New amides containing selenium as potent leishmanicidal agents targeting trypanothione reductase

Article abstract of DOI:10.1128/AAC.00524-20

Two new series of 28 selenocyanate and diselenide derivatives containing amide moieties were designed, synthesized, and evaluated for their leishmanicidal activity against Leishmania infantum axenic amastigotes, and selectivity was assessed in human THP-1

Full text of DOI:10.1128/AAC.00524-20

AAC Accepted Manuscript Posted Online 12 October 2020
Antimicrob. Agents Chemother. doi:10.1128/AAC.00524-20
Copyright © 2020 American Society for Microbiology. All Rights Reserved.
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New amides containing selenium as potent leishmanicidal agents
targeting trypanothione reductase
Mikel Etxebeste-Mitxeltorena1,2,3, Daniel Plano1,2,3, Socorro Espuelas1,2,3, Esther
Moreno1,2,3, Carlos Aydillo1,2,3, Antonio Jiménez-Ruiz4, Juan Carlos García
Soriano4, Carmen Sanmartín1,2,3
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1Universidad de Navarra, Facultad de Farmacia y Nutrición, Departamento
de Tecnología y Química Farmacéuticas, Irunlarrea, 1. E-31008 Pamplona,
Spain
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2Instituto de Salud Tropical, Universidad de Navarra, ISTUN, Irunlarrea, 1.
E-31008 Pamplona, Spain
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3Instituto de Investigaciones Sanitarias de Navarra (IdiSNA) Irunlarrea, 1. E-
31008 Pamplona, Spain
4Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá,
Carretera Madrid-Barcelona km 33,600. E-28871, Alcalá de Henares, Madrid,
Spain
* Prof. Carmen Sanmartín
Department of Pharmaceutical Technology and Chemistry
University of Navarra
Irunlarrea, 1, E-31008 Pamplona
SPAIN
+34 948 425 600 (Telephone)
+34 948 425 649 (Fax)
e-mail: sanmartin@unav.es
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Abstract
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Two new series of twenty-eight selenocyanate and diselenide derivatives
containing amide moiety were designed, synthesized and evaluated for their
leishmanicidal activity against Leishmania infantum axenic amastigotes, and
selectivity was assessed in human THP-1 cells. Eleven compounds exhibited
excellent leishmanicidal activity with EC50 values lower than the reference drug
miltefosine (EC50 = 2.84 µM). In addition, for six of them the selectivity index
ranged from 9 to > 1442, greater than both references used. The most potent
and selective compounds were 2h, 2k and 2m that displayed EC50 values of
0.52, 1.19 and 0.50 µM and a high selectivity index (SI) when tested against
THP-1 monocytic cells (SI = >1442, > 672 and >1100, respectively). These
derivatives showed an efficacy similar to that of the reference drugs but much
better SI. They also showed very interesting activity values against infected
macrophages. Trypanothione reductase (TryR) activity and intracellular thiol
level measurement assays were performed for the three best compounds in an
attempt to elucidate their mechanism of action. Even though the new analogues
exhibited comparable or better inhibitory activities than reference TryR inhibitors
more studies are necessary to confirm this target. To sum up, our findings
suggest that the three presented compounds could constitute lead
leishmanicidal drug candidates.
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Keywords: amides, diselenide, selenocyanate, thiols, trypanothione reductase
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INTRODUCTION
Leishmaniasis, caused by protozoan parasites from the Leishmania genus, is
one of the most devastating neglected tropical diseases(1). These parasites
belong to the Trypanosomatidae family and include more than 21 species with
capacity of infecting humans(2). The clinical manifestations are classified into at
least four major forms, including cutaneous, diffused cutaneous, muco-
cutaneous and visceral leishmaniasis(3).
Actually, available treatments for leishmaniasis include pentavalent antimonials
as first-line drugs, and amphotericin B, pentamidine, paromomycin, and
miltefosine as second-line drugs(4). However, most of these drugs exhibit high
toxicity, easily induce resistance, are responsible for severe side effects, and
show increased incidence of treatment failure(5). Furthermore, there is currently
no effective vaccines against this disease(6). These facts clearly emphasize the
urgent need for the development of novel chemotherapies against
leishmaniasis.
Currently, several investigations have been conducted to find alternative
treatments for leishmaniasis and the exploitation of selenium derivatives is a
viable approach. In this context, from the literature we noted the importance of
selenium for the survival of these parasites acting through immune response
modulation(7), alone or in combination therapy with amphotericin B(8) or
glucantime(9). Moreover, selenium can interfere with redox-system enzymes
such as selenophosphate synthetases(10), ascorbate peroxidases(11) or the
well-known parasitic trypanothione reductase(12) or superoxide dismutase(13).
Furthermore, computational analyses conducted over the genomes of several
Leishmania species have revealed the presence of at least three
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selenoproteins: Sel K, Sel T and a kinetoplastida specific Sel Tryp, which
evidence the dependence of the parasites for this trace element(14).
In addition, our research group has published several articles which
demonstrate that different selenium containing frameworks such as
methylseleno, selenocyanate, diselenide or selenosulfonamide, incorporated in
different organic structures showed potent leishmanicidal activity (12, 15-18).
Among these organoselenium compounds, selenocyanate and diselenide motifs
are fascinating because of the promising results in terms of activity and
selectivity(19, 20).
Prompted by the previous reported results regarding the activity of 4-
aminophenylselenocyanate and bis(4-aminophenyl)diselenide we have focused
on these nuclei to synthesize the compounds reported in this work. Herein we
describe the synthesis and characterization of two new series containing 4-
aminophenylselenocyanate
and
bis(4-aminophenyl)diselenide
entities
derivatized using an amide group as linkage. The amide based ligand has been
selected for the current study due to its hydrophilic character with the aim to
increase water solubility of the new compounds, improve their physico-chemical
properties and for its ability to generate prodrugs that may increase drug
concentration in the desired targets(21, 22). These amides have been
decorated with varied substituents in order to obtain synthetic species with
progressive degree of complexity in terms of electronic and steric properties or
3D structural characteristics. Among these scaffolds, different substituted
phenyl rings along with carbo- and hetero-cycles with probed leishmanicidal
properties have been explored(19, 20, 23). Herein we compare the effects of
the following fragments: cinnamyl(24), naphtyl(25), isoxazolyl(26) and
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adamantyl(27). In addition, the incorporation of an adamantyl moiety into
several molecules results in compounds with relatively high lipophilicity which,
in turn, can modify the biological availability of these molecules. The structures
of the reported compounds are summarized in Figure 1.
(Figure 1)
Enzymes like trypanothione reductase (TryR)(28) have emerged among the
most relevant druggable targets in Leishmania and other Trypanosomatids
because of their highly relevant role in the maintenance of the parasite’s redox
homeostasis. The enzyme fulfills most of the more relevant requirements to be
an interesting drug target since it is essential for parasite survival, it is
druggable and because of its absence in the host, were is replaced by
glutathione reductase(29). Trypanothion reductase acts catalyzing the reduction
of trypanothione disulfide to trypanothione based on the ability of sulfur to act as
donor or an acceptor of electrons. In this sense, several sulfides have
demonstrated the capacity of killing leishmanial parasites binding this enzyme
by competing with the substrate(30). Due to the chemical analogy between
sulfur and selenium we decided to explore the ability of this molecules inhibiting
TryR. Moreover, previous studies of our research group demonstrated the
interesting inhibitory activity of seleno-compounds, including selenocyanates
and diselenides, on TryR enzyme(23).
On the bases of the above mentioned, the leishmanicidal activity of the 28
resulting compounds was determined against the amastigote form of L.
infantum and the cytotoxicity of these newly synthesized molecules was also
assessed on one different complementary human cell line (THP-1) in order to
select those compounds with high selectivity. Moreover, leishmanicidal activity
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of the most active compounds was evaluated in infected macrophages. Finally,
in order to elucidate the underlying molecular mechanisms, the inhibitory activity
against trypanothione reductase (TryR) and their effect on intracellular thiol
concentration was determined.
RESULTS
Chemistry
The target compounds 1a-1n and 2a-2n were synthesized in accordance with
the procedures outlined in Figure 2.
(Figure 2)
The
intermediates
4-aminophenylselenocyanate
(0A)
and
bis-(4-
aminophenyl)diselenide (0B) were achieved as previously reported(31). The
treatment of 4-aminophenylselenocyanate with the corresponding acyl chlorides
in a molar ratio 1:1 in chloroform at room temperature during 12-24h yielded
derivatives 1a-1n. The products were isolated from reaction solutions by
filtration and purified by recrystallization from methanol or a mixture of
methanol/water.
