New amide derivatives of quinoxaline 1,4-di-N-oxide with leishmanicidal and antiplasmodial activities

Article abstract of DOI:10.3390/molecules18044718

Malaria and leishmaniasis are two of the World's most important tropical parasitic diseases. Continuing with our efforts to identify new compounds active against malaria and leishmaniasis, twelve new 1,4-di-N-oxide quinoxaline derivatives were synthesized and evaluated for their in vitro antimalarial and antileishmanial activity against Plasmodium falciparum FCR-3 strain, Leishmania infantum and Leishmania amazonensis. Their toxicity against VERO cells (normal monkey kidney cells) was also assessed. The results obtained indicate that a cyclopentyl derivative had the best antiplasmodial activity (2.9 μM), while a cyclohexyl derivative (2.5 μM) showed the best activity against L. amazonensis, and a 3-chloropropyl derivative (0.7 μM) showed the best results against L. infantum. All these compounds also have a Cl substituent in the R7 position.

Full text of DOI:10.3390/molecules18044718

Molecules 2013, 18, 4718-4727; doi:10.3390/molecules18044718
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
New Amide Derivatives of Quinoxaline 1,4-di-N-Oxide with
Leishmanicidal and Antiplasmodial Activities
Carlos Barea 1,*, Adriana Pabón 2,3, Silvia Pérez-Silanes 1, Silvia Galiano 1, German Gonzalez 4,5,
Antonio Monge 1, Eric Deharo 4,5 and Ignacio Aldana 1
1
Unidad de Investigación y Desarrollo de Nuevos Medicamentos, Centro de Investigación en
Farmacobiología Aplicada (CIFA), Universidad de Navarra, Pamplona 31080, Spain
2
Grupo Malaria, Facultad de Medicina, Universidad de Antioquia, 050010 Medellín, Colombia
3
Programa de Biología, Facultad de Ciencias Básicas, Universidad del Atlántico, 080001 Barranquilla,
Colombia
4
Université de Toulouse, UPS, UMR 152 Pharma-DEV, Université Toulouse 3,
Faculté des Sciences Pharmaceutiques, F-31062 Toulouse cedex 09, France
5
Institut de Recherche pour le Développement (IRD), UMR 152 Pharma-DEV,
F-31062 Toulouse cedex 09, France
* Author to whom correspondence should be addressed; E-Mail: cabareari@hotmail.com;
Tel.: +34-948-425-653; Fax: +34-948-425-652.
Received: 26 March 2013; in revised form: 11 April 2013 / Accepted: 18 April 2013 /
Published: 22 April 2013
Abstract: Malaria and leishmaniasis are two of the World’s most important tropical
parasitic diseases. Continuing with our efforts to identify new compounds active against
malaria and leishmaniasis, twelve new 1,4-di-N-oxide quinoxaline derivatives were
synthesized and evaluated for their in vitro antimalarial and antileishmanial activity against
Plasmodium falciparum FCR-3 strain, Leishmania infantum and Leishmania amazonensis.
Their toxicity against VERO cells (normal monkey kidney cells) was also assessed. The
results obtained indicate that a cyclopentyl derivative had the best antiplasmodial activity
(2.9 µM), while a cyclohexyl derivative (2.5 µM) showed the best activity against
L. amazonensis, and a 3-chloropropyl derivative (0.7 µM) showed the best results against
L. infantum. All these compounds also have a Cl substituent in the R7 position.
Keywords: quinoxaline; 1,4-di-N-oxide; leishmanicidal; antiplasmodial

Molecules 2013, 18
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1. Introduction
Malaria and leishmaniasis are important, social and economical health problems, particularly in the
tropical countries. Malaria is a major public health problem today in more than 106 countries and its
prevalence is estimated on the order of 216 million clinical cases annually, with a mortality estimated
at 266 thousand persons per year; leishmania is responsible for some 2 million clinical cases each year
in 88 countries. Most available drugs against malaria and leishmania are costly, highly toxic,
require long treatment regimens and are currently losing their effectiveness due to the development
of resistance on the part of the respective parasites. Therefore, new effective and affordable
antiplasmodial and leishmanicidal agents are urgently needed [1,2].
