Functionalized alkyl and aryl diselenides as antimicrobial and antiviral agents: Synthesis and properties

Article abstract of DOI:10.1016/j.farmac.2003.08.003

The different dialkyl and diaryl diselenides with carbamoyl and sulfamoyl moieties 2, 3, 5 and other substituents in the ortho position of benzene ring 4, 7, 8 as well as derivatives of 1,2,4-benzoselenadiazine (6) were designed as antiviral and antimicrobial agents and synthesized. Some of them, particularly 8a and 8b, were found in the antiviral assay in vitro to be strong inhibitors of cytopathic activity encephalomyocarditis virus (EMCV). The compound 4a and 8a were found to have a broad spectrum of acivity against bacteria, yeasts and pathogenic fungi in vitro.

Full text of DOI:10.1016/j.farmac.2003.08.003

Il Farmaco 58 (2003) 1235ꢀ1242
/
www.elsevier.com/locate/farmac
Functionalized alkyl and aryl diselenides as antimicrobial and
antiviral agents: synthesis and properties
H. Wo´jtowicz a, M. Chojnacka a, J. M lochowski a,*, J. Palus a, L. Syper a,
D. Hudecova b, M. Uher c, E. Piasecki d, M. Rybka d
a Institute of Organic Chemistry, Biochemistry and Biotechnology Wroc law University of Technology, Wyb. Wyspian´skiego 27, 50-375 Wroc law, Poland
b Department of Biochemistry and Microbiology, Faculty of Chemical and Food Technology Slovak Technical University, Radlinskeho 9, SK-81237
Bratislava, Slovak Republic
c Department of Organic Chemistry, Faculty of Chemical and Food Technology Slovak Technical University, Radlinskeho 9, SK-81237 Bratislava,
Slovak Republic
d Laboratory of Virology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland
Received 15 April 2003; accepted 8 August 2003
Abstract
The different dialkyl and diaryl diselenides with carbamoyl and sulfamoyl moieties 2, 3, 5 and other substituents in the ortho
position of benzene ring 4, 7, 8 as well as derivatives of 1,2,4-benzoselenadiazine (6) were designed as antiviral and antimicrobial
agents and synthesized. Some of them, particularly 8a and 8b, were found in the antiviral assay in vitro to be strong inhibitors of
cytopathic activity encephalomyocarditis virus (EMCV). The compound 4a and 8a were found to have a broad spectrum of acivity
against bacteria, yeasts and pathogenic fungi in vitro.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Diselenides; Ebselen; Bacteriocides; Fungicides; Virucides
1. Introduction
been presented, a little attention has been focused on
their antimicrobial properties.
Interest in the use of organoselenium compounds in
biochemistry and medicinal biology started with find-
ings that they are much less toxic compared with the
inorganic species [1]. Studies of the anti-infective orga-
noselenium compounds have been initiated about 50
years ago, when the selenium analogs of sulfonamides
were tested until last decade but no particular interest
has been paid for this problem [2,3]. Since 1984 when 2-
phenyl-benzisoselenazol-3(2H)-one named ebselen (1)
was found as an nontoxic glutathione peroxidase mimic
[4,5] a lot of effort has been directed toward the
development of stable organoselenium compounds that
could be used as antioxidants, enzyme inhibitors,
cytokine inducers, immunomodulators and antiradia-
tion agents [6,7]. Although a broad spectrum of
biological activity of organoselenium compounds has
Most recently it has been found that ebselen (1), other
benzisoselenazolones and related compounds exhibited
inhibitory activity against some strains of bacteria and
fungi [8]. Some of these compounds also exhibited
cytopathic activity against RNA and DNA viruses:
EMCV, HSV-1 and VSV [9,10]. In the present work
fourteen different dialkyl and diaryl diselenides (2ꢀ
8) are described. Some of them (compounds 5a,b) have
phenylsulfamoyl groups and other (2, 3aꢀd) have 4-
/
5, 7,
/
aminophenylsulfamoyl moiety potentially responsible
for inhibition of dihydropteridine synthetase involved
in the biosynthesis of folic acid [11]. All of them having
Seꢀ
thiol groups of peptides and enzymes in the similar way
as selenenamide SeꢀN group [6,7]. For comparison of
biological response of diselenides and selenenamides
three their representatives*ebselen (1) and two other
/
Se moiety should be able to interact covalently with
/
/
cyclic selenenamides 6d and 6e having new 1,2,4-
benzoselenadiazine ring system [12] were obtained. All
* Corresponding author.
E-mail address: mlochowski@kchf.ch.pwr.wroc.pl (J. M lochowski).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.08.003

1236
H. Wo´jtowicz et al. / Il Farmaco 58 (2003) 1235ꢀ1242
/
these compounds were tested as antibacterial, antifungal
and antiviral agents in vitro.
