Reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with phenols, aminophenols, and other amines towards diphenyl diselenides with antimicrobial and antiviral properties ?

Article abstract of DOI:10.3390/molecules22060974

A reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with various mono and bisnucleophiles such as aminophenols, phenols, and amines have been studied as a convenient general route to a series of new antimicrobial and antiviral diphenyl diselenides. The compounds, particularly bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides and reference benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive bacterial species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against Gram-negative E. coli and fungi (Candida spp., A. Niger). The majority of compounds demonstrated high activity against human herpes virus type 1 (HHV-1) and moderate activity against encephalomyocarditis virus (EMCV), while they were generally inactive against vesicular stomatitis virus (VSV).

Full text of DOI:10.3390/molecules22060974

molecules
Article
Reaction of bis[(2-chlorocarbonyl)phenyl] Diselenide
with Phenols, Aminophenols, and Other Amines
towards Diphenyl Diselenides with Antimicrobial
and Antiviral Properties †
Mirosław Giurg 1,*, Anna Goła˛b 1, Jakub Suchodolski 2, Rafał Kaleta 1, Anna Krasowska 2,
Egbert Piasecki 3 and Magdalena Pie˛tka-Ottlik 4,
*
1
Department of Organic Chemistry, Faculty of Chemistry, Wrocław University of Science and Technology,
˙
Wybrzeze Wyspian´skiego 27, 50-370 Wrocław, Poland; anna_golab@interia.pl (A.G.);
rafal.kaleta@op.pl (R.K.)
2
3
4
Department of Biotransformation, Faculty of Biotechnology, University of Wrocław, Joliot-Curie 14a,
50-383 Wrocław, Poland; jakub.suchodolski@uwr.edu.pl (J.S.); anna.krasowska@uwr.edu.pl (A.K.)
Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12,
53-114 Wrocław, Poland; piasecki@iitd.pan.wroc.pl
Department of Organic and Pharmaceutical Technology, Faculty of Chemistry, Wrocław University of
˙
Science and Technology, Wybrzeze Wyspian´skiego 27, 50-370 Wrocław, Poland
*
Correspondence: miroslaw.giurg@pwr.edu.pl (M.G.); magdalena.pietka@pwr.edu.pl (M.P.-O.);
Tel.: +48-71-320-3616 (M.G.); +48-71-320-2534 (M.P.-O.)
†
This paper is dedicated to Prof. Jacek Młochowski on the occasion of his 80th anniversary.
Academic Editors: Claudio Santi and Luana Bagnoli
Received: 1 May 2017; Accepted: 2 June 2017; Published: 12 June 2017
Abstract: A reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with various mono and
bisnucleophiles such as aminophenols, phenols, and amines have been studied as a
convenient general route to a series of new antimicrobial and antiviral diphenyl diselenides.
The compounds, particularly bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides and reference
benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive bacterial
species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against
Gram-negative E. coli and fungi (Candida spp., A. niger). The majority of compounds demonstrated
high activity against human herpes virus type 1 (HHV-1) and moderate activity against
encephalomyocarditis virus (EMCV), while they were generally inactive against vesicular stomatitis
virus (VSV).
Keywords: ebselen; organoselenium compounds; diselenides; benzisoselenazol-3(2H)-ones;
antimicrobial activity; antiviral activity; morpholine; benzohydrazide; 2-hydroxyacetanilide;
antimycin A
1. Introduction
Organoselenium chemistry and biology have been areas of continuous research over the last
few decades [
synthetic routes [
agents [ 16 22 23]. In the context of biological applications, organoselenium compounds have
received particular attention as glutathione peroxidase (GPx) mimics [ 11 14 24], antioxidant, and
anti-inflammatory agents [10 11 13 23]. Furthermore, a number of selenium-containing compounds
22 25], anti-angiogenic [26], antiviral [27 35], antimicrobial [32 46],
1–
17]. Selenium-containing molecules have been of interest as reagents in new
1
], promising catalysts of various oxidation reactions [ 18 21], and bioactive
1–7, –
8– , ,
9, , ,
,
, ,
have been studied as anticancer [12
,
,
–
–
and antileishmanial agents [47].
Molecules 2017, 22, 974; doi:10.3390/molecules22060974
www.mdpi.com/journal/molecules

Molecules 2017, 22, 974
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The development of novel anti-infective agents is an area of intense activity due to the constant
appearance of new infections, the emergence of multidrug resistance in common pathogens, as well as
the need to eliminate undesirable side effects of available drugs. The urgent need for the next generation
of antimicrobials and antivirals perpetuates research into designing novel molecules and investigating
their mechanisms of action. The tremendous efforts in this area has led to the identification of many
bioactive structures [27
–
46]. Several enzymes, among them thioredoxin reductase [36
,48], bacterial
urease [49], and plasma membrane H+-ATPase [50
–52], were suggested as targets for organoselenium
compounds with antimicrobial properties. Furthermore, it has been reported that the antiviral effects
of organoselenium compounds might be related to the inhibition of hepatitis C virus NS3 helicase [28],
HIV-1 capsid dimerization [29], and nucleocapsid protein 7 [30].
Inspired by the encouraging findings in the area of antimicrobial and antiviral properties of
organoselenium compounds and as a continuation of our interest in the chemistry and biology of
this class of compounds, we aimed to develop a series of new diphenyl diselenides and investigate
their activity against various microbial strains and viruses, along with selected cyclic analogues.
It seemed interesting to focus our attention on a group of 2,2’-dicarbamoylphenyl diselenides
4 as
the most reduced analogues of ebselen, with a phenyl ring containing hydroxy and/or methoxy
moieties. However, the rationale behind studying these compounds was based not only on the fact
that the structural modifications in the diphenyl diselenide scaffold seemed to be interesting from
the medicinal chemistry perspective; we also aimed to investigated a synthetic route to reduced
forms of ebselenols (2-(hydroxyphenyl)-1,2-benzisoselenazol-3(2H)-ones) and its derivatives via the
reaction of bis[(2-chlorocarbonyl)phenyl] diselenide
aminophenols, phenols, and amines.
3 with various mono and bisnucleophiles such as
Up to the present time, several methods have been applied for the preparation of
bis(2-arylcarbamoyl)phenyl diselenides One path is the reductive benzisoselenazolone ring
opening of ebselenols and its derivatives [53], which can be obtained from 2-(chloroseleno)benzoyl
chloride with moderate to excellent yields [54 56]. An alternative route to ebselenols requires
metalation-selenenylation of appropriate benzanilides while protecting the hydroxyl group in
the first step [57 58]. Although the reductive ring opening reaction has been successfully
4
.
–
,
applied for the reduction of ebselen and its various alkyl analogues [59], in the case of
ebselenols, yields were less satisfactory [53]. Only bis[2-(4-hydroxyphenylcarbamoyl)]phenyl
diselenide was prepared in 46% yield while bis[2-(2-hydroxyphenylcarbamoyl)]phenyl diselenide
and 2-(3-hydroxyphenylcarbamoyl)]phenyl diselenide were obtained in very low yields of 8.3% and
12%, respectively [53]. Another, general route to ebselenol-derived diselenides which we present
in this study is the reaction of bis[(2-chlorocarbonyl)phenyl)] diselenide
3 with the corresponding
anilines. In the past, this reaction was successfully applied to obtain various 2,2’-dicarbamoyldiphenyl
diselenides [60]. However, to our best knowledge, there is only one example of a reaction with an
electron-rich arene system bearing a hydroxyl group [26] but with no synthetic details. Thus, we
aimed to investigate the behaviour of bis[(2-chlorocarbonyl)phenyl)] diselenide
3 in the reaction with
N and O nucleophiles, such as aminophenols, phenols, and amines. The aminophenols were of
particular interest as the ones containing two nucleophilic centers in the molecule, with both amine
nitrogen and hydroxide oxygen potentially susceptible toward acylation. The reaction of various O
and N nucleophiles with another selenium-containing chloride, 2-(chloroseleno)benzoyl chloride 9,
showed the preference of a primary amino group toward selenenylation-acylation [55]. As chloride
9
contains both hard and soft electrophilic centers localized on the carbonyl carbon and selenium atoms,
respectively, the results of the reaction with O-nucleophiles strongly depended on the nucleophile
structure. However, in the absence of the soft electrophilic center, no side reactions should occur. Thus,
we expected that the reaction of bis[(2-chlorocarbonyl)phenyl] diselenide
3 with aminophenols could
be considered as a convenient alternative for low-efficient reductive ring opening of ebselenols [53
]
and the preparation from 2,2’-dicarboxydiphenyl diselenide using active ester coupling agents [23
,61],
where the protection of the hydroxyl group would be recommended.

Molecules 2017, 22, 974
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2. Results and Discussion
2.1. Synthesis
Herein we present our investigation of synthetic route to diselenides
4–
8
via a reaction of
(bis[(2-chlorocarbonyl)phenyl] diselenide), hereafter called chloride 3, with N and O nucleophiles,
such as a series of various aminophenols and their derivatives as well as other specific reactants such
as morpholine, benzohydrazide, 2-hydroxyacetanilide, and antimycin A (Scheme 1, Table 1). The key
chloride 3 was prepared in a three-step synthesis starting from anthranilic acid 1, which was diazotized
and reacted with disodium diselenide in a methanolic alkaline medium to give, after acidification,
2,2’-diselenobisbenzoic acid 2 which was further reacted with thionyl chloride [59,60,62].
R1
O
O
O
R2
R3
H
N
N
N
H
N
H
O
O
Se
5
Se
4a–p
Se
2
2
2
6
O
Me
vi
v
vii
viii
HN
O
COCl
COOH
NH2
COOH
i - iii
iv
O
Se
Se
Se 2
2
2
1
2
3
7
CHO
HN
ix
x
O
O
O
R1
COCl
SeCl
X
Me
O
O
xi
R
O
N
Se
O
N
O
Se
:
2
H
R2
Me
Me
10 a-g
9
8
O
O
Me
R: mixture of C3H7, C4H9, C5H11, C6H13
Reagents: i: NaNO2, HCl, -7 - +7oC, ii: Na2Se2, MeOH, NaOH, -7 - +5oC, iii: HCl, iv: 3,5 eq. SOCl2, benzene, Δ, v: Ar-NH2,
CH2Cl2, K2CO3 or Ar-NH2, MeCN, Et3N, vi: morpholine (2.5 eq.), CH2Cl2, vii: benzohydrazide, Na2CO3, CH2Cl2, viii: 2-
hydroxyacetanilide, Na2CO3, CH2Cl2, ix: Antimycin A, Na2CO3, CH2Cl2, x: SOCl2 (excess), Δ, xi: Ar-NH2, Na2CO3,
CH2Cl2 or Ar-NH2, Et3N, MeCN.
Scheme 1. Synthesis of bis(2-carbamoy)diphenyl diselenides 4a–p, 5 and 6, phenolic esters 7–8, and
benzisoselenazol-3(2H)-ones 10a–g.
In a first step, reaction of chloride
3 was tested with various free and protected aminophenols,
such as monohydroxyanilines, monomethoxyanilines, and their derivatives containing additional
substituents in the benzene ring, such as electron-donating methyl and methoxy groups and
electron-withdrawing chloro and carboxymethyl groups. As in the case of 2-(chloroseleno)benzoyl
chloride
9, which was intensively studied with various bisnucleophiles [55], the reaction occurred
on the amine group of both protected (4k
–
p
) and free phenols (4a ). In the IR spectra, there were
–j
observed signals from primary amide bonds at ca. 3300–3500 cm−1 (N–H), carbonyl groups at ca.
1600–1650 cm−1 (C=O), amide groups at ca. 1500–1550 cm−1 (CONH), and in 1H-NMR there were
present protons of N–H group at ca. 9.6–10.5 ppm (see the Supplementary Materials for details).
All compounds were prepared with very good to excellent yields, significantly higher than those
achieved by the reductive ring opening method [53]. The simplest three isomeric aminophenols reacted