The structures of the final compounds were confirmed by infrared (IR), nuclear
magnetic resonance (1H NMR, 13C NMR) as well as mass spectrometry and
elemental analysis. Aromatic signals appear at δH 6.72-8.15 ppm depending on
the nature of substituent attached. 13C NMR spectra show the typical C=O
signal between δC 156.8-176.7 ppm (see supplementary material).
The synthesis of the target compounds 2a-2c, 2e-2j, 2l and 2n was carried out
by reaction of the corresponding selenocyanates, dissolved in ethanol, and
sodium borohydride at room temperature. The reaction mixture was evaporated
under reduced pressure and the final residue was extracted with
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dichloromethane. The target compounds were purified by recrystallization.
Analogues 2d, 2k and 2m were obtained by reaction between bis-(4-
aminophenyl)diselenide and the corresponding acyl choride in chloroform. The
reaction yields are moderated ranging from 46-68%. Formation of diselenide
was confirmed by the disappearance of the SeCN peak at a wavenumber
around 2147-2226 cm-1 in infrared spectroscopy.
Biological evaluation
(i) Activity in amastigotes and cytotoxic activity in human cells. The newly
synthesized compounds along with miltefosine and edelfosine (used as positive
controls) were tested in vitro against cultured L. infantum amastigotes following
a previously described method(19). Axenic amastigotes have been chosen
because of their high similarity with the parasite stage that develops infections
in humans. Parent compounds 4-aminophenylselenocyanate (0A) and the
corresponding Bis(4-aminophenyl)diselenide (0B) were also investigated in
order to compare their biological response. Additionally, and to assess their
selectivity, these compounds were tested against a human monocytic leukemia
cell line (THP-1). The results shown in Table 1 reveal that eleven compounds
(1a, 1d, 1h, 1i, 1j, 1k, 2h, 2i, 2j, 2k and 2m) exhibit potent antileishmanial
activity with EC50 values similar or lower than those of miltefosine (EC50 = 2.84
µM) used as reference. Compared to edelfosine, the other reference drug used
in this study, 1h, 2h, 2i, 2j, and 2m also fulfil this standard. Regarding the
difference of activity between the selenocyanate and the diselenide analogs,
globally considered, it is difficult to infer a general trend that either increases or
reduces the activity. In order to get insight into the structure activity relationship,
it has been found that the diselenide compounds derived from p-substituted
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phenyl ring 2a-2g and 2n were less potent than the others (Table 1). This effect
is less marked in the corresponding selenocyanates. Compounds containing
heteroaryl rings on “R”, i.e. 1h, 1i, 1j, 2h, 2i and 2j displayed potent activities
with EC50 values between 0.52 and 1.96 µM. On the other hand, introduction of
rigid and bulky moieties such as adamantyl (1k, 2k) or naphtyl (1m, 2m)
reflected different behaviors. While the adamantyl motif increased the
leishmanicidal activity both in selenocyanates and diselenides, the naphtyl
entity was effective only with diselenide function. Compared with the chosen
parent compound selenocyanate (EC50 = 9.29 µM), twelve of the fourteen
compounds synthesized were more potent, whereas four of the diselenide
derivatives were comparable to their corresponding parent molecule.
In terms of selectivity, compounds 1d, 1h, 1i, 2h, 2k and 2m showed SI values
that ranged from 9 to > 25, greater than those of the reference drugs edelfosine
and miltefosine (SI = 6 and 7, respectively). Being 2h, 2k and 2m the most
promising derivatives (Figure 3), they were further tested against THP-1 cells at
higher concentrations in order to determine their real EC50 values. Interestingly,
derivatives 2h (EC50 = 0.52 µM, SI > 1442), 2k (EC50 = 1.19 µM, SI > 672.3)
and 2m (EC50 = 0.50 µM, SI > 1100) are between 112 and 240-fold less toxic
than edelfosine while showing similar or even better leishmanicidal activity.
(Table 1)
(Figure 3)
(ii) Leishmanicidal activity in infected macrophages. After this first
screening and taking into account activity and selectivity data, compounds 2h,
2k and 2m were selected for the analysis of their leishmanicidal activity on
infected macrophages. Again, edelfosine was used as reference drug. The ED50
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for each compound was calculated as mean of three independent experiments
and summarized in Table 2. These compounds reduced the parasite load of the
cells comparing with the non-treated infected cells (Figure 4), exhibiting ED50
values of 2.20, 3.77 and 1.03 µM respectively, with similar or better
effectiveness than that obtained for edelfosine (ED50 = 3.10 µM). Taking into
account that selectivity is one of the most relevant characteristics to be
achieved in new leishmanicidal drugs, the excellent SI values obtained for the
three selected compounds are highly remarkable.
(Figure 4)
(Table 2)
(iii) Inhibition of L. infantum trypanothione reductase activity. As last topic,
these derivatives have been considered as promising leads for a preliminary
study of their mechanism of action. Initially, we focused on the alteration of the
redox system.
In an effort to assess the mechanism by which 2h, 2k and 2m inhibit
Leishmania growth, we evaluated their activity against TryR, an essential
enzyme involved in parasite detoxification of reactive oxygen species(32).
Trypanothione reductase is an essential enzyme in trypanosomatids (T. brucei,
T. cruzi, and Leishmania species) that has a unique role in the trypanothione-
based redox metabolism and oxidant defense. Lack of this enzyme in the
mammalian hosts is one of the reasons that turned it very attractive as drug-
target(29).
The well stablished TryR inhibitor mepacrine was used as positive control.
(Table 3).
(Table 3)
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The results from the in vitro assays revealed that compounds 2h and 2k exhibit
an inhibitory capacity against TryR comparable to that of the reference drug.
Moreover, 2m derivative is 3-fold more active than mepacrine. In spite of these
results, numerous TryR inhibitors have been found to have only a modest effect
on parasite growth. A partial explanation was provided by Krieger et al. who
produced conditioned TryR knockout in T. brucei(33). They have shown that the
redox metabolism of the parasite was affected only when TryR was titrated
down to less than 5% of normal(34).
(iv) Redox system alteration. To further confirm the alteration of the parasite
redox system by these molecules, intracellular thiol levels was determined
inside the parasites by flow cytometry. As shown in Figure 5, treatment of the
amastigotes for 1 hour with the three selected molecules triplicates the levels of
intracellular thiols.
(Figure 5)
(v) ADME and Lipinski properties. Finally, considering that most of the new
drug candidates fail in clinical trials due to reduced ADME properties, we
predicted the ADME parameters for lead compounds (2h, 2k and 2m) using the
PreADMET software. The values obtained were compared with the Lipinski rule
of five although it is well-known that pharmacologically active compounds do not
necessarily fulfill all rules. Molecular weight (MW), number of hydrogen donors
(DHB), number of hydrogen acceptors (AHB), octanol/water partition coefficient
(log P), apparent Caco-2 cell permeability (PCaco), and percent of human
intestinal absorption (%HIA) are presented in Table 4(35, 36).
(Table 4)
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The three compounds violate just two of the Lipinksi’s rule of five, similar to
what occurs with the most clinically relevant reference drug (miltefosine). Oral
bioavailability according to VEBER and to EGAN was good for all compounds
too. Moreover, all of the compounds passed the three PAINS filters, which may
help to detect compounds that are unspecific assay-interfering compounds. We
also used PreADMET (http://preadmet.bmdrc.org/preadmet/index.php) web-
application for a rapid prediction of ADME/Tox data. Caco-2 permeability and
Human Intestinal Absorption (HIA) are interesting indicators of drug
absorbance. The predicted percentages of intestinal absorption are quite good
for all of the compounds, with values over 97%. Otherwise, the three
compounds presented middle permeability values in Caco-2 cells ranging from
38 to 53.
DISCUSSION
Selenium compounds are used as potential treatments for multiples diseases
such as cancer(37) Alzheimer(38) or cardiovascular diseases(39) among
others.
In addition, selenium derivatives have emerged as promising compounds
against leishmaniasis and other trypanosomiasis. Previous studies of our
research group demonstrated the interesting leishmanicidal activity of
selenocyanate and diselenide derivatives. Moreover, several selenium
containing compounds have demonstrated its trypanocidal activity against other
trypanosomiasis like Chagas(40-42) or African trypanosomiasis(7, 43) The
twenty-eight new compounds presented in this work contain the 4-
aminopheylselenocyanate (1a-n) or the bis(4-aminophenyl)diselenide (2a-n)
moieties based on the promising leishmanicidal activity reported for them.
