Novel hybrid selenosulfonamides as potent antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2013.12.030

Diselenide and sulfonamide derivatives have recently attracted considerable interest as leishmanicidal agents in drug discovery. In this study, a novel series of sixteen hybrid selenosulfonamides has been synthesized and screened for their in vitro activity against Leishmania infantum intracellular amastigotes and THP-1 cells. These assays revealed that most of the compounds exhibited antileishmanial activity in the low micromolar range and led us to identify three lead compounds (derivatives 2, 7 and 14) with IC₅₀ values ranging from 0.83 to 1.47 μM and selectivity indexes (SI) over 17, much higher than those observed for the reference drugs miltefosine and edelfosine. When evaluated against intracellular amastigotes, hybrid compound 7 emerged as the most active compound (IC₅₀ = 2.8 μM), showing higher activity and much less toxicity against THP-1 cells than edelfosine. These compounds could potentially serve as templates for future drug-optimization and drug-development efforts for their use as therapeutic agents in developing countries.

Full text of DOI:10.1016/j.ejmech.2013.12.030

European Journal of Medicinal Chemistry 74 (2014) 116e123
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel hybrid selenosulfonamides as potent antileishmanial agents
,
,
,b
Ylenia Baquedano a b, Esther Moreno a b, Socorro Espuelas b, Paul Nguewa b, María Font a
,
Kilian Jesús Gutierrez c, Antonio Jiménez-Ruiz c, Juan Antonio Palop a
,
,b,
*
Carmen Sanmartín a
,
b
a Departamento de Química Orgánica y Farmacéutica, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain
b Instituto de Salud Tropical, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain
c Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Carretera Madrid-Barcelona km 33,600, E-28871 Alcalá de Henares, Madrid,
Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Diselenide and sulfonamide derivatives have recently attracted considerable interest as leishmanicidal
agents in drug discovery. In this study, a novel series of sixteen hybrid selenosulfonamides has been
synthesized and screened for their in vitro activity against Leishmania infantum intracellular amastigotes
and THP-1 cells. These assays revealed that most of the compounds exhibited antileishmanial activity in
the low micromolar range and led us to identify three lead compounds (derivatives 2, 7 and 14) with IC50
Received 8 August 2013
Received in revised form
12 December 2013
Accepted 22 December 2013
Available online 3 January 2014
values ranging from 0.83 to 1.47
for the reference drugs miltefosine and edelfosine. When evaluated against intracellular amastigotes,
M), showing higher activity and
mM and selectivity indexes (SI) over 17, much higher than those observed
Keywords:
hybrid compound 7 emerged as the most active compound (IC50 ¼ 2.8
m
Leishmanicidal agents
Selenocompounds
Diselenides
much less toxicity against THP-1 cells than edelfosine. These compounds could potentially serve as
templates for future drug-optimization and drug-development efforts for their use as therapeutic agents
in developing countries.
Sulfonamides
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
depending on the pathogenic species; they encompass some
groups of disorders: cutaneous, diffuse cutaneous, mucocutaneous
Leishmaniasis is defined as a cluster of vector-borne diseases
with diverse clinical manifestations [1] and it constitutes one of the
six priority diseases in the “Tropical Diseases Research” program of
the World Health Organization (WHO). Leishmaniasis [2,3] are a
complex of neglected tropical diseases caused by obligate intra-
cellular protozoan parasites from the genus Leishmania that are
transmitted by around 30 species of the phlebotomine sandflies
through the bite of females infected with the pathogen [4,5].
The life cycle of the parasite is relatively simple. Sandflies
inoculate the skin with flagellated promastigotes, which are
phagocytosed by local and immediately recruited host cells. Within
the phagolysosomes of resident macrophages surviving promasti-
gotes transform and replicate as amastigotes, which infect addi-
tional macrophages either locally or in distant tissues after
dissemination [6]. The clinical manifestations of leishmaniasis vary
and visceral leishmaniasis. The latter is the most severe form of the
disease, and it is usually fatal if left untreated [7].
Leishmaniasis is endemic in broad tropical areas of the world
including many underdeveloped countries, which makes it a major
international health problem. It has high morbidity and mortality
rates and is classified as an emerging and uncontrolled disease by
the World Health Organization. The migration of population from
endemic to non-endemic regions and tourist activities in endemic
zones are contributing to the spread of the disease to new areas.
Currently, about 350 million people in 88 countries around the
world are at risk of infection; 12 million people worldwide are
infected and 500,000 new cases of visceral leishmaniasis emerge
every year, causing about 60,000 deaths [8]. At present available
chemotherapy is far from satisfactory and presents several prob-
lems including toxicity, many adverse effects, high costs and
development of drug resistance [9].
Two pentavalent antimonial [Sb(V)] compounds, sodium stibo-
gluconate
(PentostamÒ)
and
meglumine
antimoniate
* Corresponding author. Department of Organic and Pharmaceutical Chemistry,
University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain. Tel.: þ34 948 425
600; fax: þ34 948 425 649.
(GlucantimeÒ), were first introduced in the 1940s and have been
used since then as first-line chemotherapeutic agents against all
forms of leishmaniasis through parenteral administration.
E-mail address: jpalop@unav.es (J.A. Palop).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.12.030

Y. Baquedano et al. / European Journal of Medicinal Chemistry 74 (2014) 116e123
117
However, their efficacy is becoming increasingly smaller and highly
dependent on Leishmania species and endemic regional variations,
even within the same country [10]. Besides, antimonial agents may
also cause acute pancreatitis and cardiac arrhythmia.
infantum, Leishmania mexicana and Leishmania donovani [39e41].
Finally, the incorporation of the thienyl moiety has been explored
due to its demonstrated effectiveness in leishmanicidal therapy by
itself, combined, or fused with other skeletons [42,43]. In summary,
we designed a new class of diselenide derivatives by molecular
combination between 4,40-diselanediyldianiline, as the core, and
other bioactive substructures trying to make use of the “Medicinal
Chemical Hybridization” (MCH) as one of the approaches to design
a polyvalent drug. This strategy can be carried out by joining
through appropriate linkers to pharmacophores, the linkers usually
are susceptible to metabolic cleavage [44e46]. From our point of
view, molecular hybridization is a relatively new concept in drug
design and development. It is based on the combination of phar-
macophoric moieties of different bioactive substances to produce a
new hybrid compound with improved physico-chemical and
bioactive properties, in an attempt to obtain synergistic effects
through the action of some mechanistic routes and the reduction of
undesired side effects when compared to the parent compounds.
