New salicylamide and sulfonamide derivatives of quinoxaline 1,4-di-N-oxide with antileishmanial and antimalarial activities

Article abstract of DOI:10.1016/j.bmcl.2011.05.125

Continuing with our efforts to identify new active compounds against malaria and leishmaniasis, 14 new 3-amino-1,4-di-N-oxide quinoxaline-2- carbonitrile derivatives were synthesized and evaluated for their in vitro antimalarial and antileishmanial activi

Full text of DOI:10.1016/j.bmcl.2011.05.125

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4498–4502
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New salicylamide and sulfonamide derivatives of quinoxaline 1,4-di-N-oxide
with antileishmanial and antimalarial activities
Carlos Barea a, Adriana Pabón b, Denis Castillo c, Mirko Zimic d, Miguel Quiliano d, Silvia Galiano a,
Silvia Pérez-Silanes a, Antonio Monge a, Eric Deharo e,f, Ignacio Aldana a,
⇑
a Unidad en Investigación y Desarrollo de Medicamentos, Centro de Investigación en Farmacobiología Aplicada (CIFA), University of Navarra, Pamplona, Spain
b Grupo Malaria, Universidad de Antioquia, Medellín, Colombia
c Unidad de Parasitología Celular, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
d Bioinformatics Unit – Drug Design Group, Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Lima, Peru
e Université de Toulouse; UPS; UMR 152 (PHARMA-DEV, Pharmacochimie et Pharmacologie pour le Développement), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France
f Institut de Recherche pour le Développement; UMR-152; Mission IRD Casilla 18-1209 Lima, Peru
a r t i c l e i n f o
a b s t r a c t
Article history:
Continuing with our efforts to identify new active compounds against malaria and leishmaniasis, 14 new
3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivatives were synthesized and evaluated for their
in vitro antimalarial and antileishmanial activity against Plasmodium falciparum Colombian FCR-3 strain
and Leishmania amazonensis strain MHOM/BR/76/LTB-012A. Further computational studies were carried
out in order to analyze graphic SAR and ADME properties. The results obtained indicate that compounds
with one halogenous group substituted in position 6 and 7 provide an efficient approach for further
development of antimalarial and antileishmanial agents. In addition, interesting ADME properties were
found.
Received 12 May 2011
Revised 30 May 2011
Accepted 31 May 2011
Available online 12 June 2011
Keywords:
Quinoxaline
N-Oxides
Ó 2011 Elsevier Ltd. All rights reserved.
Malaria
Leishmaniasis
Malaria is by far the world’s most important tropical parasitic
disease and one of the oldest diseases known to mankind. For
nearly half a century, chloroquine (CQ) has been the primary ther-
apy of choice. However, CQ-resistant Plasmodium falciparum is now
observed in nearly all of the malaria-endemic regions and causes
the most deadly form of malaria.1 Mortality, currently estimated
at over a million people per year, has risen in recent years, probably
due to increasing resistance. Therefore, it is necessary to develop
cheaper and more effective drugs against the parasite.2,3 Leishman-
iasis is generally recognized for its cutaneous form which causes
non-fatal disfiguring lesions, although epidemics of the potentially
fatal visceral form cause thousands of deaths. Today, this disease
threatens about 350 million people, and 12 million people are be-
lieved to be currently infected, with about 1–2 million estimated
new cases occurring every year. Most available drugs against Leish-
maniasis are costly, require long treatment regimes and are becom-
ing more and more ineffective, requiring the discovery of new
drugs.4 Moreover, since the introduction of miltefosine at the
beginning of this century, no new antileishmanial compounds have
been approved for human treatment.5
Many classes of organic compounds have been tested, with spe-
cial attention being paid to nitrogen heterocycles, five- and six-
membered rings. Quinoxalines display diverse pharmacological
activities as antibacterial, antiviral, anticancer and antiparasitic
agents and more specifically, their 1,4-di-N-oxides, which increase
the biological properties enormously.6 Our research project led to
the publication of several papers, in which both synthesis and bio-
logical activity assessments have been described for a large number
of quinoxaline and quinoxaline 1,4-di-N-oxide derivatives with a
variety of substituents in positions 2, 3, 6 and 7 of the quinoxaline
ring.7–10 In an attempt to exalt antiparisitic activity of quinoxaline
derivatives, our group has synthesized different series with promis-
ing results, one of them being the introduction of a carbonitrile
group in position 2, which increases the antiparasitic activity, and
an amine group in position 3, with the aim of linking together
new molecules with interesting activities. We synthesized and
evaluated in vitro 14 new 3-amino-1,4-di-N-oxide quinoxaline-2-
carbonitrile analogues against P. falciparum Colombian FCR-3 strain
and Leishmania amazonensis MHOM/BR/76/LTB-012A strain. The
ADME properties of all molecules were calculated in silico.
