Synthesis, biological evaluation and structure-activity relationships of new quinoxaline derivatives as anti-Plasmodium falciparum agents

Article abstract of DOI:10.3390/molecules19022166

We report the synthesis and antimalarial activities of eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives, eight of which are completely novel. Compounds 1a and 2a were the most active against Plasmodium falciparum strains. Structure-activity

Full text of DOI:10.3390/molecules19022166

Molecules 2014, 19, 2166-2180; doi:10.3390/molecules19022166
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Biological Evaluation and Structure-Activity
Relationships of New Quinoxaline Derivatives as
Anti-Plasmodium falciparum Agents
Ana Gil 1,*, Adriana Pabón 2,3, Silvia Galiano 1, Asunción Burguete 1,4, Silvia Pérez-Silanes 1,
Eric Deharo 5, Antonio Monge 1 and Ignacio Aldana 1
1
Unidad de Investigación y Desarrollo de Medicamentos, Centro de Investigación en
Farmacobiología Aplicada (CIFA), Universidad de Navarra, c/ Irunlarrea 1, Pamplona 31008,
Spain; E-Mails: sgaliano@unav.es (S.G.); abpnqb@cid.csic.es (A.B.); sperez@unav.es (S.P.-S.);
amonge@unav.es (A.M.); ialdana@unav.es (I.A.)
2
Grupo Malaria, Facultad de Medicina, Universidad de Antioquia, Medellín 050010, Colombia;
E-Mail: apabon72@gmail.com
3
Programa de Biología, Facultad de Ciencias Básicas, Universidad del Atlántico, Barranquilla
080001, Colombia
4
Instituto de Química Avanzada de Cataluña, Consejo Superior de Investigaciones Científicas
(CSIC), c/ Jordi Girona 18-26, Barcelona 08034, Spain
5
PHARMA-DEV, UMR 152 IRD-UPS, Faculté des Sciences Pharmaceutiques,
Université Paul Sabatier, 35 chemin des Maraîchers, 31062 Toulouse Cedex 09, France;
E-Mail: ericdeharo@gmail.com
* Author to whom correspondence should be addressed; E-Mail: ana.gil.villar@unav.es;
Tel.: +34-948-425-653; Fax: +34-948-425-652.
Received: 10 January 2014; in revised form: 8 February 2014 / Accepted: 10 February 2014 /
Published: 18 February 2014
Abstract: We report the synthesis and antimalarial activities of eighteen quinoxaline and
quinoxaline 1,4-di-N-oxide derivatives, eight of which are completely novel. Compounds
1a and 2a were the most active against Plasmodium falciparum strains. Structure-activity
relationships demonstrated the importance of an enone moiety linked to the quinoxaline ring.
Keywords: Plasmodium falciparum; antimalarial agents; quinoxaline; quinoxaline
1,4-di-N-oxide; chalcone

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1. Introduction
Malaria is an infectious disease with an estimated 219 million cases and 660,000 deaths in 2010. Of
these cases 86% correspond to children under 5 years old. In 2012, there were a total of 104 countries
where malaria is considered to be endemic [1].
Plasmodium falciparum is the most dangerous form of the malaria parasite and it is responsible for
a very high percentage of clinical attacks [2]. Artemisinin-based combination therapies (ACTs) are the
standard treatment against uncomplicated P. falciparum malaria. Nevertheless, resistance to ACTs
containing dihydroartemisinin has been reported in Pailin (Cambodia) [3] and resistance to pyrethroids
(used as insecticides) has been detected in 64 countries around the World [1]. Other antimalarial drugs,
such as chloroquine or mefloquine, are not effective enough [4]. FCR-3 P. falciparum is a
chloroquine-resistant strain, but sensitive to pyrimethamine and sulfadoxine [5].
