Antiplasmodial and leishmanicidal activities of 2-cyano-3-(4- phenylpiperazine-1-carboxamido) quinoxaline 1,4-dioxide derivatives

Article abstract of DOI:10.3390/molecules17089451

Malaria and leishmaniasis are two of the World's most important tropical parasitic diseases. Thirteen new 2-cyano-3-(4-phenylpiperazine-1-carboxamido) quinoxaline 1,4-dioxide derivatives (CPCQs) were synthesized and evaluated for their in vitro antimalari

Full text of DOI:10.3390/molecules17089451

Molecules 2012, 17, 9451-9461; doi:10.3390/molecules17089451
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antiplasmodial and Leishmanicidal Activities of
2-Cyano-3-(4-phenylpiperazine-1-carboxamido)
Quinoxaline 1,4-Dioxide Derivatives
Carlos Barea 1,*, Adriana Pabón 2, Silvia Galiano 1, Silvia Pérez-Silanes 1, German Gonzalez 3,4,
Chloe Deyssard 3,4, Antonio Monge 1, Eric Deharo 3,4 and Ignacio Aldana 1
1
Research and Development of Novel Drugs Unit, Center for Applied Pharmacobiology Research
(CIFA), University of Navarre, Pamplona 31080, Spain; E-Mails: sgaliano@unav.es (S.G.);
sperez@unav.es (S.P.-S.); amonge@unav.es (A.M.); ialdana@unav.es (I.A.)
2
Malaria Group, University of Antioquia, Medellín, Colombia; E-Mail: apabon72@gmail.com
3
UMR 152 PharmaDev (Pharmacologie et Pharmacochimie pour le Développement),
Université de Toulouse, UPS, 118, rte de Narbonne, F-31062 Toulouse cedex 9, France; E-Mails:
germangonzalez.a@hotmail.com (G.G.); c.deyssard@gmail.com (C.D.); ericdeharo@gmail.com (E.D.)
4
UMR 152 PharmaDev Institut de Recherche pour le Développement, Faculté des Sciences
Pharmaceutiques, Université de Toulouse 3, 31062 Toulouse, France
* Author to whom correspondence should be addressed; E-Mail: cabareari@hotmail.com;
Tel.: +34-948-425-653; Fax: +34-948-425-652.
Received: 28 June 2012; in revised form: 20 July 2012 / Accepted: 31 July 2012 /
Published: 7 August 2012
Abstract: Malaria and leishmaniasis are two of the World’s most important tropical
parasitic diseases. Thirteen new 2-cyano-3-(4-phenylpiperazine-1-carboxamido) quinoxaline
1,4-dioxide derivatives (CPCQs) were synthesized and evaluated for their in vitro
antimalarial and antileishmanial activity against erythrocytic forms of Plasmodium
falciparum and axenic forms of Leishmania infantum. Their toxicity against VERO cells
(normal monkey kidney cells) was also assessed. None of the tested compounds was
efficient against Plasmodium, but two of them showed good activity against Leishmania.
Toxicity on VERO was correlated with leishmanicidal properties.
Keywords: quinoxaline; piperazine; Plasmodium; Leishmania; VERO

Molecules 2012, 17
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1. Introduction
Malaria is a major public health problem in more than 90 countries, affecting 40% of the World’s
poorest population. Mortality due to malaria is estimated to be over 1 million deaths annually and this
situation is worsened by the spread of drug-resistant strains of the parasite. Therefore, new effective
and affordable antimalarial agents are urgently needed [1,2].
Leishmaniasis threatens approximately 350 million people and almost 12 million people are
currently infected with the disease. The emergence of resistant parasites, the high cost and toxicity of
current treatments call for the discovery of new drugs [3,4].
Quinoxalines, also known as 1,4-benzodiazines, are aromatic bicycles that present two nitrogen
atoms on positions 1 and 4, described as bioisosteres of quinoline, naphtyl and some other
heteroaromatic rings including pirazine [5].
