Novel heteroaryl selenocyanates and diselenides as potent antileishmanial agents

Article abstract of DOI:10.1128/AAC.02529-15

A series of new selenocyanates and diselenides bearing interesting bioactive scaffolds (quinoline, quinoxaline, acridine, chromene, furane, isosazole, etc.) was synthesized, and their in vitro leishmanicidal activities against Leishmania infantum amastigotes along with their cytotoxicities in human THP-1 cells were determined. Interestingly, most tested compounds were active in the low micromolar range and led us to identify four lead compounds (1h, 2d, 2e, and 2f) with 50% effective dose (ED₅₀) values ranging from 0.45 to 1.27 μM and selectivity indexes of >25 for all of them, much higher than those observed for the reference drugs. These active derivatives were evaluated against infected macrophages, and in order to gain preliminary knowledge about their possible mechanism of action, the inhibition of trypanothione reductase (TryR) was measured. Among these novel structures, compounds 1h (3,5-dimethyl-4-isoxazolyl selenocyanate) and 2d [3,3′-(diselenodiyldimethanediyl)bis(2-bromothiophene)] exhibited good association between TryR inhibitory activity and antileishmanial potency, pointing to 1h, for its excellent theoretical ADME (absorption, distribution, metabolism, and excretion) properties, as the most promising lead molecule for leishmancidal drug design.

Full text of DOI:10.1128/AAC.02529-15

AAC Accepted Manuscript Posted Online 11 April 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.02529-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
2
Novel heteroaryl selenocyanates and diselenides as
potent antileishmanial agents
a,b,1
a,b,1
c
3
4
5
6
Ylenia Baquedano , Verónica Alcolea , Miguel Ángel Toro ,
c
b
a,b
Killian Jesús Gutiérrez , Paul Nguewa , María Font , Esther
a,b
b
c
Moreno , Socorro Espuelas , Antonio Jiménez-Ruiz , Juan Antonio
a,b
a,b
a,b,*
Palop , Daniel Plano , Carmen Sanmartín
a
b
7
8
9
0
1
Departamento de Química Orgánica y Farmacéutica, Instituto de Salud
Tropical, University of Navarra (ISTUN), Irunlarrea, 1. E-31008 Pamplona, Spain
c
Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá,
1
1
Carretera Madrid-Barcelona km 33,600. E-28871, Alcalá de Henares, Madrid,
Spain
1
1
2
3
1
14
15
16
17
18
19
20
21
22
23
24
25
These authors have contributed equally to this manuscript.
* Prof. Carmen Sanmartín
Department of Organic and Pharmaceutical 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
1

26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Abstract
A serie of new selenocyanates and diselenides bearing interesting bioactive
scaffolds (quinoline, quinoxaline, acridine, chromene, furane, isosazole…)
were synthesized and their in vitro leishmanicidal activity against L. infantum
amastigotes along with their cytotoxicity in human THP-1 cells were
determined. Interestingly, most tested compounds were active in the low
micromolar range and led us to identify four lead compounds (1h, 2d, 2e and
2f) with ED50 values ranging from 0.45 to 1.27 µM and selectivity indexes > 25
for all of them, much higher than those observed for the reference drugs.
These active derivatives were evaluated against infected macrophages and,
in order to gain a preliminary knowledge about their possible mechanism of
action, the inhibition of trypanothione reductase (TryR) was measured.
Among these novel structures, compounds 1h (3,5-dimethyl-4-isoxazolyl
selenocyanate)
and
2d
[3,3'-(diselenodiyldimethanediyl)bis(2-
bromothiophene)] exhibited good association between TryR inhibitory activity
and antileishmanial potency pointing 1h, for its excellent theoretical ADME
properties, as the most promising lead molecule for leishmancidal drug
design.
Keywords: Diselenide, selenocyanate, leishmanicidal, trypanothione
reductase
2

51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
Introduction
Leishmaniasis is an infectious poverty-associated disease caused by protozoan
parasites of the genus Leishmania. In fact, this term includes a wide spectrum of
vector-borne diseases with great epidemiological and clinical diversity. Even
though exact statistical data are lacking (1, 2), within the 350 million people that
live in endemic areas, approximately 12 million people get infected per year.
There are three major clinical types: cutaneous (CL), mucocutaneous (MCL), and
visceral leishmaniasis (VL; also known as kala-azar) which differ in their
immunopathologies and degrees of morbidity and mortality (3). Among the
different manifestations, VL is the most severe form with nearly 200,000 to
400,000 new cases, causing more than 20,000 deaths per year. Left untreated, it
is usually fatal within two years.
The efficacy of the different drugs available seems to vary according to the
Leishmania species and the current chemotherapy is far from being satisfactory.
Furthermore, they present several problems, including toxicity, many adverse
side-effects and high costs. The most relevant problem is related to the fact that
many of these drugs were developed many years ago, and currently, there are
resistant strains (4).
Since their discovery in the 1940s, the toxic pentavalent antimony [Sb(V)]
compounds have been the mainstay of treatment against all forms of
leishmaniasis through parenteral administration and their efficacy is progressively
decreasing owing to the development of resistance (5). For this reason, in the
last decades several drugs, such as amphotericin B and miltefosine (6),
3

7
7
7
7
8
8
8
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
paromomycin and pentamidine (7), sitamaquine (8) and edelfosine (9), have
been used in the treatment of leishmaniasis. Nevertheless, their high cost and
therapeutic complications limit their use. Nowadays, several other drugs based
on natural products have shown promising antileishmanial activity but, despite
the significant progress, an ideal drug is still awaited (10).
The development of new antiparasitic drugs has not been much of a priority for
the pharmaceutical industry because many of the parasitic diseases occur in
poor countries where the populations cannot afford to pay a high price for the
drugs. Thus, although important initiatives such as the Drugs for Neglected
Diseases Initiative (DNDi) are attracting more interest in these neglected
pathologies, an investment in drug development against parasitic diseases is
needed.
The incorporation of different functionalities bearing the Se atom (i.e.
methylseleno, selenocyanate, diselenide...) onto organic scaffolds can be
considered a promising rational design to achieve potent and selective cytotoxic
compounds (11). Several reports have shown a vast and miscellaneous types of
structures applying this approach, resulting in very promising antitumoral
compounds in pre-clinical models (12, 13). Recently, our research group has
been using this rational design in order to obtain new derivatives with potent and
selective antileishmanicidal activity. Continuing with these efforts, herein we have
designed novel Se compounds which gather two different chemical entities: the
selenium entity on its selenocyanate and diselenide forms; and different carbo-
and hetero-cyclic entities with proven leishmanicidal activity. Below in this
section, a brief description with several reported data that supports the selection
for each of these sub-units can be found.
1
00
4

101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
During the last years, various reports have shown that an increase in plasma
selenium levels has been recognized as a new defensive strategy against
Leishmania infection (14, 15). The choice of the chemical form for the selenium
derivatives can modulate the level of this element on the basis of several
metabolic routes (16). The mechanism of action for selenium is unknown though
some enzymatic pathways such as mitochondrial peroxiredoxins (17),
selenophosphate synthetases (18) or ascorbate peroxidases (19) could be
implicated. On the other hand, the incorporation of selenium into novel
nanomaterials has demonstrated effectiveness in the treatment of leishmaniasis
(20). We have reported (21-24) new selenium compounds with potent in vitro
antiparasitic activity against L. infantum and L. mayor, and selectivity indexes
higher than those observed for the reference drugs miltefosine, edelfosine or
paromomicyn. Additionally, some of them induced nitric oxide production and
alterations in gene expression profiling related to proliferation (PCNA), treatment
resistance (ABC-transporter and α-tubulin) and virulence (QDPR) (23). Among
the various antileishmanial scaffolds containing selenium earlier reported by us,
selenocyanate and diselenide showed promising activity against Leishmania
parasites (24).
We have payed special attention to quinoline, which constitutes the central
nucleus of sitamaquine (25, 26), acridine (27, 28), quinoxaline (29-31) and
coumarins (32, 33). On the other hand, nitrofuran compounds (34, 35), the most
relevant registrated as nifurtimox, and derivatives of the benzodioxol core (36)
have been selected. Besides, substituted five-membered heterocyclic rings such
as isoxazol (37) and thiophenyl (38) or pirrol (39) have been tested as
leishmanicidal agents. Finally, related to heterocycles derivatives, some fused
5