2. Chemistry
Ebselen was synthesized from anthranilic acid 9 which
was diazotized and diazonium salt reacting with dis-
odium diselenide produced 2,2?-diselenobisbenzoic acid
(10). The acid 10 was converted to 2-(chloroseleno)ben-
zoyl chloride (11) by treatment with thionyl chloride,
used in large excess in the presence of DMF as a
catalyst. The reaction of chloride 11 with aniline gave
ebselen 1. The same acid 10 treated with a stoichiometric
amount of thionyl chloride in benzene solution gave
2,2?-diselenobisbenzoyl chloride (12) (Scheme 1). For
this purpose the procedures elaborated earlier in our
laboratory were applied [13,14]. The chloride 12 was
used as the reagent for acylation of 4-aminobenzenesul-
fonamide and 2,2?diselenobis[benz(p-sulfonamido)ani-
lide] (2) was obtained in this way.
4,4?-Diselenobisbutyramides (3) were synthesized ac-
cording as it is shown in Scheme 2. The key substrate
was g-butyrolactone (13). In the first step it was treated
with dilithium diselenide generated in situ from elemen-
tal lithium and selenium, in the manner similar to
described earlier [15]. The procedure was slightly
modified since 4,4?-di(tert-butyl)biphenyl was used as
catalyst for selenenylation instead of diphenylacetylene
and the reaction was carried out using the ultrasonic
bath. It made the reaction more effective since dilithium
diselenide was produced faster [10]. The acid 14, thus
obtained, was converted into chloride 15 by treatment
with oxalyl chloride. This chlorinating reagent made the
reaction highly chemoselective and possible chlorination
of selenium was avoided. The last step of the synthesis
Scheme 2.
involved acylation of amino group of 4-aminobenzene-
sulfonamides, prepapared as described in Ref. [15], with
acyl chloride 14 in the presence of 4-methylmorpholine
(NMM) as a base.
The key substrate for synthesis of diselenides 5, 1,2,4-
benzoselenadiazin-3(4H)-one (6d) and related thione 6e
was bis(2-aminophenyl) diselenide (4). It was obtained
by selenenylation of 1-chloro-2-nitrobenzene (16) with
dilithium diselenide [15] and reduction of resulted bis(2-
nitrophenyl) diselenide (17) with hydrazine in the
presence of catalytic Ni (Raney) (Scheme 3). The amine
4 was sulfonylated and acylated to corresponding
sulfonamides (5a,b) and carboxyamide (5c). Treatment
of amine 4 with benzaldehyde gave imine 7 while its
reaction with isocyanate or isothiocyanate resulted in
bis[2-(carbamoylamino)phenyl] diselenide (5d) and
bis[2-(thiocarbamoylamino)phenyl] diselenide (5e). The
compounds 5d and 5e were converted to corresponding
benzoselenadiazinone (6d) and benzoselenadiazinthione
(6e) by oxidation with benzoyl peroxide.
Bis(2-hydroxyphenyl) diselenide (8a) was synthesized
in the way shown in the Scheme 4. The methoxymethy-
loxybenzene (18) [19] was lithiated at the ortho position
and formed 19, which was selenenylated to compound
20. It converted to selenol 21, which finally was oxidized
to diselenide 22. The intermediates 19 and 20 were not
isolated and selenol 21 was used in the crude form. The
last step involved acid hydrolysis of methoxymethyloxy
group and bis(2-hydroxyphenyl) diselenide (8a) was
produced. The reaction of propylisocyanate with 8a
gave carbamate 8b (Scheme 4) but all attempts for its
oxidative cyclization to expected 8c failed since no
individual product could be isolated from the tarry
mixtures.
3. Results and discussion
Compounds 2, 3aꢀ
/
d, 4, 5dꢀ
/
e, 6dꢀ
/
e, 7 and 8aꢀb were
/
Scheme 1.
tested against bacteria, yeasts and pathogenic fungi. The

H. Wo´jtowicz et al. / Il Farmaco 58 (2003) 1235ꢀ
/1242
1237
Scheme 3.
173 mg/ml. For the bacteria and yeasts the biological
response observed for 4 and 8a was similar to that
established for ebselen (1). Moreover, both of these
diselenides exhibited appreciable activity against fungal
strains R. oryzae, A. alternata as well as for M. gypsum
resistant toward ebselen. None of organoselenium
compound tested was active against B. cinarea. Some
other ortho-substituted bisaryl diselenides derived from
amine 4 (compounds 5aꢀc, 7) and phenol 8a (compound
/
8b) were highly active against Gram-positive S. aureus
remaining inactive toward Gram-negative E. coli. 4,4?-
Diselenobutyramides (3aꢀd), bisaryl diselenides with
/
urea or thiourea moiety (5d,e), 2-propyl-1,2,4-benzose-
lenadiazinone (6d) and 2-phenyl-1,2,4-benzoselenadia-
zin-3(4H)-thione (6e) were practically inactive against all
tested microbial strains (MIC values above 500 mg/ml).
Scheme 4.