Quinoxaline derivatives are of great interest as antimycobacterial, anti-inflammatory, anticancer and
antiparasitic agents. More specifically, their 1,4-di-N-oxides are considered to be particularly
important because they are responsible for a resulting increase in various biological properties [6].
As a result of different research projects, our group synthesized different series of quinoxaline
1,4-di-N-oxide derivatives, with a great variety of substituents in positions 2, 3, 6 and 7 [7–15]. With
the aim of improving their pharmacological properties, we synthesized compounds with a carbonitrile
group in position 2, thereby enhancing their antiparasitic activity. We also added an amine group in
position 3 in order to link together new molecules, leading to interesting activities [10,16].
In silico studies showed that piperazine derivatives could target Plasmodium plasmepsin II enzyme.
We synthesized phenylpiperazines derivatives that were active against Plasmodium falciparum [17].
In this context, we have now synthesized thirteen new 3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile
derivatives linked with phenyl piperazines analogs (Figure 1) and investigated their in vitro activity
and toxicity against Plasmodium falciparum Colombian FCR-3 strain and Leishmania infantum [18].
Figure 1. Design of new CPCQs as potential drugs against P. falciparum and L. infantum.
O-
R7
N+
CN
NH
O
R6
N+
O-
N
N
R4'
Quinoxaline
R2'
Piperazine
2. Results and Discussion
2.1. Chemistry
We prepared thirteen new 2-cyano-3-(4-phenylpiperazine-1-carboxamido) quinoxaline 1,4-dioxide
derivatives (CPCQs; Scheme 1). The benzofuroxane starting compounds, (BFX, I), were prepared

Molecules 2012, 17
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using previously described methods [19,20]. The 3-amine-1,4-di-N-oxide quinoxaline-2-carbonitrile
derivatives (cyanoamines, II) were obtained from the corresponding BFX by the Beirut reaction with
malononitrile, using N,N-dimethylformamide (DMF) as solvent and triethylamine as catalyst [21].
Scheme 1. Synthetic route to CPCQs 1–13.
O-
O-
O
R7
N+
CN
R7
N+
CN
NH
O
dry THF
50ºC, 3h
N
N
N
N
R6
N+
O-
NH2
R6
N+
O-
N
N
CDI
II
3a-m
HN
N
R4'
CHCl3, N2, 50ºC, 3h
R2'
O-
R7
N+
CN
O
R6'
N+
O-
NH
N
N
R4'
R2'
1–13
The method for synthesizing the final compounds consisted of two steps; the first one was the
reaction of cyanoamines with an excess of commercially available 1,1'-carbonyldiimidazole (CDI) in
order to obtain the intermediates 3a–m that can react in the second step with the phenylpiperazines,
thus affording the CPCQs [22].
2.2. Pharmacology and Structure-Activity Relationship
With regard to antiplasmodial activity shown in Table 1, halogen groups in R7 and/or R6 increase
the activity as shown in previous series of quinoxaline 1,4-di-N-oxide derivatives [16]. Compounds
with CF3 or CH3O groups at R4' show comparable activity, but higher activity than those which have F
in that position. In relation with position R2', the NO2 group does not increase the activity.
With regard to leishmanicidal activity shown in Table 1, among the most active compounds, 5 and
8 were also among the most cytotoxic compounds against VERO cells. Interestingly, the presence of
halogen groups in R7 and/or R6, responsible for increasing anti-malarial activity, also increased
leishmanicidal activity. Moreover, the compounds without NO2 group in R2' showed considerably
higher activity. Compounds with CF3 or F in R4' show similar activity, but higher activity than those
with CH3O in this position.

Molecules 2012, 17
Table 1. Biological characterization of the thirteen new quinoxaline 1,4-di-N-oxides.
