl-o-iodobenzamide (16). Yield: 95%, m.p. 119–121 C, [α] = +21.5 (c 1, CHCl ), H-NMR
3
(
700 MHz, CDCl3) δ = 0.90 (s, 3H, CH ), 0.97 (s, 3H, CH ), 1.00 (s, 3H, CH ), 1.17–1.22 (m, 1H),
3 3 3
1
5
7
2
.42–1.47 (m, 1H), 1.54–1.60 (m, 3H), 1.71 (t, J = 4.2 Hz, 1H), 1.78–1.83 (m, 1H), 4.42–4.46 (m, 1H),
.75 (d, J = 7.0 Hz, 1H, NH), 7.10 (dt, J = 2.1, 7.7 Hz, 1H, 1Har), 7.37–7.42 (m, 2Har), 7.87 (dd, J = 0.7
.7 Hz, 1Har) ppm; 13C-NMR (100.6 Hz, CDCl3)
= 13.9 (CH ), 18.7 (CH ), 19.8 (CH ), 28.1 (CH ),
8.3 (CH ), 37.4 (CH ), 44.9 (CH), 48.3 (C), 49.6 (C), 54.6 (CH), 92.3 (Car), 128.1 (CHar), 128.4 (CHar),
,
δ
3
3
3
2
2
2
1
30.9 (CHar), 139.8 (CHar), 142.9 (Car), 169.9 (C=O) ppm; Elemental Anal. Calcd for C H INO
17 22
(383.267): C, 53.27; H, 5.79. Found: C, 53.13; H, 5.85.
3
.2.5. General Procedure for the Synthesis of Compounds 16 and 17
To a solution of benzisoselenazolone 19 and 20 (1.0 mmol) in methanol (10 mL) cooled to 0 C,
◦
sodium borohydride (1.0 mmol) was added and the mixture was stirred for 1 h. Water (15 mL) was added
and the mixture was oxidized with air for 1 h. The formed precipitate was filtered and dried in air.
0
◦
25
D
2
,2 -Diselenobis(N-isobornylbenzamide) (17). Yield: 90%, m.p. 148–150 C, [α]
=
−
0.9 (c 1, CHCl3),
CH3),
.20–1.27 (m, 2H), 1.37–1.43 (m, 2H), 1.61–1.81 (m, 6H), 1.83 (t, J = 4.4 Hz, 2H), 2.01 (dd, J = 9.2, 13.2 Hz,
1
H-NMR (400 MHz, CDCl3)
δ
= 0.89 (s, 6H, 2
×
CH ), 0.98 (s, 6H, 2
×
CH ), 1.04 (s, 6H, 2
×
3
3
1
2
7
H), 4.17 (dt, J = 5.2, 8.8 Hz, 2H), 6.14 (d, J = 8.8 Hz, 2H, 2
×
NH), 7.22 (dt, J = 1.2, 7.2 Hz, 2Har),
.28 (dt, J = 1.6, 7.2 Hz, 2Har), 7.41 (dd, J = 1.6, 7.6 Hz, 2Har), 7.91 (dd, J = 1.2, 8.0 Hz, 2Har) ppm;
13
C-NMR (100.6 Hz, CDCl3)
δ
= 11.8 (2
CH ), 44.9 (2 CH), 47.2 (2
CHar), 131.6 (2 CHar), 133.0 (2
×
CH ), 20.2 (2
×
CH ), 20.3 (2
×
CH ), 27.0 (2× CH )
,
3
3
3
2
3
5.8 (2× CH ), 39.0 (2
×
×
×
C), 48.9 (2
×
C), 57.4 (2
×
CH), 126.0 (2
×
Car),
C=O) ppm;
2
2
1
26.1 (2
×
CHar), 131.4 (2
×
×
×
C
ar), 133.5 (2
×
C
ar), 167.4 (2
×
77
Se (76.3 MHz, DMSO),
δ
= 450.58 ppm; Elemental Anal. Calcd for C H N O Se (670.645): C, 60.89;
34
44
2
2
2
H, 6.61. Found: C, 60.52; H, 6.55.
