Synthesis and biological evaluation of new nitrogen-containing diselenides

Article abstract of DOI:10.1016/j.ejmech.2014.09.022

The antioxidant properties of organoselenium compounds have been extensively investigated with the aim of developing new drugs, since oxidative stress is responsible for a variety of chronic human diseases. Herein, we reported the synthesis of new nitrogen-containing diselenides by a simple and efficient synthetic route. The products were obtained in good to excellent yields and their identification and characterization were achieved by NMR and HRMS techniques. The new derivatives may represent promising structures with different biological activities, which can act against oxidative stress through diverse mechanisms of action. The glutathione peroxidase-like assay (GPx-like activity) of the new synthesized compounds indicated that they reduced H₂O₂to water at the expense of PhSH. The best results were obtained with diselenide 2b, which was 9 times more active than the standard organoselenium drug ebselen and, in contrast, this compound was not reduced by hepatic TrxR. All of the new compounds inhibited Fe(II)-induced TBARS.

Full text of DOI:10.1016/j.ejmech.2014.09.022

European Journal of Medicinal Chemistry 87 (2014) 131e139
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of new nitrogen-containing
diselenides
a
a
a
b
^
Vanessa Nascimento , Natasha L. Ferreira , Romulo F.S. Canto , Karen L. Schott ,
b
c
c
b
a, *
~
Emily P. Waczuk , Luca Sancineto , Claudio Santi , Joao B.T. Rocha , Antonio L. Braga
Laboratorio de Síntese de Substancias de Selenio Bioativas, Centro de Ciencias Físicas e Matematicas, Departamento de Química, Universidade Federal de
a
ꢀ
^
^
^
ꢀ
ꢀ
Santa Catarina, 88040-900 Florianopolis, SC, Brazil
b
^
Departamento de Química, Centro de Ciencias Naturais e Exatas, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil
c
ꢁ
Dipartimento di Scienze Farmaceutiche, Universita di Perugia, Via del Liceo 1, 06134 Perugia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
The antioxidant properties of organoselenium compounds have been extensively investigated with the
aim of developing new drugs, since oxidative stress is responsible for a variety of chronic human dis-
eases. Herein, we reported the synthesis of new nitrogen-containing diselenides by a simple and efficient
synthetic route. The products were obtained in good to excellent yields and their identification and
characterization were achieved by NMR and HRMS techniques. The new derivatives may represent
promising structures with different biological activities, which can act against oxidative stress through
diverse mechanisms of action. The glutathione peroxidase-like assay (GPx-like activity) of the new
synthesized compounds indicated that they reduced H2O2 to water at the expense of PhSH. The best
results were obtained with diselenide 2b, which was 9 times more active than the standard organo-
selenium drug ebselen and, in contrast, this compound was not reduced by hepatic TrxR. All of the new
compounds inhibited Fe(II)-induced TBARS.
Received 16 March 2014
Received in revised form
6 September 2014
Accepted 6 September 2014
Available online 8 September 2014
Keywords:
Diselenides
Amphetamine
Glutathione peroxidase
Antioxidant
Selenium
© 2014 Elsevier Masson SAS. All rights reserved.
Ebselen
1. Introduction
damage essential cell structures. A number of pathologies are
linked to the overproduction of ROS, among them
In the past few decades, interest in the chemistry of organo-
selenium compounds has significantly increased due to the dis-
covery of a series of selenoproteins, which are involved in a number
of physiological processes [1e4]. Selenoenzymes are an important
class of mammalian antioxidant enzymes that protect bio-
membranes and other cellular components from oxidative stress
[5e9]. This condition can be generated during oxygen metabolism
where reactive oxygen species (ROS) are produced and these can
neurodegenerative diseases such as Alzheimer's and Parkinson's
diseases, as well as inflammatory processes and physiological
signaling pathways [10e14]. Nevertheless, our organism has a
refined defense mechanism against ROS and the enzyme
glutathione peroxidase (GPx) is part of a complex detoxification
system. The structural determination of GPx revealed the presence
of selenocysteine (Sec) which, together with Trp153 (tryptophan)
and Gln79 (glutamine), form the catalytic triad. The pivotal role of
GPx relies on the reduction of endogenous peroxides using, as a
cofactor, glutathione (GSH) and affording, as byproducts, water or
alcohols depending on the nature of the initial substrate [15e19].
While the presence of Sec has been demonstrated to be funda-
mental for the enzyme being directly involved in the reduction
process, tryptophan and glutamine allow Sec to remain in its
selenol/active form.
Abbreviations: NMR, nuclear magnetic resonance; HMRS, high resolution mass
spectrometry; GPx, glutathione peroxidase; TrxR, thioredoxin reductase; TBARS,
thiobarbituric acid reactive substances; ROS, reactive oxygen species; Trp, trypto-
phan; Gln, glutamine; Sec, selenocysteine; GSH, glutathione; (PhSe)2, diphenyl
diselenide;
SAR,
structure
activity
relationship;
EDC,
1-ethyl-3-(3-
dimethylaminopropyl)carbodi-imide; DCC, N,N0-dicyclohexylcarbodiimide; CDI,
1,10-carbonyldiimidazole; m.p., melting point; T50, time required, in min, to reduce
the thiol concentration by 50%; UV, ultraviolet; LDH, lactate dehydrogenase; ALA-D,
aminolevulinic acid dehydratase; S1, brain homogenates; NADPH, nicotinamide
adenine dinucleotide phosphate hydrogen; MDA, malondialdhyde.
* Corresponding author.