Quinoxaline derivatives are a class of compounds of great interest within the field of medicinal
chemistry because they display a broad range of biological properties such as anticancer [3,4],
antimycobacterial [5,6], anti-inflammatory [7], antiviral [8], antiprotozoal [9–12] and antibacterial
activities [13]. The oxidation of both nitrogens of this heterocyclic system, carried out in order to
obtain quinoxaline 1,4-di-N-oxide derivatives, increases the range of biological properties [14].
In an attempt to intensify the antiparasitic activity of quinoxaline derivatives, our group has
synthesized different series that offer promising results; these consisted in the introduction of a
carbonitrile group in position 2, which increases the antiparasitic activity, and an amide group in
position 3, with the aim of linking together new molecules with interesting activities [15,16].
Continuing with this strategy, we have synthesized and evaluated in vitro twelve new amide
derivatives of 1,4-di-N-oxide quinoxaline against Plasmodium falciparum FCR-3 strain
(chloroquine-resistant), against Leishmania infantum, responsible for visceral forms, and against
Leishmania amazonensis, responsible for cutaneous expression of the disease.
2. Results and Discussion
2.1. Chemistry
The benzofuroxane starting compounds (BFX, I, Scheme 1) have been prepared using previously
described methods [17,18]. The 3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivatives (QX, II)
were obtained from the corresponding BFX by the Beirut reaction with malononitrile, using
N,N-dimethylformamide (DMF) as solvent and triethylamine as catalyst [19].
Scheme 1. General synthesis of new amide derivatives of quinoxaline 1,4-di-N-oxide.
O-
O-
O-
N+
R7
N+
CN
NH
R6
NC
CN
R7
N+
CN
COCl-R'
ii
O
O
R6
N+
O-
2
N
R5
i
R6
N+
O-
N
H
R'
BFX
QX
1-12

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The method for synthesizing the final compounds consists of reacting 3-amino-2-cyanoquinoxaline
1,4-dioxide derivatives with (purchased) cyclo- and aliphatic-acyl chlorides at room temperature for
two hours, using dry tetrahydrofuran as solvent.
2.2. Pharmacology and Structure-Activity Relationship
With regard to the antiplasmodial activity shown in Table 1, halogen groups at R7 increase the
activity, as shown in previous series of quinoxaline 1,4-di-N-oxide derivatives [15]. The cyclopropyl
group lowered the antiplasmodial activity almost 100-fold compared to chloroquine (0.2 µM). The
cyclopentyl group associated with Cl enhanced the activity (2.9×), but it was still fifteen times less
active than chloroquine. When this group was changed for a methyl, acetyl or chloropropyl, the activity
decreased to approximately 5 µM. A cyclohexyl group did not enhance the activity (the best one being
7.5 with a Cl substituent). None of the tested compounds showed noticeable toxicity towards VERO cells.
Table 1. Biological characterization of the final compounds.
O-
R7
N+
CN
O
N+
O-
N
H
R'
Compound
R7
H
R’
IC50 (µM) a IC50 (µM) b IC50 (µM) c CC50 (µM) d
SI e
15.3
40.8
15
1
2
cyclopropyl
cyclopropyl
cyclopropyl
cyclopropyl
cyclopentyl
cyclohexyl
cyclohexyl
cyclohexyl
methyl
18.3
13.3
31
3.6
3.5
3.5
3.9
-
-
55.1
144.6
52.8
145.6
NT
Cl
-
3
CH3
CH3O
Cl
-
4
27.8
2.9
-
14.9
-
36.7
NT
98
5
6
Cl
7.5
2.5
4.6
3.4
-
249
7
CH3
CH3O
H
21.6
12.9
6.2
-
240.1
238.5
NT
52.2
69.1
NT
8
-
9
16.6
11.9
4
10
11
12
CQ
H
acetyl
5.3
-
NT
NT
Cl
acetyl
4.3
-
NT
NT
NT
Cl
3-chloropropyl
5.7
-
0.7
NT
0.2
Amph B
0.2
0.15
13
62.1
a
b
c
IC50 against P. falciparum FCR-3; IC50 against axenic amastigotes of L. infantum; IC50 against axenic
amastigotes of L. amazonensis; d Cytotoxicity in VERO cells; e Selectivity Index (SI): CC50 drugd/IC50 drugb.
