New organoselenium compounds active against pathogenic bacteria, fungi and viruses

Article abstract of DOI:10.1248/cpb.56.1423

Different N-substituted benzisoselenazol-3(2H)-ones, analogues of ebselen were designed as new antiviral and antimicrobial agents. We report their synthesis, chemical properties as well as study on biological activity against broad spectrum of pathogenic microorganisms (Staphylococcus aureus, Staphylococcus simulans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans, Aspergillus niger) and viruses (herpes simplex virus type 1 (HSV-1), encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV)), in vitro. Most of them exhibited high activity against viruses (HSV-1, EMCV) and gram-positive bacteria strains (S. aureus, S. simulans), while their activity against gram-negative bacteria strains (E. coli, P. aeruginosa, K. pneumoniae) was substantially lower. Some of tested compounds were active against yeast C. albicans and filamentous fungus A. niger.

Full text of DOI:10.1248/cpb.56.1423

October 2008
Chem. Pharm. Bull. 56(10) 1423—1427 (2008)
1423
New Organoselenium Compounds Active against Pathogenic Bacteria,
Fungi and Viruses
a
a
a
⁄
⁄
Magdalena PIE˛TKA-OTTLIK, Halina WÓJTOWICZ-MLOCHOWSKA, Katarzyna KOLODZIEJCZYK,
b
,a
⁄
*
Egbert PIASECKI, and Jacek MLOCHOWSKI
a
⁄
Department of Organic Chemistry, Faculty of Chemistry, Wroclaw University of Technology; Wybrze˙ze Wyspian´skiego 27,
b
⁄
50–370 Wroclaw, Poland: and Institute of Immunology and Experimental Therapy, Polish Academy of Sciences; R. Weigla
12, 53–114 Wroclaw, Poland. Received April 9, 2008; accepted July 10, 2008; published online July 28, 2008
⁄
Different N-substituted benzisoselenazol-3(2H)-ones, analogues of ebselen were designed as new antiviral
and antimicrobial agents. We report their synthesis, chemical properties as well as study on biological activity
against broad spectrum of pathogenic microorganisms (Staphylococcus aureus, Staphylococcus simulans, Es-
cherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans, Aspergillus niger) and viruses
(herpes simplex virus type 1 (HSV-1), encephalomyocarditis virus (EMCV), vesicular stomatitis virus (VSV)), in
vitro. Most of them exhibited high activity against viruses (HSV-1, EMCV) and gram-positive bacteria strains (S.
aureus, S. simulans), while their activity against gram-negative bacteria strains (E. coli, P. aeruginosa, K. pneu-
moniae) was substantially lower. Some of tested compounds were active against yeast C. albicans and filamentous
fungus A. niger.
Key words benzisoselenazol-3(2H)-one; antiviral agent; antimicrobial agent; organoselenium compound; ebselen
Organoselenium compounds are currently an area of inter- molecular interaction, like hydrogen bonds, between the mol-
est for many chemists and medicinal biologists due to their ecule and active centre of the biological receptor can occur.
broad spectrum of biological activity and prospective appli- Moreover, insertion of the polar group can increase the solu-
cations as enzyme inhibitors, antiinflammatory and antitumor bility in water, thus increase the bioavailability and make
agents.1—3) It has been revealed that a simple organoselenium compounds more accessible for in vivo study.
compound 2-phenylbenzisoselenazol-3(2H)-one called ebse-
len (1a) could act against oxidative stress in similar way as a alkylbenzisoselenazol-3(2H)-ones (2), 2-hydroxyalkylben-
common enzyme—glutathione peroxidase (Gpx).4—6)
zisoselenazol-3(2H)-ones and 2-(2,2-dialkoxyalkyl)ben-
For this purpose several organoselenium compounds: 2-
In our previous papers we reported that various 2-substi- zisoselenazol-3(2H)-ones (3), 2-carboxymethylbenzisosele-
tuted benzisoselenazol-3(2H)-ones and related diaryl dise- nazol-3(2H)-one (4a) and the esters (4b, c), 2-aminoalkyl-
lenides exhibited high activity as immunostimulants inducing benzisoselenazol-3(2H)-ones (5) and 2-(N-substituted car-
cytokines such as interferon, tumor necrosis factor and inter- bamoyl)benzisoselenazol-3(2H)-ones (6) were synthesized.
leukin (IL-2) in human peripheral blood leukocytes.7,8) More- All these analogues were screened in the antiviral, antibacte-
over, some of these compounds, particularly their 7-azaana- rial and antifungal assays in vitro.