Antiviral effects of ebselen (1), diselenides 2, 3aꢀd, 4,
/
results of these tests, expressed as MIC (minimum
inhibitory dose) values, are shown in Table 1, together
with those obtained for the same strains for mentioned
5d,e, 7, 8a,b and compounds 6d,e were measured against
encephalomyocarditis virus (EMCV, non-enveloped
RNA virus), vesicular stomatitis virus (VSV, enveloped
DNA virus) and herpes simplex virus type 1 (HSV-1,
enveloped DNA virus). The results are presented in
Table 2.
Strong anti-EMCV activitiy, similar or even higher
that activity of ebselen (1) was found for bis(2-amino-
phenyl) diselenide (4), its derivatives 5d, 7 and benzose-
earlier ebselen (1)*one of the most active organosele-
/
nium compounds [7,8].
The broadest spectra of activity against tested micro-
organisms were observed for bis(2-aminophenyl) dis-
elenide (4) and bis(2-hydroxyphenyl) diselenide (8a).
Both of them have similar MIC values in the range 3.4ꢀ
/

1238
H. Wo´jtowicz et al. / Il Farmaco 58 (2003) 1235ꢀ1242
/
Table 1
Antimicrobial activity of organoselenium compounds 1ꢀ
/
8 against bacteria, yests and filamentous fungi characterized by values of MIC (mg/ml)
Filamentous fungi
R. oryzae A. alternata
Compound
Bacteria
Yests
S. aureus
E. coli
137.0 a
C. parapsilosis
B. cinaerea
M. gipsum
1
13.7 a
35.4 a
13.7 b
ꢀ
/
500.00
500.00
500.00
500.00
500.00
500.00
17.3 b
ꢀ/
500.00
500.00
500.00
500.00
500.00
500.00
17.3 a
ꢀ
/
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
500.00
274 a
2
ꢀ
/
500.00
500.00
500.00
500
ꢀ
/
500.00
500.00
500.00
500.00
500.00
17.3 a
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
500.00
500.00
500.00
500.00
500.00
3.5 a
3a
3b
3c
3d
4
ꢀ
/
500.00
500.00
500.00
500.00
17.3 b
29.7 a
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
500.00
173.0 b
500.00
500.00
500.00
500.00
500.00
500.00
500.00
259.0 b
172.0 a
500.00
ꢀ/
ꢀ/
ꢀ/
ꢀ
/
ꢀ/
ꢀ/
5a
5b
5c
5d
5e
6d
6e
7
ꢀ
/
ꢀ
/
500.00
321.0 b
500.00
500.00
500.00
500.00
500.00
2.6 b
ꢀ
/
500.00
312.0 b
500.00
500.00
500.00
500.00
500.00
51.8 b
ꢀ
/
500.00
500.00
500.00
500.00
500.00
500.00
500.00
51.8 b
ꢀ
/
ꢀ
/
500.00
156.0 b
500.00
500.00
500.00
500.00
500.00
5.2 a
124.8 b
27.5 b
500.00
500.00
500.00
500.00
25.9 b
17.2 b
25.7 b
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ
/
ꢀ/
ꢀ
/
8a
8b
17.2 b
51.4 b
34.4 b
17.2 a
ꢀ
/
3.4 a
ꢀ/
ꢀ/
500.00
ꢀ/
500.00
ꢀ/
51.4 a
a
Microbicidal effect.
Microbistatical effect.
b
lanadiazinone (6d) as well as for phenol 8a and its
carbamate 8b. Contrary to highly active ebselen, among
all organoselenium compounds tested, only 4 and 5a
were moderate and 6d weak inhibitors of cytopathic
activity of herpex simplex virus (HSV-1), other were
inactive. None of the compounds tested had appreciable
anti-VSV activity. Only phenol 8a and its derivative 8b
exhibited weak activity against this virus.
Cytotoxicity of the compounds reported in this work,
determined on human A 549 cells, presented in Table 2
falls in a broad range from 2.5 mg/ml for 6d to 275 mg/ml
for low toxic compound 3d. Because of cytotoxicity only
a few compounds*/ebselen (1) and diselenides 3c, 7,
8a,b have appreciable indices ꢀ/1 (inhibiting virus dose
is lower than cytotoxic dose) for EMCV and only
ebselen for HSV-1.