NT: Not tested. CQ: chloroquine. Amph B: amphotericin B.
With regard to leishmanicidal activity, also shown in Table 1, some compounds were tested against
L. infantum and others against L. amazonensis. This choice was made according to preliminary testing
results. All compounds assayed against L. infantum had some activity and most of them presented low
toxicity, especially compounds 6 and 8, which showed better selectivity indexes than amphotericin B.
Compound 6 was the most active, but it was ten times less active than amphotericin B. Among the

Molecules 2013, 18
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compounds assayed against Leishmania amazonensis, compound 12 showed an activity which was
only 5 times lower than the activity shown by amphotericin B. Interestingly, the presence of halogen
groups at R7 and an increase in the length of the aliphatic chain are correlated with increasing anti-
malarial and leishmanicidal activity. As Leishmania species are known to harbor different sensitivity
against leishmanicidal compounds we plan to perform supplementary studies for all the compounds in
both models. Additional cytotoxic evaluation must be conducted prior to in vivo testing [20].
3. Experimental
3.1. Chemical Synthesis
3.1.1. General Remarks
All of the synthesized compounds were chemically characterized by thin layer chromatography
(TLC), infrared spectroscopy (IR), proton nuclear magnetic resonance (1H-NMR) and elemental
microanalyses (CHN). Alugram SIL G/UV254 (Layer: 0.2 mm) (Macherey-Nagel GmbH & Co. KG.,
Düren, Germany) was used for TLC and Silica gel 60 (0.040–0.063 mm, Merck) was used for Flash
Column Chromatography. Automated Flash Column Chromatography was developed on an automated
Flash Chromatography System CombiFlash® Rf (TELEDYNE ISCO, Lincoln, NE, USA) instrument
with Silica RediSep® Rf columns (average particle size: 35 to 70 microns; average pore size: 60 Å).
Purification methods were developed using dichloromethane and methanol to run suitable gradient
1
conditions. The H-NMR spectra were recorded on a Bruker 400 Ultrashield instrument (400 MHz),
using TMS as internal standard and with DMSO-d6 as solvent; the chemical shifts are reported in ppm
(δ) and coupling constant (J) values are given in Hertz (Hz). Signal multiplicities are represented by: s
(singlet), bs (broad singlet), d (doublet), t (triplet), dd (doublet of doublets) and m (multiplet). The IR
spectra were recorded on a Nicolet Nexus FTIR (Thermo, Madison, WI, USA) in KBr pellets.
Elemental microanalyses were obtained on a CHN-900 Elemental Analyzer (Leco, Tres Cantos, Spain)
from vacuum-dried samples. The analytical results for C, H and N, were within ±0.5 of the theoretical
values. Chemicals were purchased from Panreac Química S.A. (Barcelona, Spain), Sigma-Aldrich
Química S.A. (Alcobendas, Spain), Acros Organics (Janssen Pharmaceutical, Geel, Belgium) and
Lancaster (Bischheim-Strasbourg, France).
3.1.2. General Procedure for the Synthesis of Quinoxalines II
Malononitrile (18.0 mmol) was added to a solution of the appropriate benzofuroxane (I, 15.0 mmol)
in DMF (10 mL). The mixture was allowed to stand at 0 °C. Triethylamine (1.5 mL) was added dropwise,
and the reaction mixture was stirred at room temperature in darkness for 1 day. The resulting
precipitate was filtered off and washed by adding diethyl ether, affording the target compound. The
obtained red solid was used in the next step without further purification [21]. The yield of this reaction
depends on the substituents in positions 5 and 6 in the benzofuroxane. When quinoxalines were prepared
from monosubstituted-BFX, the formation of isomeric quinoxalines 1,4-di-N-oxide was observed. In
most cases, the 7-substituted isomer prevailed over 6-substituted isomer, and when the methoxy
substituted quinoxalines were prepared, only the 7-isomer was obtained, as previously described [22,23].