0
◦
25
D
2
,2 -Diselenobis(N-bornylbenzamide) (18). Yield: 91%, m.p. 208–210 C, [α]
=
−
80.0 (c 1, CHCl3),
CH3),
1
H-NMR (700 MHz, CDCl3)
δ
= 0.94 (s, 6H, 2
×
CH ), 1.00 (s, 6H, 2
×
CH ), 1.09 (s, 6H, 2
×
3
3
1
2
5
δ
4
.41–1.47 (m, 6H), 1.55–1.59 (m, 2H), 1.81 (t, J = 4.2 Hz, 2H), 1.92–1.95 (m, 2H), 2.00–2.05 (m, 2H),
.49–2.55 (m, 2H), 5.06 (ddd, J = 2.1, 4.9, Hz, 2H, 2 NH), 7.44 (dt, J = 0.7, 7.0 Hz, 2Har), 7.60 (dt, J = 1.4
.6 Hz, 2Har), 7.62–7.65 (m, 2Har), 8.06 (dd, J = 0.7, 7.7 Hz, 2Har) ppm; 13C-NMR (100.6 Hz, CDCl3)
= 13.8 (2 CH ), 18.6 (2 CH ), 19.8 (2 CH ), 28.2 (2 CH ), 28.4 (2 CH ), 37.8 (2 CH2),
4.9 (2× CH), 48.3 (2 CHar), 131.4 (2× CHar)
31.6 (2 CHar), 132.8 (2
C=O) ppm; 77Se (76.3 MHz, DMSO),
δ = 450.03 ppm; Elemental Anal. Calcd for C H N O Se (670.645): C, 60.89; H, 6.61. Found: C, 60.63;
×
,
×
×
×
×
×
×
3
3
3
2
2
×
C), 49.8 (2
×
C), 54.5 (2
×
CH), 126.0 (2
ar), 168.3 (2
×
CHar), 126.4 (2
×
,
1
×
×
C
ar), 133.8 (2
×
C
×
3
4
44
2
2
2
H, 6.64.
3
.2.6. General Procedure for the Synthesis of Compounds 19 and 20
Selenium powder (1.2 mmol) and lithium hydroxide (3.6 mmol) were weighed into a single
neck flask under argon atmosphere and dissolved in DMF (3 mL). Hydrazine hydride (8.0 mmol)
◦
was added dropwise, and the mixture was heated to 120 C and stirred for 15 min. After cooling to
room temperature, the amide (1.0 mmol) dissolved in DMF (2 mL) was added. The reaction mixture
◦
was heated to 120 C and stirred for 20 h under argon atmosphere. The solution was cooled to room
temperature and 25 mL of brine was added. The mixture was stirred for 20 h. The obtained precipitate
was filtered under vacume, washed with water, and dried in air. The crude product was purified by
column chromatography (silica gel, DCM:MeOH 0.8:99.2).
◦
25
N-Isobornyl-1,2-benzisoselenazol-3(2H)-one (19). Yield: 92%, m.p. 205–207 C, [α]D
=
−
59.0 (c 1, CHCl3),
1
H-NMR (400 MHz, CDCl3) δ = 0.91 (s, 3H, CH ), 0.93 (s, 3H, CH ), 1.17 (s, 3H, CH ), 1.25–1.34 (m, 2H),
3 3 3

Molecules 2017, 22, 492
10 of 15
1
8
.63–1.71 (m, 1H), 1.80–1.88 (m, 2H), 2.00–2.07 (m, 2H), 4.77 (t, J = 8.4 Hz, 1H), 7.42 (ddd, J = 2.0, 6.0,
.0 Hz, 1Har), 7.55–7.60 (m, 2Har), 8.04 (d, J = 7.6 Hz, 1Har) ppm; 13C-NMR (100.6 Hz, CDCl3)
δ = 11.3 (CH3), 21.2 (CH ), 22.0 (CH ), 26.5 (CH ), 37.1 (CH ), 39.3 (CH ), 45.2 (CH), 46.7 (C),
3
3
2
2
2
5
1.4 (C), 63.3 (CH), 123.2 (CHar), 126.0 (CHar), 128.5 (Car), 128.7 (CHar), 131.7 (CHar), 138.2 (Car),
7
7Se (76.3 MHz, CDCl3),
1
68.6 (C=O) ppm
;
δ
= 866.07 ppm; Elemental Anal. Calcd for C H NOSe
17
21
(334.315): C, 61.08; H, 6.33. Found: C, 61.29; H, 6.40.