Besides GPx, thioredoxin reductase (TrxR) is another
selenoenzyme which plays a key role within the antioxidant ma-
chinery in the cell context. Mammalian TrxR mantains thioredoxin
in the reduced form which, in turn, converts disulfide bridges into
thiols, allowing target proteins to retain the native conformation.
E-mail addresses: albraga1@gmail.com, braga.antonio@ufsc.br (A.L. Braga).
http://dx.doi.org/10.1016/j.ejmech.2014.09.022
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

132
V. Nascimento et al. / European Journal of Medicinal Chemistry 87 (2014) 131e139
TrxR is characterized by a broad substrate specificity, being able to
reduce not only thioredoxin but also a number of substrates
including organic and inorganic selenium compounds, increasing
the pool of free selenols within the cell context and thus improving,
through peroxides degradation, the antioxidant capacity of the
cellular environment [20e22].
pivotal interaction and gain some insight into the structure activity
relationship (SAR) of this class of compounds, we designed a small
set of phenylethylamine derivatives, selecting, as the joining moi-
ety, the amidic group, mainly because of its presence in ebselen. In
this way, our compound is designed to be a diselenide substituted
with biological relevant amines in the form of amides that can
interact with selenium and improve its antioxidant activity.
Moreover, amide-diselenides obtained from simple amines [53] or
amino acids [54e56] have been previously described as good GPx
mimics, indicating that the amide group chelation with the
diselenide function has a positive influence on the antioxidant
activity (Fig. 1).
In particular, the aromatic derivative 2a and its methyl analog 2b
were designed in order to constrain the amidic nitrogen and keep it
close to the diselenide group, thus increasing the probability of the
occurrence of the non-covalent interaction and allowing its effect
on the antioxidant activity to be verified. Aliphatic derivatives 3aeb
were designed with the interatomic distance of only a methylene
linker. 3ced were conceived, keeping the distance between the
selenium and nitrogen constant but introducing a certain degree of
flexibility. The aim of this approach was to provide some insight
into the interaction vs. activity relationship of the diselenides with
amide bonds.
Modulation of multiple targets along the same biological
pathway could potentially lead to disease modification rather than
just control of symptoms and the development of new drugs is
being focused on multi-potent molecules acting in a complemen-
tary manner [57]. The whole set of compounds was investigated in
depth in terms of the antioxidant capacity, through different in vitro
tests, and the inherent SAR is also discussed. In particular, the
glutathione peroxidase-like activity was initially assayed and the
inhibition of lipid peroxidation in brain homogenates was then
evaluated. Furthermore, all of the compounds were investigated for
their capacity to act as substrates for the enzyme thioredoxin
reductase (TrxR). The antioxidant action of the new nitrogen-
containing diselenides (2aeb and 3aed) was compared with that
Several researchers have dedicated considerable effort to the
design and synthesis of organoselenium compounds capable
of reproducing the catalytic activity of GPx and, at the same time, act
as a substrate for TrxR, thus operating as strong antioxidants, in
an attempt to mimic the activity of endogenous antioxidant
machinery and provide therapeutic options for oxidative stress-
related diseases [23e30]. In this regard, the synthetic
organoselenium derivative first reported and most extensively
studied is ebselen [31e33]. This compound exhibits antioxidant and
anti-inflammatory activities besides other therapeutic applications,
for instance, anti-atherosclerotic and cytoprotective behavior [1,33].
Inspired by these studies, a number of new selenium-containing
compounds with antioxidant activity have been successfully
developed as potential therapeutic agents and also, from a purely
synthetic point of view, as newgreen bio-mimetic catalysts [34e41].
The antioxidant properties of ebselen and other organoselenium
compounds, such as diphenyl diselenide (PhSe)2, are mainly related
to their glutathione peroxidase-like activities. Some studies have
also shown that these compounds are substrates for the mammalian
TrxR, which allows the formation of selenol intermediates, hence
improving the degradation of peroxides [42,43].
However, the exact contribution to the antioxidant activity of
each pathway is still unclear, but from a theoretical point of view
the operation of the two routes can be considered advantageous in
living cells, because it increases the range of reducing equivalent
donors (i.e., thiols and NADPH) capable of synthesize selenol in-
termediates. Theoretically, the operation of the two pathways is
expected to increase the steady state levels of selenol intermediate
of given diselenide in the cellular environment. Therefore, these
organoselenium compounds might represent novel therapeutic
agents for the treatment of diseases caused by oxidative stress [44].
Among organoselenium derivatives, diselenides are particularly
attractive given their relatively easy synthesis and stability;
consequently, they have often been object of biological in-
vestigations [45]. Selenoamides [46,47], such as ebselen or sele-
nides [48e50], have been widely explored in terms of antioxidant
activity. Diselenides containing side moiety with heteroatoms in a
suitable position to establish with the selenium atom a non-
covalent interaction are a potentially fertile field of research in
several aspects, including some biological issues such as antioxi-
dant and anticancer activities and toxicity.
In this context, we recently reported some ephedrine-based
seleno derivatives in an attempt to develop novel chiral catalysts
while assessing their biological profile [51]. Among the derivatives,
diselenide 1 appeared to be of interest since it showed a GPx-like
activity better to that displayed by other derivatives and even
superior than that of diphenyl diselenide, in the same assay. It has
been found that selenium atom often interacts with a nearby
heteroatom (N, O, S etc.) [52] and in the case of glutathione
peroxidase mimics, the increased activity of diselenide derivatives
is attributed to this beneficial non-covalent interactions, which
occur during the course of the reaction.