Over the last years, our group has been working on the development and synthesis of new
quinoxaline derivatives. As a result of this line of research, the activities of quinoxaline 1,4-di-N-oxide
derivatives against Mycobacterium tuberculosis [6–13], Trypanosoma cruzi [14,15], Leishmania
amazonensis [16], L. infantum [17], P. falciparum [16–23] and different tumor cells [24–26] have been
reported. The presence of two N-oxides is associated with a significant increase in some biological
properties, such as anticancer [27] or antioxidant [28] activity. On the other hand, some reduced
quinoxalines showed good antitubercular activity [29].
In this article, we report the synthesis and antimalarial activity of some quinoxaline analogs of
chalcones and other compounds derived from them (Figure 1). Chalcones or 1,3-diaryl-2-propen-1-
ones are ,-unsaturated ketones with a large number of biological activities [30]. Different chalcones have
been reported as anti-inflammatory [31], anticancer [32], antitubercular [33,34] or antimalarial [34–38]
agents. With regard to antiplasmodial activity, previous structure-activity relationship studies found
that the enone linker [38] and its trans-configuration [39] are essential in active chalcones.
Some of the compounds belonging to this study were previously tested against L. amazonensis [40]
(series 1), T. cruzi and L. peruviana [41] (series 3) and as cytotoxic agents [42] (compound 1a) and
anti-inflammatory/antioxidant [28,43] agents (series 1, 3 and 7). According to the structure-activity
relationships, these reports affirm that compounds in series 3 have better antioxidant activity than their
analogs in series 1 due to the fact that the former group of molecules lacks the N-oxide groups [28]. On
the other hand, compounds 1d and 3d have an interesting activity against different Leishmania strains,
so the activity is associated with R6/R7 = Me/Me substitution [40,41]. Compound 1a stands out as both
a good anti-inflammatory and cytotoxic agent [42,43].
With the aim of expanding the SAR study of these chalcone analogs and obtaining new compounds
with improved antimalarial activity, we describe herein the synthesis and the relationships between
structure and antiplasmodial activity against the FCR-3 P. falciparum strain of eighteen quinoxaline
and quinoxaline 1,4-di-N-oxide derivatives.

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Figure 1. (a) Design of quinoxaline derivatives as potential antimalarial drugs.
(b) Examples of two numbered compounds obtained in this synthesis.
n=0 / n=1
(a)
O-
n
R7
R6
N+
R
O
O
N+
O-
n
O
O
R6/R7: H/H
H/Cl
H/OMe
O
O
Me/Me
O-
O
(b)
b
N+
1
8
7
1'
4'
8'
5'
2'
3'
2
3
7'
6'
a
6
5
4
N+
O-
O-
O
b
N+
O
O
1
8
7
6'
3'
1'
2'
a
2
3
5'
4'
c+c'
6
5
4
N+
O-
O
2. Results and Discussion
2.1. Chemistry
Synthesis of compounds in series 1, 3 and 7 were previously reported [25,40]. Benzofuroxans I
with Ra/Rb = H/H, H/Cl and H/OMe are commercially available. Benzofuroxan with Ra/Rb = Me/Me
was synthesized by an oxidative cyclization of 4,5-dimethyl-2-nitroaniline according to a previously
described method [44].

Molecules 2014, 19
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Scheme 1 shows the entire synthetic route for obtaining compounds 1a–d, 2a and 3a, 3c and their
cyclopropyl derivatives 4a–d, 5a and 6a, 6c. The starting reagents used, quinoxaline 1,4-di-N-oxides
II and reduced quinoxalines III, were obtained following previously reported procedures [7,27].
Quinoxaline 1,4-di-N-oxides II were synthesized by a classic Beirut reaction [45], using CaCl2 and
1-aminoethanol as the catalysts [46]. Their reduced analogs III were prepared by reduction of the
N-oxide groups [27], for which a reaction temperature of 65 °C and a mixture of ethyl acetate/methanol 1:1
as the solvent [47] was established.
Scheme 1. Synthesis of series 1, 2, 3, 4, 5 and 6.