In light of these findings, and as a part of our efforts to develop
new compounds for the treatment of leishmaniasis, we present the
synthesis and the leishmanicidal activity of sixteen new hybrids
(Fig. 2) based on the 4,40-diselanediyldianiline with sulfonamide
moiety bound to different rings. Their activity was evaluated
against both axenic and, for the most interesting compounds,
intracellular amastigotes. Cytotoxicity against the human cell line
THP-1 was also assessed in order to exclude those molecules
showing unfavorable toxicological profile from further
development.
In the recent years, new potential therapies have been intro-
duced for visceral leishmaniasis. They include an amphotericin B
liposome formulation [11]; oral miltefosine [12]; a parenteral
formulation of aminosidine (paromomycin) [13] and oral sitama-
quine [14]. Edelfosine [15e17], another alkyl-phospholipid, has also
been tested and found to display higher in vitro activity than mil-
tefosine. However, all current treatments suffer from limitations
derived either from their high cost, route of administration, drug
resistance or extended treatment regimens and, especially, serious
side effects such as nephrotoxicity, hypokalemia, hepatic and
pancreatic toxicity, hypotension and dysglycemia among others
[18,19]. Therefore, there is an urgent need for the development of
improved treatments for leishmaniasis that are safe, inexpensive
and easily available to the patients. Furthermore, the discovery of
new lead compounds for this disease is a pressing concern for
global health programs.
During the last few years, various reports have shown the
relevance of the trace element selenium, whose increased con-
centration in plasma has been recognized as a new defensive
strategy against Leishmania infection [20,21]. Selenium derivatives
have antioxidant, cancer preventing, and antiviral activities and
also appear to improve the immune response of hosts against
various bacterial and viral species [22,23]. Recently, we reported
[24,25] new selenium compounds with in vitro antiparasitic activity
against Leishmania infantum. Some of them possess a powerful
activity with selectivity indexes higher than the reference drugs
miltefosine and edelfosine. Additionally, their leishmanicidal ac-
tivity in infected macrophages (THP-1 cells) was comparable to that
of edelfosine. Among others, compounds referable to formula
(Fig. 1) which contains as essential pharmacophore the diselenide
group within the framework of molecular symmetry that, in our
opinion, appear as a critical factor for activity. In view of its
remarkable performance, we considered interesting to explore the
modulation by derivatization of amine groups in order to adjust
polarity and solubility, facilitate cellular uptake, optimize anti-
leishmanial activity and reduce cytotoxicity improving the level of
selectivity. With this aim, we chose the sulfonamide group,
because: 1. Hydrolysis of this group inside the cell could release the
active moieties; 2. This functional group assists drug transport to
the cell and it could improve the permeability through lipid
membranes by transitory partial loss of basicity of aminic nitrogen;
3. Sulfonamide, according to literature, shows intrinsic anti-
leishmanial activity and is an important structural core in leish-
manicidal therapy [26e31]; 4. The sulfonamide group can be used
as a chemical link that allows binding of other potential “active
components” such as aromatic and heteroaromatic systems with
demonstrated antiparasitic activity [32e34]. Concerning theses
aromatic rings, we have added several different substituents in
order to explore new characteristics related to volume, rigidity and
lipophilicity in the new molecules. On the other hand, some het-
erocycles are interesting molecular scaffolds in the design of new
efficient leishmanicidal compounds; it is well established that
many quinoline derivatives act as antiprotozoal agents [35e38] and
also that imidazo derivatives are highly active against Leishmania
2. Results and discussion
2.1. Chemistry
We have previously described the syntheses of various dis-
elenide derivatives by reduction of the corresponding selenocya-
nates with sodium borohydride in ethanol [25]. This method was
employed for the preparation of 4,40-diselanediyldianiline, which
was used as template and coupled to the corresponding sulfonyl
chlorides (1:2 or 1:2.5 M ratio) to give the target compounds. The
reaction was carried out in dry ether under N2 atmosphere, at room
temperature during 48e72 h. This synthesis was based on a pub-
lished procedure with modifications [47] following our own pro-
tocol. The compounds were obtained in yields ranging from 3.6 to
38.5% and were purified by recrystallization from ethanol/water,
washed with dichloromethane, ethyl ether, or water, and one of
them was purified by column chromatography. The purity of the
compounds was assessed by TLC and elemental analyses and their
structures were identified from spectroscopic data. IR, 1H NMR, 13
C
NMR, mass spectrometry and elementary analysis methods were
used for structure elucidation (Scheme 1). All the signals were fully
consistent with proposed structures. In this regard we found an
interesting observation in the 1H NMR spectra obtained for com-
pounds 7 and 10, as they did not show doublets corresponding to
the coupling of fluorine and hydrogen signals in the FCaCH
Fig. 1. Formula of 4,40-diselanediyldianiline.
Fig. 2. General structure of hybrid compounds.

118
Y. Baquedano et al. / European Journal of Medicinal Chemistry 74 (2014) 116e123
(SI ¼ 18, 14, 9.5, 17, 11, 15, and 30, respectively) outperformed
edelfosine and miltefosine (SI ¼ 6 and 7 respectively) with regards
to this therapeutic index (Table 1).
2.2.3. Leishmanicidal activity in infected macrophages
Because of their activity and selectivity, three compounds (2, 7
and 14) were selected for testing their leishmanicidal activity in
amastigote-infected THP-1 cells. The IC50 for each compound was
calculated and displayed in Table 2. The potency of the analogs was
compared with edelfosine,
(IC50 ¼ 3.1 ꢀ 0.1 M). We observed that these derivatives are active
against the intracellular form of the parasite and reduce the para-
a current antileishmanial agent
m
site load of the cells exhibiting IC50 values of 4.1, 2.8 and 6.2
respectively.
mM,
Because of its activity on amastigotes and infected macrophages
and on its selectivity against the parasites (therapeutic index > 17),
analog 7 seems to be the best compound, which makes it a suitable
lead structure for the development of future antiparasitic drugs.
3. Conclusions
A series of sixteen diselenide-sulfonamide hybrids has been
designed, synthesized, completely characterized and bioevaluated
as a new series of in vitro leishmanicides whose activities were
compared with the standard drugs miltefosine and edelfosine. All
the synthetic derivatives, with the exception of compounds 12 and
13, exhibited a remarkable inhibition of L. infantum amastigotes
growth. Compounds 2, 7 and 14 combined a very good anti-
leishmanial activity with low toxicity against the human cell line
THP-1, two properties essential for the development of new drug
candidate prototypes. Regarding their structure/activity analysis,
no clear-cut correlation was found but, taken together these results
suggest that the sulfonamide scaffold could be a valuable linker for
the parent diselenide-containing antileishmanial compounds
Scheme 1. Synthesis of compounds 1e16.
moieties. The strategy for the preparation of sulfonamide de-
rivatives (compounds 1e16) is outlined in Scheme 1.