126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
aryl azo and triazo molecules have been described (34, 40). Furthermore, some
carbocycles, such as adamantane ring (41) and anthraquinone structure (42, 43).
Among the potential molecular targets for the treatment of leihshmaniasis,
trypanothione reductase (TryR) is considered an ideal enzyme since it is involved
in the unique thiol-based metabolism observed in the trypanosomatidae family
and is a validated target for the search of antitrypanosomatidae drugs. TryR
catalyzes the reduction of trypanothione disulfide to trypanothione (44).
Therefore, during the last years a great number of inhibitors of this key enzyme
have been reported (45-47). Based on the chemical analogy of sulphur and
selenium, we decided to explore the relevance of this trace element to generate
new TryR inhibitors.
In summary, and as a continuation of an ongoing program aiming to find new
structural leaders with potential leishmanicidal activity, we have constructed a
new class of selenoderivatives. They were designed by incorporating
selenocyanate or diselenide moieties onto other bioactive carbo or heterocycles
selected on the basis of the above mentioned findings. In this work, we present
the synthesis of twenty-three new Se compounds (Figure 1) and their
leishmanicidal activity against the amastigote form of L. infantum. In parallel, the
cytotoxicity of these newly synthesized molecules was assessed. Moreover,
leishmanicidal activity of the most active compounds was evaluated in L.
infantum-infected macrophages. Finally, in order to elucidate a preliminary
mechanism of action, their inhibitory activity against trypanothione reductase was
determined.
149
MATERIALS AND METHODS
6

150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
Chemistry. Melting points (mp) were determined with a Mettler FP82+FP80
1
13
apparatus (Greifense, Switzerland) and are not corrected. The H NMR and
C
TM
NMR spectra were recorded on a Bruker 400 Ultrashield
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 (0.040−0.063
mm) 1.09385.2500 (Merck KGaA, 64271 Darmstadt, Germany) was used for
®
Column Chromatography and Alugram 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), Sigma-Aldrich Química,
S.A. (Alcobendas, Madrid, Spain), Acros Organics (Janssen Pharmaceuticalaan
3a, 2440 Geel, België) and Lancaster (Bischheim-Strasbourg, France).
General procedure for the synthesis of compounds 1a−o. The synthesis of
compounds 1a−o was carried out according to the procedure described in the
literature (48-50) with few modifications. Briefly, KSeCN (4 mmol) was added to a
solution of the appropriate halyl derivative (4 mmol) in acetone (50 mL) and the
mixture was heated under reflux for 2−4 h. The resulting precipitate (KBr) was
filtered off. The filtrate was evaporated under vacuum and the residue was
treated with water (2×50 mL) and dried. The target compounds were obtained in
high purity.
7

174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
(Quinolin-8-yl)methyl selenocyanate (1a). From 8-bromomethylquinoline and
potassium selenocyanate. The compound was washed with ethyl ether (2×50
-
1
mL). Brown solid. Yield: 71.5%; mp: 49.5−50.5 ºC. IR (KBr) cm : 2138 (s, C≡N);
1
1593 (f, C=N). H NMR (400 MHz, DMSO-d
6
) δ: 4.89 (s, 2H, CH
); 7.97 (dd, 1H, H , J5-6 = 8.1 Hz, J5-7=1.6 Hz);
, J4-3 = 8.4 Hz, J4-2 = 2.2 Hz); 8.94 (dd, 1H, H
2.2 Hz). C NMR (100 MHz, DMSO-d ) δ: 29.5 (CH -Se); 106.1 (CN); 123.6 (C
127.3 (C ); 129.5 (C , C ); 131.7 (C ); 136.0 (C ); 138.1 (C ); 146.9 (C
(C10). MS (m/z, % abundance): 222 (58); 158 (35); 142 (100); 130 (13); 115 (18);
2
-Se); 7.58−7.63
(m, 2H, H
6
+ H
7
); 7.88 (m, 1H, H
3
5
8.44 (dd, 1H, H
4
2
, J2-3 = 4.3 Hz, J2-4
=
);
1
3
6
2
3
7
5
6
8
9
4
2
); 151.4
89 (8); 63 (6). Elemental Analysis for C11
53.44/53.35; H: 3.33/3.49; N: 11.33/11.06.
8 2
H N Se, Calcd/Found (%): C:
(Quinolin-2-yl)methyl selenocyanate (1b). From 2-chloromethylquinoline
hydrochloride and potassium selenocyanate. In first time, the quinoline
hydrochloride was treated with an aqueous solution of NaOH (1N) in
water/methanol (40:20) during 15 minutes in order to obtain 2-
chloromethylquinoline. The white powder obtained was washed with water (4×25
mL) and dried. The compound was washed with ethyl ether (2×50 mL). Brown
-
1
solid. Yield: 27.5%; mp: 83−84 ºC. IR (KBr) cm : 2142 (m, C≡N); 1591 (m, C=N).
1
H NMR (400 MHz, DMSO-d
7-8 = 11.0 Hz); 7.79 (d, 1H, H
11.0 Hz, J5-7 = J8-6 = 9.4 Hz); 8.41 (d, 1H, H
DMSO-d ) δ: 35.8 (CH -Se); 105.9 (CN); 122.6 (C
); 131.7 (C ); 138.2 (C ); 147.8 (C10); 158.0 (C ). MS (m/z, % abundance): 248
(25); 142 (100); 115 (35); 89 (8); 63 (5); 51 (4). Elemental Analysis for
6
) δ: 4.73 (s, 4H, 2CH
2
); 7.62 (t, 2H, H
6
+ H
7
, J6-5
=
J
3
, J3-4 = 9.4 Hz); 7.98 (dd, 2H, H
5
+ H
8
, J5-6 = J8-7
=
13
4
, J4-3 = 9.4 Hz). C NMR (100 MHz,
6
2
3
); 127.3 (C , C ); 129.0 (C ,
5
7
6
C
9
8
4
2
C
11
H
8
N
2
Se, Calcd/Found (%): C: 53.44/53.52; H: 3.33/3.62; N: 11.33/11.10.
8

199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
Acridin-9-ylmethyl selenocyanate (1c). From 9-(bromomethyl)acridine and
potassium selenocyanate. The compound was washed with ethyl ether (2×50
-
1
mL). Yellow solid. Yield: 92.3%; mp: 137−138 ºC. IR (KBr) cm : 2145 (m, C≡N);
1
1625 (d, C=N). H NMR (400 MHz, DMSO-d
6
) δ: 5.47 (s, 2H, CH
2
-Se); 7.69 (t,
2H, H
2H, H
NMR (100 MHz, DMSO-d
(C , C11); 127.0 (C , C10); 130.7 (C
). MS (m/z, % abundance): 204 (12); 193 (100); 177 (5); 165 (9); 87 (7); 63 (4).
2
+ H
7
, J2-1 = J7-8 = 9.3 Hz); 7.87 (t, 2H, H
, J4-3 = J5-6 = 9.1 Hz); 8.58 (d, 2H, H
) δ: 24.4 (CH
, C
3
+ H
6
, J3-4 = J6-5 = 9.1 Hz); 8.19 (d,
1
3
4
+ H
5
1
+ H
8
, J1-2 = J8-7 = 9.3 Hz).
C
6
2
-Se); 104.0 (CN); 124.9 (C12, C14); 125.3
1
2
4
8
); 132.2 (C , C ); 142.1 (C13); 149.4 (C ,
3
9
5
C
7
Elemental Analysis for C15
3.36/3.36; N: 9.42/9.13.
10 2
H N Se, Calcd/Found (%): C: 60.60/60.74; H:
Quinoxalin-2,3-diyldimethanediyl bisselenocyanate (1d). From 2,3-
bis(bromomethyl)quinoxaline and potassium selenocyanate. The compound was
washed with ethyl ether (2×50 mL). Brown solid. Yield: 46.5%; mp: 153−154 ºC.
-
1
1
IR (KBr) cm : 2153 (s, C≡N); 1608 (m, C=N). H NMR (400 MHz, DMSO-d
4.91 (s, 4H, CH -Se); 7.87−7.89 (m, 2H, H + H ); 8.06−8.08 (m, 2H, H + H
C NMR (100 MHz, DMSO-d ) δ: 32.3 (CH -Se); 105.0 (CN); 129.9 (C , C
132.2 (C , C ); 141.3 (C , C10); 151.0 (C , C
(100); 235 (44); 156 (72); 129 (27); 102 (21); 76 (20). Elemental Analysis for
Se , Calcd/Found (%): C: 39.34/39.16; H: 2.18/2.17; N: 15.30/15.06.
6
) δ:
2
6
7
5
8
).
);
1
3
6
2
6
9
7
8
5
2
3
). MS (m/z, % abundance): 262
C
12
H
8
N
4
2
(6,7-Dimethoxy-2-oxo-2H-chromen-4-yl)methyl selenocyanate (1e). From
6,7-dimethoxy-4-bromomethyl-2H-chromen-2-one and potassium selenocyanate.
The compound was washed with ethyl ether (2×50 mL). Yellow solid. Yield:
-
1
1
28.1%; mp: 197−199 ºC. IR (KBr) cm : 2150 (m, C≡N); 1709 (s, C=O). H NMR
(400 MHz, DMSO-d
6 3 3
) δ: 3.83 (s, 3H, OCH ); 3.87 (s, 3H, OCH ); 4.42 (s, 2H,
9