It is know from the literature that diselenides are better anti-
oxidants than ebselen and that diselenides with non-covalent
interaction between selenium and a heteroatom present even
better antioxidant activity [1]. This non-covalent interaction play a
major role in the stabilization of the selenium atom, mainly by
preventing the over oxidation and increasing the electrophilicity of
the selenium in the dimer [52]. In an attempt to reproduce this
Fig. 1. Structures of nitrogen-containing diselenides reported herein.

V. Nascimento et al. / European Journal of Medicinal Chemistry 87 (2014) 131e139
133
of the standard organoselenium drug ebselen, used as the positive
2.2. Biological evaluation
control.
Reactive oxygen species (ROS), such as superoxides (Oꢀ2 ꢁ), hy-
droxyl radicals (ꢁOH) and hydrogen peroxide (H2O2) are produced
in small quantities during cell metabolism. These species are
known for their high reactivity and their tendency to initiate and
participate in chain reactions, causing extensive damage to cells
[62]. Organism defense against ROS may occur through antioxidant
enzymes and naturally occurring antioxidants [63]. The aim of the
study reported herein was to investigate the antioxidant properties
of the synthesized compounds and shed light on their mechanisms
of action. This was performed by carrying out three different anti-
oxidant in vitro assays, aiming to show that the compounds can act
as antioxidants via multiple mechanisms, increasing their final
antioxidant activity. From these studies we can show that some
factors, such as the size of the chain, steric hindrance and strength
of the Se-heteroatom interaction, may be of overriding importance
in relation to one mechanism but not to another.
2. Results and discussion
2.1. Chemistry
Initially, we synthesized the carboxylic acid diselenide de-
rivatives that were used as starting materials to prepare the target
compounds. The aromatic acid diselenide 5 was synthesized in
good yield through diazotization of anthranilic acid and substitu-
tion with a freshly prepared solution of Na2Se2, as shown in Scheme
1 [58]. The aliphatic acid diselenides 7aeb were synthesized by
reacting the appropriate bromo-carboxylic acids with Na2Se2 as
outlined in Scheme 2 [59]. The 1-phenylpropan-2-amine used in
this study was prepared as described by Swern et al. [60] and
Collins et al. [61].
After preparing the starting materials we promoted amide bond
formation between the acid diselenides and amines, through (1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC)-mediated
coupling reactions (Schemes 1 and 2). It is important to note that on
using other coupling reagents, such as N,N0-Dicyclohex-
ylcarbodiimide (DCC) and 1,10-Carbonyldiimidazole (CDI), the re-
actions were not successful, resulting in a complex mixture of
byproducts and the starting materials.
The target molecules were obtained in good to excellent yields
ranging from 55 to 96%. As an example, compound 2a was obtained
in 61% yield (Scheme 1). When the amine used was 1-
phenylpropan-2-amine (R ¼ Me), the yields of the compounds
were slightly lower in comparison to those obtained using
phenylethylamine, as can be seen by comparing 2a and 2b (Table 1,
entries 1e2). Not surprisingly, exchanging aromatic for aliphatic
acid-diselenides led to a pronounced increase in the yields, as
shown in Table 1, entries 3e6. Additionally, we found that a change
in the chain length between the selenium atom and the amide
group did not produce a strong effect on the product yields (Table 1,
entries 3 and 5 or 4 and 6). Overall yields of the synthetic routes
from the bromo carboxylic acids or anthranilic acid ranged from 44
to 96%.
By applying this strategy many different biologically active
amines can be easily transformed into organoselenium compounds
and the biological activities of these amide-diselenides can be
explored. Furthermore, the modification of the chain length or
conformation of the diselenides gives us some evidences of the
influence of the Se-heteroatom interaction between selenium and
the amide group on the antioxidant activity of the molecules
synthesized.
The structure and purity of all products obtained were satis-
factorily confirmed by 1H and 13C nuclear magnetic resonance
(NMR) spectroscopy and HRMS spectrometry. Their molecular
structures are all reported for the first time and selected physical
properties are shown in Table 1.
2.2.1. Glutathione peroxidase-like activity
After the synthesis, we explored the potential antioxidant ac-
tivity of all of the novel diselenide derivatives by measuring the
time required to reduce the concentration of the thiol by 50% (T50),
determined according to the Tomoda method [64,65] using ben-
zenethiol (PhSH) as a glutathione alternative. In this method, the
reduction of H2O2 was monitored through the ultraviolet (UV) ab-
sorption increase at 305 nm, due to diphenyl disulfide formation.
The literature contains reports of some compounds with chelating
groups, such as amines, amides or alcohols, close to selenium that
showed a positive influence on the GPx-like activity [66e74] and
we anticipated that our proposed structures would show the same
influence due to the presence of the amide group.
Thus, we examined the catalytic activities of our compounds
(2aeb and 3aed) and the results are outlined in Table 2. Linear
increases in absorbance (Fig. 2) were observed by mixing MeOH,
the catalyst (2aeb and 3aed), PhSH and H2O2. The reduction of
H2O2 was observed at catalyst concentrations of 25, 50, 100 and
200 mM, and the concentration of 100 mM was selected for further
studies. The control reaction was performed in the absence of
catalyst and did not show notable activity. On slightly modifying
the method, that is, replacing the methanol solvent with ethanol
for reasons of solubility, once again no appreciable thiophenol
oxidation in the absence of the catalysts or hydrogen peroxide was
observed, as well as for both references (ebselen and diphenyl
diselenide). In addition, in the first 120 s, the reactions were carried
out in the absence of H2O2 and in all the cases the oxidation of PhSH
(via an exchange reaction with diselenide compounds) was not
detected and there was no appreciable formation of PhSSPh. The
addition of H2O2 produced significant increase in the absorbance at
305 nm (PhSSPh formation), characterizing the GPx mimetic ac-
tivity (For details, see the Supporting Information).