9454
O-
R7
N+
CN
O
R6
N+
O-
NH
N
N
R4'
R2'
Compd. MW
R 6 R 7
R 2'
R 4' IC50 (µM) a IC50 (µM) b CC50 (µM) c SI d
1
2
3
4
5
6
7
8
9
537.5
517
521
572
492.5
472
476
527
422
426
454.5
434
489
320
543.5
H
H
H
Cl
H
H
H
Cl
H
H
H
H
Cl
Cl
NO2
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
F
24.5
44.7
14.6
13.9
18.6
30.5
30.9
18.5
36.3
34.3
12.8
30.4
26.1
0.1
21.8
36.3
41.1
22.7
7.6
23.3
28.8
5.7
23.0
31.3
18.8
30.0
10.9
7.0
17.7
11.0
1.6
0.3
0.5
0.3
0.1
0.8
0.5
0.4
0.4
1.1
0.8
2.5
6.1
1.3
CH3 NO2
F
Cl
Cl
CH3
F
Cl
CH3
F
Cl
CH3
Cl
NO2
NO2
H
H
H
H
H
H
H
6.4
12.1
12.2
2.2
24.1
24.3
47.5
183.5
14.0
10
11
12
13
CQ
DOX
F
CH3O
CH3O
CH3O
H
H
6.4
a
0.4
>10
Selectivity index (SI): DT50 drug/IC50 drug. MW: molecular weight; IC50 against P. falciparum
FCR-3; b IC50 against L. Infantum; c Cytotoxicity in VERO cells; d Selectivity index.
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 (IR), proton nuclear magnetic resonance (1H-NMR), melting point and elemental
microanalyses (CHN). Alugram SIL G/UV254 (0.2 mm layer, Macherey-Nagel GmbH & Co. KG.,
Düren, Germany) was used for TLC and silica gel 60 (0.040–0.063 mm, Merck, Darmstadt, Germany)
1
was used for Flash Column Chromatography. The H-NMR spectra were recorded on a Bruker 400
Ultrashield instrument (400 MHz, Bruker, Billerica, MA, USA), using TMS as internal standard and
with DMSO-d6 as solvent; the chemical shifts are reported in ppm (δ) and coupling constants (J)
values are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), bs (broad singlet), d
(doublet), t (triplet), q (quadruplet), dd (double doublet) 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

Molecules 2012, 17
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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 Cyanoamines 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 [10]. The yield of this reaction
depends on the substituents in position 5 and 6 in the benzofuroxane.
3.1.3. General Procedure for the Synthesis of 2-Cyano-3-(4-phenylpiperazine-1-carboxamido)
Quinoxaline 1,4-Dioxide (CPCQs)
The corresponding cyanoamine (5.0 mmol) was reacted with a slight excess (1.5 equiv.) of
1,1'-carbonyldiimidazole (CDI) in dry tetrahydrofuran (40 mL) during 3hours at 50 °C. The solvent
was removed in vacuo. The solid was then purified by column chromatography with toluene/dioxane
(6:4) as the solvent; this solvent was subsequently removed in vacuo. The intermediate (2.5 mmol) was
stirred with an excess of phenylpiperazine (1.2 equiv.) in chloroform during 3 h at 50 °C and under
nitrogen atmosphere. Next, the solvent was removed in vacuo and the solid was collected and purified
by column chromatography using dichlorometane/methanol (9:1). Finally, the solvent was removed
in vacuo and the solid precipitated with cold diethyl ether, and filtered off to obtain a solid [22].
