New hydrazine and hydrazide quinoxaline 1,4-di-N-oxide derivatives: In silico ADMET, antiplasmodial and antileishmanial activity

Article abstract of DOI:10.1016/j.bmcl.2017.02.049

We report the design (in silico ADMET criteria), synthesis, cytotoxicity studies (HepG-2 cells), and biological evaluation of 15 hydrazine/hydrazide quinoxaline 1,4-di-N-oxide derivatives against the 3D7 chloroquine sensitive strain and FCR-3 multidrug re

Full text of DOI:10.1016/j.bmcl.2017.02.049

Accepted Manuscript
New hydrazine and hydrazide quinoxaline 1,4-di-N-oxide derivatives: In sili-
co ADMET, antiplasmodial and antileishmanial activity
Miguel Quiliano, Adriana Pabón, Gustavo Ramirez-Calderon, Carlos Barea,
Eric Deharo, Silvia Galiano, Ignacio Aldana
PII:
S0960-894X(17)30180-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.049
BMCL 24721
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 December 2016
16 February 2017
17 February 2017
Please cite this article as: Quiliano, M., Pabón, A., Ramirez-Calderon, G., Barea, C., Deharo, E., Galiano, S., Aldana,
I., New hydrazine and hydrazide quinoxaline 1,4-di-N-oxide derivatives: In silico ADMET, antiplasmodial and
antileishmanial activity, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.
2017.02.049
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New hydrazine and hydrazide quinoxaline 1,4-di-N-oxide
derivatives: In silico ADMET, antiplasmodial and antileishmanial
activity
Miguel Quilianoa,b,, Adriana Pabónc, Gustavo Ramirez-Calderonc, Carlos Bareab, Eric
Deharoe, Silvia Galianoa,b, Ignacio Aldanaa,b
a Universidad de Navarra, Institute of Tropical Health (ISTUN), Campus Universitario,
31008, Pamplona, Spain.
b Universidad de Navarra, Facultad de Farmacia y Nutrición, Department of Organic
and Pharmaceutical Chemistry, Campus Universitario, 31008, Pamplona, Spain
c Malaria Group, Universidad de Antioquía, Medellín, 1226, Colombia
e Institut de Recherche pour le Développement (IRD), Université Paul Sabatier
Toulouse III, UMR 152 PHARMA-DEV, 31059, Toulouse, France
ABSTRACT
We report the design (in silico ADMET criteria), synthesis, cytotoxicity studies (HepG-
2 cells), and biological evaluation of 15 hydrazine/hydrazide quinoxaline 1,4-di-N-oxide
derivatives against the 3D7 chloroquine sensitive strain and FCR-3 multidrug resistant
strain of Plasmodium falciparum and Leishmania infantum (axenic amastigotes).
Fourteen of derivatives are novel quinoxaline 1,4-di-N-oxide derivatives. Compounds
18 (3D7 IC50 = 1.40 μM, FCR-3 IC50 = 2.56 μM) and 19 (3D7 IC50 = 0.24 μM, FCR-3
IC50 = 2.8 μM) were identified as the most active against P. falciparum, and they were
the least cytotoxic (CC50-values > 241 μM) and most selective (SI > 86). None of the
compounds tested against L. infantum were considered to be active. Additionally, the
functional role of the hydrazine and hydrazide structures were studied in the
quinoxaline 1,4-di-N-oxide system.
Keywords: Malaria; Leishmaniasis; Quinoxaline 1,4-di-N-oxide; Hydrazine; Hydrazide
Abbreviations: ACTs, artemisinin-based combination therapies; QdNO, quinoxaline
1,4-di-N-oxide; SAR, structure-activity relationship; VL, virtual library; SI, selectivity
index; TPSA, Topological polar surface area; %ABS, human intestinal absorption
*Corresponding author. E-mail address: mquiliano@alumni.unav.es (M. Quiliano)