1
3
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
CH
MHz, DMSO-d
(CN); 107.8 (C
154.1 (C ); 161.5 (CO). MS (m/z, % abundance): 325 (59); 219 (73); 191 (100);
2
-Se); 6.33 (s, 1H, CH-CO); 7.10 (s, 1H, H
) δ: 29.5 (CH -Se); 56.3 (OCH
); 110.0 (C ); 114.6 (C ); 147.2 (C10); 150.3 (C
5
); 7.43 (s, 1H, H
); 58.7 (OCH ); 100.1 (C
); 152.2 (C
8
). C NMR (100
); 102.7
);
6
2
3
3
9
6
3
5
7
8
4
163 (12); 147 (25); 119 (7); 69 (8). Elemental Analysis for C13
Calcd/Found (%): C: 48.15/48.02; H: 3.40/3.40; N: 4.32/4.20.
4
H11NO Se,
(5-Nitrofuran-2-yl)methyl
selenocyanate
(1f).
From
5-nitro-2-
bromomethylfurane and potassium selenocyanate. The compound was washed
-
1
with ethyl ether (2×50 mL). Yellow solid. Yield: 42%; mp: 87-88 ºC. IR (KBr) cm :
1
2152 (m, C≡N). H NMR (400 MHz, DMSO-d
6
) δ: 4.45 (s, 2H, CH
2
-Se); 6.80 (d,
, J4-3 = 7.8 Hz). C NMR (100 MHz, DMSO-
-Se); 101.1 (CN); 109.4 (C ); 111.2 (C ); 150.0 (C ); 156.3 (C ). MS
Se,
13
1H, H
3 4
, J3-4 = 7.8 Hz); 7.70 (d, 1H, H
d
6
) 23.0 (CH
2
3
4
5
2
6 4 2 3
(m/z, % abundance): 126 (100); 113 (85). Elemental Analysis for C H N O
Calcd/Found (%): C: 31.17/31.28; H: 1.73/2.02; N: 12.12/11.72.
(6-Bromo-1,3-benzodioxol-5-yl)methyl selenocyanate (1g). From 5-bromo-6-
(bromomethyl)-1,3-benzodioxole and potassium selenocyanate. The compound
was washed with ethyl ether (2×50 mL). White solid. Yield: 78.6%; mp: 110−111
-
1
1
ºC. IR (KBr) cm : 2150 (s, C≡N); 1033 (s, C-Br). H NMR (400 MHz, DMSO-d
4.36 (s, 2H, CH -Se); 6.10 (s, 2H, O-CH -O); 7.11 (s. 1H, H ); 7.27 (s, 1H,
). C NMR (100 MHz, DMSO-d ) δ: 34.3 (CH -Se); 103.5 (O-CH -O); 105.9
(CN); 111.3 (C ); 113.8 (C ); 115.1 (C ); 130.6 (C ); 148.2 (C ); 149.4 (C ). MS
(m/z, % abundance): 213 (100); 157 (7); 75 (19); 50 (15). Elemental Analysis for
BrNO Se, Calcd/Found (%): C: 33.86/34.02; H: 1.88/2.05; N: 4.39/4.13.
6
) δ:
2
2
6
1
3
H
3
6
2
2
6
4
3
5
2
1
C
9
H
6
2
3,5-Dimethyl-4-isoxazolyl selenocyanate (1h). From 4-chloromethyl-3,5-
dimethylisoxazole and potassium selenocyanate. The brown oil obtained after
1
0

249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
washed with water was extracted with dichloromethane (3×50 mL). The organic
layer was dried with Na SO . The dichloromethane was removed under vacuum
and the residue was treated with ethyl ether (3×25 mL) and a clear brown powder
2
4
-
1
was obtained. Yield: 21.3%; mp: 69−70 ºC. IR (KBr) cm : 2143 (s, C≡N); 1628
1
(s, C=N). H NMR (400 MHz, DMSO-d
6
) δ: 2.23 (s, 3H, CH
-Se). C NMR (100 MHz, DMSO-d
-Se-); 105.9 (C ); 112.3 (CN); 159.9 (C
3
-C
) δ: 10.6 (C
); 168.2 (C
5
5
); 2.40 (s, 3H,
1
3
CH
3
-C
3
); 4.19 (s, 2H, CH
2
6
3
-CH
3
);
).
11.8 (C
5
-CH ); 21.4 (-CH
3
2
4
3
MS (m/z, % abundance): 156 (4); 110 (100); 68 (89); 52 (5); 43 (45). Elemental
7 8 2
Analysis for C H N OSe, Calcd/Found (%): C: 39.08/39.14; H: 3.75/3.88; N:
13.02/12.89.
(2-Bromothiophene-3-yl)methyl selenocyanate
(1i). From 2-bromo-3-
bromomethylthiophene and potassium selenocyanate. The compound was
washed with ethyl ether (2×50 mL). Brown solid. Yield: 88%; mp: 53−55 ºC. IR
-
1
1
(KBr) cm : 2148 (m, C≡N). H NMR (400 MHz, DMSO-d
Se); 7.09 (d, 1H, H , J1-2 = 5.7 Hz); 7.63 (d, 1H, H , J2-1 = 5.7 Hz). C NMR (100
MHz, DMSO-d ) δ: 26.5 (CH ); 105.3 (CN); 112.5 (C ); 128.4 (C ); 129.7 (C );
138.3 (C ). MS (m/z, % abundance): 281 (M , 3); 175 (100). Elemental Analysis
for C BrNSSe, Calcd/Found (%): C: 25.62/25.44; H: 1.42/1.38; N: 4.98/4.59.
6 2
) δ: 4.27 (s, 2H, CH -
1
3
2
1
6
2
2
5
4
+·
3
6
H
4
(2-Chlorothiophene-5-yl)methyl selenocyanate (1j). From 5-bromo-2-chloro-
methylthiophene and potassium selenocyanate. The brown oil obtained after
washed with water was extracted with ethyl ether (3×50 mL). The organic layer
was washed with water (3×50 mL) and dried with Na
2 4
SO . The ethyl ether was
removed under vacuum and a brown solid was obtained. Brown solid. Yield:
-
1
1
60%; mp: 37−39 ºC. IR (KBr) cm : 2149 (m, C≡N). H NMR (400 MHz, DMSO-
d
6
) δ: 4.52 (s, 2H, CH -Se); 6.98 (d, 1H, H3, 3-4 = 7.8 Hz); 7.00 (d, 1H, H
2
J
4
, J4-3 =
1
1

1
3
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
7.8 Hz). C NMR (100 MHz, DMSO-d
6 2 4
) δ: 28.6 (CH ); 106.5 (CN); 127.5 (C );
128.4 (C ); 129.3 (C ); 141.4 (C ). MS (m/z, % abundance): 131 (100); 95 (7); 87
3
2
5
(5); 69 (5); 45 (5). Elemental Analysis for C
30.44/30.04; H: 1.69/1.82; N: 5.92/5.80.
6 4
H ClNSSe, Calcd/Found (%): C:
4-(1H-Pyrrol-1-yl)benzyl selenocyanate (1k). From 1-[4-(bromomethyl)phenyl]-
1H-pyrrole and potassium selenocyanate. The solid obtained after the treatment
with water was solved in THF and the insoluble fraction was rejected. The THF
was removed under vacum and the residue was washed with water (3×25 mL)
and with hexane (3×25 mL). Brown solid. Yield: 73.3%; mp: 154−155 ºC. IR (KBr)
-
1
1
cm : 2145 (m, C≡N). H NMR (400 MHz, DMSO-d
(t, 2H, H + H , J3-2 = J4-5 = 8.5 Hz); 7.38 (t, 2H, H
(d, 2H, H3’ + H5’, J3’-2’ = J5’-6’ = 8.5 Hz); 7.58 (d, 2H, H2’ + H6’, J2’-3’ = J6’-5’ = 8.5 Hz.
6
2
) δ: 4.35 (s, 2H, CH -Se); 6.27
3
4
2
+ H , J2-3 = J5-4 = 8.5 Hz); 7.45
5
1
3
6 2 3 4
C NMR (100 MHz, DMSO-d ) δ: 33.1(-CH -Se-); 105.8 (CN); 111.5 (C , C );
119.8 (C
2
, C
5
); 120.1 (C2’, C6’); 131.1 (C3’, C5’); 136.1 (C4’); 140.2 (C1’). MS (m/z,
+
% abundance): 262 (M+1 , 3); 156 (100); 128 (17); 89 (8); 78 (4). Elemental
Analysis for C12
10 2
H N Se, Calcd/Found (%): C: 55.18/55.59; H: 3.86/4.01; N:
10.73/10.07.
1H-Benzotriazol-1-ylmethyl selenocyanate (1l). From 1-chloromethyl-1H-
benzotriazole and potassium selenocyanate. The orange oil obtained after
washed with water was extracted with dichloromethane (3×50 mL). The organic
2 4
phase was washed with water (3×20 mL) and dried over Na SO . The
dichloromethane was removed under vacuum and the residue was recrystallized
-
from ethanol to give an orange solid. Yield: 19.7%; mp: 158−160 ºC. IR (KBr) cm
1
1
: 2151 (m, C≡N). H NMR (400 MHz, DMSO-d
6
) δ: 6.51 (s, 2H, CH
2
-Se); 7.49 (t,
1H, H , J2-1 = 7.5 Hz, J2-3 = 7.6 Hz); 7.66 (t, 1H, H
2
3
, J3-4 = 7.5 Hz, J3-2 = 7.6 Hz);
1
2