NO2
NH2
a
O-
N+
O
O
O-
N+
Rb
Ra
Rb
Ra
N
N
Rb
Ra
b
c
O
N
N+
O-
III
I
II
e
d
f
O-
O
O-
N+
O
O
Rb
Ra
N+
O
O
Rb
Ra
Rb
Ra
N
N
O
O
N+
O-
N+
O-
O
O
Series 2
2a
Series 1
Series 3
3a, 3c
1a, 1b, 1c, 1d
g
g
g
O-
N+
O
O-
N+
O
O
Rb
Ra
O
O
Rb
Ra
Rb
Ra
N
N
O
O
N+
O-
N+
O-
O
O
Series 5
5a
Series 4
Series 6
6a, 6c
4a, 4b, 4c, 4d
Ra/Rb: H/H (a); H/Cl (b); H/OMe (c); Me/Me (d)
Reagents and conditions: (a) NaClO, N,N-DMF, 0 °C; (b) 2,4-pentanedione, CaCl2, 1-aminoethanol;
(c) Na2S2O4, AcOEt/MeOH 1:1, 65 °C; (d) 3,4,5-trimethoxybenzaldehyde, 3% NaOH/MeOH, −10 °C;
(e) 2-naphthaldehyde, 5% NaOH/MeOH; (f) 3,4,5-trimethoxybenzaldehyde, 3% NaOH/MeOH, r.t.;
(g) TBAB, TMSOI, 15% aq. NaOH.

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As previously described [48], in reactions of monosubstituted benzofuroxans 7-substituted
quinoxaline 1,4-di-N-oxide derivatives prevail over the 6-isomers. While compound II with R6/R7=
H/Cl was obtained as the major isomer and was isolated with a subsequent purification [25], 2-acetyl-
7-methoxy-3-methylquinoxaline 1,4-di-N-oxide was obtained as the sole isomer [7].
Chalcone analogs 1a–d were synthesized by a previously reported base-catalyzed Claisen-Schmidt
condensation [49], using 3% NaOH in methanol as the catalyst and establishing an optimum reaction
temperature of −10 °C [43]. Their reduced analogs 3a, 3c were synthesized using the same catalyst,
but in this case, the condensation was performed at room temperature, as previously reported [28]. The
compounds 1a–d and 3a, 3c obtained have been previously described [28,47]. Compound 2a was first
synthesized according to a previously reported method [50]. It was a spontaneous reaction that gives
the desired compound in good yield.
New cyclopropyl derivatives 4a–d, 5a and 6a, 6c were obtained according to a previously reported
method [51]. These reactions were carried out between compounds 1a–d, 2a and 3a, 3c and
trimethylsulfoxonium iodide (TMSOI) in the presence of an aqueous solution of NaOH, with
tetrabutylammonium bromide (TBAB) as a phase transfer catalyst and dichloromethane as the solvent.
With regard to the reaction mechanism, Corey and Chaykovsky [52] demonstrated that
trimethylsulfoxonium halides in the presence of a strong base allow the formation of a reactive
compound named dimethylsulfoxonium methylide (DMSY), commonly known as Corey’s reagent [53].
The reaction of DMSY with an ,-unsaturated ketone involves the cyclopropanation of C=C double
bond [54].
As their coupling constant values in the 1H-NMR spectra showed J values ≈ 16 Hz [47], the double
bond geometries of compounds 1a–d and 3a, 3c were trans, just like the newly synthesized 2a.
According to previous reports [55,56], the Corey-Chaykovsky reaction with TMSOI ocurrs with
retention of stereochemistry, giving trans-cyclopropyl derivatives 4a–d, 5a and 6a, 6c [51].
Scheme 2 shows the synthetic route for obtaining compounds 7a–d. These chalcone analogs were
obtained by a microwave-assisted Wittig reaction [28] between 2-formyl-3-methylquinoxaline 1,4-di-
N-oxide derivatives V and a phosphonium ylide VII, both previously synthesized [28,45,57].
Scheme 2. Synthesis of series 7 [28].