◦
25
N-Bornyl-1,2-benzisoselenazol-3(2H)-one (20). Yield: 90%, m.p. 228–230 C, [α]D
=
−
64.3 (c 0.7, CHCl3),
1
H-NMR (700 MHz, CDCl3)
.78 (t, J = 4.2 Hz, 1H), 1.88–1.93 (m, 1H), 1.97–2.01 (m, 1H), 2.45–2.50 (m, 1H), 5.03 (ddd, J = 2.1
.9, 11.9 Hz, 1H), 7.41 (dt, J = 1.4, 7,7 Hz, 1Har), 7.57 (dt, J = 1.4, 7,0 Hz, 1Har), 7.60–7.63 (m, 1H),
.02 (d, J = 7.7 Hz, 1Har) ppm; 13C-NMR (100.6 Hz, CDCl3)
= 14.2 (CH ), 18.9 (CH ), 19.9 (CH ),
8.3 (CH ), 28.6 (CH ), 38.1 (CH ), 45.2 (CH), 48.4 (C), 52.1 (C), 60.1 (CH), 123.2 (CHar), 126.0 (CHar),
δ = 0.91 (s, 3H, CH ), 0.96 (s, 3H, CH ), 1.06 (s, 3H, CH ), 1.38–1.44 (m, 3H),
3 3 3
1
,
4
8
2
δ
3
3
3
2
2
2
1
28.3 (Car), 128.7 (CHar), 131.7 (CHar), 138.0 (Car), 168.3 (C=O) ppm; 77Se (76.3 MHz, CDCl3),
δ
= 877.38 ppm;
Elemental Anal. Calcd for C H NOSe (334.315): C, 61.08; H, 6.33. Found: C, 61.24; H, 6.31.
17
21
3
.2.7. Conversion of Diselenides to Benzisoselenazolones
To a solution of diselenide 17 and 18 (1.0 mmol) in DMF (3 mL), under argon atmosphere,
◦
potassium iodate (1.0 mmol) was added and the mixture was stirred at 120 C for 24 h. The mixture
was cooled to ambient temperature and brine (5 mL) was added. Mixture was stirred for 3 h and the
formed precipitate was filtered and dried in air. Yield: 83% (19), 86% (20).
3
.2.8. Synthesis of 2,2-Diselenobis(benzoic acid) 6 and 2-(Chloroseleno)benzoyl Chloride 7
To a suspension of selenium (12.66 mmol) in water (7 mL) sodium borohydride (25.32 mmol)
◦
was added under argon atmosphere. Mixture was stirred for 0.5 h at 0 C, selenium (12.66 mmol)
was added, and stirring was continued at the same temperature for 1 h. Mixture was warmed to
room temperature and stirred for 18 h. After adding 10% NaOH (5 mL) the reaction was cooled
◦
to 5 C and the diazonium salt of anthranilic acid was added dropwise. (The diazonium salt was
prepared in advance—to a solution of anthranilic acid (25.96 mmol) and concentrated hydrochloric
◦
acid (5 mL) in water (15 mL) cooled to 5 C a cooled solution of sodium nitrite (27.54 mmol) in
◦
water (15 mL) was 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 filtred off and
◦
the solution was acidified to pH = 1 by 36% HCl. The formed precipitate was filtrated. The crude
product
6
was purified by washing with boiling water and dried in air. Acid 6 (0.01 mmol) was further
◦
converted to 2-(chloroseleno)benzoyl chloride
7
by heating 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.