2.2. Biological evaluation
2.2.1. Activity in axenic amastigotes
The antileishmanial activity of the sixteen synthesized sulfon-
amides was analyzed against L. infantum axenic amastigotes using
miltefosine and edelfosine as standard drugs according to a pre-
viously described procedure [48]. Analyses were carried out with a
minimum of three independent experiments and results were
expressed as IC50 values. Biological data evidenced that most of the
screened compounds (twelve of them) show similar or even higher
Table 1
IC50
(
mM) ꢀ SEM values for the compounds on amastigotes and cytotoxic activity in
THP-1 cell line.
bioactivity than miltefosine (IC50 ¼ 2.84
mM). From a structural
point of view, introduction of different substituents on the pendent
phenyl group does not modify the activity with the exception of the
4-nitro and 4-chloro substitutions which slightly decrease it. In
general, both electron-donating and electron-withdrawing groups
are tolerated. We also checked the influence of the introduction of
other bulky aryl rings and heteroaryl moieties with known anti-
parasitic activity. Our results demonstrate that biphenyl (12) and
naphthyl (13) derivatives are inactive whereas the 8-quinolinyl
(14), 2-thienyl (15) and 2-(1-methyl-1H-imidazolyl) (16) analogs
display high activity. Based on the comparison of compounds 7 and
Compound
R
Amastigote
THP-1
SIa
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
phenyl
1.64 ꢀ 0.05 11.9 ꢀ 1.0
1.40 ꢀ 0.09 >25
7.3
4-methylphenyl
4-methoxyphenyl
4-cyanophenyl
4-nitrophenyl
4-trifluoromethylphenyl
4-fluorophenyl
4-chlorophenyl
4-bromophenyl
2-fluorophenyl
2-chlorophenyl
4-biphenyl
2-naphtyl
8-quinolinyl
2-thienyl
2-(1-methyl-1H-imidazolyl)
e
>18
14
2
4.6
9.5
>17
>7
2
10.7
15
1.30 ꢀ 0.08 18.1 ꢀ 0.2
1.76 ꢀ 0.30
3.6 ꢀ 0.3
3.74 ꢀ 0.11 17.1 ꢀ 0.8
2.01 ꢀ 0.62 19.1 ꢀ 0.8
1.47 ꢀ 0.07 >25
8 (IC50 values of 1.47 and 3.63
mM, respectively) with analogs 10 and
11 (IC50 values of 1.66 and 1.17
mM) location of the atom of halogen
3.63 ꢀ 0.12 >25
2.80 ꢀ 0.29
5.2 ꢀ 0.05
in the para (7 and 8) or ortho (10 and 11) position does not seem to
markedly influence the antileishmanial activity.
1.66 ꢀ 0.25 17.8 ꢀ 0.6
1.17 ꢀ 0.08 17.4 ꢀ 1.5
13.81 ꢀ 0.32 >25
5.65 ꢀ 0.22 >25
>2
>4.4
>30
2.2.2. Cytotoxicity
0.83 ꢀ 0.04 >25
Both high leishmanicidal activity and low cytotoxicity are
required in a good antileishmanial drug. Accordingly, cytotoxicity
against the THP-1 human cell line was evaluated and the selectivity
index (SI) of the compounds was calculated as the ratio of cyto-
toxicity (IC50 value on THP-1 cells) to activity (IC50 value on
L. infantum amastigotes). Compounds 2, 3, 6, 7, 10, 11, and 14
1.54 ꢀ 0.14
1.05 ꢀ 0.07
0.82 ꢀ 0.13
8.6 ꢀ 1.1
4.4 ꢀ 0.02
4.9 ꢀ 0.1
5.6
4
6
Edelfosine
Miltefosine
e
2.84 ꢀ 0.10 18.5 ꢀ 0.6
7
a
Selectivity index (SI) is the ratio of IC50 values of compounds against THP-1 cells
relative to those against L. infantum amastigotes.

Y. Baquedano et al. / European Journal of Medicinal Chemistry 74 (2014) 116e123
119
Table 2
IC50
M) ꢀ SEM values for the compounds in amastigote-infected THP-1 cells.
(
m
Compound
R
IC50
2
7
14
4-methylphenyl
4-fluorophenyl
8-quinolinyl
e
4.1 ꢀ 0.1
2.8 ꢀ 0.1
6.2 ꢀ 0.9
3.1 ꢀ 0.1
Edelfosine
Fig. 3. Assignment for the chemical shifts in NMR spectroscopy.
resulting in hybrid derivatives that can allow synergistic effects. To
sum up, the potent leishmanicidal activity, synthetic accessibility
and good selectivity strongly encourage further optimization of 2, 7
and 14 as leads to develop more potent leishmanicidal agents.
Furthermore, this work contributes to validate the choice of the
molecular symmetry as a useful template to design new active
compounds.
The precipitate was washed with water (150 mL), ethyl ether
(4 ꢁ 25 mL) and recrystallized from ethanol:water (50:50). Yield:
26%; mp: 162e164 ꢂC. IR (KBr) cmꢃ1: 3238 (m, NeH), 1160 (s, SO2),
820 (m, SeeSe). 1H NMR (400 MHz, DMSO-d6)
d
: 7.02 (d, 4H, A þ A0,
H3 þ H5, J3e2 ¼ J5e6 ¼ 8.6 Hz); 7.38 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e
¼ 8.6 Hz); 7.56 (t, 4H, B þ B0, H3 þ H5); 7.61 (dd, 2H, B þ B0, H4, J4e
5
¼ 7.4 Hz, J4e2 ¼ 1.2 Hz); 7.77 (dd, 4H, B þ B0, H2 þ H6, J2e3 ¼ J6e
3
¼ 7.4 Hz, J2e4 ¼ J6e4 ¼ 1.2 Hz); 10.50 (s, 2H, 2NH). 13C NMR
5
4. Experimental protocols
(100 MHz, DMSO-d6)
d
: 121.1 (4C, A þ A0, C3, C5); 125.6 (2C, A þ A0,
C1); 127.5 (4C, B þ B0, C2, C6); 130.2 (4C, B þ B0, C3, C5); 133.9 (4C,
A þ A0, C2, C6); 134.2 (2C, B þ B0, C4); 138.8 (2C, A þ A0, C4); 140.1 (2C,
B þ B0, C1). MS (m/z, % abundance): 136 (100). Elemental Analysis
for C24H20N2O4S2Se2, Calcd/Found (%): C: 46.30/46.21; H: 3.21/3.62;
N: 4.50/4.46.
