Synthesis of new alkylated and methoxylated analogues of ebselen with antiviral and antimicrobial properties

Article abstract of DOI:10.24820/ark.5550190.p009.797

A series of new mono and disubstituted alkylated and methoxylated benzisoselanzol-3(2H)-ones and bis(2carbamoylaryl)diselenides were prepared in yields ranging from 55% to 95% starting from anthranilic acid and were evaluated for antiviral and antimicrobial activity. The compounds exhibited antiviral activity against Human herpes virus 1 and Encephalomyocarditis virus as well as antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Candida albicans. R¹ O O R¹ COOH N N R² H Se R² NH₂ Se 2 R¹, R² = Me, OMe, Cl, t-Bu, H.

Full text of DOI:10.24820/ark.5550190.p009.797

The Free Internet Journal
for Organic Chemistry
Paper
Archive for
Organic Chemistry
Arkivoc 2017, part ii, 546-556
Synthesis of new alkylated and methoxylated analogues of ebselen with antiviral
and antimicrobial properties
Magdalena Piętka-Ottlik,*a Małgorzata Burda-Grabowska,b Marta Woźna,a Joanna Waleńska,a Rafał Kaleta,b
Ewa Zaczyńska,c Egbert Piasecki,c and Mirosław Giurgb
aWrocław University of Science and Technology, Department of Organic and Pharmaceutical Technology,
Wybrzeże Wyspiańskiego 27, 50-370 Wrocław, Poland,
bWrocław University of Science and Technology, Department of Organic Chemistry, Wybrzeże Wyspiańskiego
27, 50-370 Wrocław, Poland,
cHirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114
Wrocław, Poland
E-mail: magdalena.pietka@pwr.edu.pl
Dedicated to Prof. Jacek Młochowski on the occasion of his 80th anniversary
Received 07-13-2016
Accepted 09-20-2016
Published on line 02-06-2017
Abstract
A series of new mono and disubstituted alkylated and methoxylated benzisoselanzol-3(2H)-ones and bis(2-
carbamoylaryl)diselenides were prepared in yields ranging from 55% to 95% starting from anthranilic acid and
were evaluated for antiviral and antimicrobial activity. The compounds exhibited antiviral activity against
Human herpes virus 1 and Encephalomyocarditis virus as well as antimicrobial activity against Staphylococcus
aureus, Bacillus subtilis, Escherichia coli and Candida albicans.
R1
O
O
R1
COOH
N
R2
N
H
Se
Se
NH2
R2
2
R1, R2 = Me, OMe, Cl, t-Bu, H
Keywords: Ebselen, organoselenium compounds, benzisoselenazol-3(2H)-ones, diselenides, antiviral activity,
antimicrobial activity
DOI: https://doi.org/10.24820/ark.5550190.p009.797
Page 546
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
Introduction
Over last few decades organoselenium compounds have been extensively studied in organic and medicinal
chemistry.1-15 Due to their unique properties, they attracted a wide interest as reagents in new synthetic
routes,3 promising catalysts for various oxidation reactions1-7,16-19 and potential pharmaceuticals.8-15,20,21
Ebselen (2-phenylbenzisoselenazol-3(2H)-one) and its analogues have received particular attention as
glutathione peroxidase mimics,8,11,15 antioxidant and anti-inflammatory agents.13-15 Furthermore, the
continuing interest in this class of compounds has led to the development of various structures with
antiviral22-26 and antimicrobial24-27 properties. In this work we report a convenient synthetic route to a series of
new alkylated and methoxylated benzisoselanazol-3(2H)-ones 4 and bis(2-carbamoylaryl)diselenides 6 which
were tested for antiviral and antimicrobial activity in biological studies.
Results and Discussion
Searching for new antiviral and antimicrobial agents, we report here the synthesis of new alkylated and
methoxylated analogues of ebselen – an organoselenium compound well known for its variety of biological
and oxygen transfer properties. 2-Phenylbenzisoselanazol-3(2H)-ones alkylated and methoxylated on the
benzene ring 4a-l were prepared through a tandem selenenylation-acylation of mono and disubstituted
anilines with 2-(chloroseleno)benzoyl chloride (3), in the presence of triethylamine as a base, whereas
corresponding bis(2-carbamoylaryl) diselenides 6c,e,g-h,j-l were prepared via the reaction of the benzoyl
chloride 5 with appropriate anilines in the presence of sodium carbonate (Scheme 1). Both benzoyl chlorides 3
and 5 were obtained in a three-step synthesis starting from anthranilic acid (1), which was diazotized and
reacted with dilithium diselenide in aprotic medium (THF) to give after acidification 2,2'-diselenobisbenzoic
acid 2 with 78 % yield.28-30 Efficient cooling is important to reduce by-product formation such as salicylic acid.
Dilithium diselenide was generated in situ from elemental lithium and selenium in the presence of 4,4'-di(t-
butyl)biphenyl, as a catalyst.31 Alternatively, disodium diselenide in protic medium (MeOH) can be used to
introduce the diselenide moiety, however the yield was reported to be lower (18-67 %).24,32 The procedure for
preparation of disodium diselenide is critical to obtain 2,2'-diselenobisbenzoic acid (2) with good yields. The
type of reducing agent, such as hydrazine monohydrate,24 and sodium tetrahydroborate21 as well as reaction
times up to 48 hours, are of particular importance. Some authors report yields even up to 95% however no
synthetic details were provided.20
The acid 2 was easily converted into 2-(chloroseleno)benzoyl chloride (3) (85%) with thionyl chloride,
used in excess, in the presence of catalytic amounts of N,N’-dimethylformamide.29 When thionyl chloride was
used in excess (2.5 equivalents), chloride 5 (64%) was produced.29 The reaction of benzoyl chlorides 3 and 5
with anilines substituted at ortho, meta or para positions of the phenyl ring by electron-donating methyl
and/or strong electron-donating methoxy groups, (in some cases with a chlorine substituent) provides a
straightforward way to obtain new mono and disubstituted 2-phenylbenzisoselanzol-3(2H)-ones 4 and bis(2-
carbamoylaryl) diselenides 6, respectively. All products were prepared with good to excellent yields (Scheme
1). An ortho effect of methyl and methoxy groups, and a steric hindrance effect of the tert-butyl substituent in
the case of formation of compound 4f, were not observed, only in the cases of selenazolones 4d, and 4j were
minor effects of electron-withdrawing chlorine substituent observed. This confirms that the general approach
presented here can be successfully used for preparation of new substitued analogues of ebselen. To our best
Page 547
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
knowledge compounds 4a-h, 4j, 6c, 6e, 6g, 6h, 6j, 6k are new structures and were not previously described in
the literature. All compounds were fully characterized by spectroscopic methods (see Experimental Section).
