Selenocyanates and diselenides: A new class of potent antileishmanial agents

Article abstract of DOI:10.1016/j.ejmech.2011.04.054

Thirty five selenocyanate and diselenide compounds were subjected to in vitro screening against Leishmania infantum promastigotes and the most active ones were also tested in an axenic amastigote model. In order to establish the selectivity indexes (SI) t

Full text of DOI:10.1016/j.ejmech.2011.04.054

European Journal of Medicinal Chemistry 46 (2011) 3315e3323
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Selenocyanates and diselenides: A new class of potent antileishmanial agents
Daniel Plano a, Ylenia Baquedano a, David Moreno-Mateos c, María Font b, Antonio Jiménez-Ruiz c,
,
a
Juan Antonio Palop a , Carmen Sanmartín
*
a Sección de Síntesis, Departamento de Química Orgánica y Farmacéutica, University of Navarra, Irunlarrea, 1, E-31008 Pamplona, Spain
b Sección de Modelización Molecular, Departamento de Química Orgánica y Farmacéutica, 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:
Thirty five selenocyanate and diselenide compounds were subjected to in vitro screening against
Leishmania infantum promastigotes and the most active ones were also tested in an axenic amastigote
model. In order to establish the selectivity indexes (SI) the cytotoxic effect of each compound was also
assayed against Jurkat and THP-1 cell lines.
Thirteen derivatives exhibit better IC50 values than miltefosine and edelfosine. Bis(4-aminophenyl)
diselenide exhibits the best activity when assayed in infected macrophages and one of the lowest
cytotoxic activities against the human cell lines tested, with SI values of 32 and 24 against Jurkat and
THP-1 cells, respectively. This compound thus represents a new lead for further studies aimed at
establishing its mechanism of action.
Received 1 October 2010
Received in revised form
20 April 2011
Accepted 21 April 2011
Available online 28 April 2011
Keywords:
Selenocyanate
Diselenide
Ó 2011 Elsevier Masson SAS. All rights reserved.
Antileishmanial activity
1. Introduction
administration required e as well as their high renal and cardiac
toxicity e are all major drawbacks. On the other hand, the high
The leishmaniases are a group of diseases with a broad range of
clinical manifestations. These diseases are caused by different
species of the kinetoplastid parasite Leishmania and affect man and
other mammals in tropical and subtropical regions of the world.
The World Health Organization (WHO) estimates that the disease
results in 2 million new cases a year, threatens 350 million people
in 88 countries and that there are 12 million people currently
infected worldwide [1,2]. This pathology is transmitted by blood-
feeding female sandflies. Parasites cycle between two different
forms: extracellular flagellated promastigotes in the vector and
intracellular non-flagellated amastigotes within mononuclear
phagocytes in the mammalian host [3,4].
Maintenance of parasites at dermal sites or subsequent dispersal
to internal tissues is responsible for the different pathologies of the
disease, giving rise to cutaneous, mucocutaneous, diffuse cuta-
neous and visceral leishmaniases (CL, MCL, DCL and VL, respec-
tively) [5]. Conventional therapies are based on treatment with
pentavalent antimonials (sodium stibogluconate and meglumine
antimonate), pentamidine, or amphotericin B [6]. The high resis-
tance rates developed against pentavalent antimonials, their high-
dose regimens, and the long treatment courses of parentheral
toxicity and declining efficacy of pentamidine have restricted its
use. Amphotericin B and its lipid formulation proved to be very
effective in the treatment of leishmaniasis, but the cost of this drug
is still prohibitively high and out of the reach of poor people. More
recently, the alkyl-phospholipid miltefosine [7] has emerged as an
oral drug that is highly effective in children but occasionally pres-
ents gastrointestinal complications and also shows teratogenic
effects. Edelfosine [8], another alkyl-phospholipid, has also been
tested and was found to display a higher in vitro activity than
miltefosine. Other drugs, such as paromomycin and sitamaquine,
have been reported to give variable cure rates [9].
The serious problems still associated with the treatment of
leishmaniasis have led many research groups to search for novel,
more efficient and safe chemotherapeutic agents against Leish-
mania infections.
Recently, the development and use of metal complexes as
potent chemotherapeutic agents against leishmaniasis has been
explored. Thus, some organometallic and coordination complexes
of palladium, gold, iridium, rhodium, platinum and zinc sulfate
have been tested against the Leishmania parasite [10]. In addition,
some metallointercalators of DNA based on platinum, copper and
silver have been evaluated and were shown to possess remarkable
antileishmanial activity [11]. Another relevant trace element is
selenium, whose increased concentration in plasma has been rec-
ognised as a new defensive strategy against Leishmania infection
* Corresponding author. Tel.: þ34 948 425 600; fax: þ34 948 425 649.
E-mail address: jpalop@unav.es (J.A. Palop).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.054

3316
D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
[12,13]. Recently, a study has implicated the human selenium-
containing selenoprotein P (Sepp1) in the protection against
illness caused by Trypanosoma due to its protective action against
oxidative damage [14]. The biochemical pathways that constitute
the cellular targets for selenium are still under investigation.
For these reasons, in the selection of the chemical groups for the
new molecules described here we have considered, as a first and
promising approach, the importance of possible potential interfer-
ence of selenium derivatives with the redox system of the parasites.
It is well known [15] that in trypanosomatids the glutathione system
is replaced by a trypanothione system where the key enzyme is
trypanothione reductase (TrypR) which catalyzes the reduction of
trypanothione disulfide to trypanothione [16]. In contrast to its
human counterpart, the enzymatic system that regulates the redox
status in Trypanosomatids is not based in the activity of selenopro-
teins [17] so, we propose to incorporate structural moieties such as
diselenide function in an attempt to find more potent and selective
leishmanicidal compounds.
acetic anhydride. Compounds 1eeq were obtained in variable yields
(15e81%) by reaction of haloarenes with potassium selenocyanate in
acetone under reflux [24]. Neither of these methods gave good
results in the case of derivative 1r and nor did they allow us to carry
out the synthesis in sufficient amounts to study the biological
behavior. The subsequent reduction of compounds 1aeq with
sodium borohydride [25] in ethanol afforded derivatives 2aeq. A
different synthetic pathway was developed to prepare compound 2r.
The strategy [26] employed 100% hydrazine hydrate and sodium
hydroxide in DMF to reduce elemental selenium and generate
sodium diselenide and this was followed by reaction with
p-methoxybenzyl chloride.
All of the compounds prepared during the course of these
investigations (Table 1) are stable and they were characterized by
spectroscopic methods such as IR and NMR and also by elemental
analysis. The spectroscopic data for the new synthesized deriva-
tives were shown in Tables 2 and 3.
Taking into account all of the aforementioned and due to the
urgent need for innovative drugs based on new molecular scaffolds,
and as an extension of our earlier work on selenium derivatives
[18e22], we decided to test the efficiency of the chemical forms
selenocyanate and diselenide with different functionalities in the
aryl ring as leishmanicidal agents. The substitutions of groups at
various positions of the phenyl ring allowed us to explore the effect
of the position on biological activity. Further studies are necessary
to verify parasite selenium dependence and determine the
concentration and mechanism of action of this trace element that is
required for their viability.
2.2. Biological evaluation
2.2.1. Activity in axenic promastigotes and amastigotes
The synthesized selenocyanates (1aeq) and diselenides (2aer)
were initially tested in vitro at concentrations of 25, 12.5 and
6.25 mM against cultured promastigotes of Leishmania infantum
(MCAN/ES/89/IPZ229/1/89) according to a previously described
procedure [27]. All the analyses were carried out with a minimum
of three independent experiments.