logues, exhibited high inhibitory activity against pathogenic
bacteria, fungi and viruses.9)
Results and Discussion
In this paper we report synthesis, chemical properties as
Chemistry Since it is believed that Se–N bond in benz-
well as inhibitory activity against viruses, bacteria and fungi isoselenazol-3(2H)-ones is responsible for their biological
of ebselen analogues (2—6), having in 2-position alkyl activity we wanted to check the influence of different sub-
groups (2) or containing oxaalkyl or azaalkyl functions (3— stituents at nitrogen atom on biological activity. We supposed
6). We wanted to answer the question whether the introduc- that character of substituent, mainly polarity and shape could
tion of additional polar functions to alkyl substituent in 2-po- have a significant influence on biological activity. Thus,
sition can have the influence on biological activity of tested among designed N-substituted benzisoselenazol-3(2H)-ones
compounds. It was predicted that in this case an extra inter- there were derivatives with short non-polar groups such as
methyl, ethyl, propyl and butyl, long non-polar chains like
dodecyl and octacedyl which make molecule more lipophilic,
branched non-polar isopropyl, tert-butyl and adamantyl sub-
stituents which can be a steric hinderence. Compounds with
polar substituents containing more polar groups such as hy-
droxyl, carboxyl, amino and carbamoyl and the less polar
like acetal and ester groups fell to the second series. These
groups can act as proton donors or acceptors in hydrogen
bond formation. Some of them like carboxyl and amino
group also can take part in ionic interations, similarly as
polar ammonium salt. In control experiment we used nonsub-
stituted benzisoselenazol-3(2H)-one and ebselen which were
proved to exhibit biological activity. All considered com-
Fig. 1. General Formula of the Analogues of Ebselen 1a
1: 1a: RꢀPh, 1b: RꢀH
2: 2a: RꢀMe; 2b: RꢀEt; 2c: Rꢀn-Pr; 2d: Rꢀi-Pr; 2e: Rꢀn-Bu; 2f: Rꢀt-Bu; 2g:
Rꢀc-Hex; 2h: adamantyl; 2i: Rꢀ(CH2)11CH3; 2j: Rꢀ(CH2)17CH3
3: 3a: RꢀCH2OH; 3b: RꢀCH2CH2OH; 3c: RꢀCH(CH2OH)2 3d: RꢀCH2CH(OEt)2;
3e: RꢀCH(CH3)CH(OMe)2
4: 4a: RꢀCH2COOH ; 4b: RꢀCH2COOCH3; 4c: COOCH2CH3
5: 5a: RꢀCH2CH2N(CH3)2; 5b: CH2CH2N(CH3)3ꢁIꢂ
6: 6a: RꢀCꢀO(NHPr); 6b: CꢀO(NH-c-Hex), 6c: CꢀO(NHPh); 6d: CꢀO(NHPh-
3Cl)
pounds contain endocyclic Se–N bond which is responsible
∗ To whom correspondence should be addressed. e-mail: jacek.mlochowski@pwr.wroc.pl
© 2008 Pharmaceutical Society of Japan

1424
Vol. 56, No. 10
for transformation of thiols to selenosulphides or disul- tested against pathogenic bacteria, yeast and filamentous fun-
phides.10)
gus. The results expressed as minimal inhibitory concentra-
In this work we present two synthetic approaches to N- tion (MIC) values, are shown in Table 1.
substituted benzisoselenazol-3(2H)-ones. First method was
The majority of tested compounds 2—6 was highly active
based on the selenenylation-acylation of the primary amines against gram-positive bacteria strains, particularly S. aureus,
with chloride 10, and the second new one, on the substitution having MIC values in a range 2.0—32.0 mg/ml, close to these
of hydrogen at 2-position of benzisoselenazol-3(2H)-one evaluated for control compounds 1a, b and penicillin G
(1b) in reactions with formaldehyde, ethyl chloroformate and (MICꢀ1.0 mg/ml). Generally, the compounds 2—6 were in-
isocyanates. Ebselen (1a) and unsubstituted benzisoselena- active or weakly active against gram-negative bacteria strains
zol-3(2H)-one (1b) were prepared by the treatment of 2- (E. coli, P. aeruginosa and K. pneumoniae). Only 3a and 3b,
chloroselenobenzoyl chloride (10) with aqueous ammonia or having in 2-position of heterocyclic ring hydroxyl group
aniline, respectively, the same way as reported in our previ- as substituent were moderately active against E. coli
ous works.7) Chloride 10 was obtained in a four-step synthe- (MICꢀ14.0 mg/ml and 29.0 mg/ml). Strong fungicidal activ-
sis starting from anthranilic acid.11) The compound 10 treated ity against yeast strain C. albicans was shown by benziso-
with various primary alkylamines produced 2-alkylbenz- selenazol-3(2H)-ones 2a—h substituted in 2-position with
isoselenazol-3(2H)-ones 2a—j, while treated with 2-amino- alkyl groups (MICꢀ1.0—3.0 mg/ml). The same compounds
ethanol and serinol gave corresponding 2-hydroxyalkylbenz- 2a—h were moderately active against A. niger with MIC val-
isoselenazol-3(2H)-ones 3b and 3c. The reaction of chloride ues in a range of 8.0—28.0 mg/ml.