O
R1
COCl
SeCl
v
N
Se
R2
3
4
iv
COOH
NH2
COOH
i-iii
Se
2
2
5
1
R1
vi
O
COCl
N
H
vii
R2
Se
Se
2
2
6
4 / 6
R1
R2
a
2-Me
4-OMe
67
b
c
3-Me
4-Cl
70
d
e
f
g
2-Cl
5-Me
70
h
2-Cl
4-Me
95
i
j
k
l
2-OMe
5-Me
63
2-Me
3-Cl
55
2-OMe 2-OMe
4-Me
2-Me
5-Cl
60
3-OMe 4-OMe
5-Cl
79
5-t-Bu
H
72
-
H
H
Yield 4 (%)
Yield 6 (%)
82
-
63
74
70
83
-
-
95
-
85
75
62
86
Reagents: i: NaNO2, HCl, -7 – 5oC, ii: Li2Se2, NaOHaq, -7 – 0oC, iii: HCl, iv: SOCl2 (excess), DMF (cat.), Δ, v: R1R2-
C6H3-NH2, MeCN, Et3N, vi: 3,5 eq. SOCl2, DMF (cat.), benzene, Δ, vii: R1R2-C6H3-NH2, CH2Cl2, Na2CO3.
Scheme 1. Synthesis of alkylated and methoxylated benzisoselenazol-3(2H)-ones 4 and bis(2-carbamoyl-
aryl)diselenides 6.
Antiviral and antimicrobial evaluation of organoselenium compounds
The alkylated and methoxylated analogues of ebselen 4 and 6 were evaluated for antiviral activity against
Human herpes virus 1 (HHV-1, Herpesviridae, enveloped virus), Encephalomyocarditis virus (EMCV,
Picornaviridae, non-enveloped virus) and Vesicular stomatitis virus (VSV, Rhabdoviridae, enveloped virus) as
well as antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Candida
albicans by serial dilution techniques.26 Their activities were compared with those of ebselen.
The compounds exhibited high antiviral activity against HHV-1 and EMCV but in almost all cases were inactive
towards VSV (Table 1). A similar tendency was observed in our previous studies.24-26 Generally,
benzisoselenazol-3(2H)-ones 4 were more active than corresponding diselenides 6. The results of screening of
benzisoselenazol-3(2H)-ones 4 toward HHV-1 are particularly interesting. These compounds were more active
than ebselen, with minimal inhibitory concentration (MIC) values in a range of 2 – 6 μg/mL. The most
Page 548
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
promising as antiviral agents were compounds 4b-d and 6c with higher chemotherapeutic indices (I),
indicating that the concentration active against virus was lower than that showing a cytotoxic effect.
Table 1. Antiviral activity and cytotoxicity of compounds 4 and 6
a
Compound TCCD50
HHV-1
MICb
4
2
4
6
4
6
4
4
EMCV
MICb
VSV
Ic
Ic
0.4
0.8
0.3
-
MICb
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
400
n.a
n.a
n.a
n.a
Ic
-
-
-
-
-
-
-
-
-
-
-
[μg/mL]
4a
4b
4c
7.3
8.5
19.5
78
1.8
4.2
4.9
13
20
10
60
n.a
20
8
4d
4e
4f
4g
8.5
4.9
11
2.1
0.8
2.7
1.8
1.8
2.1
5.8
0.5
2.9
0.6
0.1
1.5
0.4
0.6
1.4
0.7
0.09
0.9
-
8
4h
4i
4j
6c
7.3
3.7
8.5
58.5
22
10
40
10
n.a
200
60
n.a
800
10
2
4
10
40
20
100
200
8
6e
0.1
1.0
-
0.06
6g
6h
6j
58.5
58.5
19.5
12
-
-
-
-
0.02
1.2
Ebselen
a TCCD50 – Tissue Culture Cytotoxic Dose (µg/mL). b MIC – Minimal Inhibitory Concentration (µg/mL).
c I= TCCD50/MIC. n.a – non-active, MIC > 1000 µg/mL.
Table 2. Antimicrobial activity of compounds 4 and 6
MICa [μg/mL]
E. coli
80
Compound
S. aureus
20
B. subtilis
10
P. aeruginosa
n.a.
C. albicans
4a
4b
80
80
20
20
80
n.a.
4e
85
85
85
n.a.
42
4f
33
33
33
n.a.
65
4k
76
38
38
n.a.
68
4l
6e
6k
6l
10
152
340
n.a
n.a
17
152
n.a
n.a
n.a
32
n.a.
n.a.
n.a.
n.a.
76
n.a
305
n.a
17
n.a
n.a
n.a
128
Ebselen
n.a.
a MIC – Minimal Inhibitory Concentration (µg/mL). n.a. – non-active, MIC > 512 µg/mL.
Benzisoselenazol-3(2H)-ones 4 were found to be active against S. aureus, B. subtilis, E. coli and C. albicans
(Table 2). None of compounds exhibited antibacterial activity against P. aeruginosa which is in agreement with
previous observations.26 Usually this gram-negative bacterial strain is not sensitive to benzisoselenazol-3(2H)-
Page 549
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
ones and only a few structures were found to exhibit very low antibacterial activity towards it.24,26 Contrary to
their cyclic analogues, diselenides appeared to be practically inactive against all tested microorganisms. Only
6e and 6k showed low activity against B. subtilis and S. aureus, respectively.