Malaria and leishmaniasis are serious tropical diseases with far-reaching and
important global implications. According to the WHO, an estimated 212 million cases
and 429,000 deaths were attributed to malaria in 2015.1 Meanwhile, there have been 1.3
million cases and 20,000–30,000 deaths annually attributed to leishmaniasis.2 Due to
the high number of cases reported in developing countries in Asia, South America, and
Africa, these diseases are commonly associated with poverty. Both human diseases are
caused by protozoan parasites of the genus Plasmodium spp. and Leishmania spp..1,2
The clinical manifestation of malaria includes fever, chills, headache, and muscular
weakness, and it is the most severe and deadly form caused by Plasmodium falciparum
(P. falciparum).1 The common clinical forms of leishmaniasis involve a cutaneous,
mucocutaneous, and visceral form (also known as kala-azar). Visceral leishmaniasis is
the most severe form caused by Leishmania infantum (L. infantum) and Leishmania
donovani, and it mainly affects internal organs.2-4 Currently, chemotherapy is used to
treat malaria, which includes artemisinin-based combination therapies (ACTs),1,5
whereas pentavalent antimonials, pentamidine, amphotericin B, paramomycin, and
miltefosine are used to treat leishmaniasis. However, the high toxicity, route of
administration, and high cost limit their use.4,6 Additionally, in recent years parasite
drug resistance has become more common.6,7
In an effort to address this situation, a variety of initiatives have emerged at the civil,
public, and pharmaceutical levels to generate new and more effective drugs to treat
these diseases.8 To achieve this goal, drug repurposing and novel drug discovery
strategies have been applied in recent decades to develop new potential drug candidates
for malaria and leishmaniasis.9-14 Therefore, there is a need to search for new types of
chemical entities that have remarkable potency against these diseases with minimum
side effects.
One scaffold that has received attention in recent years due to its diverse
pharmacological profile is quinoxaline 1,4-di-N-oxide (QdNO) (Figure 1A), which is a
derivative of the quinoxaline system.

O-
N+
O-
A
B
H
R7
R6
N
O
R7
R6
N+ R2
N
N+
O-
N+ R3
O-
N
M. tuberculosis H37Rv (IC50 < 1.51 µM)
QdNO
O-
N+
C
H
New SAR
N
N
R
exploration
N+
O-
Figure 1. A) General structure of quinoxaline 1,4-di-N-oxide (QdNO); B) Previous
exploration at positions 6 and 7 of QdNO isonicotinic acid hydrazide derivatives; C) New
hydrazine and hydrazide QdNO derivatives that are proposed here
Its different forms, N-oxide and N,N’-dioxide, have been studied by our group and
others around the world with good results against infectious diseases, such as malaria,
tuberculosis, leishmaniasis, among others.15-19 As part of our ongoing search for
biologically active antimalarial and leishmanicidal compounds, we have published
several articles reporting a large number of QdNO derivatives with a variety of
substituents at positions 2, 3, 6 and 7.18 With regard to position 2, the chemical
exploration was focused on carbonitrile,20-25 carboxylic acid,26 ester,26 and ketone
derivatives.27-31
Similarly, hydrazine and hydrazide derivatives have been shown to possess a wide
variety of biological activities such as antiplasmodial and leishmanicidal activities.32,33
In 2011, our group synthesized the first hydrazide QdNO derivatives with anti-M.
tuberculosis activity using the hybridization concept as a guide.34 The compounds were
designed based on the fusion of QdNO derivatives with isonicotinic acid hydrazide
(Figure 1B). Since the chemical exploration was only focused on positions 6 and 7 of
the QdNO core, only one hydrazide scaffold was probed at position 2. Therefore, the
entire structure-activity relationship (SAR) between the QdNO core and
hydrazine/hydrazide remains unexplored.
To develop new antimalarial and leishmanicidal drugs, the synthesis and SAR of new
hydrazine and hydrazide QdNO derivatives were the main goals in this study (Figure