1
3
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
8.02 (d, 1H, H
DMSO-d ) δ: 45.4 (CH
(C ); 132.9 (C
1
, J1-2 = 7.5 Hz); 8.12 (d, 1H, H
4
, J4-3 = 7.5 Hz). C NMR (100 MHz,
); 105.3 (CN); 112.3 (C ); 120.4 (C ); 125.6 (C ); 128.8
). MS (m/z, % abundance): 132 (62); 77 (100).
Se, Calcd/Found (%): C: 40.51/40.47; H:
6
2
8
5
6
7
); 146.41 (C
9 4
Elemental Analysis for C
2.53/2.75; N: 23.63/23.95.
8 6 4
H N
2-Adamant-1-yl-2-oxoethyl selenocyanate (1m). From 1-adamant-1-yl-2-
bromoethanone and potassium selenocyanate. The compound was washed with
-
1
ethyl ether (2×50 mL). Yellow solid. Yield: 77%; mp: 108−110 ºC. IR (KBr) cm :
1
2149 (s, C≡N); 1678 (s, C=O). H NMR (400 MHz, DMSO-d
6
) δ: 1.63−1.67 (m,
-C-CO); 1.99 (s, 3H, CH); 4.63 (s, 2H, CH
) δ: 27.1 (CH -Se); 29.0 (CH); 36.7 (CH -CH);
-C-CO); 47.1 (C-CO); 104.5 (CN); 210.2 (C=O). MS (m/z, %
abundance): 163 (4); 135 (100); 107 (8); 93 (17); 79 (18); 67 (7). Elemental
6H, CH
2
-CH); 1.80−1.81 (m, 6H, CH
2
2
-
13
Se). C NMR (100 MHz, DMSO-d
6
2
2
38.4 (CH
2
Analysis for C13
H17NOSe, Calcd/Found (%): C: 55.31/55.38; H: 6.03/6.19; N:
4.96/4.70.
(9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl selenocyanate (1n). From 2-
chloromethylanthraquinone and potassium selenocyanate. The compound was
washed with ethyl ether (2×50 mL). Yellow solid. Yield: 75.3%; mp: 168−169 ºC.
-
1
1
IR (KBr) cm : 2145 (m, C≡N); 1674 (s, C=O). H NMR (400 MHz, DMSO-d
6
) δ:
4.51 (s, 2H, CH
2H, H + H
); 8.22−8.25 (m, 4H, H
) δ: 33.3 (CH
10); 135.0 (C , C
256 (9); 221 (100); 207 (4); 165 (22); 139 (5); 76 (4); 63 (4). Elemental Analysis
NO Se, Calcd/Found (%): C: 58.89/58.74; H: 2.76/2.86; N: 4.29/4.25.
2
-Se); 7.90 (dd, 1H, H
3
, J3-4 = 8.1 Hz, J3-1 = 2.5 Hz); 7.93−7.96 (m,
1
3
1
4
5
+ H
6
+ H
, C
); 183.4 (CO). MS (m/z, % abundance):
7
+ H
8
). C NMR (100 MHz, DMSO-
d
6
2
-Se); 106.5 (CN); 128.5 (C
, C12, C14); 146.9 (C
1
4
, C
8
, C11); 133.2 (C ); 134.1 (C
3
9
,
C
5
7
2
for C16
H
9
2
1
3

324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
(1,3-Dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl selenocyanate (1o). From N-
bromomethylphthalimide and potassium selenocyanate. The oil obtained after
washed with water was extracted with dichloromethane (3×50 mL). The organic
2 4
phase was washed with water (3×20 mL) and dried over Na SO . The
dichloromethane was removed under vacuum and the residue was recrystallized
from ethanol to give a white-pink solid. Yield: 50.4%; mp: 162−164 ºC. IR (KBr)
-
1
1
cm : 2155 (m, C≡N); 1778 (CO); 1718 (CO). H NMR (400 MHz, DMSO-d
6
) δ:
). C NMR (100 MHz,
); 132.2 (C ); 136.0 (C ); 166.8
). MS (m/z, % abundance): 160 (100); 104 (22). Elemental Analysis for
10 6 2 2
C H N O Se, Calcd/Found (%): C: 45.29/44.93; H: 2.26/2.49; N: 10.57/10.37.
1
3
5.28 (s, 2H, CH
DMSO-d ) δ: 35.0 (CH
(C
2
); 7.89−7.98 (m, 4H, H
3 4 5 6
+ H + H + H
6
2
); 105.2 (CN); 124.5 (C
4
3
5
2
General procedure for the synthesis of compounds 2a−g. The appropriate
selenocyanate derivative (3 mmol) was solved in absolute ethanol (40 mL) and
NaBH
4
(6.2 mmol) was added in small portions with caution to the solution. For
CN (6.2 mmol) was used. The mixture was
the obtention of compound 2g, NaBH
3
stirred at room temperature for 2 h. The solvents were removed under vacuum by
rotary evaporation and the residue was treated with water (50 mL) and purified in
order to obtain the target compounds.
8,8'-(Diselenodiyldimethanediyl)diquinoline (2a). From (quinolin-8-yl)methyl
selenocyanate (1a) and sodium borohydride. The resultant solid was washed
with ethyl ether (3×50 mL) and recystallized from ethanol to give a yellow solid.
-
1
1
Yield: 60.3%; mp: 103−104 ºC. IR (KBr) cm : 1591 (s, C=C); 790 (s, Se-Se). H
NMR (400 MHz, DMSO-d
6
) δ: 4.58 (s, 4H, 2 CH
2
-Se); 7.43−7.48 (m, 4H, H
6
+ H
7
,
,
H
6’ + H7’); 7.57 (dd, 2H, H
3
, H3’, J3-4 = 8.1 Hz, J3-2 = 4.6 Hz); 7.89 (dd, 2H, H , H5’
5
4
J5-6 = 8.1 Hz, J5-7 = 2.5 Hz); 8.38 (dd, 2H, H , H4’, J4-3 = 8.1 Hz, J4-2 = 2.5 Hz); 8.97
1
4

1
3
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
(dd, 2H, H
30.2 (CH -Se); 122.5 (C
8’); 137.9 (C , C9’); 138.4 (C
2
, H2’, J2-3= 4.6 Hz, J2-4 = 2.5 Hz). C NMR (100 MHz, DMSO-d
6
) δ:
, C5’, C6’); 130.7 (C
, C2’); 150.1 (C10, C10’). MS (m/z, %
2
3
, C3’); 127.1 (C
7
, C7’); 128.0 (C
5
, C
6
8
,
C
9
4
, C4’); 146.2 (C
2
+·
abundance): 442 (M , 5); 222 (75); 142 (100); 130 (10); 115 (17); 89 (7); 63 (5).
Elemental Analysis for C20
3.62/4.00; N: 6.33/6.10.
16 2 2
H N Se , Calcd/Found (%): C: 54.30/54.80; H:
9,9'-(Diselenodiyldimethanediyl)diacridine (2b). From acridin-9-ylmethyl
selenocyanate (1c) and sodium borohydride. The resultant solid was washed
with ethyl ether (3×50 mL) and recystallized from ethanol to give an orange solid.
-
1
1
Yield: 45%; mp: 108−109 ºC. IR (KBr) cm : 1552 (s, C=C); 752 (s, Se-Se). H
NMR (400 MHz, DMSO-d
6
) δ: 5.22 (s, 4H, CH
2-3 = J7-6 = 9.0 Hz); 7.84 (t, 4H, H + H , J3-2 = J6-7 = 9.0 Hz); 8.14 (d, 4H, H
4-3 = J5-6 = 9.0 Hz); 8.41 (d, 4H, H + H
) δ: 26.3 (CH -Se); 123.1 (C12, C14, C12’, C14’); 125.0 (C
, C10, C2’, C10’); 130.0 (C , C , C4’, C8’); 133.4 (C , C , C3’, C9’); 141.8
(C13, C13’); 149.9 (C , C5’, C7’). MS (m/z, % abundance): 204 (100); 165 (47);
2
-Se, CH2’-Se); 7.63 (t, 4H, H
2
+ H
7
,
J
J
3
6
4
+ H
5
,
13
1
8
, J1-2 = J8-7 = 9.1 Hz). C NMR (100 MHz,
DMSO-d
6
2
1
, C11, C1’, C11’);
128.6 (C
2
4
8
3
9
5
, C
7
63 (6). Elemental Analysis for C28
3.69/4.03; N: 5.16/5.12.
20 2 2
H N Se , Calcd/Found (%): C: 61.99/61.62; H:
5,5'-(Diselenodiyldimethanediyl)bis(6-bromo-1,3-benzodioxole) (2c). From
(6-bromo-1,3-benzodioxol-5-yl)methyl selenocyanate (1g) and sodium
borohydride. The resultant solid was washed with ethyl ether (3×50 mL) and
recystallized from ethanol to give a yellow solid. Yield: 70.3%; mp: 90−91 ºC. IR
-
1
1
(KBr) cm : 766 (s, Se-Se). H NMR (400 MHz, DMSO-d
CH2’-Se); 6.00 (s, 4H, O-CH -O, O-CH2’-O); 6.77 (s, 2H, H
3’). C NMR (100 MHz, DMSO-d ) δ: 33.3 (CH -Se); 103.1 (O-CH
2
6
) δ: 4.03 (s, 4H, CH
, H6’); 7.01 (s, 2H, H
-O); 111.6
2
-Se,
2
6
3
,
1
3
H
6
2
1
5