Conclusions
A
series of new alkylated and methoxylated 2-phenylbenzisoselanzol-3(2H)-ones 4 and bis(2-
carbamoylaryl)diselenides 6 were prepared in good to excellent yields starting from anthranilic acid. This
general approach proved to be convenient for the preparation of various mono- and disubstituted analogues
of ebselen. The studies of antiviral and antimicrobial activity revealed new potential antiviral agents against
HHV-1 and EMCV, and antimicrobial agents against Staphylococcus aureus, Bacillus subtilis, Escherichia coli
and Candida albicans.
Experimental Section
General. Melting points were determined on an Electrothermal IA 91100 digital melting-point apparatus using
13
the standard open capillary method. 1H and C NMR spectra were recorded in CDCl3 or DMSO-d6 on a Bruker
DRX 300 Spectrometer (1H: 300.1 MHz, 13C: 75.4 MHz) or on a Bruker Avance 600 Spectrometer (1H: 600.6
MHz, 13C: 151.0 MHz) at 295 K. Chemical shifts (δ) are given in parts per million (ppm) downfield relative to
TMS, and coupling constants (J) are in Hz. Residual solvent central signals were recorded as follows: CDCl3, δH =
7.263, δC = 77.00; DMSO-d6, δH = 2.50, δC = 39.43. High-resolution mass spectra (HRMS) were recorded on a
Waters LCD Premier XE instrument, and only the [M + H]+ or [M + Na]+ molecular species are reported.
Selenium powder (100 mesh) with purity ≥99.5% used for Li2Se2 or Na2Se2 preparation was purchased from
Aldrich. Freshly distilled acetonitrile (MeCN) was redistilled twice over P2O5 to obtain the anhydrous MeCN for
preparation of the ebselen derivatives 4. Methylene chloride (CH2Cl2) was distilled over P2O5 before
preparation of diselenides 6. Distilled triethylamine was stored over NaOH pellets. Other reagents and starting
materials were directly used as obtained commercially. Purity of the products was confirmed by comparison of
their melting point with data given in the literature and by spectroscopic methods.
2,2'-Diselenobisbenzoic acid (2) was prepared from anthranilic acid, using dilithium diselenide,30 following the
method previously reported28 with some modifications. The crude product was recrystallized from 1,4-dioxane
by slow evaporation under ca. 200 mmHg from a water bath at ca 75 °C to give 2,2'-diselenobisbenzoic acid (2)
with 85% yield as pale crystals, mp 299-300 °C (lit.30 300-303 °C).
2-(Chloroseleno)benzoyl chloride (3) was prepared from 2,2’-diselenobisbenzoic acid (2) using excess of
thionyl chloride, following the method previously reported.29 Crude oily product was recrystallized from n-
hexane and dried under vacuum (200 mmHg) to give 2-(chloroseleno)benzoyl chloride (3) as yellow needles,
mp 65-67 °C (lit.29 65-66 °C).
Bis[(2-chlorocarbonyl)phenyl)] diselenide (5) was prepared from 2,2'-diselenobisbenzoic acid (2), using
thionyl chloride in excess of 2.5 equivalents, following the method previously reported.29 The crude oily
product was washed with n-hexane, recrystallized from CH2Cl2 and dried under vacuum to give bis[(2-
chlorocarbonyl)phenyl)] diselenide (5) as pale greenish yellow prisms, mp 180-181 °C (lit.29 174-175 °C).
Page 550
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
General synthetic procedure for benzisoselenazol-3(2H)-ones 4. To a stirred solution of substituted aniline
(5.0 mmol) and dry triethylamine (1.8 ml, 1.02 g, 12.5 mmol) in anhydrous MeCN (50 mL) a solution of (2-
chloroseleno)benzoyl chloride 3 (1.27 g, 5.0 mmol) in anhydrous MeCN (25 mL) was slowly added dropwise for
1 h. The reaction was continued for 1-48 h. The progress of reaction was monitored by TLC. After the reaction
finished, the solvent was evaporated and water was added dropwise (100 mL) resulting in the product
precipitation. Crude product was washed with water and 3.5% HCl if TLC indicated unreacted amine, and left
to dry on the air.
o
2-(4-Methoxy-2-methylphenyl)benzisoselenazol-3(2H)-one (4a). White flakes, yield 67%, mp 179–180 C
1
(from acetonitrile-water, 1:1 v/v); H NMR (300 MHz, DMSO-d6): δH 2.07 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 6.84
(dd, J 8.6 Hz, J 2.9 Hz, 1H, H-5’), 6.92 (d, J 2.9 Hz, 1H, H-3’), 7.20 (d, J 8.6 Hz, 1H, H-6’), 7.47 (dd, J 8.3 Hz, J 6.9
Hz, 1H, H-6), 7.67 (ddd, J 8.0 Hz, J 6.9 Hz, J 1.3 Hz, 1H, H-5), 7.88 (dd, J 8.3 Hz, J 1.3 Hz, 1H, H-7), 8.08 (d, J 8.0
Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 18.0, 55.2, 111.9, 115.7, 125.9, 126.0, 127.3, 127.8, 129.7, 129.9,
131.8, 137.9, 140.0, 158.8, 165.2. HRMS (TOF MS ESI): m/z for (C15H13NO2Se + H)+ calculated: 320.0190; found:
320.0190.
2-(2-Methoxy-5-methylphenyl)benzisoselenazol-3(2H)-one (4b). Pale brown needles, yield 63%, mp 172–173
oC (from water); 1H NMR (300 MHz, DMSO-d6): δH 2.27 (s, 3H, CH3), 3.73 (s, 3H, OCH3), 7.03 (d, 1 H, J 6.4 Hz, H-
3’), 7.16-7.18 (m, 2H, H-4’, H-6’), 7.45 (ddd, J 7.9 Hz, J 7.5 Hz, J 0.8 Hz, 1H, H-6), 7.66 (ddd, J 8.2 Hz, J 7.0 Hz, J
1.4 Hz, 1H, H-5), 7.86 (dd, J 7.7 Hz, J 0.9 Hz, 1H, H-7), 8.06 (d, J 8.0 Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6):
δC 19.8, 55.7, 112.5, 125.7, 125.8, 126.8, 127.3, 127.7, 129.3, 129.4, 130.0, 131.8, 140.3, 153.0, 165.7. HRMS
(TOF MS ESI): m/z for (C15H13NO2Se + H)+ calculated: 320.0190; found: 320.0201.