Table 2
Virucidal activity of organoselenium compounds 1ꢀ
/
8
EMCV
MIC b
a
Compound
Cytotoxicity
HSV-1
MIC b
VSV
Index c
1.50
Index c
7.50
MIC b
Index c
1
15.0
60.0
20.0
40.0
234.0
275.0
7.0
10.0
2.0
ꢀ
/
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
1000.0
400.0
B
/
0.02
0.06
0.02
0.04
0.23
0.77
0.01
0.03
0.01
0.05
0.01
0.12
0.01
0.12
0.01
0.02
0.04
2
600.0
1000.0
1000.0
100.0
1000.0
10.0
0.10
0.02
0.04
2.34
ꢀ
/
1000.0
1000.0
1000.0
1000.0
1000.0
40.0
B
/
0.06
0.02
0.04
0.23
0.27
0.18
0.30
ꢀ
/
B
/
3a
3b
3c
3d
4
ꢀ
ꢀ/
/
B/
ꢀ
/
B
/
ꢀ
/
B
/
ꢀ
/
B
/
ꢀ
/
B
/
ꢀ
/
B
/
ꢀ
/
B
/
ꢀ/
B/
027
0.70
ꢀ/
B/
ꢀ
/
B
/
ꢀ
/
B
/
5a
5b
5c
5d
5e
6d
6e
7
30.0
10.0
50.0
3.0
200.0
60.0
1000.0
0.15
0.17
0.05
0.30
0.12
0.62
0.12
1.25
1.88
3.75
100.0
600.0
ꢀ
/
B
/
B/002
ꢀ
/
B
/
ꢀ/
B
/
ꢀ
/
1000.0
1000.0
1000.0
400.0
B
/
0.05
0.01
0.12
0.01
0.12
0.01
0.01
0.02
ꢀ
/
B
/
10.0
1000.0
ꢀ
/
B
/
ꢀ
/
B
/
117.0
2.5
ꢀ/
B/
ꢀ/
B
/
ꢀ
/
B
/
4.0
B
/
ꢀ
/
B
/
117.0
10.0
7.5
100.0
8.0
4.0
B/
ꢀ
/
1000.0
1000.0
1000.0
1000.0
B
/
ꢀ
/
B
/
ꢀ
/
B
/
ꢀ/
B
/
8a
8b
ꢀ
/
B
/
B
/
15.0
4.0
ꢀ/
B/
400.0
B/
a
Cytotoxicity determined on human A549 cells (mg/ml).
Minimal inhibiting virus concentration (mg/ml).
b
c
Indexꢁcytotoxicity/MIC.
/

H. Wo´jtowicz et al. / Il Farmaco 58 (2003) 1235ꢀ
/1242
1239
1
It may be concluded that the structural modifications
of organodiselenides induce their improvement in the
antimicrobial and antiviral activity. Some of them
particularly ortho-substituted bisphenyl diselenides
were found to be highly active inhibitors of cytopathic
activity encephalomyocarditis virus EMCV (compound
8a and 8b) and to have activity against broad spectrum
of microorganisms (compounds 4, 8a). It make evidence
that introduction of amino- or hydroxyl-groups in ortho
positions of phenyl rings of bisaryl diselenides plays an
important role in enhancement of the antimicrobial and
antiviral activity. On the contrary, introduction of
sulfonyl group into diselenide molecule or selenenamide
moiety into heterocyclic ring, another than benzisosele-
nazonyl one, had no influence on the antimicrobial or
antiviral activity such modified organoselenium com-
pounds.
cmꢂ1 (Cꢀ
6H, Jꢁ6.6 Hz, CH3), 1.21ꢀ
(quintet, 4H, Jꢁ7.2 Hz, CH2), 2.54 (t, 4H, Jꢁ
CH2), 2.75 (q, 4H, Jꢁ6.5 Hz, CH2), 3.04 (t, 4H, Jꢁ
Hz, CH2), 7.44 (t, 2H, Jꢁ6.0 Hz, SO2NH), 7.79 (dd,
8H, Jꢁ13.8 and 9.0 Hz, ArH), 10.36 (s, 2H, CONH).
/
O), 1153 cmꢂ1 (Sꢀ
1.40 (m, 24H, CH2), 2.08
7.3 Hz,
7.2
/
O): H NMR d 0.89 (t,
/
/
/
/
/
/
/
/
4.1.4. bis(2-Nitrophenyl) diselenide (17)
Compound 17 was obtained by selenenylation of 1-
chloro-2-nitrobenzene (16) with dilithium diselenide in
the way reported in Ref. [15]. Yield 86%, m.p. 219ꢀ
/
220 8C, Ref. [16] 220 8C.
4.1.5. bis(2-Aminophenyl) diselenide (4)
Raney nickel (ca. 10 g) was added to a solution of
diselenide 17 (18.0 g, 45 mmol) in propanol (150 ml),
heated to 95 8C and then hydrazine (15 ml, 300 mmol)
was added dropwise and the mixture was stirred for 3 h.
After additional 2 h the hot reaction mixture was
filtered, the solvent was evaporated in vacuo and the
residue was recrystallized from ethanol; yield 70%, m.p.
83.2 8C, Ref. [17] 83.5 8C.