We have prepared 14 new quinoxaline 1,4-di-N-oxide deriva-
tives. The benzofuroxane starting compounds (BFX, I, Schemes 1
and 2) have been prepared using previously described methods.11,12
The 3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivatives
(cyanoamines, II) were obtained by the Beirut reaction from the
⇑
Corresponding author. Tel.: +34 948 425653; fax: +34 948 425652.
E-mail address: ialdana@unav.es (I. Aldana).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.125

C. Barea et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4498–4502
4499
Scheme 1. General synthesis of sulfonamides. Reagents and conditions: (i) malononitrile, DMF, triethylamine, 15–90%; (ii) pyridine, 0° C, 13-15%.
Scheme 2. General synthesis of acetoxybenzamides. Reagents and conditions: (i) malononitrile, DMF, triethylamine, 15–90%; (ii) dry THF, 25%.
corresponding BFX with malononitrile using N,N-dimethylformam-
ide (DMF) as solvent and triethylamine as catalyst.13
With regard to Leishmania, compounds 4 and 5 inhibited 50% of
parasite growth at approximately 3 M, being almost 10–15 times
l
With regard to Scheme 1, the next stage consists of reacting
3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile derivatives
with aromatic sulfonyl chloride derivatives in order to synthesize
sulfonamides which have shown interesting antileishmanial
activity.14
These quinoxaline derivatives were tested for their activity
against two parasites: P. falciparum (responsible for malaria)
and L. amazonensis (responsible for cutaneous New World leish-
maniasis), and against non-tumorigenic cell line (Murine Perito-
neal Macrophages: MPM). Biological activities and graphical
representation of the structure–activity relationship for series 1
and 2 are presented in Table 1, Table 2 and Figure 1(a,b). None
of the sulfonamides (compounds 1–7) showed interesting activ-
ity against malaria parasite, being 60 to >80 times less active
than chloroquine. Similarly, most of acetoxybenzamides were
scarcely active, being 90 to >100 times less active than chloro-
quine. Only compounds 9 and 14 showed some activity, with
less active than the reference drug amphotericin B. As we men-
tioned before, in order to be active, the R7 position must be occu-
pied by one electronegative atom Cl, because when missing (7),
the activity drops dramatically (86 lM). The paranitrophenyl sub-
stituent is also responsible for the activity in association with Cl in
R7 position, because once it is charged by a naphtyl group (1), the
activity lowers ten times. Interestingly, the presence of withdraw-
ing groups, such as o-nitro and p-nitro, in the sulfonamide region is
correlated to the activity (see Fig. 1b). Nevertheless, compounds 4
and 5 showed a low Selectivity Index (2–4), almost 10 times lower
than the SI of amphotericin B. Although compound 2 and 6 were
almost five times less active than compounds 4 and 5, we decided
to test them against Leishmania-infected macrophages, because
these products were not toxic against non-infected macrophages
(>245 and 89 lM respectively).
In infected macrophages, compound 2 was 7 times more active
than 6 and showed the best Selectivity Index (>15), being very
close to that of amphotericin B. In the second series, the most ac-
tive compound against Leishmania (14) was 2–3 times less active
than compounds 4 and 5. It appears that in order to be leishman-
icidal, the R7 position must be occupied by a CH3 or a F. The activity
drops when Cl or CH3O are in R7 position. Nevertheless, when the
active compounds were tested on non-infected macrophage, they
showed some toxicity, being almost as toxic on macrophage as
on Leishmania.
A computational study designed to predict the ADME properties
of our salicylamide and sulfonamide derivatives of quinoxaline
was performed (results are presented in Table 3). Topological polar
surface area (TPSA) is a good indicator of drug absorption in the
intestines, Caco-2 monolayer penetration, and blood–brain barrier
crossing.15 TPSA was used to calculate the percentage of absorption
(%ABS) following the equation: %ABS = 109 À 0.345 Â TPSA, as re-
ported.16 In addition, the number of rotable bonds (n-ROTB), and
Lipinski’s rule of five were also calculated.17 The most active
compounds found for antileishmanial and antimalarial bioassays
an IC50 of approximately 10 lM (although they were almost 30
times less active than CQ). Interestingly, a Cl (9) or a F (14) in
R7 position increased the activity 4–7 times when compared to
a H or a CH3 in the same position. As shown in Figure 1(b),
the position of one electronegative atom (F, Cl or O), represented
as a blue field, is clearly important in order for the atoms to act
as possible hydrogen bond acceptors. This contrasts with the
groups CH3 and H, which projected hydrophobic regions (repre-
sented as golden fields).