10 with 2-aminoacetaldehyde diethylacetal and 2-aminopro-
Antiviral Activity and Cytotoxicity All compounds
pionaldehyde dimethylacetal produced compounds 3d and 1—6 were tested as inhibitors of cytophatogenicity of en-
3e. 2-Carboxymethylbenzisoselenazol-3(2H)-one 4a and its cephalomyocarditis virus (EMCV, noneveloped RNA virus),
methyl ester 4b were obtained in the reaction of chloride 10 herpes simplex virus type 1 (HSV-1, enveloped DNA virus)
with glycine and methyl ester of its hydrochloride, respec- and vesicular stomatitis virus (VSV, enveloped RNA virus).
tively. The reaction of chloride 10 with N,N-dimethylethyl- The virus titer was measured in human cell line A549 and
enediamine resulted in 2-aminoethylbenzisoselenazol-3(2H)- MIC (mg/ml) was determined. The results are given in Table
one 5a, which heated in excess of methyl iodide gave com- 2.
pound 5b. For the synthesis of compounds 3a and 6a—d
High activity against HSV-1 and EMCV (MICꢀ4.0—
benzisoselenazol-3(2H)-one (1b) was used as an initial mate- 8.0 mg/ml) similar to the activity of the reference compounds
rial. The reaction of 1b with formaldehyde gave another hy- 1a, b was observed for majority of tested 2-substituted ben-
droxy derivative—2-hydroxymethylbenzisoselenazol-3(2H)- zisoselenazol-3(2H)-ones (2a—h, 3a—d, 4b, c, 5a, 6a, c, d).
one (3a). In the reaction of the compound 1b with different The 2-(2,2-dialkoxyethyl)benzisoselenazol-3(2H)-ones 3d, e
isocyanates new carbamoyl derivatives 6a—d were obtained. and carbamoyl derivatives were moderately active against
Potassium salt of 1b treated with ethyl chloroformate gave EMCV virus (MICꢀ20—60 mg/ml). The long alkyl chains
ester 4c (Fig. 2).
–(CH2)11CH3 and –(CH2)17CH3 made compounds 2i and 2j
Biological Activity The compounds 1—6 were tested as practically inactive against these viruses, probably because of
potential antimicrobial and antiviral agents using in vitro as- their low solubility in culture medium. Almost all tested
says.
compounds 1—6 were completely inactive against VSV, the
Antimicrobial Activity The compounds 1—6 were only one exception was compound 4c.
Fig. 2. Synthesis of the Compounds 1—6

October 2008
1425
Table 1. Antimicrobial Activity of the Organoselenium Compounds 1—6
Bacteria
Compound
Gram-(ꢁ)
No.
Yeasts
C. albicans
Fungi
A. niger
Gram-(ꢂ)
S. aureus
S. simulans
E. coli
P. aeruginosa
K. pneumoniae
1a
1b
2a
2b
2c
2d
2e
2f
34
6.0
7.0
7.0
7.0
7.0
8.0
32
9.0
332
n.a
n.a
2.0
15
8.0
9.0
9.0
32
4
32
55
12
13
14
8.0
15
8.0
8.0
4.0
42
274
25
53
n.a
99
n.a
50
8.0
3.0
3.0
2.0
2.0
2.0
1.0
2.0
2.0
5.0
11
n.a
228
242
272
314
n.a
n.a
135
129
n.a
n.a
9
17
396
27
28
15
15
16
16
9.0
10
n.a
n.a
228
242
n.a
63
300
512
17
129
n.a
n.a
1
162
127
352
424
452
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
512
n.a
n.a
135
206
n.a
n.a
n.a
88
106
226
480
n.a
508
n.a
n.a
n.a
n.a
n.a
27
121
272
n.a
n.a
n.a
270
n.a
135
206
n.a
n.a
n.a
176
57
120
120
32
127
280
n.a
n.a
n.a
14
2g
2h
2i
46
2j
n.a
7.0
8.0
32
39
75
128
34
64
67
26
3a
3b
3c
3d
3e
4a
4b
4c
5a
5b
6a
6b
6c
6d
29
68
n.a
n.a
512
135
129
67
34
51
71
10
40
2.7
51
71
10
10
11
283
n.a
n.a
22
10
79
n.a
MICꢀminimal inhibitory concentration (mg/ml). n.a: non-active, MICꢃ512 mg/ml.