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from
Aldrich or Fluka. Melting points were determined with
a digital melting point apparatus Electrothermal IA
9100. IR spectra were measured on a Perkinꢀ
/
Elmer
4.1.6. bis[(2-Phenylsulfonylamino)phenyl] diselenide
(5a) and bis[2-(4-methylphenyl-sulfonylamino)phenyl]
diselenide (5b)
1
2000 FT spectrometer in KBr pellets. H NMR spectra
were recorded in DMSO-d6 on a Bruker DRX spectro-
meter 300 MHz. Chemical shifts d are reported in ppm
relative to TMS. Reaction progress was monitored by
thin layer chromatography (TLC) on silica gel 60F254
coated aluminium TLC plates from Merck. Elemental
analyses were performed in our analytical laboratory
and agreed with theoretical values to within 0.3%.
A solution of benzenesulfonyl chloride (3.51 g, 20
mmol) or p-toluenesulfonyl chloride (3.81g, 20 mmol) in
pyridine (5 ml) was added dropwise, to the vigorously
stirred solution of bis(2-aminophenyl) diselenide (4, 3.42
g, 10 mmol) in pyridine (10 ml) and cooled on the ice/
salt bath for 20 min. Then mixture was stirred addition-
ally for 20 h at r.t. After, pyridine was evaporated in
vacuo and the residue was stirred with 5% solution of
hydrochloric acid (15 ml) until the bright-yellow pre-
cipitate was formed. It was filtered and recrystallized
4.1.1. Ebselen (1)
This compound was prepared from anthranilic acid
(9) following the synthetic route described previously
[12].
from ethanol. Compound 5a (89%): m.p. 146ꢀ
IR n 3261ꢀ H), 1581,
3237 cmꢂ1 (NꢀH), 3062 cmꢂ1 (Cꢀ
1471ꢀ CꢀH), 1399ꢀ
1447 cmꢂ1 (Ä 1388, 1332 cmꢂ1
(SO2, sulfonamide), 1168, 1157 cmꢂ1 (SO2, sulfona-
mide): 1H NMR d 6.61 (d, 2H, Jꢁ
7.56 Hz, ArH), 7.11
(t, 2H, Jꢁ7,50 Hz, ArH), 7.18 (t, 2H, Jꢁ7.2 Hz, ArH),
7.49 (d, 2H, Jꢁ7.62 Hz, ArH), 7.59 (t, 4H, Jꢁ7.47 Hz,
ArH), 7.67 (t, 6H, Jꢁ6.84 Hz, ArH), 10.10 (s, 2H,
ArNH): Compound 5b (92%): m.p. 144ꢀ145 8C: IR n
3293ꢀ Har), 2956
3243 cmꢂ1 (NꢀH), 3060 cmꢂ1 (Cꢀ
cmꢂ1 (Cꢀ
Har),
/
147 8C:
/
/
/
4.1.2. 2,2?-Diselenobis[benz(4-sulfonamido)]anilide (2)
The solution of acid chloride 12, obtained in the way
reported in Ref. [13], (0.47 g, 2.3 mmol) sulfanilamide (
0.86 g, 5 mmol) and pyridine (1 ml) in toluene (40 ml)
was refluxed for 4 h until chloride was consumed. The
solvent was evaporated in vacuo and the residue was
washed with 5% HCl and then with water. The crude
product was recrystallized from dimethylformamide;
/
/
/
/
/
/
/
/
/
/
/
/
/
/
yield 62%, m.p. 316ꢀ
/
318 8C, ref. [10] 315ꢀ
/
318 8C.
/
Haliph), 1582, 1472ꢀ
/
1442 cmꢂ1 (Ä
/
Cꢀ
/
1398ꢀ
/
1385, 1337 cmꢂ1 (SO2, sulfonamide), 1156 cmꢂ1
1
(SO2, sulfonamide): H NMR d 2.37 (s, 6H, CH3), 6.63
4.1.3. 4,4?-Diselenobisbutyramides (3aꢀ
/
d)
These compounds were prepared from g-butyrolac-
tone (13) following the procedures described for 3a,b,d
in our earlier paper [10]. Compound 3c (34%): m.p.
(d, 2H, Jꢁ
Hz, ArH), 7.38 (d, 2H, Jꢁ
Jꢁ7.41 Hz, ArH), 7.56 (d, 2H, Jꢁ
(s, 2H, ArNH).