Compound 9, with an acetoxybenzamide in position 3, is almost
twice as active as compound 4, the most active compound of the
first series. From a spatial point of view, according to Figure
1(a,b), this could mean that a strong concentration of electronega-
tive atoms (potential hydrogen bond acceptors) in one same plane
is a good feature for acetoxybenzamides. Therefore, we can say that
o-nitrophenyl, p-nitrophenyl and 2-naphtyl sulfonamides lower the
activity. The most active compound against malaria was compound
14 (IC50 7,4 lM), followed by compound 9 (10.8 lM).

4500
C. Barea et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4498–4502
Table 1
Biological characterization of sulfonamides
a
c
e
Compd
MW
R6
R7
Ar
IC50
(l
M)
CQb index
IC50 amas (
l
M)
Anfd index
CC50 Mø (
lM)
SIf
1
2
3
4
5
6
7
426.6
406.1
420.1
421.6
421.6
456.1
387.1
320
H
H
CH3
H
H
Cl
2-Naphtyl
2-Naphtyl
2-Naphtyl
o-Nitrophenyl
p-nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
>23.4
>24.6
>23.8
17.4 0.5
>23.7
>78
>82
>79
58
>79
62
20 1.4
16.3 0.8
>100
3.1 0.1
2.1 0.1
15.9 1.3
86.3 8.5
20 1.4
0.2
100
81.5
>100
15.5
10.5
À15
430
100
1
88.6 5.6
>245.6
NT
6.8 0.8
7.9 0.3
89.1 6.4
NT
4.4
>15.1
NT
2.2
3.8
5.6
—
CH3
CH3
Cl
Cl
Cl
Cl
H
18.7
20
1
H
2
67
1
CQ
Anf B
0.2 0.1
88.6 5.6
4.4
4.4
22
320
Selectivity index (SI): CC50 drug/IC50 drug.
MW molecular weight; NT: not tested.
a
IC50 against P. falciparum FCR-3.
CQ Index: IC50 drug/IC50 CQ.
b
c
IC50 against axenic amastigotes of L. amazonensis.
Anf Index: IC50 of compounds /IC50 amphotericin B.
Cytotoxicity in murine peritoneal macrophages.
Selectivity index.
d
e
f
Table 2
Biological characterization of acetoxybenzamides
a
c
e
Compd
MW
R6
R7
IC50
(lM)
CQb index
IC50 amas (
lM)
Anf indexd
CC50 Mø (
lM)
SIf
8
9
364
398.5
378
394
400
392
382
320
H
H
H
H
F
CH3
H
H
Cl
41.4 1.1
10.8 1.8
52.8 1.4
27 1.7
138
36
176
90
120
198
25
111.8 3.2
33.6 1.3
18.8 0.3
42.4 1.7
14.8 0.1
17.6 0.6
7.3 0.1
559
168
94
212
74
NT
NT
—
—
10
11
12
13
14
CQ
Anf B
CH3
CH3O
F
CH3
F
21.7 1.9
NT
10.5 0.7
12.5 1.1
13.6 1.6
1.1
—
0.7
0.7
1.8
36 0.9
59.4 2.0
7.4 0.6
0.3 0.01
88
36.5
1
0.2 0.01
1
4.4 0.1
22
Selectivity Index (SI): CC50 drug/ IC50 drug.MW molecular weight; NT: not tested.
a
IC50 against P. falciparum FCR-3.
CQ Index: IC50 drug/ IC50 CQ.
IC50 against axenic amastigotes of L. amazonensis.
Anf Index: IC50 of compounds /IC50 amphotericin B.
Cytotoxicity in murine peritoneal macrophages.