O-
O-
O
O-
N+
N+
Rb
Ra
Rb
Ra
N+
Rb
Ra
H
a
b
O
N
N+
O-
N+
O-
V
O-
O
IV
I
Rb
Ra
N+
O
O
e
N+
O-
O
Series 7
7a, 7b, 7c, 7d
O
O
O
PPh3
O
O
Br
O
O
O
H
d
c
Ra/Rb: H/H (a); H/Cl (b); H/OMe (c); Me/Me (d)
O
O
O
O
VII
VI
Reagents and conditions: (a) 2-butanone, morpholine; (b) SeO2, acetonitrile, MW: 5 min, 200 W;
(c) Br2, acetic acid; (d) PPh3, NaH/H2O, toluene; (e) MeOH, MW: 5 min, 25 W.

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2.2. Biological Results
In this study, eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives were tested against a
chloroquine-resistant FCR-3 strain of P. falciparum. All the synthesized compounds and their
biological activities are shown in Tables 1, 2 and 3.
Table 1. In vitro activities of chalcone derivatives.
O-
O
n
R7
R6
N+
R
N+
O-
n
Compound R6
R7
n
R
IC50 (M) a SD b
1a
1b
1c
1d
2a
H
H
H
H
1 3,4,5-trimethoxyphenyl
6.2
27.3
31.1
24.4
5.8
21.5
26.4
0.173
1.7
3.6
20.4
14.0
0.8
5.3
0.3
0.003
Cl 1 3,4,5-trimethoxyphenyl
OMe 1 3,4,5-trimethoxyphenyl
Me Me 1 3,4,5-trimethoxyphenyl
H
H
H
-
H
H
1
naphthyl
3a
0 3,4,5-trimethoxyphenyl
3c
OMe 0 3,4,5-trimethoxyphenyl
CQ c
-
-
-
a
IC50, concentration required for inhibiting growth of FCR-3 P. falciparum strain by 50%
(in M). Mean values of two independent experiments performed in triplicate; b SD, standard
deviation; c Chloroquine, control.
Table 2. In vitro activities of cyclopropyl derivatives.
O-
O
n
n
R7
R6
N+
R
N+
O-
Compound R6
R7
n
R
IC50 (M) a SD b
4a
4b
4c
4d
5a
H
H
H
H
1 3,4,5-trimethoxyphenyl
56.4
20.1
NA d
NA
62.1
NA
6.4
6.9
-
-
20.0
-
Cl 1 3,4,5-trimethoxyphenyl
OMe 1 3,4,5-trimethoxyphenyl
Me Me 1 3,4,5-trimethoxyphenyl
H
H
H
-
H
H
1
naphthyl
6a
0 3,4,5-trimethoxyphenyl
6c
OMe 0 3,4,5-trimethoxyphenyl
NA
0.173
-
CQ c
-
-
-
0.003
a
IC50, concentration required for inhibiting growth of FCR-3 P. falciparum strain by 50%
b
(in M). Mean values of two independent experiments performed in triplicate; SD,
standard deviation; c Chloroquine, control; d NA, not active.

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Table 3. In vitro activities of inverted chalcone derivatives.
O-
O
R7
R6
N+
O
O
N+
O-
O
Compound
R6
H
H
R7
H
Cl
IC50 (M) a
29.0
SD b
3.5
7a
7b
24.2
0.8
7c
H
Me
-
OMe
Me
-
34.3
24.5
0.173
2.0
4.7
0.003
7d
CQ c
a
IC50, concentration required for inhibiting growth of FCR-3 P. falciparum strain by 50%
(in M). Mean values of two independent experiments performed in triplicate; b SD, standard
deviation; c Chloroquine, control.
Almost all the synthesized compounds exhibited some activity against P. falciparum, but none of
them had better IC50 values than chloroquine itself. Compounds 1a and 2a have been chosen as lead
compounds with interesting activities against the parasite. Although both leads were chalcone analogs
with R6/R7 = H/H, compounds 7b and 4b had the best activities among inverted chalcone and
cyclopropyl derivatives. In these cases, the presence of a halogen atom in position 7 led to an increase
in the activity. In fact, 4b was the third most active compound of the study. These results coincide with
previous reports [18], where the most active compounds were those without any substitution on the
quinoxaline ring and with monosubstitution by an electron withdrawing group.