1C). Additionally, important factors were considered advantages when performing the
study. These include synthetic accessibility, our “in-house” hydrazine and hydrazide
chemical library, acceptable yields as a result of our method of using microwave-
assisted synthesis,34 cost and time efficiency, and our vast experience with these tropical
diseases and QdNO derivatives.18
In the present study, a preliminary virtual library (VL) of hydrazine and hydrazide
QdNO derivatives was generated. Then, using in silico ADMET filters, 15 virtual
compounds were selected and synthesized. Additionally, to obtain a more efficient SAR
exploration, we performed the re-synthesis of 4 QdNO derivatives that were previously
published by our group. Therefore, 19 QdNO derivatives were evaluated in vitro against
infectious pathogens, including the 3D7 chloroquine sensitive strain and FCR-3
multidrug resistant (chloroquine and cycloguanil) strain of P. falciparum and L.
infantum. Their cytotoxicity and selectivity were also evaluated against HepG-2 cells.
Because the exploration of a new chemical family is a difficult process and the
chemical space is vast,35 the work involved two well-defined steps: (1) SAR exploration
of new hydrazine and hydrazide QdNO derivatives and (2) the specific synthesis of
potential candidates using acquired data.
The first part of the study began with the generation of a VL that contained potential
candidates to be synthesized. Using our in-house library of commercial hydrazine and
hydrazide reagents, a VL of 48 hydrazine and hydrazide QdNO derivatives were
generated and enumerated (VL01-VL48) (see Supplementary data). Since the number of
potential variants could increase considerably if substitutions at position 6 and 7 of the
QdNO scaffold were explored, only variants at position 2 were explored using the
hydrazine and hydrazide scaffolds. Thus, our first aim was the establishment of the best
substitution before varying the groups at position 6 and 7 (second part of the study). To
determine which compounds of the VL should be synthesized and screened, common
drug-likeness criteria were used, such as oral bioavailability,36,37 toxicity,38 membrane
permeability,39 blood-brain barrier penetration,39 CYP mechanism-based inhibitor,40 and
general developability.41 These rules were applied based on the physicochemical
parameters and molecular descriptors calculated for the entire VL (see Supplementary
data). As a result, the number of potential candidates for the synthesis process was
reduced from 48 to 28; however, the cost and time requirements remained too high to
obtain preliminary results efficiently. Thus, an informative strategy of the SAR was

applied. For this reason, an economical set of test compounds was synthesized. This set
included 11 compounds: 5 (VL45), 6 (VL23), 7 (VL18), 8 (VL05), 9 (VL31), 10
(VL43), 11 VL39), 12 (VL48), 13 (VL26), 14 (VL28), and 15 (VL37). Although three
compounds (6, 7, and 10) were initially discarded in the drug-likeness study due to their
potential behavior as CYP mechanism-based inhibitors, they were proposed because
they could provide important information on the variety of biological activity of the
hydrazine or hydrazyde derivatives, (e.g., halogen atoms and their steric and electronic
influence). Additionally, four classical intermediates (1-4) in the synthetic route of
QdNO were added to complete the SAR landscape. The synthetic route for compound 4
in relation to this study was also explored.
The synthesis of the selected and designed compounds (1–15) was published
previously and is presented in Scheme 1.34 The synthetic route involved three steps:
first, 2,3-dimethylquinoxaline 1,4-di-N-oxide derivatives 1-3 were prepared using a
variation of the Beirut reaction in which the appropriate benzofuroxane reacts with
butanone in the presence of triethylamine and pyrrolidine. A new 2-methyl-3-
trifluoromethylquinoxaline 1,4-di-N-oxide derivative, 4, was prepared in a similar
manner using 1,1,1-trifluoro-2-butanone, triethylamine, and toluene. In this first step, all
5-substituted benzofuroxanes or 5,6-disubstituted benzofuroxane that were used were
commercially available; second, the oxidation of the methyl group at position 2 of the
QdNO ring was performed using microwave-assisted synthesis and selenium dioxide
(SeO2) as the oxidant agent to obtain the corresponding carboxaldehydes, 1a-3a.
However, the respective formation of the carboxaldehyde for compound 4 was not
observed; third, the new hydrazine and hydrazide derivatives, 5–15, were synthesized
using the microwave-assisted synthesis method in which the corresponding aldehyde
derivatives, 1a-3a, were reacted with selected hydrazine/hydrazide derivatives from our
in-house library using tetrahydrofuran as the solvent and Na2S2O5 as the catalyst.