Table 2. Antiviral Activity and Cytotoxicity of the Organoselenium Compounds 1—6
HSV-1
EMCV
VSV
Compound
TCCD50
No.
MIC
I
MIC
10.0
I
MIC
I
1a
1b
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
3a
12.0
3.0
2.5
3.0
3.5
3.5
5.0
6.0
3.0
78
6.0
12.0
4.0
4.0
15.0
7.3
2.5
78
8.0
8.0
8.0
8.0
6.0
6.0
6.0
6.0
6.0
1.5
0.4
0.3
0.4
0.6
0.6
0.8
1.0
0.5
0.8
1.5
ꢄ0.02
1.0
1.0
3.8
1.2
0.3
2.0
1.0
1.7
0.6
3.9
2.5
0.6
5.9
0.7
—
1.2
0.8
0.6
0.8
0.6
0.6
0.8
1.5
0.4
ꢄ0.1
ꢄ0.006
ꢄ0.02
1.0
1.0
1.9
0.4
0.04
2.0
0.8
1.7
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
600
ꢄ0.012
ꢄ0.003
ꢄ0.003
ꢄ0.003
ꢄ0.004
ꢄ0.004
ꢄ0.005
ꢄ0.006
ꢄ0.003
ꢄ0.078
ꢄ0.006
ꢄ0.012
ꢄ0.004
ꢄ0.004
ꢄ0.015
ꢄ0.007
ꢄ0.003
ꢄ0.078
ꢄ0.006
0.028
4.0
4.0
4.0
6.0
6.0
6.0
4.0
8.0
100
4.0
ꢃ500
ꢃ1000
ꢃ1000
ꢃ500
4.0
4.0
4.0
6.0
8.0
40
6
10
2
40
4
60
10
4.0
3b
3c
3d
3e
4.0
8.0
20.0
60.0
40
8
10
10
20
40
100
10
20
—
4a
4b
4c
6.1
17
5a
5b
6a
6b
6c
1.22
156
9.8
34
58.5
4.3
0.1
7.8
0.2
0.3
5.9
0.2
—
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
ꢃ1000
—
ꢄ0.001
ꢄ0.156
ꢄ0.01
ꢄ0.034
ꢄ0.059
ꢄ0.004
—
6d
Acyclovir
6
ꢃ230
ꢃ1000a)
TCCD50ꢀtissue culture cytotoxic dose determined on human A549 cells (mg/ml). MICꢀminimal inhibitory concentration (mg/ml). IꢀTCCD50/MIC. a) Acyclovir was inac-
tive in virucidal assay, but it inhibited viral replication at 20 mg/ml.

1426
Vol. 56, No. 10
2-(1-Adamantyl)benzisoselenazol-3(2H)-one (2h) 75% yield, yellow
prisms, mp 218—221 °C, 1H-NMR (DMSO-d6) d: 1.65—1.74 (6H, m), 2.11
(3H, s), 2.33 (6H, d, Jꢀ2.0 Hz), 7.38 (1H, t, Jꢀ7.4 Hz), 7.57 (1H, t, Jꢀ7.6),
7.73 (1H, d, Jꢀ7.4 Hz), 7.99 (1H, d, Jꢀ8.0 Hz). 13C-NMR (DMSO-d6) d:
29.3, 35.7, 40.7, 58.0, 125.1, 125.5, 126.9, 130.4, 130.9, 138.1, 165.6. 77Se-
The cytotoxic effect of the compounds 1—6 was deter-
mined in human cell line A549 and minimal concentration
which was toxic to approximately 50% of cells was taken as
TCCD50 (mg/ml). Values of the cytotoxicity of synthesized
compounds are presented in Table 2. Generally, they were NMR (DMSO-d6) d: 843. IR (KBr) cmꢂ1: 3054, 2906, 1596, 1445, 1328,
731.
high (2.5—15 mg/ml) with few exceptions for compounds
2h, 4a, 5b, 6b, c.
In consequence, although most of compounds tested
2-(1,3-Dihydroxyisopropyl)benzisoselenazol-3(2H)-one (3c) 79%
1
yield, orange prisms, mp 174 °C, H-NMR (DMSO-d6) d: 3.59—3.63 (2H,
m), 3.66—3.70 (2H, m), 4.52 (1H, q, Jꢀ5.2 Hz), 5.06 (2H, t, Jꢀ5.4 Hz),
against HSV-1 and EMCV exhibited appreciable antiviral ac-
tivity, their high cytotoxicity caused that chemotherapeutic
indices (I) definied as TCCD50/MIC ratio, in most cases were
below 1.0 which means that cytotoxic dose was lower than
inhibitory dose. The compounds 3c, 4a, 4c, 5b and 6c acted
more selectively and their I values were above 1.0 that made
them more prospective antiviral agents.