4.1. Chemistry
Melting points were determined with a Mettler FP82 þ FP80
apparatus (Greifensee, Switzerland) and are not corrected. The 1H
NMR and 13C NMR spectra were recorded on a Bruker 400 Ultra-
shieldÔ spectrometer (Rheinstetten, Germany) using TMS as the
internal standard. The IR spectra were obtained on a Thermo
Nicolet FT-IR Nexus spectrophotometer with KBr pellets. Mass
spectrometry was carried out on a MS-DIP, system MSD/DS 5973N
(G2577A) Agilent. Elemental microanalyses were carried out on
vacuum-dried samples using a LECO CHN-900 Elemental Analyzer.
Silica gel 60 (0.040e0.063 mm) 1.09385.2500 (Merck KGaA, 64271
Darmstadt, Germany) was used for Column Chromatography and
AlugramÒ SIL G/UV254 (Layer: 0.2 mm) (MachereyeNagel GmbH &
Co. KG. Postfach 101352, D-52313 Düren, Germany) was used for
Thin Layer Chromatography. Chemicals were purchased from E.
Merck (Darmstadt, Germany), Scharlau (F.E.R.O.S.A., Barcelona,
Spain), Panreac Química S.A. (Montcada i Reixac, Barcelona, Spain),
SigmaeAldrich Química, S.A. (Alcobendas, Madrid, Spain), Acros
Organics (Janssen Pharmaceuticalaan 3a, 2440 Geel, België) and
Lancaster (Bischheim-Strasbourg, France).
4 .1.1. 2 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
methylbenzenesulfonamide) (2). From 4,40-diselanediyldianiline
and 4-methylbenzenesulfonyl chloride. The precipitate was
washed with dichloromethane (20 mL) and the dichloromethane
was evaporated to reduced pressure. Yield: 4%; mp: 89e90 ꢂC. IR
(KBr) cmꢃ1: 3235 (m, NeH), 1159 (s, SO2), 812 (m, SeeSe). 1H NMR
(400 MHz, DMSO-d6)
d
: 2.33 (s, 6H, 2CH3); 7.02 (d, 4H, A þ A0,
H3 þ H5, J3e2 ¼ J5e6 ¼ 8.7 Hz); 7.34 (d, 4H, B þ B0, H3 þ H5, J3e2 ¼ J5e
¼ 8.2 Hz); 7.38 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.7 Hz); 7.64
6
(d, 4H, B þ B0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.2 Hz); 10.43 (s, 2H, 2NH). 13
C
NMR (100 MHz, DMSO-d6)
d
: 21.8 (1C, CH3); 121.1 (4C, A þ A0, C3,
C5); 125.4 (2C, A þ A0, C1); 127.6 (4C, B þ B0, C2, C6); 130.6 (4C, B þ B0,
C3, C5); 134.1 (4C, A þ A0, C2, C6); 137.3 (2C, A þ A0, C4); 138.9 (2C,
B þ B0, C1); 144.3 (2C, B þ B0, C4). MS (m/z, % abundance): 184 (100).
Elemental Analysis for C26H24N2O4S2Se2, Calcd/Found (%): C: 48.00/
47.77; H: 3.69/4.09; N: 4.30/4.20.
4.1.1. General procedure for the synthesis of compounds 1e16
To a stirred solution of compound 4,40-diselanediyldianiline
(1 mmol) solved in dry ether (50 mL), different sulfonyl chlorides (2
or 2.5 mmol) were added dropwise (when the sulfonyl chloride was
solid, previously, it was solved in dry ether 10 mL). The reaction
mixture was stirred at different time intervals (48e72 h) at room
temperature under N2 atmosphere (progress and completion of
reaction was confirmed by TLC). Then, a precipitate was produced
which was collected by filtration.
4 .1.1. 3 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
methoxybenzenesulfonamide) (3). From 4,40-diselanediyldianiline
and 4-methoxybenzenesulfonyl chloride. The precipitate was
washed with dichloromethane (20 mL) and the dichloromethane
was evaporated under reduced pressure. Yield: 5%; mp: 78e79 ꢂC.
IR (KBr) cmꢃ1: 3251 (m, NeH), 1156 (s, SO2), 822 (m, SeeSe). 1H
NMR (400 MHz, DMSO-d6) d: 3.79 (s, 6H, 2OCH3); 7.02 (d, 4H,
A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.7 Hz); 7.05 (d, 4H, B þ B0, H3 þ H5, J3e
¼ J5e6 ¼ 9.0 Hz); 7.40 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.7 Hz);
2
One of the following work-up methods was used:
7.70 (d, 4H, B þ B0, H2 þ H6, J2e3 ¼ J6e5 ¼ 9.0 Hz); 10.36 (s, 2H, 2NH).
Work-up method A: After 72 h of stirring at room temperature,
the precipitate was filtered off, washed and purified in order to
obtain the target compounds (1e4, 6e8, 12, 14 and 16).
Work-up method B: Once filtered the solid, the organic layer
reaction mixture was treated suitably in order to obtain derivatives
5, 9e11, 13 and 15.
In order to assign the chemical shifts in NMR spectroscopy the
following assignment has been done (Fig. 3): Central rings A and A0;
External Rings B and B0. For derivative 12, which possesses two
rings in the external position have been named B1, B2, B10 and B20,
respectively.
13C NMR (100 MHz, DMSO-d6)
d: 56.5 (1C, OeCH3); 115.3 (4C,
B þ B0, C3, C5); 121.0 (4C, A þ A0, C3, C5); 125.3 (2C, A þ A0, C1); 129.7
(4C, B1 þ B10, C2, C6); 131.7 (2C, B þ B0, C1); 134.1 (4C, A þ A0, C2, C6);
139.0 (2C, A þ A0, C4); 163.4 (2C, B þ B0, C4). MS (m/z, % abundance):
172 (100). Elemental Analysis for C26H24N2O6S2Se2, Calcd/Found
(%): C: 45.75/45.36; H: 3.52/3.82; N: 4.10/3.75.
4 .1.1. 4 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
cyanobenzenesulfonamide) (4). From 4,40-diselanediyldianiline and
4-cyanobenzenesulfonyl chloride. The precipitate was washed with
ethyl ether (4 ꢁ 20 mL) and dried. Yield: 27%; mp: 194e195 ꢂC. IR
(KBr) cmꢃ1: 3251 (m, NeH), 1159 (s, SO2), 834 (m, SeeSe). 1H NMR
4.1.1.1. N,N0-(Diselanediyldibenzene-4,1-diyl)dibenzenesulfonamide
(1). From 4,40-diselanediyldianiline and benzenesulfonyl chloride.