Selectivity index.
b
c
d
e
f
(compounds 4, 5, 9 and 14) showed medium percentages of
intestinal absorption, with mean values of 58%, and an excellent
n-ROTB, ranging only from 3 to 4.18 None of these compounds
violated any of the Lipinski’s parameters, an important characteris-
tic for future drug-development.
and sulfonamides derivatives were found. Simple Pearson
correlation values, which are useful for measuring the association
between two variables, were calculated between biological
activities reported in Table 1 and Table 2, and physical chemical
properties in Table 3. In contrast to previously reported results8,9
,
On the other hand, correlations between molecular descriptors
such as molecular weight, log P and activities of acetoxybenzamides
whose derivatives only correlate between log P and activities, in
our case, an important element for the activity was also conferred

C. Barea et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4498–4502
4501
Figure 1. Graphical representation of the structure-activity relationship for sulfonamides (a) and acetoxybenzamides (b). Field Points are colored as follows: Blue; negative
field points (like to interact with positives/H-bond donors on a protein), red; positive field points (like to interact with negatives/H-bond acceptors on a protein), yellow; van
der Waals surface field points (describing possible surface/vdW interactions), gold/orange; Hydrophobic field points (describe regions with high
polarizability/hydrophobicity).
by MW of the derivatives, an important parameter that is directly
related with the size of the molecule, which is useful to illustrate
the influence of the shape and structural features of a molecule.
In the second series, the reported values showed a correlation
between MW and antimalarial activity and antileishmanial
activity respectively, expressed as log (1/IC50), in which higher
values exponentially indicate greater potency (the logarithm of
inverse of the IC50 is classically used in QSAR studies). Compounds
in the second series followed a positive linear relationship
(r = 0.47 for malaria, r = 0.64 for Leishmania and undetermined
for cytotoxicity in murine peritoneal macrophages) while the
first series only followed a negative linear relationship between
log P and antimalarial activity (r = À0.56), also expressed as log
(1/IC50).
The seven new sulfonamides presented in this paper were pre-
pared through the synthetic route illustrated in Scheme 1.

4502
C. Barea et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4498–4502
Table 3
Physical Chemical Properties of quinoxaline derivativesa
ID
rule
%ABS TPSA (Å)
n-
RT
Molecular weight
<500
mi Log P
<5
Lipophilicity descriptorb
n-OHNH donors
65
n-NO acceptors
610
Lipinski’s violations
61
6140
(R
p)
1
2
3
4
5
6
7
8
67.28 120.9
67.27 120.8
67.27 120.8
51.48 166.7
51.48 166.7
51.48 166.7
51.48 166.7
69.98 113.1
64.11 130.1
64.11 130.1
60.94 139.3
64.11 130.1
64.11 130.1
64.11 130.1
3
3
3
4
4
4
4
4
4
4
5
4
4
4
426.84
406.42
420.45
421.79
421.79
456.22
387.33
364.32
398.76
378.34
394.34
400.29
392.37
396.33
1.19
0.96
1.34
0.65
0.43
0.80
0.65
0.65
1.25
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
8
8
8
11
11
11
11
9
9
9
10
9
9
0
0
0
1
1
1
1
0
0
0
0
0
0
0
À0.08
À0.03
0.57
À0.68
À0.47
0.18
À0.04
À0.43
À0.23
0.33
0
9
0.65
0.43
0.04
0.24
0.80
0.14
10
11
12
13
14
À0.33
9
a
%ABS, percentage of absorption, calculated by:%ABS = 109 À (0.345 Â TPSA); TPSA, topological polar surface area; n-RT, number of rotable bonds; mi log P, logarithm of
compounds partition coefficient between n-octanol and water.
b
Hydrophilic–lipophilic fragments determined for changes in R7 and R6, calculated by the sum of
Rp values, using compounds 7 and 8, and the fragment constant method
as Ref.20.
With regard to Scheme 2, the final synthetic route consists in
combining 3-amino-1,4-di-N-oxide quinoxaline-2-carbonitrile
derivatives with o-acetylsalicyloyl chloride, used for the treatment
of infections caused by protozoans, bacteria and viruses.19
In the last stage of the synthesis of sulfonamides, the tempera-
ture is critical and must remain below 0 °C, whereas the last stage
of synthesis of acetoxybenzamides is carried out at room temper-
ature. In addition, there is a loss of yield due to the purification
by column chromatography, decreasing from 50% to 25% (acet-
oxybenzamides) and 13–15% (sulfonamides).
A computational study for prediction of ADME properties of all
the molecules was performed using Molinspiration online property
calculation toolkit (MolinspirationCheminformatics). Parameters
such as solubility (mi log P), Topological Polar Surface Area
(TPSA)15, and absorption (%ABS) were calculated using the formula:
% ABS = 109 À (0.345 Â TPSA).16 Violations of Lipinski’s rule-of-five
were evaluated.17
Analysis of structure-activity relationship was performed using
Cresset’s field technology (http://www.cresset-group.com/) which
condenses the molecular fields down to a set of points around the
molecule, termed ‘Field Points’.25 Field Points are the local extrema
of the electrostatic, van der Waals and hydrophobic potentials of
the molecule. Throughout FielViewÒ software, the Field Points are
colored as follows: Blue; negative field points (like to interact with
positives/H-bond donors on a protein), red; positive field points
(like to interact with negatives/H-bond acceptors on a protein), yel-
low; van der Waals surface field points (describing possible surface/
vdW interactions), or gold/orange; Hydrophobic field points (de-
scribe regions with high polarizability/hydrophobicity).