7.38 (1H, t, Jꢀ7.4 Hz), 7.57 (1H, t, Jꢀ7.6 Hz), 7.82 (1H, d, Jꢀ7.7 Hz), 8.01
(1H, d, Jꢀ8.0 Hz). 13C-NMR (DMSO-d6) d: 56.2, 59.9, 125.2, 125.3, 127.0,
127.6, 131.0, 140.9, 166.8. 77Se-NMR (DMSO-d6) d: 877. IR (KBr) cmꢂ1
3217, 2890, 1604, 1451, 1343, 738.
:
2-(2,2-Diethoxyethyl)benzisoselenazol-3(2H)-one (3d) 87% yield, col-
orless crystals, mp 102—103 °C, 1H-NMR (DMSO-d6) d: 1.16 (6H, t,
Jꢀ7.0 Hz), 3.51—3.56 (2H, m), 3.65—3.70 (2H, m), 3.83 (2H, d,
Jꢀ5.2 Hz), 4.62 (1H, t, Jꢀ5.2 Hz), 7.40 (1H, t, Jꢀ7.4 Hz), 7.60 (1H, t,
Jꢀ7.6 Hz), 7.83 (1H, d, Jꢀ7.7 Hz), 8.03 (1H, d, Jꢀ8.0 Hz). 13C-NMR
(DMSO-d6) d: 15.2, 46.0, 62.2, 99.4, 100.7, 125.4, 125.5, 127.2, 127.3,
131.4, 140.2, 166.6.77Se-NMR (DMSO-d6) d: 903. IR (KBr) cmꢂ1: 3069,
2975, 2884, 1587, 1442, 1370, 1122, 1062, 745.
2-(2,2-Dimethoxy-1-methylethyl)benzisoselenazol-3(2H)-one (3e)
84% yield, colorless crystals, mp 117 °C, 1H-NMR (DMSO-d6) d: 1.18 (3H,
d, Jꢀ6.8 Hz), 3.38 (3H, s), 3.41 (3H, s), 4.43 (1H, d, Jꢀ4.0 Hz), 4.69—4.73
(1H, m), 7.40 (1H, t, Jꢀ7.4 Hz), 7.58 (1H, t, Jꢀ7.6 Hz), 7.81 (1H, d,
Jꢀ7.6 Hz), 8.07 (1H, d, Jꢀ8.0 Hz). 13C-NMR (DMSO-d6) d: 15.2, 49.8,
55.1, 55.6, 105.7, 125.4, 125.6, 127.1, 127.8, 131.2, 140.1, 166.3. 77Se-
Conclusions
It has been shown that the structural modification of parent
ebselen structure by a replacement of phenyl substituent in 2-
position of heterocyclic ring by a small non-polar alkyl
group, plays a crucial role in strong enhancement of antimi-
crobial and antiviral activity. However, high cytotoxicity of
these compounds resulted in undesirable low chemotherapeu-
tic indices (I) values. Generally, heteroatom (nitrogen, oxy- NMR (DMSO-d6) d: 855. IR (KBr) cmꢂ1: 3089, 2931, 2830, 1596, 1446,
gen) built in the 2-substituent didn’t enhance antimicrobial or
antiviral activity significantly, but sometimes diminished cy-
totoxicity that resulted in indices above 1.
1346, 1124, 1055, 740.
2-Carboxymethylbenzisoselenazol-3(2H)-one Methyl Ester (4b) 75%
yield, white powder, mp 141—145 °C, 1H-NMR (DMSO-d6) d: 3.68 (3H, s),
4.56 (2H, s), 7.43 (1H, t, Jꢀ7.4 Hz), 7.64 (1H, t, Jꢀ7.6 Hz), 7.84 (1H, d,
Jꢀ7.7 Hz), 8.07 (1H, d, Jꢀ8.0 Hz). 13C-NMR (DMSO-d6) d: 45.0, 52.4,
126.2, 126.4, 127.4, 127.8, 132.6, 140.8, 167.5, 170.0. 77Se-NMR (DMSO-
Experimental
d6) d: 909. IR (KBr) cmꢂ1: 3073, 2952, 1747, 1595, 1444,1208, 733.
Chemistry All reagents and solvents were purchased from Aldrich and
Fluka. Melting points were determined with a digital melting point appara-
tus Electrothermal IA 9100. IR spectra were measured on a Perkin-Elmer
2-[2-(N,N-Dimethylamino)ethyl]benzisoselenazol-3(2H)-one (5a) 68%
yield, pale yellow powder, mp 89—93 °C, 1H-NMR (DMSO-d6) d: 2.28 (6H,
s), 2.53 (2H, t, Jꢀ5.7 Hz), 3.82 (2H, t, Jꢀ5.6 Hz), 7.37 (1H, t, Jꢀ7.4 Hz),
7.55 (1H, t, Jꢀ7.6 Hz), 7.80 (1H, d, Jꢀ7.7 Hz), 8.00 (1H, d, Jꢀ8.0 Hz). 13C-
NMR (DMSO-d6) d: 40.8, 44.6, 58.1, 125.3, 125.4, 126.8, 127.7, 130.9,
142.1, 166.5. 77Se-NMR (DMSO-d6) d: 890. IR (KBr) cmꢂ1: 3085, 2935,
2852, 2818, 1600, 1445, 751.
