Antileishmanial effect of new indeno-1,5-naphthyridines, selective inhibitors of Leishmania infantum type IB DNA topoisomerase

Article abstract of DOI:10.1016/j.ejmech.2016.09.017

Visceral leishmaniasis is a neglected disease of poor and developing countries. The current therapeutic approach is based on pentavalent antimonial (Sb^V) drugs and amphotericin B, both nephrotoxic and parenterally administered drugs. Therefore, there is a real need of new antileishmanial drugs. Eukaryotic type I DNA topoisomerases (TopIB) have been identified as druggable targets against leishmaniasis. These enzymes are involved in solving topological problems generated during replication, transcription and recombination of DNA. Leishmanial TopIB is a unique heterodimeric protein structurally different than that found in the mammalian host, thus making it an interesting target for drug discovery. Tetrahydro indeno-1,5-naphthyridines 5 and indeno[1,5]naphthyridines 6 were synthesized. The inhibition of Leishmania and human TopIB of these polycyclic heterocycles were studied and their antileishmanial activity on promastigotes and amastigote-infected splenocytes of Leishmania infantum were evaluated. In this regard, it is noteworthy that some of the prepared heterocycles, as compounds 6b, 6i and 5 h, showed selective inhibition of LtopIB while no inhibition of hTopIB was observed at evaluated conditions. In addition, the cytotoxic effects of newly synthesized compounds were assessed on host murine splenocytes in order to calculate the corresponding selective indexes (SI). Tetrahydro indeno-1,5-naphthyridines 5e and 5h showed good antileishmanial activity (IC₅₀values of 0.67 ± 0.06 and 0.54 ± 0.17 μM) with similar activity than the standard drug amphotericin B (0.32 ± 0.05 μM) and even tetrahydro indeno-1,5-naphthyridine 5h showed higher (SI) towards L. Infantum amastigotes. Likewise, in the family of indeno-[1,5]-naphthyridines 6, compound 6b showed good antileishmanial activity (IC₅₀value 0.74 ± 0.08 μM) and higher selective index (SI) towards L. Infantum amastigotes than amphotericin B.

Full text of DOI:10.1016/j.ejmech.2016.09.017

European Journal of Medicinal Chemistry 124 (2016) 740e749
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Antileishmanial effect of new indeno-1,5-naphthyridines, selective
inhibitors of Leishmania infantum type IB DNA topoisomerase
a
a
a
a, **
ꢀ
~
Ana Tejería , Yolanda Perez-Pertejo , Rosa M. Reguera , Rafael Balana-Fouce
,
b
b
b
b
ꢀ
ꢀ
Concepcion Alonso , María Fuertes , María Gonzalez , Gloria Rubiales ,
Francisco Palacios b
,
*
a
ꢀ
ꢀ
ꢀ
Departamento de Ciencias Biomedicas, Universidad de Leon, Campus de Vegazana s/n, 24071 Leon, Spain
Departamento de Química Organica I, Facultad de Farmacia and Centro de Investigacion Lascaray (Lascaray Research Center), Universidad del País Vasco/
Euskal Herriko Unibertsitatea (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain
b
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Visceral leishmaniasis is a neglected disease of poor and developing countries. The current therapeutic
approach is based on pentavalent antimonial (SbV) drugs and amphotericin B, both nephrotoxic and
parenterally administered drugs. Therefore, there is a real need of new antileishmanial drugs. Eukaryotic
type I DNA topoisomerases (TopIB) have been identified as druggable targets against leishmaniasis. These
enzymes are involved in solving topological problems generated during replication, transcription and
recombination of DNA. Leishmanial TopIB is a unique heterodimeric protein structurally different than
that found in the mammalian host, thus making it an interesting target for drug discovery. Tetrahydro
indeno-1,5-naphthyridines 5 and indeno[1,5]naphthyridines 6 were synthesized. The inhibition of
Leishmania and human TopIB of these polycyclic heterocycles were studied and their antileishmanial
activity on promastigotes and amastigote-infected splenocytes of Leishmania infantum were evaluated. In
this regard, it is noteworthy that some of the prepared heterocycles, as compounds 6b, 6i and 5 h,
showed selective inhibition of LtopIB while no inhibition of hTopIB was observed at evaluated conditions.