2-(4-Chloro-3-methylphenyl)benzisoselenazol-3(2H)-one (4c). Shiny orange crystals, yield 70%, mp 209–210
oC (from water); 1H NMR (300 MHz, DMSO-d6): δH 2.38 (s, 3H, CH3), 7.47-7.53 (m, 3H, H-2’,5’,6’), 7.66-7.73 (m,
13
2H, H-5,6), 7.91 (d, J 7.7 Hz, 1H, H-7), 8.10 (d, J 8.0 Hz, 1H, H-4). C NMR (75 MHz, DMSO-d6): δC 19.6, 123.6,
125.8, 126.2, 127.0, 127.9, 128.3, 129.3, 130.0, 132.3, 136.3, 138.5, 138.8, 165.0. HRMS (TOF MS ESI): m/z for
(C14H10ClNOSe + H)+ calculated: 323.9692; found: 323.9692.
2-(3-Chloro-2-methylphenyl)benzisoselenazol-3(2H)-one (4d). Yellow crystals, yield 55%, mp 282–283 C
o
(from 1,2-dichloroethane); 1H NMR (300 MHz, DMSO-d6): δH 2.11 (s, 3H, CH3), 7.29-7.36 (m, 2H, H-5’,6’), 7.47-
7.54 (m, 2H, H-6,4’), 7.70 (ddd, J 8.0 Hz, J 7.0 Hz, J 1.4 Hz, 1H, H-5), 7.90 (dd, J 7.7 Hz, J 1.2 Hz, 1H, H-7), 8.10 (d,
J 8.0 Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 15.3, 126.0, 126.1, 126.9, 127.5, 127.9, 128.0, 128.7, 132.1,
134.1, 134.8, 139.0, 140.2, 165.3. HRMS (TOF MS ESI): m/z for (C14H10ClNOSe + H)+ calculated: 323.9692; found:
323.9685.
2-(5-Chloro-2-methoxyphenyl)benzisoselenazol-3(2H)-one (4e). Light yellow prisms, yield 79%, mp 205–206
1
oC (from acetonitrile-water, 85:15 v/v); H NMR (300 MHz, DMSO-d6): δH 3.79 (s, 3H, OCH3), 7.19 (d, J 8.9 Hz,
1H, H-3’), 7.41-7.49 (m, 3H, H-6,4’,6’), 7.67 (ddd, J 8.2 Hz, J 7.2 Hz, J 1.4 Hz, 1H, H-5), 7.87 (dd, J 7.8 Hz, J 0.8 Hz,
1H, H-7), 8.07 (d, J 8.1 Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 56.6, 114.6, 124.1, 126.3, 126.4, 127.5,
128.4, 129.0, 129.1, 129.6, 132.6, 141.0, 154.6, 166.5. HRMS (TOF MS ESI): m/z for (C14H10ClNO2Se + H)+
calculated: 339.9641; found: 339.9636.
o
2-(5-t-Butyl-2-methoxyphenyl)benzisoselenazol-3(2H)-one (4f). Purple powder, yield 82%, mp 207–208 C
(purified by column chromatography, chloroform); 1H NMR (300 MHz, CDCl3): δH 1.31 (s, 9H, t-Bu), 3.82 (s, 3H,
OCH3), 6.94 (d, J 8.7 Hz, 1H, H-3’), 7.37 (dd, J 8.7 Hz, J 2.5 Hz, 1H, H-4’), 7.42-7.47 (m, 2H, H-6,6’), 7.62-7.65 (m,
2H, H-5,7), 8.12 (d, J 7.6 Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 31.7, 33.7, 55.7, 112.2, 125.6 (2C),
125.7, 126.2, 126.5, 127.3, 127.7, 131.8, 140.3, 142.9, 152.9, 165.7. HRMS (TOF MS ESI): m/z for (C18H19NO2Se
+ H)+ calculated: 362.0660; found: 362.0659.
Page 551
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
o
2-(2-Chloro-5-methylphenyl)benzisoselenazol-3(2H)-one (4g). White powder, yield 70%, mp 184–185 C
(purified by column chromatography (chloroform/ethyl acetate, 5:1); 1H NMR (300 MHz, CDCl3): δH 2.35 (s, 3H,
CH3), 7.16 (d, J 8.2 Hz, 1H, H-4’), 7.27 (s, 1H, H-6), 7.38 (d, J 8.2 Hz, 1H, H-3’), 7.47 (dd, J 7.6 Hz, J 6.8 Hz, 1H, H-
5), 7.62-7.70 (m, 2H, H-6,7), 8.13 (d, J 7.6 Hz, 1H, H-4). 13C NMR (75 MHz, CDCl3): δC 20.5, 124.4, 125.8, 126.1,
129.0, 129.9, 130.4, 130.6, 131.4, 132.3, 135.1, 137.8, 139.5, 166.6. HRMS (TOF MS ESI): m/z for
C14H10ClNOSe+H+ calculated: 323.9692; found: 323.9607.
o
2-(2-Chloro-4-methylphenyl)benzisoselenazol-3(2H)-one (4h). White powder, yield 95%, mp 208–209 C
(from ethyl acetate); 1H NMR (300 MHz, CDCl3): δH 2.36 (s, 3H, CH3), 7.25 (dd, J 8.0 Hz, J 1.1 Hz, 1H, H-5’), 7.37
(d, J 8.0 Hz, 1H, H-6’), 7.45-7.50 (m, 2H, H-6,3’), 7.69 (ddd, J 8.0 Hz, J 7.3 Hz, J 1.3 Hz, 1H, H-5), 7.89 (dd, J 7.7
Hz, J 0.8 Hz, 1H, H-7), 8.09 (d, J 8.0 Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 20.3, 125.9, 126.0, 126.8,
127.9, 128.5, 130.3, 130.6, 132.1 (2C), 133.3, 139.8, 140.3, 165.6. HRMS (TOF MS ESI): m/z for
C14H10ClNOSe+H+ calculated: 323.9692; found: 323.9694.