Molecules 2017, 22, 974
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with 73–92% yield towards 4a
–
c. Reaction with 2-aminophenols containing electron-withdrawing
substituents (Cl and COOMe) at the vicinity carbon atom generally resulted in slightly lower
yields (4g ; 57–63%), when compared to those containing electron-donating ones (4d ; 60–89%).
As expected [35], methoxy derivatives 4j were also prepared with very good yields (72–99%). As a
–
i
–f
–p
result of our studies, seven new open-chain analogues of ebselenols 4d–j and two new methoxy
derivatives 4n and 4p were developed.
Table 1. Characteristics of compounds 4a–p, 5–8, and 10a–g.
Rx Time
(h)
Yield
(%)
◦
◦
Compound
R1
R2
R3
X
m.p. ( C)
m.p. lit ( C)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
5
6
7
8
10a
10b
10c
10d
10e
10f
10g
2-OH
3-OH
4-OH
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
3-OH
2-OMe
3-OMe
4-OMe
2-OMe
3-OMe
3-OMe
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
96
60
120
24
96
168
96
48
72
120
168
72
92
84
73
60
89
88
59
63
57
94
94
98
94
99
72
76
85
100
36
97
40
65
71
65
70
65
68
233.5–236.5
285.5–287.0
271.0–272.0
221.0–223.5
191.5–194.5
264.0–267.0
230.0–234.0
219.5–221.5
281.0–282.0
232.5–236.5
211.0–214.0
221.0–223.0
285.5–287.5
211.0–213.5
231.5–234.5
193.0–195.0
149.0–152.0
296.5 dec.
178.0–181.0
>300
195.5–197.5
229.0 dec.
189.0–191.5
166.0–168.0
180.0–181.0
240.5–242.5
159.0–161.0
235–238 [53]
285–286 [53]
282–284 [53]
4-Me
5-Me
6-Me
4-Cl
5-Cl
5-COOMe
4-OMe
-
-
-
-
-
-
-
-
-
-
216–217 [53]
221–223 [35]
290–292 [53]
48
168
96
4-Ome
4-OMe
4-OMe
-
235 [53]
5-OMe
96
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
120
120
96
240
20
2
24
2
20
-
-
-
-
-
-
-
2-OH
6-OH
2-OMe
3-OMe
4-OMe
2-OMe
3-OMe
CH
N
CH
CH
CH
CH
CH
194–196 [54]
199–201 [55] a
172–174 [63]
165.5–167.5 [35]
180.5–181.5 [35]
-
-
4-Ome
4-Ome
6
48
-
a
Phase transfer was observed at 199 ◦C.
Reaction of benzoyl chloride
secondary amide bond formation to produce compound
compound containing piperazine moiety was prepared with a comparable yield [64].
Another highly nucleophilic benzohydrazide reacted quantitatively with chloride
nitrogen atom to form bishydrazide . Encouraged by this result, we continued our studies with
3
with secondary amines such as morpholine also resulted in
5
with a very good yield of 85%. A similar
3
on its terminal
6
other N-nucleophiles, focusing on hydrazine, ethylene diamine, tri(hydroxymethyl)aminomethane,
and isoniazyde (isonicotinic hydrazide). The resulting oligomeric products were, however, difficult
to identify.
Further efforts were directed into the investigation of the behavior of aminophenol derivatives
containing blocked amine groups such a 2-hydroxyacetanilide and natural antimycin A. In this case,
reaction occurred on the free hydroxyl group only, resulting in phenolic ester derivatives
however, highly sterically hindered antimycin A required an extremely long reaction time. In the IR
spectrum of compound
, a characteristic phenolic ester bond was observed at 1760 cm−1. Antimycin
7 and 8,
7
A is a mixture of at least four closely related compounds, containing alkyl or acyl substituents of
different lengths or degrees of branching (usually C3–C6) in the dilactone portion of the molecule [65].
Thus, the spectroscopic analysis is complicated and only diagnostic signals could be reported in
13C-NMR spectra. Fortunately, in HRMS spectra, seven diagnostic molecular species (M + Na+)

Molecules 2017, 22, 974
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were observed with R=C5H11 being a predominant one, which was consistent with the results of the
elemental analysis.
Ebselen and several other benzisoselenazol-3(2H)-ones 10a
and 10g, were prepared as a reference for biological tests. The 2,2’-diselenobisbenzoic acid
converted to the 2-(chloroseleno)benzoyl chloride that, upon reaction with corresponding anilines,
formed ebselen and benzisoselenazol-3(2H)-ones 10a through a tandem selenenylation-acylation
sequence (Scheme 1, Table 1) [55 59]. To our best knowledge, compounds 4d 4n 4p 10f are
–
g, including two new molecules, 10f
2
was
9
–
g
,
–j
,
,
,
5–8
,
–g
new structures and were not previously described in the literature.
2.2. Antimicrobial Activity
The in vitro antimicrobial activity of compounds 4–7, 10 was screened against four Gram-positive
bacterial strains (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus hirae),
two Gram-negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa), two yeast-like
fungi (Candida albicans, Candida glabrata), and filamentous fungus (Aspergillus niger) in a range
of 0.125–128
µg/mL. The minimum inhibitory concentration values (MIC) for organoselenium
compounds are summarized in Tables 2 and 3, along with reference substances.
Table 2. Antibacterial activity of compounds 4–7, and 10.
Gram-Positive Bacteria a
Gram-Negative Bacteria a
Compound
S. aureus
S. epidermidis
E. faecalis
E. hirae
E. coli
P. aeruginosa
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
5
6
16
16
16
>128
16
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
8
8
16
128
16
4
2
2
2
1
8
8
8
4
2
>128
>128
>128
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
64
4
2
8
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
128
128
>128
128
>128
128
128
128
128
128
>128
4
>128
>128
>128
>128
>128
8
4
16
4
>128
4
>128
2
1
2
16
4
2
2
>128
>128
>128
>128
>128
>128
64
64
32
>128
8
>128
4
2
8
32
32
4
8
8
7
10a
10b
10c
10d
10e
10f
10g
Ebs
AMC
AMP
CHK
ERM
STR
NAT
a
8
32
>128
>128
64
4
32
128
128
64
>128
128
>128
>128
>128
>128
128
>128
16
2
64
64
32
32
2
>128
4
>128
>128
32
>128
>128
>128
>128
128
>128
>128
16
128
>128
>128
>128
>128
>128
128
>128
>128
>128
64
32
16
>128
32
MIC—Minimum Inhibitory Concentration
(µg/mL); AMC—antimycin A; AMP—ampicillin;
CHK—chloramphenicol; ERM—erythromycin; STR—streptomycin; NAT—nourseothricin.

Molecules 2017, 22, 974
6 of 22
Table 3. Antifungal activity of compounds 4–7, and 10.
Yeasts a
Fungi a
Compound
C. albicans
C. glabrata
A. niger
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
5
6
>128
>128
>128
>128
32
>128
>128
>128
>128
32
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
-
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
16
>128
32
>128
>128
64
64
64
>128
>128
>128
64
-
-
7
10a
10b
10c
10d
10e
10f
10g
Ebselen
AMC
KLK
ITC
NAT
AMB
>128
>128
64
-
-
64
-
16
>128
-
>128
16
>128
64
16
32
32
>128
>128
>128
64
-
-
-
1
1
a
MIC—Minimum Inhibitory Concentration (µg/mL); AMC—antimycin A; KLK—ketoconazole; ITC—itraconazole;
NAT—nourseothricin; AMB—amphotericin B.
Generally, there is a noticeable tendency that the compounds tested have been more active against
Gram-positive bacterial strains than against Gram-negative ones. These results are in line with our
previous observations [33–35] and might indicate the possible mode of action of the whole group.
Gram-positive bacteria are more susceptible to plasma membrane-binding agents, absorbing them
directly into the plasma membrane, whereas a Gram-negative outer membrane may capture such
agents before they penetrate through a cell wall and bind into the inner membrane, which usually
results in subsequent cell permeabilization [66]. Using propidium iodide, no significant plasma
membrane permeabilization was observed in S. aureus after treating with ebselen [67], however,
membrane binding is not always sufficient for its permeabilization [68]. Membrane binding agents
alter plasma membrane biophysics, which may be toxic itself.
The antibacterial activity towards Gram-positive species varied in a range of 1 to >128
Particularly interesting is the strong activity towards E. faecalis and E. hirae. These enterococci display
highest vulnerability to most of the tested compounds and only compounds 4h and (as well as
µg/mL.
–l,
5
7
4m in the case of E. hirae) exhibit no activity in the tested range of concentrations. Enterococci seem
to be significantly more susceptible towards organoselenium compounds than reference antibiotics,
with the exception of 4o and 10d, displaying same MIC as ampicillin towards E. faecalis. This makes
organoselenium compounds promising for the treatment of enterococci infections, which although

Molecules 2017, 22, 974
7 of 22
being low virulent bacteria, often cause infections in immunocompromised patients [69]. In the case of
staphylococci, it can be noticed that compounds 4a and 10a , containing hydroxyl substituent in the
phenyl ring, were the most active against both S. aureus and S. epidermidis. Benzisoselenazol-3(2H)-ones
10a and 10b were slightly more active than their open-chain analogues 4a . Only two compounds, 4e
–
c
–b
–
c
and 10a, showed inhibitory effect against E. coli, similar to commonly known antibiotics. None of the
compounds showed activity towards P. aeruginosa in the screening range. However, it is worth pointing
out that this microorganism was also not susceptible toward the majority of reference antibiotics in the
tested range.
Contrary to their cyclic analogues, diselenides appeared to be practically inactive towards both
Candida spp., with the exception of compound 4e, which also displayed a high inhibitory effect against
Gram-positive bacteria and moderate effects against Gram-negative E. coli (Table 1). The probable lack
of inhibitory effect of the majority of compounds tested might be due to the fact that the antifungal
activity of diphenyl diselenides is dependent on the presence of functional groups [40]. However,
diphenyl diselenides may still display high activity in reducing germ tube formation, one of the
crucial virulent factors of C. albicans [38]. Azoles (ketoconazole and itraconazole), commonly used
in antifungal therapy, also did not show activity under the test conditions. However, these two
compounds are considered static drugs [70], therefore it is difficult to obtain MIC90 values for them.
Thus, it is possible that organoselenium compounds which lack inhibitory effects may still display
synergistic effects. Benzisoselenazol-3(2H)-ones 10a
, 10c, 10f–g, and 10e (in the case of C. albicans
only) inhibited fungal growth in a range of 16–64 g/mL (Table 2), comparable to nourseothricin.
µ
Amphotericin B exhibited a much higher activity than all the benzizsoelenazol-3(2H)-ones tested,
however, this highly cytotoxic compound is considered as a drug of choice for the most severe fungal
infections, when the patients are close to death [71]. The majority of diselenides have shown no activity
against A. niger in the tested range. Only compound 4e showed weak activity against this filamentous
fungus. Benzisoselenazol-3(2H)-ones 4c and 4f showed moderate activity towards A. niger, comparable
to other previously tested cyclic analogues of ebselen [33,34].
The compound 5, prepared by reaction with a secondary amine, as well as phenolic ester 7 were
inactive against all tested microorganisms.
2.3. Antiviral Activity
The antiviral activity of compounds 4, 5, 7, and 10 has been evaluated in vitro towards HHV-1
(human herpes virus type 1, Herpesviridae, enveloped virus), EMCV (encephalomyocarditis virus,
Picornaviridae, non-enveloped virus), and VSV (vesicular stomatitis virus, Rhabdoviridae, enveloped
virus) in the human cell line A549. The minimum inhibitory concentrations (MICs) for the
abovementioned viruses along with cytotoxicity against the testing cell line are summarized in Table 4.
The compounds exhibited high antiviral activity against HHV-1 and moderate to high activity
against EMCV, but in almost all cases were inactive towards VSV (Table 4). A similar tendency was
observed in our previous studies [32
of 2–40 g/mL; only the one containing morpholine moiety showed lower activity at 200
The benzisoselenazol-3(2H)-ones also showed high activity against HHV-1, in a range of 2–4
comparable to ebselen. The most promising anti-HHV-1 agents would be compounds 4k
–35]. The anti-HHV-1 activity of most diselenides was in a range
µ
µ
g/mL.
g/mL,
µ
,
4n, and
10f with higher chemotherapeutic indices (I), indicating that the concentration active against the
virus was lower than that showing a cytotoxic effect. Up to the present time, there were only several
organoselenium compounds identified with such high chemotherapeutic indices [33]. Generally, the
compounds were less active against EMCV and only compounds 4e, 4o–p, and 10c showed activity
below 10 µg/mL.