In addition, the cytotoxic effects of newly synthesized compounds were assessed on host murine sple-
nocytes in order to calculate the corresponding selective indexes (SI). Tetrahydro indeno-1,5-
Received 12 July 2016
Received in revised form
1 September 2016
Accepted 6 September 2016
Available online 9 September 2016
Keywords:
Leishmania
Indeno-naphthyridines
DNA-topoisomerase
naphthyridines 5e and 5h showed good antileishmanial activity (IC50 values of 0.67
0.54 0.17 M) with similar activity than the standard drug amphotericin B (0.32 0.05
0.06 and
M) and even
m
m
tetrahydro indeno-1,5-naphthyridine 5h showed higher (SI) towards L. Infantum amastigotes. Likewise,
in the family of indeno-[1,5]-naphthyridines 6, compound 6b showed good antileishmanial activity (IC50
value 0.74 0.08
B.
m
M) and higher selective index (SI) towards L. Infantum amastigotes than amphotericin
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
transmitted by phlebotominae insects. From an epidemiological
point of view, leishmaniasis is the second most widespread
Visceral leishmaniasis (VL) is a serious zoonotic disease caused
by digenetic protozoan parasites of Leishmania Genus, which are
neglected tropical disease, after malaria [1]. The disease is fatal in
Old World countries, especially in some Northern states of India
(Bihar) where more than 90% of the estimated 40,000 deaths all
over the world occur [2]. However, despite the good sanitary con-
ditions of European countries, VL is endemic at the Northern shore
of the Mediterranean Basin [3]. The treatment of this disease is
based on chemotherapy [4], since potential human vaccines are in
preclinical or early clinical stages of development [5].
Abbreviations: VL, Visceral Leishmaniasis:; TopIB, Type IB DNA Topoisomerase;
IRFP, Infra Red Fluorescent Protein; FCS, Foetal Calf Serum; DMSO, Dimethyl sulf-
oxide; SI, Selectivity Index; CPT, camptothecin; CPTs, camptothecin derivatives;
DDQ, dichloro-5,6-dicyanobenzoquinone; HDAr, hetero-Diels-Alder reaction; MCR,
multicomponent reactions; TLC, thin layer chromatography.
* Corresponding author.
Drugs for Neglected Diseases initiative (DNDi) recommends
combinations of pentavalent antimony (SbV) with the polyene
fungicide amphotericin B (AMB), the aminoglycoside antibiotic
** Corresponding author.
E-mail address: francisco.palacios@ehu.eus (F. Palacios).
http://dx.doi.org/10.1016/j.ejmech.2016.09.017
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
741
paromomycin and the phospholipid milefosine to face VL, in
developing countries [6]. However, with the exception of miltefo-
sine, these compounds have nephrotoxic side effects or require
parenteral administration [7], which limit their use in countries
where the frequently overwhelmed health services are very far
away from the residences of the affected people [8]. Therefore, the
search for new drugs [9] based on validated targets against VL re-
mains urgently needed, especially when Big Pharma have waived
funds and efforts in R þ D to treat other more profitable pathologies
of wealthy countries [10].
Eukaryotic type I DNA topoisomerase IB (TopIB) is a nuclear
enzyme that resolves DNA tangles during replication, transcription
and repair processes. All these processes are very active during cell
division [11] For these reasons, TopIB is considered a validated
druggable target for the treatment of different tumours in humans
[12]. The most studied TopIB inhibitors are camptothecin (CPT,
Fig. 1) derivatives [13], which are being used clinically against
certain tumours. However, many others polynuclear heterocycle
compounds such as indenoisoquinolines have improved ability to
inhibit the enzyme from human cell cultures [14].
friendlier aspects [24], diversity with atom-economy and
enantiocontrol.
In addition, among the organic reactions the Hetero-Diels-
Alder-reaction (HDAr) is an excellent choice for the six-
membered heterocycle formation [25]. Among them, Povarov re-
action [26,27] can be considered as an example of HDAr and rep-
resents an excellent method for the preparation of nitrogen-
containing heterocyclic compounds, in which aldimines derived
from anilines and aldehydes react with electron-rich alkenes in the
presence of a Lewis acid catalyst. This process has found applica-
tions in total synthesis of natural biologically active compounds
[28]. In this sense, if pyridyl amines are used in Povarov reactions
this strategy can be also applied to the synthesis of nitrogen poly-
heterocyclic derivatives, such as 1,5-naphthyridines [29]. This
structure can be found in the skeleton of a wide range of biologi-
cally active derivatives [30]. In this sense, 7H-indeno[2,1-c][1,5]-
naphthyridine derivatives may be good candidates with interesting
pharmacological activity as new enzyme inhibitors [31]. Conse-
quently, their synthesis and biological evaluation seems to be an
attractive task.