O-
N+
H
H
N
N
R7
R6
NH2
R
N
R
iii
N+
O-
O-
O-
O
O
5-10, 16-19
R7
R6
N+
R7
R6
N+
SeO2
H
ii
ia
N+
O-
N+
O-
O-
N+
H
1a-3a
1-3
R6
R5
N
O
O-
N+
NH2
H
N
O
R7
R6
O
R
N
N
R
BFX
iii
N+
O-
O-
N+
O
11-15
SeO2
ii
F3C
N+ CF3
O-
ib
4
Scheme 1. General synthesis of quinoxaline 1,4-di-N-oxide derivatives 1-19. Reagents and
conditions: (ia) triethylamine, pyrrolidine; (ib) toluene, triethylamine; (ii) ethyl acetate, microwave-
assisted synthesis, 70 ºC; (iii) tetrahydrofuran, sodium metabisulfite, microwave-assisted synthesis,
65 ºC.
In the first part of the study, the activity of 15 synthesized QdNO derivatives, 1-15,
were determined against the 3D7 chloroquine sensitive strain and FCR-3 multidrug
resistant strain of P. falciparum and the axenic amastigotes of L. infantum (Table 1).
Chloroquine (3D7 IC50 = 0.03 μM, FCR-3 IC50 = 0.14 μM) and amphotericin B (IC50 =
0.05 μM) were used as reference drugs in all of the experiments. Although nine of these
compounds were shown to possess no antiplasmodial activity (1-3, 5, 6, 8, 9, 12, and
15), five of them exhibited a weak activity (7, 10, 11, 13, and 14; IC50-values ≤ 87 μM).
Only compound 4 was identified as potentially interesting for further studies (3D7 IC50
= 7.9 μM, FCR-3 IC50 = 4.2 μM); however, as mentioned above, its respective
carboxaldehyde was not obtained via the same synthetic route making it impossible to
explore with hydrazine or hydrazide variations. However, only four compounds
exhibited weak leishmanicidal activity (4, 10, 11, and 14; IC50 ≤ 52 μM), whereas the
remaining compounds were inactive.

Table 1. Quinoxaline 1,4-di-N-oxide derivatives 1-19 produced via Scheme 1
O-
R7
R6
N+ R2
N+ R3
O-
IC50 (µM)b
SI-Pc
IC50 (µM)b
L.infantum
Amastigotes
>100
SI-Ld
CC50 (µM)e
HepG-2f
Code
VLa
Substituents
P. falciparum
-
R2
R3
R6
H
H
H
H
R7
H
OCH3
Cl
3D7
FCR-3
1*
2*
3*
4
-
-
-
-
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CF3
>100g
>100
>100
>100
<3.21
<7.44
<2.28
0.42
<3.21
<7.44
<2.28
0.05
321.80
744.30
228.00
1.80
>100
>100
30.50
>100
>100
H
7.90 ± 1
4.20 ± 1
H
N
N
5
VL45
CH3
H
H
>100
>100
<0.35
>100
<0.35
35.40
H
N
Cl
N
N
6
7
VL23
VL18
CH3
CH3
H
H
H
H
>100
76.70
>100
87.10
<0.47
6.32
>100
>100
<0.47
<5.50
47.34
H
N
Cl
Cl
550.70
Cl
H
N
N
8
VL05
CH3
H
H
>100
>100
<1.24
>100
<1.24
124.20
Cl
Cl
O
H
N
N
9
VL31
VL43
CH3
CH3
H
H
H
H
>100
85.30
>100
>100
<3.72
<2.41
>100
53.40
<3.72
4.51
372.80
241.20
OH
H
N
O
S
N
10
O
H
N
O
N
11
VL39
VL48
CH3
CH3
H
H
H
H
55.70
>100
36.5
1.11
59.10
>100
0.68
40.70
90.90
H
N
O
N
12*
>100
<0.90
<0.90
N
H
N
O
N
13
14
VL26
VL28
CH3
CH3
H
H
H
H
27.80
54.90
30.0
2.22
0.81
>100
52.30
<0.66
0.54
66.60
28.30
OCH3
O
H
N
N
N
34.80
Cl
H
N
O
15
16
VL37
-
CH3
CH3
H
H
H
>100
>100
>100
>100
<0.87
<2.09
>100
>100
<0.87
<2.08
87.20
Cl
Cl
H
N
N
OCH3
208.90
Cl
Cl
H
N
N
17
-
CH3
H
Cl
>100
61.10
0.20
>100
>100
<0.12
12.20
Cl
Cl
Cl
H
N
18
19
-
-
N
CH3
CH3
H
H
OCH3
Cl
1.40
0.24
2.56
2.80
>95.30
>86.07
NDh
ND
>244
>241
N
Cl
H
N
N
>100
ND
N
CQi
Apj
-
-
-
-
-
-
-
-
-
-
0.03
ND
0.14
ND
ND
-
-
-
ND
ND
0.05
aVL: Virtual library code; IC50: Concentration that produces 50% of the inhibitory effect; SI-P: Selectivity index in
b
c
P.falciparum = CC50 HepG-2 cells/FCR-3 IC50 P. falciparum; dSI-L: Selectivity index in L.infantum = CC50 HepG-2
e
f
cells/IC50 L. infantum; CC50: Concentration that produces 50% of cytotoxicity; HepG-2: Human hepatocellular