1
2000 FT spectrometer in KBr pellets. H-, 13C- and 77Se-NMR spectra were
recorded in DMSO-d6 or CDCl3 on a Bruker DRX spectrometer 300 MHz or
600 MHz. Chemical shifts are reported in ppm relative to TMS or dimethyl
selenide. Reaction progress was monitored by a thin layer chromatography
(TLC) on silica gel 60F254 coated aluminium TLC plates from Merck.
Reaction of Chloride 10 with Amines. General Procedure A solution
of chloride 10 (1.27 g, 5 mmol) in dry acetonitrile (1b, 2a—h, 3b—e, 4a, b)
or dichloromethane (1a, 2i, j, 5a) was added dropwise at room temperature
or in ice/NaCl bath (4b) over 30 min to a stirred solution of corresponding
amine (16.5 mmol) (or its hydrochloride (5 mmol) and triethylamine 1.67 g,
16.5 mmol for 2h, 4b) in dry acetonitrile (1b, 2a—h, 3b—e, 4a, b) or
dichloromethane (1a, 2i, j, 5a) and the reaction was continued for additional
2—16 h. When the reaction was completed, the solvent was evaporated in
vacuo and the crystalline residue was treated with water (100 ml) and stirred
for 2—3 h. The insoluble solid was filtered off, washed with water and dried
in the air. Crude products were recrystallized from methanol (1a, 2h, 4a),
dioksan (1b), acetonitrile (2a, 3c), cyclohexane (2b, d, 5a), hexane (2c),
hexane/toluene (2e—f), methanol/H2O (2g) ethyl acetate/heksan (3d, e) or
purified by chromatography on silica gel with chloroform (2i), dichloro-
methane/ethyl acetate (20 : 1) (2j) or chloroform/acetone (20 : 1) (4b) as
the eluent and then recrystallized from hexane (2i, j) or hexane/chloroform
(4b).
The compound 5b was obtained by heating of 5a with methyl iodide, used
in excess, during 1h. After this time the yellow solid precipitated.
2-[2-(N,N-Dimethylamino)ethyl]benzisoselenazol-3(2H)-one Methyl
1
Iodide (5b) 92% yield, yellow powder, mp 210 °C, H-NMR (DMSO-d6)
d: 3.15 (9H, s), 3.59 (2H, t, Jꢀ6.8 Hz), 4.19 (2H, t, Jꢀ6.7 Hz), 7.44 (1H, t,
Jꢀ7.3 Hz), 7.63 (1H, t, Jꢀ7.6 Hz), 7.83 (1H, d, Jꢀ7.5 Hz), 8.23 (1H, d,
Jꢀ8.0 Hz). 13C-NMR (DMSO-d6) d: 37.3, 52.6, 63.4, 125.9, 126.7, 127.1,
127.3, 131.6, 139.4, 166.7. 77Se-NMR (DMSO-d6) d: 881. IR (KBr) cmꢂ1
3012, 2959, 1618, 1442, 742.
:
Reaction of Benzisoselenazol-3(2H)-one (1b) with Formaldehyde
Benzisoselenazol-3(2H)-one (1b) (0.39 g; 2 mmol) and 38% aqueous solu-
tion of formaldehyde in excess (1.5 ml) were placed in a hermetically closed
test-tube. The reaction was carried out in reflux for 20 min until all 1b com-
pletely dissolved. After cooling, water (2 ml) was added. The insoluble solid
was filtered off, washed with water, dried and recrystallized from chloro-
form to give pure 3a.
2-Hydroxymethylbenzisoselenazol-3(2H)-one (3a) 81% yield, white
Compounds 1a (88% yield, mp 181—182 °C),7) 1b (86% yield, mp 231—
232 °C),7) 2a (84% yield, mp 156—158 °C),7) 2b (68% yield, mp 98—
99 °C),12) 2d (83% yield, mp 99—101 °C),13) 2e (55% yield, mp 90—
91 °C),14) 2f (94% yield, mp 151 °C),7) 2g (96% yield, mp 148—151 °C),13)
2i (96% yield, mp 80—82 °C),7) 2j (95% yield, mp 87—89 °C),7) 3b (85%
yield, mp 148—150 °C),15) 4a (85% yield, mp 185—187 °C)7) have been
known and are reported in references cited.