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These cores have been joined by a linker fragment, which involves a new amide
bond, to a different substituted phenyl rings, heterocycles, and bulky
carbocycles such as adamantyl or naphtyl. Overall, if we compared with the
parent compound 0A, thirteen of the fourteen (with the exception of 1e)
selenocyanates exhibited similar or better leishmanicidal activity than 0A. If we
focused in diselenides only four derivatives (2h, 2i, 2j and 2m) showed
comparable activity to the parent 0B in amastigotes form. However, it is
remarkable the excellent SI for diselenides, especially for 2h, 2k and 2m. These
data helped to select these three compounds as the best candidates for further
studies. Compounds 2k and 2m share a bulky carbocyclic substituent
(adamantyl and naphtyl, respectively) in their structure, while 2h possess a
heteroaryl monocyclic entity. The studies in infected macrophages revealed that
all of them exhibited the same (2k) or even better (2h, 2m) ED50 values than
edelfosine. Regarding their possible mechanism of action and taking into
account that selenium compounds used to alter the redox system(44),
intracellular thiol levels were evaluated in treated parasites. Similarly, their
inhibitory activity against L. infantum trypanothione reductase (TryR) was
determined. Trypanothion reductase emerged as an interesting target for its
critical role in the trypanosomatidae family parasite’s redox system control and
because humans lack this enzyme(29, 45). Strong correlation between
intracellular thiols disturbance, TryR inhibition and antiparasitic activity was
observed for the three compounds, which may be considered as an evidence of
their possible mechanism of action. Parasites, in response to an oxidative
stress, seem to induce the novo synthesis of trypanothion and that could be the
reason why the concentrations of thiols rise up. Moreover, presented
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compounds improved the results of activity against TryR obtained so far among
previous selenocompounds of our research group(23). The calculated ADME
parameters for compounds 2h, 2k and 2m predicted a good bioavailability, with
low Lipinski’s rule violations, similar or even better (2h) solubility (LogP), and
high intestinal absorption prediction values. It is well-known that current
available drugs for leishmania treatment are not fully effective in all cases,
having only parenteral use and only miltefosine is the oral drug approved.
However, the appearance of resistance led to change its use from monotherapy
to combination therapy(46). For these reasons, the incorporation of new
strategies or compounds that can improve the oral bioavailability is an urgent
need(47).
In this context, the compounds described herein are promising candidates.
Moreover, they are also easily affordable and economical in their synthesis.
Further in vivo toxicity and efficiency studies will be carried out with these
promising molecules in order to confirm their potent and selective
antileishmanial activity. A graphical summary of the discussion drawn from this
work is depicted in Figure 6.
(Figure 6)
MATERIAL AND METHODS
Chemistry
Melting points (mp) were determined with a Mettler FP82 + FP80 apparatus
(Greifensee, Switzerland). Proton (1H), carbon (13C) and selenium (77Se) NMR
spectra were recorded on a Bruker 400 Avance Neo spectrometer
(Rheinstetten, Germany) using DMSO-d6 as solvent. IR spectra were recorded
on a Thermo Nicolet FTIR Nexus spectrophotometer using KBr pellets for solid
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samples or NaCl plates for liquid compounds. Elemental analysis was
performed on a LECO CHN-900 Elemental Analyzer. Purity of all final
compounds was 95 % or higher. TLC assays were carried out in Alugram SIL
G7UV254 sheets (Macherey-Nagel, Düren, Germany). Chemicals were
purchased from E. Merck (Darmstadt, Germany), Panreac Química S.A.
(Montcada
i
Reixac, Barcelona, Spain), Sigma-Aldrich Quimica, S.A.
(Alcobendas, Madrid, Spain) and Acros Organics (Janssen Pharmaceuticalaan,
Geel, Belgium).
General procedure of synthesis for compounds 1a-n
Compounds
1a-n
were
obtained
by
reaction
between
4-
aminophenylselenocyanate and the corresponding acyl chloride, in a 1:1 molar
ratio and in dry chloroform (50 mL) as solvent. The mixture was stirred at room
temperature during 12-24 h. A precipitate was formed that was filtered and
recrystallized from methanol or a mixture of methanol/water.
(Figure 7)
N-(4-selenocyanatophenyl)benzamide (1a). From benzoyl chloride.
Recrystallized from methanol. White powder; mp: 169-170 ºC. Yield: 56 %. IR
(KBr) cm-1: 3342 (N-H); 2154 (CN); 1663 (C=O). 1H NMR (400 MHz, DMSO-d6)
δ: 7.52 – 7.59 (m, 3H, B, H3+H4+H5), 7.72 (d, 2H, A, J2-3 = J5-6 = 8.4 Hz, H2+H6),
7.89 (d, 2H, A, H3+H5), 7.97 (d, 2H, B, H2+H6), 10.46 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6) δ: 105.8 (1C, SeCN), 117.5 (1C, A, C1), 122.0 (2C, A, C3+C5),
128.2 (2C, B, C2+C6), 128.9 (2C, B, C3+C5), 132.3 (2C, B, C1+C4), 135.1 (2C, A,
C2+C6), 141.0 (1C, A, C4), 166.3 (1C, C=O). MS (m/z % abundance): 302 (55;
M+·), 105 (98), 77 (100). Elemental Analysis for C14H10N2OSe,
Calculated/Found (%): C: 55.81/55.47; H: 3.32/3.69; N: 9.30/9.21.
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4-chloro-N-(4-selenocyanatophenyl)benzamide (1b). From 4-chlorobenzoyl
chloride. Recrystallized from methanol. White powder; mp: 226-227 ºC. Yield:
1
62 %. IR (KBr) cm-1: 3322 (N-H); 2151 (CN); 1674 (C=O). H NMR (400 MHz,
DMSO-d6) δ: 7.63 (d, 2H, A, J2-3 = J5-6 = 8.3 Hz, H2+H6), 7.72 (d, 2H, B, J2-3 = J5-
6 = 8.5 Hz, H3+H5), 7.87 (d, 2H, B, H2+H6), 8.00 (d, 2H, A, H3+H5), 10.51 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 105.8 (1C, SeCN), 117.7 (1C, A, C1),
122.1 (2C, A, C3+C5), 129.0 (2C, B, C3+C5), 130.2 (2C, B, C2+C6), 133.7 (1C, B,
C1), 135.0 (2C, A, C2+C6), 137.1 (1C, B, C4), 140.8 (1C, A, C4), 165.2 (1C,
C=O). MS (m/z % abundance): 336 (20; M+·), 139 (100). Elemental Analysis for
C14H9ClN2OSe, Calculated/Found (%): C: 50.07/50.36; H: 2.68/3.05; N:
8.34/8.48.
4-methoxy-N-(4-selenocyanatophenyl)benzamide
(1c).
From
4-
methoxybenzoyl chloride. Recrystallized from methanol. Green powder; mp:
167-168 ºC. Yield: 52 %. IR (KBr) cm-1: 3394 (N-H); 2963 (C-H); 2164 (CN);
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1662 (C=O). H NMR (400 MHz, DMSO-d6) δ: 3.85 (s, 3H, OCH3), 7.08 (d, 2H,
B, J2-3 = J5-6 = 8.7 Hz, H3+H5), 7.71 (d, 2H, B, J2-3 = J5-6 = 8.6 Hz, H3+H5), 7.88
(d, 2H, B, H2+H6), 7.98 (d, 2H, A, H3+H5), 10.30 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6) δ: 55.9 (OCH3), 105.8 (1C, SeCN), 114.1 (2C, B, C3+C5),
117.1 (1C, A, C1), 122.0 (2C, A, C3+C5), 127.0 (1C, B, C1), 130.2 (2C, B,
C2+C6), 135.1 (2C, A, C2+C6), 141.2 (1C, A, C4), 162.6 (1C, B, C4), 165.6 (1C,
C=O). MS (m/z % abundance): 332 (25; M+·), 135 (100), 77 (45). Elemental
Analysis for C15H12N2O2Se, Calculated/Found (%): C: 54.38/54.49; H:
3.62/3.88; N: 8.46/8.32.