7-Chloro-2-cyano-3-(4-(2-nitro-4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline 1,4-
dioxide (1). Yield 30%; 1H-NMR δ ppm: 8.30 (s, 1H, H11'); 8.24 (d, 2H, H2', J2'−3' = 2.44 Hz); 8.22 (dd,
1H, H9', J9'−11' = 2.73 Hz, J9'−8' = 3.84 Hz); 8.19 (dd, 2H, H6', J6'−2' = 2.96 Hz, J6'−5' = 3.70 Hz); 8.11 (s,
NH); 7.96 (dd, 2H, H3', J3'−5' = 8.95 Hz, J3'−2' = 9.02 Hz); 7.90 (d, 1H, H8', J8'−9' = 2.01 Hz); 7.88 (d, 1H,
H8, J8−6 = 2.68 Hz); 7.62 (d, 1H, H6, J6−5 = 9.27 Hz); 7.54 (d, 1H, H5, J5−6 = 8.40 Hz); 7.49 (dd, 2H,
H5', J5'−3' = 4.21 Hz, J5'−6' = 8.77 Hz). IR ν cm−1: 3,105 (w, NH); 2,211 (w, C≡N); 1,533 (s, C=O); 1,326
(s, N+O−). Anal. Calc. for C21H15N7O5F3Cl: C: 46.88%; H: 2.79%; N: 18.23%. Found: C: 46.74%; H:
2.94%; N: 18.18%.
2-Cyano-7-methyl-3-(4-(2-nitro-4-(trifluoromethyl)phenyl)piperazine-1-carboxamido)
quinoxaline
1
1,4-dioxide (2). Yield 50%; H-NMR δ ppm: 8.21 (d, 1H, H5, J5−6 = 3.85 Hz); 8.20 (d, 1H, H8,
J8−6 = 1.66 Hz); 8.17 (s, 1H, NH); 7.91 (d, 1H, H6, J6−5 = 2.04 Hz); 7.88 (d, 1H, H8', J8'−9' = 2.43 Hz);
7.51 (s, 1H, H11'); 7.49 (s, 1H, H9'); 3.69 (d, 2H, H2', J2'−3' = 2.66 Hz); 3.25 (dd, 2H, H6', J6'−5' = 2.65 Hz,
J6'−2' = 3.11 Hz); 3.11 (d, 2H, H3', J3'−2' = 5.27 Hz); 2.56 (dd, 2H, H5', J5'−3' = 2.79 Hz, J5'−6' = 3.40 Hz);
2.54 (s, 3H, CH3-C7). IR ν cm−1: 3,098 (w, NH); 2,231 (w, C≡N); 1,549 (s, C=O); 1,328 (s, N+O−). Anal.
Calc. for C22H18N7O5F3: C: 51.06%; H: 3.48%; N: 18.95%. Found: C: 51.28%; H: 3.46%; N: 19.03%.
2-Cyano-7-fluoro-3-(4-(2-nitro-4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline 1,4-
1
dioxide (3). Yield 15%; H-NMR δ ppm: 8.38 (dd, 2H, H3', J3'−5' = 4.94 Hz, J3'−2' = 9.74 Hz); 8.20

Molecules 2012, 17
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(dd,2H, H2', J2'−6' = 2.68 Hz, J2'−3 = 3.37 Hz); 8.13 (d, 1H, H11', J11'−9' = 1.79 Hz); 8.11 (dd, 1H, H9',
J9'−11' = 1.42 Hz, J9'−8' = 2.01 Hz); 7.90 (d, 2H, H5', J5'−6' = 2.64 Hz); 7.88 (dd, 2H, H6', J6'−2' = 2.81 Hz,
J6'−5' = 1.47 Hz); 7.69 (dd, 1H, H6, J6−8 = 5.87 Hz, J6−5 = 9.23 Hz); 7.51 (d, 1H, H8, J8−6 = 2.62 Hz);
7.49 (d, 1H, H8', J8'−9' = 2.67 Hz); 7.44 (d, 1H, H5, J5−6 = 2.13 Hz); 3.55 (s, NH). IR ν cm−1: 3,108 (w,
NH); 2,362 (w, C≡N); 1,533 (s, C=O); 1,321 (s, N+O−). Anal. Calc. for C21H15N7O5F4: C: 48.36%; H:
2.87%; N: 18.80%. Found: C: 47.92%; H: 2.94%; N: 18.31%.