Molecules 2013, 18
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3.1.3. General Procedure for the Synthesis of New Amide Derivatives of Quinoxaline 1,4-di-N-Oxide
An excess of the corresponding carbonyl chloride (1:1.2) was added to a stirred solution of
3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivative (5 mmol) in dry tetrahydrofuran (60 mL).
The resulting mixture was stirred at room temperature for 2 h and the solid was collected and purified
by column chromatography (dichloromethane/methanol 97:3 or toluene/dioxane 6:4). Finally, the
solvents were removed in vacuo and the solid precipitated with cold diethyl ether, filtered off in order
to obtain a yellow or orange solid [15].
1
2-Cyano-3-(cyclopropanecarboxamido)quinoxaline 1,4-dioxide (1). Yield 21%; H-NMR δ ppm: 11.54
(s, 1H, NH); 8.52 (d, 1H, H8, J8-7 = 8.3 Hz); 8.45 (d, 1H, H5, J5-6 = 8.3 Hz); 8.08 (t, 1H, H7, J7-8 = 8.3 Hz,
J
7-6 = 8.3 Hz); 7.99 (t, 1H, H6, J6-7 = 8.3 Hz, J6-5 = 8.3 Hz); 2.26 (m, 1H, CH); 0.98 (d, 2H, CH2); 0.92
(d, 2H, CH2); IR ν cm−1: 3250 (m, NH); 2373 (w, C≡N); 1692 (s, C=O); 1332 (s, N+O−); Anal. Calc.
for C13H10N4O3: C: 57.77%; H: 3.70%; N: 20.74%. Found: C: 57.28%; H: 3.82%; N: 20.44%.
1
7-Chloro-2-cyano-3-(cyclopropanecarboxamido)quinoxaline 1,4-dioxide (2). Yield 20%; H-NMR δ
ppm: 11.64 (s, 1H, NH); 8.50 (d, 1H, H8, J8-6 = 2.2 Hz); 8.45 (d, 1H, H5, J5-6 = 9.3 Hz); 8.02 (dd, 1H,
H6, J6-8 = 2.2 Hz, J6-5 = 9.3 Hz); 2.27 (m, 1H, CH); 0.99 (d, 2H, CH2); 0.93 (d, 2H, CH2); IR ν cm−1:
3245 (m, NH); 2371 (w, C≡N); 1687 (s, C=O); 1326 (s, N+O−); Anal. Calc. for C13H9N4O3Cl: C:
51.23%; H: 2.95%; N: 18.39%. Found: C: 50.86%; H: 3.28%; N: 18.37%.
1
2-Cyano-3-(cyclopropanecarboxamido)-7-methylquinoxaline 1,4-dioxide (3). Yield 23%; H-NMR δ
ppm: 11.49 (s, 1H, NH); 8.39 (d, 1H, H5, J5-6 = 8.6 Hz); 8.32 (d, 1H, H6, J6-5 = 8.6 Hz); 8.26 (s, 1H,
H8); 2.58 (s, 3H, CH3-C7); 2.25 (bs, 1H, CH); 0.97 (bs, 2H, CH2); 0.91 (bs, 2H, CH2); IR ν cm−1: 3247
(m, NH); 2312 (w, C≡N); 1685 (s, C=O); 1328 (s, N+O−); Anal. Calc. for C14H12N4O3: C: 59.15%; H:
4.22%; N: 19.71%. Found: C: 59.09%; H: 4.65%; N: 19.30%.