4-cyano-N-(4-selenocyanatophenyl)benzamide (1d). From 4-cyanobenzoyl
chloride. Recrystallized from methanol. Yellow cotton-like powder; mp: 218-219
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ºC. Yield: 63 %. IR (KBr) cm-1: 3303 (N-H); 2226 (CN); 2157 (Se-CN); 1674
1
(C=O). H NMR (400 MHz, DMSO-d6) δ: 7.74 (d, 2H, A, J2-3 = J5-6 = 8.5 Hz,
H2+H6), 7.87 (d, 2H, A, H3+H5), 8.04 (d, 2H, B, J2-3 = J5-6 = 8.2 Hz, H2+H6), 8.12
(d, 2H, B, H3+H5), 10.68 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 105.8
(1C, SeCN), 114.5 (1C, B, C4), 118.1 (1C, CN), 118.7 (1C, A, C1), 122.1 (2C, A,
C3+C5), 129.1 (2C, B, C2+C6), 133.0 (2C, B, C3+C5), 135.0 (2C, A, C2+C6),
139.0 (1C, B, C1), 140.5 (1C, A, C4), 164.9 (1C, C=O). MS (m/z % abundance):
327 (20; M+·), 130 (100), 102 (40). Elemental Analysis for C15H9N3OSe,
Calculated/Found (%): C: 55.24/54.82; H: 2.76/2.79; N: 12.88/12.44.
4-nitro-N-(4-selenocyanatophenyl)benzamide (1e). From 4-nitrobenzoyl
chloride. Recrystallized from methanol/water (1:1). Green powder; mp: 235-236
ºC. Yield: 54 %. IR (KBr) cm-1: 3304 (N-H); 2154 (CN); 1676 (C=O); 1350 (NO2).
1H NMR (400 MHz, DMSO-d6) δ: 7.74 (d, 2H, A, J2-3 = J5-6 = 8.6 Hz, H2+H6),
7.87 (d, 2H, A, H3+H5), 8.20 (d, 2H, B, J2-3 = J5-6 = 8.6 Hz, H3+H5), 8.39 (d, 2H,
B, H2+H6), 10.77 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 106.2 (1C,
SeCN), 118.7 (1C, A, C1), 122.5 (2C, A, C3+C5), 124.5 (2C, B, C3+C5), 130.2
(2C, B, C2+C6), 135.5 (2C, A, C2+C6), 140.9 (1C, B, C1), 141.0 (1C, A, C4),
150.1 (1C, B, C4), 165.1 (1C, C=O). MS (m/z % abundance): 347 (10; M+·), 188
(100), 127 (50), 105 (32). 77 (25). Elemental Analysis for C14H9N3O2Se,
Calculated/Found (%): C. 48.55/48.50; H: 2.60/2.66; N: 12.13/12.23.
4-trifluoromethyl-N-(4-selenocyanatophenyl)benzamide (1f). From 4-
trifluoromethylbenzoyl chloride. Recrystallized from methanol/water (1:1). Green
powder; mp: 214-215 ºC. Yield: 51 %. IR (KBr) cm-1: 3324 (N-H); 2154 (CN);
1675 (C=O); 1176-1339 (CF3). 1H NMR (400 MHz, DMSO-d6) δ: 7.73 (d, 2H, B,
J2-3 = J5-6 = 7.1 Hz, H3+H5), 7.87 (d, 2H, B, H2+H6), 7.93 (d, 2H, A, J2-3 = J5-6
=
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7.8 Hz, H3+H5), 8.15 (d, 2H, A, H2+H6), 10.68 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 106.2 (1C, SeCN), 118.4 (1C, A, C1), 122.5 (2C, A, C3+C5), 124.7
1
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(q, JC-F = 272.6 Hz, 1C, CF3), 126.3 (q, JC-F = 3.6 Hz, 2C, B, C3+C5), 129.7
2
(2C, B, C2+C6), 132.5 (q, JC-F = 31.9 Hz, 1C, B, C4), 135.5 (2C, A, C2+C6),
139.2 (1C, B, C4), 141.0 (1C, A, C1), 165.5 (1C, C=O). MS (m/z % abundance):
370 (22; M+·). 173 (100). Elemental Analysis for C15H9F3N2OSe,
Calculated/Found (%): C: 48.78/48.91; H: 2.43/2.27; N: 7.58/7.62.
4-methyl-N-(4-selenocyanatophenyl)benzamide (1g). From 4-methylbenzoyl
chloride. Recrystallized from methanol. Blue powder; mp: 235-236 ºC. Yield: 43
1
%. IR (KBr) cm-1: 3334 (N-H); 2913 (C-H); 2155 (CN); 1662 (C=O). H NMR
(400 MHz, DMSO-d6) δ: 2.40 (s, 3H, CH3), 7.35 (d, 2H, B, J2-3 = J5-6 = 8.3 Hz,
H3+H5), 7.70 (d, 2H, A, J2-3 = J5-6 = 8.7 Hz, H3+H5), 7.88 (d, 2H, B, H2+H6), 7.90
(d, 2H, A, H2+H6), 10.37 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 21.9
(1C, CH3), 106.2 (1C, SeCN), 117.7 (1C, A, C1), 122.4 (2C, A, C3+C5), 128.7
(2C, B, C2+C6), 129.8 (2C, B, C3+C5), 132.5 (1C, B, C1), 135.5 (2C, A, C2+C6),
141.6 (1C, A, C4), 142.8 (1C, B, C4), 166.5 (1C, C=O). MS (m/z % abundance):
316 (25; M+·), 119 (100), 91 (32), 65 (20). Elemental Analysis for C15H12N2OSe,
Calculated/Found (%): C: 57.14/56.77; H: 3.80/3.98; N: 8.88/8.67.
N-(4-selenocyanatophenyl)furan-2-carboxamide (1h). From furoyl chloride.
Recrystallized from methanol. Blue powder; mp: 146-147 ºC. Yield: 38 %. IR
(KBr) cm-1: 3309 (N-H); 2148 (CN); 1676 (C=O). 1H NMR (400 MHz, DMSO-d6)
δ: 6.72 (dd, 1H, B, J2-3= 3.5 Hz, J3-4= 1.7 Hz, H3), 7.38 (d, 1H, B, H2), 7.70 (d,
2H, A, J2-3 = J5-6 = 8.7 Hz, H2+H6), 7.86 (d, 2H, A, H3+H5), 7.97 (d, 1H, B, H4),
10.42 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 105.8 (1C, SeCN), 112.7
(1C, B, C3), 115.8 (1C, B, C2), 117.6 (1C, A, C1), 122.0 (2C, A, C3+C5), 135.1
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(2C, A, C2+C6), 140.4 (1C, A, C4), 146.5 (1C, B, C4), 147.6 (1C, B, C1), 156.8
(1C, C=O). MS (m/z % abundance): 292 (48; M+·), 95 (100). Elemental Analysis
for C12H8N2O2Se, Calculated/Found (%): C: 49.48/49.40; H: 2.75/3.10; N:
9.62/9.34.
N-(4-selenocyanatophenyl)thiophene-2-carboxamide
(1i).
From
2-
thiophenecarbonyl chloride. Recrystallized from methanol. Blue powder; mp:
205-206 ºC. Yield: 56 %. IR (KBr) cm-1: 3347 (N-H); 2147 (CN); 1657 (C=O). 1H
NMR (400 MHz, DMSO-d6) δ: 7.25 (t, 1H, B, J2-3 = J3-4 = 4.3 Hz, H3), 7.72 (d,
2H, A, J2-3 = J5-6 = 8.6 Hz, H2+H6), 7.83 (d, 2H, A, H3+H5) 7.89 (d, 1H, B, H4),
8.05 (d, 1H, B, H2), 10.42 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 105.8
(1C, SeCN), 117.6 (1C, A, C1), 122.0 (2C, A, C3+C5), 128.6 (1C, B, C4), 130.0
(1C, B, C3), 132.8 (1C, B, C2), 135.1 (2C, A, C2+C6), 140.0 (1C, A, C4), 140.6
(1C, B, C1), 160.6 (1C, C=O). MS (m/z % abundance): 308 (40; M+·), 111 (100).
Elemental Analysis for C12H8N2OSSe, Calculated/Found (%): C: 46.90/46.73; H:
2.60/2.67; N: 9.12/9.26.
N-(4-selenocyanatophenyl)isoxazole-5-carboxamide (1j). From isoxazole-5-
carbonyl chloride. Recrystallized from methanol/water (1:1). White powder; mp:
185-186 ºC. Yield: 52 %. IR (KBr) cm-1: 3297 (N-H); 2156 (CN); 1675 (C=O). 1H
NMR (400 MHz, DMSO-d6) δ: 7.29 (d, 1H, B, J2-3 = 1.6 Hz, H2), 7.76 (d, 2H, A,
J2-3 = J5-6 = 8.7 Hz, H2+H6), 7.86 (d, 2H, A, H3+H5), 8.84 (d, 1H, B, H3), 10.94 (s,
1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 105.7 (1C, SeCN), 107.6 (1C, B,
C2), 118.9 (1C, A, C1), 122.4 (2C, A, C3+C5), 135.0 (2C, A, C2+C6), 139.5 (1C,
A, C4), 152.4 (1C, B, C3), 154.7 (1C, B, C1), 162.7 (1C, C=O). MS (m/z %
abundance): 293 (45; M+·), 96 (100). Elemental Analysis for C11H7N3O2Se,
Calculated/Found (%): C: 45.20/44.97; H: 2.40/2.62; N:14.38/14.33.