For comparison purposes, ebselen and (PhSe)2, well-known
GPx-like mimetics [1,75], were used as controls in our study and
their activities were ascribed the value of 1.0, in each case (Entries
1e2). Most of the tested compounds showed catalytic activity in
this screening which was at least comparable to that shown by the
controls, confirming that the nitrogen-containing diselenide is a
viable group in relation to obtaining GPx-mimetic agents. The
aromatic derivatives 2a and 2b provided the best results (Entries
3e4), with compound 2b being capable of reducing the
concentration of thiol to half its value (T50) in only 16.87 min (Entry
4), approximately 9 times faster than ebselen (T50 ¼ 154.26 min)
(Entry 1)and 3 times than (PhSe)2. In addition, for compound 2a it
was found that T50 ¼ 29.73 min, which is around 5 and 2 times
Scheme 1. Reagents and conditions: i. 1 M HCl, NaNO2, H2O, 20 min, r.t. ii. Se0, NaOH,
NaBH4, H2O, 2 h, r.t. iii. CH2Cl2, amine (N2HCH(R)CH2Ph), DMAP, Et3N, 0 ꢂC, EDC, 24 h,
r.t.

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V. Nascimento et al. / European Journal of Medicinal Chemistry 87 (2014) 131e139
Scheme 2. Reagents and conditions: i. EtOH, NaBH4, Se0, 18 h ii.CH2Cl2, amine (N2HCH(R)CH2Ph), DMAP, Et3N, 0 ꢂC, EDC, 24 h, r.t.
faster than our references, ebselen and (PhSe)2 respectively (Entry
3). Compounds 3aed were tested under the same conditions.
The phenylethylamine derivative 3a, characterized by a shorter
chain length, also showed good catalytic activity (T50 ¼ 44.90 min)
(Entry 5) while diselenide 3b (Entry 6) was not able to promote the
reduction of H2O2 at an appreciable rate (T50 ¼ 299.63 min). It
should be noted that when the carbon chain length between the
amide and selenium is increased (n ¼ 2, derivative 3c and 3d), only
a moderate activity in the reduction of hydrogen peroxide (Entries
7e8) was observed, such activity is however 2 times better than
that of ebselen, but slower than diphenyl diselenide. The length of
the carbon chain is thus important with regard to the interaction
between the selenium atom and the amidic nitrogen, with two
carbons being the ideal distance, forming a five membered ring.
Aromatization of this side chain restricts the possible conforma-
tions forcing the interaction to occur. The presence or absence of
the methyl group within the phenylethylamine fragment seems to
be less important in terms of the GPx-like activity.
Table 1
Physical properties of the synthesized compounds 2aeb and 3aed.
Entry Product
Overall
yield (%)a (m.p.) (ꢂC)b
Melting point HRMSc found/
calculated ([Mþ])
1
2a 48
199e201
200e201
609.0561/609.0560
2
2b 44
637.0879/637.0873
3
4
5
3a 92
3b 76
93e94
485.0249/485.0245
513.0558/513.0558
Oil
3c 96
3d 83
114e115
95e96
513.0558/513.0565
541.0885/541.0871
2.2.2. Inhibition of thiobarbituric acid reactive substances (TBARS)
production in brain homogenates
6
Lipid peroxidation is an important mechanism involved in
cellular toxicity and it can be either a primary response to peroxide
attack to biomembranes via Fenton or HabereWeiss reactions or
can be secondary toxic response caused by oxidative stress induced
by electrophiles agents [76]. The presence of lipid peroxidation
indicates the potential disruption of biomembrane integrity and is
an important biomarker of oxidative stress. Consequently, the
protective effect against lipid peroxidation has a fundamental
a
Yields of compounds isolated in two steps from the corresponding carboxylic
acids.
b
c
Melting points are uncorrected.
HMRS: High Resolution Mass Spectrometry.
Table 2
Glutathione peroxidase-like activity of organoselenium catalysts 2aeb, 3aed,
ebselen and (PhSe)2.
Entrya,b Catalystc T50 (min)d,e
Efficiency relative to Efficiency relative to
ebselen
(PhSe)2
1f
2f
3g
4g
5
6
7
8
Ebselen 154.26 ( 6.35) 1.00
(PhSe)2 55.04 ( 3.50) 2.80
e
1.00
1.85
3.26
1.23
e
2a
2b
3a
3b
3c
3d
29.73 ( 0.16) 5.20
16.87 ( 0.33) 9.14
44.90 ( 2.15) 3.43
299.63 ( 8.00) e
81.43 ( 1.77) 1.89
76.00 ( 2.57) 2.03
e
e
a
Under this condition addition of H2O2 in the absence of the organoselenium
compound did not produce any significant oxidation of PhSH for both solvents
(MeOH and EtOH).
b
Under this condition in the absence of the H2O2 (MeOH or
EtOH þ catalyst þ PhSH) did not observe any significant oxidation of PhSH.
c
MeOH (1 mL); catalyst (0.1 mM); PhSH (2 mM); H2O2 (5 mM).
T50 is the time required, in min, to reduce the thiol concentration by 50% after
d
the addition of H2O2.
e
Data in parentheses: experimental error.
Both solvents are used (MeOH and EtOH) and it was not observe any appreciable
f
difference in the T50 values.
Fig. 2. Glutathione peroxidase-like behavior of catalysts 2aeb, 3a, 3ced, ebselen and
(PhSe)2.
g
EtOH was used in place of MeOH.