Leishmania TopIB (LTopIB) is a promising target for developing
antileishmanial drugs due to two main reasons: i) the enzyme has
an increased expression during the division cycle of rapidly
growing Leishmania and especially ii) LTopIB is structurally
different from that of the host [15]. LTopIB is an unusual hetero-
dimeric enzyme, constituted by a large subunit that contains the
four amino acids of the active site, and a small subunit that contains
the catalytic Tyr222 involved in breaking one DNA strand [16]. In
addition, LTopIB displays two non-conserved regions; one at the C-
terminal end of the large protomer and the other at the N-terminal
end of the small protomer, which are important in enzyme as-
sembly, in conferring sensitivity to topoisomerase inhibitors [17]
and in enzyme translocation towards the nucleus. All these
distinctive features are also present in other trypanosomatids of
medical interest i.e. Trypanosoma brucei [18] and T. cruzi [19], which
makes this enzyme remarkably interesting for drug discovery of
other neglected diseases.
Some compounds showing good human TopIB inhibition, such
as CPT derivatives, have been studied as LTopIB inhibitors [20].
From a chemical point of view, CPT derivatives present fused ring
heterocycles and this feature may be important towards their
effectiveness as TopIB inhibitors. Taking this into account, other
quasi-flat condensed heterocycles, such as naphthyridine de-
rivatives (Fig. 1), that are effective hTopIB inhibitors [21] might be
also adequate candidates to act as LTopIB inhibitors and overcome
some of CPTs inherent limitations.
This manuscript describes for the first time the synthesis and
antileishmanial effect of new synthetic indeno-1,5-naphthyridines
on L. infantum, the aetiological agent responsible for VL in
humans and dogs in Mediterranean countries. For this purpose an
ex vivo intracellular screening on macrophages isolated from
naturally infected BALB/c mice with an infrared-emitting
L. infantum strain was used [32]. In addition, the inhibitory effect
of these compounds has been studied on recombinant TopIB from
both Leishmania and human sources, showing a selective effect over
the parasite enzyme. These results point indeno-1,5-
naphthyridines as promising antileishmanial drugs.
2. Chemistry
The synthesis of tetrahydroindeno-1,5-naphthyridines was
developed by means of a Povarov type [4 þ 2]-cycloaddition re-
action by both step by step and multicomponent strategies (MCRs)
and in this article only the best reaction conditions and yields are
reported. Thus, the preparation of novel tetrahydroindeno-1,5-
naphthyridine 5b (R ¼ 4-F-C6H4) was accomplished by reaction of
N-(3-pyridyl)aldimine 3b, prepared in situ by reaction of 3-
pyridylamine 1 and aldehyde 2b (R ¼ 4-F-C6H4, Scheme 1, route
A1), and indene 4 in the presence of 2 equivalents of BF3$Et2O in
refluxing chloroform (Scheme 1, route A2, Chart 1) with excellent
yield (93%).
The structure of compound 5b was assigned on the basis of the
1D and 2D spectroscopy., including HMQC and HMBC experiments
and mass spectral data. Thus, the 1H NMR spectrum showed one
singlet at 4.68 ppm corresponding to proton of C-6, one doublet at
4.61 ppm with coupling constant of 3JHH ¼ 6.9 Hz corresponding to
proton of C-11b, one singlet at 3.73 ppm corresponding to proton of
In order to prepare such heterocyclic compounds multicompo-
nent reactions (MCR) [22], widely applied in Medicinal Chemistry
[23], represent a very appropriate strategy. MCR have some ad-
vantages over classic divergent reaction strategies, including lower
costs, shorter reaction time, and energy, as well as environmentally
Fig. 1. Structure of camptothecin (left) and of newly synthesized 7H-indeno[2,1-c][1,5]-naphthyridines (right).