g
h
i
j
carcinoma cells; IC50 > 100 µM = Not active; ND: Not determined, CQ: Chloroquine; Ap: Amphotericin B.
*Previously synthesized by our group.34 The IC50 and CC50 results are the mean of three independent experiments with
an SD <10% in all cases.
The cytotoxicity of the fifteen QdNO analogues (1-15) was determined against
HepG-2 cells (see Supplementary data) (Table 1). The cytotoxicity assays for QdNO
cores, 1, 2, and 3, exhibited a low toxicity against the HepG-2 cells (1 vs. 2 vs. 3, CC50-
values ≥ 228 μM). The substitution of the methoxy group at position 7 on the QdNO
core caused the least cytotoxic effect (2 CC50 = 744 μM, OCH3 > H > Cl groups). In
contrast, modification of the methyl group at position 3 to a trifluoromethyl group was
not well tolerated (1 vs. 4, 4 CC50 = 1.76 μM). For hydrazine and hydrazide derivatives
(5-15), compounds 7, 8, 9, and 10 were less cytotoxic (CC50-values ≥ 100 μM).
However, in terms of selectivity index (SI) against P. falciparum and L. infantum (Table
1), QdNO derivatives 1-15 exhibited a low selectivity (SI < 7.44 μM).
From the first results obtained, the general SAR analysis showed that hydrazine
derivatives are less active than hydrazide derivatives in P. falciparum (5-10 vs. 11-15).
The presence of an aromatic system in the hydrazide scaffold increases the activity (11
vs. 15). Additionally, electron-donating group at the para position on the phenyl ring
such as -OCH3 was well tolerated and resulted in an active compound (11 vs. 13 vs. 14)
In the second part of the study, based on previously obtained data ((a) the collected
antimalarial activity data for hydrazine derivatives in the literature,32,33 (b) the SAR
exploration for QdNO derivatives (1-15), and (c) our previous studies with hydrazide
QdNO derivatives in tuberculosis using isonicotinic acid (Figure 1B),34) the use of
hydrazine and hydrazide derivatives should effectively ensure the obtaining of active
compounds due to the versatility of the scaffold itself or the use of a hybridization
concept. To study this hypothesis in detail, we decided to: (1) synthesize four final
compounds (16, 17, 18 and 19) involving substitutions at positions 7 (not tested in the
first part of the study) and (2) compare two hydrazine scaffolds: one previously tested
and another completely new. Substitutions at position 7 were performed using chloro
and methoxy groups due to the low cytotoxicity values showed in this study with HepG-
2 cells (2-3, Table 1) and due to their well-documented modulation of the biological
activity on QdNO derivatives against infectious diseases.15-19 Comparisons of the
hydrazine derivatives were performed between 2,4,6-trichlorophenyl hydrazine and 7-
chloro-4-hydrazinoquinoline (compounds 16 vs. 18 and 17 vs. 19). The first one,