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N-(4-selenocyanatophenyl)adamantamide (1k). From adamantanoyl chloride.
Recrystallized from methanol/water (1:1). Yellow powder; mp: 180-181 ºC.
Yield: 72 %. IR (KBr) cm-1: 3306 (N-H); 2899 (C-H); 2147 (CN); 1661 (C=O). 1H
NMR (400 MHz, DMSO-d6) δ: 1.71 (s, 6H, B, 2H6+2H8+2H10), 1.92 (s, 6H, B,
2H2+2H3+2H5), 2.03 (s, 3H, B, H4+H7+H9), 7.64 (d, 2H, A, J2-3 = J5-6 = 8.6 Hz,
H2+H6), 7.76 (d, 2H, A, H3+H5), 9.32 (s, 1H, NH). 13C NMR (100 MHz, DMSO-
d6) δ: 28.1 (3C, B, C4+C7+C9), 36.4 (3C, B, C6+C8+C10), 38.6 (3C, B,
C2+C3+C5), 41.6 (1C, B, C1), 105.7 (1C, SeCN), 116.7 (1C, A, C1), 121.8 (2C,
A, C3+C5), 134.9 (2C, A, C2+C6), 141.2 (1C, A, C4), 176.7 (1C, C=O). MS (m/z
% abundance): 360 (22; M+·). 135 (100). Elemental Analysis for C18H20N2OSe,
Calculated/Found (%): C: 60.17/60.16; H: 5.57/5.75; N: 7.79/7.70.
N-(4-selenocyanatophenyl)cinnamamide (1l). From cinnamoyl chloride.
Recrystallized from methanol/water (1:1). Blue powder; mp: 186-187 ºC. Yield:
67 %. IR (KBr) cm-1: 3306 (N-H); 2899 (C-H); 2147 (CN); 1661 (C=O). 1H NMR
(400 MHz, DMSO-d6) δ: 6.86 (d, 1H, B, Ja-b = 15.7 Hz, CHa=CHPh), 7.36 – 7.50
(m, 3H, B, H3+H4+H5), 7.59 – 7.67 (m, 3H, B, H2+H6, CH=CHbPh), 7.73 (d, 2H,
A, J2-3 = J5-6 = 8.4 Hz, H2+H6), 7.82 (d, 2H, A, H3+H5), 10.45 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6) δ: 105.8 (1C, SeCN), 117.0 (1C, A, C1), 121.0 (2C,
A, C3+C5), 122.4 (1C, CaH=CH), 128.2 (2C, B, C3+C5), 129.5 (2C, B, C2+C6),
130.4 (1C, B, C4), 135.1 (1C, B, C1), 135.3 (2C, A, C2+C6), 141.0 (1C, A, C4),
141.3 (1C, CH=CbH), 164.3 (1C, C=O). MS (m/z % abundance): 328 (22; M+·),
131 (100), 103 (75), 77 (40). Elemental Analysis for C16H12N2O2Se,
Calculated/Found (%): C: 58.71/58.40; H: 3.67/4.06; N: 8.56/8.56.
N-(4-selenocyanatophenyl)-1-naphthamide (1m). From
1-naphthoyl
chloride. Recrystallized from methanol/water (1:1). Green cotton-like powder;
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mp: 216-217 ºC. Yield: 56 %. IR (KBr) cm-1:3310 (N-H); 2145 (CN); 1668
(C=O). 1H NMR (400 MHz, DMSO-d6) δ: 7.56 – 7.66 (m, 3H, B), 7.75 (d, 2H, A,
J2-3 = J5-6 = 8.5 Hz, H2+H6), 7.78 (d, 1H, B, J = 7.0 Hz,), 7.91 (d, 2H, A, H3+H5),
8.00 – 8.06 (m, 1H, B), 8.10 (d, 1H, B, J = 8.2 Hz), 8.16 – 8.22 (m, 1H, B), 10.81
(s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 105.8 (1C, SeCN), 117.5 (1C, A,
C1), 121.6 (2C, A, C3+C5), 125.5 (2C, B), 126.1 (1C, B), 126.9 (1C, B), 127.6
(1C, B), 128.9 (1C, B), 130.1 (1C, B), 130.9 (1C, B), 133.6 (1C, B), 134.8 (1C,
B), 135.3 (2C, A, C2+C6), 141.1 (1C, A, C4), 168.0 (1C, C=O). MS (m/z %
abundance): 352 (20; M+·), 155 (100), 127 (98), 77 (10). Elemental Analysis for
C18H12N2OSe, Calculated/Found (%): C: 61.54/61.15; H: 3.42/3.64; N:
7.97/8.00.
4-methylthio-N-(4-selenocyanatophenyl)benzamide
(1n).
From
4-
methylthiobenzoyl chloride. Recrystallized from methanol. White powder; mp:
202-203 ºC. Yield: 68 %. IR (KBr) cm-1: 3391 (N-H); 2913 (C-H); 2162 (CN);
1
1662 (C=O). H NMR (400 MHz, DMSO-d6) δ: 2.55 (s, 3H, SCH3), 7.40 (d, 2H,
B, J2-3 = J5-6 = 8.3 Hz, H3+H5), 7.70 (d, 2H, A, J2-3 = J5-6 = 8.6 Hz, H2+H6), 7.88
(d, 2H, A, H3+H5), 7.94 (d, 2H, B, H2+H6), 10.38 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6) δ: 14.6 (1C, SCH3), 105.8 (1C, SeCN), 117.3 (1C, A, C1),
122.0 (2C, A, C3+C5), 125.4 (2C, B, C3+C5), 128.7 (2C, B, C2+C6), 130.8 (1C, B,
C1), 135.1 (2C, A, C2+C6), 141.0 (1C, A, C4), 144.0 (1C, B, C4), 165.6 (1C,
C=O). MS (m/z % abundance): 348 (7; M+·), 151 (100), 77 (10). Elemental
Analysis for C15H12N2OSSe, Calculated/Found (%): C: 49.19/48.80; H:
3.66/3.73; N: 7.66/7.52.
General procedure of synthesis for compounds 2a-2c, 2e-2j, 2l and 2n.
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To a solution of the corresponding selenocyanate (Compounds 1a-1c, 1e-1j, 1l
and 1n) in ethanol (50 mL) was added NaBH4 (0.25 eq). The resulting mixture
was stirred at room temperature during 2 h. Upon completion the solvent was
removed under vacuum and the residue was treated with water (2 x 50 mL) and
extracted with dichloromethane (2 x 50 mL). The combined organic phases
were dried over Na2SO4, filtered and the solvent was removed by rotary
evaporation. The solid residue was purified by recrystallization from ethanol.
General procedure of synthesis for compounds 2d, 2k and 2m.
To a solution of bis(4-aminophenyl)diselenide in dry chloroform (50 mL) the
corresponding acyl chloride was added (1:2 molar ratio), and the mixture was
kept at room temperature for 12-24 h. Compounds precipitated after the
addition of water. After filtration, the compounds were washed with diethyl ether
for purification.
N,N’-(4,4’-diselanediylbis(4,1-phenylene))bisbenzamide (2a). From N-(4-
selenocyanatophenyl)benzamide. Yellow powder; mp: 255-256 ºC. Yield: 62 %.