(400 MHz, DMSO-d6)
d
: 7.03 (d, 4H, A þ A0, H3 þ H5, J3e2 ¼ J5e

120
Y. Baquedano et al. / European Journal of Medicinal Chemistry 74 (2014) 116e123
¼ 8.5 Hz); 7.44 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.5 Hz); 7.92
¼ J6e5 ¼ 8.6 Hz); 7.63 (d, 4H, B þ B0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.5 Hz);
6
3
(d, 4H, B þ B0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.4 Hz); 8.05 (d, 4H, B þ B0,
7.75 (d, 4H, B þ B0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.5 Hz); 10.57 (s, 2H, 2NH).
H2 þ H6, J2e3 ¼ J6e5 ¼ 8.4 Hz); 10.76 (s, 2H, 2NH). 13C NMR
13C NMR (100 MHz, DMSO-d6)
d
: 121.8 (4C, A þ A0, C3, C5); 125.9 (2C,
(100 MHz, DMSO-d6)
d
: 116.4 (2C, B þ B0, C4); 118.3 (2C, CN); 121.6
A þ A0, C1); 129.5 (4C, B þ B0, C2, C6); 130.4 (4C, B þ B0, C3, C5); 134.1
(4C, A þ A0, C2, C6); 138.4 (2C, B þ B0, C4); 138.6 (2C, A þ A0, C4);
138.9 (2C, B þ B0, C1). MS (m/z, % abundance): 57 (100). Elemental
Analysis for C24H18Cl2N2O4S2Se2, Calcd/Found (%): C: 41.68/41.46;
H: 2.60/2.68; N: 4.05/3.96.
(4C, A þ A0, C3, C5); 126.3 (2C, A þ A0, C1); 128.3 (4C, B þ B0, C2, C6);
133.9 (4C, A þ A0, C2, C6); 134.4 (4C, B þ B0, C3, C5); 138.0 (2C, A þ A0,
C4); 144.2 (2C, B þ B0, C1). MS (m/z, % abundance): 57 (100).
Elemental Analysis for C26H18N4O4S2Se2 $0.5 HCl, Calcd/Found (%):
C: 45.20/45.33; H: 2.75/2.74; N: 8.11/7.90.
4 .1.1. 9 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
bromobenzenesulfonamide) (9). From 4,40-diselanediyldianiline
and 4-bromobenzenesulfonyl chloride. The organic layer was
evaporated under reduced pressure and the residue was recrys-
tallized from ethanol:water (80:20).Yield: 9%; mp: 173e174 ꢂC. IR
(KBr) cmꢃ1: 3252 (m, NeH), 1160 (s, SO2), 816 (w, SeeSe). 1H NMR
4 .1.1. 5 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
nitrobenzenesulfonamide) (5). From 4,40-diselanediyldianiline and
4-nitrobezenesulfonyl chloride. The organic layer was evaporated
under reduced pressure and the residue was stirred in water
(50 mL) at room temperature during 24 h. The resultant solid was
filtered and dried. Yield: 38%; mp: 215e216 ꢂC. IR (KBr) cmꢃ1: 3268
(m, NeH), 1164 (s, SO2), 835 (m, SeeSe). 1H NMR (400 MHz, DMSO-
(400 MHz, DMSO-d6)
d
: 7.03 (d, 4H, A þ A0, H3 þ H5, J3e2 ¼ J5e
¼ 7.1 Hz); 7.43 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 7.1 Hz); 7.67 (d,
6
d6)
d
: 7.05 (d, 4H, A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.7 Hz); 7.45 (d, 4H,
4H, B þ B0, H3 þ H5, J3e2 ¼ J5e6 ¼ 7.3 Hz); 7.78 (d, 4H, B þ B0, H2 þ H6,
J2e3 ¼ JHFeHE ¼ 7.3 Hz); 10.36 (s, 2H, 2NH). 13C NMR (100 MHz,
A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.7 Hz); 8.00 (d, 4H, B þ B0, H2 þ H6, J2e
¼ J6e5 ¼ 8.9 Hz); 8.37 (d, 4H, B þ B0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.9 Hz);
DMSO-d6)
d
: 121.6 (4C, A þ A0, C3, C5); 126.0 (2C, A þ A0, C1); 127.9
3
10.81 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-d6)
d: 121.7 (4C,
(2C, B þ B0, C4); 129.5 (4C, B þ B0, C2, C6); 133.3 (4C, B þ B0, C3, C5);
134.0 (4C, A þ A0, C2, C6); 138.4 (2C, A þ A0, C4); 139.4 (2C, B þ B0, C1).
A þ A0, C3, C5); 125.6 (2C, A þ A0, C1); 128.4 (4C, B þ B0, C3, C5); 129.1
(4C, B þ B0, C2, C6); 133.9 (4C, A þ A0, C2, C6); 137.9 (2C, A þ A0, C4);
145.5 (2C, B þ B0, C1); 150.8 (2C, B þ B0, C4). MS (m/z, % abundance):
57 (100). Elemental Analysis for C24H18N4O8S2Se2, Calcd/Found (%):
C: 40.45/40.62; H: 2.53/2.78; N: 7.87/7.90.
MS (m/z,
% abundance): 57 (100). Elemental Analysis for
C24H18Br2N2O4S2Se2$1HCl, Calcd/Found (%): C: 34.80/35.27; H:
2.36/2.32; N: 3.26/3.42.