Apart from compound 4b, changing the double bond in chalcones (compounds 1a–d, 2a and 3a, 3c)
for a cyclopropyl structure led to a dramatic drop in the antiplasmodial activity in each case
(compounds 4a–d, 5a and 6a, 6c). The same trend was noted when comparing cyclopropyl derivatives
4a–d with their analogs in series 7.
With regard to series 1 and 7, the inversion of the chalcone did not lead to a change in the
antimalarial activity, except for compound 1a, whose IC50 was significantly lower than that of the
others. Therefore, the orientation of the enone moiety does not appear to be a determining factor for
the antimalarial activity. Finally, only reduced chalcones 3a and 3c showed some interesting activity,
while reduced cyclopropyl derivatives 6a and 6c were completely inactive. According to previous
reports [58], a minimum requirement for the antimalarial activity was the oxygenation of N-1 and N-4
of the quinoxaline ring, because mono and reduced compounds were not active. Nevertheless, in this
study the presence of N-oxides was not decisive for the synthesized compounds to be active.
3. Experimental
3.1. General Information
All of the synthesized compounds were chemically characterized by thin layer chromatography
(TLC), melting point, proton nuclear magnetic resonance (1H-NMR), infrared spectroscopy (IR) and

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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 column chromatography. The 1H-NMR spectra were recorded on a Bruker 400
Ultrashield instrument (400 MHz, Bruker, Billerica, MA, USA), using TMS as the internal standard
and DMSO-d6 as the solvent. Chemical shifts are reported in ppm () and coupling constants (J) values
are given in Hertz (Hz). Signal multiplicities are represented by: s (singlet), d (doublet), t (triplet), q
(quadruplet), dd (double doublet) and m (multiplet). The IR spectra were recorded on a Thermo
Nicolet Nexus FTIR (Madison, WI, USA) in KBr pellets. Frequencies () are reported in cm−1 and
peak intensities are represented by: w (weak), m (medium), s (strong) and vs (very strong). Elemental
microanalyses were obtained on a CHN-900 Elemental Analyzer (Leco, Tres Cantos, Madrid, Spain)
from vacuum-dried samples. The analytical results for C, H and N were within ±0.4 of the theoretical
values, indicating a purity of >95%. All reagents and solvents were purchased from commercial
sources: E. Merck (Darmstadt, Germany), Panreac Química S.A. (Barcelona, Spain), Sigma-Aldrich
Química S.A. (Alcobendas, Madrid, Spain), Acros Organics (Janssen Pharmaceuticalaan, Geel,
Belgium), Scharlau (F.E.R.O.S.A., Barcelona, Spain) and Lancaster (Bischheim-Strasbourg, France).
3.2. General Procedure of Synthesis
3.2.1. Synthesis of 3-Methyl-2-[3-(naphth-2-yl-prop-2-enoyl)] quinoxaline 1,4-di-N-oxide Derivatives
(Series 2)
The appropriate quinoxaline di-N-oxide II (2-acetyl-3-methylquinoxaline 1,4-di-N-oxide, 3 mmol)
and 2-naphthaldehyde (3 mmol) were dissolved in methanol (30 mL). 5% NaOH in methanol (10 mL)
was added dropwise and the reaction mixture was magnetically stirred at room temperature for 15 min,
until a yellow precipitate appeared. The solid obtained was filtered off and washed with diethyl ether.
3-Methyl-2-[3-(naphth-2-yl-prop-2-enoyl)] quinoxaline 1,4-di-N-oxide (2a). Yield 57%. mp 184 °C.