Molecules 2017, 22, 974
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Table 4. Antiviral activity and cytotoxicity of compounds 4, 5, 7, and 10.
HHV-1
MIC b
EMCV
VSV
a
Compound
TCCD50
I c
I c
I c
MIC b
MIC b
4a
4c
4d
4e
4f
4g
4h
4i
4j
4k
4n
4o
4p
5
7
10c
10f
4.9
3.7
4.9
4.9
9.8
4.9
4.9
7.3
4.9
312
58
4.9
2.4
4.9
15
9.8
156
15
40
2
2
2
4
2
6
20
4
6
2
2
4
200
20
4
2
2
0.12
1.8
2.5
2.5
2.5
10
60
>1000
6
>1000
40
>1000
20
0.49
0.06
<0.005
0.82
<0.01
0.12
<0.005
0.37
0.25
0.78
0.10
0.61
0.30
0.49
0.04
1.2
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
200
<0.005
<0.004
<0.005
<0.005
<0.01
<0.005
<0.005
<0.07
<0.005
<0.31
<0.06
<0.005
0.01
2.5
0.82
0.37
1.2
52
29
20
400
600
8
8
10
400
8
200
10
2.5
0.61
0.02
0.75
2.5
78
7.5
>1000
600
>1000
>1000
>1000
<0.005
0.03
<0.01
<0.16
<0.02
0.78
1.5
Ebselen
a
b
TCCD50—Tissue Culture Cytotoxic Dose (
I = TCCD50/MIC.
µg/mL); MIC—Minimum Inhibitory Concentration (µg/mL);
c
3. Materials and Methods
3.1. Synthesis
General Information
Melting points were determined on an Electrothermal IA 91100 digital melting-point apparatus
using the standard open capillary method. 1H- and 13C-NMR spectra were recorded in CDCl3 or
DMSO-d6 on a Bruker DRX 300 Spectrometer (Bruker, Rheinstetten, Germany) (1H: 300.1 MHz, 13C:
75.4 MHz), on a Joel 400 Spectrometer (Joel, Tokyo, Japan) (1H: 399.8 MHz, 13C: 100.5 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 tetramethylsilane (TMS, Si(CH3)4), 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. FT-IR spectra were recorded between 4000 and 400 cm−1
on a Perkin-Elmer 2000 FT-IR spectrometer. High-resolution mass spectra (HRMS) were recorded on a
Waters LCD Premier XE instrument (Waters, Manchester, UK), and only the [M + H]+ or [M + Na]+
diagnostic molecular species are reported. Preparative column chromatography was performed on
Merck Si60 silica gel (63–200 µm) (Sigma-Aldrich, Saint Louis, MO, USA) as the stationary phase.
Analytical thin-layer chromatography (TLC) was performed on PET foils precoated with silica gel
(Merck silica gel, 60 F254) (Sigma-Aldrich, Saint Louis, MO, USA), and visualised using UV light
(λ
max
= 254 nm), or by staining with iodine vapours. Selenium powder (100 mesh) (Sigma-Aldrich,
Saint Louis, MO, USA) used for Na2Se2 preparation had a purity of
≥
99.5%. Acetonitrile (MeCN)
(POCH, Gliwice, Poland) and methylene chloride (CH2Cl2) (POCH, Gliwice, Poland) were distilled
over P2O5, and CH2Cl2 was stored over sodium hydride (NaH) pills. Triethylamine (Et3N) (POCH,
Gliwice, Poland), distilled over NaOH, was stored over NaOH pellets. Methanol (MeOH) (POCH,
Gliwice, Poland) was refluxed over magnesium shavings (Mg) in the presence of elemental iodine
(I2) and distilled over Mg(OMe)2 was formed and stored over 3A molecular sieves (Sigma-Aldrich,
Saint Louis, MO, USA). Diethyl ether (DEE) (POCH, Gliwice, Poland) was distilled over a CaH2 and
◦
LiAlH4 mixture from water batch at ca. 45 C. Morpholine (POCH, Gliwice, Poland) was distilled over
NaOH pellets (POCH, Gliwice, Poland) before use. Anhydrous sodium carbonate (Na2CO3) (POCH,
Gliwice, Poland) was ground in a mortar before use. Antimycin A natural product (Sigma-Aldrich,

Molecules 2017, 22, 974
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Saint Louis, MO, USA) was used as a mixture of co-crystalized n-alkylated compounds with R = C3–C6
predominantly. 2-Hydroxy-5-carboxymethylaniline was prepared from 3-amino-4-hydroxybenzoic
acid (Sigma-Aldrich, Saint Louis, MO, USA) by direct metoksylation in a hot methanol environment
and in the presence of H2SO4 (POCH, Gliwice, Poland), as mentioned below. Ebselen was prepared
according to the procedure described earlier [59]. Other reagents and starting materials were directly
used as obtained commercially. Purity of the products was confirmed by the comparison of their
melting point with data given in literature and by spectroscopic methods. The new compounds were
fully characterized.
2-Hydroxy-5-carboxymethylaniline. To 2-amino-4-hydroxybenzoic acid (1.53 g, 10 mmol) in dry MeOH
(25 mL) was added H2SO4 (1.0 mL, 1.8 g, 19 mmol) portionwise during stirring, and the solution formed
was stirred in solvent reflux (+65 ◦C) for 20 h. The reaction mixture was cooled and poured into 0.5%
NaOH (200 mL) and kept in an ice/water bath, NaHCO3 (3.6 g, 44 mmol) was added and the mixture
was washed with DEE (100 mL) followed by the addition of 3.5% HCl to a pH of 8.0, and was then
extracted with degased DEE. The collected DEE solution was washed with water, dried over Na2SO4,
and the solvent was distilled off from a water bath to obtain crude 2-hydroxy-5-carboxymethylaniline.
◦
◦
−1
Yield: (1.60 g, 96%); m.p.: 114.5–115.0 C (110 C [72]); as prisms (benzene, 40 mL/g). IR (KBr, cm ) ν
3390, 3300 (NH), 2500–3200 (br OH), 1702 (C=O), 1610, 1429, 1306, 1294, 1284 (C–N), 1201 and 1112
1
(C–O), 858, 764, 421. H-NMR (300 MHz, CDCl3:DMSO-d6, 5:1, v/v):
δ 3.81 (s, 3H, OCH3), 4.24 (br s,
2H, NH2), 6.75 (d, J = 8.2 Hz, 1H, Ar-H), 7.24 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H, Ar-H), 7.33 (d, J = 2.0 Hz,
1H, Ar-H), 9.50 (br s, 1H, OH).
2,2’-Diselenobisbenzoic acid
2. The compound was prepared from anthranilic acid
1 using disodium
diselenide with a modified literature procedure [59
,
60 62]. The anthranilic acid (20.5 g, 0.15 mol) was
,
1
dissolved in a solution of 37% aq. HCl (30 mL) in distilled water (90 mL) at ca. 75 ◦C. The solution
was cooled in an ice-water bath (0–5 ◦C) to form anthranilic acid hydrochloride precipitate. To the
resulting thick suspension, a cold solution of NaNO2 (11.4 g, 0.165 mol) in distilled water (90 mL) at ca.
−
5 ◦C was added while stirring for 0.5 h, and the reaction was continued for an additional 15 min.
The prepared cold solution of anthranilic acid diazonium chloride was added dropwise for 2.5 h to
◦
the freshly prepared methanolic solution of disodium diselenide (NaSeSeNa) at
−
10–7 C during
stirring with nitrogen evolution accompanied by the formation of red selenium . The reaction mixture
was warmed to RT (room temperature) and stored overnight. (NaSeSeNa solution preparation: to
the suspension of selenium element (11.2 g, 0.15 mol) in NaOH (18 g, 0.45 mol) solution with MeOH
(300 mL) in a round-bottom flask equipped with an oil closure, hydrazine monohydrate (1.9 mL,
0.040 mol) was added and gently stirred at RT for 60 h.) The formed grey selenium was filtered,
washed with water, and air-dried to obtain selenium powder (6.20 g, 0.083 mol) with 55% yield, which
can be reused. The collected filtrates were acidified with 37% aq. HCl (25 mL), gently heated with
mixing and left at RT overnight. The solid was filtered off, washed with hot water (ca. 1 L) (until no
odor of salicylic acid in the filtrate was detected), and air-dried. The crude product was recrystallized
from hot 1,4-dioxane (400 mL) by slow concentration under ca. 200 mmHg to give acid
37%); m.p.: 298–300 ◦C (300–303 ◦C [59]); as a pale brick powder (1,4-dioxane).
2. Yield: (11.1 g,
Bis[(2-chlorocarbonyl)phenyl)] diselenide 3. To 2,2’-diselenobisbenzoic acid (2) (20 g, 50 mmol) suspended
in benzene (300 mL) in a round-bottom flask equipped with an oil closure and gas trapping, thionyl
◦
chloride (SOCl2) (15.5 g, 0.13 mol) was added. The mixture was gently refluxed at ca. +80 C
with stirring for 4 h and the evolution of gaseous HCl and SO2, following the method previously
reported [60]. Benzene and excess of SOCl2 were removed under reduced pressure, then the residue
was washed with dry n-hexane (3
×
25 mL) and crystallized from dry CH2Cl2 (15 mL/g) to obtain 3.
Yield: (16.4 g, 75%); m.p.: 174–175 ◦C (m.p.: 174–175 ◦C [60]); as green yellow prisms.
General procedure for the synthesis of bis(2-carbamoylaryl)phenyldiselenides 4a . To a stirred mixture
–p
of substituted aniline (1.05 mmol) and sodium carbonate (Na2CO3) powdered in a mortar (0.265 g,