The percentages of growth inhibition obtained at three different
concentrations are summarized in Table 4. This procedure allowed
us to select the compounds that caused more than 50% cell growth
inhibition at 25 mM. These compounds were subsequently screened
against amastigotes. Edelfosine and miltefosine were used as the
reference drugs in these assays.
Of the thirty five compounds evaluated against promastigotes,
twenty of them caused more than 50% cell growth inhibition at
2. Results and discussion
2.1. Chemistry
The synthetic strategy for the preparation of selenocyanate
derivatives (compounds 1aeq) and diselenides (compounds 2aer) is
outlined in Scheme 1. Compounds 1aec were prepared by addition
of selenium dioxide [23] to malononitrile in DMSO at room
temperature followed by reaction with the corresponding aminoaryl
derivatives. Compound 1d was obtained from 1c by reaction with
25
mM; thirteen retained inhibition over 50% at 12.5
mM and nine at
6.25
mM. The IC50 values for promastigotes and amastigotes ob-
tained with the fifteen most active compounds are shown in
Table 5.
Table 1
Structures of compounds 1aeq and 2aer.
DMSO
NC-Se-Se-Se-CN
SeO2
+ NC-CH2-CN
Ar-(CH2)n-SeCN
Ar-(CH2)n-Se-Se-(CH2)n-Ar
Ar-H
For 1a-c
1a-q
2a-r
Acetone
Ar-(CH2)n-Br + KSeCN
1a-q
Ar-(CH2)n-SeCN
Compounds
Aryl ring (Ar)
n
For 1e-q
1a/2a
1b/2b
1c/2c
1d/2d
1e/2e
1f/2f
1g/2g
1h/2h
1i/2i
1j/2j
1k/2k
1l/2l
1m/2m
1n/2n
1o/2o
1p/2p
1q/2q
2r
4-Aminophenyl
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
2
1
4-(N,N-dimethylamino)phenyl
4-Amino-3-carboxyphenyl
4-Acetamido-3-carboxyphenyl
4-Nitrophenyl
3-Nitrophenyl
2-Nitrophenyl
NaBH4
2Se
NH2NH2
For 2a-q
NaOH
4-Bromophenyl
CH2-Cl
OMe
4-Trifluoromethylphenyl
4-Methylthiophenyl
4-Methylphenyl
4-Cyanophenyl
3-Cyanophenyl
2-Cyanophenyl
Phenyl
Naphthyl
4-Nitrophenyl
4-Methoxyphenyl
For 2r
Na2Se2
+
Ar-(CH2)n-Se-Se-(CH2)n-Ar
2a-r
Scheme 1. Synthesis of compounds 1aeq and 2aer. Compound 1d was obtained from
1c (see Experimental section).

D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
3317
Table 2
Physical constants of the new synthesized compounds.
Ar-(CH2)n-SeCN
Ar-(CH2)n-Se-Se-(CH2)n-Ar
1a-q
2a-r
Comp.
Aryl ring (Ar)
n
Yield (%)
Mp (ꢀC)
PMa
Molecular formula
10H8N2O3Se
C8H6O2N2Se
C8H6O2N2Se
C9H6F3NSe
C9H9NSSe
C9H6N2Se
C9H6N2Se
C9H6N2Se
Anal (%) calcd/found
1d
1f
1g
1i
1j
1l
1m
1n
1p
1q
2c
2d
2e
2f
2g
2h
2i
2j
2l
2m
2n
2q
4-Acetamino-3-carboxyphenyl
3-Nitrophenyl
2-Nitrophenyl
4-Trifluoromethylphenyl
4-Methylthiophenyl
4-Cyanophenyl
3-Cyanophenyl
2-Cyanophenyl
Naphthyl
4-Nitrophenyl
4-Amino-3-carboxyphenyl
4-Acetamino-3-carboxyphenyl
4-Nitrophenyl
3-Nitrophenyl
2-Nitrophenyl
0
1
1
1
1
1
1
1
1
2
0
0
1
1
1
1
1
1
1
1
1
2
89
30
83
67
41
80
80
63
43
70
63
84
35
22
65
67
38
15
30
33
11
83
172e173
84e86
72e74
54e55
81e83
129e131
60e62
120e121
119e120
67e68
231e232
142e143
99e100
86e88
99e100
102e103
64e66
C
B
B
B
C
B
B
B
C
C
B
B
B
B
B
B
B
B
B
B
B
B
C
C, 42.40/42.20; H, 2.83/3.10; N, 9.89/9.55.
C, 39.83/39.95; H, 2.49/2.60; N, 11.62/11.24.
C, 39.83/39.56; H, 2.49/2.54; N, 11.62/11.40.
C, 40.90/41.10; H, 2.27/2.27; N, 5.30/4.94.
C, 44.62/44.63; H, 3.71/3.80; N, 5.78/5.55.
C, 49.05/48.95; H, 2.76/2.69; N, 12.47/12.33.
C, 49.05/48.83; H, 2.76/2.68; N, 12.47/12.26.
C, 49.05/48.83; H, 2.76/2.80; N, 12.47/12.40.
C, 58.54/58.31; H, 3.66/3.90; N, 5.69/5.42.
C, 42.35/42.44; H, 3.14/3.31; N, 10.98/11.08.
C, 39.07/38.73; H, 2.79/2.78; N, 6.51/6.14.
C, 42.02/41.69; H, 3.11/3.45; N, 5.45/5.03.
C, 39.07/39.09; H, 2.79/2.78; N, 6.51/6.37.
C, 39.07/39.25; H, 2.79/2.73; N, 6.51/6.46.
C, 39.07/39.08; H, 2.79/2.76; N, 6.51/6.56.
C, 33.73/33.37; H, 2.41/2.31.
C12H9NSe
C9H8N2O2Se
C14H12N2O4Se2
C18H16N2O6Se2
C14H12O4N2Se2
C14H12O4N2Se2
C14H12O4N2Se2
C14H12Br2Se2
C16H12F6Se2
4-Bromophenyl
4-Trifluoromethylphenyl
4-Methylthiophenyl
4-Cyanophenyl
3-Cyanophenyl
2-Cyanophenyl
C, 40.34/40.84; H, 2.52/2.53.
C, 44.44/44.12; H, 4.16/4.20.
C, 49.23/49.04; H, 3.07/3.11; N, 7.18/7.07.
C, 49.23/48.88; H, 3.07/3.27; N, 7.18/7.29.
C, 49.23/49.63; H, 3.07/3.11; N, 7.18/7.07.
C, 41.92/41.77; H, 3.49/3.52; N, 6.11/5.97.
99e100
136e137
118e120
103e105
86e88
C
16H18S2Se2
C16H12N2Se2
C16H12N2Se2
C16H12N2Se2
C16H16N2O4Se2
4-Nitrophenyl
A ¼ Recrystallized from a mixture of MeOH:H2O (1:1); B ¼ Recrystallized from EtOH; C ¼ Washed with H2O; D ¼ Recrystallized from 1,4-dioxane; E ¼ Washed with hot
EtOH:N,N-DMF (1:1); F ¼ Recrystallized from n-hexane.
a
PM ¼ Purification method.