374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
(C
(C
6
, C6’); 113.7 (C4’, C4’); 115.2 (C
3 5 2
, C3’); 132.9 (C , C5’); 147.0 (C , C2’); 148.8
1
, C1’). MS (m/z, % abundance): 213 (100); 157 (9); 135 (9); 75 (10); 50 (5).
Elemental Analysis for C16
2 4 2
H12Br O Se , Calcd/Found (%): C: 32.76/32.99; H:
2.04/2.02.
3,3'-(Diselenodiyldimethanediyl)bis(2-bromothiophene) (2d). From (2-
bromothiophene-3-yl)methyl selenocyanate (1i) and sodium borohydride. The
mixture was extracted with ethyl ether (3×50 mL). The organic phase was
washed with water (3×50 mL) and dried with anhydrous Na
2 4
SO . The ethyl ether
was removed under vacuum and a yellow powder was obtained. Yield: 29%; mp:
-
1
1
49−50 ºC. IR (KBr) cm : 3099 (w, C-H); 722 (s, Se-Se). H NMR (400 MHz,
DMSO-d ) δ: 4.04 (s, 4H, CH -Se, CH2’-Se); 7.00 (d, 2H, H + H2’, J1-2 = 5.6 Hz);
7.58 (d, 2H, H + H1’, J2-1 = 5.6 Hz). C NMR (100 MHz, DMSO-d ) δ 26.5 (CH
Se); 111.1 (C2, 2’); 128.4 (C4’, C4’); 130.6 (C , C5’); 140.0 (C , C3’). MS (m/z, %
abundance): 510 (M , 3); 175 (100); 96 (19); 69 (9); 45 (8). Elemental Analysis
Br Se , Calcd/Found (%): C: 23.55/23.49; H: 1.58/1.52.
1,1'-(Diselenodiyldimethanediyl)bis(1H-benzotriazole) (2e).
6
2
2
13
1
6
2
-
C
5
3
+.
for C10
H
8
2
S
2
2
From
1H-
benzotriazol-1-ylmethyl selenocyanate (1l) and sodium borohydride. The mixture
was extracted with dichloromethane (3×50 mL). The organic phase was washed
with water (3×50 mL) and dried with anhydrous Na
2 4
SO . The dichloromethane
was removed under vacuum and a white powder was obtained. Yield: 25%; mp:
-
1
1
197−199 ºC. IR (KBr) cm : 744 (s, Se-Se). H NMR (400 MHz, DMSO-d
(s, 4H, N-CH -Se, N-CH2’-Se); 7.44 (t, 2H, H + H3’, J3-2 = 8.2 Hz, J3-4 = 8.2 Hz);
7.57 (t, 2H, H + H2’, J2-1 = 8.2 Hz, J2-3 = 8.2 Hz); 7.87 (d, 2H, H + H4’, J3-4 = 8.2
Hz); 8.07 (d, 2H, H 1-2 = 8.2 Hz). C NMR (100 MHz, DMSO-d
(CH -Se); 112.2 (C , C14, C12’, C14’); 120.2 (C ,); 125.3 (C ); 128.3 (C
6
) δ: 6.19
2
3
2
4
13
1
+ H1’,
J
6
) δ: 43.6
2
8
5
6
7
); 133.0
1
6

399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
(C
9
); 146 (C
4
). MS (m/z, % abundance): 132 (86); 77 (100). Elemental Analysis
for C14
H
12
N
6
Se ·H O, Calcd/Found (%): C: 38.80/38.40; H: 2.77/2.74; N:
2
2
19.61/19.39.
2,2'-(Diselenodiyldimethanediyl)di(9,10-dihydroanthracene-9,10-dione) (2f).
From (9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl selenocyanate (1n) and
sodium borohydride. The resultant solid was washed with ethyl ether (3×50 mL)
and recystallized from ethanol to give a yellow solid. Yield: 52%; mp: 186−187
-
1
1
ºC. IR (KBr) cm : 1666 (CO); 710 (m, Se-Se). H NMR (400 MHz, DMSO-d
6
) δ:
4.21 (s, 4H, CH
4H, H + H , H5’ + H8’); 7.95 (s, 2H, H
8.09−8.11 (m, 4H, H
(CH -Se); 127.6 (C , C
9’, C10’); 135.3 (C , C
MS (m/z, % abundance): 177 (100); 149 (13); 96 (22); 69 (17); 51 (8). Elemental
Analysis for C30 Se ·½H O, Calcd/Found (%): C: 59.11/59.12; H: 3.12/3.44.
2
-Se, CH2’-Se); 7.70 (d, 2H, H
3
, H3’, J3-4 = 8.1 Hz); 7.86−7.89 (m,
5
8
1
, H1’); 8.06 (d, 2H, H
4
, H4’, J4-3 = 8.1 Hz);
) δ: 31.4
, C3’); 134.1 (C
, C12, C14, C5’, C7’, C12’, C14’); 148.0 (C , C2’); 183.2 (CO).
13
6
+ H
, C
7
, H6’ + H7’). C NMR (100 MHz, DMSO-d
6
2
1
4
8
, C11, C1’, C4’, C8’, C11’); 133.2 (C
3
9
, C10,
C
5
7
2
H
18
O
4
2
2
2,2'-(Diselenodiyldimethanediyl)bis(1H-isoindole-1,3(2H)-dione) (2g). From
(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl selenocyanate and sodium
cyaneborohydride. A white solid was obtained. Yield: 28%; mp: 162−164 ºC. IR
-
1
1
(KBr) cm : 1774 and 1715 (vs, C=O); 719 (m, Se-Se). H NMR (400 MHz,
DMSO-d ) δ: 5.07 (s, 4H, N-CH -Se, N-CH2’-Se); 7.86 (s, 8H, H
+ H + H6’). C NMR (100 MHz, DMSO-d ) δ: 34.0 (CH
(C ); 132.3 (C ); 135.6 (C ); 167.3 (CO). MS (m/z, % abundance): 478 (M , 2);
6
2
3 4
+ H3’ + H + H4’
1
3
5
+ H5’ + H
6
6
2
-Se); 124.2
+·
4
3
5
160 (100). Elemental Analysis for C18
12 2 4 2
H N O Se , Calcd/Found (%): C:
45.19/45.25; H: 2.51/2.70; N: 5.86/5.64.
1
7

423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
2,2'-(Selenodiyldimethanediyl)bis(1H-isoindole-1,3(2H)-dione) (3). From (1,3-
dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl selenocyanate (1o) (0.75 mmol) and
-
1
sodium borohydride (0.75 mmol). Yield: 47%; mp: 230−232ºC. IR (KBr) cm :
1
1772 and 1718 (vs, C=O). H NMR (400 MHz, DMSO-d
6
) δ: 5.13 (s, 4H, N-CH
2
-
1
3
Se, N-CH2’-Se); 7.87−7.89 (m, 8H, H
NMR (100 MHz, DMSO-d ) δ: 31.5 (CH
167.8 (CO). MS (m/z, % abundance): 400 (M+1 , 2); 160 (100). Elemental
3
+ H3’ + H
4
+ H4’ + H
5
+ H5’ + H
6
+ H6’). C
6
2
-Se); 124.2 (C
4
); 132.5 (C
3
); 135.6 (C );
5
+
Analysis for C18
7.02/7.07.
12 2 4
H N O Se, Calcd/Found (%): C: 54.13/54.13; H: 3.00/2.79; N:
Biological evaluation. (i) Cells and culture conditions. L. infantum
promastigotes (MCAN/ES/ 89/IPZ229/1/89) were grown in RPMI-1640 medium
(Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% heat-inactivated
o
fetal calf serum (FCS), antibiotics, and 25 mm HEPES (pH 7.2) at 26 C.
6
L. infantum axenic amastigotes were obtained by incubation of 4.5x10 late
logarithmic promastigotes in 5 mL of M199 medium (Invitrogen, Leiden, The
-
1
Netherlands) supplemented with 10% heat-inactivated FCS, 1 gL β-alanine, 100
-
1
-1
-1
-1
mgL L-asparagine, 200 mgL sucrose, 50 mgL sodium pyruvate, 320 mgL
-
1
-1
-1
malic acid, 40 mgL fumaric acid, 70 mgL succinic acid, 200 mgL
α
-
1
-1
-1
ketoglutaric acid, 300 mgL citric acid, 1.1 gL sodium bicarbonate, 5 gL MES,
-
1
-1
o
0.4 mgL hemin, and 10 mgL gentamicine, pH 5.4 at 37 C. After 48 hours of
incubation, all parasites had a rounded morphology without flagellum and divided
during several weeks under the described conditions.
THP-1 cells were grown in RPMI 1640 medium (Gibco, Leiden, The Netherlands)
supplemented with 10% heat inactivated FCS, antibiotics, 1 mM HEPES, 2 mM
o
glutamine and 1 mM sodium pyruvate, pH 7.2 at 37 C and 5% CO
2
.
1
8