1H-NMR (DMSO-d6)  ppm: 2.40 (s, 3H, CH3); 7.37 (d, 1H, Hb, Jb-a,trans = 16.4 Hz); 7.58 (m, 2H,
H6+H7); 7.91 (d, 1H, H3'); 8.01 (m, 5H, H4'+H5'+H6'+H7'+H8'); 8.03 (d, 1H, Ha, Ja-b,trans = 16.0 Hz); 8.24 (s,
1H, H1'); 8.47 (d, 1H, H8, J8–7 = 8.6 Hz); 8.58 (d, 1H, H5, J5–6 = 8.4 Hz). IR (KBr)  cm−1: 3091 (w,
arC-H); 3055 (w, alkC-H); 3018 (w, C-H); 1675 (m, C=O); 1600 (s, arC=C); 1331 (vs, N-O). Anal. Calc.
for C22H16N2O3: C = 74.14%, H = 4.53%; N = 7.86%; Found: C = 74.19%, H = 4.40%; N = 7.85%.
3.2.2. Synthesis of 3-Methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline 1,4-di-
N-oxide Derivatives (Series 4), 3-Methyl-2-[2-(2-naphthyl)cyclopropanecarbonyl] quinoxaline 1,4-di-
N-oxide Derivatives (Series 5) and 3-Methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl]
quinoxaline Derivatives (Series 6)
The appropriate chalcone 1a–d, 2a and 3a, 3c (1 mmol), trimethylsulfoxonium iodide (TMSOI,
2 mmol) and tetrabutylammonium bromide (TBAB, 0.2 mmol) were dissolved in dichloromethane
(40 mL) and stirred magnetically at room temperature for 15 min. Then a 15% aqueous solution of
NaOH (10 mL) was added dropwise and the reaction mixture was stirred magnetically at room
temperature for 24 h, until the solution turned dark. At this point, excess dichloromethane and water

Molecules 2014, 19
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were added to the reaction. The organic phase was extracted, dried with anhydrous Na2SO4, filtered
and concentrated under reduced pressure. The residue was purified by column chromatography using
ethyl acetate as the eluent. The desired compounds 4a–d, 5a and 6a, 6c were obtained after
recrystallization from ethanol.
3-Methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline 1,4-di-N-oxide (4a). Yield
56%. mp 178–179 °C. 1H-NMR (DMSO-d6)  ppm: 1.86 (m, 2H, Hc+Hc'); 2.40 (s, 3H, CH3); 2.78 (m,
1H, Ha); 2.91 (m, 1H, Hb); 3.59 (s, 3H, p-OCH3); 3.75 (s, 6H, m-OCH3); 6.55 (s, 2H, H2'+H6'); 7.98 (m,
2H, H6+H7); 8.44 (d, 1H, H8, J8–7 = 8.2 Hz); 8.49 (d, 1H, H5, J5–6 = 8.2 Hz). IR (KBr)  cm−1: 3071 (w,
arC-H); 2998 and 2965 (w, C-H); 1690 (m, C=O); 1590 (m, arC=C); 1329 (s, N-O); 1128 (s, C-O-C). Anal.
Calc. for C22H22N2O6: C = 64.37%, H = 5.41%; N = 6.83%; Found: C = 64.08%, H = 5.74%; N =
6.69%.
7-Chloro-3-methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline 1,4-di-N-oxide
1
(4b). Yield 9%. mp 143–144 °C. H-NMR (DMSO-d6)  ppm: 1.88 (m, 2H, Hc+Hc'); 2.39 (s, 3H,
CH3); 2.79 (m, 1H, Ha); 2.87 (m, 1H, Hb); 3.60 (s, 3H, p-OCH3); 3.76 (s, 6H, m-OCH3); 6.54 (s, 2H,
H2'+H6'); 8.01, 8.02 (dd, 1H, H6, J6–8 = 2.3 Hz, J6–5 = 9.2 Hz); 8.42 (d, 1H, H8, J8–6 = 2.2 Hz); 8.48 (d,
1H, H5, J5–6 = 9.2 Hz). IR (KBr)  cm−1: 3101 (w, arC-H); 2935 (w, C-H); 1695 (m, C=O); 1592 (m,
arC=C); 1326 (s, N-O); 1128 (s, C-O-C). Anal. Calc. for C22H21N2O6Cl: C = 59.39%, H = 4.77%; N =
6.30%; Found: C = 59.52%, H = 4.93%; N = 6.15%.