Molecules 2017, 22, 974
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2.5 mmol) in dry CH2Cl2 (5 mL), the solution of bis[(2-chlorocarbonyl)phenyl)] diselenide (
3
) (0.218 g,
0.50 mmol) in dry CH2Cl2 was added dropwise for 30 min at RT. The reaction progress was monitored
using TLC (CHCl3:EtOAc, 4:1, v/v). The stirring was continued for 24–168 h. Over a 3-day period,
chloride
3 (10.9 mg, 25.0 µmol) was added. When the reaction was completed, the product was filtered
off and washed with CH2Cl2 (2
obtain products 4a–p.
×
5 mL), water (5 mL), and 3.5% aq. HCl (3
×
5 mL), and air-dried to
Bis[2-(2-hydroxyphenylcarbamoyl)]phenyl Diselenide 4a
.
The general procedure starting from
2-hydroxyaniline (0.115 g, 1.05 mmol) at RT was employed, with a 96-h reaction time to obtain
4a. Yield: (0.28 g, 92% calculated on 2-hydroxyaniline used); m.p.: 233.5–236.5 ◦C (235–238 ◦C [53]);
as a white solid (MeOH, 0.45 g/L). IR (KBr, cm−1
(CONH), 1456, 1351, 1286 (C–N), 1235 (C–O), 1194, 1029, 757, 729, 622 (C–Se), 594. H-NMR (300 MHz,
)
ν
3403 (NH), 3176 (br OH), 1636 (C=O), 1549
1
DMSO-d6):
δ 6.86 (ddd, J = 7.8 Hz, J = 7.3 Hz, J = 1.0 Hz, 2H, Ar-H), 6.95 (dd, J = 7.5 Hz, J = 1.0 Hz,
2H, Ar-H), 7.09 (ddd, J = 7.5 Hz, J = 7.3 Hz, J = 1.5 Hz, 2H, Ar-H), 7.40 (ddd, J = 7.3 Hz, J = 6.5 Hz,
J = 1.5 Hz, 2H, Ph-H), 7.46 (ddd, J = 7.8 Hz, J = 6.5 Hz, J = 1.5 Hz, 2H, Ph-H), 7.63 (dd, J = 7.8 Hz,
J = 1.5 Hz, 2H, Ar-H), 7.79 (dd, J = 7.8 Hz, J = 1.5 Hz, 2H, Ph-H), 8.03 (dd, J = 7.3 Hz, J = 1.5 Hz, 2H,
Ph-H), 9.76 (s, 2H, NH), 9.78 (s, 2H, OH). 13C-NMR (75 MHz, DMSO-d6):
δ
115.8 (2
×
CH), 118.9 (2
×
CH), 125.1 (2
×
CH), 126.2 (2
×
CH), 126.3 (2
×
CH), 128.4 (2
×
CH), 130.1 (2 CH), 132.0 (2
×
×
C),
132.1 (2 CH), 132.2 (2
×
×
C), 133.0 (2
×
C), 150.0 (2
×
C), 166.1 (2
×
C=O). HRMS (TOF MS ESI): m/z
for C26H20N2O4Se2 + Na+ calculated: 604.9654; found: 604.9662.
Bis[2-(3-hydroxyphenylcarbamoyl)]phenyl Diselenide 4b
3-hydroxyaniline (109 mg, 1.00 mmol) in anhydrous MeCN (5 mL) and benzoyl chloride
.
The general procedure starting from
in anhydrous
3
MeCN (10 mL) at RT was employed, with a 60-h reaction time followed by the dropwise addition of
H2O (10 mL) during stirring. Solid precipitate was filtered off, washed with aq. HCl and EtOH:H2O
◦
◦
(19:1, v/v), and air-dried to obtain 4b. Yield: (244 mg, 84%); m.p.: 285.5–287.0 C (m.p.: 285–286 C [53]);
as a pale yellow powder. H-NMR (400 MHz, DMSO-d6):
1
δ 6.51–6.59 (m, 2H, Ar-H), 7.11–7.19 (m,
4H, Ar-H), 7.37–7.39 (m, 2H, Ar-H), 7.40 (ddd, J = 7.5 Hz, J = 7.4 Hz, J = 1.0 Hz, 2H, Ph-H), 7.46 (ddd,
J = 7.9 Hz, J = 7.4 Hz, J = 1.4 Hz, 2H, Ph-H), 7.79 (dd, J = 7.9 Hz, J = 1.0 Hz, 2H, Ph-H), 7.93 (dd,
J = 7.5 Hz, J = 1.4 Hz, 2H, Ph-H), 9.50 (s, 2H, OH), 10.45 (s, 2H, NH). 13C-NMR (101 MHz, DMSO-d6):
δ
107.56 (2
131.93 (2
×
CH), 111.23 (4
CH), 131.95 (2
×
CH), 126.38 (2
C), 133.93 (2
×
CH), 128.60 (2
×
CH), 129.35 (2
×
CH), 130.15 (2
×
CH),
×
×
×
C), 139.65 (2 C), 157.55 (2
×
×
C), 166.23 (2
×
C=O). HRMS
(TOF MS ESI): m/z for C26H20N2O4Se2 + Na+ calculated: 604.9654; found: 604.9660. Anal. Calcd. for
C26H20N2O4Se2 (582.37): C, 53.62; H, 3.46; N, 4.81. Found: C, 53.87; H, 3.25; N, 4.89.
Bis[2-(4-hydroxyphenylcarbamoyl)]phenyl Diselenide 4c
.
The general procedure starting from
4-hydroxyaniline (0.115 g, 1.05 mmol) at RT was employed, with a 120-h reaction time to obtain
4c as a beige powder. Yield: (222 mg, 73% calculated on the 4-hydroxyaniline used). An analytically
pure sample was obtained by column chromatography on silica gel using a mixture of CHCl3 and
EtOAc (4:1, v/v) as an eluent to obtain pure 4c; m.p.: 271–272 ◦C (282–284 ◦C [53]); as a pale solid
(CH2Cl2). IR (KBr, cm−1
1352, 1280 (C–N), 1235 (C–O), 828, 732, 670 (C–Se), 517. H-NMR (300 MHz, DMSO-d6):
)
ν
3260 (br NH), 3071 (br OH), 1640 (C=O), 1619, 1589, 1510 (CONH), 1444,
1
δ 6.83 (d,
J = 8.7 Hz, 4H, Ar-H), 7.35 (d, J = 8.7 Hz, 4H, Ar-H), 7.46 (dd, J = 7.5 Hz, J = 7.2 Hz, 2H, Ph-H), 7.66
(dd, J = 8.0 Hz, J = 7.2 Hz, 2H, Ph-H), 7.87 (d, J = 7.5 Hz, 2H, Ph-H), 8.06 (d, J = 8.0 Hz, 2H, Ph-H),
9.60 (s, 4H, 2
×
NH and 2
CH), 126.70 (4
C), 155.66 (2
×
OH). 13C-NMR (75 MHz, DMSO-d6):
δ
115.49 (4
×
CH), 125.67 (2
C), 131.84 (2
×
CH),
CH),
126.04 (2
138.91 (2
×
×
CH), 127.74 (2
×
CH), 128.21 (2
×
C), 130.53 (2
×
×
×
×
C), 164.82 (2
×
C=O). HRMS (TOF MS ESI): m/z for C26H20N2O4Se2 + Na+
calculated: 606.9646; found: 606.9676.
Bis[2-(2-hydroxy-4-methylphenylcarbamoyl)]phenyl Diselenide 4d. The general procedure starting from
2-hydroxy-4-methylaniline (129 mg, 1.05 mmol) at RT was employed, with a 24-h reaction time to
◦
obtain 4d. Yield: (183 mg, 60% calculated on benzoyl chloride
3
used); m.p.: 221.0–223.5 C; as a beige

Molecules 2017, 22, 974
11 of 22
powder (CH2Cl2). IR (KBr, cm−1
(C–N), 1228 (C–O), 809, 736, 647 (C–Se), 589. H-NMR (300 MHz, DMSO-d6):
)
ν
3394 (NH), 3250 (br OH), 1640 (C=O), 1603, 1541 (CONH), 1288
1
δ 2.25 (s, 6H, CH3), 6.66
(dd, J = 8.2 Hz, J = 1.2 Hz, 2H, Ar-H), 6.76 (d, J = 1.2 Hz, 2H, Ar-H), 7.39 (ddd, J = 7.2 Hz, J = 7.0 Hz,
J = 1.0 Hz, 2H, Ph-H), 7.43 (d, J = 8.2 Hz, 2H, Ar-H), 7.44 (ddd, J = 7.8 Hz, J = 7.0 Hz, J = 1.6 Hz, 2H,
Ph-H), 7.77 (dd, J = 7.8 Hz, J = 1.0 Hz, 2H, Ph-H), 8.00 (dd, J = 7.2 Hz, J = 1.6 Hz, 2H, Ph-H), 9.60 (s, 2H,
OH), 9.72 (s, 2H, NH). 13C-NMR (75 MHz, DMSO-d6):
CH), 122.5 (2 C), 125.0 (2 CH), 126.3 (2 CH), 128.4 (2
132.2 (2 C), 133.0 (2 C), 135.7 (2 C), 149.9 (2
δ
20.7 (2
×
CH3), 116.4 (2
×
CH), 119.5 (2
×
×
×
×
×
CH), 130.0 (2 CH), 131.9 (2
×
×
CH),
×
×
×
×
C), 166.1 (2
×
C=O). HRMS (TOF MS ESI): m/z
for C28H24N2O4Se2 + Na+ calculated: 634.9959; found: 634.9949. Anal. Calcd. for C28H24N2O4Se2
(610.42): C, 55.09; H, 3.96; N, 4.59. Found: C, 55.18; H, 3.82; N, 4.52.
Bis[2-(2-hydroxy-5-methylphenylcarbamoyl)]phenyl Diselenide 4e. The general procedure starting from
2-hydroxy-5-methylaniline (129 mg, 1.05 mmol) and benzyl chloride
3 (240 mg, 0.55 mmol) at
RT was employed, with a 96-h reaction time ◦to obtain 4e. Yield: (285 mg, 89% calculated on
2-hydroxy-5-methylaniline); m.p.: 191.5–194.5 C; as a beige powder (CH2Cl2). IR (KBr, cm−1
) ν
3409 (NH), 3235 (br OH), 1640 (C=O), 1599, 1547 (CONH), 1504, 1279 (C–N), 1236 (C–O), 1200, 1027,
1
809, 740, 619 (C–Se), 452. H-NMR (300 MHz, DMSO-d6):
δ 2.25 (s, 6H, CH3), 6.83 (d, J = 8.2 Hz, 2H,
Ar-H), 6.89 (dd, J = 8.2 Hz, J = 1.7 Hz, 2H, Ar-H), 7.39 (ddd, J = 7.5 Hz, J = 7.1 Hz, J = 1.0 Hz, 2H,
Ph-H), 7.46 (d, J = 1.7 Hz, 2H, Ar-H), 7.47 (ddd, J = 7.8 Hz, J = 7.1 Hz, J = 1.4 Hz, 2H, Ph-H), 7.78 (dd,
J = 7.8 Hz, J = 1.0 Hz, 2H, Ph-H), 8.00 (dd, J = 7.5 Hz, J = 1.4 Hz, 2H, Ph-H), 9.49 (s, 2H, NH), 9.71 (s, 2H,
OH). 13C-NMR (75 MHz, DMSO-d6):
δ
20.2 (2
CH), 127.5 (2 C), 128.4 (2
C), 166.1 (2 C=O). HRMS (TOF MS ESI): m/z for C28H24N2O4Se2 +
×
CH3), 115.7 (2
×
CH), 124.8 (2
×
C), 125.2 (2
×
CH),
126.4 (2
×
CH), 126.5 (2
×
×
×
CH), 130.1 (2
×
CH), 132.0 (2
×
CH), 132.2
(2 C), 133.0 (2
×
×
C), 147.5 (2
×
×
Na+ calculated: 632.9967; found: 632.9987. Anal. Calcd. for C28H24N2O4Se2 (610.42): C, 55.09; H, 3.96;
N, 4.59. Found: C, 55.01; H, 3.88; N, 4.64.
Bis[2-(2-hydroxy-6-methylphenylcarbamoyl)]phenyl Diselenide 4f. The general procedure starting from
2-hydroxy-6-methylaniline (129 mg, 1.05 mmol) at RT was employed, with a 168-h reaction time to
obtain 4f. Yield: (282 mg, 88% calculated−o1n 2-hydroxy-6-methylaniline used); m.p.: 264–267 ◦C; as a
pale beige powder (CH2Cl2). IR (KBr, cm
1472, 1294 (C–N), 1191 (C–O), 1026, 781, 737, 549 (C–Se). H-NMR (300 MHz, DMSO-d6):
)
ν
3360 (NH), 3251 (OH), 1622 (C=O), 1592, 1515 (CONH),
1
δ 2.19 (s,
6H, CH3), 6.74 (d, J = 7.7 Hz, 2H, Ar-H), 6.78 (d, J = 8.2 Hz, 2H, Ar-H), 7.05 (dd, J = 8.2 Hz, J = 7.7 Hz,
2H, Ar-H), 7.39 (dd, J = 7.6 Hz, J = 6.0 Hz, 2H, Ph-H), 7.45 (dd, J = 6.8 Hz, J = 6.0 Hz, 2H, Ph-H), 7.77
(d, J = 7.6 Hz, 2H, Ph-H), 8.09 (d, J = 6.8 Hz, 2H, Ph-H), 9.39 (s, 2H, NH), 9.78 (s, 2H, OH). 13C-NMR
(75 MHz, DMSO-d6):
127.3 (2 CH), 128.5 (2
(2 C), 153.3 (2 C), 166.5 (2
632.9967; found: 632.9971. Anal. Calcd. for C28H24N2O4Se2 (610.42): C, 55.09; H, 3.96; N, 4.59. Found:
C, 55.16; H, 4.09; N, 4.45.
δ
17.9 (2
CH), 129.9 (2
C=O). HRMS (TOF MS ESI): m/z for C28H24N2O4Se2 + Na+ calculated:
×
CH3), 113.5 (2
×
CH), 120.4 (2
×
CH), 123.4 (2
×
C), 126.1 (2
×
CH),
×
×
×
CH), 131.7 (2
×
CH), 132.4 (2
×
C), 133.0 (2
×
C), 136.8
×
×
×
Bis[2-(2-hydroxy-4-chlorophenylcarbamoyl)]phenyl Diselenide 4g. The general procedure starting from
2-hydroxy-4-chloroaniline (151 mg, 1.05 mmol) at RT was employed, with a 96-h reaction time to obtain
◦
4g. Yield: (202 mg, 59% calculated on 2-hydroxy-4-chloroaniline used); m.p.: 230–234 C; as a brownish
gray powder (CH2Cl2). IR (KBr, cm−1
)
ν
3398 (NH), 3176 (br OH), 1639 (C=O), 1532 (CONH), 1414,
1342, 1265 (C–N), 1226 (C–O), 911, 734, 648, 590 (C–Se), 452. H-NMR (300 MHz, DMSO-d6):
1
δ 6.92 (dd,
J = 8.5 Hz, J = 2.2 Hz, 2H, Ar-H), 6.98 (d, J = 2.2 Hz, 2H, Ar-H), 7.40 (dd, J = 7.2 Hz, J = 7.0 Hz, 2H,
Ph-H), 7.46 (ddd, J = 7.9 Hz, J = 7.0 Hz, J = 1.2 Hz, 2H, Ph-H), 7.63 (d, J = 8.5 Hz, 2H, Ar-H), 7.76 (d,
J = 7.9 Hz, 2H, Ph-H), 8.02 (dd, J = 7.2 Hz, J = 1.2 Hz, 2H, Ph-H), 9.86 (s, 2H, NH), 10.37 (s, 2H, OH).
13C-NMR (75 MHz, DMSO-d6): δ 115.5 (2 × CH), 118.7 (2 × CH), 124.3 (2 × C), 126.4 (2 × CH), 126.6
(2
×
CH), 128.6 (2
×
CH), 129.6 (2
×
×
C), 130.0 (2
×
CH), 132.1 (2
×
CH), 132.3 (2
×
C), 132.7 (2
×
C),
151.3 (2
×
C), 166.2 (2
C=O). HRMS (TOF MS ESI): m/z for C26H18Cl2N2O4Se2 + Na+ calculated:

Molecules 2017, 22, 974
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672.8874; 674.8866; found: 672.8876; 674.8884. Anal. Calcd. for C26H18Cl2N2O4Se2 (651.26): C, 47.95;
H, 2.79; N, 4.30. Found: C, 48.08; H, 2.54; N, 4.50.
Bis[2-(2-hydroxy-5-chlorophenylcarbamoyl)]phenyl Diselenide 4h. The general procedure starting from
2-hydroxy-5-chloroaniline (151 mg, 1.05 mmol) at RT was employed, with a 48-h reaction time to
◦
obtain 4h. Yield: (206 mg, 63% calculated on benzoyl chloride
3
); m.p.: 219.5–221.5 C; as a gray-brown
powder (CH2Cl2). IR (KBr, cm−1
)
ν
3400 (NH), 3218 (br OH), 1638 (C=O), 1596, 1543 (CONH), 1428,
1270, 1239 (C–N), 1195 (C–O), 1027, 731 (C–Cl), 655 (C–Se), 614. H-NMR (400 MHz, DMSO-d6):
1
δ 6.96
(d, J = 8.7 Hz, 2H, Ar-H), 7.13 (dd, J = 8.7 Hz, J = 2.6 Hz, 2H, Ar-H), 7.41 (dd, J = 7.6 Hz, J = 7.3 Hz,
2H, Ph-H), 7.47 (dd, J = 7.9 Hz, J = 7.3 Hz, 2H, Ph-H), 7.77 (d, J = 2.6 Hz, 2H, Ar-H), 7.78 (d, J = 7.6 Hz,
2H, Ph-H), 8.01 (d, J = 7.9 Hz, 2H, Ph-H), 9.82 (s, 2H, OH), 10.18 (s, 2H, NH). 13C-NMR (101 MHz,
DMSO-d6):
δ
116.89 (2
CH), 128.61 (2
C), 166.19 (2
×
CH), 121.93 (2
×
C), 124.06 (2
×
CH), 125.56 (2
×
CH), 126.38 (2
C), 132.68 (2
×
C),
C),
126.41 (2
148.76 (2
×
×
CH), 130.10 (2
×
CH), 132.18 (2
×
CH), 132.30 (2
×
×
×
×
C=O). HRMS (TOF MS ESI): m/z for C26H18Cl2N2O4Se2 + Na+ calculated:
674.8866; found: 674.8862. Anal. Calcd. for C26H18Cl2N2O4Se2 (651.26): C, 47.95; H, 2.79; N, 4.30.
Found: C, 48.03; H, 2.56; N, 4.25.
Bis[2-(2-hydroxy-5-carboxymethylphenylcarbamoyl)]phenyl Diselenide 4i. The general procedure starting
from 2-hydroxy-5-carboxymethylaniline (175 mg, 1.05 mmol) at RT was employed, with a 72-h reaction
time to obtain 4i. Yield: (209 mg, 57% calculated on 2-hydroxy-5-carboxymethylaniline used); m.p.:
281–282 ◦C; as a cream powder (CH2Cl2). IR (KBr, cm−1
1639 (NC=O), 1604, 1537 (CONH), 1430, 1298 (C–N), 1236 (C–O), 1120 (C–O), 737, 606 (C–Se). H-NMR
)
ν
3323 (br NH and OH), 1709, 1691 (OC=O),
1
(400 MHz, DMSO-d6):
δ 3.83 (s, 6H, OCH3), 7.05 (d, J = 8.5 Hz, 2H, Ar-H), 7.41 (ddd, J = 7.6 Hz,
J = 7.3 Hz, J = 1.2 Hz, 2H, Ph-H), 7.48 (ddd, J = 7.9 Hz, J = 7.3 Hz, J = 1.2 Hz, 2H, Ph-H), 7.74 (dd,
J = 8.5 Hz, J = 2.2 Hz, 2H, Ar-H), 7.79 (dd, J = 7.9 Hz, J = 1.2 Hz, 2H, Ph-H), 8.03 (dd, J = 7.6 Hz,
J = 1.2 Hz, 2H, Ph-H), 8.31 (d, J = 2.2 Hz, 2H, Ar-H), 9.89 (s, 2H, NH), 10.86 (s, 2H, OH). 13C-NMR
(101 MHz, DMSO-d6):
δ
51.76 (2
CH), 127.95 (2
C), 154.64 (2 C), 165.83 (2
×
OCH3), 115.58 (2
CH), 128.61 (2
C=O), 166.25 (2
×
CH), 120.14 (2
CH), 130.10 (2
C=O). HRMS (TOF MS ESI): m/z
×
C), 125.09 (2
×
C), 126.27
CH), 132.30
(2
(2
×
CH), 126.42 (2
C), 132.74 (2
×
×
×
×
CH), 132.16 (2
×
×
×
×
×
×
for C30H24N2O8Se2 + Na+ calculated: 720.9763; found: 720.9755. Anal. Calcd. for C30H24N2O8Se2
(698.44): C, 51.59; H, 3.46; N, 4.01. Found: C, 51.43; H, 3.60; N, 3.95.
Bis[2-(3-hydroxy-4-methoxyphenylcarbamoyl)]phenyl Diselenide 4j. The general procedure starting from
3-hydroxy-4-methoxyaniline (146 mg, 1.05 mmol) at RT was employed, with a 120-h reaction time to
obtain 4j. Yield: (317 mg, 94% calculated−o1n 3-hydroxy-4-methoxyaniline used); m.p.: 232.5–236.5 ◦C;
as a white powder (CH2Cl2). IR (KBr, cm
1270 (C–N), 1239 (C–O), 1175 (C–O), 1024 (C–O), 738, 565 (C–Se). H-NMR (300 MHz, DMSO-d6):
)
ν
3500 (NH), 3270 (OH), 1670, 1636 (C=O), 1513 (CONH),
1
δ
3.76 (s, 6H, 2OCH3), 6.91 (d, J = 8.8 Hz, 2H, Ar-H), 7.13 (dd, J = 8.8 Hz, J = 2.5 Hz, 2H, Ar-H), 7.37 (d,
J = 2.5 Hz, 2H, Ar-H), 7.36–7.46 (m, 4H, Ph-H), 7.78 (dd, J = 7.7 Hz, J = 1.2 Hz, 2H, Ph-H), 7.93 (dd,
J = 7.3 Hz, J = 1.4 Hz, 2H, Ph-H), 7.06 (br s, 2H, OH), 10.32 (s, 2H, NH). 13C-NMR (75 MHz, DMSO-d6):
δ
55.8 (2
×
OCH3), 108.9 (2
CH), 131.7 (2 CH), 131.9 (2
C=O). HRMS (TOF MS ESI): m/z for C28H24N2O6Se2 + Na+ calculated: 664.9865; 666.9857;
×
CH), 111.4 (2
×
CH), 112.3 (2
×
CH), 126.3 (2
×
CH), 128.4 (2
×
CH),
130.0 (2
165.8 (2
×
×
×
C), 132.1 (2
×
C), 133.9 (2
×
C), 144.4 (2
×
C), 146.3 (2
×
C),
×
found: 664.9854; 666.9868. Anal. Calcd. for C28H24N2O6Se2 (642.42): C, 52.35; H, 3.77; N, 4.36. Found:
C, 52.30; H, 3.52; N, 4.45.
Bis[2-(2-methoxyphenylcarbamoyl)]phenyl Diselenide 4k
.
The general procedure starting from
2-methoxyaniline (129 mg, 1.05 mmol) at RT was employed, with a 168-◦h reaction time to obtain
◦
4k. Yield: (0.301 g, 94% calculated on 2-methoxyaniline used); m.p.: 214–215 C (m.p.: 216–217 C [53]);
as yellow crystals (MeOH, 2 L/g). IR (KBr, cm−1
)
ν
3411 (NH), 1646 (C=O), 1602, 1528 (CONH),
1462, 1434, 1340, 1289 (C–N), 1251 and 1026 (C–O), 745, 629 (C–Se), 591, 561, 457. H-NMR (300 MHz,
1
DMSO-d6):
δ 3.84 (s, 6H, OCH3), 7.00 (ddd, J = 7.7 Hz, J = 7.4 Hz, J = 1.0 Hz, 2H, Ar-H), 7.13 (dd,