V. Nascimento et al. / European Journal of Medicinal Chemistry 87 (2014) 131e139
135
Table 3
importance for the design of new clinically effective antioxidant
drugs and it is an active field of drug research [45]. TBARS are
formed by the reaction of thiobarbituric acid (TBA) with byproducts
of lipid peroxidation, mainly malondialdehyde. Thus, the stable
colored product of TBA with malondialdehyde (TBARS) can be used
to quantify lipid peroxidation in biological samples [77]. Brain ho-
mogenates (S1) can be used as a source of biomembranes and we
have been using this model for some time as an index of lipid
peroxidation (for details see references in 43 and 28). In this study,
we used Fe(II) as an inducer of brain lipid peroxidation as it caused
a considerable increase in the TBARS production. All of the new
compounds inhibited Fe(II)-induced TBARS formation. However,
compounds 2a and 2b were more effective than ebselen and they
inhibited TBARS formation at lower concentration than did ebselen.
Compounds 3aed were also inhibitors of lipid peroxidation;
however, compounds 3aed were less effective than ebselen as
inhibitors of Fe(II)-induced TBARS formation in brain homogenates
(Fig. 3). Notably, the results obtained here indicated that the aro-
matic derivatives 2a and 2b were more effective than ebselen
confirming the trend already observed during the GPx-mimic test.
Although the exact molecular mechanism involved in the inhibition
of lipid peroxidation by diselenide compounds is still unknown, we
have proposed that their transformation to selenol intermediates is
involved in the degradation of organic peroxides formed in bio-
logical membranes [35,78]. Consequently, in brain homogenates,
diselenide compounds can react with both low molecular thiol
molecules and with thiol-containing proteins, such as lactate de-
hydrogenase (LDH) and aminolevulinic acid dehydratase (ALA-D)
[43,79] resulting in the formation of selenol intermediates.
NADPH oxidation by partially purified hepatic TrxR obtained from rats in the
presence of organoselenium catalysts 2aeb, 3aed and ebselen.a
Entry Catalyst Substrate of TrxR (nmolNADPH/min/ Efficiency relative to
mg)
ebselen
1
2
3
4
5
6
7
Ebselenb 3.4 ( 1.0)
1.0
e
2and
2bnd
3a
0
0
e
19.1 ( 2.2)
0
16.1 ( 3.7)
13.7 ( 7.1)
5.6
e
3bnd
3c
4.7
4.0
3d
n.d.Not detected during 10 min of assay.
a
NADPH oxidation was monitored at 340 nm for 10 min, in the absence or
presence of organoselenium compounds.
b
The oxidation of NADPH determined in the presence of ebselen was inhibited by
more than 95% by the addition of 5 mmol/L of AuCl3.
compound to act as a substrate for TrxR are unknown, and this issue
is very complex. In fact, we have previously observed that small
modifications to simple diselenides can decrease or completely
inhibit the ability of a given compound to act as a substrate for TrxR
[81]. From this study, it clearly emerges that the aromatic
substituents are detrimental to the diselenide functionality, at least
with regard to the affinity for TrxR.
In contrast, three out of four aliphatic derivatives (compounds
3a, 3c and 3d) are able to properly interact with the enzyme, being
fully converted into their selenol counterparts. In this case we can
speculate that the less hindered and more flexible structure allows
the diselenide moiety to be accessible and easily transformed.
However, further studies are clearly needed to better
understand the structural determinants indispensable for the
interaction of selenium compounds with TrxR. In fact, different
selenium compounds can be reduced by TrxR, including inorganic
compounds [83].
2.2.3. Substrate for rat liver thioredoxin reductase (TrxR)
Ebselen, its diselenide and various diselenide compounds act as
substrates for mammalian TrxR [80e82]. In this study the potential
reduction of new diselenides by partially purified rat liver enzyme
was investigated by determining the consumption of Nicotinamide
adenine dinucleotide phosphate hydrogen (NADPH) in the
presence and absence of new synthesized compounds (for details
see Ref. [81]). In contrast to the GPx-like assay, compounds 2a and
2b were not reduced by hepatic TrxR (Table 3). Compounds 3a, 3c
and 3d were good substrates for TrxR and their relative activity was
higher than that of ebselen (Table 3). Indeed, the consumption of
NADPH by partially purified hepatic TrxR was around 4e5 times
higher in the presence of compounds 3a, 3c and 3d than in the
presence of ebselen. The structural requirements for a selenium
3. Conclusions
In summary, in this paper, we have described the synthesis, in
good to excellent yields, of novel diselenide derivatives. The
compounds were fully characterized by 1H, 13C NMR and by HRMS.
All diselenides were evaluated for their antioxidant capacity in
order to better understand the structural determinants essential for
the antioxidant properties and shed light on the SAR of this class of
compounds. The new derivatives showed antioxidant activity
Fig. 3. Effect of amphetamine diselenide derivatives (2aeb and 3aed) and ebselen on TBARS production in rat brain homogenates (S1). All compounds were tested at final
concentrations ranging from 1 to 120 mol/L. Results are expressed as nmol of malondialdhyde (MDA) per g of brain.
m

136
V. Nascimento et al. / European Journal of Medicinal Chemistry 87 (2014) 131e139
through a GPx-like mechanism, they provide protection against
lipid peroxidation and, some of them can act as substrates for TrxR.