7-Methoxy-3-methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline 1,4-di-N-oxide
1
(4c). Yield 13%. mp 174–175 °C. H-NMR (DMSO-d6)  ppm: 1.84 (m, 2H, Hc+Hc'); 2.37 (s, 3H,
CH3); 2.79 (m, 1H, Ha); 2.90 (m, 1H, Hb); 3.61 (s, 3H, p-OCH3); 3.77 (s, 6H, m-OCH3); 3.98 (s, 3H,
7-OCH3); 6.54 (s, 2H, H2'+H6'); 7.58, 7.61 (dd, 1H, H6, J6–8 = 2.8 Hz, J6–5 = 9.5 Hz); 7.74 (d, 1H, H8,
J8–6 = 2.7 Hz); 8.40 (d, 1H, H5, J5–6 = 9.5 Hz). IR (KBr)  cm−1: 3005 (w, arC-H); 2945 (w, C-H); 1689
(m, C=O); 1587 (m, arC=C); 1323 (s, N-O); 1120 (m, C-O-C). Anal. Calc. for C23H24N2O7: C = 62.71%,
H = 5.50%; N = 6.36%; Found: C = 62.55%, H = 5.83%; N = 6.64%.
3,6,7-Trimethyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline 1,4-di-N-oxide (4d).
1
Yield 24%. mp 187–189 °C. H-NMR (DMSO-d6)  ppm: 1.84 (m, 2H, Hc+Hc'); 2.37 (s, 3H, CH3);
2.47–2.51 (m, 6H, 6,7-CH3); 2.76 (m, 1H, Ha); 2.90 (m, 1H, Hb); 3.59 (s, 3H, p-OCH3); 3.75 (s, 6H,
m-OCH3); 6.54 (s, 2H, H2'+H6'); 8.18 (s, 1H, H8); 8.25 (s, 1H, H5). IR (KBr)  cm−1: 2932 (w, C-H);
1683 (m, C=O); 1589 (m, arC=C); 1326 (s, N-O); 1132 (s, C-O-C). Anal. Calc. for C24H26N2O6: C =
65.73%, H = 5.99%; N = 6.39%; Found: C = 65.41%, H = 5.79%; N = 6.40%.
3-Methyl-2-[2-(2-naphthyl)cyclopropanecarbonyl] quinoxaline 1,4-di-N-oxide (5a). Yield 66%. mp
176–177 °C. 1H-NMR (DMSO-d6)  ppm: 1.93 (m, 1H, Hc); 1.99 (m, 1H, Hc'); 2.40 (s, 3H, CH3); 3.01
(m, 2H, Ha+Hb); 7.38 (d, 1H, H3’); 7.47 (m, 2H, H6'+H7'); 7.80 (s, 1H, H1'); 7.85 (m, 3H, H4'+H5'+H8');
7.96 (m, 2H, H6+H7); 8.42 (d, 1H, H8, J8–7 = 8.2 Hz); 8.48 (d, 1H, H5, J5–6 = 8.5 Hz). IR (KBr)  cm−1:
3091 (w, arC-H); 2959 (w, C-H); 1689 (s, C=O); 1597 (w, arC=C); 1328 (s, N-O). Anal. Calc. for
C23H18N2O3: C = 74.57%, H = 4.91%; N = 7.56%; Found: C = 74.57%, H = 4.90%; N = 7.57%.