6-7-Dichloro-2-cyano-3-(4-(2-nitro-4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline
1,4-dioxide (4). Yield 20%; 1H-NMR δ ppm: 8.37 (d, 1H, H11', J11'−9' = 1.47 Hz); 8.31 (s, NH); 8.25 (d,
1H, H8, J8−5 = 1.98 Hz); 8.18 (d, 1H, H5, J5−8 = 2.41 Hz); 7.95 (dd, 1H, H9', J9'−11' = 3.31 Hz,
J
9'−8' = 9.84 Hz); 7.88 (d,2H, H2', J2'−3' = 6.93 Hz); 7.73 (d,2H, H6', J6'−5' = 1.89 Hz); 7.51 (m, 2H, H5');
3.59 (d,2H, H3', J3'−2' = 4.38 Hz); 1.09 (t, 1H, H8', J8'−9' = 6.73 Hz, J8'−11' = 6.73 Hz). IR ν cm−1: 3,104
(w, NH); 2,240 (w, C≡N); 1,532 (s, C=O); 1,330 (s, N+O−). Anal. Calc. for C21H14N7O5F3Cl2: C:
44.05%; H: 2.44%; N: 17.13%. Found: C: 43.87%; H: 2.50%; N: 17.36%.
7-Chloro-2-cyano-3-(4-(4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline 1,4-dioxide
1
(5). Yield 65%; H-NMR δ ppm: 8.13 (t,1H, H9'); 7.99 (t, 1H, H8', J8'−9' = 9.06 Hz, J8'−12' = 9.06 Hz );
7.78 (dd, 1H, H12', J12'−8' = 3.86 Hz, J12'−11' = 9.00 Hz); 7.62 (dd, 2H, H6', J6'−2' = 1.21 Hz, J6'−5' = 8.84 Hz);
7.58 (s, NH); 7.54 (d, 2H, H3', J3'−2' = 8.50 Hz); 7.50 (dd, 1H, H11', J11'−9' = 2.30 Hz, J11'−12' = 9.19 Hz);
7.41 (dd, 1H, H6, J6−8 = 8.44 Hz, J6−5 = 9.06 Hz); 7.10 (dd, 2H, H5', J5'−3' = 8.39 Hz, J5'−6' = 9.06 Hz);
6.74 (d, 1H, H5, J5−6 = 7.39 Hz); 3.57 (d, 1H, H8, J8−6 = 1.47 Hz); 3.10 (d, 2H, H2', J2'−3' = 5.43 Hz).
IR ν cm−1: 3,107 (w, NH); 2,234 (w, C≡N); 1,524 (s, C=O); 1,333 (s, N+O−). Anal. Calc. for
C21H16N6O3F3Cl: C: 51.16%; H: 3.24%; N: 17.05%. Found: C: 50.85%; H: 3.12%; N: 16.93%.
2-Cyano-7-methyl-3-(4-(4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline 1,4-dioxide
1
(6). Yield 23%; H-NMR δ ppm: 8.24 (d, 1H, H5, J5−6 = 8.08 Hz); 8.17 (d, 1H, H6, J6−5 = 6.82 Hz);
8.09 (s, 1H, H11'); 8.02 (s, 1H, H9'); 7.94 (s, NH); 7.82 (d, 1H, H8', J8'−9' = 9.78 Hz); 7.54 (d, 2H, H2',
J
2'−3' = 2.85 Hz); 7.53 (d, 2H, H5', J5'−6' = 4.83 Hz); 7.39 (d, 1H, H12', J12'−11' = 4.54 Hz); 7.32 (s, 1H, H8);
7.12 (d, 2H, H3', J3'−2' = 2.91 Hz); 7.10 (d, 2H, H6', J6'−5' = 1.88 Hz); 2.44 (s, 3H, CH3-C7). IR ν cm−1:
3,082 (w, NH); 2,233 (w, C≡N); 1,547 (s, C=O); 1,329 (s, N+O−). Anal. Calc. for C22H19N6O3F3: C:
55.93%; H: 4.02%; N: 17.79%. Found: C: 55.67%; H: 3.88%; N: 18.27%.