/
7.32 Hz, ArH), 7.00ꢀ
8.13 Hz, ArH), 7.50 (d, 2H,
8.01 Hz, ArH), 9.99
/
7.20 (q, 4H, Jꢁ8.30
/
/
/
/
172ꢀ
/
173 8C (acetonitrile); IR n 3272 cmꢂ1 (Nꢀ
H), 1667
/

1240
H. Wo´jtowicz et al. / Il Farmaco 58 (2003) 1235ꢀ1242
/
4.1.7. bis[(2-Benzoylamino)phenyl] diselenide (5c)
A solution of benzoyl chloride (2.84 g, 20 mmol) in
pyridine (5 ml) was added dropwise to the vigorously
stirred solution of bis(2-aminophenyl) diselenide (4; 3.42
g, 10 mmol) in pyridine (10 ml) and cooled on the ice/
salt bath for 20 min. Then the mixture was stirred
additionally for 20 h at r.t. After, pyridine was
evaporated in vacuo and the residue was stirred with
5% solution of hydrochloric acid (15 ml) until the
bright-yellow precipitate was formed. It was filtered
and recrystallized from ethanol. Compound 5c (90%):
7.6 Hz, ArH), 7.15 (t, 1H, Jꢁ
Jꢁ7.6 Hz, ArH), 7.29 (s, 1H, ArNH); Compound 6e
(52%): m.p. 128ꢀ H), 3061
129 8C: IR n 3179 cmꢂ1 (Nꢀ
cmꢂ1 (CꢀH), 1625 cmꢂ1 (CÄ
S, thiourea), 1466ꢀ1428
(NꢀCSꢀN, thiourea), 1241 (CÄS): H NMR d 6.99ꢀ
7.05 (m, 2H, ArH), 7.27ꢀ7.38 (m, 3H, ArH), 7.57 (d,
1H, Jꢁ8.1 Hz, ArH), 7.82 (t, 3H, Jꢁ8.4 Hz, ArH),
10.43 (s, 1H, ArNH).
/
7.3 Hz, ArH), 7.49 (d, 1H,
/
/
/
/
/
/
1
/
/
/
/
/
/
/
4.1.10. bis[(2-Benzylimino)phenyl] diselenide (7)
A solution of bis(2-aminophenyl) diselenide (4; 3.42 g,
10 mmol) and benzaldehyde (2.12 g, 20 mmol) in
benzene (50ml) was refluxed under azeotropic adapter,
until the appropriate amount of water was formed. The
solvent was evaporated in vacuo and the solid residue
was recrystallized from ethanol. Compound 7 (90%):
m.p. 168ꢀ
cmꢂ1 (Cꢀ
(NꢀH, Cꢀ
NMR d 7.16ꢀ
Hz, ArH), 7.54 (t, 4H, Jꢁ
Jꢁ6.72 Hz, ArH), 7.72 (d, 2H, Jꢁ
(d, 4H, Jꢁ7.65 Hz, ArH), 10.15 (s, 2H, ArNH).
/
169 8C: IR n 3365ꢀ
H), 1677 cmꢂ1 (CÄ
N, amide) 1305ꢀ
1252 cmꢂ1 (amide): 1H
7.21 (m, 2H, ArH), 7.31 (t, 4H, Jꢁ7.30
7.50 Hz, ArH), 7.62 (d, 2H,
7.56 Hz, ArH), 7.97
/
3319 cmꢂ1 (Nꢀ
/
H), 3057
/
/
/
O), 1576ꢀ
/
1519 cmꢂ1
/
/
/
/
/
/
/
/
m.p. 169ꢀ
/
170 8C: IR n 3057 cmꢂ1, 2879 cmꢂ1 (Cꢀ
NÄCHꢀAr), 1565, 1458ꢀ
1437 cmꢂ1 (Ä
H): H NMR d 7.18 (t, 2H, Jꢁ7.53 Hz, ArH), 7.31
(t, 2H, Jꢁ7.14 Hz, ArH), 7.38 (d, 2H, Jꢁ7.77 Hz,
ArH), 7.51 (d, 2H, Jꢁ7.80 Hz, ArH), 7.57 (m, 6H,
4.62 Hz, ArH), 8.80 (s, 2H,
/
H),
/
1621 cmꢂ1 (Arꢀ
/
/
/
/
/
1
Cꢀ
/
/
4.1.8. bis[[2-(N-Propylcarbamoyl)amino]phenyl]
diselenide (5d) and bis[[2-(N-phenyl-
thiocarbamoyl)amino]phenyl] diselenide (5e)
/
/
/
ArH), 8.02 (t, 4H, Jꢁ
/
A mixture of amine 4 (3.42 g, 10 mmol) with propyl
isocyanate (2.55 g, 30 mmol) or with phenyl isothiocya-
nate (4.06 g, 30 mmol) was heated at 80 8C until it
solidified (2 h). The excess of reagent was evaporated in
vacuo and the residue was recrystallized from toluene.
ArNH).