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3-Methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline (6a). Yield 41%. mp
155–156 °C. 1H-NMR (DMSO-d6)  ppm: 1.77 (s, 1H, Hc); 1.84 (s, 1H, Hc'); 2.69 (s, 1H, Ha); 2.86 (s,
3H, CH3); 3.57 (m, 1H, Hb); 3.61 (s, 3H, p-OCH3); 3.77 (s, 6H, m-OCH3); 6.59 (s, 2H, H2'+H6'); 7.88
(m, 1H, H6); 7.96 (m, 1H, H7); 8.08 (m, 1H, H8); 8.18 (m, 1H, H5). IR (KBr)  cm−1: 2961 and 2935
(w, C-H); 1679 (s, C=O); 1589 (m, arC=C); 1132 (s, C-O-C). Anal. Calc. for C22H22N2O4: C = 69.81%,
H = 5.87%; N = 7.40%; Found: C = 69.86%, H = 5.83%; N = 7.40%.
7-Methoxy-3-methyl-2-[2-(3,4,5-trimethoxyphenyl)cyclopropanecarbonyl] quinoxaline (6c). Yield
29%. mp 163–164 °C. 1H-NMR (DMSO-d6)  ppm: 1.76 (m, 1H, Hc); 1.84 (m, 1H, Hc'); 2.69 (m, 1H,
Ha); 2.83 (s, 3H, CH3); 3.58 (m, 1H, Hb); 3.63 (s, 3H, p-OCH3); 3.80 (s, 6H, m-OCH3); 3.96 (s, 3H,
7-OCH3); 6.60 (s, 2H, H2'+H6'); 7.52 (d, 1H, H8, J8–6 = 2.8 Hz); 7.58, 7.60 (dd, 1H, H6, J6–8 = 2.8 Hz,
J6–5 = 9.2 Hz); 7.98 (d, 1H, H5, J5–6 = 9.2 Hz). IR (KBr)  cm−1: 2936 (w, C-H); 1680 (s, C=O); 1587
(m, arC=C); 1130 (s, C-O-C). Anal. Calc. for C23H24N2O5: C = 67.62%, H = 5.93%; N = 6.86%; Found:
C = 67.23%, H = 6.13%; N = 6.74%.
3.3. In Vitro Antiplasmodial Drug Assay
Chloroquine-resistant FCR-3 strain of P. falciparum was cultivated at 37 °C in 5% CO2, 5% O2 in a
balanced N2 atmosphere environment on RPMI 1640 medium supplemented with gentamicin
0.1 mg/mL and 10% heat-inactivated A+ human serum, as previously described [59]. The drugs,
dissolved in dimethyl sulfoxide, were added at final concentrations ranging from 250 to 0.1 M. 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 [60]. Briefly,
250 L of total culture medium with the diluted drug and the suspension of human red blood cells in
medium (A+ group, 5% hematocrit) with 1% parasitemia 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 was calculated.
4. Conclusions
Eighteen quinoxaline and quinoxaline 1,4-di-N-oxide derivatives have been synthesized with the
aim of studying their antimalarial activity. The SAR study suggested that the chalcone and inverted
chalcone moieties can act as useful linkers in the search for antimalarial ligands. Moreover, the similar
activities of series 1, 3 and 7 allow us to affirm that the enone moiety plays an important role in the
antimalarial activity of these compounds, but not its orientation. Compounds 1a and 2a showed the
best antiplasmodial activity. In general, the addition of cyclopropyl moiety dramatically reduces the
biological activity in each case. We are not able to unequivocally confirm that N-oxides are essential
for the antiplasmodial activity. These results show that further structural modifications may provide
better antimalarial compounds.

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Acknowledgments
We wish to express our gratitude to the PIUNA Project of the University of Navarra for the
financial support provided. Ana Gil is grateful to the Navarra Government for a grant.
Author Contributions
The listed authors contributed to this work as described in the following. Ana Gil together with
Silvia Galiano carried out much of the synthetic work and prepared the manuscript. Asunción
Burguete carried out the synthesis of chalcones. Adriana Pabón together with Eric Deharo conducted
assays against P. falciparum and helped interpreting the results. Ignacio Aldana proposed the subject
and monitored the progress of the ongoing research. Silvia Pérez-Silanes and Antonio Monge
contributed with valuable discussions and scientific input. All authors helped preparing the manuscript
and approved the final version.
Conflicts of Interest
The authors declare no conflict of interest.
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