Molecules 2017, 22, 974
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J = 8.3 Hz, J = 1.0 Hz, 2H, Ar-H), 7.24 (ddd, J = 8.3 Hz, J = 7.4 Hz, J = 1.6 Hz, 2H, Ar-H), 7.40 (dd,
J = 7.4 Hz, J = 7.4 Hz, 2H, Ph-H), 7.47 (dd, J = 7.9 Hz, J = 7.4 Hz, 2H, Ph-H), 7.73 (dd. J = 7.7 Hz,
J = 1.6 Hz, 2H Ar-H), 7.77 (d, J = 7.9 Hz, 2H, Ph-H), 8.01 (d, J = 7.4 Hz, 2H, Ph-H), 9.83 (s, 2H, NH).
13C-NMR (75 MHz, DMSO-d6):
126.16 (2 C), 126.39 (2 C), 126.42 (2
(2 C), 132.96 (2 C), 152.04 (2 C), 166.12 (2
literature data [26].
δ
55.56 (2
×
OCH3), 111.56 (2
C), 128.49 (2 C), 130.06 (2
C=O). The HRMS (TOF MS ESI) is in agrement with
×
C), 120.17 (2
×
C), 125.15 (2
×
C),
×
×
×
×
×
C), 132.04 (2
×
C), 132.30
×
×
×
×
Bis[2-(3-methoxyphenylcarbamoyl)]phenyl Diselenide 4l. Preparation according to Reference [35]. The
general procedure starting from 3-methoxyaniline (129 mg, 1.05 mmol) at RT was employed, with
a 72-h reaction time to obtain 4l. Yield: (0.300 g, 98% calculated on benzoyl chloride
3 used); m.p.:
221–223 ◦C (221–223 ◦C [35]); as a colorless powder (CHCl3:EtOAc, 1:2, v/v). Yield of the analytically
1
pure sample: (226 mg, 74%). The H-NMR, 13C-NMR, and HRMS (TOF MS ESI) spectra are in
agreement with literature data [35].
Bis[2-(4-methoxyphenylcarbamoyl)]phenyl Diselenide 4m. Preparation according to a modified literature
procedure [35]. The general procedure starting from 4-methoxyaniline (129 mg, 1.05 mmol) and
bis[(2-chlorocarbonyl)phenyl)] diselenide
3 (0.229 g, 0.525 mmol) at RT was employed, with a 48-h
reaction time to obtain 4m. Yield: (0.300 g, 94%); m.p.: 285.5–287.5 ◦C (290–292 ◦C [53]); as a colorless
powder (CHCl3:EtOAc, 1:2, v/v). Yield of the analytically pure sample: (266 mg, 83%). The 1H-NMR,
13C-NMR, and HRMS (TOF MS ESI) spectra are in agreement with literature data [35].
Bis[2-(2,4-dimethoxyphenylcarbamoyl)]phenyl Diselenide 4n
.
The general procedure starting from
2,4-dimethoxyaniline (161 mg, 1.05 mmol) at RT was employed, with a 168-h reaction time, with
separation by acidification with 3.5% HCl to a pH of 2.5, extraction with CH2Cl2, dry with anhydrous
Na2SO4, filtration, and solvent evaporation to obtain 4n
.
Yield: (348 mg, 99% calculated on
2,4-dimethoxyaniline used); m.p.: 211.0–213.5 C; as a white powder (CH2Cl2). IR (KBr, cm−1
3436 (NH), 1640 (C=O), 1531 (CONH), 1414, 1283 (C–N), 1205, and 1036 (C–O), 831, 732, 578 (C–Se),
◦
)
ν
1
453. H-NMR (300 MHz, DMSO-d6):
δ 3.79 (s, 6H, 2OCH3), 3.82 (s, 6H, 2OCH3), 6.57 (dd, J = 8.8 Hz,
J = 2.6 Hz, 2H, Ar-H), 6.69 (d, J = 2.6 Hz, 2H, Ar-H), 7.35–7.48 (m, 4H, Ph-H), 7.47 (d, J = 8.8 Hz, 2H,
Ar-H), 7.75 (d, J = 7.3 Hz, 2H, Ph-H), 8.01 (d, J = 7.2 Hz, 2H, Ph-H), 9.76 (s, 2H, NH). 13C-NMR (75 MHz,
DMSO-d6):
(2 C), 126.79 (2
153.74 (2 C), 158.28 (2
δ
55.33 (2
C), 128.39 (2
C), 166.20 (2
×
OCH3), 55.69 (2
C), 129.98 (2
C=O). HRMS (TOF MS ESI): m/z for C30H28N2O6Se2 + Na+
×
OCH3), 98.94 (2
×
C), 104.25 (2
×
C), 119.06 (2
×
C), 126.28
×
×
×
×
C), 131.88 (2
×
C), 132.30 (2
×
C), 132.93 (2
×
C),
×
×
×
calculated: 693.0178; found: 693.0176. Anal. Calcd. for C30H28N2O6Se2 (670.47): C, 53.74; H, 4.21; N,
4.18. Found: C, 53.62; H, 4.13; N, 4.27.
Bis[2-(3,4-dimethoxyphenylcarbamoyl)]phenyl Diselenide 4o
.
The general procedure starting from
3,4-dimethoxyaniline (161 mg, 1.05 mmol) at RT was employed, with a 96-h reaction time to obtain
4o. Yield: (253 mg, 72%); m.p.: 231.5–234.5 ◦C (m.p.: 235 ◦C [53]); as a pale yellow powder (CH2Cl2).
IR (KBr, cm−1
)
ν
3291 (NH), 1641 (C=O), 1513 (CONH), 1452, 1263 (C–N), 1237 (C–O), 1134, 1026
1
(C–O), 741, 681, 574 (C–Se), 454. H-NMR (300 MHz, DMSO-d6):
δ 3.76 (s, 6H, 2OCH3), 3.77 (s, 6H,
2OCH3), 6.96 (d, J = 8.8 Hz, 2H, Ar-H), 7.30–7.45 (m, 6H, Ph-H and Ar-H), 7.47 (s, 2H, Ar-H), 7.83 (s,
br., 2H, Ph-H), 7.95 (d, J = 7.7 Hz, 2H, Ph-H), 10.51 (s, 2H, NH). 13C-NMR (75 MHz, DMSO-d6):
(2 OCH3), 55.67 (2 OCH3), 105.81 (2 CH), 111.84, (2 CH), 112.72 (2 CH), 126.08 (2
CH), 131.59 (2 CH), 132.19 (2 C), 132.30 (2
δ
55.41
CH),
×
×
×
×
×
×
128.36 (2
145.46 (2
×
CH), 130.10 (2
C), 148.42 (2
×
×
×
×
C), 133.63 (2
×
C),
×
×
C), 165.78 (2
×
C=O). HRMS (TOF MS ESI): m/z for C30H28N2O6Se2 + Na+
calculated: 693.0178. Found: 693.0178.
Bis[2-(3,4,5-trimethoxyphenylcarbamoyl)]phenyl Diselenide 4p. The general procedure starting from
3,4,5-trimethoxyaniline (192 mg, 1.05 mmol) at RT was employed, with a 96-h reaction time to obtain
4p. Yield: (0.291 g, 76%); m.p.: 193–195 ◦C; as a white powder (CH3OH, 100 g/L). IR (KBr, cm−1
) ν
3322 (NH), 1605 (C=O), 1507 (CONH), 1450, 1411, 1313, 1235 (C–N), 1128 (C–O), 1027 (C–O), 1004

Molecules 2017, 22, 974
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1
(C–O), 738, 623 (C–Se). H-NMR (300 MHz, DMSO-d6):
δ
3.65 (s, 6H, 2OCH3), 3.77 (s, 12H, 4OCH3),
7.20 (s, 4H, Ar-H), 7.38 (dd, J = 7.3 Hz, 2H, Ph-H), 7.44 (dd, J = 7.3 Hz, 2H, Ph-H), 7.80–8.00 (m, 4H,
Ph-H), 10.56 (s, 2H, NH). 1H-NMR (600 MHz, DMSO-d6):
δ
3.67 (s, 6H, 2OCH3), 3.80 (s, 12H, 4OCH3),
7.21 (s, 4H, Ar-H), 7.42 (dd, J = 7.3 Hz, J = 6.6 Hz, 2H, Ph-H), 7.46 (dd, J = 7.9 Hz, J = 6.6 Hz, 2H, Ph-H),
7.81 (d, J = 7.9 Hz, 2H, Ph-H), 7.95 (d, J = 7.3 Hz, 2H, Ph-H), 10.47 (s, 2H, NH). 13C-NMR (75 MHz,
DMSO-d6):
130.5 (2 CH), 132.1 (2
(2
C=O). 13C-NMR (151 MHz, DMSO-d6):
126.31 (2 CH), 128.53 (2 CH), 130.12 (2
(2 C), 134.75 (2 C), 152.64 (4
δ
56.33 (4
×
OCH3), 60.64 (2
×
OCH3), 99.21 (4
×
CH), 126.6 (2
C), 135.8 (2
OCH3), 60.10 (2
CH, 2
C=O).HRMS (TOF MS ESI): m/z for C32H32N2O8Se2
×
CH), 128.9 (2
C), 153.2 (4 C), 166.5
OCH3), 98.24 (4 CH),
C), 133.71 (2 C), 134.05
×
CH),
×
×
CH, 2 C), 134.4 (2
×
×
C), 134.6 (2
×
×
×
×
δ
55.75 (4
×
×
×
×
×
×
CH), 131.97 (2
×
×
×
×
×
×
C), 166.10 (2
×
+ Na+ calculated: 755.0381; found: 755.0381. Anal. Calcd. for C32H32N2O8Se2 (730.53): C, 52.61; H,
4.42; N, 3.83. Found: C, 52.46; H, 4.56; N, 3.72.
Bis[2-(N-morpholinecarbamoyl)]phenyl Diselenide
5.0 mmol) in dry CH2Cl2 (10 mL), the solution of bis[(2-chlorocarbonyl)phenyl]diselenide
1.0 mmol) in dry CH2Cl2 was added and the reaction was continued at RT (+20 ◦C) for 120 h until
no spot of chloride was observed during TLC control (silica gel, eluent CHCl3:EtOAc, 4:1, v/v).
When the reaction was completed, the mixture was poured into water (50 mL), acidified with 3.5%
HCl to a pH of 1.0, extracted with CH2Cl2 (3 25 mL), and dried with Na2SO4. After evaporation of
the solvent, the residue was washed with n-hexane and DEE (25 mL) was added to the oily residue,
followed by stirring until the product solidified and was filtered off to obtain . Yield: (458 mg, 85%
calculated on diselenide
used); m.p.: 149–152 ◦C; as a pale yellow powder (DEE). IR (KBr, cm−1
5
. To a stirred solution of morpholine (436 mg, 0.44 mL,
3
(437 mg,
3
×
5
3
) ν
3059, 2972, 2959, 2902, 2854 (CH), 1625 (C=O), 1608, 1430, 1279, 1248, 1111 (C–N), 1021 (C–O), 1014
1
(C–O), 757, 596, 559 (C–Se), 461. H-NMR (300 MHz, DMSO-d6):
δ 3.57 (s, 16H, 8CH2), 7.28–7.37 (m,
4H, Ph-H), 7.39 (ddd, J = 7.7 Hz, J = 6.4 Hz, J = 2.7 Hz, 2H, Ph-H), 7.74 (dd, J = 8.2 Hz, J = 1.2 Hz, 2H,
Ph-H). 13C-NMR (75 MHz, DMSO-d6):
66.04 (8 CH2), 126.84 (2 CH), 127.36 (2 CH), 129.05
(2 C), 130.38 (2 CH), 131.71 (2 CH), 136.22 (2 C), 167.48 (2 C=O). HRMS (TOF MS ESI): m/z
δ
×
×
×
×
×
×
×
×
for C22H24N2O4Se2 + Na+ calculated: 558.9986; 560.9967; 562.9959; found: 559.0029; 560.9987; 562.9982.
Anal. Calcd. for C22H24N2O4Se2 (538.36): C, 49.08; H, 4.49; N, 5.20. Found: C, 48.96; H, 4.62; N, 5.29.
Bis[2-(N-benzamidecarbamoyl)]phenyl Diselenide
6
. The general procedure starting from benzhydrazide
. Yield: (335 mg, 100%);
m.p.: 296.5 C (with decomp.); as a pale yellow wool (AcOH, 1.5 g/L). 1H-NMR (601 MHz, DMSO-d6):
7.42 (ddd, J = 7.7 Hz, J = 7.3 Hz, J = 0.8 Hz, 2H, Ph-H), 7.50 (ddd, J = 8.0 Hz, J = 7.3 Hz, J = 1.1 Hz, 2H,
(143 mg, 1.05 mmol) at RT was employed, with a 120-h reaction time to obtain
6
◦
δ
Ph-H), 7.54 (dd, J = 7.8 Hz, J = 7.4 Hz, 4H, Ar-H), 7.62 (t, J = 7.3 Hz, 2H, Ar-H), 7.78 (dd, J = 8.0 Hz,
J = 0.8 Hz, 2H Ph-H), 7.97 (d, J = 7.8 Hz, 4H, Ar-H), 8.00 (dd, J = 7.7 Hz, J = 1.1 Hz, 2H, Ph-H), 10.68
(s, 2H, NH), 10.83 (s, 2H, NH). 13C-NMR (151 MHz, DMSO-d6):
δ
126.40 (2
C), 131.94 (2
C=O). HRMS (TOF MS ESI): m/z for
×
CH), 127.45 (4
×
CH),
128.30 (2
132.44 (2
×
CH), 128.51 (4
CH), 132.47 (2
×
CH), 130.12 (2
C), 165.77 (2
×
CH), 130.70 (2
×
×
CH), 132.30 (2
×
C),
×
×
×
C=O), 166.86 (2
×
C28H22N4O4Se2 + Na+ calculated: 656.9891; 658.9872; found: 656.9968; 658.9862. Anal. Calcd. for
C28H22N4O4Se2 (636.42): C, 52.84; H, 3.48; N, 8.80. Found: C, 53.02; H, 3.29; N, 8.79.
2-Hydroxyacetanilide phenolic ester of bis(2-carboxy)phenyl diselenide
from 2-hydroxyacetanilide (151 mg, 1.0 mmol) dissolved in dry CH2Cl2 (10 mL) at RT was employed,
with a 96-h reaction time to obtain . Yield: (119 mg, 36% calculated on 2-hydroxyacetanilide used);
m.p.: 178–181 ◦C; as a pale solid (CH2Cl2). IR (KBr, cm−1
7. The general procedure starting
7
)
ν
3402 (NH), 1760 (OC=O), 1659 (NC=O),
1
1545 (CONH), 1455, 1370, 1285 (C–N), 1216 (C–O), 1116, 767, 622 (C–Se), 457. H-NMR (300 MHz,
DMSO-d6):
δ 2.09 (s, 6H, CH3), 6.75 (dd, J = 7.9 Hz, J = 7.1 Hz, 2H, Ph-H), 6.85 (d, J = 7.0 Hz, 2H, Ph-H),
6.93 (dd, J = 7.9 Hz, J = 6.6 Hz, 2H, Ph-H), 7.36 (dd, J = 7.6 Hz, J = 7.0 Hz, 2H, Ph-H), 7.49 (dd, J = 7.2 Hz,
J = 7.0 Hz, 2H, Ph-H), 7.66 (d, J = 6.6 Hz, 2H, Ar-H), 7.67 (d, J = 7.9 Hz, 2H, Ph-H), 8.33 (d, J = 7.2 Hz,
2H, Ph-H), 9.72 (s, 2H, NH). HRMS (TOF MS ESI): m/z for C30H24N2O6Se2 + Na+ calculated: 688.9865;