4.1.1.10 . N, N 0-(Dis ela ned iyld ibenz ene- 4,1- diyl )bi s(2 -
fluorobenzenesulfonamide) (10). From 4,40-diselanediyldianiline
and 2-fluorobenzenesulfonyl chloride. The organic layer was
evaporated under reduced pressure and the residue was solved in
water (50 mL). The water phase was extracted with dichloro-
methane (3 ꢁ 50 mL). The combined organic layers were dried with
anhydrous sodium sulfate and evaporated to dryness. The sticky
residue was recrystallized from ethanol:water (80:20). Yield: 13%;
mp: 137e138 ꢂC. IR (KBr) cmꢃ1: 3237 (s, NeH), 1160 (s, SO2), 816
4.1.1.6. N,N0-(Diselanediyldibenzene-4,1-diyl)bis[4-(trifluoromethyl)
benzenesulfonamide] (6). From 4,40-diselanediyldianiline and 4-
(trifluoromethyl)benzenesulfonyl chloride. The precipitate was
washed with water (150 mL) and recrystallized from ethanol:water
(80:20). Yield: 28%; mp: 243e244 ꢂC. IR (KBr) cmꢃ1: 3256 (m, Ne
H), 1161 (s, SO2), 840 (m, SeeSe). 1H NMR (400 MHz, DMSO-d6)
d:
7.04 (d, 4H, A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.3 Hz); 7.44 (d, 4H, A þ A0,
H2
þ
H6, J2e3
¼
J6e5
¼
8.3 Hz); 7.96 (s, 8H,
B
þ
B0,
H2 þ H3 þ H5 þ H6); 10.72 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-
(m, SeeSe). 1H NMR (400 MHz, DMSO-d6)
d
: 7.03 (d, 4H, A þ A0,
d6)
d
: 121.5 (4C, A þ A0, C3, C5); 122.8 (2C, CF3); 125.5 (2C, A þ A0, C1);
H3 þ H5, J3e2 ¼ J5e6 ¼ 7.9 Hz); 7.37e7.44 (m, 8H, A þ A0, H2 þ H6,
126.2 (4C, B þ B0, C3, C5); 127.5 (4C, B þ B0, C2, C6); 128.5 (2C, B þ B0,
C4); 134.1 (4C, A þ A0, C2, C6); 138.2 (2C, A þ A0, C4); 144.1 (2C, B þ B0,
C1). MS (m/z, % abundance): 172 (100). Elemental Analysis for
B þ B0, H3 þ H5); 7.67e7.71 (m, 2H, B þ B0, H4); 7.82e7.87 (m, 2H,
B þ B0, H6); 10.82 (s, 2H, 2NH). 13C NMR (100 MHz, DMSO-d6)
d:
118.1 (2C, B þ B0, C3); 120.9 (4C, A þ A0, C3, C5); 125.7 (2C, A þ A0, C1);
127.6 (2C, B þ B0, C5); 131.3 (2C, B þ B0, C1); 134.1 (4C, A þ A0, C2, C6);
137.0 (2C, B þ B0, C6); 138.2 (2C, A þ A0, C4); 157.7 (2C, B þ B0, C4);
160.2 (2C, B þ B0, C2). MS (m/z, % abundance): 57 (100). Elemental
Analysis for C24H18F2N2O4S2Se2, Calcd/Found (%): C: 43.76/44.23;
H: 2.74/3.20; N: 4.25/4.23.
C
26H18F6N2O4S2Se2, Calcd/Found (%): C: 41.16/41.46; H: 2.37/2.53;
N: 3.69/3.48.
4 .1.1. 7 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
fluorobenzenesulfonamide) (7). From 4,40-diselanediyldianiline and
4-fluorobenzenesulfonyl chloride. The precipitate was washed with
water (150 mL) and recrystallized from ethanol:water (80:20).
Yield: 13%; mp: 181e182 ꢂC. IR (KBr) cmꢃ1: 3279 (m, NeH), 1157 (s,
4.1.1.11. N , N 0- ( D i s e l a n e d i yl di b e nz e ne - 4 ,1 - d i yl ) b i s ( 2 -
chlorobenzenesulfonamide) (11). From 4,40-diselanediyldianiline
and 2-chlorobenzenesulfonyl chloride. The organic layer was
evaporated under reduced pressure and the residue was solved in
water (50 mL). The water phase was extracted with dichloro-
methane (3 ꢁ 50 mL). The combined organic layers were dried with
anhydrous sodium sulfate and evaporated to dryness. The sticky
residue was treated with water (50 mL) during 24 h and the
resultant solid was recrystallized from ethanol:water (80:20).
Yield: 14%; mp: 200e201 ꢂC. IR (KBr) cmꢃ1: 3279 (m, NeH), 1164 (s,
SO2), 816 (w, SeeSe). 1H NMR (400 MHz, DMSO-d6)
d: 7.03 (d, 4H,
A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.6 Hz); 7.37e7.43 (m, 8H, A þ A0,
H2 þ H6, B þ B0, H3 þ H5); 7.82 (m, 4H, B þ B0, H2 þ H6); 10.52 (s, 2H,
2NH). 13C NMR (100 MHz, DMSO-d6)
d
: 116.8 (4C, B þ B0, C3, C5);
122.1 (4C, A þ A0, C3, C5); 125.8 (2C, A þ A0, C1); 130.7 (4C, B þ B0, C2,
C6); 133.6 (4C, A þ A0, C2, C6); 136.5 (2C, B þ B0, C1); 138.6 (2C, A þ A0,
C4); 166.5 (2C, B þ B0, C4). MS (m/z, % abundance): 172 (100).
Elemental Analysis for C24H18F2N2O4S2Se2, Calcd/Found (%): C:
43.76/43.31; H: 2.73/2.83; N: 4.25/4.21.
SO2), 814 (w, SeeSe). 1H NMR (400 MHz, DMSO-d6)
d: 7.01 (d, 4H,
A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.6 Hz); 7.36 (d, 4H, A þ A0, H2 þ H6, J2e
4 .1.1. 8 . N , N 0- ( D i s e l a n e d i yl d i b e n z e n e - 4 ,1 - d i yl ) b i s ( 4 -
chlorobenzenesulfonamide) (8). From 4,40-diselanediyldianiline and
4-chlorobenzenesulfonyl chloride. The precipitate was washed
with water (150 mL) and recrystallized from ethanol:water (80:20).
Yield: 7%; mp: 199e200 ꢂC. IR (KBr) cmꢃ1: 3254 (m, NeH), 1158 (s,
¼ J6e5 ¼ 8.6 Hz); 7.53 (m, 4H, B þ B0, H4 þ H5); 7.62 (d, 2H, B þ B0,
3
H3, J3e4 ¼ 3.7 Hz); 8.05 (d, 2H, B þ B0, H6, J6e5 ¼ 7.8 Hz); 10.82 (s, 2H,
2NH). 13C NMR (100 MHz, DMSO-d6)
d
: 120.5 (4C, A þ A0, C3, C5);
125.4 (2C, A þ A0, C1); 128.6 (2C, B þ B0, C5); 131.5 (2C, B þ B0, C6);
132.5 (2C, B þ B0, C3); 132.8 (2C, B þ B0, C2); 134.2 (4C, A þ A0, C2,
C6); 135.7 (2C, B þ B0, C4); 137.1 (2C, A þ A0, C4); 138.2 (2C, B þ B0,
C1). MS (m/z, % abundance): 57 (100). Elemental Analysis for
SO2), 806 (w, SeeSe). 1H NMR (400 MHz, DMSO-d6)
d: 7.02 (d, 4H,
A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.6 Hz); 7.43 (d, 4H, A þ A0, H2 þ H6, J2e

Y. Baquedano et al. / European Journal of Medicinal Chemistry 74 (2014) 116e123
121
C24H18Cl2N2O4S2Se2, Calcd/Found (%): C: 41.68/41.61; H: 2.60/3.01;
N: 4.05/3.71.