1
2,2-Diselenobis(benzoic acid) (
6
). Yield: 90%, H-NMR (700.27 MHz, DMSO)
δ
= 7.35–7.37 (m, 2Har),
7
.48–7.50 (m, 2Har), 7.67 (dd, J = 8.4, 0.7, 2H, 2
×
CHar), 8.03 (dd, J = 7.7, 1.4, 2H, 2
×
CHar),
CHar),
1
3.72 (s, 2H, 2
×
OH); 13C-NMR (100.6 Hz, DMSO)
δ
= 126.9 (2
×
CHar), 129.4 (2 ar), 129.9 (2
×
C
×
131.9 (2 CHar), 133.93 (2
×
×
C
ar), 133.97 (2
×
CHar), 169.0 (2
×
C=O) ppm; 77Se (76.3 MHz, DMSO),
δ = 441.09 ppm.
1
2
7
(
1
-(Chloroseleno)benzoyl chloride (
7
). Yield 98%; H-NMR (400.13 MHz, CDCl )
δ
= 7.50–7.54 (m, 1Har),
3
13
.80–7.84 (m, 1Har), 8.12 (dd, J = 8.4, 0.8, 1H, CHar), 8.37 (dd, J = 8.0, 1.2, 1H, CHar); C-NMR
CDCl , 100.62 MHz) = 126.7 (CHar), 127.1 (CHar), 129.0 (Car), 134.5 (CHar), 136.2 (CHar), 146.3 (Car),
72.6 (Car); Se-NMR (CDCl , 76.35 MHz) δ = 1060.46 ppm.
δ
3
77
3
3
.2.9. General Procedure for the Synthesis of Compounds 21–25
To a solution of amine (2.0 mmol) and triethylamine (4.0 mmol) in dichloromethane
-(chloroseleno)benzoyl chloride (1.0 mmol) was added. The mixture was stirred for 24 h at room
2

Molecules 2017, 22, 492
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temperature, poured on water and extracted with DCM. The combined organic layers were dried over
anhydrous magnesium sulfate and evaporated [22].
1
N-(Glycine methyl ester)-benzisoselenazol-3(2H)-one (21). Yield 68%; H-NMR (400.13 MHz, CDCl )
3
δ = 3.80–3.81 (m, 3H), 4.61–4.62 (d, J = 4.0, 2H), 7.43–7.45 (m, 1Har), 7.61–7.68 (m, 2Har), 8.06–8.08 (m, 1Har);
1
3C-NMR (176.10 MHz, CDCl3)
28.6 (CHar), 132.0 (CHar), 138.5 (Car), 167.4 (C=O), 168.7 (C=O); Se-NMR (76.35 MHz, CDCl3)
δ = 45.0 (CH ), 52.2 (-OCH ), 123.6 (CHar), 125.6 (Car), 125.9 (CHar),
2 3
77
1
δ = 937.49 ppm.
1
N-(Alanine methyl ester)-benzisoselenazol-3(2H)-one (22). Yield; 84%; H-NMR (700.27 MHz, CDCl )
3
δ = 1.58 (d, J = 7.0, 3H, CH ), 3.74 (s, 3H, -OCH ), 5.42 (q, J = 7.0, 1H, CH), 7.40–7.42 (m, 1Har), 7.58–7.60
3
3
13
(
m, 1Har), 7.64–7.65 (m, 1Har), 8.05 (d, J = 7.7, 1H, CHar); C-NMR (100.62 MHz, CDCl3) δ = 18.7 (CH3)
,
5
1.6 (-OCH ), 52.7 (CH), 123.8 (CHar), 126.2 (CHar), 126.9 (Car), 128.8 (CHar), 132.2 (CHar), 139.2 (Car),
3
77
1
67.5 (C=O), 172.2 (C=O); Se-NMR (76.35 MHz, CDCl ) δ = 888.79 ppm.