Quinoxaline derivatives harboring a halogenous group in posi-
tions 6 and 7 present interesting activity against Plasmodium and
Leishmania parasite and show interesting in silico ADME character-
istics. These products should be the starting point for the synthesis
of antiprotozoal compounds.
Denis Castillo. Carlos Barea is indebted to the University of Navarra
(Spain) for PhD scholarship.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.05.125.
References and notes
1. Vicente, E.; Charnaud, S.; Bongard, E.; Villar, R.; Burguete, A.; Solano, B.; Ancizu,
S.; Perez-Silanes, S.; Aldana, I.; Vivas, L.; Monge, A. Molecules 2008, 13, 69.
2. Biot, C.; Chibale, K. Infect. Disord. Drug Targets 2006, 2, 173.
3. WHO:
html.
http://www.who.int/malaria/world_malaria_report_2010/en/index.
4. WHO: http://www.who.int/leishmaniasis/en/. Accessed 27 March, 2011.
5. Kedzierski, L.; Sakthianandeswaren, A.; Curtis, J. M.; Andrews, P. C.; Junk, P. C.;
Kedzierska, K. Curr. Med. Chem. 2009, 16, 599.
6. Carta, A.; Corona, P.; Loriga, M. Curr. Med. Chem. 2005, 12, 2259.
7. Monge, A.; Palop, J. A.; Piñol, A.; Martínez-Crespo, F. J.; Narro, S.; González, M.;
Sáinz, Y.; López de Ceráin, A. J. Heterocycl. Chem. 1994, 31, 1135.
8. Burguete, A.; Estevez, Y.; Castillo, D.; González, G.; Villar, R.; Solano, B.; Vicente,
E.; Pérez-Silanes, S.; Aldana, I.; Monge, A.; Sauvain, M.; Deharo, E. Memorias do
Oswaldo Cruz 2008, 103, 778.
9. Estevez, Y.; Quiliano, M.; Burguete, A.; Zimic, M.; Málaga, E.; Verástegui, M.;
Pérez-Silanes, S.; Aldana, I.; Monge, A.; Castillo, D.; Deharo, E. Exp. Parasitol.
2011, 127, 745.
10. Ancizu, S.; Moreno, E.; Torres, E.; Burguete, A.; Perez-Silanes, S.; Benitez, D.;
Villar, R.; Solano, B.; Marin, A.; Aldana, I.; Cerecetto, H.; Gonzalez, M.; Monge, A.
Molecules 2009, 14, 2256.
11. Ortega, M. A.; Sainz, Y.; Montoya, M. E.; Jaso, A.; Zarranz, B.; Aldana, I.; Monge,
A. Arzneim.-Forsch. 2002, 52, 113.
12. González, M.; Cerecetto, H. Springer 2007, 10, 265.
13. Ley, K.; Seng, F. Synthesis 1975, 415.
14. Dea-Ayuela, M. A.; Castillo, E.; González-Álvarez, M.; Vega, C.; Rolón, M.; Bolás-
Fernández, F.; Borrás, J.; González-Rosende, M. E. Bioorg. Med. Chem. 2009, 17,
7449.
15. Ertl, P.; Rohde, B.; Selzer, P. J. Med. Chem. 2000, 43, 3714.
16. Zhao, Y. H.; Abraham, M. H.; Le, J.; Hersey, A.; Luscombe, C. N.; Beck, G.;
Sherborne, B.; Cooper, I. Pharm. Res. 2002, 19, 1446.
17. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Delivery Rev.
2001, 46, 3.
18. Veber, D. F.; Johnson, S. R.; Cheng, H. Y.; Smith, B. R.; Ward, K. W.; Kopple, K. D.
J. Med. Chem. 2002, 45, 2615.
19. De Carvalho, L. P. S.; Lin, G.; Jiang, X.; Nathan, C. J. Med. Chem. 2009, 52, 5789.
20. Gordon, L. F. J. Pharm. Sci. 1980, 69, 1109.
25. Cheeseright, T.; Mackey, M.; Rose, S.; Vinter, J. G. J. Chem. Inf. Model. 2006, 46,
665.
Acknowledgements
The authors gratefully acknowledge the ‘Oficina de Cooperación
de la Embajada de Bélgica’ for funding a Master Scholarship to