1
crystals, mp 227—228 °C, H-NMR (DMSO-d6) d: 5.13 (2H, d, Jꢀ7.3 Hz),
6.45 (1H, t, Jꢀ7.3 Hz), 7.42 (1H, t, Jꢀ7.4 Hz), 7.61 (1H, t, Jꢀ7.6 Hz), 7.83
(1H, d, Jꢀ7.7 Hz), 8.05 (1H, d, Jꢀ8.0 Hz). 13C-NMR (DMSO-d6) d: 67.0,
125.7, 125.8, 127.4, 128.2, 131.6, 139.5, 165.9. 77Se-NMR (DMSO-d6) d:
859. IR (KBr) cmꢂ1: 3261, 3059, 2950, 1635, 1597, 1450, 1348, 731.
Reaction of Benzisoselenazol-3(2H)-one Potassium Salt (11) with
Ethyl Chloroformate Benzisoselenazol-3(2H)-one potassium salt (11)
(1.18 g; 5 mmol), ethyl chloroformate in excess (5 ml) and DMF (5 ml) were
heated in reflux for 16 h. Then the mixture was poured into water (100 ml).
The insoluble product was filtered off, dried and recrystallized from ethyl
acetate.
2-Propylbenzisoselenazol-3(2H)-one (2c) 82% yield, yellow powder,
1
mp 78—79 °C, H-NMR (DMSO-d6) d: 0.90 (3H, t, Jꢀ7.4 Hz), 1.59—1.71
(2H, m), 3.68 (2H, t, Jꢀ7.1 Hz), 7.41 (1H, t, Jꢀ7.4 Hz), 7.60 (1H, t, Jꢀ7.6,
1.3 Hz), 7.80 (1H, d, Jꢀ7.6 Hz), 8.04 (1H, d, Jꢀ8.0 Hz). 13C-NMR (DMSO-
d6) d: 11.0, 23.2, 44.8, 125.6, 125.8, 127.2, 128.0, 131.3, 139.0. 77Se-NMR
(DMSO-d6) d: 868. IR (KBr) cmꢂ1: 3090, 2959,1640, 1591, 1444, 1355,
741.
2-Carboxybenzisoselenazol-3(2H)-one Ethyl Ester (4c) 81% yield,
colorless crystals, mp 180.5—181.5 °C, 1H-NMR (DMSO-d6) d: 1.31 (3H, t,
Jꢀ7.1 Hz), 4.30 (2H, q, Jꢀ7.1 Hz), 7.46 (1H, t, Jꢀ7.4 Hz), 7.72 (1H, t,

October 2008
1427
Jꢀ7.6 Hz), 7.87 (1H, d, Jꢀ7.6 Hz), 8.00 (1H, d, Jꢀ8.0 Hz). 13C-NMR
(DMSO-d6) d: 14.1, 62.9, 125.8, 126.3, 128.1, 128.3, 133.8, 139.0, 151.3,
164.4. 77Se-NMR (DMSO-d6) d: 926. IR (KBr) cmꢂ1: 3086, 2992, 1751,
1639, 1446, 1259, 731.
Reaction of Benzisoselenazol-3(2H)-one (1b) with Isocyanates. Gen-
eral Procedure Benzisoselenazol-3(2H)-one (1b) (0.2 g; 1 mmol), corre-
sponding liquid isocyanate in excess (2 ml) and toluene (3 ml) for 6b, d were
placed in a hermetically closed test-tube. The reaction was carried out in re-
flux for 1 h until all 1b completely dissolved. Then the mixture was cooled
down and the insoluble product was filtered off, washed with hexane, dried
and recrystallized from methanol (6a, b) or DMSO/H2O (6c, d).
2-Propylcarbamoylbenzisoselenazol-3(2H)-one (6a) 99% yield, or-
ange crystals, mp 112—114 °C, 1H-NMR (DMSO-d6) d: 0.91 (3H, t, Jꢀ7.4
Hz), 1.52—1.58 (2H, m), 3.26—3.29 (2H, m), 7.48 (1H, t, Jꢀ7.5 Hz), 7.72
(1H, t, Jꢀ7.6 Hz), 7.91 (1H, d, Jꢀ7.7 Hz), 8.07 (1H, d, Jꢀ8.0 Hz), 8.88 (1H,
t, Jꢀ5.7 Hz). 13C-NMR (DMSO-d6) d: 11.1, 22.3, 41.4, 125.9, 126.1, 128.0,
128.9, 133.4, 139.3, 152.7, 165.8. 77Se-NMR (DMSO-d6) d: 906. IR (KBr)
cmꢂ1: 3043, 2934, 1693, 1631, 1444, 1317, 1226, 732.