1
IR (KBr) cm-1: 3341 (N-H); 1665 (C=O). H NMR (400 MHz, DMSO-d6) δ: 7.52
(d, 4H, A+A’, J2-3 = J5-6 = 8.7 Hz, 2H2+2H6), 7.55 – 7.63 (m, 6H, B+B’,
2H3+2H4+2H5), 7.77 (d, 4H, A+A’, 2H3+2H5), 7.94 (d, 4H, B+B’, J2-3 = J5-6 = 7.1
Hz, 2H2+2H6), 10.45 (s, 2H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 121.3 (4C,
A+A’, 2C3+2C5), 124.4 (2C, A+A’, 2C1), 127.8 (4C, B+B’, 2C2+2C6), 128.6 (4C,
B+B’, 2C3+2C5), 131.9 (2C, B+B’, 2C4), 132.9 (4C, A+A’, 2C2+2C6), 134.7 (2C,
B+B’, 2C1), 139.5 (2C, A+A’, 2C4), 165.9 (2C, 2C=O). MS (m/z % abundance):
302 (5), 155 (55), 105 (100), 77 (80). Elemental Analysis for C26H20N2O2Se2,
Calculated/Found (%): C: 56.73/56.48; H: 3.64/4.03; N: 5.09/4.83
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N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis(4-chlorobenzamide) (2b). δ:
From 4-chloro-N-(4-selenocyanatophenyl)benzamide. Green powder; mp: 252-
253 ºC. Yield: 63 %. IR (KBr) cm-1: 3307 (N-H); 1646 (C=O). 1H NMR (400 MHz,
DMSO-d6) δ: 7.56 – 7.67 (m, 8H, A+A’, 2H2+2H6, B+B’, 2H3+2H5), 7.77 (d, 4H,
A+A’, J2-3 = J5-6 = 8.6 Hz, 2H3+2H5), 7.99 (d, 4H, B+B’, J2-3 = J5-6 = 8.5 Hz,
2H2+2H6) 10.45 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-d6) δ: 121.6 (4C,
A+A’, 2C3+2C5), 124.9 (2C, A+A’, 2C1), 129.0 (4C, B+B’, 2C3+2C5), 130.1 (4C,
B+B’, 2C2+2C6), 133.3 (4C, A+A’, 2C2+2C6), 133.9 (2C, B+B’, 2C1), 137.0 (2C,
B+B’, 2C4), 139.7 (2C, A+A’, 2C4), 165.0 (2C, 2C=O). MS (m/z % abundance):
344 (7), 231 (15), 172 (52), 139 (100), 93 (42), 77 (33), 65 (40). Elemental
Analysis for C26H18Cl2N2O2Se2, Calculated/Found (%): C: 50.40/50.71; H:
2.91/3.14; N: 4.52/4.61.
N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis(4-methoxybenzamide)
(2c).
From 4-methoxy-N-(4-selenocyanatophenyl)benzamide. Yellow powder; mp:
261-262 ºC. Yield: 61 %. IR (KBr) cm-1: 3350 (N-H); 2961 (C-H); 1652 (C=O).
1H NMR (400 MHz, DMSO-d6) δ: 3.84 (s, 6H, 2OCH3), 7.05 (d, 4H, B+B’, J2-3
=
J5-6 = 8.9 Hz, 2H3+2H5), 7.59 (d, 4H, A+A’, J2-3 = J5-6 = 8.7 Hz, 2H2+2H6), 7.82
(d, 4H, A+A’, 2H3+2H5), 8.02 (d, 4H, B+B’, 2H2+2H6), 10.38 (s, 2H, 2NH). 13C
NMR (100 MHz, DMSO-d6) δ: 55.9 (2C, 2OCH3), 114.1 (4C, B+B’, 2C3+2C5),
121.6 (4C, A+A’, 2C3+2C5), 124.4 (2C, A, C1), 127.1 (2C, B+B’, C1), 130.3 (4C,
B+B’, 2C2+2C6), 133.4 (4C, A+A’, 2C2+2C6), 140.3 (2C, A+A’, 2C4), 162.5 (2C,
B+B’, 2C4), 165.5 (2C, 2C=O). MS (m/z % abundance): 378 (5), 211 (15), 172
(76), 105 (90), 77 (100). Elemental Analysis for C28H24N2O4Se2,
Calculated/Found (%): C: 55.08/55.23; H: 3.93/3.86; N: 4.59/4.48.
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N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis(4-cyanobenzamide)
(2d).
From 4-cyano-N-(4-selenocyanatophenyl)benzamide. Yellow powder; mp: >
300 ºC. Yield: 65 %. IR (KBr) cm-1: 3258 (N-H); 2237 (CN); 1661 (C=O). H
1
NMR (400 MHz, DMSO-d6) δ: 7.64 (d, 4H, A+A’, J2-3 = J5-6 = 7.3 Hz, 2H2+2H6),
7.77 (d, 4H, A+A’, 2H3+2H5), 8.04 (d, 4H, B+B’, J2-3 = J5-6 = 6.6 Hz, 2H2+2H6),
8.10 (d, 4H, B+B’, 2H3+2H5), 10.61 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-
d6) δ: 114.4 (2C, B+B’, 2C4), 118.7 (2C, 2CN), 121.6 (4C, A+A’, 2C3+2C5),
125.2 (2C, A+A’, 2C1), 129.1 (4C, B+B’, 2C2+2C6), 132.9 (4C, B+B’, 2C3+2C5),
133.3 (4C, A+A’, 2C2+2C6), 139.2 (2C, B+B’, 2C1), 139.4 (2C, A+A’, 2C4), 164.7
(2C, 2C=O). MS (m/z % abundance): 342 (10), 262 (5), 172 (75), 131 (75), 93
(100), 77 (90), 55 (85). Elemental Analysis for C28H18N4O2Se2,
Calculated/Found (%): C: 56.00/55.62; H: 3.00/2.80; N: 9.33/9.22.
N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis4-nitrobenzamide (2e). From
4-nitro-N-(4-selenocyanatophenyl)benzamide. Yellow powder; mp: 247-246 ºC.
1
Yield: 68 %. IR (KBr) cm-1: 3333 (N-H); 1648 (C=O); 1350 (NO). H NMR (400
MHz, DMSO-d6) δ: 7.65 (d, 4H, A+A’, J2-3 = J5-6 = 8.7 Hz, 2H2+2H6), 7.78 (d, 4H,
A+A’, 2H3+2H5), 8.18 (d, 4H, B+B’, J2-3 = J5-6 = 8.8 Hz, 2H2+2H6), 8.37 (d, 4H,
B+B’, 2H3+2H5), 10.70 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-d6) δ: 121.1
(4C, A+A’, 2C3+2C5), 123.5 (4C, B+B’, 2C3+2C5), 124.8 (2C, A+A’, 2C1), 129.2
(4C, B+B’, 2C2+2C6), 132.7 (4C, A+A’, 2C2+2C6), 138.9 (2C, B+B’, 2C1), 140.3
(2C, A+A’, 2C4), 149.1 (2C, B+B’, 2C4), 163.9 (2C, 2C=O). MS (m/z %
abundance): 342 (5), 172 (90), 131 (100), 55 (88). Elemental Analysis for
C26H18N4O6Se2, Calculated/Found (%): C:47.85/47.41; H: 2.79/3.18; N:
8.58/8.48.
23

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N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis-4-trifluoromethylbenzamide
(2f). From 4-trifluoromethyl-N-(4-selenocyanatophenyl)benzamide. Yellow
powder; mp: 247-248 ºC. Yield: 64 %. IR (KBr) cm-1: 3317 (N-H); 1647 (C=O);
1176-1339 (CF3). 1H NMR (400 MHz, DMSO-d6) δ: 7.66 (d, 4H, A+A’, J2-3 = J5-6
= 8.5 Hz, 2H2+2H6), 7.78 (d, 4H, A+A’, 2H3+2H5), 7.93 (d, 4H, B+B’, J2-3 = J5-6
=
8.2 Hz, 2H3+2H5), 8.15 (d, 4H, B+B’, 2H2+2H6), 10.59 (s, 2H, 2NH). 13C NMR
(100 MHz, DMSO-d6) δ: 121.3 (4C, A+A’, 2C3+2C5), 123.9 (q, 2C, 1JC-F = 272.5
3
Hz, 2CF3) 124.8 (2C, A+A’, 2C1), 125.4 (q, 4C, JC-F = 3.6 Hz, B+B’, 2C3+2C5),
128.7 (4C, B+B’, 2C2+2C6), 131.5 (q, 2C, 2JC-F = 32.0 Hz, B+B’, C4), 132.9 (4C,
A+A’, 2C2+2C6), 138.6 (2C, A+A’, 2C4), 139.1 (2C, B+B’, 2C1), 164.6 (2C,
2C=O). MS (m/z % abundance): 370 (10), 173 (90), 145 (50), 121 (100).
Elemental Analysis for C28H18F6N2O2Se2, Calculated/Found (%): C:
48.98/48.41; H: 2.62/2.92; N: 4.08/4.15.
N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis-4-methylbenzamide
(2g).