o
2-(4-Methylphenyl)benzisoselenazol-3(2H)-one (4i). Pale powder, yield 72%, mp 163–164 C (from water)
o
1
(lit.33 163–164 C); H NMR (300 MHz, DMSO-d6): δH 2.32 (s, 3H, CH3), 7.24 (d, J 8.1 Hz, 2H, H-2’,6’), 7.47-7.53
(m, 3H, H-6,3’,5’), 7.67 (ddd, J 8.0 Hz, J 7.5 Hz, J 1.3 Hz, 1H, H-5), 7.90 (d, J 7.0 Hz, 1H, H-7), 8.09 (d, J 8.0 Hz,
1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 20.5, 124.5, 125.7, 126.2, 127.8, 128.4, 129.5, 132.0, 135.1, 137.0,
138.8, 164.8. HRMS (TOF MS ESI): m/z for (C14H11NOSe + H)+ calculated: 290.0085; found: 290.0084.
2-(5-chloro-2-methylphenyl)benzisoselenazol-3(2H)-one (4j). White powder, yield 60%, mp 189–190 oC (from
chloroform); 1H NMR (300 MHz, DMSO-d6): δH 2.08 (s, 3H, CH3), 7.36-7.42 (m, 3H, H-3’,4’,6’), 7.49 (dd, J 7.5 Hz,
J 7.3 Hz, 1H, H-6), 7.69 (ddd, J 8.0 Hz, J 7.3 Hz, J 1.3 Hz, 1H, H-5), 7.89 (d, J 7.6 Hz, 1H, H-7), 8.10 (d, J 8.0 Hz, 1H,
H-4). 13C NMR (151 MHz, DMSO-d6): δC 17.8, 126.5, 126.6, 127.5, 128.4, 128.5, 128.6, 129.1, 130.8, 132.6,
132.7, 139.4, 140.8, 165.8. HRMS (TOF MS ESI): m/z for (C14H10ClNOSe + H)+ calculated: 323.9692; found:
323.9687.
2-(3-Methoxyphenyl)benzisoselenazol-3(2H)-one (4k). Pale yellow crystals, yield 63%, mp 165.5–167.5 °C
(from acetonitrile) (lit.34 162–164 °C); 1H NMR (300 MHz, DMSO-d6): δH 3.77 (s, 3H, OCH3), 6.82 (dd, J 8.3 Hz, J
0.8 Hz, 1H, H-6’), 7.15 (dd, J 8.0 Hz, J 0.8 Hz, 1H, H-4’), 7.31-7.36 (m, 2H, H-2’,5’), 7.45 (ddd, J 7.8 Hz, J 7.2 Hz, J
0.9 Hz, 1H, H-6), 7.64 (ddd, J 8.0 Hz, J 7.2 Hz, J 1.4 Hz, 1H, H-5), 7.88 (dd, J 7.7 Hz, J 0.9 Hz, 1H, H-7), 8.36 (d, J
8.0 Hz, 1H, H-4). 13C NMR (75 MHz, DMSO-d6): δC 55.2, 110.3, 111.3, 116.6, 125.7, 126.2, 127.9, 128.5, 129.9,
132.2, 138.8, 140.8, 159.6, 164.9. HRMS (TOF MS ESI): m/z for (C14H11NO2Se + H)+ calculated: 306.0034; found:
306.0030.
2-(4-Methoxyphenyl)benzisoselenazol-3(2H)-one (4l). Pale needles, yield 70%, mp 180.5–181.5 °C (from
water) (lit.28 181–184 °C); 1H NMR (300 MHz, DMSO-d6): δH 3.78 (s, 3H, OCH3), 7.01 (dd, J 9.0 Hz, J 2.3 Hz, 2H,
H-3’,5’), 7.47 (ddd, J 7.8 Hz, J 7.1 Hz, J 1.0 Hz, 1H, H-6), 7.50 (dd, J 9.0 Hz, J 2.3 Hz, 2H, H-2’,6’), 7.67 (ddd, J 7.9
Hz, J 7.2 Hz, J 1.4 Hz, 1H, H-5), 7.89 (dd, J 7.8 Hz, J 0.8 Hz, 1H, H-7), 8.08 (d, J 7.9 Hz, 1H, H-4). 13C NMR (75
MHz, DMSO-d6): δC 55.3, 114.3 (2C), 125.7, 126.1, 126.5 (2C), 127.8, 128.2, 131.9, 132.1, 138.9, 157.2, 164.9.
HRMS (TOF MS ESI): m/z for (C14H11NO2Se + H)+ calculated: 306.0034; found: 306.0039.
General synthetic procedure for bis(2-carbamoylaryl) diselenides 6. To a stirred solution of substituted
aniline (1.0 mmol) in dry CH2Cl2 (5 mL) Na2CO3 (2.5 mmol, 0.265 g) was added. Next, the solution of chloride 5
in dry CH2Cl2 was added dropwise for 30 min. The desired product, diselenide precipitated immediately. The
reaction progress was controlled by TLC. The stirring was continued for 1-48 h. The product was filtered off
and washed with dry CH2Cl2 (2 x 5 mL), water (1 x 5 mL) and 3.5% HCl (3 x 5 mL).
Bis[2-(4-chloro-3-methylphenylcarbamoyl)]phenyl diselenide (6c). White solid, yield 95%, mp 239–240 °C
1
(from 1,2-dichloroethane); H NMR (300 MHz, DMSO-d6): δH 2.35 (s, 6H, CH3), 7.38-7.50 (m, 6H, ArH-5, PhH-
Page 552
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
4,5), 7.63 (d, J 8.6 Hz, 2H, ArH-6), 7.76-7.82 (m, 4H, PhH-6, ArH-2), 7.96 (d, J 7.2 Hz, 2H, PhH-3), 10.61 (s, 2H,
13
NH). C NMR (151 MHz, DMSO-d6): δC 19.81 (CH3), 119.64 (CH), 122.83 (CH), 126.37 (CH), 128.01 (C), 128.62
(CH), 128.93 (CH), 130.14 (CH), 131.99 (C), 132.07 (CH), 133.45 (C), 135.54 (C), 137.48 (C), 166.26 (C=O). HRMS
(TOF MS ESI): m/z for (C28H21Cl2N2O2Se2 + Na)+ calculated: 668.9297, 670.9284, 672.9272; found: 668.9341,
670.9288, 672.9265.