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A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
Scheme 1. Preparation of novel indeno-1,5-naphthyridine derivatives 5 and 6. aReagents and conditions: (Route A) route A1: 3-pyridylamine 1 (1 equiv), aldehyde 2 (1 equiv),
molecular sieves 4 Å, chloroform, reflux, 8e24 h; route A2: indene 4 (1.5 equiv), BF3$Et2O (2 equiv), chloroform, reflux, 8e36 h. (Route B) 3-pyridylamine 1 (1 equiv), aldehyde 2 (1
equiv), indene 4 (1.5 equiv), molecular sieves 4 Å, BF3$Et2O (2 equiv), chloroform, reflux, 24e48 h. (Route C): DDQ (1 equiv), toluene, microwave irradiation at 150 W, 25 ꢀC, 120min;
(Route D): Mn(AcO)3 (10 equiv), acetic acid (7 mL), 25 ꢀC, 24 h. bYields obtained through route A. cYields obtained through route B. dYields obtained through route C. eYields obtained
f
through route D. Compound 5h was obtained along with 3% of compound 6h through route A.
Chart 1. Structures of tetrahydroindeno-1,5-naphthyridines 5. aReference [29b]. bReference [29a]. cReference [21].
NH, one multiplet at 3.26e3.19 ppm corresponding to proton of C-
signals between NH and methylenic carbon C-6a which confirms
the regiochemistry of the process.
When the reaction was carried out between N-(3-pyridyl)aldi-
mines 3h (R ¼ 3-MeO-C6H4) and 3k (R ¼ 4-Me-C6H4), prepared in
situ (Scheme 1, route A1), and indene 4 in the presence of BF3$Et2O
6a and one of methylenic protons of C-7 and a double doublet at
2
2.37 ppm with coupling constants of JHH
¼
12.4 Hz and
3JHH ¼ 7.3 Hz corresponding to the other methylenic proton of C-7.
HMBC experiment for compound 5b showed among others cross

A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
743
the corresponding novel tetrahydroindeno-1,5-naphthyridines 5h
and 5k were obtained (80% and 41% respectively, Scheme 1, route
A2, Chart 1) along with different amounts of corresponding dehy-
drogenated indeno-1,5-naphthyridines 6h (3%) and 6k (35%)
respectively (Chart 2).
Formation of tetrahydroindeno-1,5-naphthyridines 5 may be
explained by a regio- and stereospecific [4 þ 2]-cycloaddition re-
action between aldimines 3 and indene 4 followed by prototropic
tautomerization. Subsequent dehydrogenation of tetrahy-
corresponding endo-1,2,3,4-tetrahydronaphthyridine 5m with
good yield (50%, Scheme 1, route B) along with indeno-1,5-
naphthyridine 6m (10%, Chart 2).
Dehydrogenation
of
1,2,3,4-tetrahydroindeno-1,5-
naphthyridines was performed by reaction with stoichiometric
amount of DDQ as previously reported [21]. Following this meth-
odology, new tetrahydroindeno-1,5-naphthyridines 5b and 5m
were treated with 1 equivalent of DDQ in toluene at 25 ꢀC under
microwave irradiation for 120 min producing in good yields the
corresponding 7H-indeno[2,1-c][1,5]naphthyridines 6b (62%), and
6m (60%, Scheme 1, route C, Chart 2).
droindeno-1,5-naphthyridine
5 under the reaction conditions
would afford derivative 6.
In this fashion, following procedures previously reported [21,29]
some other endo-1,2,3,4-tetrahydroindeno-1,5-naphthyridines 5,
such as naphthyridines 5a,g [29a] 5c-f,l,n [21] as well as 5o [29b]
(Chart 1) were prepared for testing their biological activity
against promastigotes and L. infantum amastigotes-infected
macrophages.