previously tested in compound 8, was selected due to its good synthetic yield, HepG-2
CC50 value = 124 μM, and null activity against both P. falciparum and L. infantum. The
second, a quinoline derivative, was selected because of its reported high antimalarial
activity and low toxicity.42
Final compounds 16-19 were synthesized and evaluated against P. falciparum (3D7
and FCR-3 strains) and L. infantum (amastigotes) in the same manner as compounds 5-
15 (see Scheme 1, Table 1, and Supplementary data). In contrast to a methoxy group (8
vs. 16), substitution at position 7 with a chlorine atom increases the antimalarial activity
(8 vs. 17); however, an increase in toxicity by ten-fold was also observed. Compounds
18 (3D7 IC50 = 1.40 μM, FCR-3 IC50 = 2.56 μM) and 19 (3D7 IC50 = 0.24 μM, FCR-3
IC50 = 2.80 μM) showed a significant increase in activity in both strains of P.
falciparum in comparison with their analogues (16 vs. 18 and 17 vs. 19). No
leishmanicidal activity was reported for compounds 16-19. As expected, based on the
preliminary data obtained for compound 8, compounds 16 and 17 that had the same
substituent as 8 did not show activity as antileishmanial agents. In terms of toxicity,
compound 17 (CC50 = 12 μM) was more cytotoxic than 16, 18, and 19 (CC50-values ≥
208 μM). In addition, 18 and 19 exhibited high values of SI (SI > 86).
An analysis of the results obtained in the second part of the study showed that
different substitutions on the phenyl ring that were attached to hydrazine and hydrazide
structures did not contribute to the biological activity itself (16 vs. 18 and 17 vs. 19).
This suggested that the real function of the hydrazine and hydrazide structures was to
act as linkers between the QdNO core and aromatic fragment attached to them. Thus,
when the appended aromatic scaffold is more active, the new derivative is more active
(e.g., the quinoline scaffold in compounds 18 and 19 and its well-known antimalarial
activity). This hypothesis is also reinforced by compound 12, previously tested in M.
tuberculosis (H37Rv IC50 = 1.17 μM),34 that includes an antimycobacterial aromatic
fragment but is completely inactive against P. falciparum and L. infantum (Table 1).
Finally, a brief summary of the ADMET properties (oral bioavailability) of the
synthesized QdNO derivatives (1-19) is shown in Table 2. The topological polar surface
area (TPSA) was used to calculate the percentage of human intestinal absorption
(%ABS) according to the equation: %ABS = 109 − (0.345 × TPSA) (see Supplementary
data), as described by Zhao et al.43 In addition, Lipinski's rule of five36 was also
calculated to evaluate the “drug-likeness”. From these parameters, all compounds

exhibited a %ABS between 70 and 91%. None of the compounds violated Lipinski's
rule of five. Thus, the in silico oral bioavailability of QdNO derivatives could be
considered promising, especially for the most active compounds in P. falciparum (18
and 19).
Table 2. In silico physicochemical properties of the tested compounds, 1-19 (ADME
profile)
n-OHNH
Donors
n-ON
ID
%ABS
LogP
MW
LV
acceptors
Rule
1-17
18
<5
≤3.5
2.3
<500
≤432
409
<5
≤2
1
<10
≤8
8
≤1
0
≤91
75
0
19
79
2.8
414
1
7
0
%ABS: human intestinal absorption, calculated by: %ABS = 109 − (0.345 × TPSA); LogP (ALOGPs
2.1): logarithm of the compound partition coefficient between n-octanol and water; n-OHNH: number of
hydrogen bond donors; n-ON: number of hydrogen bond acceptors; LV: Lipinski`s violations: molecular
weight expressed in Daltons.
In conclusion, this study included the design (in silico ADMET criteria), synthesis,
cytotoxicity studies (HepG-2 cells), and biological evaluation of 15 hydrazine/hydrazide
QdNO derivatives (14 were novel QdNO derivatives) against the 3D7 chloroquine
sensitive strain and FCR-3 multidrug resistant strain of P. falciparum and L. infantum
(axenic amastigotes). Novel compounds 18 (3D7 IC50 = 1.40 μM, FCR-3 IC50 = 2.56
μM) and 19 (3D7 IC50 = 0.24 μM, FCR-3 IC50 = 2.80 μM) were identified as the most
active against P. falciparum, least cytotoxic (CC50-values > 241 μM), and most
selective (SI >86). None of compounds tested against L. infantum were considered to be
active. The low SI, antiplasmodial and leishmanicidal activity exhibited by the
compounds (5-17) led us to discontinue the synthesis and biological studies of this
family of compounds; however, the use of the QdNO core and its fusion with an active
antimalarial scaffold, such as 18 and 19, to form hybrid compounds remains a valid
approach to develop new chemical entities. Therefore, in-depth studies are in progress
by our group.
Acknowledgments
This work was supported by the PIUNA Project (Universidad de Navarra) and
Foundation CAN (grant number: 70391). The authors are grateful to Universidad de
Antioquia for its Sustainability Strategic Plan and the Institute of Tropical Health

(ISTUN) of Universidad de Navarra for their financial support and assistance. Miguel
Quiliano is grateful to “Programa Nacional de Innovación para la competitividad y
productividad” (Innóvate-Perú) for his PhD scholarship (grant 065-FINCYT-BDE-
2014). The authors thank Dr. Kim Y. Fong for critical reading of this manuscript.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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Graphical abstract