4.1.11. bis[(2-Methoxymethyloxy)phenyl] diselenide
(22)
5d (80%): m.p. 174ꢀ
amide), 3097, 2958, 2931, 2872 cmꢂ1 (Cꢀ
(CÄ
O, urea): 1H NMR d 0.88 (t, 6H, Jꢁ
1.40ꢀ1.47 (m, 4H, CH2), 2.99ꢀ3.31 (m, 4H, CH2N), 6.76
(t, 2H, Jꢁ7.4 Hz, NH) 6.89 (t, 2H, Jꢁ7.5 Hz, ArH),
7.21 (t, 2H, Jꢁ7.5 Hz, ArH), 7.49 (d, 2H, Jꢁ8.0 Hz,
ArH), 7.59 (d, 2H, Jꢁ8.0 Hz, ArH), 8.01 (s, 2H,
ArNH); Compound 5e (82%): m.p. 121ꢀ122 8C: IR n
3179 cmꢂ1 (NꢀH, thioamide), 3047 cmꢂ1 (Cꢀ
Har),
1624 cmꢂ1 (CÄ 1445 cmꢂ1 (Nꢀ
S, thiourea), 1450ꢀ CSꢀ
N, thiourea), 1241 (CÄ 6.99 (m, 4H,
S): 1H NMR d 6.94ꢀ
ArH), 7.23ꢀ7.30 (m, 6H, ArH), 7.45 (d, 4H, Jꢁ7.7 Hz,
ArH), 7.55 (d, 2H, Jꢁ7.7 Hz, ArH), 7.70 (d, 2H, Jꢁ8.1
Hz, ArH), 8.33 (s, 2H, ArNH), 9.23 (s, 2H, ArNH).
/
175 8C: IR n 3306 cmꢂ1 (Nꢀ
H), 1639 cmꢂ1
7.3 Hz, CH3),
/
H
A solution of n-buthyllithium (10 ml, 2.7 M) in
hexane was added dropwise to a vigorously stirred
solution of methoxymethyloxybenzene (18) [18] (2.76
g, 20 mmol) in tetrahydrofuran (50 ml) and cooled on
the ice/salt bath under nitrogen for 10 min. The mixture
was stirred additionally for 2 h while the white solid 19
was precipitated. Finally powdered gray selenium (2.0 g,
25 mmol) was added in three portions to the mixture
and the reaction was continued on the ice/salt bath for 1
h and then at r.t. for additional 1 h. Then, saturated
aqueous ammonium chloride (50 ml) was added and
after stirring for 10 min the layers were separated and
the water layer was shaken with ether. The organic
layers were combined, washed with saturated aqueous
sodium chloride, dried with magnesium sulfate and the
solvent was evaporated in vacuo. The oily crude selenol
21 was dissolved in methanol (25 ml), sodium hydroxide
(one pellet) was added to the stirred solution and stream
of air was passed over the reaction mixture for 3 h. Then
water was added and the mixture was shaken with three
portions of dichloromethane (30, 20 and 20 ml). The
combined extracts were dried with magnesium sulfate,
solvent was evaporated in vacuo and pure compound 22
was isolated from the residue on silica gel column, using
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
4.1.9. 2-Propyl-1,2,4-benzoselenadiazin-3(4H)-one (6d)
and 2-Phenyl-1,2,4-benzoselenadiazin-3(4H)-thione (6e)
A magnetically stirred solution of 5d (0.441 g, 1
mmol) or 5e (0.495 g, 1 mmol) and benzoyl peroxide
(0.27 g, 1.1 mmol) in dry benzene (30 ml) was heated to
80 8C for 20 h. After this period the solvent was
evaporated in vacuo and product 6d or 6e was isolated
from the residue by column chromatography on silica
gel using hexaneꢀ
recrystallized from toluene. Compound 6d (65%): m.p.
106ꢀ H, amide), 3097, 2957,
107 8C: IR n 3305 cmꢂ1 (Nꢀ
2933, 2871 cmꢂ1 (CꢀH), 1664 cmꢂ1 (CÄ
NMR d 0.85 (t, 3H, Jꢁ7.3 Hz, CH3), 1.42ꢀ
2H, CH2), 3.25 (t, 2H, Jꢁ7.3, CH2N), 6.97 (t, 2H, Jꢁ
/
ethyl acetate (4:1) as eluent and
hexaneꢀ
(62%): m.p. 34ꢀ
H), 1575, 1468ꢀ
/
ethyl acetate (9:1) as an eluent. Compound 22
35 8C: IR n 3060 cmꢂ1, 2954 cmꢂ1 (Cꢀ
1439 cmꢂ1 (Ä
CꢀH), 1123, 1083, 987
C): H NMR 3.39 (s, 6H, CH3), 5.30 (s,
4H, CH2), 6.97 (t, 2H, Jꢁ7.50 Hz, ArH), 7.10 (d, 2H,
/
/
/
/
1
/
/
O, urea): H
/
/
/
1
/
/1.55 (m,
cmꢂ1 (Cꢀ
/
Oꢀ
/
/
/
/

H. Wo´jtowicz et al. / Il Farmaco 58 (2003) 1235ꢀ
/1242
1241
Jꢁ
(d, 2H, Jꢁ
/
8.04 Hz, ArH), 7.22 (t, 2H, Jꢁ
/7.17 Hz, ArH), 7.45
agar (dermatophytes) and malt agar (other filamentous
fungi) during cultivation at 25 8C and the diameter of
groving colonies was measured at intervals [19].