2-sulfonyl chloride. The organic layer was evaporated under
reduced pressure and the residue was stirred in water (50 mL) at
room temperature during 24 h. The resultant solid was chromato-
graphed on silica gel (toluene:dioxane, 1:1). Yield: 5%; mp: 114e
115 ꢂC. IR (KBr) cmꢃ1: 3246 (m, NeH), 1154 (s, SO2), 814 (w, SeeSe).
4.1.1.12. N,N0-(Diselanediyldibenzene-4,1-diyl)bis(biphenyl-4-
sulfonamide) (12). From 4,40-diselanediyldianiline and biphenyl-4-
sulfonyl chloride. The precipitate was washed with water (150 mL)
and recrystallized from ethanol:water (80:20). Yield: 18%; mp:
197e198 ꢂC. IR (KBr) cmꢃ1: 3250 (s, NeH), 1157 (s, SO2), 806 (m,
1H NMR (400 MHz, DMSO-d6)
d
: 7.08 (d, 4H, A þ A0, H3 þ H5, J3e
¼ J5e6 ¼ 8.6 Hz); 7.12 (dd, 2H, B þ B0, H4, J4e3 ¼ 4.9 Hz, J4e
2
¼ 3.8 Hz); 7.45 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.6 Hz); 7.56
5
SeeSe). 1H NMR (400 MHz, DMSO-d6)
d
: 7.07 (d, 4H, A þ A0, H3 þ H5,
(dd, 2H, B þ B0, H5, J5e4 ¼ 3.8 Hz, J5e3 ¼ 1.3 Hz); 7.90 (dd, 2H, B þ B0,
J3e2 ¼ J5e6 ¼ 8.6 Hz); 7.43 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e
H3, J3e4 ¼ 4.9 Hz, J3e5 ¼ 1.3 Hz). 13C NMR (100 MHz, DMSO-d6)
d:
¼ 8.6 Hz); 7.47 (m, 6H, B2 þ B20, H3 þ H4 þ H5); 7.69 (dd, 4H,
121.6 (4C, A þ A0, C3, C5); 126.0 (2C, A þ A0, C1); 128.5 (2C, B þ B0, C5);
133.5 (4C, A þ A0, C2, C6); 134.2 (2C, B þ B0, C4); 134.4 (2C, B þ B0, C3);
138.6 (2C, A þ A0, C4); 140.6 (2C, B þ B0, C2). MS (m/z, % abundance):
177 (100). Elemental Analysis for C20H16N2O4S4Se2, Calcd/Found
(%): C: 37.85/38.02; H: 2.52/2.86; N: 4.41/4.09.
5
B2 þ B20, H2 þ H6, J2e3 ¼ J6e5 ¼ 7.1 Hz, J2e4 ¼ J6e4 ¼ 1.5 Hz); 7.84e
7.87 (m, 8H, B1 þ B10, H2 þ H3 þ H5 þ H6); 10.57 (s, 2H, 2NH). 13
C
NMR (100 MHz, DMSO-d6)
d
: 121.8 (4C, A þ A0, C3, C5); 125.6 (2C,
A þ A0, C1); 127.9 (4C, B1 þ B10, C3, C5); 129.0 (2C, B2 þ B20, C4);
129.4 (4C, B1 þ B10, C2, C6); 129.6 (4C, B2 þ B20, C2, C6); 130.3 (4C,
B2 þ B20, C3, C5); 133.7 (4C, A þ A0, C2, C6); 134.6 (2C, B1 þ B10, C4);
138.8 (2C, A þ A0, C4); 139.0 (2C, B1 þ B10, C1); 145.3 (2C, B2 þ B20,
C1). MS (m/z, % abundance): 169 (100). Elemental Analysis for
4.1.1.16. N,N0-(Diselanediyldibenzene-4,1-diyl)bis(1-methyl-1H-imid-
azole-2-sulfonamide)$0.5
hydrochloride
(16). From 4,40-dis-
elanediyldianiline and 1-methyl-1H-imidazole-2-sulfonyl chloride.
The precipitate was washed with water (150 mL) and recrystallized
from ethanol:water (80:20). Yield: 8%; mp: 244e245 ꢂC. IR (KBr)
cmꢃ1: 3249 (m, NeH), 1145 (s, SO2), 825 (m, SeeSe). 1H NMR
C
36H28N2O4S2Se2, Calcd/Found (%): C: 55.81/55.35; H: 3.62/3.51; N:
3.61/3.58.
4.1.1.13. N,N0-(Diselanediyldibenzene-4,1-diyl)bis(naphthalene-2-
sulfonamide)$0.5 hydrochloride (13). From 4,40-diselanediyldiani-
line and naphthalene-2-sulfonyl chloride. The organic layer was
evaporated under reduced pressure and the residue was stirred in
water (50 mL) at room temperature during 24 h. The resultant solid
was recrystallized from ethanol:water (80:20). Yield: 36%; mp: 97e
98 ꢂC. IR (KBr) cmꢃ1: 3249 (m, NeH), 1158 (s, SO2), 806 (m, SeeSe).
(400 MHz, DMSO-d6)
d
: 3.80 (s, 6H, NeCH3); 7.02 (s, 2H, B þ B0, H4),
7.10 (d, 4H, A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.1 Hz); 7.41 (s, 2H, B þ B0,
H5); 7.47 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.1 Hz); 10.99 (s, 2H,
2NH). 13C NMR (100 MHz, DMSO-d6)
d: 35.4 (2C, NeCH3); 121.5 (4C,
A þ A0, C3, C5); 125.8 (2C, A þ A0, C1); 127.5 (2C, B þ B0, C5); 128.7 (2C,
B þ B0, C4); 133.9 (4C, A þ A0, C2, C6); 138.2 (2C, A þ A0, C4); 142.5 (2C,
B þ B0, C2). MS (m/z, % abundance): 57 (100). Elemental Analysis for
1H NMR (400 MHz, DMSO-d6)
d
: 7.04 (d, 4H, A þ A0, H3 þ H5, J3e
C20H20N6O4S2Se2$0.5HCl, Calcd/Found (%): C: 36.99/36.90; H: 3.15/
¼ J5e6 ¼ 8.6 Hz); 7.33 (d, 4H, A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.6 Hz);
3.51; N: 12.94/12.52.