The formation of compounds 6 was determined by 1H NMR
spectroscopy, where signal corresponding to the protons of tetra-
hydronaphthyridine ring of starting compounds 5 disappeared
while the appearance of only one signal as a singlet around
4.00 ppm, corresponding to the methylene group present in the
dehydrogenated derivatives 6, was observed. In a similar way,
indeno-1,5-naphthyridines 6a,c-f,l,n were also prepared [21] (Chart
On the other hand, when aldimine 3i (R ¼ 4-MeO-C6H4) and
aldimine 3j (R ¼ 4-MeS-C6H4) were used for the Povarov reaction
the corresponding tetrahydroindeno-1,5-naphthyridines 5 were
not obtained and, instead, dehydrogenated indeno-1,5-
naphthyridines 6i and 6j were isolated, respectively (Scheme 1,
route A, Chart 2). As before, formation of these derivatives could be
reasoned by a [4 þ 2]-cycloaddition reaction between aldimines 3
2).
However,
dehydrogenation
of
tetrahydroindeno-1,5-
naphthyridine 5o was performed by using Mn(AcO)3 as oxidant
in acetic acid at room temperature and 7H-indeno[2,1-c][1,5]
naphthyridine 6o was obtained in excellent yield (79%, Scheme 1,
route D, Chart 2).
A wide scope of indeno-1,5-naphthyridines 5 and 6 with
electron-donating and electron-withdrawing aromatic sub-
stituents, including fluorine substituents [33], has been achieved,
and in order to increase the diversity of these compounds also
derivatives with heterocyclic substituents, such as pyridine (R ¼ 4-
pyridyl), and carboxylic substituents, such as ethyl carboxylate
(R ¼ CO2Et) were also obtained. Biological activity against pro-
mastigotes and amastigotes-infected macrophages of L. infantum,
has been studied for all these mentioned heterocyclic compounds,
and indene
4 and subsequent dehydrogenation of tetrahy-
droindeno-1,5-naphthyridines 5. In both cases, the presence of
electron-donating groups (OMe and SMe) seems to favour the
direct formation of indeno-1,5-naphthyridines 6.
When the reaction was performed by a three-component syn-
thetic protocol, the reaction of commercially available 3-
pyridylamine 1, aromatic aldehyde 2m (R ¼ 2-naphthyl) and
indene 4 in the presence of 2 equivalents of BF3$Et2O gave the
Chart 2. Structures of indeno-1,5-naphthyridines 6. aReference [21].

744
A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
tetrahydroindeno-1,5-naphthyridines 5 (Chart 1) and indeno-1,5-
naphthyridines 6 (Chart 2).
Sigma-Plot statistical package, are listed in Table 1 as IC50 values.
Selectivity indexes of each compound were calculated as the ratio
between the IC50 values on the uninfected explants system vs. the
IC50 values on infected ex vivo splenic explants.
3. Biological results and discussion
Interestingly, the values of antiparasitic activity obtained with
both tetrahydroindeno-1,5-naphthyridines
5 and indeno-1,5-
3.1. In vitro screening of indeno-1,5-naphthyridines
naphthyridines 6 derivatives were generally better on intracel-
lular amastigotes than on free-living promastigotes (Table 1), with
the exception of compound 5c. This effect can be explained by the
cell composition of the splenic explant used in our experiments.
An initial comparison the IC50 values of tetrahydroindeno-1,5-
naphthyridines 5 and indeno-1,5-naphthyridines 6 with anti-
leishmanial effect against intracellular amastigotes in the context of
molecular structure-activity correlation revealed that the presence
of the aryl substituent in position 6 (Scheme 1, vide supra) is very
relevant in the cytotoxic outcomes of the resulting compounds. In
such a way, tetrahydroindeno-1,5-naphthyridines 5a (R ¼ C6H5), 5l
(R ¼ 4-NO2-C6H4), 5n (R ¼ 4-pyridyl, Table 1, entries 2, 10 and 11)
and their corresponding counterparts compounds 6a, 6l and 6n
respectively (Table 1, entries 13, 22 and 24), had high IC50 values
The antileishmanial effect of the newly synthesized tetrahy-
droindeno-1,5-naphthyridines 5 and indeno-1,5-naphthyridines 6
was investigated on both forms of Leishmania cell cycle: in vitro
free-living promastigotes and ex vivo L. infantum iRF1.4 infected
splenocytes (amastigotes). This alternative screening-method
supposes a series of advantages over the traditional cultures of
axenic amastigotes and in vitro infections on standard macrophages
cell lines. With respect to axenic amastigotes, the ex vivo infecting
amastigotes resemble better the actual parasite form that threat
host cells. In addition, the infection of the target cells e murine
macrophages e has been produced under naturally-occurring
conditions; regardless in vitro infections that are developed over
differentiated monocyte cell lines most of them from tumoral
origin. Last but not least, splenic explants have the insurmountable
advantage of containing all the immunological cell populations
involved in host response that can help to destroy the invading cell
within the macrophage [32]. On the other hand, the infrared
emission fluorescence of the transgenic parasites permits the rapid
assessment of cell viability and growth progression of the parasite
forms thanks to rapid fluorescence readout at 708 nm that
correlates-well with the number of living promastigotes and
amastigotes.