3
1
N-(Leucine methyl ester)-benzisoselenazol-3(2H)-one (23). Yield 83%; H-NMR (700.27 MHz, CDCl )
3
δ = 0.96 (d, J = 6.3, 3H, CH ) 0.97 (d, J = 7, 3H, CH ), 1.53–1.59 (m, 1H), 1.75–1.85 (m, 2H),
3
3
3
7
.75 (s, 3H, -OCH3), 5.44–5.46 (m, 1H), 7.40–7.42 (m, 1Har), 7.59 (dt, J = 7.0, 1.4, 1H, CHar),
.63 (d, J = 7.7, 1H, CHar), 8.05 (dd, J = 7.7, 0.7, 1H, CHar); C-NMR (176.10 MHz, CDCl3)
13
δ = 22.6 (CH ), 23.9 (CH ), 25.6 (CH), 43.2 (CH ), 53.5 (-OCH ), 55.2 (CH), 124.9 (CHar), 127.1 (CHar),
3
3
2
3
77
1
27.9 (Car), 129.9 (CHar), 133.2 (CHar), 140.0 (Car), 168.6 (C=O), 173.1 (C=O); Se-NMR (76.35 MHz,
CDCl ) δ = 886.93 ppm.
3
1
N-(Phenylalanine methyl ester)-benzisoselenazol-3(2H)-one (24). Yield 98%; H-NMR (700.27 MHz, CDCl )
3
δ
= 3.15–3.18 (m, 1H), 3.33–3.36 (m, 1H), 3.70 (s, 3H, -OCH ), 5.62–5.64 (m, 1H), 7.19–7.261 (m, 5H),
3
13
7
.38 (dt, J = 7.0, 0.7, 1H, CHar), 7.56–7.62 (m, 2Har), 8.00 (dd, J = 7.7, 0.7, 1H, CHar); C-NMR
(100.62 MHz, CDCl3)
δ
= 39.1 (CH ), 52.5 (-OCH ), 57.2 (CH), 123.7 (CHar), 126.1 (CHar), 126.6 (Car),
2
3
1
1
27.2 (CHar), 128.6 (2
67.5 (C=O), 171.4 (C=O); Se-NMR (76.35 MHz, CDCl ) δ = 904.16 ppm.
×
CHar), 128.8 (CHar), 129.1 (2
×
CHar), 132.2 (CHar), 135.5 (Car), 139.3 (Car),
77
3
1
N-(Alanine carboxyl acid)-benzisoselenazol-3(2H)-one (25). Yield 63%; H-NMR (400.13 MHz, CDCl )
3
δ = 2.73–2.78 (m, 2H), 4.10 (t, J = 6.4, 2H, CH ), 7.44 (t, J = 7.2, 1H, CHar), 7.60–7.64 (m, 1Har), 7.91–7.94
2
1
3
(
1
m, 2Har); C-NMR (100.62 MHz, CDCl3)
27.2 (Car), 127.3 (CHar), 131.7 (CHar), 168.0 (C=O), 172.1 (C=O); Se-NMR (76.35 MHz, CDCl3)
δ = 922.33 ppm.
δ = 34.0 (CH ), 40.1 (CH ), 124.8 (CHar), 125.7 (CHar),
2 2
77
3
.3. Antioxidant Activity Assay
red
To a solution of compounds and 17
–
25 (0.015 mmol) and dithiothreitole DTT (0.15 mmol) in
1
1
.1 mL of CD OD 30% H O (0.15 mmol) was added. H-NMR spectra were measuared right after
3
2
2
addition of hydrogen peroxide and then in specific time intervals. The concentration of the substrate
1
was determined according to the changes in the integration on the H-NMR spectra [43].
3
.4. Cell Viability Assays
3
.4.1. MTT Viability Assay
Cell Culture
The HL-60, MCF-7, and HepG2 cell lines were purchased from the European Collection of
Cell Cultures (ECACC, Salisbury, UK). HL-60 cells were cultured in RPMI 1640 plus GlutaMax
I medium (Invitrogen, Grand Island, NY, USA), supplemented with 10% heat-inactivated FBS
(
Biological Industries, Beit-Haemek, Israel) and antibiotics (100 µg/mL streptomycin and 100 U/mL
penicillin) (Sigma Aldrich, St. Louis, MO, USA). MCF-7 and HepG2 cells were cultured in
MEME (Sigma–Aldrich, St. Louis, MO, USA), supplemented with 10% FBS (Biological Industries,

Molecules 2017, 22, 492
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Beit-Haemek, Israel), 2 mM glutamine, Men Non-essential amino acid solution and antibiotics
(
100 mg/mL streptomycin and 100 U/mL penicillin), all from Sigma Aldrich (St. Louis, MO, USA).