2-Cyano-7-fluoro-3-(4-(4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline 1,4-dioxide
1
(7). Yield 31%; H-NMR δ ppm: 7.89 (dd, 1H, H5, J5-8 = 2.75 Hz, J5-6 = 8.98 Hz); 7.71 (dd, 1H, H6,
J6−8 = 2.75 Hz, J6−5 = 7.96 Hz); 7.67 (s, 1H, H8); 7.52 (dd, 2H, H6', J6'−2' = 7.83 Hz, J6'−5' = 9.14 Hz);
7.45 (s, 2H, H2'); 7.38 (d, 1H, H9', J9'−8' = 8.55 Hz); 7.10 (dd, 2H, H5', J5'−3' = 8.94 Hz, J5'−6' = 9.57 Hz);
6.97 (d, 1H, H11', J11'−12' = 9.11 Hz); 6.74 (dd, 2H, H3', J3'−5' = 7.94 Hz, J3'−2' = 9.35 Hz); 3.70 (s, NH);
2.98 (d, 1H, H12', J12'−11' = 7.19 Hz); 2.59 (d, 1H, H8', J8'−9' = 6.56 Hz). IR ν cm−1: 3,106 (w, NH); 2,232
(w, C≡N); 1,532 (s, C=O); 1,332 (s, N+O−). Anal. Calc. for C21H16N6O3F4: C: 52.94%; H: 3.36%; N:
17.64%. Found: C: 52.53%; H: 3.32%; N: 17.76%.
6,7-Dichloro-2-cyano-3-(4-(4-(trifluoromethyl)phenyl)piperazine-1-carboxamido) quinoxaline 1,4-
1
dioxide (8). Yield 17%; H-NMR δ ppm: 8.31 (s, 1H, H5); 7.88 (s, 1H, H8); 7.73 (d, 2H, H6',

Molecules 2012, 17
9457
J6'−5' = 1.75 Hz); 7.54 (dd, 2H, H3', J3'−5' = 3.60 Hz, J3'−2' = 9.32 Hz); 7.11 (dd, 2H, H5', J5'−3' = 4.68 Hz,
J5'−6' = 8.37 Hz); 6.74 (dd, 1H, H8', J8'−12' = 5.93 Hz, J8'−9' = 8.95 Hz); 3.68 (dd, 1H, H12', J12'−8' = 2.93 Hz,
J12'−11' = 5.26 Hz); 3.57 (s, NH); 3.10 (dd, 2H, H2', J2'−6' = 3.92 Hz, J2'−3' = 5.90 Hz); 2.59 (d, 1H, H9',
J9'−8' = 1.89 Hz); 2.48 (dd, 1H, H11', J11'−9' = 1.55 Hz, J11'−12' = 2.72 Hz). IR ν cm−1: 3,111 (w, NH);
2,364 (w, C≡N); 1,525 (s, C=O); 1,331 (s, N+O−). Anal. Calc. for C21H15N6O3F3Cl2: C: 47.81%; H:
2.84%; N: 15.93%. Found: C: 47.32%; H: 2.97%; N: 16.07%.