(63e90 mM range) and had not selective killing effect on intracel-
lular parasites (SI ca.1), which can be explained as a consequence of
macrophage destruction rather than parasite death due to drug
effects. Nevertheless, the inclusion of one to three fluorine atoms
[33] in the phenyl substituent at position 6 of the indeno-1,5-
naphthyridine cores 5 and 6, very much improved both the anti-
leishmanial effect (amastigote) and the corresponding SI values. In
such a way, the tetrahydroindeno-1,5-naphthyridine 5b (R ¼ 4-F-
C6H4) and the corresponding indeno-1,5-naphthyridine 6b showed
much better IC50 (2.97 and 0.74
mM, respectively) and SI values
The antiparasitic effect was compared with that found for CPT as
model of TopIB poison and with AMB as current leishmanicide drug
in clinical use.
(19.5 and 68.2, respectively, Table 1, entries 3 and 14).
Similarly, the presence of a second ortho-fluorine atom substi-
tution (R ¼ 2,4-F2-C6H3) in the phenyl substituent at position 6 of
Dose response curves were fitted by nonlinear analysis with the
Table 1
Structures and bioactivity results for tetrahydroindeno-1,5-naphthyridines 5 and indeno-1,5-naphthyridines 6.a
Entry
Compound
R
IC50
(
mM)
CC50
(m
M)
SI*
L. infantum
L. infantum
Murine
Promastigotes
Amastigotes
splenocytes
1
2
3
4
5
6
7
8
Amphotericin B (AMB)
0.77 0.15
>100
0.32 0.05
65.12 12.36
2.97 0.5
94.74 32.76
un
>20
>62.5
1.45
19.5
0.9
5a
5b
5c
5d
5e
5f
5g
5h
5l
5n
5o
6a
6b
6d
6e
6f
6h
6i
6j
6k
6l
6m
6n
6o
CPTb
C6H5
94.28 0.48
57.9 9.20
85.27 1.27
4.6 0.20
32.1 1.1
77.48 3.26
>100
35.8 2.30
>100
94.64 1.05
>100
90.20 2.01
50.5 20.60
125.8 18.10
76.9 11.90
74.20 4.89
>100
116.4 27.40
80.94 4.67
68.25 3.29
88,11 0,18
75.8 7.70
19.9 1.50
59.4 19.60
0.62 0.13
4-F-C6H4
3-F-C6H4
4-CF3-C6H4
2,4-F2-C6H3
3,4-F2-C6H3
Me
3-MeO-C6H4
4-NO2-C6H4
4-pyr
9.8 0.30
89.90 9.23
6.1 0.80
3.7 0.10
86.81 3.24
>100
un
0.67 0.06
76.23 1.36
89.37 4.57
0.54 0.17
91.34 3.05
65.25 3.58
78.45 4.11
63.28 1.06
0.74 0.08
3.35 0.07
6.07 1.45
78.15 3.94
93.26 8.30
5.8 0.43
47.9
1.02
>1.12
66.3
>1.09
1.45
>1.27
1.43
68.2
37.6
12.7
0.95
>1.07
20.1
1.39
1.17
1,29
6.4
9
5.4 1.60
>100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
82.47 3.78
90.77 1.36
70.06 8.54
34.9 4.35
84.7 3.61
un
86.12 3.69
>100
un
75.07 1.03
69.36 8.11
>100
34.6 14.05
33.9 3.05
33.3 5.20
1.12 0.13
CO2Et
C6H5
4-F-C6H4
4-CF3-C6H4
2,4-F2-C6H3
3,4-F2-C6H3
3-MeO-C6H4
4-MeO-C6H4
4-MeS-C6H4
4-Me-C6H4
4-NO2-C6H4
2-naphthyl
4-pyr
58.14 8.31
58.29 3.54
68,26 1,96
11.90 2.80
un
un
2.4
20.7
CO2Et
25.05 7.20
0.03 0.01
a
Antileishmanial effects (IC50 SD) on promastigotes and amastigotes of L. infantum. Cytotoxicity effects (CC50 SD) on murine splenocytes. *SI: Selective Index. un:
undefined.
b
Ref. [34].