The aromatic derivatives 2a and 2b show a high potential to cata-
lyze the reduction of H2O2 to water at the expense of thiophenol,
with diselenide 2b exhibiting a T50 value of 16.87 min, whereas for
the known GPx mimetic ebselen a T50 value of 154.26 min was
observed. The improved ability to act as a GPx-mimic may be due to
the non-covalent interaction between selenium and amidic-
nitrogen, this being particularly evident on comparing the activity
of aliphatic and aromatic diselenides. In particular, in the aromatic
derivative the structural constraint forces such interactions to occur
and enhances the GPx-like activity.
reaction was kept under argon atmosphere and stirred at room
temperature for 1 h. The mixture was then slowly warmed to 110 ꢂC
and left at this temperature for an additional 30 min. The resulting
dark red solution was used without further treatment. The diazo-
nium salt solution was added dropwise to the sodium diselenide
solution. The resulting mixture was stirred for 0.5 h at room tem-
perature, then slowly warmed to 100 ꢂC, cooled to room temper-
ature and filtered over Celite. The red/orange solution was acidified
with 10% hydrochloric acid and the precipitate so formed was
collected, resuspended in methanol and refluxed. The suspension
was filtered off, and the filtrate was evaporated in vacuo yielding
0.9 g (80% yield) of the target acid.
The inhibition of lipid peroxidation can be in part explained by
the transformation of diselenides into selenol intermediates
through the reaction with thiol groups present in brain homoge-
nate. However, under the conditions of the TrxR assay, the absence
of appreciable concentrations of NADPH may have hampered the
reduction of some of the diselenides by TrxR. Nevertheless, the
results obtained in this study indicate that all of the new com-
pounds can exhibit important antioxidant properties, which are
mostly superior to those displayed by ebselen, evidencing that
these preliminary results are encouraging and indicate that these
new nitrogen-containing diselenides merit further investigation,
aimed at the development of new therapeutic drugs for the treat-
ment of oxidative stress-related disease.
4.2.1. 2,20-diselanediyldibenzoic acid (5)
Pale yellow solid. M.p. 289e292 ꢂC, (lit. [85] m.p. 292e293 ꢂC)
80% Yield; 1H NMR (DMSO-d6, 400 MHz)
d
¼ 8.48 (d, J ¼ 4 Hz, 1H),
8.14 (d, J ¼ 8 Hz, 1H), 7.91 (t, J ¼ 8 Hz, 1H), 7.84e7.77 (m, 1H); 13C
(DMSO-d6, 100 MHz)
128.83, 126.59.
d
¼ 168.63, 133.96, 133.59, 131.65, 129.57,
4.3. General procedure for the synthesis of diselenides 7aeb
The literature procedure [59] was slightly modified. To a two-
necked round-bottom flask, under argon atmosphere, Se0 (1
equivalent) and ethanol (5 mL/mmol) were added followed by the
addition of NaBH4 (2 equivalents). The mixture was left under
stirring until the solution became colorless. After this time, Se0 (1
equivalent) was added and the mixture was warmed briefly using a
hot air gun until boiling started. After 30 min, the bromo acid (2
equivalents) dissolved in ethanol (1 mL/mmol) was added to the
brownish-red ethanolic solution of Na2Se2 and the mixture was
stirred for 12 h. The mixture was then diluted in water and
extracted with ethyl acetate (3 ꢃ 20 mL). The organic layer was
dried over MgSO4 and concentrated under vacuum to give the
products as solids. The crude diselenides 7aeb were used without
further purification.
4. Experimental protocols
4.1. General methods and materials
NMR spectras (1H NMR and 13C NMR) were recorded on a Varian
AS-400 or Bruker Avance 200 spectrometer. Chemical shifts (
d) are
reported (in ppm) relative to the TMS (1H NMR) and the solvent (13
C
NMR). ESI-micrOTOF-Q II measurements were performed with a
micrOTOF Q-II (Bruker Daltonics) mass spectrometer equipped
with an automatic syringe pump (KD Scientific) for sample injec-
tion. The mass spectrometer was operated in the positive ion mode.
The sample was injected using a constant flow (3uL/min). The
solvent was a chloroform/methanol mixture. The ESI-micrOTOF-Q
II instrument was calibrated in the mass range of 50e3000 m/z
using an internal calibration standard (low concentration tuning
mix solution) supplied by Agilent Technologies. Data were pro-
cessed employing Bruker Compass Data Analysis software version
4.0. Column chromatography was carried using Merck Silica Gel
(230e400 mesh). Thin layer chromatography (TLC) was conducted
using Merck Silica Gel GF254, 0.25 mm thickness. For visualization,
the TLC plates were either placed under ultraviolet light, or stained
with iodine vapor or acidic vanillin. The melting points were
determined using a microscopy coverslip on a Micro Chemical MQA
PF digital apparatus and are uncorrected. All common reagents and
solvents were used as purchased unless otherwise noted. The
product yields included in all tables refer to isolated yields. Ebselen
was prepared as described in a previous report [84].
4.3.1. 2,20-diselanediyldiacetic acid (7a)
Yellow solid. M.p. 100e103 ꢂC, (lit. [59] m.p. 102e105 ꢂC) 96%
Yield; 1H NMR (DMSO-d6, 400 MHz)
d
¼ 13.60 (sl, 2H), 3.76 (s, 4H);
13C NMR (DMSO-d6, 100 MHz)
d
¼ 172.14, 23.92.
4.3.2. 3,30-diselanediyldipropanoic acid (7b)
Pale yellow solid. M.p. 127e130 ꢂC, (lit. [59] m.p. 130e132 ꢂC)
Quantitative yield; 1H NMR (DMSO-d6, 400 MHz)
d
¼ 3.05 (t,
J ¼ 8 Hz, 4H), 2.71 (t, J ¼ 8 Hz, 4H); 13C NMR (DMSO-d6, 100 MHz)
d
¼ 173.17, 35.50, 24.01.