2-Cyano-3-(cyclopropanecarboxamido)-7-methoxyquinoxaline 1,4-dioxide (4). Yield 25%; 1H-NMR δ
ppm: 11.42 (s,1H, NH); 8.43 (d, 1H, H5, J5-6 = 9.4 Hz); 7.74 (d, 1H, H8, J8-6 = 2.7 Hz); 7.69 (dd, 1H,
H6, J6-8 = 2.7 Hz, J6-5 = 9.4 Hz); 4.01 (s, 3H, CH3O); 2.51 (bs, 1H, CH); 0.97 (d, 2H, CH2); 0.91 (d,
2H, CH2); IR ν cm−1: 3256 (m, NH); 2370 (w, C≡N); 1691 (s, C=O); 1327 (s, N+O−); Anal. Calc. for
C14H12N4O4: C: 56.00%; H: 4.00%; N: 18.66%. Found: C: 55.61%; H: 4.12%; N: 18.19%.
1
7-Chloro-2-cyano-3-(cyclopentanecarboxamido)quinoxaline 1,4-dioxide (5). Yield 25%; H-NMR δ
ppm: 11.24 (s, 1H, NH); 8.49 (d, 1H, H5 QX, J5-6 = 9.2 Hz); 8.46 (d, 1H, H8 QX, J8-6 = 2.2 Hz); 8.10
(dd, 1H, H6 QX, J6-8 = 2.2 Hz, J6-5 = 9.2 Hz); 3.18 (m, 1H, CH); 1.90 (bs, 2H, H2+H5 eq. cyclo); 1.81
(bs, 2H, H2+H5 ax. cyclo); 1.68 (bs, 2H, H3+H4 eq. cyclo); 1.60 (bs, 2H, H3+H4 ax. cyclo); IR ν cm−1:
3307 (m, NH); 2315 (w, C≡N); 1701 (s, C=O); 1327 (s, N+O−); Anal. Calc. for C15H13N4O3Cl: C:
54.13%; H: 3.90%; N: 16.84%. Found: C: 53.90%; H: 3.77%; N: 17.10%.
1
7-Chloro-2-cyano-3-(cyclohexanecarboxamido)quinoxaline 1,4-dioxide (6). Yield 17%; H-NMR δ
ppm: 11.19 (s, 1H, NH); 8.48 (d, 1H, H5 QX, J5-6 = 9.2 Hz); 8.46 (d, 1H, H8 QX, J8-6 = 2.2 Hz); 8.10
(dd, 1H, H6 QX, J6-8 = 2.2 Hz, J6-5 = 9.2 Hz); 2.72 (m, 1H, CH); 1.86 (d, 2H, H2+H6 eq. cyclo); 1.78
(d, 2H, H3+H5 eq. cyclo); 1.65 (d, 2H, H2+H6 ax. cyclo); 1.43 (m, 2H, H3+H5 ax. cyclo); 1.25 (d, 2H,

Molecules 2013, 18
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4
CH2 cyclo); IR ν cm−1: 3286 (m, NH); 2236 (w, C≡N); 1696 (s, C=O); 1327 (s, N+O−); Anal. Calc. for
C16H15N4O3Cl: C: 55.41%; H: 4.32%; N: 16.16%. Found: C: 54.95%; H: 4.59%; N: 16.00%.
1
2-Cyano-3-(cyclohexanecarboxamido)-7-methylquinoxaline 1,4-dioxide (7). Yield 11%; H-NMR δ
ppm: 11.07 (s, 1H, NH); 8.03 (d, 1H, H5 QX, J5-6 = 8.7 Hz); 7.32 (s, 1H, H8 QX); 7.27 (dd, 1H, H6
QX, J6-8 = 1.3 Hz, J6-5 = 8.7 Hz); 2.71 (m, 1H, CH); 2.51 (s, 3H, CH3-C7 QX); 1.87 (d, 2H, H2+H6 eq.
cyclo); 1.78 (d, 2H, H3+H5 eq. cyclo); 1.65 (d, 2H, H2+H6 ax. cyclo); 1.44 (d, 2H, H3+H5 ax. cyclo);
4
1.27 (dd, 2H, CH2 cyclo); IR ν cm−1: 3248 (m, NH); 2374 (w, C≡N); 1691 (s, C=O); 1327 (s, N+O−);
Anal. Calc. for C17H18N4O3: C: 62.57%; H: 5.52%; N: 17.17%. Found: C: 62.09%; H: 5.43%; N: 16.76%.