Bis[2-(5-chloro-2-methoxyphenylcarbamoyl)]phenyl diselenide (6e). Grey powder, yield 85%, mp 194–195 oC
1
(from methylene chloride); H NMR (300 MHz, DMSO-d6): δH 3.86 (s, 6H, OCH3), 7.15 (d, J 8.9 Hz, 2H, ArH-3),
7.29 (dd, J 2.6 Hz, J 8.8 Hz, 2H, ArH-4), 7.38-7.51 (m, 4H, PhH-4,5), 7.78 (d, J 7.8 Hz, 2H, PhH-6), 7.84 (d, J 2.6
Hz, 2H, ArH-6), 7.99 (d, J 7.5 Hz, 2H, PhH-3), 9.93 (s, 2H, NH). HRMS (TOF MS ESI): m/z for (C28H22Cl2N2O4Se2 +
H)+ calculated: 680.9363; found: 680.9400.
Bis[2-(2-chloro-5-methylphenylcarbamoyl)]phenyl diselenide (6g). White solid, yield 75%, mp 203–204 °C
1
(from methylene chloride); H NMR (300 MHz, DMSO-d6): δH 2.35 (s, 6H, CH3), 7.18 (dd, J 8.1 Hz, J 1.1 Hz, 2H,
ArH-4), 7.43-7.46 (m, 4H, PhH-5, ArH-6), 7.47 (d, J 8.2 Hz, 2H, ArH-3), 7.50 (dd, J 7.4 Hz, J 1.0 Hz, 2H, PhH-4),
7.80 (d, J 8.0 Hz, 2H, PhH-6), 8.06 (d, J 7.5 Hz, 2H, PhH-3), 10.36 (s, 2H, NH). 13C NMR (75 MHz, DMSO-d6): δC
20.4, 126.5, 126.7, 128.6, 128.7, 129.0, 129.3, 130.2, 132.3, 132.5, 132.6, 134.2, 137.3, 166.5. HRMS (TOF MS
ESI): m/z for (C28H21Cl2N2O2Se2 + Na)+ calculated: 670.9284; found: 670.9287.
Bis[2-(2-chloro-4-methylphenylcarbamoyl)]phenyl diselenide (6h). White powder, yield 62%, mp 260–261 °C
1
(from methylene chloride); H NMR (300 MHz, DMSO-d6): δH 2.35 (s, 6H, CH3), 7.23 (d, J 8.4 Hz, 2H, ArH-5),
7.40-7.51 (m, 8H, PhH-4,5, ArH-3,6), 7.77 (d, J 7.4 Hz, 2H, PhH-6), 8.05 (d, J 6.9 Hz, 2H, PhH-3), 10.35 (s, 2H,
NH). 13C NMR (151 MHz, DMSO-d6): δC 20.8, 126.9, 128.7, 129.0, 129.1, 130.1, 130.3, 130.6, 132.3, 132.7,
133.0, 133.1, 138.3, 167.0. HRMS (TOF MS ESI): m/z for (C28H22Cl2N2O2Se2 + H)+ calculated: 648.9465; found:
648.9327.
Bis[2-(5-chloro-2-methylphenylcarbamoyl)]phenyl diselenide (6j). White powder, yield 86%, mp 247–248 °C
1
(from 1,2-dichloroethane); H NMR (300 MHz, DMSO-d6): δH 2.28 (s, 6H, CH3), 7.28 (dd, J 8.3 Hz, J 2.2 Hz, 2H,
ArH-4), 7.34 (d, J 8.3 Hz, 2H, ArH-3), 7.40-7.52 (m, 4H, PhH-4,5), 7.53 (d, J 2.2 Hz, 2H, ArH-6), 7.80 (dd, J 7.8 Hz,
J 1.3 Hz, 2H, PhH-6), 8.02 (dd, J 7.4 Hz, J 1.4 Hz, 2H, PhH-3), 10.28 (s, 2H, NH). 13C NMR (75 MHz, DMSO-d6): δC
17.4, 125.85, 125.9, 126.4, 128.6, 129.8, 130.1, 131.9, 132.1, 132.3, 132.5, 132.9, 137.2, 166.5. HRMS (TOF MS
ESI): m/z for (C28H21Cl2N2O2Se2 + Na)+ calculated: 670.9284; found: 670.9291.
Bis[2-(3-methoxyphenylcarbamoyl)]phenyl diselenide (6k). White powder, yield 74%, mp 221–223 °C (from
1
methylene chloride); H NMR (300 MHz, DMSO-d6): δH 3.78 (s, 6H, OCH3), 6.74 (dd, J 8.2 Hz, J 1.7 Hz, 2H, ArH-
4), 7.30 (dd, J 8.2 Hz, J 8.1 Hz, 2H, ArH-5), 7.40 (dd, J 8.1 Hz, J 1.2 Hz, 2H, ArH-6), 7.42 (ddd, J 8.0 Hz, J 7.0 Hz, J
1.2 Hz, 2H, PhH-5), 7.45-7.48 (m, 4H, PhH-4, ArH-2), 7.81 (dd, J 8.0 Hz, J 1.1 Hz, 2H, PhH-6), 7.96 (dd, J 7.6 Hz, J
1.3 Hz, 2H, PhH-3), 10.54 (s, 2H, NH). 13C NMR (151 MHz, DMSO-d6): δC 55.0, 106.2, 109.5, 112.7, 126.4, 128.6,
129.5, 130.1, 131.9, 132.0, 133.7, 139.8, 159.4, 166.3. HRMS (TOF MS ESI): m/z for (C28H24N2O4Se2 + H)+
calculated: 612.0189; found: 612.0232.