Molecules 2017, 22, 974
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690.9857; found: 688.9866; 690.9855. Anal. Calcd. for C30H24N2O6Se2 (666.44): C, 54.07; H, 3.63; N,
4.20. Found: C, 53.85; H, 3.90; N, 4.06.
(2R,3S,6S,7R,8R)-3-(3-Formamido-2-hydroxybenzamido)-8-n-alkyl-2,6-dimethyl-4,9-dioxo-1,5-dioxonan-7-yl
3-Methylbutanoate (antimycin A) phenolic ester of bis(2-carboxy)phenyl diselenide
starting from naturally occurred antimycin A (0.21 g, 0.40 mmol) dissolved in dry CH2Cl2 (5 mL),
and Na2CO3 (0.16 g, 1.5 mmol), diselenide (87 mg, 0.20 mmol) at RT was employed, with a 10-day
reaction time to obtain phenolic ester as a co-crystallizable mixture of different alkyl substituents
8. The general procedure
3
8
with R=C5H11 predominantly. Yield: (0.277 g, 97% calculated on antimycin A used); m.p.: >300 ◦C;
as a pale solid. In 13C-NMR, a number of signals is observed and only diagnostic ones are reported
13C-NMR (101 MHz, DMSO-d6):
δ 13.88, 17.72, 18.92, 19.00, 19.02, 20.28, 21.85, 21.94, 22.85, 24.43, 25.66,
26.81, 27.18, 28.10 and 30.89 (Caliphatic); 58.05, 58.49, 66.77, 66.80 and 77.35 (CHO and CNO); 107.05,
107.47, 117.11, 117.21, 117.28, 118.94, 124.16, 130.59, 131.94 and 136.79 (CAr, CAr-H); 151.23 (CAr-CON),
166.95, 167.16, 167.26, 167.43, 170.42, 174.84, 174.98, 175.09 and 175.11 (C=O). HRMS (TOF MS ESI):
m/z for C64H74N4O20Se2 + Na+ calculated: 1401.3119; found: 1401.3433; for C65H76N4O20Se2 + Na+
calculated: 1411.3291; 1413.3283; found: 1411.3070; 1413.3112; for C66H78N4O20Se2 + Na+ calculated:
1427.3440; 1429.3432; found: 1427.3264; 1429.3787; for C67H80N4O20Se2 + Na+ calculated: 1441.3596;
found: 1441.3926; for C68H82N4O20Se2 + Na+ calculated: 1455.3753; 1457.3745; found: 1455.4171;
1457.4158; for C69H84N4O20Se2 + Na+ calculated: 1469.3909; 1471.3902; found: 1469.4357; 1471.4440;
for C70H86N4O20Se2 + Na+ calculated: 1481.4085; 1483.4066; 1485.4058; 1486.4092; found: 1481.3927;
1483.4473; 1485.5210; 1486.4746. Anal. Calcd. for C68H82N4O20Se2 (1433.31): C, 56.98; H, 5.77; N, 3.91.
Found: C, 57.01; H, 5.82; N, 3.77.
2-(Chloroseleno)benzoyl chloride
(8.0 g, 20 mmol) using excess of thionyl chloride (6.0 g, 50 mmol) and DMF (1.5 g, 20 mmol) in
boiling benzene (150 mL), following the method previously reported [60]. Crude oily product was
9. The compound was prepared from 2,2’-diselenobisbenzoic acid
2
recrystallized from n-hexane (ca. 15 mL/g) and dried under vacuum (20 mmHg) to give
(8.1 g, 80%); m.p.: 65–67 ◦C (65–66 ◦C [60]); as yellow needles (n-hexane).
9. Yield:
General synthetic procedure for benzisoselenazol-3(2H)-ones 10a
–g. To a stirred solution of substituted
aniline (13.0 mmol) in anhydrous CH2Cl2 (5 mL) a solution of (2-chloroseleno)benzoyl chloride
9
(1.02 g, 4.0 mmol) in anhydrous CH2Cl2 (25 mL) was added dropwise. The reaction was continued
for 2–24 h. The progress of reaction was controlled by TLC (silica gel, eluent CHCl3:EtOAc, 4:1, v/v).
After the reaction was finished, the solvent was evaporated, HCl (3.5%, w/w, 40 mL) was added, and
the mixture was stirred at RT overnight, then filtered off and left to dry in the air. Crude products were
purified by column chromatography on silica gel (70–230 mesh) eluted with CHCl3 to obtain pure
10a–g.
2-(2-Hydroxyphenyl)-1,2-benzisoselenazol-3(2H)-one 10a
.
The general procedure starting from
2-hydroxyaniline (0.44 g, 4.0 mmol) and anhydrous Et3N (1.01 g, 1.4 mL, 10 mmol) at RT was employed,
with a 20-h reaction time with separation of the product from the crude reaction mixture by column
chromatography using EtOAc as an eluent to obtain 10a. Yield: (0.46 g, 40% calculated on benzoyl
chloride
1H-NMR (400 MHz, CDCl3):
J = 1.5 Hz, 1H, Ar-H), 7.24–7.30 (m, 2H, Ar-H), 7.47–7.54 (m, 1H, Ph-H), 7.65–7.71 (m, 2H, Ph-H), 8.13
(d, J = 8.1 Hz, 1H, Ar-H), 8.55 (s, 1H, OH) [57]. 13C-NMR (101 MHz, DMSO-d6):
116.94 (2 CH),
CH), 125.97 (C), 127.52 (C), 127.76 (CH), 128.76 (CH),
9
used); m.p.: 195.5–197.5 ◦C (194–196 ◦C [54]); as orange flakes (MeCN:H2O, 84:16, v/v).
δ
6.99 (ddd, J = 8.0 Hz, J = 7.2 Hz, J = 1.5Hz, 1H, Ar-H), 7.13 (dd, J = 8.6 Hz,
δ
×
119.22 (2
×
CH), 125.73 (2
×
CH), 125.81 (2
×
129.32 (CH), 131.82 (CH), 140.50 (C), 153.25 (C), 165.76 (C=O) [57]. The 77Se NMR, and HRMS are in
agreement with literature data [26,57].
2-(3-Hydroxypyridin-2-yl)-1,2-benzisoselenazol-3(2H)-one 10b. The general procedure starting from
2-amino-3-hydroxypyridine (0.44 g, 4.0 mmol) and anhydrous Et3N (0.95 g, 1.3 mL, 9.4 mmol) at
anhydrous MeCN (40 mL) using benzoyl chloride
9 in anhydrous MeCN was employed for a 2-h