◦
Cells were maintained at 37 C in a 5% CO atmosphere and were grown until 80% confluent.
2
MTT Assay
The MTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay, which measures
activity of cellular dehydrogenases, was based on the method of Mosmann [46]. Briefly, cells were
seeded into 96-well plates (about 1.5
grow for 24 h. Subsequently, 100 L of the tested compounds in the medium were added to a final
concentration of 0–250 M, and kept for 48 h, followed by the addition of 100 L MTT, 3 mg/mL in PBS,
for the next 3 h. After the incubation, the medium was removed. Remaining insoluble formazan crystals
were dissolved in 100 L DMSO. The absorbance of the blue formazan product was measured at 570 nm
×
104 cells per well, in 100 µL) and then left to adhere and
µ
µ
µ
µ
in the plate reader spectrophotometer Infinite M200 (Tecan, Grödig, Austria) and compared with
control (untreated cells). All experiments were performed three times in triplicate. The concentration
of tested compounds required to inhibit cell viability by 50% (IC ) was calculated using Microsoft
50
Excel software for semi-log curve fitting with linear regression analysis.
3
.4.2. SRB Viability Assay
Cell Culture
The prostate cancer cell line DU-145 was purchased from the American Type Culture Collection
ATTC, Manassas, VA, USA). The DU-145 cells were cultured in MEME medium supplemented with
(
1
0% fetal bovine 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 at 37 C in an atmosphere containing 5% CO . Stock solutions
◦
2
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-confluent
3
levels at the certain culture medium and then seeded into 96-well plates at 6.0 × 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, and 40 µM) of N-substituted ebelsen derivatives for the next 24 h. After incubation,
the cells were fixed in 20% trichloroacetic acid for an 1 h. The plates were washed with distilled water,
and 0.4% SRB (Sigma-Aldrich, St. Louis, MO, USA) in 1% acetic acid solution was added to the plates
for 15 min. 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.
4
. Conclusions
In this paper, we synthetized the first optically active ebselen analogues derived from terpenes 17
Additionally, we presented an improved version of the known procedure involving the synthesis
of 2,2-diselenobis(benzoic acid) and proved that the cyclization of the Se-N bond depends
on the nucleophilic selenium reagent used. Antioxidant and anticancer activities of N-bornyl 20
and N-isobornyl 19 benzisoselenazol-3(2H)-ones and corresponding diselenides 17 18 were
evaluated and compared to a series of N-amino acid ebselen analogues 21 25. The highest
–20.
6
,
–
antioxidant potential was observed for benzisoselenazol-3(2H)-ones substituted with N-bornyl 20
and N-leucine methyl ester 23 moieties. Select reactive derivatives were tested as anticancer
agents on four cancer cell lines. The highest antiproliferative activity was observed for N-leucine
methyl ester benzisoselenazol-3(2H)-one 23. Additionally, we tested antiproliferative activity for
ebselen with 3-methylbutyl carbon chain 26. The data suggest that the basic structural skeleton

Molecules 2017, 22, 492
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connecting 3-methylbutyl carbon chain can be a pharmacophore necessary for the antioxidant and
anticancer activity.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This work was supported by the National Science Centre, Poland, grant No. UMO-2015/17/
B/NZ7/03058.
´
Author Contributions: A.J.P. and J.S. conceived and designed the experiments, discussed the results, and wrote the
paper; K.B.K. and J.A. performed the SRB viability assays; A.J., A.D. and T.J. performed the MTT viability assays;
A.W. performed and analyzed X-ray data. All authors read and approved the final version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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