2-Cyano-3-(4-(4-fluorophenyl)piperazine-1-carboxamido)-7-methylquinoxaline 1,4-dioxide (9). Yield
79%; 1H-NMR δ ppm: 8.23 (d, 1H, H5, J5−6 = 8.39 Hz); 8.16 (s, 1H, H9); 7.94 (s, 1H, H11); 7.81 (d, 1H,
H6, J6−5 = 8.46 Hz); 7.52 (d, 1H, H8’, J8'−9' = 8.36 Hz); 7.39 (s, 1H, H8); 7.33 (s, 1H, NH); 7.17 (d, 1H,
H12', J12'−11' = 8.17 Hz); 7.08 (d, 2H, H2', J2'−3' = 6.02 Hz); 7.06 (d, 2H, H6', J6'−5' = 7.13 Hz); 7.01 (d, 2H,
H3', J3'−2' = 4.73 Hz); 6.99 (d, 2H, H5', J5'−6' = 6.17 Hz); 3.19 (s, 3H, CH3-C7). IR ν cm−1: 3,079 (w,
NH); 2,231 (w, C≡N); 1,538 (s, C=O); 1,344 (s, N+O−). Anal. Calc. for C21H19N6O3F: C: 59.71%; H:
4.50%; N: 19.90%. Found: C: 59.80%; H: 4.55%; N: 19.51%.
2-Cyano-7-fluoro-3-(4-(4-fluorophenyl)piperazine-1-carboxamido)quinoxaline 1,4-dioxide (10). Yield
1
23%; H-NMR δ ppm: 8.41 (d, 1H, H5, J5−6 = 5.14 Hz); 8.38 (d, 1H, H6, J6−5 = 5.96 Hz); 8.14 (s, 1H,
H9'); 8.12 (s, 1H, H11'); 7.91 (t, 2H, H5', J5'−6' = 8.92 Hz, J5'−3' = 8.92 Hz); 7.45 (s, 1H, H8); 7.10 (s, 1H,
H8'); 7.07 (s, 2H, H2'); 7.05 (s, 1H, H12'); 7.01 (d, 2H, H3', J3'−2' = 3.69 Hz); 7.00 (d, 2H, H6', J6'−5' = 3.25
Hz); 6.99 (s, 1H, NH). IR ν cm−1: 3,109 (w, NH); 2,231 (w, C≡N); 1,534 (s, C=O); 1,354 (s, N+O−).
Anal. Calc. for C20H16N6O3F2: C: 56.33%; H: 3.75%; N: 19.71%. Found: C: 55.84%; H: 3.68%; N:
19.53%.
7-Chloro-2-cyano-3-(4-(4-methoxyphenyl)piperazine-1-carboxamido)quinoxaline 1,4-dioxide (11).
1
Yield 12%; H-NMR δ ppm: 8.24 (s, 1H, H8); 8.19 (d, 1H, H6, J6−5 = 8.47 Hz); 8.13 (d, 1H, H5,
J5−6 = 8.63 Hz); 8.07 (s, 1H, NH); 7.90 (d, 1H, H9', J9'−8' = 7.83 Hz); 7.63 (d, 1H, H12', J12'−11' = 6.55 Hz);
7.58 (s, 1H, H11'); 7.53 (d, 1H, H8', J8'−9' = 8.73 Hz); 6.93 (d, 2H, H6', J6'−5' = 6.87 Hz); 6.91 (d, 2H, H2',
J2'−3' = 3.39 Hz); 6.84 (d, 2H, H3', J3'−2' = 7.41 Hz); 6.82 (d, 2H, H5', J5'−6' = 6.48 Hz); 3.63 (s,3H,
CH3O). IR ν cm−1: 3,115 (w, NH); 2,228 (w, C≡N); 1,540 (s, C=O); 1,351 (s, N+O−). Anal. Calc. for
C21H19N6O4Cl: C: 55.44%; H: 4.18%; N: 18.48%. Found: C: 54.97%; H: 3.95%; N: 18.21%.
2-Cyano-3-(4-(4-methoxyphenyl)piperazine-1-carboxamido)-7-methylquinoxaline 1,4-dioxide (12).