The results presented in Table 4 indicate that most of the aryl
rings confer higher activity against Leishmania promastigotes when
present in the selenocyanate derivatives compared to the same
moiety in the diselenide scaffold. However, a detailed analysis of
the IC50 values for amastigotes (see Table 5) reveals that the rings a,
b, e and o, with the substituent group in para position generate
more active compounds when present in the diselenide forms. In
fact, all of the IC50 values obtained for compounds 2a, 2b, 2e and 2o
respectively) support this conclusion, with the effect being more
evident for the diselenide function. Finally, for diselenide
compounds, when n ¼ 1, the activity is improved by the presence in
the aromatic moiety of strongly electron-withdrawing substituents
(NO2 in 2e) compared to electron-donating ones (for example, CH3 in
2k, OCH3 in 2r). However, for selenocyanate compounds the activity
does not seem to correlate with modulation of the electronic
distribution through the aromatic ring. In order to evaluate the role
in the biological activity of the position of substituent group in the
aryl ring we synthesized various ortho and meta derivatives of some
of the most active para compounds. The selection of the new
compounds to be synthesized was made according to the commer-
cial availability so as the synthetic accessibility. So, the functionali-
zation of positions 2 and 3, in order to obtain the corresponding
ortho and meta derivatives from 1a, 2a, 1b and 2b, that requires an
electrophilic substitution on a deactivated position, does not occur
[23]. This difficulty has not been overcome, using other synthetic
methods [24e26]. In general, the structureeactivity relationship
(SAR) information indicates that in the promastigote model the
highest activity among selenocyanate compounds is obtained with
the cyano group in the ortho position whereas the nitro group
located at the para position in the aryl ring results in the best activity
among diselenides. However, a very slight increase in the activity for
ortho nitro and meta cyano related to their corresponding para is
obtained in the amastigote model.
are below 1 mM when tested against amastigotes. The cytotoxic
activity of these four compounds was further confirmed in an
additional assay by direct counting of living cells in a neubauer
chamber after staining of the dead cells with trypan blue
(Supplementary data).
Comparison of the IC50 values of our compounds with those
obtained for miltefosine in the promastigote model reveals that
nineteen of them are more potent than miltefosine, with IC50 values
in the range 0.96e11.9
thirteen of the compounds tested (1b,1e,1f,1g,1i,1l,1m,1o, 2a, 2b,
2e, 2g and 2o) show IC50 values (IC50 ¼ 0.29e2.79 M) better than
that obtained for miltefosine (IC50 ¼ 2.84 M). Moreover, ten of
them (1e,1f,1g,1l,1m, 2a, 2b, 2e, 2g and 2o), with IC50 values in the
range 0.29e0.77 M, show stronger in vitro antileishmanial activity
than edelfosine (IC50 ¼ 0.82 M). It is remarkable that 2g, 1g and 2e
mM. When considering the amastigote form,
m
m
m
m
are 9.8, 8.3 and 7.5 times more effective respectively than milte-
fosine and 2.8, 2.4 and 2.2 times more potent than edelfosine.
Given the relatively small number of derivatives tested, a detailed
SAR analysis is not feasible but some observations and comparisons
on the effect of the size of the linker on activity can be made. For
example, analysis of the nitro derivatives (compounds 1e, 1q, 2e and
2q) that contain chains with one or two carbons reveals that an
increase in chain length has a detrimental effect on the activity. The
results for compounds 1e and 2e (aryl ring ¼ 4-nitrophenyl; n ¼ 1,
2.2.2. Cytotoxic activity in human cells
In order to determine the toxicity/activity index, the most active
compounds against Leishmania promastigotes were also tested
against two leukemia cell lines derived from either lymphoblasts
(Jurkat) or monocytes (THP-1) at 0.08, 0.4, 2.0, 10.0 and 50.0
mM.
The IC50 values obtained are gathered in Table 5. The selectivity
index (SI) was defined as the ratio of the IC50 values of compounds
against either Jurkat or THP-1 cells relative to those obtained
IC50 amastigotes ¼ 0.68 and 0.38
m
M, respectively) and 1q and 2q (aryl
ring ¼ 4-nitrophenyl; n ¼ 2, IC50
¼ 3.80 M and inactive,
m
amastigotes

3318
D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
Table 3
Spectroscopic data for the new synthesized compounds.
Comp.
IR (KBr; cmꢁ1
)
1H NMR (400 MHz, DMSO-d6,
d, J in Hz)
13C NMR (400 MHz, DMSO-d6,
d)
1d
3245 (m, NeH),
2154 (m, C^N), 1694
(s, C]O(OH)), 1654 (C O)
2.16 (s, 3H, CH3); 7.91 (dd, 1H, J4e3 ¼ 8.8, J4e6 ¼ 2.2, H4);
8.25 (d, 1H, H6);
8.51 (d, 1H, H3); 11.09 (s, 1H, NH)
25.8 (1C, CH3); 106.3 (1C, CN); 117.4 (1C, C3-phenyl);
118.7 (1C, C1-phenyl); 122.2 (1C, C5-phenyl);
137.2 (1C, C2-phenyl); 140.0 (1C, C6-phenyl);
142.4 (1C, C4-phenyl); 169.2 (1C, CONH); 169.7 (1C, COOH)