A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
745
both pharmacophores, originated two active compounds 5e with a
IC50 value of 0.67
M and SI ¼ 47.9 and 6e with a IC50 value of
6.07
M and SI ¼ 12.7 (Table 1, entries 6 and 16). It is worth
5l, 6d and 6l were discarded of further analysis. Therefore, a
handful of compounds displayed interesting IC50 values inhibiting
LTopIB with no effect on hTopIB namely: 5b, 5e, 5h, 6b, 6h, 6i and
6m (Table 2, entries 3, 6, 9, 14, 18, 19 and 23).
It is very striking that the selected compounds are those that
showed better SI in the in vitro/ex vivo assays. Effectively, the para-
fluorophenyl substituent at position 6 either in the tetrahy-
droindeno-1,5-naphthyridine 5b (R ¼ 4-F-C6H4, Table 2, entry 3) or
indeno-1,5-naphthyridine 6b pharmacophores (R ¼ 4-F-C6H4,
Table 2, entry 14) resulted in two selective LTopIB inhibitors with
interesting SI range of 20e68. The introduction of a second adjacent
fluorine atom in the phenyl ring resulted in an interesting com-
pound 5e (R ¼ 2,4-F2-C6H3, Table 2, entry 6) that selectively
m
m
mentioning that the position of fluorine atoms in the phenyl group
was crucial to selectively kill the parasites. Unlike 2,4-
difluorophenylsubstituted tetrahydroindeno-1,5-naphthyridine 5e
and indeno-1,5-naphthyridine 6e, those compounds with adjacent
fluorine atoms in the phenyl group (R ¼ 3,4-F2-C6H3), namely 5f
and 6f, respectively, had much higher IC50 values for killing
amastigotes, and poor SI values (ca. 1, Table 1, entries 7 and 17). This
loss of effectiveness was also observed in tetrahydroindeno-1,5-
naphthyridine 5c (R ¼ 3-F-C6H4, Table 1, entry 4), pointing this
position disadvantageous to obtain active compounds. On the other
hand, the 4-trifluoromethylphenyl substituted derivatives in 6
position resulted in two compounds with opposite antileishmanial
behavior and toxicity. Whereas compound 5d (R ¼ 4-CF3-C6H4) was
extremely toxic for host cells, the indeno-1,5-naphthyridine 6d
(R ¼ 4-CF3-C6H4) was effective killing amastigotes and displayed an
interesting SI ¼ 37 (Table 1, entries 5 and 15).
inhibited LTopIB (IC50 ¼ 41
m
M) and that killed intracellular
amastigotes at submicromolar concentrations (EC50 ¼ 0.67
m
M;
SI ¼ 48). A similar argumentation can be done with the methoxy-
lated ringed compounds 5h (R ¼ 3-MeO-C6H4, Table 2, entry 9) and
6i (R ¼ 4-MeO-C6H4, Table 2, entry 19) that showed good inhibitory
effects on LTopIB (IC50 ¼ 21.78 and 14.11
mM) and SI of 66 and 20,
The presence of the electron donating methoxy group in the
phenyl substituent at 6 position of the indeno-1,5-naphthyridines
had paradoxical antileishmanial effects depending upon the se-
ries of compounds. The tetrahydroindeno-1,5-naphthyridine 5h
respectively.
4. Conclusions
(R
(IC50 ¼ 0.54
sponding indeno-1,5-naphthyridine counterpart 6h had poorer
IC50 ¼ 93.26 M and was no selective at all (SI ¼ 1.07, Table 1, entry
18). This situation changed in case of the indeno-1,5-naphthyridine
¼
3-MeO-C6H4) was very effective killing amastigotes
Some of these compounds showed selective inhibitory effects
on LTopIB mediated relaxation assays compatible to those observed
for the natural inhibitor CPT. All the prepared derivatives have been
further subjected to evaluation of their therapeutic efficacy against
both parasite forms of L. infantum, the responsible agent causing VL
in the Mediterranean Basin. These preliminary studies revealed
that some of newly synthesized compounds exhibited significant
antileishmanial effect in both promastigotes and amastigotes.