From 4-methyl-N-(4-selenocyanatophenyl)benzamide. Yellow powder; mp: 266-
1
267 ºC. Yield: 59 %. IR (KBr) cm-1: 3366 (N-H); 2915 (C-H); 1654 (C=O). H
NMR (400 MHz, DMSO-d6) δ: 2.39 (s, 6H, 2CH3), 7.35 (d, 4H, B+B’, J2-3 = J5-6
=
8.1 Hz, 2H3+2H5), 7.60 (d, 4H, A+A’, J2-3 = J5-6 = 8.7 Hz, 2H2+2H6), 7.78 (d, 4H,
A+A’, 2H3+2H5), 7.87 (d, 4H, B+B’, 2H2+2H6), 10.30 (s, 2H, 2NH). 13C NMR
(100 MHz, DMSO-d6) δ: 21.1 (2C, 2CH3), 121.1 (4C, A+A’, 2C3+2C5), 124.2
(2C, A+A’, 2C1), 127.8 (4C, B+B’, 2C2+2C6), 129.1 (4C, B+B’, 2C3+2C5), 132.0
(2C, B+B’, 2C1), 133.0 (4C, A+A’, 2C2+2C6), 139.7 (2C, A+A’, 2C4), 141.9 (2C,
B+B’, 2C4), 165.6 (2C, 2C=O). MS (m/z % abundance): 370 (5), 173 (100), 133
(80), 57 (90). Elemental Analysis for C28H24O2N2Se2.HCl, Calculated/Found
(%): C: 54.60/54.23; H: 4.10/4.22; N: 4.55/4.46.
24

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N,N’-(4,4'-diselanediylbis(4,1-phenylene))bisfuran-2-carboxamide
(2h).
From N-(4-selenocyanatophenyl)furan-2-carboxamide. Yellow powder; mp: 222-
223 ºC. Yield: 61 %. IR (KBr) cm-1: 3352 (N-H); 1660 (C=O). 1H NMR (400 MHz,
DMSO-d6) δ: 6.71 (dd, 2H, B+B’, J2-3= 3.5 Hz, J3-4= 1.7 Hz, 2H3), 7.35 (d, 2H,
B+B’, 2H2), 7.60 (d, 4H, A+A’, J2-3 = J5-6 = 8.7 Hz, 2H2+2H6), 7.74 (d, 4H, A+A’,
2H3+2H5), 7.94 (d, 2H, B+B’, 2H4), 10.32 (s, 2H, 2NH). 13C NMR (100 MHz,
DMSO-d6) δ: 112.1 (2C, B+B’, 2C3), 114.9 (2C, B+B’, 2C2), 121.0 (4C, A+A’,
2C3+2C5), 124.3 (2C, A+A’, 2C1), 132.7 (4C, A+A’, 2C2+2C6), 138.7 (2C, A+A’,
2C4), 145.8 (2C, B+B’, 2C4), 147.2 (2C, B+B’, 2C1), 156.1 (2C, 2C=O). MS (m/z
% abundance): 370 (5), 173 (100), 145 (70). Elemental Analysis for
C22H16N2O4Se2, Calculated/Found (%): C: 49.81/49.71; H: 3.02/3.37; N:
5.28/5.21.
N,N’-(4,4'-diselanediylbis(4,1-phenylene))bisthiophene-2-carboxamide (2i).
From N-(4-selenocyanatophenyl)thiophene-2-carboxamide. Yellow powder; mp:
167-168 ºC. Yield: 57 %. IR (KBr) cm-1: 3354 (N-H); 1666 (C=O). 1H NMR (400
MHz, DMSO-d6) δ: 7.23 (dd, 2H, B+B’, J2-3= 4.7 Hz, J3-4= 3.9 Hz, 2H3), 7.61 (d,
4H, A+A’, J2-3 = J5-6 = 8.4 Hz, 2H2+2H6), 7.74 (d, 4H, A+A’, 2H3+2H5), 7.87 (d,
2H, B+B’, 2H2), 8.09 (d, 2H, B+B’, 2H4), 10.42 (s, 2H, 2NH). 13C NMR (100
MHz, DMSO-d6) δ: 121.6 (4C, A+A’, 2C3+2C5), 124.8 (2C, A+A’, 2C1), 128.6
(2C, B+B’, 2C3), 129.9 (2C, B+B’, 2C4), 132.6 (2C, B+B’, 2C2), 133.3 (4C, A+A’,
2C2+2C6), 139.5 (2C, A+A’, 2C4), 140.2 (2C, B+B’, 2C1), 160.4 (2C, 2C=O). MS
(m/z % abundance): 342 (5), 264 (20), 184 (100). Elemental Analysis for
C22H16N2O2S2Se2.2HCl, Calculated/Found (%): C: 41.57/41.56; H: 2.83/2.86; N:
4.40/4.47.
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N,N’-(4,4'-diselanediylbis(4,1-phenylene))bisisoxazole-5-carboxamide (2j).
From N-(4-selenocyanatophenyl)isoxazole-5-carboxamide. Yellow powder; mp:
204-205 ºC. Yield: 64 %. IR (KBr) cm-1: 3347 (N-H); 1662 (C=O). 1H NMR (400
MHz, DMSO-d6) δ: 7.31 (d, 2H, B+B’, J2-3 =1.7 Hz, H2), 7.65 (d, 4H, A+A’, J2-3
=
J5-6 = 7.6 Hz, 2H2+2H6), 7.77 (d, 4H, A+A’, 2H3+2H5), 8.82 (d, 2H, B+B’, H3),
10.93 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-d6) δ: 107.5 (2C, B+B’, 2C2),
121.9 (4C, A+A’, 2C3+2C5), 125.9 (2C, A+A’, 2C1), 133.1 (4C, A+A’, 2C2+2C6),
138.5 (2C, A+A’, 2C4), 152.3 (2C, B+B’, 2C3), 154.6 (2C, B+B’, 2C1), 162.8 (2C,
2C=O). MS (m/z % abundance): 264 (5), 184 (50), 93 (100), 66 (45). Elemental
Analysis for C20H14N4O4Se2.2HCl, Calculated/Found (%): C: 39.66/39.90; H:
2.64/2.67; N: 9.25/9.26.
N,N’-(4,4'-diselanediylbis(4,1-phenylene))Bisadamantamide (2k). From N-
(4-selenocyanatophenyl)adamantamide. Yellow powder; mp: 230-231 ºC. Yield:
1
54 %. IR (KBr) cm-1: 3386 (N-H); 2915 (C-H); 1665 (C=O). H NMR (400 MHz,
DMSO-d6) δ: 1.69 (s, 12H, B+B’, 4H4+4H6+4H10), 1.89 (s, 12H, B+B’,
4H2+4H8+4H9), 2.01 (s, 6H, B+B’, 2H3+2H5+2H7), 7.50 (d, 4H, A+A’, J2-3 = J5-6
=
8.8 Hz, 2H2+2H6), 7.6 (d, 4H, A+A’, 2H3+2H5), 9.28 (s, 2H, 2NH). 13C NMR (100
MHz, DMSO-d6) δ: 28.1 (6C, B+B’, 2C4+2C7+2C9), 36.4 (6C, B+B’,
2C6+2C8+2C10), 38.6 (6C, B+B’, 2C2+2C3+2C5), 41.5 (2C, B+B’, 2C1), 121.3
(4C, A+A’, 2C3+2C5), 124.1 (2C, A+A’, C1), 133.4 (4C, A+A’, 2C2+2C6), 140.2
(2C, A+A’, 2C4), 176.6 (2C, 2C=O). MS (m/z % abundance): 344 (6), 184 (100),
165 (95), 93 (90), 65 (65). Elemental Analysis for C34H40N2O2Se2,
Calculated/Found (%): C: 61.26/61.35; H: 6.05/5.95; N: 4.20/4.23.
N,N'-(4,4'-diselanediylbis(4,1-phenylene))bis(3-phenylacrylamide) (2l). δ:
From N-(4-selenocyanatophenyl)cinnamamide. Yellow powder; mp: 234-235
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ºC. Yield: 67 %. IR (KBr) cm-1: 3307 (N-H); 1662 (C=O). H NMR (400 MHz,
DMSO-d6) δ: 6.98 (d, 2H, B+B’, Ja-b = 15.7 Hz, CHa=CHPh), 7.37 – 7.49 (m, 6H,
B+B’, H3+H4+H5), 7.54 – 7.67 (m, 10H, A+A’+B+B’, H2+H6+CH=CHbPh), 7.75
(d, J2-3 = J5-6 = 7.3 Hz, 4H, A+A’, 2H3+2H5), 10.68 (s, 2H, 2NH). 13C NMR (100
MHz, DMSO-d6) δ: 120.5 (4C, A+A’, 2C3+2C5), 122.7 (2C, 2CaH=CH), 124.3
(2C, A+A’, 2C1), 128.2 (4C, B+B’, 2C3+2C5), 129.5 (4C, B+B’, 2C2+2C6), 130.3
(2C, B+B’, 2C4), 133.6 (4C, A+A’, 2C2+2C6), 135.2 (2C, B+B’, 2C1), 139.1 (2C,
A+A’, 2C4), 140.7 (2C, 2CH=CbH), 164.2 (2C, 2C=O). MS (m/z % abundance):
538 (5), 278 (30), 108 (100), 77 (98). Elemental Analysis for
C30H24N2O2Se2.HCl, Calculated/Found (%): C: 56.25/56.55; H: 3.90/4.05; N:
4.37/4.93.
