molecules Molecules Molecules Molecules 1420-3049 MDPI 10.3390/molecules17089451 molecules-17-09451 Article Antiplasmodial and Leishmanicidal Activities of 2-Cyano-3-(4-phenylpiperazine-1-carboxamido) Quinoxaline 1,4-Dioxide Derivatives Barea Carlos 1 * Pabón Adriana 2 Galiano Silvia 1 Pérez-Silanes Silvia 1 Gonzalez German 3 4 Deyssard Chloe 3 4 Monge Antonio 1 Deharo Eric 3 4 and Aldana Ignacio 1 Research and Development of Novel Drugs Unit, Center for Applied Pharmacobiology Research (CIFA), University of Navarre, Pamplona 31080, Spain; Email: sgaliano@unav.es (S.G.); sperez@unav.es (S.P.-S.); amonge@unav.es (A.M.); ialdana@unav.es (I.A.) Malaria Group, University of Antioquia, Medellín, Colombia; Email: apabon72@gmail.com UMR 152 PharmaDev (Pharmacologie et Pharmacochimie pour le Développement), Université de Toulouse, UPS, 118, rte de Narbonne, F-31062 Toulouse cedex 9, France; Email: germangonzalez.a@hotmail.com (G.G.); c.deyssard@gmail.com (C.D.); ericdeharo@gmail.com (E.D.) 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; Email: cabareari@hotmail.com; Tel.: +34-948-425-653; Fax: +34-948-425-652. 07 08 2012 08 2012 17 8 9451 9461 28 06 2012 20 07 2012 31 07 2012 © 2012 by the authors; licensee MDPI, Basel, Switzerland. 2012

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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.

 quinoxaline piperazine Plasmodium Leishmania VERO 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 [7,8,9,10,11,12,13,14,15]. 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].

Design of new CPCQs as potential drugs against P. falciparum and L. infantum.

 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 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].

molecules-17-09451-scheme1_Scheme 1

Synthetic route to CPCQs 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-17-09451-t001_Table 1

Biological characterization of the thirteen new quinoxaline 1,4-di-N-oxides.

Compd. MW R 6 R 7 R 2' R 4' IC50 (µM) a IC50 (µM) b CC50 (µM) c SI d
1 537.5 H Cl NO2 CF3 24.5 21.8 7.0 0.3
2 517 H CH3 NO2 CF3 44.7 36.3 17.7 0.5
3 521 H F NO2 CF3 14.6 41.1 11.0 0.3
4 572 Cl Cl NO2 CF3 13.9 22.7 1.6 0.1
5 492.5 H Cl H CF3 18.6 7.6 6.4 0.8
6 472 H CH3 H CF3 30.5 23.3 12.1 0.5
7 476 H F H CF3 30.9 28.8 12.2 0.4
8 527 Cl Cl H CF3 18.5 5.7 2.2 0.4
9 422 H CH3 H F 36.3 23.0 24.1 1.1
10 426 H F H F 34.3 31.3 24.3 0.8
11 454.5 H Cl H CH3O 12.8 18.8 47.5 2.5
12 434 H CH3 H CH3O 30.4 30.0 183.5 6.1
13 489 Cl Cl H CH3O 26.1 10.9 14.0 1.3
CQ 320 0.1
DOX 543.5 6.4 0.4 >10

Selectivity index (SI): DT50 drug/IC50 drug. MW: molecular weight; a 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) was used for Flash Column Chromatography. The 1H-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 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 <bold>II</bold>

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 1,4-dioxide (2). Yield 50%; 1H-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-dioxide (3). Yield 15%; 1H-NMR δ ppm: 8.38 (dd, 2H, H3', J3'−5' = 4.94 Hz, J3'−2' = 9.74 Hz); 8.20 (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, J9'−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 (5). Yield 65%; 1H-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 (6). Yield 23%; 1H-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', J2'−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 (7). Yield 31%; 1H-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-dioxide (8). Yield 17%; 1H-NMR δ ppm: 8.31 (s, 1H, H5); 7.88 (s, 1H, H8); 7.73 (d, 2H, H6', 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 23%; 1H-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). Yield 12%; 1H-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). Yield 32%; 1H-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', J5'−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). Yield 11%; 1H-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, 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. <italic>In Vitro</italic> 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. <italic>In Vitro</italic> 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.<italic>In Vitro</italic> 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.

 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.