448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
(ii) Leishmanicidal activity and cytotoxicity assays. Drug treatment of
amastigotes was performed during the logarithmic growth phase at
a
6
6
concentration of 2×10 parasites/mL at 26 ºC or 1×10 parasites/mL at 37 ºC for
24 h, respectively. Drug treatment of THP-1 cells was performed during the
5
logarithmic growth phase at a concentration of 4×10 cells/mL at 37 ºC and 5%
2
CO for 24 h. The percentage of living cells was evaluated by flow cytometry by
the propidium iodide (PI) exclusion method (51). Drug concentrations ranged
from 0.2 µM to 25 µM.
(iii) Leishmania infection assay. Human THP-1 monocytic cells were seeded at
5
1.2 x 10 cells/mL in 24 multidishes plates (Nunc, Roskilde, Denmark) and
differentiated to macrophages for 24 h in 1mL of RPMI-1640 medium containing
10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO,
6
USA). Medium culture was removed and 1.2×10 Leishmania amastigotes in 1
mL of THP-1 medium were added to each well. 4 h later all medium with non-
infective amastigotes was removed, washed 3 times with 1X phosphate buffered
saline (1X PBS) and replaced with new THP-1 medium and corresponding
treatment. After 48 h treatment, medium was removed; THP-1 cells were washed
TM
3 times with 1X PBS and detached with TrypLE Express (Invitrogen, Leiden,
The Netherlands) according to the manufacturer’s indications. Infection
quantization was measured by flow cytometry. Drug concentrations ranged from
0.2 µM to 25 µM.
(iv) Trypanothione reductase assay. Oxidoreductase activity was determined
according to the method described by Toro et al. (52). Briefly, reactions were
carried out at 26ºC in 250 µL of 40 mM pH 8.0 HEPES buffer containing 1 mM
EDTA, 150 µM NADPH, 30 µM NADP+, 25 µM DTNB, 1 µM T[S]2, 0.02%
1
9

473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
glycerol, 1.5% DMSO and 7 nM of recombinant Li-TryR. 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-lineal regression model with the Grafit6 software
(Erithacus, Horley, Surrey, UK).
Drug-likeness parameters. The drug-likeness and drug score values along with
the TPSA values and the properties described in the Lipinski’s Rule of Five
[molecular weight (MW) ≤500 Da, log P ≤ 5, H-bond donors (HBD) ≤5 and H-
bond acceptors (HBA) ≤10] were calculated using the online available Osiris (53)
and Molinspiration property calculation programs (54), respectively. Topological
polar surface area was used to calculate the percentage of absorption (%ABS)
according to the equation: %ABS = 109 − [0.345 × TPSA] (55).
RESULTS
Chemistry. The synthetic approaches adopted to obtain the target compounds
are depicted in Figure 2. The selenocyanate derivatives (compounds 1a−o) were
obtained in variable yields (27–92%) by reaction between the commercially
available haloalkyl carbo or heterocyclic reactives with potassium selenocyanate
in a molar ratio 1.1 in acetone under reflux during 3−4 h (24). The subsequent
reduction of compounds 1a−o with sodium borohydride or sodium
cyanoborohydride in ethanol afforded derivatives 2a−f and 2g, respectively (24)
in yields ranging from 25 to 70%. Unfortunately, for some selenocyanates (1a,
1d, 1e, 1f, 1h, 1i, 1k and 1m) several difficulties were found and this procedure
failed to afford the expected products. This prompted us to seek alternative
routes to prepare the corresponding diselenides. Surprisingly, modification of the
2
0

498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
reaction conditions (temperature, solvents or molar ratio) resulted in
decomposition of the starting materials by rupture of the bond between
heterocycle and methylene.This hypothesis was confirmed by the disappearance
1
of the signal corresponding to the methylene group in H NMR. Additionally, an
undesired mixture of side compounds was identified in TLC. The alternative
strategy employing 100% hydrazine hydrate and sodium hydroxide in DMF to
reduce elemental selenium and generate sodium diselenide followed by reaction
with the corresponding haloalkyl reactives (56) did not allow the synthesis of the
corresponding diselenides. These results can be explained by the greater steric
hindrance so as the quick degradation of the starting materials in the reduction
process.
Finally, and contrary to our expectations, the reduction of 1o with NaBH
ethanol yielded compound 3, an unexpected compound instead of the
corresponding diselenide that was obtained by reaction with NaBH CN.
4
in
3
All of the compounds prepared during the course of these investigations are
stable and their purity was assessed by TLC and elemental analyses and their
1
13
structures were identified from spectroscopic data. IR, H NMR, C NMR, mass
spectrometry and elementary analysis methods were used for structure
elucidation (Figure 2).
-
The IR spectra of compounds 1a−o illustrate sharp peaks around 2138−2155 cm
1
due to CN group. Derivatives 2a−g showed multiple bands in the range
-
1
710−790 cm attributable to the Se-Se group and lacked the CN band,
confirming the reduction. In NMR all the signals were fully consistent with
1
13
proposed structures. Copies of the registered H- and C-NMR spectra for the
selected compounds (1h, 2d and 2e) can be found as supplementary material.
2
1

523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
Biological evaluation. (i) Activity in amastigotes and cytotoxic activity in
human cells. Compounds 1a−o, 2a−g and 3 were tested for their antiprotozoal
activity against the pathogenic Leishmania infantum amastigotes using
miltefosine and edelfosine as standard drugs according to a previously described
procedure (57). Although most of the studies on the in vitro biological activity of
new compounds against Leishmania spp. are performed on promastigote forms,
this assay must be considered as preliminary because this stage of parasite is
significantly more susceptible to drug-induced effects than the amastigote form.
Moreover, promastigotes are not the developed forms of the parasite in
vertebrate hosts so the evaluations made with them are merely indicative of the
potential leishmanicidal activity of the compounds tested. Accordingly, because
amastigotes are responsible for all clinical manifestations in humans, the
intracellular amastigote model has been cited as the golden standard for in vitro
Leishmania drug discovery research. Taking this into account, all the analyses
were carried out in the amastigote form with a minimum of three independent
experiments and the results are expressed as ED50 values. In addition, for a
compound to be a candidate for antileishmanial drug, it is required both high
leishmanicidal activity and low cytotoxicity. Cytotoxicity on THP-1 cell line was
evaluated for all compounds in order to identify drugs with low toxicity in human
cells and as a prelude to selecting drugs for in vitro assay on the relevant clinical
Leishmania amastigote stage. The selectivity index (SI) of the compounds is
expressed by the ratio between cytotoxicity (ED50 value on THP-1 cells) and
activity (ED50 value on L. infantum amastigotes).
Table 1 shows the ED50 values obtained after 24 h of exposure to the
compounds in L. infantum axenic amastigotes. Values for the reference drugs
2
2

548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
miltefosine and edelfosine are included in all cases for comparison. Biological
data evidenced that most of the screened compounds (fifteen out of twenty-three)
showed high bioactivity (ED50 ≤ 2.53 µM) against L. infantum, being more potent
than miltefosine (ED50 = 2.84 µM). In addition, under our experimental conditions
seven compounds (1d, 1e, 1h, 1m, 1n, 2e and 2f) displayed comparable or
higher in vitro potency than edelfosine (ED50 = 0.82 µM).
Different authors have claimed that compounds having SI values greater than 20
can be considered ideal candidates for further development as leishmanicidal
drugs (58). However, in this study and with rigorous criteria, we have considered
the SI threshold > 25 to further analyse their activity in amastigote-infected THP-
1 cells. This requirement is satisfied by compounds 1h, 2d, 2e and 2f considering
them as the lead ones in this series due to their excellent biological behaviour.
(ii) Leishmanicidal activity in infected macrophages. According to their
activity and selectivity, four compounds (1h, 2d, 2e and 2f) were advanced for
testing leishmanicidal activity in amastigote-infected THP-1 cells. Compound 2f
(ED50 = 0.68 µM, SI = 36.8) was not further tested due to the reproducibility
issues showed by this derivative pertaining to its lack of solubility in the assay
conditions. The ED50 values for the other selected derivatives (1h, 2d and 2e)
were calculated and summarized in Table 2. The potency of the analogues was
compared with edelfosine, a current antileishmanial agent (ED50 = 3.1 ± 0.1 µM).
These compounds reduced the parasite load of the cells exhibiting ED50 values
of 23.2, 14.0 and 14.4 µM respectively, the members of diselenide family being
the most potent compounds.
(iii) Inhibition of L. infantum trypanothione reductase activity. In an attempt
to investigate a possible mechanism of action, the ability to inhibit the
2
3