Molecules 2017, 22, 974
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reaction time at RT. Solvent was evaporated, water (100 mL) was added portionwise, and the solution
was stirred for 1 h. The solid was filtered off, washed with H2O, and air-dried to obta◦in 10b. Yield:
◦
◦
(0.76 g, 65%); m.p.: 229 C (decomp.) with phase transfer at 199 C (m.p.: 199–201 C [55]); as a
greenish yellow powder (DMSO). 1H-NMR (DMSO-d6): δ 7.26 (dd, J = 8.0 Hz, J = 4.6 Hz, 1H, Pyr-H),
7.38 (dd, J = 8.0 Hz, J = 1.4 Hz, 1H, Pyr-H), 7.50 (ddd, J = 7.8 Hz, J = 7.2 Hz, J = 0.9 Hz, 1H, Ar-H), 7.72
(ddd, J = 8.0 Hz, J = 7.2 Hz, J = 1.3 Hz, 1H, Ar-H), 7.95 (dd, J = 7.8 Hz, J = 0.9 Hz, 1H Ar-H), 7.98 (dd,
J = 4.6 Hz, J = 1.4 Hz, 1H, Pyr-H), 8.09 (dd, J = 8.0 Hz, J = 1.3 Hz, 1H, Ar-H), 12.18 (s, 1H, OH). The
13C-NMR is in agreement with literature data [55].
2-(2-Methoxyphenyl)-1,2-benzisoselenazol-3(2H)-one 10c
.
The general procedure starting from
2-methoxyaniline (1.60 g, 13 mmol) at RT was employed, with a 24-h reaction time followed by
separation by column chromatography to obtain 10a. Yield: (0.86 g, 71% calculated on the benzoyl
chloride
1621 (C=O), 1270 (C–N), 1108 (C–O), 1020 (C–O). 1H-NMR (300 MHz, CDCl3) [63]:
9 )
); m.p.: 189.0–191.5 ◦C (m.p.: 172–174 ◦C [63]); as yellow prisms (CHCl3). IR (KBr, cm−1
ν
δ 3.85 (s, 3H,
OCH3), 6.98–7.07 (m, 2H, Ph-H), 7.36 (ddd, J = 8.2 Hz, J = 7.6 Hz, J = 1.7 Hz, 1H, Ph-H), 7.44 (ddd,
J = 8.0 Hz, J = 6.4 Hz, J = 1.7 Hz, 1H, Ar-H), 7.48 (dd, J = 8.0 Hz, J = 1.7 Hz, 1H, Ph-H), 7.58–7.70 (m,
2H, Ar-H), 8.13 (d, J = 7.8 Hz, 1H, Ar-H). 13C-NMR (75 MHz, CDCl3):
δ 55.80 (OCH3), 112.22 (CH),
120.74 (CH), 123.86 (CH), 126.01 (CH), 126.44 (C), 126.74 (C), 129.21 (CH), 129.66 (CH), 129.89 (CH),
132.17 (CH), 139.31 (C), 155.39 (C), 166.70 (C=O).
2-(3-Methoxyphenyl)-1,2-benzoisoselenazole-3(2H)-one 10d
.
Prepared according to a modified
procedure [35]. The general procedure starting from 3-methoxyaniline (0.493 g, 4.0 mmol) and dry
Et3N (1.02 g, 1.4 mL, 10 mmol) in anhydrous MeCN (40 mL) at RT with a 2-h reaction time was
employed. Water was added portionwise and the solution was left in the refrigerator for crystallization.
Then, the solid was filtered off, washed with water and MeCN, and dried in the air to obtain 10d
.
Yield: (0.79 g, 65%), m.p: 166–168 ◦C (165.5–167.5◦C [35]); as yellow prisms in (MeCN:H2O, 1:1, v/v).
The 1H-NMR and 13C-NMR are in agreement with literature data [35].
2-(4-Methoxyphenyl)-1,2-benzoisoselenazole-3(2H)-one 10e
.
Prepared according to a modified
procedure [35]. The general procedure starting from 3-methoxyaniline (0.49 g, 4.0 mmol) and
Et3N (1.4 mL, 1.02 g, 10 mmol) in anhydrous MeCN (40 mL) at RT with a 20-h reaction time was
employed. Water (40 mL) was added portionwise and the solution was left in the refrigerator for
crystallization. Then, the solid was filtered off, washed with water, dried in the air, and the crude
product was recrystalized from EtOAc (30 mL/g) to obtain 10e. Yield: (0.85 g, 70%); m.p.: 180–181 ◦C
(180.5–181.5◦C [35]); as yellow prisms (EtOAc). The 1H-NMR and 13C-NMR data are in agreement
with literature data [26,35].
2-(2,4-Dimethoxyphenyl)benzisoselenazol-3(2H)-one 10f
.
The general procedure starting from
2,4-dimethoxyaniline (1.99 g, 13 mmol) at RT was employed, with a 6-h reaction time with separation
by column chromatography using CHCl3 as an eluent to obtain 10f. Yield: (0.87 g, 65% calculated on
the benzoyl chloride
9
); m.p.: 239–240 ◦C; as pale yellow prisms (CHCl3, 75 mL/g). IR (KBr, cm−1
)
ν
1
3050, 1601 (C=O), 1588, 1560, 1510, 1442, 1208 (C–N), 1162 (C–O), 1044 (C–O), 747 (C–Se). H-NMR
(300 MHz, CDCl3):
δ 3.79 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.54 (dd, J = 8.6 Hz, J = 2.6 Hz, 1H, Ph-H),
6.59 (d, J = 2.6 Hz, 1H, Ph-H), 7.25 (d, J = 8.6 Hz, 1H, Ph-H), 7.41 (ddd, J = 7.8 Hz, J = 7.2 Hz, J = 1.2 Hz,
1H, Ar-H), 7.60 (ddd, J = 7.8 Hz, J = 7.2 Hz, J = 1.1 Hz, 1H, Ar-H), 7.95 (dd, J = 7.2 Hz, J = 1.2 Hz, 1H,
Ar-H), 7.99 (dd, J = 8.0 Hz, J = 1.1 Hz, 1H, Ar-H). 1H-NMR (400 MHz, DMSO-d6):
δ 3.75 (s, 3H, OCH3),
3.81 (s, 3H, OCH3), 6.58 (dd, J = 8.6 Hz, J = 2.7 Hz, 1H, Ph-H), 6.70 (d, J = 2.7 Hz, 1H, Ph-H), 7.23 (d,
J = 8.6 Hz, 1H, Ph-H), 7.45 (ddd, J = 7.6 Hz, J = 7.3 Hz, J = 1.1 Hz, 1H, Ar-H), 7.65 (ddd, J = 7.9 Hz,
J = 7.3 Hz, J = 1.5 Hz, 1H, Ar-H), 7.85 (ddd, J = 7.6 Hz, J = 1.5 Hz, J = 0.6 Hz, 1H, Ar-H), 8.05 (ddd,
J = 7.9 Hz, J = 1.1 Hz, J = 0.6 Hz, 1H, Ar-H). 13C-NMR (101 MHz, DMSO-d6):
δ
55.45 (OCH3), 55.68
(OCH3), 99.39, 104.86, 119.75, 125.74, 125.78, 127.30, 127.74, 130.33, 131.77, 140.29, 156.29, 160.09, 165.76

Molecules 2017, 22, 974
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(C=O). HRMS (TOF MS ESI): m/z for C15H13NO3Se + Na+ calculated: 357.9953; found: 357.9961. Anal.
Calcd. for C15H13NO3Se (334.2286): C, 53.90; H, 3.92; N, 4.19. Found: C, 53.94; H, 3.75; N, 4.11.
2-(3,4-Dimethoxyphenyl)-1,2-benzoselenazol-3(2H)-one 10g [53]. The general procedure starting from
3,4-dimethoxyaniline (0.61 g, 4.0 mmol) and anhydrous Et3N (0.81 g, 1.1 mL, 8 mmol) in anhydrous
MeCN (30 mL) at RT was employed, with a 48-h reaction time. Distilled water (60 mL) was added
portionwise and left to refrigerator overnight to crystallise. The formed solid was filtered off, washed
with water, and dried in the air to obtain 10g. Yield: (0.91 g, 68%). The analytically pure sample was
obtained by column chromatography using CHCl3:EtOAc in gradient as an eluent with 40% yield;
1
m.p.: 159–161◦C; as colorless crystals (EtOAc). H-NMR (300 MHz, DMSO-d6):
δ 3.78 (s, 6H, 2OCH3),
6.99 (d, J = 8.6 Hz, 1H, Ph-H), 7.03 (dd, J = 8.6 Hz, J = 1.9 Hz, 1H, Ph-H), 7.29 (d, J = 1.9 Hz, 1H, Ph-H),
7.47 (dd, J = 7.8 Hz, J = 7.2 Hz, 1H, Ar-H), 7.67 (dd, J = 8.1 Hz, J = 7.2 Hz, 1H, Ar-H), 7.90 (d, J = 7.8 Hz,
1H, Ar-H), 8.09 (d, J = 8.1 Hz, 1H, Ar-H). 13C-NMR (75 MHz, DMSO-d6): 55.5 (OCH3), 55.6 (OCH3),
109.5 (C–H), 111.8 (C–H), 117.2 (C–H), 125.8 (C–H), 126.1 (C–H), 127.8 (C–H), 128.4 (C), 132.0 (C–H),
132.4 (C), 139.0 (C), 147.1 (C), 148.7 (C), 164.9 (C=O). HRMS (TOF MS ESI): m/z for C15H13NO3Se +
Na+ calculated: 357.9953; found: 357.9951. Anal. Calcd. for C15H13NO3Se (334.2282): C, 53.90; H, 3.92;
N, 4.19. Found: C, 53.79; H, 4.05; N, 4.08.
3.2. Evaluation of Antimicrobial Activity
The antimicrobial activity of compounds
4–6, 7, 10 was determined by the 96-well microdilution
assay, according to Clinical and Laboratory Standards Institute (2008), 3rd ed. M27-A3 [73
]
with slight modifications, using Gram-positive bacterial strains: Staphylococcus aureus ATCC 6538,
Staphylococcus epidermidis ATCC 14990, Enterococcus faecalis ATCC 29212, Enterococcus hirae ATCC 10541;
Gram-negative bacterial strains: Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 15442;
yeast-like fungi: Candida albicans ATCC 10231, Candida glabrata ATCC 90030; and filamentous fungus
Aspergillus niger. Standard strains were supplied by American Type Culture Collection (ATCC), while
A. niger was obtained from the Collection of Microorganisms of Wroclaw Medical University. Bacterial
◦
strains were cultured in Lysogeny broth (LB) at 37 C and yeast strains in Sabouraud dextrose broth at
30 ◦C.
Briefly, stock solutions in DMSO were serially diluted in LB or Sabouraud dextrose broth to
obtain a final range of 0.125–128 µg/mL concentration in microdilution sterile plates (Sarstedt, Stare
Babice, Poland). Microbial inocula were prepared to obtain a final optical◦density at
30 ◦C (fungi). Afterwards, OD600 was measured using a microplate reader ASYS UVM 340 (Biogenet,
λ = 600 nm
(OD600) = 0.1
±
10% in a well. The plates were cultivated for 24 h at 37 C (bacteria) or 48 h at
Józefów, Poland). The concentration which resulted in
minimal inhibitory concentration (MIC90; µg/mL).
≥
90% growth inhibition was determined as the
3.3. Evaluation of Antiviral Activity and Cytotoxicity
The compounds 4–5, 7, 10 at various concentrations were incubated with the following viruses:
HHV-1 (human herpes virus type 1), EMCV (encephalomyocarditis virus), and VSV (vesicular
stomatitis virus) for 1 h at RT. HHV-1 was used at the dose of 106 TCID50/mL, whereas EMCV and
VSV were used 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 the minimal inhibitory concentration, MIC (µg/mL).
In parallel, the cytotoxicity of the compounds was determined in the A549 cell line (cell line used
in antiviral activity test) by incubation at various concentrations for 48 h at 37 ◦C in the atmosphere
of 5% CO2 in air. The minimal concentration which was toxic to approximately 50% of the cells was
taken as the TCCD50 (Tissue Culture Cytotoxic Dose).

Molecules 2017, 22, 974
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Both, cytotoxicity and virus titers were determined using MTT assay. To ensure that colourful
compounds did not interfere with MTT reagents, the extent of cell damage was evaluated by
microscopic examination prior to MTT assay.
4. Conclusions
The reaction of bis[(2-chlorocarbonyl)phenyl] diselenide
derivatives is a convenient synthetic route to bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides
(reduced forms of ebselenols) and other compounds containing protected hydroxyl groups. This
3 with various aminophenols and their
4
general approach allows the production of ebselenol-derived diselenides with higher yields than
the alternative method based on the reductive benzisoselenazolone ring opening of ebselenols.
The secondary amine such as morpholine and benzohydrazide also reacted on their nitrogen
atoms to form bis[2-(N-morpholinecarbamoyl)]phenyl and bis[2-(N-benzamidecarbamoyl)]phenyl
diselenides, respectively, while reaction with aminophenol derivatives containing a blocked amine
group such a 2-hydroxyacetanilide and natural antimycin A led to corresponding phenolic esters of
bis(2-carboxy)phenyl diselenide. As a result of our studies, 15 new structures have been developed,
including 13 diselenides and two benzisoselenazol-3(2H)-ones.
The investigation of the antimicrobial and antiviral activities of bis[2-(hydroxyphenylcarbamoyl)]
phenyl diselenides and their derivatives as well as cyclic analogues have revealed new biologically
active structures. The compounds, particularly bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides
and reference benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive
bacterial species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against
Gram-negative E. coli and fungi (Candida spp., A. niger). The antiviral activity evaluation allowed
the identification of substances with high anti-HHV-1 and moderate anti-EMCV activities. The
chemotherapeutic indices for some of these compounds were significantly higher (concentration
active against virus was lower than the one showing a cytotoxic effect) than for other organoselenium
compounds previously reported in the literature.
As novel molecules with the ability to inhibit microbial and viral functions are in high demand,
the results of our studies are encouraging for further exploration of organoselenium compounds as
anti-infective agents.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: This work was supported by the National Science Centre (NCN) through grant no.
UMO-2013/09/D/ST5/03814 (evaluation of antimicrobial activity 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). The publication fee
was supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW)
for years 2014–2018. The authors wish to acknowledge the kind help of Kip Hillshafer for his valuable advice
during the manuscript preparation.
Author Contributions: M.G. and M.P.-O. conceived and designed the experiments; A.G., J.S. and R.K. performed
the experiments; M.G., J.S. and M.P.-O. analyzed the data; M.G., J.S. and M.P.-O. wrote the paper; A.K. and E.P.
reviewed the paper. All authors read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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