2-Cyclohexylcarbamoylbenzisoselenazol-3(2H)-one (6b) 96% yield,
white powder, mp 187.5—191.5 °C, 1H-NMR (DMSO-d6) d: 1.21—1.24
(1H, m), 1.29—1.37 (4H, m), 1.50—1.52 (1H, m), 1.63—1.65 (2H, m),
1.84—1.86 (2H, m), 3.65—3.71 (1H, m), 7.46 (1H, t, Jꢀ7.5 Hz), 7.70 (1H,
t, Jꢀ7.7 Hz), 7.89 (1H, d, Jꢀ7.7 Hz), 8.06 (1H, d, Jꢀ8.0 Hz), 8.86 (1H, d,
Jꢀ7.6 Hz). 13C-NMR (DMSO-d6) d: 23.9, 24.9, 32.1, 48.6, 125.9, 126.1,
128.0, 128.9, 133.4, 139.2, 151.7, 166.0. 77Se-NMR (DMSO-d6) d: 907. IR
(KBr) cmꢂ1: 3098, 2921, 1699, 1614, 1533, 1444, 1319, 736.
medium) were incubated with tested compounds at 28 °C for 24 h. After-
wards, small amounts of assay medium were transfered with platinum loop
on Petri dishes containing solid agar medium and incubated at 28 °C for 24
or 48 h. The microbial activity was characterized by MIC value (minimal
concentration of the compound in mg/ml which inhibits growth of the mi-
croorganisms by 100%).
Antiviral Assay The compounds 1—6 at various concentrations (1—
1000 mg/ml) were incubated with following viruses: HSV-1 (herpes simplex
virus type 1, Herpesviridae, enveloped virus), EMCV (encephalomyocarditis
virus, Picornaviridae, non-enveloped virus) and VSV (vesicular stomatitis
virus, Rhabdoviridae, enveloped virus). Viruses EMCV and VSV were used
at the dose of 105 tissue culture infectious dose for 50% (TCID50)/ml, HSV-1
at the dose of 104 TCID50/ml. After 1 h incubation at room temperature, the
virus titer was measured in human cell line A549. Concentration of com-
pound causing 100 times (for HSV-1) or 1000 times (for EMCV and VSV)
decrease of virus titer was taken as minimal inhibitory concentration, MIC
(mg/ml).
Cytotoxicity Cytotoxicity of the compounds 1—6 was determined in
human lung adenocarcinoma cell line A549 (ATCC 185). The experiment
was performed in 96-well microplates. The cells were treated with various
doses of compounds and cultivated for 48 h at 37 °C in the atmosphere of
5% CO2 in air. Then, cultures were examined under microscope. The mini-
mal concentration which was toxic to approximately 50% of cells was taken
as TCCD50.
Acknowledgments This work was supported by PBZ-Min-015/P05/
2004 grant.
2-Phenylcarbamoylbenzisoselenazol-3(2H)-one (6c) 87% yield, color-
less crystals, mp 222—224 °C, H-NMR (DMSO-d6) d: 7.14 (1H, t, Jꢀ7.3
Hz), 7.38 (2H, t, Jꢀ7.8 Hz), 7.52 (1H, t, Jꢀ7.5 Hz), 7.59 (2H, d, Jꢀ8.1 Hz),
7.76 (1H, t, Jꢀ7.6 Hz), 7.97 (1H, d, Jꢀ7.8 Hz), 8.10 (1H, d, Jꢀ8.1 Hz),
11.08 (1H, s). 13C-NMR (DMSO-d6) d: 118.1, 119.7, 124.0, 126.0, 126.4,
128.3, 128.6, 128.7, 129.0, 133.8, 137.1, 139.3, 150.3, 166.4. 77Se-NMR
(DMSO-d6) d: 919. IR (KBr) cmꢂ1: 3076, 1699, 1441, 1301, 744.
2-(3-Chlorophenylcarbamoyl)benzisoselenazol-3(2H)-one (6d) 98%
yield, beige powder, mp 215—218 °C, 1H-NMR (DMSO-d6) d: 7.20 (1H, d,
Jꢀ7.9 Hz), 7.37—7.55 (3H, m), 7.74—7.83 (2H, m), 7.97 (1H, d, Jꢀ7.4
Hz), 8.10 (1H, d, Jꢀ8.1 Hz), 11.16 (1H, s). 13C-NMR (DMSO-d6) d: 116.8,
117.7, 121.6, 125.5, 126.2, 127.1, 127.5, 130.3, 131.4, 133.1, 140.9, 141.4,
152.2, 168.6. 77Se-NMR (DMSO-d6) d: 807. IR (KBr) cmꢂ1: 3058, 2894,
1602, 1559, 1440, 1348, 737.
1
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