4.4. General procedure for the synthesis of nitrogen-containing
diselenides 2aeb and 3aed
4.2. General procedure for the synthesis of diselenide 5
The literature procedure was modified [86]. The mixture of
diselenide acid (5 or 7aeb) (1 equivalent), amine (1.1 equivalent)
and 4-dimethylaminopyridine (DMAP) (0.2 equivalent) in dry
CH2Cl2 (5 mL) was cooled to 0 ꢂC, triethylamine (1.1 equivalent),
and 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride
(EDC) (1.1 equivalent) were then added. The solution was stirred for
24 h. The reaction mixture was diluted with CH2Cl2 and washed
with 0.5 N HCl (3 ꢃ 20 mL), 1% NaOH solution (3 ꢃ 20 mL) and
brine. After drying over MgSO4, the solvent was evaporated off
under reduced pressure and the crude product was purified by flash
chromatography (acetate:hexane).
Sodium nitrite (0.456 g, 6.61 mmol) in 6 mL of water was added
dropwise to a stirred solution of anthranilic acid (0.856 g,
6.24 mmol) in 1 mL of 37% hydrochloric acid and 7 mL of water
cooled in an ice bath. The solution of the resulting diazonium salt
was stirred for 20 min while a solution of sodium diselenide was
prepared in the following fashion. To a suspension of selenium (1 g,
12.6 mmol) and sodium hydroxide (0.5 g, 14.67 mmol), a 0 ꢂC so-
lution of sodium borohydride (0.06 g, 1.57 mmol) and sodium hy-
droxide (0.083 g, 2.07 mmol) in 1.2 mL water was added. The

V. Nascimento et al. / European Journal of Medicinal Chemistry 87 (2014) 131e139
137
4.4.1. 2,20-diselanediylbis(N-phenethylbenzamide) (2a)
H2O2 (final concentration: 5 mM). The reduction of H2O2 was
monitored by UV spectrophotometry, at 305 nm, via the formation
of PhSSPh. The reaction was monitored for more 150 s, more than
three times under the same conditions. Activities of the compounds
were compared with the activity of ebselen and (PhSe)2 (positives
controls).
White solid. M.p. 199e200 ꢂC, (25:75 acetate:hexane) 61% Yield;
1H NMR (CDCl3, 200 MHz)
d
¼ 7.87 (d, J ¼ 8 Hz, 2H), 7.38e7.13 (m,
16H), 6.23 (t, J ¼ 6 Hz, 2H), 3.76 (q, J ¼ 6 Hz, 4H), 2.98 (t, J ¼ 8 Hz,
4H); 13C NMR (CDCl3, 50 MHz)
d
¼ 168.16, 138.73, 133.10, 131.73,
128.87, 128.78, 126.69, 126.46, 126.08, 41.32, 35.62. HRMS (ESI) m/z
calculated for C30H28N2O2Se2 [Mþ]: 609.0560, found 609.0561.
4.5.1.1. Calculations for the T50 values. The T50 value is done by
mean of extrapolation or a rule of 3. The velocity of PhSSPh for-
mation is determined from the linear portion of peroxidase-like
activity spectrophotometric quantification. Thus, using a generic
example, for a compound that exhibited a rate of PhSSPh formation
4.4.2. 2,20-diselanediylbis(N-(1-phenylpropan-2-yl)benzamide)
(2b)
White solid. M.p. 200e201 ꢂC, (15:85 acetate:hexane) 55% Yield;
1H NMR (CDCl3, 400 MHz)
d
¼ 7.87 (d, J ¼ 8 Hz, 2H), 7.35e7.17 (m,
16H), 5.99 (d, J ¼ 4 Hz, 2H), 4.55e4.48 (m, 2H), 2.99 (dd, J1 ¼ 12 Hz,
J2 ¼ 4 Hz, 2H), 2.91 (dd, J1 ¼ 12 Hz, J2 ¼ 8 Hz, 2H), 1.27 (d, J ¼ 8 Hz,
of 0.1 mmol per min or per 60 s (in 1 mL, this means 0.1 mmol and
indicates the consumption of 0.2 mmol of PhSH/min or 60 s),
knowing that the total concentration of PhSH used was 5 mmol/L),
it is possible calculate that at 10 min or 600 s the expected con-
sumption will be 2 mmol, and that specifically 1.25 mmol of PhSSPh
(i.e., 2.5 mmol consumption of PhSH, which is 50% of total PhSH)
will occur at 12.5 min or 750 s. Thus, the linear portion of PhSSPh
formation was used to calculate the velocity of reaction and then
extrapolate it to 50% consumption of PhSH.
6H); 13C NMR (CDCl3, 100 MHz)
d
¼ 167.47, 137.59, 131.66, 129.58,
128.48, 126.61, 126.38, 126.02, 46.76, 42.25, 19.95. HRMS (ESI) m/z
calculated for C32H32N2O2Se2 [Mþ]: 637.0873, found 637.0879.