573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
trypanothione reductase activity for the most active compounds were first
screened at six different concentrations between 0.1 and 75 μM. Mepacrine, a
well known TryR inhibitor, was used as positive control (59) and DMSO as
vehicle. The IC50 values obtained are gathered in Table 3.
The compounds 1h and 2d were able to inhibit TryR with IC50 values of 0.46 and
6.85 μM, respectively. It is remarkably, that derivative 1h was 37-fold more active
than the positive control. 1h and 2d exhibited good association between TryR
inhibitory activity and antileishmanial potency (intracellular forms of the parasite).
The results for 2e, that did not show inhibitory activity, suggest an alternative
mechanism of action for its potent leishmanicidal activity. Compound 1h, as well
as compound 2d, can be considered as promising antileishmanial lead
candidates because they show a strong inhibitory activity against axenic
amastigotes (IC50 values of 0.73 and 1.27 µM), excellent SI (29.9 and 25.8) and a
marked inhibitory activity against TryR.
Drug-likeness properties. Employing the Molinspiration (54) and Osiris (53)
software the selected compounds (1h, 2d and 2e) were subjected to the
Lipinski’s rule of five analyses (drug-likeness), which helps to predict and explain
the biological behavior of small molecules. This preliminary analysis allows
prediction of the physicochemical properties related to their absorption and
bioavailability. We have found that the derivatives 1h and 2e show no violations
of Lipinski’s Rule of Five (Table 4). Among leishmanicidal drugs available on the
market, only miltefosine does not violate Lipinski’s Rule of Five; all other drugs
have, at least, 1 violation (edelfosine). It has been well established that optimal
lipophilicity range along with low logP (< 5) and low topological polar surface area
(TPSA) are the major driving forces that lead to good absorption, including
2
4

598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
622
intestinal absorption, bioavailability, Caco-2 permeability, and blood-brain barrier
2
penetration. Molecules with TPSA < 140 Å are indicative of excellent
bioavailability (60). According to the theoretical study carried out by De Toledo et
al. (61), the TPSA of most leishmanicidal drugs currently on the market is higher
than this limit, which probably restricts their absorption and bioavailability. The
logP and TPSA values for compounds 1h, 2d and 2e range from 0.75 to 4.68 and
0 to 61.44 respectively, suggesting that these compounds are potentially able to
cross cell membranes in a permeation process, which could explain the ability to
reach the amastigotes inside the phagolysosome.
The drug score uses and relates other molecular parameters, such as drug-
likeness, logP, molecular weight, and toxicity risks, being considered a
convenient value that may be used to judge the overall potential of a compound
to become a drug. A value of 0.5 or more is indicative of a promising lead for
future development of a safe and efficient drug (62). Compound 1h possess the
maximum drug-score value (0.5) for the selected compounds whereas edelfosine
presented a drug score value 0.3.
DISCUSSION
There are many available antileishmanial agents, but the drug of choice is still
awaited by several limitations of current drugs such as high cost, poor
compliance, drug resistance, low efficacy and poor safety. The high prevalence
and severity of this illness justify the urgency for the discovery of new drugs. In
the last two decades, several “interesting drug targets” have been proposed
including many proteins and enzymes that differ from mammalian counterpart
which can interfere with the redox system. Among the promising targets that
scientific community considers for the design of useful therapies, enzymes
2
5

623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
(trypanothione reductase, proteinases, superoxide dismutase, dihydrofolate
reductase, metacaspase, topoisomerase, kinases, sirtuins …) are one of the
most representatives. In this context, we notice that selenium plays an important
role in medicinal chemistry particularly as antioxidant, antitumoral,
chemopreventive or antiparasitic agents. Thus, we have described here the
synthesis and leishmanicidal activity of novel selenocyanate and diselenide
compounds.
For the novel selenoderivatives presented in this work it seems to exist a
tendency suggesting that analogues with the diselenide unit were more active
than those with the selenocyanate moiety (1a vs 2a, 1c vs 2b, 1g vs 2c, 1i vs 2d,
1l vs 2e and 1o vs 2g) against L. infantum amastigotes. Regarding the selectivity
index, the addition of the diselenide scaffold clearly improved the selectivity, for
example in compounds 1g/2c, 1i/2d, 1l/2e, 1n/2f. In general, it was observed
that tricyclic nitrogenated rings such as acridine (1c and 2b) are detrimental to
the biological activity and selectivity compared with bicyclic nitrogenated rings
(1b, 1d, 1l, 1o, 2e and 2g). Furthermore, no regular order of decrease or
increase in the activity among the rest derivatives can be concluded.
Taking into account the results related to activity and selectivity and considering
our exigent criteria for both parameters (ED50 < 2.5 µM and SI > 25), four
derivatives, one selenocyanate, 1h, and two diselenides (2d and 2e) were
selected for further studies. Despite compound 2f fullfiled these criteria, it could
not be tested due to solubility problems. Then, when we performed intracellular
form tests these derivatives did not improve the activity compared to edelfosine.
However, their lack of toxicity against THP-1 cells (Table 1) represents a
remarkable advantage over the reference drug.
2
6

648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
669
670
671
672
We hypothesized that TryR inhibition may be related with the leishmanicidal
activity observed for selenoderivatives. The results for in vitro assays revealed
that compounds 1h and 2d exhibited a good correlation between leishmanicidal
activity and TryR inhibition confirming our previous proposal. On the other hand,
2e did not show inhibitory activity, suggesting that not only this enzyme is
involved in its potent leishmanicidal activity, but also other mechanisms can be
implicated.
Finally, in silico prediction studies were performed in order to determine the drug-
like properties for the lead compounds. Considering these properties, derivatives
1h and 2e showed to meet the Lipinski’s Rule of Five, indicating favorable
properties for drug development. The in silico toxicity profile, drug-likeness, and
drug-score (0.5) data for compound 1h make it a promising leader for future
development of safer and more efficient leishmanicidal drugs.
In conclusion, the present study reports the synthesis of new selenocyanates and
diselenides bearing interesting bioactive scaffolds (quinoline, quinoxaline,
acridine, chromene, furane, isosazole...) and their in vitro leishmanicidal activity
against L. infantum amastigotes along with their cytotoxicity in THP-1 cells.
Fifteen of such compounds exhibited better potency against axenic amastigotes
than the standard drug miltefosine. Based on its antiparasitic activity and low
toxicity in THP-1 cells, compounds 1h, 2d, 2e and 2f were identified as the best
candidates for further studies in infected macrophages. Although their potency
against intracellular amastigotes is lower than that observed for the reference
drug, these compounds combined a potent leishmanicidal activity with excellent
selectivity index (>25) resulting in promising therapeutic utility. In order to get
further insight into their putative mechanism of action, their activity against L.
2
7

673
674
675
676
677
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
infantum TryR was determined. A clear correlation between enzyme inhibition
and antiparasitic activity was observed for compounds 1h and 2d, which may be
considered as an evidence for one of their many possible mechanisms of action.
No correlation was detected for 2e, which suggest the existence of different
targets in this family of compounds. The ADME parameters calculated for
derivatives 1h and 2e predict a good bioavailability. In silico ADME profiling and
drug score results along with in vitro leishmanicidal, cytotoxicity and TryR
inhibitory activity make 1h a promising lead compound for the development of
more potent antiparasitary drugs. A graphical resume of the conclusions drawn
from this work is depicted in Figure 3.
Moreover, to the best of our knowledge, this is in itself the first study that reports
new selenoderivatives as leishmanicidal and TryR inhibitors and opens new
possibilities in the field of neglected diseases.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to the Foundation for Applied Medical
Investigation (FIMA), University of Navarra. The authors also acknowledge
financial support from the Ministerio de Educación y Ciencia, Spain (grant
SAF2012-39760-C02-02). The authors have no other relevant affiliations or
financial involvement with any organization or entity with a financial interest in or
financial conflict with the subject matter or materials discussed in the manuscript
apart from those disclosed.
REFERENCES
695
696
697
698
699
1.
Louzir H, Aoun K, Spath GF, Laouini D, Prina E, Victoir K, Bouratbine A. 2013.
Leishmania epidemiology, diagnosis, chemotherapy and vaccination approaches in the
international network of Pasteur Institutes. Med Sci (Paris) 29:1151-1160.
McGwire BS, Satoskar AR. 2014. Leishmaniasis: clinical syndromes and treatment. QJM
107:7-14.
2.
2
8