N,N’-(4,4'-diselanediylbis(4,1-phenylene))bisnaphthamide (2m). From N-(4-
selenocyanatophenyl)-1-naphthamide. Yellow powder; mp: 243-247 ºC. Yield:
1
54 %. IR (KBr) cm-1: 3392 (N-H); 2915 (C-H); 1665 (C=O). H NMR (400 MHz,
DMSO-d6) δ: 7.57 – 7.65 (m, 6H, B+B’), 7.67 (d, 4H, A+A’, J2-3 = J5-6 = 8.6 Hz,
2H2+2H6), 7.75 – 7.79 (m, 2H, B+B’), 7.83 (d, 4H, A+A’, 2H3+2H5), 7.99 – 8.06
(m, 2H, B+B’), 8.08 (d, 2H, B+B’, J = 8.2 Hz), 8.16 – 8.25 (m, 2H, B+B’), 10.74
(s, 2H, 2NH). 13C NMR (100 MHz, DMSO-d6) δ: 121.1 (4C, A+A’, 2C3+2C5),
124.8 (2C, A+A’, 2C1), 125.5 (2C, B+B’), 125.6 (2C, B+B’), 126.0 (2C, B+B’),
126.9 (2C, B+B’), 127.5 (2C, B+B’), 128.8 (2C, B+B’), 130.1 (2C, B+B’), 130.7
(2C, B+B’), 133.6 (4C, A+A’, 2C2+2C6), 133.6 (2C, B+B’), 135.0 (2C, B+B’),
140.1 (2C, A+A’, 2C4), 167.9 (2C, 2C=O). MS (m/z % abundance): 344 (5), 184
(75), 172 (92), 127 (95), 57 (100). Elemental Analysis for C34H24N2O2Se2,
Calculated/Found (%): C: 62.78/62.86; H: 3.72/3.59; N: 4.31/4.52.
27

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N,N’-(4,4'-diselanediylbis(4,1-phenylene))bis-4-methylthiobenzamide (2n).
From 4-methylthio-N-(4-selenocyanatophenyl)benzamide. Yellow powder; mp:
289-290 ºC. Yield: 46 %. IR (KBr) cm-1: 3355 (N-H); 2930 (C-H); 1648 (C=O).
1H NMR (400 MHz, DMSO-d6) δ: 2.55 (s, 6H, 2CH3), 7.39 (d, 4H, B+B’, J2-3 = J5-
6 = 8.5 Hz, H3+H5), 7.61 (d, 4H, A+A’, J2-3 = J5-6 = 8.7 Hz, 2H2+2H6), 7.77 (d, 4H,
A+A’, 2H3+H5), 7.91 (d, 4H, B+B’, 2H2+2H6), 10.30 (s, 2H, 2NH). 13C NMR (100
MHz, DMSO-d6) δ: 14.6 (2C, 2SCH3), 121.5 (2C, A+A’, 2C1), 122.0 (4C, A+A’,
2C3+2C5), 125.4 (4C, B+B’, 2C3+2C5), 128.7 (4C, B+B’, 2C2+2C6), 130.8 (2C,
B+B’, 2C1), 135.1 (4C, A+A’, 2C2+2C6), 141.0 (2C, A+A’, 2C4), 144.0 (2C, B+B’,
2C4), 165.6 (2C, 2C=O). MS (m/z % abundance): 292 (35), 107 (42), 77 (100).
Elemental Analysis for C28H24N2O2S2Se2.2HCl, Calculated/Found (%): C:
47.12/46.91; H: 3.65/3.75; N: 3.92/3.90.
Biological evaluation
(i) Cells and Culture conditions. L. infantum axenic amastigotes were grown
in M199 medium (Invitrogen, Leiden, Netherlands) supplemented with 10% heat
inactivated FBS, 1g/L β-alanine, 100mg/L L-asparagine, 200mg/L sacarose, 50
mg/L sodium pyruvate, 320 mg/L malic acid, 40mg/L fumaric acid, 70mg/L
succinic acid, 200mg/L α-ketoglutaric acid, 300 mg/L citric acid, 1.1 g/L sodium
bicarbonate, 5 g/L MES, 0.4 mg/L hemin, 10 mg/L gentamicin, pH 5.4 at 37ºC
and 5% CO2. THP-1 cells were kindly provided by Dr. Michel /Université Nice
Sophia Antipolis, Nice, France) and were grown in RMPI-1640 medium (Gibco,
Lieden, Netherlands) supplemented with 10% heat inactivated FBS, 5%
penicillin/streptomycin, 1nM HEPES, 2mM glutamine and 1mM sodium
pyruvate, pH 7.2 at 37ºC and 5% CO2 atmosphere(20).
28

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(ii) Leishmanicidal activity and cytotoxicity in vitro assays. Drug treatment
of amastigotes and THP-1 cells was performed during the logarithmic growth
phase at a concentration of 2x106 parasites/ml and 4x105 cells/mL, respectively,
at 37ºC and 5% CO2 for 24 h. Compounds were tested at five different
concentrations between 1.56 and 25 µM. In some cases, compounds
concentrations needed to be decrease until 0.2 µM. The number and
percentage of living parasites/cells was figured out by flow cytometry by the
propidium iodide (PI) exclusion method(48). After the selection of the parasite
population based on their forward scatter (FSC) and side scatter (SSC) values,
live and dead parasites cells were identified by their permeability to PI. This is a
conservative procedure that may underestimate LC50 values as parasites that
became fragmented as a consequence of cell death are excluded from the
analysis. To minimize the presence of fragmented parasites drug treatment
never exceeded 24 h. The selectivity index (SI) was defined as the ratio of the
EC50 values of compounds against THP-1 cells relative to those obtained
against L. infantum axenic amastigotes.
(iii) Leishmania infection assay. 120.000 cells/mL THP-1 cells were seeded
in 24 multidishes plates (Nunc, Roskilde, Denmark) and differentiated to
macrophages in 1 mL of RPMI-1640 medium containing 10 ng/mL phorbol 12-
myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA). After 24 h,
medium was removed and 1.2 x 106 Leishmania infantum eGFP-amastigotes in
1 mL of THP-1 medium were added to half of the plate wells. 4 h later, all
medium with non-infecting amastigotes was removed, washed 3 times with 1X
phosphate buffered saline (PBS) and replaced with the corresponding treatment
dissolved in 1 mL THP-1 medium. 48 h after the treatment, medium was
29

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removed; Cells were washed 3 times with 1X PBS and detached with TrypLETM
Express (Invitrogen, Leiden, Netherlands) following manufacturer’s protocol.
Infection quantization was measured by flow cytometry(49).
(iv) Trypanothione reductase assay. The oxidorreductase activity against
trypanothione reductase enzyme was determined following the method
described by Toro et al.(50) Reaction was done at room temperature in 250 µL
of HEPES pH 8.0 (40 mM), containing EDTA (1 mM), NADPH (150 µM), NADP+
(30 µM), DTNB (25µM), T[S]2 (1 µM), glycerol (0.02%), DMSO (1.5%) and
recombinant Li-TryR (7 nM). For IC50 values determination, the enzyme was
pre-incubated with the compounds (concentrations ranging from 75 μM to 0.29
μM) for 10 min prior the addition of T[S]2 and NADPH. Enzyme activity was
monitored by the increase in absorbance at 412 nm for 1 h at 26°C in a
VERSAmax microplate reader (Molecular Devices, California, USA). All the
assays were conducted in triplicate in at least three independent experiments.
Data were analyzed using a non-linear regression model with the Grafit6
software (Erithacus, Horley, Surrey, UK). Mepacrine was used as positive
control (same concentrations).
(v) Measurement of intracellular thiols. The disturbance of the intracellular
thiol levels is an evidence of an oxidative stress. For its measurement, 0.5
mL/plate amastigotes at a concentration of 1x106/mL were seeded in 6
multidishes plate and treated with DMSO (5 µL) as negative control, Menadione
(25 µM) as positive control and compounds (25 µM). Amastigotes were
incubated at 37ºC and 5% CO2 for 1 hour. 30 minutes after the treatment
CMFDA (5-chloromethylfluorescein diacetate) was added (20 µM) and
30