Bis[2-(4-methoxyphenylcarbamoyl)]phenyl diselenide (6l). White powder, yield 62%, mp 285.5–287.5 °C
(from methylene chloride) (lit.35 290–292 °C; 1H NMR (300 MHz, DMSO-d6): δH 3.76 (s, 6H, OCH3), 6.97 (d, J 9.0
Hz, 4H, ArH-3,5), 7.40 (ddd, J 7.1 Hz, J 6.9 Hz, J 0.9 Hz, 2H, PhH-4), 7.44 (ddd, J 8.0 Hz, J 7.1 Hz, J 1.3 Hz, 2H,
PhH-5), 7.68 (d, J 9.0 Hz, 4H, ArH-2,6), 7.79 (dd, J 8.0 Hz, J 0.8 Hz, 2H, PhH-6), 7.95 (d, J 6.8 Hz, 2H, PhH-3),
10.44 (s, 2H, NH). 13C NMR (151 MHz, DMSO-d6): δC 55.7, 114.3, 122.7, 126.9, 129.0, 130.6, 132.2, 132.4,
132.5, 134.3, 156.4, 166.4. HRMS (TOF MS ESI): m/z for (C28H24N2O4Se2 + H)+ calculated: 612.0189; found:
612.01823.
Page 553
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
Evaluation of antiviral activity and cytotoxicity. The compounds 4 and 6 at various concentrations were
incubated with following viruses: HHV-1 (human herpes virus type 1), EMCV (encephalomyocarditis virus) and
VSV (vesicular stomatitis virus) for 1 hr at RT. The HHV-1 was used at the dose of 106 TCID50/mL, whereas
EMCV and VSV at the dose of 108 TCID50/mL. The TCID50 (tissue culture infectious dose) determines the
cytopathic effect caused by the virus in about 50% of infected cells. The virus titer was measured in human cell
line A549 (human lung adenocarcinoma cell line, ATCC 185) which was observed for cytopathic effect after 48
h. The minimal concentration that caused 1000-fold decrease of virus titer was taken as minimal inhibitory
concentration, MIC (μg/mL).
In parallel, the cytotoxicity of the compounds was determined in A549 cell line (cell line used in antiviral
activity test) by incubation at various concentrations for 48 h at 37 oC in the atmosphere of 5% CO2 in air. The
minimal concentration which was toxic to approximately 50% of the cells was taken as TCCD50.
Both, cytotoxicity and virus titers were determined using MTT assay. To ensure that colourful compounds did
not interfere with MTT reagents, extent of cell damage was evaluated by microscopic examination prior to
MTT assay.
Evaluation of antimicrobial activity. Antimicrobial activity of compounds 4 and 6 was determined by serial
dilution method using gram-positive bacterial strains: Staphylococcus aureus PCM 1944, Bacillus subtilis PCM
1949, gram-negative bacterial strains: Escherichia coli PCM 2057, Pseudomonas aeruginosa PCM 2058 and
yeast Candida albicans ATCC 90028 obtained from the Polish Collection of Microorganisms, following
procedure previously described.26 The antimicrobial activity was characterized by MIC value which is minimal
concentration of the compound in μg/ml that totally inhibits growth of the microorganisms.
Acknowledgements
This work was supported by the National Science Centre (NCN) through grant no. UMO-2013/09/D/ST5/03814
(antimicrobial studies of organoselenium compounds) and a statutory activity of subsidy from the Polish
Ministry of Science and Higher Education for the Faculty of Chemistry of Wrocław University of Science and
Technology (synthesis of organoselenium compounds).
References
1.
2.
Młochowski, J.; Wójtowicz-Młochowska, H. Molecules 2015, 20, 10205-10243 and references therein.
http://dx.doi.org/10.3390/molecules200610205
Młochowski, J.; Kloc. K.; Lisiak, R.; Potaczek, P.; Wójtowicz, H. Arkivoc 2007, (vi), 14-46 and references
therein.
http://dx.doi.org/10.3998/ark.5550190.0008.603
3.
4.
Wirth, T. Angew. Chem. Int. Ed. 2000, 39, 3740-3749 and references therein.
http://dx.doi.org/10.1002/1521-3773(20001103)39:21<3740::AID-ANIE3740>3.0.CO;2-N
Freudendahl, D. M; Santoro, S.; Shahzad, S. A.; Santi, C.; Wirth, T. Angew. Chem. Int. Ed. 2009, 48, 8409-
8411 and references therein.
http://dx.doi.org/10.1002/anie.200903893
Page 554
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
5.
6.
7.
8.
Guo, R.; Huang, J.;
http://dx.doi.org/10.1021/acs.orglett.5b03543
Kumar, A.; Rao, G. K.; Saleem, F.; Singh, A. K. Dalton Trans. 2012, 41, 11949-11977.
http://dx.doi.org/10.1039/c2dt31198d
Back, T. Curr. Green Chem. 2016, 3, 76-91.
http://dx.doi.org/10.2174/2213346103666160127003954
Pacuła, A. J.; Mangiavacchi, F.; Sancineto, L.; Lenardão, E. J.; Ścianowski, J.; Santi, C. Curr. Chem. Biol.
2015, 9, 97-112 and references therein.
Huang, H.; Zhao, X. Org. Lett. 2016,
18,
504–507.
http://dx.doi.org/10.2174/2212796810666160120220725
Santi C. Organoselenium Chemistry: Between Synthesis and Biochemistry, Bentham Science Publishers,
2014.
9.