700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
3.
4.
5.
Sundar S, Chakravarty J. 2013. Leishmaniasis: an update of current pharmacotherapy.
Expert Opin Pharmacother 14:53-63.
Yasinzai M, Khan M, Nadhman A, Shahnaz G. 2013. Drug resistance in leishmaniasis:
current drug-delivery systems and future perspectives. Future Med Chem 5:1877-1888.
Fernandes FR, Ferreira WA, Campos MA, Ramos GS, Kato KC, Almeida GG, Correa JDJ,
Melo MN, Demicheli C, Frezard F. 2013. Amphiphilic antimony(V) complexes for oral
treatment of visceral leishmaniasis. Antimicrob Agents Chemother 57:4229-4236.
Solomon M, Pavlotzky F, Barzilai A, Schwartz E. 2013. Liposomal amphotericin B in
comparison to sodium stibogluconate for Leishmania braziliensis cutaneous
leishmaniasis in travelers. J Am Acad Dermatol 68:284-289.
Seifert K, Munday J, Syeda T, Croft SL. 2011. In vitro interactions between sitamaquine
and amphotericin B, sodium stibogluconate, miltefosine, paromomycin and pentamidine
against Leishmania donovani. J Antimicrob Chemother 66:850-854.
Loiseau PM, Cojean S, Schrevel J. 2011. Sitamaquine as a putative antileishmanial drug
candidate: from the mechanism of action to the risk of drug resistance. Parasite 18:115-
119.
Varela MR, Villa-Pulgarin JA, Yepes E, Muller I, Modolell M, Munoz DL, Robledo SM,
Muskus CE, Lopez-Aban J, Muro A, Velez ID, Mollinedo F. 2012. In vitro and in vivo
efficacy of ether lipid edelfosine against Leishmania spp. and SbV-resistant parasites.
PLoS Negl Trop Dis 6:e1612.
Singh N, Mishra BB, Bajpai S, Singh RK, Tiwari VK. 2014. Natural product based leads to
fight against leishmaniasis. Bioorg Med Chem 22:18-45.
Arsenyan P, Paegle E, Domracheva I, Gulbe A, Kanepe-Lapsa I, Shestakova I. 2014.
Selenium analogues of raloxifene as promising antiproliferative agents in treatment of
breast cancer. Eur J Med Chem 87:471-483.
Fernandez-Herrera MA, Sandoval-Ramirez J, Sanchez-Sanchez L, Lopez-Munoz H,
Escobar-Sanchez ML. 2014. Probing the selective antitumor activity of 22-oxo-26-
selenocyanocholestane derivatives. Eur J Med Chem 74:451-460.
Gowda R, Madhunapantula SV, Desai D, Amin S, Robertson GP. 2013. Simultaneous
targeting of COX-2 and AKT using selenocoxib-1-GSH to inhibit melanoma. Mol Cancer
Ther 12:3-15.
Araujo AP, Rocha OG, Mayrink W, Machado-Coelho GL. 2008. The influence of copper,
selenium and zinc on the response to the Montenegro skin test in subjects vaccinated
against American cutaneous leishmaniasis. Trans R Soc Trop Med Hyg 102:64-69.
da Silva MT, Silva-Jardim I, Thiemann OH. 2014. Biological implications of selenium and
its role in trypanosomiasis treatment. Curr Med Chem 21:1772-1780.
Weekley CM, Harris HH. 2013. Which form is that? The importance of selenium
speciation and metabolism in the prevention and treatment of disease. Chem Soc Rev
42:8870-8894.
Castro H, Teixeira F, Romao S, Santos M, Cruz T, Florido M, Appelberg R, Oliveira P,
Ferreira-da-Silva F, Tomas AM. 2011. Leishmania mitochondrial peroxiredoxin plays a
crucial peroxidase-unrelated role during infection: insight into its novel chaperone
activity. PLoS Pathog 7:e1002325.
Faim LM, Rosa e Silva I, Bertacine Dias MV, D'Muniz Pereira H, Brandao-Neto J, Alves
da Silva MT, Thiemann OH. 2013. Crystallization and preliminary X-ray diffraction
analysis of selenophosphate synthetases from Trypanosoma brucei and Leishmania
major. Acta Crystallogr Sect F Struct Biol Cryst Commun 69:864-867.
Pal S, Dolai S, Yadav RK, Adak S. 2010. Ascorbate peroxidase from Leishmania major
controls the virulence of infective stage of promastigotes by regulating oxidative stress.
PLoS One 5:e11271.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
2
9

750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
766
767
768
769
770
771
772
773
774
775
776
777
778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
20.
21.
22.
23.
Beheshti N, Soflaei S, Shakibaie M, Yazdi MH, Ghaffarifar F, Dalimi A, Shahverdi AR.
2013. Efficacy of biogenic selenium nanoparticles against Leishmania major: in vitro and
in vivo studies. J Trace Elem Med Biol 27:203-207.
Moreno D, Plano D, Baquedano Y, Jimenez-Ruiz A, Palop JA, Sanmartin C. 2011.
Antileishmanial activity of imidothiocarbamates and imidoselenocarbamates. Parasitol
Res 108:233-239.
Baquedano Y, Moreno E, Espuelas S, Nguewa P, Font M, Gutierrez KJ, Jimenez-Ruiz A,
Palop JA, Sanmartin C. 2014. Novel hybrid selenosulfonamides as potent antileishmanial
agents. Eur J Med Chem 74:116-123.
Fernandez-Rubio C, Campbell D, Vacas A, Ibanez E, Moreno E, Espuelas S, Calvo A,
Palop JA, Plano D, Sanmartin C, Nguewa PA. 6 July 2015. Exploring leishmanicidal
activities of novel methylseleno imidocarbamates. Antimicrob Agents Chemother
doi:10.1128/aac.00997-15.
Plano D, Baquedano Y, Moreno-Mateos D, Font M, Jimenez-Ruiz A, Palop JA,
Sanmartin C. 2011. Selenocyanates and diselenides: a new class of potent
antileishmanial agents. Eur J Med Chem 46:3315-3323.
Gopinath VS, Pinjari J, Dere RT, Verma A, Vishwakarma P, Shivahare R, Moger M,
Kumar Goud PS, Ramanathan V, Bose P, Rao MV, Gupta S, Puri SK, Launay D, Martin D.
2013. Design, synthesis and biological evaluation of 2-substituted quinolines as potential
antileishmanial agents. Eur J Med Chem 69:527-536.
Bompart D, Nunez-Duran J, Rodriguez D, Kouznetsov VV, Melendez Gomez CM, Sojo F,
Arvelo F, Visbal G, Alvarez A, Serrano-Martin X, Garcia-Marchan Y. 2013. Anti-
leishmanial evaluation of C2-aryl quinolines: mechanistic insight on bioenergetics and
sterol biosynthetic pathway of Leishmania braziliensis. Bioorg Med Chem 21:4426-4431.
Di Giorgio C, Shimi K, Boyer G, Delmas F, Galy JP. 2007. Synthesis and antileishmanial
activity of 6-mono-substituted and 3,6-di-substituted acridines obtained by acylation of
proflavine. Eur J Med Chem 42:1277-1284.
Carole DG, Michel DM, Julien C, Florence D, Anna N, Severine J, Gerard D, Pierre TD,
Jean-Pierre G. 2005. Synthesis and antileishmanial activities of 4,5-di-substituted
acridines as compared to their 4-mono-substituted homologues. Bioorg Med Chem
13:5560-5568.
24.
25.
26.
27.
28.
29.
30.
31.
Lizarazo-Jaimes EH, Reis PG, Bezerra FM, Rodrigues BL, Monte-Neto RL, Melo MN,
Frezard F, Demicheli C. 2014. Complexes of different nitrogen donor heterocyclic
III
ligands with SbCl
3
and PhSbCl
2
as potential antileishmanial agents against Sb -sensitive
and -resistant parasites. J Inorg Biochem 132:30-36.
Villalobos-Rocha JC, Sanchez-Torres L, Nogueda-Torres B, Segura-Cabrera A, Garcia-
Perez CA, Bocanegra-Garcia V, Palos I, Monge A, Rivera G. 2014. Anti-Trypanosoma
cruzi and anti-leishmanial activity by quinoxaline-7-carboxylate 1,4-di-N-oxide
derivatives. Parasitol Res 113:2027-2035.
Ronga L, Del Favero M, Cohen A, Soum C, Le Pape P, Savrimoutou S, Pinaud N, Mullie
C, Daulouede S, Vincendeau P, Farvacques N, Agnamey P, Pagniez F, Hutter S, Azas N,
Sonnet P, Guillon J. 2014. Design, synthesis and biological evaluation of novel 4-
alkapolyenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents--part III. Eur J Med
Chem 81:378-393.
Arango V, Robledo S, Seon-Meniel B, Figadere B, Cardona W, Saez J, Otalvaro F. 2010.
Coumarins from Galipea panamensis and Their Activity against Leishmania panamensis.
J Nat Prod 73:1012-1014.
Vila-Nova NS, de Morais SM, Falcao MJ, Alcantara TT, Ferreira PA, Cavalcanti ES, Vieira
IG, Campello CC, Wilson M. 2013. Different susceptibilities of Leishmania spp.
promastigotes to the Annona muricata acetogenins annonacinone and corossolone, and
the Platymiscium floribundum coumarin scoparone. Exp Parasitol 133:334-338.
32.
33.
3
0