1
2-Cyano-3-(cyclohexanecarboxamido)-7-methoxyquinoxaline 1,4-dioxide (8). Yield 30%; H-NMR δ
ppm: 11.01 (s, 1H, NH); 8.41 (d, 1H, H5 QX, J5-6 = 9.4 Hz); 7.74 (d, 1H, H8 QX, J8-6 = 2.7 Hz); 7.69
(dd, 1H, H6 QX, J6-8 = 2.7 Hz, J6-5 = 9.4 Hz); 4.01 (s, 3H, CH3O); 2.69 (m, 1H, CH); 1.87 (d, 2H,
H2+H6 eq. cyclo); 1.77 (d, 2H, H3+H5 eq. cyclo); 1.65 (d, 1H, H2+H6 ax. cyclo); 1.44 (m, 2H, H3+H5
ax. cyclo); 1.26 (d, 2H, CH2 cyclo); IR ν cm−1: 3245 (m, NH); 2373 (w, C≡N); 1691 (s, C=O); 1327
4
(s, N+O−); Anal. Calc. for C17H18N4O4: C: 59.64%; H: 5.26%; N: 16.37%. Found: C: 59.20%; H: 5.34%;
N: 16.33%.
3-Acetamido-2-cyanoquinoxaline 1,4-dioxide (9). Yield 15%; 1H-NMR δ ppm: 11.29 (s, 1H, NH); 8.50
(d, 1H, H5, J5-6 = 8.5 Hz); 8.45 (d, 1H, H8, J8-7 = 8.5 Hz); 8.08 (t, 1H, H6, J6-7 = 7.6 Hz); 7.99 (t, 1H, H7);
2.27 (s, 3H, CH3); IR ν cm−1: 3256 (m, NH); 2374 (w, C≡N); 1524 (s, C=O); 1331 (s, N+O−); Anal.
Calc. for C11H8N4O3: C: 54.09%; H: 3.27%; N: 22.95%. Found: C: 53.74%; H: 3.02%; N: 23.43%.
1
2-Cyano-3-propionamidoquinoxaline 1,4-dioxide (10). Yield 15%; H-NMR δ ppm: 11.26 (s, 1H,
NH); 8.51 (d, 1H, H5, J5-6 = 8.6 Hz); 8.46 (d, 1H, H8, J8-7 = 8.6 Hz); 8.08 (t, 1H, H7); 8.01 (dd, 1H, H6,
J6-5= 8.6 Hz); 2.58 (d, 2H, CH2, JCH2-CH3 = 7.4 Hz); 1.13 (t, 3H, CH3, JCH3-CH2 = 7.4 Hz); IR ν cm−1:
3250 (m, NH); 2236 (w, C≡N); 1524 (s, C=O); 1333 (s, N+O−); Anal. Calc. for C12H10N4O3: C:
55.81%; H: 3.87%; N: 21.70%. Found: C: 56.05%; H: 3.66%; N: 22.10%.
1
7-Chloro-2-cyano-3-propionamidoquinoxaline 1,4-dioxide (11). Yield 5%; H-NMR δ ppm: 11.28 (s,
1H, NH); 8.49 (d, 1H, H5, J5-6 = 8.6 Hz); 8.46 (d, 1H, H8); 8.09 (dd, 1H, H6, J6-8 = 2.4 Hz, J6-5 = 8.6 Hz);
2.58 (d, 2H, CH2, JCH2-CH3 = 7.2 Hz); 1.13 (t, 3H, CH3, JCH3-CH2 = 7.2 Hz); IR ν cm−1: 3254 (m, NH);
2366 (w, C≡N); 1517 (s, C=O); 1321 (s, N+O−); Anal. Calc. for C12H9N4O3Cl: C: 49.23%; H: 3.07%;
N: 19.14%. Found: C: 49.18%; H: 2.84%; N: 19.18%.