31.9 (1C, CH2); 105.6 (1C, CN); 123.5 (1C, C4-benzyl);
124.3 (1C, C2-benzyl); 131.1 (1C, C5-benzyl);
136.3 (1C, C6-benzyl); 141.8 (1C, C1-benzyl); 148.6 (1C, C3-benzyl
29.6 (1C, CH2); 104.9 (1C, CN); 126.3 (1C, C3-benzyl);
130.5 (1C, C4-benzyl); 133.5 (1C, C6-benzyl);
133.9 (1C, C1-benzyl); 135.1 (1C, C5-benzyl); 148.0 (1C, C2-benzyl
32.3 (1C, CH2); 105.7 (1C, CN); 124.1 (1C, CF3);
126.4 (2C, C-3, C5-benzyl); 129.2 (1C, C4-benzyl);
1f
2147 (m, C^N),
1520 (s, CeNO2)
4.44 (s, 2H, CH2); 7.67e7.71 (m, 1H, H5); 7.83
(d, 1H, J6e5 ¼ 7.7, H6);
8.18 (d, 1H, J4e5 ¼ 8.1, H4); 8.27 (s, 1H, H2)
4.60 (s, 2H, CH2); 7.60e7.64 (m, 1H, H4); 7.67
(d, 1H, J6e5 ¼ 7.6, H6);
)
)
1g
1i
2146 (m, C^N),
1515 (s, CeNO2)
7.77e7.81 (m, 1H, H5); 8.12 (d, 1H, J3e4 ¼ 8.2, H3)
4.37 (s, 2H, CH2); 7.59 (d, 2H, J2e3 ¼ J6e5 ¼ 8.1, H2 þ H6);
7.75 (d, 2H, H3 þ H5)
2155 (s, C^N),
1326 (s, CeF)
130.5 (2C, C-2, C6-benzyl); 144.2 (1C, C1-benzyl
)
1j
2143 (s, C^N),
1490 (s, SeCH3)
2.47 (s, 3H, CH3); 4.29 (s, 2H, CH2); 7.24,
(d, 2H, J2e3 ¼ J6e5 ¼ 8.2, H2 þ H6);
7.31 (d, 2H, H3 þ H5)
15.4 (1C, SCH3); 33.4 (1C, CH2); 105.8 (1C, CN);
126.7 (2C, C3, C5-benzyl); 130.3 (2C, C2, C6-benzyl);
135.6 (1C, C1-benzyl); 138.8 (1C, C4-benzyl
)
1l
2229 (s, C^N),
2144 (s, C^N)
4.36 (s, 2H, CH2); 7.56 (d, 2H, J2e3 ¼ J6e5 ¼ 8.3, H2 þ H6);
7.85 (d, 2H, H3 þ H5)
32.5 (1C, CH2); 105.6 (1C, SeCN); 111.3 (1C, C4-benzyl);
119.5 (1C, CN); 130.7 (2C, C2, C6-benzyl);
133.4 (2C, C3, C5-benzyl); 145.2 (1C, C1-benzyl
)
1m
1n
1p
1q
2c
2226 (s, C^N),
2150 (s, C^N)
4.35 (s, 2H, CH2); 7.59e7.62 (m, 1H, H5); 7.72
(d, 1H, J6e5 ¼ 8.2, H6);
32.0 (1C, CH2); 105.6 (1C, SeCN); 112.3 (1C, C3-benzyl);
119.4 (1C, CN); 130.8 (1C, C5-benzyl); 132.4 (1C, C4-benzyl);
133.1 (1C, C2-benzyl); 134.7 (1C, C6-benzyl); 141.2 (1C, C1-benzyl
30.5 (1C, CH2); 104.9 (1C, SeCN); 112.3 (1C, C2-benzyl);
117.9 (1C, CN); 129.6 (1C, C4-benzyl); 131.7 (1C, C6-benzyl);
134.1 (1C, C3-benzyl); 134.3 (1C, C5-benzyl); 142.2 (1C, C1-benzyl
34.0 (1C, CH2); 105.9 (1C, CN); 127.2 (1C, C6); 127.3 (1C, C7);
127.8 (1C, C1); 128.3 (1C, C4); 128.5 (1C, C8); 128.6 (1C, C5);
129.3 (1C, C3); 133.2 (1C, C10); 133.5 (1C, C9); 136.6 (1C, C2)
30.4 (1C, CH2Se); 36.9 (1C, CH2); 105.2 (1C, CN);
7.78 (d, 1H, J4e5 ¼ 7.7, H4); 7.80 (s, 1H, H2)
4.47 (s, 1H, CH2); 7.49e7.53, (m, 1H, H4); 7.63
(d, 1H, J6e5 ¼ 7.7, H6);
)
)
2230 (s, ChN),
2151 (s, ChN)
7.71e7.75, (m, 1H, H5); 7.86 (d, 1H, J3e4 ¼ 7.6, H3)
(CDCl3): 4.63 (s, 2H, CH2); 7.54e7.56 (m, 2H, H6 þ H7);
7.62 (dd, 1H, J3e4 ¼ 8.4, J3e1 ¼ 1.9, H3);
7.91e7.95 (m, 4H, H1 þ H4 þ H5 þ H8)
2148 (s, C^N)
2145 (m, C^N),
1508, 1343 (s, NO2)
3.42 (t, 2H, JCH
¼ 7:5, CH2eSeCN); 3.54
2eCH2
(t, 1H, PheCH2);
7.66 (d, 2H, J3e2 ¼ J5e6 ¼ 8.7, H3 þ H5); 8.24 (d, 2H, H2 þ H6)
124.4 (2C, C3, C5-benzyl); 130.9 (2C, C2, C6-benzyl);
147.2 (1C, C4-benzyl); 148.4 (1C, C1-benzyl
)
0
0
0
0
0
3490, 3406, 3379,
3275 (m, NH2),
1682 (s, C]O),
824 (m, SeeSe)
1690 [s, C]O(OH)],
1664 (s, C]O),
836 (w, SeeSe)
6.63 (d, 2H, J3e4 ¼ J3 e4 ¼ 8.5, H3 þ H3 ); 7.29
114.3 (2C, C3, C3 -phenyl); 114.6 (2C, C5, C5 -phenyl);
0
0
0
0
0
(dd, 2H, J4e6 ¼ J4 e6 ¼ 2.2, H4 þ H4 );
117.8 (2C, C1, C1 -phenyl); 139.2 (2C, C2, C2 -phenyl);
0
0
0
7.98 (d, 2H, H6 þ H6
)
139.6 (2C, C6, C6 -phenyl); 152.5 (2C, C4, C4 -phenyl);
170.7 (2C, COOH)
0
0
0
2d
2.14 (s, 6H, 2CH3); 7.80 (dd, 2H, J4e3 ¼ J4 e3 ¼ 8.8,
25.9 (2C, CH3); 118.3 (2C, C3, C3 -phenyl);
0
0
0
0
0
J4e6 ¼ J4 e6 ¼ 2.2, H4 þ H4 );
121.6 (2C, C1, C1 -phenyl); 123.7 (2C, C5, C5 -phenyl);
0
0
0
8.13 (d, 2H, H6 þ H6 );
135.9 (2C, C2, C2 -phenyl); 138.7 (2C, C6, C6 -phenyl);
0
0
8.42 (d, 2H, H3 þ H3 ); 11.07 (s, 2H, 2NH)
141.7 (2C, C4, C4 -phenyl); 169.2 (2C, CONH); 169.5 (2C, COOH)
0
0
2e
2f
1516 (s, NO2),
741 (m, SeeSe)
4.12 (s, 4H, 2CH2); 7.47 (d, 4H, J2e3 ¼ J6e5 ¼ 8.5,
30.9 (2C, CH2); 124.3 (4C, C3, C5, C3 , C5 -benzyl);
0
0
0
0
0
H2 þ H6 þ H2 þ H6 );
130.9 (4C, C2, C6, C2 , C6 -benzyl); 147.1 (2C, C1, C1 -benzyl);
0
0
0
8.17 (d, 4H, H3 þ H5 þ H3 þ H5
)
148.5 (2C, C4, C4 -benzyl
)
0
0
1521 (s, CeNO2),
737 (m, SeeSe)
4.13 (s, 4H, 2CH2); 7.58e7.62 (m, 2H, H5 þ H5 ); 7.67
30.5 (2C, CH2); 122.6 (2C, C4, C4 -benzyl);
0
0
0
0
0
(d, 2H, J6e5 ¼ J6 e5 ¼ 7.7, H6 þ H6 );
124.1 (2C, C2, C2 -benzyl); 130.6 (2C, C5, C5 -benzyl);
0
0
0
0
0
0
8.05 (s, 2H, H2 þ H2 ); 8.09 (d, 2H, J4e5 ¼ J4 e5 ¼ 8.2, H4 þ H4
)
136.3 (2C, C6, C6 -benzyl); 142.7 (2C, C1, C1 -benzyl);
0
148.4 (2C, C3, C3 -benzyl
)
0
0
0
0
2g
1511 (s, CeNO2),
741 (m, SeeSe)
4.27, (s, 4H, 2CH2); 7.36 (d, 2H, J6e5 ¼ J6 e5 ¼ 7.3, H6 þ H6 );
30.4 (2C, CH2); 126.2 (2C, C3, C3 -benzyl);
0
0
0
7.51e7.55 (m, 2H, H4 þ H4 );
129.6 (2C, C4, C4 -benzyl); 133.1 (2C, C6, C6 -benzyl);
134.6 (2C, C1, C1 -benzyl); 135.5 (2C, C5, C5 -benzyl);
0
0
0
7.66e7.69 (m, 2H, H5 þ H5 );
0
0
0
0
8.05 (d, 2H, J3e4 ¼ J3 e4 ¼ 8.0, H3 þ H3
)
148.0 (2C, C2, C2 -benzyl
)
0
2h
2i
1008 (s, CeBr),
710 (m, SeeSe)
3.93 (s, 4H, 2CH2); 7.19 (d, 4H, J2e3 ¼ J6e5 ¼ 8.4,
31.4 (2C, CH2); 120.9 (2C, C4, C4 -benzyl);