10. Nogueira, C. W.; Rocha J. B. Arch. Toxicol. 2011, 85, 1313-1359 and references therein.
http://dx.doi.org/10.1007/s00204-011-0720-3
11. Wirth, T. Angew. Chem. Int. Ed. 2015, 54, 10074-10076 and references therein.
http://dx.doi.org/10.1002/anie.201505056
12. Fernandes, A. P. ; Gandin, V. Biochim. Biophys. Acta. 2015, 1850, 1642-1660 and references therein.
http://dx.doi.org/10.1016/j.bbagen.2014.10.008
13. Azad, G. K.; Tomar,. R. S. Mol. Biol. Rep. 2014, 41, 4865-4879 and references therein.
http://dx.doi.org/10.1007/s11033-014-3417-x
14. Soriano-Garcia, M. Curr. Med. Chem. 2004, 11, 1657-1669 and references therein.
http://dx.doi.org/10.2174/0929867043365053
15. Mugesh, G; du Mont, W. W; Sies, H. Chem. Rev. 2001, 101, 2125-2179 and references therein.
http://dx.doi.org/10.1021/cr000426w
16. Granda, J. M.; Piekielska, K.; Wąsińska, M.; Kawecka, N.; Giurg, M. Synthesis 2015, 47, 3321–3332.
http://dx.doi.org/10.1055/s-0034-1381011
17. Denmark,
S.
E;
Chi,
H.
M.
J.
Am.
Chem.
Soc.
2014,
136,
8915–8918.
http://dx.doi.org/10.1021/ja5046296
18. Kawamata, Y.; Hashimoto, T.; Maruoka, K. J. Am. Chem. Soc. 2016, 138, 5206–5209.
http://dx.doi.org/10.1021/jacs.6b01462
19. Sancineto, L.; Tidei, C.; Bagnoli, L.; Marini, F.; Lenardão, E.J.; Santi, C. Molecules 2015, 20, 10496-10510.
http://dx.doi.org/10.3390/molecules200610496
20. He, J.; Li, D.; Xiong, K.; Ge, Y.; Jin, H.; Zhang, G.; Hong, M.; Tian, Y.; Yin, J.; Zeng, H. Bioorg. Med. Chem.
2012, 20, 3816-3827.
http://dx.doi.org/10.1016/j.bmc.2012.04.033
21. Nascimento, V.; Ferreira, N. L.; Canto, R. F.; Schott, K. L.; Waczuk, E. P.; Sancineto, L.; Santi, C.; Rocha, J.
B.; Braga, A. L. Eur. J. Med. Chem. 2014, 87, 131-139.
http://dx.doi.org/10.1016/j.ejmech.2014.09.022
22. Thenin-Houssier, S.; de Vera, I.M.; Pedro-Rosa, L.; Brady, A.; Richard, A.; Konnick, B.; Opp, S.; Buffone, C.;
Fuhrmann, J.; Kota, S.; Billack, B.; Pietka-Ottlik, M.; Tellinghuisen, T.; Choe, H.; Spicer, T.; Scampavia, L.;
Diaz-Griffero, F.; Kojetin, D. J.; Valente, S. T. Antimicrob. Agents Chemother. 2016, 60, 2195-2208.
http://dx.doi.org/10.1128/AAC.02574-15
23. Sancineto, L.; Mariotti, A.; Bagnoli, L.; Marini, F.; Desantis, J.; Iraci, N.; Santi, C.; Pannecouque, C.;
Tabarrini, O. J. Med. Chem. 2015, 58, 9601–9614.
http://dx.doi.org/10.1021/acs.jmedchem.5b01183
Page 555
©ARKAT USA, Inc

Arkivoc 2017, part ii, 546-556
Piętka-Ottlik, M. et al
24. Wójtowicz, H.; Chojnacka, M.; Młochowski, J.; Palus, J.; Syper, L.; Hudecova, D.; Uher, M.; Piasecki, E. ;
Rybka, M. Farmaco 2003, 58, 1235-1242.
http://dx.doi.org/10.1016/j.farmac.2003.08.003
25. Wójtowicz, H.; Kloc, K.; Maliszewska, I.; Młochowski, J.; Piętka, M.; Piasecki, E. Farmaco 2004, 59, 863-
868.
http://dx.doi.org/10.1016/j.farmac.2004.07.003
26. Piętka-Ottlik, M.; Wójtowicz-Młochowska, H.; Kołodziejczyk, K.; Piasecki, E.; Młochowski, J. Chem.
Pharm. Bull. 2008, 56, 1423–1427.
http://dx.doi.org/10.1248/cpb.56.1423
27. Chan, G.; Hardej, D.; Santoro, M.; Lau-Cam, C.; Billack, B. J. Biochem. Mol. Toxicol. 2007, 21, 252-264.
http://dx.doi.org/10.1002/jbt.20189
28. Kamigata, N.; Iizuka, H.; Izuoka, A.; Kobayashi M. Bull. Chem. Soc. Jpn. 1986, 59, 2179-2183.
http://dx.doi.org/10.1246/bcsj.59.2179
29. Mlochowski, J.; Gryglewski, R. J.; Inglot, A. D.; Jakubowski, A.; Juchniewicz, L.; Kloc, K. Liebigs Ann. 1996,
1751–1755.
http://dx.doi.org/10.1002/jlac.199619961108
30. Młochowski, J.; Kloc, K.; Syper, L.; Inglot, A. D.; Piasecki, E. Liebigs Ann. Chem. 1993, 1239–1244.
http://dx.doi.org/10.1002/jlac.1993199301201
31. Giurg, M.; Syper, L. Phosphorus Sulfur Silicon Relat. Elem. 2008, 183, 970-985.
http://dx.doi.org/10.1080/10426500801900956
32. Küppers, J.; Schulz-Fincke, A. C.; Palus, J.; Giurg, M.; Skarżewski, J.; Gütschow, M. Pharmaceuticals 2016,
9, 43.
http://dx.doi.org/10.3390/ph9030043
33. Chang, T. C.; Huang, M. L.; Hsu, W.L.; Hwang, J. M.; Hsu, L. Y. Chem. Pharm. Bull. 2003, 51, 1413-1416.
http://dx.doi.org/10.1248/cpb.51.1413
34. Welter, A.; Christiaens, L.; Wirtz-Peitz Eur. Pat. Appl. 44 453, 1982; Chem. Abstr. 1982, 96, 199 699.
35. Welter, A.; Fischer, H.; Christiaens, L.; Wendel, A.; Etschenberg, E. Ger. Offen. 3 513 070, 1986.
Page 556
©ARKAT USA, Inc