1
7-Chloro-3-(4-chlorobutanamido)-2-cyanoquinoxaline 1,4-dioxide (12). Yield 5%; H-NMR δ ppm:
11.44 (s, 1H, NH); 8.50 (d, 1H, H5, J5-6 = 9.1 Hz); 8.47 (d, 1H, H8, J8-6 = 2.2 Hz); 8.11 (dd, 1H, H6,
3
2
J6-8 = 2.2 Hz, J6-5 = 9.1 Hz); 3.73 (t, 2H, CH2 ); 2.75 (t, 2H, CH2 , J2-3 = 7.0 Hz, J2-1 = 7.0 Hz); 2.08 (t,
1
2H, CH2 ); IR ν cm−1: 3256 (m, NH); 2373 (w, C≡N); 1517 (s, C=O); 1324 (s, N+O−); Anal. Calc. for
C13H10N4O3Cl2: C: 45.74%; H: 2.93%; N: 16.42%. Found: C: 45.39%; H: 2.83%; N: 16.48%.

Molecules 2013, 18
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3.2. Pharmacology
3.2.1. In Vitro Antiplasmodial Drug Assay
Chloroquine-resistant FCR-3 strain of P. falciparum was cultivated at 37 °C in a 5% CO2
environment in glucose-enriched RPMI 1640 medium supplemented with gentamicin 0.1 mg/mL and
10% heat-inactivated A+ human serum, as previously described [24]. The drugs, dissolved in
dimethylsulfoxide (DMSO), were added at final concentrations ranging from 250 to 0.1 µM. The final
DMSO concentration was never greater than 0.1%. In vitro antimalarial activity was measured using
the [3H]-hypoxanthine (MP Biomedicals, Santa Ana, CA, USA) incorporation assay [25]. Briefly,
250 µL of total culture medium with the diluted drug and the suspension of human red blood cells in
medium (A+ group, 5% hematocrit) with 1% parasitaemia were placed into the wells of 96-well
microtiter plates. On the third day of the test, radioactivity was assessed. All experiments were
performed in triplicate. Results were expressed as the concentration resulting in 50% inhibition (IC50),
which was calculated by a nonlinear regression logistic dose response model; the mean IC50 values and
standard deviation for each compound were calculated.
3.2.2. In Vitro Cytotoxicity
Toxicity was determined using Vero cells (normal monkey kidney cells) cultured under the same
conditions as P. falciparum, except for the replacement of 5% human serum with 10% fetal calf serum.
After the addition of compounds at increasing concentrations, cell growth was measured by
[3H]-hypoxanthine incorporation after a 48-h incubation period and then compared with a control
sample [26].
3.2.3. In Vitro Antileishmanial Drug Assay
Leishmanicidal activity was determined on axenic cultures of L. infantum and amazonensis
amastigotes. In order to estimate the 50% inhibitory concentration (IC50) of the drugs, the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) micromethod was used as previously
described [27]. Briefly, Leishmania strain was maintained in promastigote stage in a biphasic medium
(blood agar with 0.89% NaCl, pH 7.4) at 24 °C, with sub-passage every 3–4 days. Promastigotes
(5 × 106 parasites) were then transferred to M199 medium supplemented with 10% fetal bovine serum,
pH 7.4. After 4 days, exponential phase promastigotes were centrifuged for 10 min at 1,500 g and
4 °C. The supernatant was discarded and replaced by fresh M199 medium supplemented with 20%
FBS, pH 5.5. Axenic amastigote transformation was then induced by increasing the temperature to
34 °C. Drugs were then tested at increasing concentrations.
4. Conclusions
Compounds 5, 6 and 12 were the most active against Plasmodium falciparum, Leishmania infantum
and L. amazonensis, respectively. The presence of a halogenous atom at position 7 and the increase of
the aliphatic chain length increase the level of activity. Therefore, these compounds have been selected
as lead compounds in the future design of new compounds against Plasmodium and Leishmania.

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Acknowledgments
Carlos Barea is indebted to the “Asociación de Amigos de la Universidad de Navarra” (Spain) for a
PhD scholarship. This work was supported by PiUNA.
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Sample Availability: Samples of the compounds are available from the authors.
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