0
0
0 0
131.9 (4C, C2, C6, C2 , C6 -benzyl);
H2 þ H6 þ H2 þ H6 );
7.51 (d, 4H, H3 þ H5 þ H3 þ H5
4.04 (s, 4H, 2CH2); 7.43 (d, 4H, J2e3 ¼ J6e5 ¼ 7.9,
0
0
0
0
0
)
132.1 (4C, C3, C5, C3 , C5 -benzyl); 139.6 (2C, C1, C1 -benzyl
31.2 (2C, CH2); 123.8 (2C, CF3);
126.0 (4C, C3, C5, C3 , C5 -benzyl); 128.2 (2C, C4, C4 -benzyl);
130.5 (4C, C2, C6, C2 , C6 -benzyl); 145.1 (2C, C1, C1 -benzyl
15.6 (2C, SCH3); 32.1 (2C, CH2);
)
1325 (s, CeF),
746 (d, SeeSe)
0
0
0
0
0
H2 þ H6 þ H2 þ H6 );
7.67 (d, 4H, H3 þ H5 þ H3 þ H5
2.45 (s, 6H, 2CH3); 3.90 (s, 4H, 2CH2); 7.19
0
0
0
0
0
)
)
2j
1491 (s, SeCH3),
718 (w, SeeSe)
0
0
0
0
0
0
(s, 8H, H2 þ H2 þ H3 þ H3 þ H5 þ H5 þ H6 þ H6
)
126.7 (4C, C3, C5, C3 , C5 -benzyl);
0
0
0
130.4 (4C, C2, C6, C2 , C6 -benzyl); 136.5 (2C, C1, C1 -benzyl);
0
137.6 (2C, C4, C4 -benzyl
)
0
2l
2229 (s, C^N),
729 (w, SeeSe)
4.02 (s, 4H, 2CH2); 7.40 (d, 4H, J2e3 ¼ J6e5 ¼ 8.2,
31.3 (2C, CH2); 110.3 (2C, C4, C4 -benzyl);
0
0
0
0
H2 þ H6 þ H2 þ H6 );
119.8 (2C, CN); 130.7 (4C, C2, C6, C2 , C6 -benzyl);
133.1 (4C, C3, C5, C3 , C5 -benzyl); 146.2 (2C, C1, C1 -benzyl)
0
0
0
0
0
7.77 (d, 4H, H3 þ H5 þ H3 þ H5
)
0
0
2m
2223 (s, C^N);
687 (w, SeeSe)
4.01 (s, 4H, 2CH2); 7.51e7.55 (m, 2H, H5 þ H5 );
30.6 (2C, CH2); 112.0 (2C, C3, C3 -benzyl);
0
0
0
0
7.57 (d, 2H, J6e5 ¼ J6 e5 ¼ 8.0, H6 þ H6 ,);
119.5 (2C, CN); 130.5 (2C, C5, C5 -benzyl);
0
0
0
7.64 (s, 2H, H2 þ H2 );
131.0 (2C, C4, C4 -benzyl); 133.0 (2C, C2, C2 -benzyl);
134.6 (2C, C6, C6 -benzyl); 141.9 (2C, C1, C1 -benzyl)
0
0
0
0
0
7.72 (d, 2H, J4e5 ¼ J4 e5 ¼ 7.3, H4 þ H4
)
0
0
0
2n
2q
2224 (s, C^N);
762 (w, SeeSe)
4.18 (s, 4H, 2CH2); 7.43e7.48 (m, 4H, H4 þ H6 þ H4 þ H6 );
30.0 (2C, CH2); 112.2 (2C, C2, C2 -benzyl); 118.5 (2C, CN);
0
0
0
7.65e7.69 (m, 2H, H5 þ H5 );
128.8 (2C, C4, C4 -benzyl); 131.2 (2C, C6, C6 -benzyl);
133.9 (2C, C3, C3 -benzyl); 134.1 (2C, C5, C5 -benzyl);
0
0
0
0
0
7.82 (d, 2H, J3e4 ¼ J3 e4 ¼ 7.9, H3 þ H3
)
0
143.4 (2C, C1, C1 -benzyl
)
1508, 1340 (s, NO2),
(CDCl3): 3.16e3.19 (m, 8H, 4CH2);
29.8 (2C, SeeCH2); 37.0 (2C, CH2ePh);
0
0
0
0
0
0
747 (m, SeeSe)
7.38 (d, 4H, J2e3 ¼ J6e5 ¼ J2 e3 ¼ J6 e5 ¼ 8.8,
124.3 (4C, C3, C5, C3 , C5 -phenyl);
0
0
0
0
0
H2 þ H6 þ H2 þ H6 );
130.7 (4C, C2, C6, C2 , C6 -phenyl); 146.9 (2C, C1, C1 -phenyl);
0
0
0
8.19 (d, 4H, H3 þ H5 þ H3 þ H5
)
149.7 (2C, C4, C4 -phenyl
)

D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
3319
Table 4
Table 5
In vitro activities of compounds against L. infantum promastigotes expressed as
growth inhibition (%).
IC50 ꢂ SEM (
mM) values for the most active compounds on promastigotes, amasti-
gotes and cytotoxic activity in Jurkat and THP-1 cell lines.
Compound
Promastigote Amastigote Jurkat
SIa
THP-1
SI
Ar-(CH2)n-SeCN
Ar-(CH2)n-Se-Se-(CH2)n-Ar
1a
1b
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
1p
1q
2a
2b
2e
2g
11.2 ꢂ 1.1
9.24 ꢂ 0.33
4.30 ꢂ 0.74
4.18 ꢂ 1.06
3.32 ꢂ 0.16
11.6 ꢂ 0.6
11.3 ꢂ 0.4
11.6 ꢂ 0.7
11.8 ꢂ 0.3
4.14 ꢂ 0.23
6.06 ꢂ 1.7
7.75 ꢂ 0.2
7.95 ꢂ 0.34
11.2 ꢂ 1.2
11.9 ꢂ 0.9
0.96 ꢂ 0.07
3.18 ꢂ 0.23
4.04 ꢂ 0.38
12.5 ꢂ 2.8
17.8 ꢂ 1.9
3.65 ꢂ 0.19
9.29 ꢂ 1.16 43.8 ꢂ 6.2
2.79 ꢂ 0.17 29.3 ꢂ 2.2
0.68 ꢂ 0.08 6.86 ꢂ 1.3
0.57 ꢂ 0.08 12.2 ꢂ 0.7
0.34 ꢂ 0.19 12.9 ꢂ 1.4
7.95 ꢂ 1.02 12.7 ꢂ 2.1
2.14 ꢂ 1.05 7.31 ꢂ 0.4
3.02 ꢂ 0.59 5.07 ꢂ 1.0
3.33 ꢂ 0.79 14.4 ꢂ 1.7
0.55 ꢂ 0.21 13.4 ꢂ 2.5
0.53 ꢂ 0.11 8.71 ꢂ 2.6
3.16 ꢂ 0.02 17.1 ꢂ 1.9
2.58 ꢂ 0.21 10.7 ꢂ 1.7
3.72 ꢂ 1.08 14.0 ꢂ 1.4
3.80 ꢂ 0.27 2.40 ꢂ 1.2
0.65 ꢂ 0.02 20.4 ꢂ 2.8
0.77 ꢂ 0.03 > 50
5
> 50
>5
16
25
16
21
4
9
6
7
15
14
5
10
5
10 44.7 ꢂ 3.4
10 16.8 ꢂ 1.8
21 9.32 ꢂ 0.1
38 7.10 ꢂ 0.2
1a-q
2a-r
Compound Aryl ring
n
25
m
M
12.50
mM
6.25 mM
2
3
2
4
28.9 ꢂ 3.9
19.8 ꢂ 2.7
17.0 ꢂ 1.8
24.0 ꢂ 2.2
1a
1b
1c
1d
1e
1f
4-Aminophenyl
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
2
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
2
1
76.3
97.3
NEa
38.2
86.7
e
14.4
54.4
e
4-(N,N-dimethylamino)phenyl
4-Amino-3-carboxyphenyl
4-Acetamino-3-carboxyphenyl
4-Nitrophenyl
NE
e
e
24 7.97 ꢂ 1.0
16 7.60 ꢂ 2.0
98.0
97.0
98.6
97.1
98.8
97.2
98.6
97.0
93.0
90.2
99.4
91.5
88.9
98.0
77.6
NE
95.0
80.5
89.8
80.8
97.2
92.9
82.8
80.4
71.2
62.4
64.1
81.1
81.8
97.6
67.7
e
82.3
47.2
74.0
49.2
79.4
77.3
52.8
51.5
42.2
31.4
46.5
37.1
46.6
95.8
51.5
e
3-Nitrophenyl
5
4
4
1
17.1 ꢂ 1.1
25.0 ꢂ 2.9
16.8 ꢂ 3.1
17.2 ꢂ 2.2
1g
1h
1i
1j
1k
1l
2-Nitrophenyl
4-Bromophenyl
4-Trifluoromethylphenyl
4-Methylthiophenyl
4-Methylphenyl
4-Cyanophenyl
5
32 15.3 ꢂ 0.8
>65 22.1 ꢂ 1.4
71 29.1 ꢂ 2.3
24
29
77
8
51
6
0.38 ꢂ 0.03 26.8 ꢂ 3.3
0.29 ꢂ 0.10 2.62 ꢂ 0.5
0.63 ꢂ 0.05 26.1 ꢂ 5.9
0.82 ꢂ 0.13 12.2 ꢂ 2.4
2.84 ꢂ 0.10 48.2 ꢂ 4.8
1m
1n
1o
1p
1q
2a
2b
2c
2d
2e
2f
3-Cyanophenyl
2-Cyanophenyl
Phenyl
Naphthyl
4-Nitrophenyl
4-Aminophenyl
4-(N,N-dimethylamino)phenyl
4-Amino-3-carboxyphenyl
4-Acetamino-3-carboxyphenyl
4-Nitrophenyl
9
2.42 ꢂ 0.1
2o
41 32.2 ꢂ 2.5
15 4.96 ꢂ 0.16
17 18.5 ꢂ 0.6
Edelfosine
Miltefosine 15.0 ꢂ 0.8
7
a
Selectivity index (SI) is the ratio of IC50 values of compounds against either