4.4.3. 2,20-diselanediylbis(N-phenethylacetamide) (3a)
Yellow solid. M.p. 93e94 ꢂC, (35:65 acetate:hexane) 96% Yield;
1H NMR (CDCl3, 400 MHz)
d
¼ 7.30e7.27 (m, 4H), 7.21e7.19 (m, 6H),
7,00 (t, J ¼ 4 Hz, 2H), 3.58e3.46 (m, 8H), 2.87e2.79 (m, 4H); 13C
NMR (CDCl3, 100 MHz)
41.18, 35.46, 31.66. HRMS (ESI) m/z calculated for C20H24N2O2Se2
d
¼ 170.09, 138.66, 128.68, 128.49, 126.42,
4.5.2. Thiobarbituric acid reactive substances (TBARS) assay
TBARS was quantified by the method of Ohkawa et al. [87] as
modified by Puntel et al. [88]. In short, brain homogenates (1/5, g/v)
were prepared in 10 mmol/L Tris buffer, pH 7.4 and centrifuged for
10 min at 3.6000 rpm. The supernatant (S1) was used as a source of
[Mþ]: 485.0245, found 485.0249.
4.4.4. 2,20-diselanediylbis(N-(1-phenylpropan-2-yl)acetamide)
(3b)
lipids in the assay. An aliquot of 20
60 min at 37 ꢂC with freshly prepared Fe2SO4 (final concentration of
10 Min at a final volume of 30 L) in 10 mmol/L Tris buffer (pH 7.4)
in the presence or absence of compounds 2a-b, 3a-d or ebselen. In
the next step, 40 L of 8.1% sodium dodecyl sulfate (SDS), 100 L of
buffered acetic acid (pH 3.4) and 100 L of 0.8% thiobarbituric acid
mL of S1 was incubated for
Dark yellow oil, (30:70 acetate:hexane) 79% Yield; 1H NMR
(CDCl3, 200 MHz)
d
¼ 7.28e7.19 (m, 10H), 6.82 (d, J ¼ 6 Hz, 2H),
m
m
4.30e4.17 (m, 2H), 3.53e3.52 (m, 2H), 2.98e2.97 (m, 2H),
2.94e2.61 (m, 4H), 1.17 (d, J ¼ 8 Hz, 6H); 13C NMR (CDCl3, 100 MHz),
m
m
d
¼ 169.42, 138.12, 129.28, 128.28, 126.33, 47.20, 42.50, 31.86, 20.08.
m
HRMS (ESI) m/z calculated for C22H28N2O2Se2 [Mþ]: 513.0558,
(TBA) were added sequentially. The mixture was incubated at
found 513.0558.
100 ꢂC for 1 h. The samples were then centrifuged at 6.000 rpm for
3 min and the color developed was measured in 200 mL of the su-
4.4.5. 3,30-diselanediylbis(N-phenethylpropanamide) (3c)
pernatant using an Elisa plate reader at 532 nm. TBARS levels are
expressed as nmol of MDA/g of tissue calculated against a standard
curve of MDA.
Dark yellow solid. M.p. 114e115 ꢂC (40:60 acetate:hexane) 96%
Yield; 1H NMR (CDCl3, 400 MHz)
d
¼ 7.32e7.19 (m, 10H), 6.06 (sl,
2H), 3.52 (q, J ¼ 8 Hz, 4H), 3.10 (t, J ¼ 8 Hz, 4H), 2.83 (t, J ¼ 8 Hz, 4H),
2.59 (t, J ¼ 8 Hz, 4H); 13C NMR (CDCl3, 100 MHz),
¼ 171.32, 138.74,
d
4.5.3. Thioredoxin reductase (TrxR) assay
128.73, 128.60, 126.49, 40.72, 37.37, 35.58, 24.38. HRMS (ESI) m/z
calculated for C22H28N2O2Se2 [Mþ]: 513.0565, found 513.0558.
TrxR from rat liver was purified essentially as described by Hill
et al. [22]. TrxR activity was determined according to Zhao and
Holmgren [80]. The activity was investigated in a buffer containing
4.4.6. 3,30-diselanediylbis(N-(1-phenylpropan-2-yl)propanamide)
(3d)
30 mM TriseHCl, 1.2 mM EDTA, pH 7.4, 100
TrxR and 120 M of NADPH.
mL of partially purified
Pale yellow solid. M.p. 94e95 ꢂC, (30:70 acetate:hexane) 83%
m
Yield; 1H NMR (CDCl3, 400 MHz)
d
¼ 7.30e7.17 (m, 10H), 6.03 (d,
Acknowledgments
J ¼ 8 Hz, 2H), 4.32e4.21 (m, 2H), 3.09 (t, J ¼ 8 Hz, 4H), 2.86 (dd,
J1 ¼ 12 Hz, J2 ¼ 4 Hz, 2H), 2.71 (dd, J1 ¼ 16 Hz, J2 ¼ 8 Hz, 2H),
2.59e2.56 (m, 4H), 1.13 (d, J ¼ 8 Hz, 6H); 13C NMR (CDCl3, 100 MHz),
ꢀ
The authors thank CEBIME, CNPq, INCT-Catalise, CAPES, and
FAPESC-Pronex for financial support.
d
¼ 170.57, 137.94, 129.34, 128.32, 126.37, 46.30, 42.40, 37.43, 24.47,
19.97. HRMS (ESI) m/z calculated for C24H32N2O2Se2 [Mþ]: 541.0871,
found 541.0885.
Appendix A. Supplementary data
4.5. Pharmacological evaluation
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.09.022.
4.5.1. Glutathione-peroxidase-like activity assay
The catalytic activity of the nitrogen-containing diselenides as a
GPx model enzyme was evaluated according to the Tomoda method
[64,65]. Initially, were mixed the selenium catalyst (final concen-
tration: 0.1 mM) and a methanol (or ethanol when was necessary)
solution of thiophenol (PhSH) (final concentration 2 mM) at 25 ( 3)
ꢂC. After around 120 s, the catalytic GPx model reaction
(H2O2 þ 2PhSH / 2H2O þ PhSSPh) was initiated by the addition of
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