/
7.77 Hz, ArH).
4.1.12. bis(2-Hydroxyphenyl) diselenide (8a)
The microbial activity was characterized by MIC
values (minimal concentration of the compounds which
inhibits growth by 100%). The MIC values were read
from toxicity curves. MIC experiments on subculture
dishes were used to assess of the minimal microbicidal
concentration. Subcultures were prepared separately in
Petri dishes containing an appropriate agar medium and
icubated at 30 8C for 2 days (bacteria, yeasts) and at
25 8C for 4 days (filamentous fungi). The minimal
microbicidal concentration value was the lowest con-
centration which completely suppressed growth of
microbial colonies in the subculture dishes.
A stirred mixture of diselenide 22 (2.72 g, 6.3 mmol),
methanol (25 ml) and concentrated hydrochloric acid
was heated to 60 8C for 3 h. Water (50 ml) was added to
the cooled mixture and compound 8a was isolated in the
some manner as described for 22. Finally it was
recrystallized from cyclohexaneꢀ
8a (81%): m.p. 65.0ꢀ
66.5 8C: IR n 3419 cmꢂ1 (Oꢀ
3063 cmꢂ1 (Cꢀ 1450 cmꢂ1 (Ä
H), 1589ꢀ1573, 1463ꢀ
H), 1332, 1182, 1022 cmꢂ1 (Cꢀ
1H, Jꢁ7.53 Hz, ArH), 6.79 (d, 1H, Jꢁ
7.07 (t, 1H, Jꢁ7.83 Hz, ArH), 10.31 (s, 1H, OH).
/
toluene. Compound
H),
Cꢀ
O): H NMR d 6.75 (t,
7.95 Hz, ArH),
/
/
/
/
/
/
/
1
/
/
/
/
4.1.13. bis[2-(N-Propylcarbamate)phenyl] diselenide
(8b)
4.2.2. Cytotoxicity
Cytotoxicity of the compounds was determined in
human lung adenocarcinoma cell line A549 (ATCC 185)
and mouse fibroblast-like cells L929 (ATCC CCL 1).
The experiment was performed in 96-cells microplates.
The cells were treated with various doses of the
compounds for 48 h at 37 8C in the atmosphere of 5%
CO2 in air. Then, the cultures were examined under
microscope and next stained with MTT. The minimal
concentration of a compound which was toxic to
approximately 50% of the cells was taken as TCCD50
[20].
A mixture of bis(2-hydroxyphenyl) diselenide (8a,
3.44 g, 10 mmol) with n-propyl isocyanate (2.55 g, 30
mmol) was heated at 70 8C until it solidified. The excess
of reagent was evaporated in vacuo and the solid residue
was recrystallized from ethanol. Compound 8b (90%):
m.p. 110ꢀ
2971, 2932 cmꢂ1 (Cꢀ
1462ꢀ Cꢀ
1444 cmꢂ1 (Ä
1H NMR 0.88 (t, 6H, Jꢁ
4H, CH2CH3), 3.07ꢀ3.13 (m, 4H, NCH2), 5.08 (s, 2H,
NHCH2), 6.09ꢀ7.09 (m, 4H, ArH), 7.18ꢀ7.23 (q, 2H,
Jꢁ8, 1 Hz, ArH), 7.58 (d, 2H, Jꢁ7.1 Hz, ArH).
/
111 8C: IR n 3318 cmꢂ1 (Nꢀ
H), 1716 cmꢂ1(CÄ
H), 1246, 1206 cmꢂ1 (COO):
7.3 Hz, CH3), 1.44ꢀ1.56 (m,
/
H), 3061 cmꢂ1
,
/
/O), 1546,
/
/
/
/
/
/
/
/
/
/
4.2.3. Antiviral assay
The compounds in various concentrations were in-
cubated with the following viruses: VSV (vesicular
stomatis virus, Rhabdoviridae, enveloped virus),
EMCV (encephalomyocarditis virus, Picornaviridae,
naked virus) and HSV-1 (herpes simplex virus type 1,
Herpesviridae, enveloped virus). Viruses VSV and
EMCV were used at dose of 105 TCID50/ml, HSV-1 at
dose of 104 TCID50/ml. After 2 h incubation at r.t., the
virus titer was measured in human A549 cells and
minimal virus-inhibiting concentration (MIC) was de-
termined.
4.2. Biological activity
4.2.1. Antimicrobial assay
Bacterial strains: Staphylococcus aureus CCM 3953,
Escherichia coli CCM 3988, Candida parapsilosis (re-
ceived from the laboratory of Medical Mycology,
postgraduate Medical Institute, Bratislava), filamentous
fungi Rhizopus oryzae, Alternaria alternata (obtained
from the Collection of Microorganisms of the Slovak
University of Technology), Botrytis cinaerea CCMF 16,
Microsporum gypseum 1/94 (isolated from patient) were
used.
Tested compounds 1ꢀ8 were dissolved in dimethyl
/
sulfoxide, its final concentration in broth medium never
exceeded 1% (V/V) and the same volume of solvent was
present in both control and treated samples. The
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/
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