Jurkat or THP-1 cells relative to those against L. infantum amastigotes.
NE
e
e
98.7
41.2
77.2
12.2
12.2
17.5
2.5
32.8
44.3
31.5
60.4
NE
96.3
29.4
58.9
1.9
6.0
NE
81.8
18.1
50.0
NE
11.0
NE
cytometry. Accordingly, compounds 2a, 2b, 2e and 2o were assayed
for 48 h in differentiated THP-1 cells infected with L. infantum
amastigotes expressing the green fluorescent protein (eGFP).
Infected cells were identified by the green fluorescence emitted by
their intracellular amastigotes (Fig. 1) [28]. Our results indicate that
whereas 35% of the cells were infected in untreated controls, only
5% of them remained infected after treatment with 3
mM edelfosine
and 9%, 23%, 17% or 24% of them contained parasites after treatment
with compounds 2a, 2b, 2e or 2o respectively.
It is important to emphasize that treatment of the cells with
compound 2a at 3 mM for 48 h is able to reduce infection rates by 74%
while showing a negligible cytotoxic effect over the host cells.
Cytotoxic activity of this compound was also tested by microscopic
3-Nitrophenyl
2g
2h
2i
2j
2k
2l
2-Nitrophenyl
4-Bromophenyl
4-Trifluoromethylphenyl
4-Methylthiophenyl
4-Methylphenyl
4-Cyanophenyl
NE
NE
21.9
35.4
16.5
43.6
e
10.9
22.6
7.3
33.2
e
2m
2n
2o
2p
2q
2r
3-Cyanophenyl
2-Cyanophenyl
Phenyl
Naphthyl
4-Nitrophenyl
4-Methoxyphenyl
28.4
31.7
3.2
9.9
1.2
0.5
a
NE ¼ no effect.
against L. infantum amastigotes. It can be deduced that selenocya-
nate analogs displayed a strong inhibitory activity in the cultured
amastigote model but the SI values of all the compounds were
below those obtained for the diselenides with the exception of 2g.
The selectivity of the diselenide compounds for amastigotes
increased in the order 2a < 2o < 2b < 2e with SI > 32 for Jurkat and
SI > 24 for THP-1 in all of them. Among selenocyanates, the most
selective was 1g (SI ¼ 38 and 21 when compared with Jurkat and
THP-1 respectively) followed by the derivatives 1f (SI ¼ 21 and 16)
and 1l (SI ¼ 24 and 15). Taken all the results together, the more
promising candidates were, in this order, 2e, 2b and 2o. Compar-
ison with the SI values obtained for edelfosine (SI ¼ 15 for Jurkat
and SI ¼ 6 for THP-1) and miltefosine (SI ¼ 17 for Jurkat and SI ¼ 7
for THP-1) reveals that compound 2e is 4.7 and 12.7 times more
selective than edelfosine for Jurkat and THP-1 cells, respectively,
and 4 and 11 times more selective than miltefosine for the same cell
lines. The selectivity index for 2b is 4.3 and 4.8 higher than edel-
fosine and 3.8 and 4.1higher than miltefosine, whereas the indexes
for 2o are 2.7 and 8.5 better than edelfosine and 2.4 and 7.3
superior to those obtained for miltefosine.
Fig. 1. The percentage of infected THP-1 cells after 48 h of drug-treatment was eval-
uated by flow cytometry. THP-1 cells were infected for 24 h with amastigotes
expressing the green fluorescent protein (eGFP) and then treated with the different
compounds for 48 additional hours. Infected macrophages were detected by the
fluorescence emitted by the intracellular GFP-expressing amastigotes. Results are
presented as the percentage of GFP positive cells in the THP-1 population. Control:
non-infected/non-treated THP-1 cells. GFPD: infected/non-treated THP-1 cells. Edel-
fosine, 2a, 2b, 2e and 2o: infected THP-1 cells treated with the indicated drugs.
2.2.3. Leishmanicidal activity in infected macrophages
Leishmanicidal activity was assayed in amastigote-infected
THP-1 cells. The four molecules that showed the best SI in these
cells were selected and their activity was determined by flow

3320
D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
visualization of promastigotes, amastigotes, Jurkat and Thp-1 cells.
The results presented in Fig. 2 confirm the absence of cytotoxicity of
criteria: ꢃ5 hydrogen donors (nOHNH), ꢃ10 hydrogen acceptors
(nON), MW ꢃ 500 and clogP ꢃ 5. Compounds 2a (clogP ¼ 0.36,
MW ¼ 344, nOHNH ¼ 4, nON ¼ 2), 2b (clogP ¼ 1.08, MW ¼ 400,
nOHNH ¼ 0, nON ¼ 2), 2e (clogP ¼ 1.85, MW ¼ 432, nOHNH ¼ 0,
nON ¼ 6), and 2o (clogP ¼ 2.11, MW ¼ 342, nOHNH ¼ 0, nON ¼ 0)
meet these claims. However, one of the reference drugs for phar-
macological testing, miltefosine (clogP ¼ 5.67, MW ¼ 509,
nOHNH ¼ 0, nON ¼ 7), which was also subjected to OSIRIS Explorer
Properties does not fulfil two out of four Lipinski descriptors. In
addition, it is well known that a lot of drug candidates have failed
during clinical tests because of the problems related to ADME
(absorption, distribution, metabolism and excretion) properties. So,
we utilized a web-based application called PreADMET (http://
preadmet.bmdrc.org/preadmet/index.php) which has been devel-
oped for rapid prediction of ADME/Tox data. We present the results
2a at 1
mM and 3 mM in the human cell lines tested. Remarkably, this
compound is able to kill 100% of the axenic amastigotes at 3
mM
without almost any deleterious effect in the Jurkat or Thp-1 cell lines.