2
7.61e7.68 (m, 4H, B þ B0, H4 þ H5); 7.75 (dd, 2H, B þ B0, H8, J8e
¼ 8.0 Hz, J8e2 ¼ 2.0 Hz); 7.99 (d, 2H, B þ B0, H7, J7e8 ¼ 8.0 Hz); 8.09
4.2. Biological evaluation
7
(dd, 4H, B þ B0, H3 þ H6, J3e4 ¼ J6e5 ¼ 13.6 Hz, J3e5 ¼ J6e4 ¼ 8.4 Hz);
8.45 (s, 2H, B þ B0, H2); 10.59 (s, 2H, 2NH). 13C NMR (100 MHz,
4.2.1. Cells and culture conditions
L. infantum axenic amastigotes were grown in M199 (Invitrogen,
Leiden, The Netherlands) medium supplemented with 10% heat
DMSO-d6)
d
: 120.6 (4C, A þ A0, C3, C5); 122.7 (2C, B þ B0, C3); 125.8
(2C, A þ A0, C1); 128.7 (2C, B þ B0, C1); 129.8 (2C, B þ B0, C6); 130.0
(2C, B þ B0, C9); 130.1 (2C, B þ B0, C7); 130.3 (2C, B þ B0, C8); 130.5
(2C, B þ B0, C4); 132.3 (2C, B þ B0, C10); 133.5 (4C, A þ A0, C2, C6);
134.6 (2C, B þ B0, C2); 135.1 (2C, B þ B0, C5); 138.7 (2C, A þ A0, C4).
inactivated FCS, 1 g/L
sacarose, 50 mg/L sodium pyruvate, 320 mg/L malic acid, 40 mg/L
fumaric acid, 70 mg/L succinic acid, 200 mg/L -ketoglutaric acid,
b-alanine, 100 mg/L L-asparagine, 200 mg/L
a
MS (m/z,
%
abundance): 57 (100). Elemental Analysis for
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 37 ꢂC. THP-1 cells were kindly
provided by Dr. Michel (Université Nice Sophia Antipolis, Nice,
France) and were grown in RPMI-1640 medium (Gibco, Leiden, The
Netherlands) supplemented with 10% heat inactivated FCS, antibi-
otics, 1 mM HEPES, 2 mM glutamine and 1 mM sodium pyruvate,
pH 7.2 at 37 ꢂC and 5% CO2.
C
32H24N2O4S2Se2$0.5HCl, Calcd/Found (%): C: 51.87/51.63; H: 3.37/
3.77; N: 3.78/3.96.
4.1.1.14. N,N0-(Diselanediyldibenzene-4,1-diyl)diquinoline-8-
sulfonamide (14). From 4,40-diselanediyldianiline and quinoline-8-
sulfonyl chloride. The precipitate was recrystallized from etha-
nol:water (80:20). Yield: 4%; mp: 226e228 ꢂC. IR (KBr) cmꢃ1: 3268
(m, NeH), 1164 (s, SO2), 835 (m, SeeSe). 1H NMR (400 MHz, DMSO-
4.2.2. Leishmanicidal activity and cytotoxicity assays
d6)
d
: 6.95 (d, 4H, A þ A0, H3 þ H5, J3e2 ¼ J5e6 ¼ 8.5 Hz); 7.17 (d, 4H,
Drug treatment of amastigotes was performed during the log-
arithmic growth phase at a concentration of 2 ꢁ 106 parasites/mL at
26 ꢂC or 1 ꢁ 106 parasites/mL at 37 ꢂC for 24 h, respectively. Drug
treatment of THP-1 cells was performed during the logarithmic
growth phase at a concentration of 4 ꢁ 105 cells/mL at 37 ꢂC and 5%
CO2 for 24 h. The percentage of living cells was evaluated by flow
cytometry by the propidium iodide (PI) exclusion method [49].
A þ A0, H2 þ H6, J2e3 ¼ J6e5 ¼ 8.5 Hz); 7.68e7.72 (m, 4H, B þ B0,
H6 þ H3); 8.27 (dd, 2H, B þ B0, H5, J5e6 ¼ 8.2 Hz, J5e7 ¼ 1.3 Hz); 8.37
(dd, 2H, B þ B0, H7, J7e6 ¼ 7.3 Hz, J7e5 ¼ 1.3 Hz); 8.50 (dd, 2H, B þ B0,
H4, J4e3 ¼ 8.4 Hz, J4e2 ¼ 1.7 Hz); 9.11 (dd, 2H, B þ B0, H2, J2e
¼ 4.2 Hz, J2e4 ¼ 1.7 Hz); 10.30 (s, 2H, 2NH). 13C NMR (100 MHz,
3
DMSO-d6)
d
: 120.2 (2C, B þ B0, C3); 121.6 (4C, A þ A0, C3, C5); 123.5
(2C, B þ B0, C8); 125.0 (2C, A þ A0, C1); 126.5 (2C, B þ B0, C7); 129.2
(2C, B þ B0, C5); 133.2 (4C, A þ A0, C2, C6); 134.4 (2C, B þ B0, C6); 135.8
(2C, B þ B0, C4); 138.9 (2C, A þ A0, C4); 143.5 (2C, B þ B0, C9); 151.9
(2C, B þ B0, C10); 152.7 (2C, B þ B0, C2). MS (m/z, % abundance): 57
(100). Elemental Analysis for C30H22N4O4S2Se2, Calcd/Found (%): C:
49.72/49.56; H: 3.03/3.11; N: 7.73/7.69.
4.2.3. Leishmania infection assay
THP-1 cells were seeded at 120.000 cells/mL in 24 multidishes
plates (Nunc, Roskilde, Denmark) and differentiated to macro-
phages for 24 h in 1 mL of RPMI-1640 medium containing 10 ng/mL
phorbol 12-myristate 13-acetate (PMA) (SigmaeAldrich, St. Louis,
MO, USA). Medium culture was removed and 1.2 ꢁ 106 Leishmania
amastigotes expressing the eGFP protein were added to each well
reaching a final volume of 1 mL. 4 h later all medium with non
4.1.1.15. N,N0-(Diselanediyldibenzene-4,1-diyl)ditiophene-2-
sulfonamide (15). From 4,40-diselanediyldianiline and thiophene-

122
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with phosphate buffered saline (PBS) and cells were grown in the
presence of new THP-1 medium and the corresponding treatment.
After 48 h of treatment, medium was removed; THP-1 cells were
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Acknowledgments
The authors wish to express their gratitude to the Foundation
for Applied Medical Investigation and the Institute of Tropical
Health, University of Navarra. The authors also thank the Spanish
MEC/MICINN (Project SAF2009-13914-C02), the Comunidad de
Madrid (Project BIPEDD-2-CM ref S-2010/BMD-2457) and the Junta
de Comunidades de Castilla la Mancha (Project POII10-0180-7897)
for financial support.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.12.030.
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