1
Yield 32%; H-NMR δ ppm: 8.02 (d, 1H, H5, J5−6 = 8.69 Hz); 7.94 (d, 1H, H8, J8−6 = 2.45 Hz); 7.61
(dd, 1H, H12', J12'−8' = 1.78 Hz, J12'−11' = 8.64 Hz); 7.52 (d, 1H, H6, J6−5 = 8.61 Hz); 7.39 (d, 2H, H5',
J
5'−6' = 3.60 Hz); 7.32 (s, 2H, H6'); 7.27 (dd, 1H, H8', J8'−12'' = 1.61 Hz, J8'−9' = 8.82 Hz); 6.92 (d, 1H, H9',
J9'−8' = 8.44 Hz); 6.84 (d, 2H, H3’, J3'−2' = 8.67 Hz); 3.69 (t, 3H, CH3O, J7−8 = 3.39 Hz, J7−6 = 3.39 Hz);
3.62 (s, 2H, H2'); 3.13 (s, 1H, NH); 2.55 (s, 3H, CH3-C7); 1.23 (s, 1H, H11'). IR ν cm−1: 3,120 (w, NH);
2,230 (w, C≡N); 1,546 (s, C=O); 1,328 (s, N+O−). Anal. Calc. for C22H22N6O4: C: 60.82%; H: 5.06%;
N: 19.35%. Found: C: 61.20%; H: 4.61%; N: 19.84%.
6-7-Dichloro-2-cyano-3-(4-(4-methoxyphenyl)piperazine-1-carboxamido)quinoxaline 1,4-dioxide (13).
1
Yield 11%; H-NMR δ ppm: 8.46 (d, 1H, H8', J8'−9' = 8.85 Hz); 8.29 (s, 1H, NH); 8.19 (d, 1H, H12',
J12'−11' = 1.33 Hz); 7.87 (s, 1H, H8); 7.73 (d, 2H, H6', J6'−5' = 2.11 Hz); 6.94 (s, 1H, H5); 6.91 (s, 1H,

Molecules 2012, 17
9458
H9'); 6.85 (s, 1H, H11'); 6.83 (s, 2H, H2'); 3.69 (s, 3H, CH3O); 3.18 (d, 2H, H3', J3'−2' = 3.00 Hz); 3.17 (d,
2H, H5', J5'−6' = 2.12 Hz). IR ν cm−1: 3,115 (w, NH); 2,229 (w, C≡N); 1,542 (s, C=O); 1,352 (s, N+O−).
Anal. Calc. for C21H18N6O4Cl2: C: 51.53%; H: 3.68%; N: 17.17%. Found: C: 51.05%; H: 3.49%;
N: 17.02%.
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 on glucose-enriched RPMI 1640 medium supplemented with gentamicin 0.1 mg/mL and
10% heat-inactivated A+ human serum, as previously described [23]. 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 [24]. Briefly, 250 µL
of total culture medium with the diluted drug and the suspension of human red blood cell in medium
(A+ group, 5% haematocrit) with 1% parasitaemia, were placed into the wells of 96-well microtitre
plates. After 48 h of incubation at 37 °C in a 5% O2, 5% CO2 and 90% N2 atmosphere. 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 was 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-hour incubation period and then compared with a control
sample [25].
3.2.3. In Vitro Antileishmanial Drug Assay
Leishmanicidal activity was determined on axenic cultures of L. infantum 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 [26].
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 in 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
amastigotes transformation was then induced by increasing the temperature to 34 °C. Drugs were then
tested at increasing concentrations.

Molecules 2012, 17
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4. Conclusions
All the tested compounds were almost 100 times less active against Plasmodium than chloroquine.
Consequently they did not deserve further examination as antimalarials. Against Leishmania
compounds 5 and 8 showed good activity and the most cytotoxic compound is four times less toxic
than the reference drug. Unfortunately, these compounds show a low selectivity index. In this context,
we suggest that synthesis should be focused on compounds with halogens at R7 and/or R6 in order to
improve activity and lower toxicity.
Acknowledgments
Carlos Barea is indebted to the Asociación de Amigos de la Universidad de Navarra (Spain) for a
PhD scholarship.
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Sample Availability: Samples of the compounds are available from the authors.
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