2.3. Physico-chemical and absorption properties calculations
Analysis of the Lipinski descriptors for bioavailability estimation
using the freely accessible program OSIRIS Explorer Properties
(http://www.chemexper.com/tools/propertyExplorer/main.html)
was also carried out (Table 6). These parameters describe molecular
properties important for drug pharmacokinetics in the human body,
especially for their oral absorption. According to “Lipinski rules”, an
orally active drug must not violate more than one of the following
Fig. 2. Microscopic visualization of L. infantum axenic promastigotes, L. infantum axenic amastigotes, Jurkat and Thp-1 human cell lines. Cells were treated with compound 2a at
1
5
m
M, 3
mM and 15 mM. Non-treated control cells exposed to the maximum DMSO concentration of the assay are also shown. Propidium iodide was added to a final concentration of
mg/mL and the cells were immediately analyzed by phase contrast (Ph) and fluorescence microscopy (PI). Propidium iodide is excluded from viable cells but stains the nucleic acids
from dead cells.

D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
3321
Table 6
Physico-chemical and absorption properties for the most active compounds.
Compound
cLogPa
MWb
n-OHNHc donors
n-ONd acceptors
Lipinski’s violations
Absorption
HIAe (%)
Caco-2f (nm/s)
2a
2b
2e
2o
0.36
1.80
1.85
2.11
4.34
5.67
344
400
432
342
407
509
4
0
0
0
1
0
2
2
6
0
5
7
0
0
0
0
0
2
97.64
100.00
98.33
100.00
98.43
22.89
56.07
20.14
58.33
39.38
21.74
Edelfosine
Miltefosine
98.96
a
cLogP: logarithm of compound partition coefficient between n-octanol and water.
MW: molecular weight.
n-OHNH: number of hydrogen bond donors.
n-ON: number of hydrogen bond acceptors.
HIA: Human Intestinal Absorption.
b
c
d
e
f
Caco-2, cells derived from human colon adenocarcinoma.
of some properties of our active and selective compounds, (2a, 2b,
2e, and 2o) in Table 6. Human Intestinal Absorption (HIA) and Caco-2
permeability are good indicators of drug absorbance in the intestines
and Caco-2 monolayers respectively. Human Intestinal Absorption
data are the sum of bioavailability and absorption evaluated from the
ratio of excretion or cumulative excretion in urine, bile and feces
[29]. The predicted percentages of intestinal absorption are excellent
for all the compounds tested, with values above 97% in all cases. The
compounds present middle permeability values in Caco-2 cells
ranging from 20 to 58 [30].
an assessment of their possible use in developing countries, where
the cost of drug therapies is a major factor.
4. Experimental protocols
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. Elemental micro-
analyses 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) (Macherey-Nagel 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 Pharmaceu-
ticalaan 3a, 2440 Geel, België) and Lancaster (Bischheim-Strasbourg,
France).
3. Conclusions
In summary, we have presented the synthesis of thirty five
compounds that contain either a selenocyanate (1aeq) or a dis-
elenide (2aer) moiety. These compounds were evaluated as
a potential novel class of antileishmanial agents in the promasti-
gote and amastigote models and their cytotoxic activity against the
cancer cell lines Jurkat and THP-1 was investigated. Twenty analogs
exhibit promising leishmanicidal activity in both Leishmania
models, nineteen and thirteen of them being more active than
miltefosine in promastigotes and amastigotes respectively. More-
over, ten compounds (1e, 1f, 1g, 1l, 1m, 2a, 2b, 2e, 2g and 2o) are
more potent than edelfosine in amastigotes. In particular, based on
IC50 and SI values, seven compounds (1f, 1g, 1l, 2a, 2b, 2e and 2o)
combined high potency and low cytotoxicity with SI values higher
than those obtained for the reference drugs.
Taken together, our results and the predicted values for the
Lipinski parameters indicate that the diselenide compounds 2a
(with amino group), 2e (with a 4-NO2 aryl moiety), 2b (with an N,N-
dimethylamino group) and 2o (with an unsubstituted aromatic
ring) have the best profiles, with IC50 values ranging from 0.4 to
4.1.1. General procedure for the synthesis of compounds 1aec
Selenium dioxide (6 mmol) was added with stirring to solution
of malononitrile (3 mmol) in DMSO (15 mL). The mixture was
stirred at room temperature for 15 min in order to obtain trisele-
nium dicyanide. When the exothermic reaction had finished the
appropriate aromatic amine (5 mmol) was added. The mixture was
stirred for 30e60 min. Water (150 mL) was added to the reaction
mixture and the resulting precipitate was filtered off, dried and
crystallized according to the method shown in Table 2.
4-Aminophenylselenocyanate (1a): From aniline. The synthesis of
this compound has been previously reported by Kachanov
et al. [23].
0.8 mM against L. infantum amastigotes. These compounds show SI
values that are two- to four-times and four- to twelve-times higher
than those obtained for the reference drugs miltefosine and edel-
fosine respectively. Furthermore, compound 2a emerges as the
most promising derivative, taking into account that it is able to
reduce in vitro infection rates by 74% showing a negligible cytotoxic
effect over the host cells.
Despite this encouraging advance, there is still a need to develop
safe, efficient and affordable new treatments for the different
clinical forms of leishmaniasis. In this respect, extensive
mechanism-based studies are required to gain a better under-
standing of how these compounds exert their cytotoxicity. The
series of compounds described here not only show promising drug-
like properties but they are also easy and economical to prepare.
These are important points that support further in vivo studies and
4-(N,N-dimethylamino)phenylselenocyanate (1b): From N,N-
dimethylaniline. The synthesis of this compound has been previ-
ously reported by Kachanov et al. [23].
4-Amino-3-carboxyphenylselenocyanate (1c): From o-amino-
benzoic acid. The synthesis of this compound has been previously
reported by Kachanov et al. [23].
4-Acetamido-3-carboxyphenylselenocyanate (1d): From compound
1c by reaction with acetic anhydride.

3322
D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
4.1.2. General procedure for the synthesis of compounds 1eeq
KSeCN (4 mmol) was added to a solution of the appropriate
benzyl bromide derivative (4 mmol) in acetone (50 mL) and the
mixture was heated under reflux for 2e4 h. The resulting precipi-
tate (KBr) was filtered off. The filtrate was evaporated under
vacuum and the residue was treated with water (50 mL), filtered
and purified according to Table 2.
Bis(4-trifluoromethylbenzyl)diselenide (2i): From 1i.
Bis(4-methylthiobenzyl)diselenide (2j): From 1j.
Bis(4-methylbenzyl)diselenide (2k): From 1k. The synthesis of
this compound has been previously reported by Tian et al. [40].
Bis(4-cyanobenzyl)diselenide (2l): From 1l. The synthesis of this
compound has been previously reported by Prabhu et al. [35]
Bis(3-cyanobenzyl)diselenide (2m): From 1m.
4-Nitrobenzylselenocyanate (1e): From 4-nitrobenzyl bromide.
The synthesis of this compound has been previously reported by
Maartmann-Moe et al. [31].
3-Nitrobenzylselenocyanate (1f): From 3-nitrobenzyl bromide.
The synthesis of this compound has been previously reported by
Grung et al. [32].
Bis(2-cyanobenzyl)diselenide (2n): From 1n.
Bisbenzyldiselenide (2o): From 1o. The synthesis of this
compound has been previously reported by Tian et al. [40].
Bis(2-methylnaphthyl)diselenide (2p): From 1p. The synthesis of
this compound has been previously reported by Tian et al. [40].
Bis[2-(4-nitrophenyl)ethyl]diselenide (2q): From 1q.
2-Nitrobenzylselenocyanate (1g): From 2-nitrobenzyl bromide.
The synthesis of this compound has been previously reported by
Grung et al. [32]
4.1.4. Procedure for preparation of bis(4-methoxybenzyl)diselenide
(2r)
4-Bromobenzylselenocyanate (1h): From 4-bromobenzyl bromide.
The synthesis of this compound has been previously reported by
Jacob et al. [33].
4-Trifluoromethylbenzylselenocyanate (1i): From 4-trifluoro-
methylbenzyl bromide.
4-Methylthiobenzylselenocyanate (1j): From 4-methylthiobenzyl
bromide.
4-Methylbenzylselenocyanate (1k): From 4-methylbenzyl
bromide. The synthesis of this compound has been previously re-
ported by Jacob et al. [34].
Hydrazine hydrate (11 mmol) was added to a mixture of NaOH
(15 mmol), gray selenium (10 mmol) and DMF (5 mL) under
a nitrogen atmosphere and the mixture was stirred for 6 h. A
solution of 1-chloromethyl-4-methoxybenzene (5 mmol) in DMF
(3 mL) was added dropwise to the reaction mixture and this was
stirred for 45 min. Water (150 mL) was added and the mixture was
extracted with dichloromethane (3 ꢄ 50 mL). The organic phase
was washed with a 6 N HCl (20 mL) and water (2 ꢄ 50 mL). The
organic phase was dried with anhydrous Na2SO4, filtered and the
dichloromethane was removed under vacuum. The residue was
purified by recrystallization from n-hexane. The synthesis of this
compound has been previously reported by Tian et al. [36].
4-Cyanobenzylselenocyanate (1l): From 4-bromomethylbenzo-
nitrile. The synthesis of this compound has been previously re-
ported by Prabhu et al. [35]
3-Cyanobenzylselenocyanate (1m): From 3-bromomethyl-
benzonitrile.
4.2. Biological evaluation
2-Cyanobenzylselenocyanate (1n): From 2-bromomethyl-
benzonitrile.
Benzylselenocyanate (1o): From benzyl bromide. The synthesis of
this compound has been previously reported by Krief et al. [25].
2-Selenocyanatemethylnaphthalene (1p): From 2-bromomethyl-
naphthalene.
4.2.1. Cells and culture conditions
L. infantum promastigotes (MCAN/ES/89/IPZ229/1/89) were
kindly provided by Dr. Colmenares (Centro de Investigaciones Bio-
lógicas, CIB, Madrid, Spain) and were grown in RPMI-1640 medium
(Sigma, St. Louis, MO, USA) supplemented with 10% heat inactivated
fetal calf serum (FCS), antibiotics and 25 mM HEPES, pH 7.2 at 26 ꢀC.
L. infantum axenic amastigotes were grown in M199 (Invitrogen,
Leiden, The Netherlands) medium supplemented with 10% heat
2-(4-Nitrophenyl)ethylselenocyanate (1q): From 4-nitrophenethyl
bromide.
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
4.1.3. General procedure for the synthesis of compounds 2aeq
NaBH4 (3 mmol) was added in small portions with caution to
a solution of the appropriate selenocyanate derivative 1aeq
(3 mmol) in absolute ethanol (40 mL). The mixture was stirred at
room temperature for 2 h. The solvents were removed under
vacuum by rotary evaporation and the residue was treated with
water. The mixture was extracted with dichloromethane
(3 ꢄ 50 mL). The organic phase was washed with water (3 ꢄ 50 mL)
and dried with anhydrous Na2SO4. The dichloromethane was
removed under vacuum and the residue was purified according to
the method shown in Table 2.
Bis(4-aminophenyl)diselenide (2a): From 1a. The synthesis of this
compound has been previously reported by Banks et al. [36].
Bis[4-(N,N-dimethylamino)phenyl]diselenide (2b): From 1b. The
synthesis of this compound has been previously reported by Pinto
et al. [37].
a
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 ꢀC. Jurkat cells were kindly
provided by Dr. Mollinedo [Centro de Investigación del Cáncer,
Instituto de Biología Molecular y Celular del Cáncer, Consejo Supe-
rior de Investigaciones Científicas (C.S.I.C.) e Universidad de Sala-
manca, Salamanca, Spain] and were grown in RPMI-1640 medium
(Sigma, St. Louis, MO, USA) supplemented with 10% heat inactivated
FCS, antibiotics and 10 mM HEPES, pH 7.2 at 37 ꢀC and 5% CO2. 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, antibiotics, 1 mM HEPES, 2 mM glutamine and
1 mM sodium pyruvate, pH 7.2 at 37 ꢀC and 5% CO2.
Bis(4-amino-3-carboxyphenyl)diselenide (2c): From 1c.
Bis(4-acetamido-3-carboxyphenyl)diselenide (2d): From 1d.
Bis(4-nitrobenzyl)diselenide (2e): From 1e. The synthesis of this
compound has been previously reported by Prabhu et al. [35] and
Degrand et al. [38].
Bis(3-nitrobenzyl)diselenide (2f): From 1f.
Bis(2-nitrobenzyl)diselenide (2g): From 1g.
Bis(4-bromobenzyl)diselenide (2h): From 1h. The synthesis of
this compound has been previously reported by Wang et al. [39].
4.2.2. Leishmanicidal activity and cytotoxicity assays
Drug treatment of promastigotes and amastigotes was per-
formed during the logarithmic growth phase at a concentration of
2 ꢄ106 parasites/mL at 26 ꢀC or 1 ꢄ106 parasites/mL at 37 ꢀC for
24 h, respectively. Drug treatment of Jurkat and THP-1 cells was
performed during the logarithmic growth phase at a concentration
of 4 ꢄ105 cells/mL at 37 ꢀC and 5% CO2 for 24 h. The percentage of
living cells was evaluated by flowcytometry by the propidium iodide
(PI) exclusion method [J.F. Alzate, A.A. Arias, D. Moreno-Mateos,

D. Plano et al. / European Journal of Medicinal Chemistry 46 (2011) 3315e3323
3323
A. Alvarez-Barrientos, A. Jiménez-Ruiz. Mitochondrial superoxide
mediates heat-induced apoptotic-like death in L. infantum. Mol.
Biochem. Parasitol. 152 (2007) 192e202]. Microscopic evaluation of
the cells was performed by direct observation of the parasites in 96
well plates with a Nikon eclipse TieS microscope either by phase
contrast or by fluorescence microscopy.
2009-13914-CO2-O2). We also thank Kilian Gutierrez for his
assistance in the cytotoxicity assays.
Appendix. Supplementary data
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.04.054.
4.2.3. Eukaryotic green fluorescent protein (eGFP) sequence cloning
and construct design
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Acknowledgments
The authors wish to express their gratitude to the University
of Navarra Research Plan (Plan de Investigación de la Universidad
de Navarra, PIUNA) and CAN Foundation for financial support for
the project. The authors also acknowledge financial support
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