Generally speaking some of the fluorinated compounds (5b and 5e)
and 3-methoxylated derivatives (5h) of 4-phenyl ring substituted
at 6 position of tetrahydroindeno-[1,5]-naphthyridines showed low
micromolar and submicromolar antileishmanial effect and inter-
esting SI values. In addition, their corresponding fluorinated
indeno-[1,5]-naphthyridine counterparts (6b, 6e) were also the
most active and selective compounds of this series, which also
should include the 4-methoxyphenyl substituted derivative 6i.
Among all new compounds it is noteworthy that compound 6b, 6i
and 5h showed a selective good inhibition on LTopIB activity that
correlated-well with their antileishmanial effect, thus indicating
this enzyme as putative mechanism of action of these compounds.
Nevertheless, authors consider that the more than one of magni-
tude of difference between the LTopIB inhibition and the anti-
mM) and SI ¼ 63 (Table 1, entry 9), whereas its corre-
m
6i (R ¼ 4-MeO-C6H4) which displayed a good IC50 ¼ 5.8
mM and
SI ¼ 20 (Table 1, entry 19).
The antileishmanial effect of some of these compounds
comparing with the TopIB inhibitor CPT (IC50 ¼ 0.03
m
M; SI ¼ 20.7)
and with drugs in clinical use such as AMB (IC50 ¼ 0.32
m
M;
SI > 62.5) are very promising and deserve further optimization
steps in order to improve the killing effect on the parasite preser-
ving host safety [34].
3.2. Inhibition of leishmanial and human TopIB
In a second extent we investigated if the synthesized tetrahy-
droindeno-1,5-naphthyridines
5 and indeno-1,5-naphthyridine
derivatives 6 could act as inhibitors of L. infantum and human
TopIB. For this purpose a conventional DNA relaxation assay was
used to determine if the compounds could inhibit both enzymes
from converting supercoiled plasmid DNA to relaxed DNA. We used
recombinant LTopIB and hTopIB enzymes expressed in a TopIB-
deficient Saccharomyces cerevisiae strain and purified as described
elsewhere [16].
The effect of indeno-1,5-naphthyridines on relaxation of
supercoiled circular DNA was investigated in a time course exper-
iment using an experimental setup with preincubation of the
compounds with LTopIB for 15 min. The drug concentration was
leishmanial effect, points to
a secondary target of these
compounds. This question together with the mechanism behind
the observed inhibition of TopI is currently under investigation and
is beyond the scope of the present study. The interesting
biochemical and biological features found for these derivatives
provide a promising basis for further development of biologically
active naphthyridines.
fixed at 100
mM. CPT was used as a positive control. After this time,
0.5 g of pSK circular supercoiled DNA was added for an extended
m
5. Experimental protocols
incubation time of 1 min, 5 min and 15 min, respectively. Reactions
were stopped adding SDS and topoisomers were resolved by
agarose gel electrophoresis at the conditions established in Mate-
rial and Methods section (Table 2).
According to the results of Table 2, only the tetrahydroindeno-
1,5-naphthyridines 5a, 5b, 5d, 5e, 5h and 5l (entries 2, 3, 5, 6, 9 and
10) and indeno-1,5-naphthyridines 6b, 6d, 6h, 6i, 6l and 6m (en-
tries 14, 15, 18, 19, 22 and 23) showed significant ability to prevent
relaxation of supercoiled DNA by conventional LTopIB assay. Since
selective inhibition of LTopIB and no inhibition of hTopIB, is a
desirable condition for antileishmanial activity, compounds 5a, 5d,
5.1. Chemistry
The preparation of only some representative compounds is
described in this section. All new compounds prepared have been
described in the supporting information of this communication.
5.1.1. Synthesis of 6-(4-fluorophenyl)-6,6a,7,11b-tetrahydro-5H-
indeno[2,1-c][1,5]naphthy-ridine (5b) by Povarov reaction
Indene 4 (15 mmol, 1.735 mL) and 2 equivalents of BF3$Et2O
(20 mmol, 2.461 mL) were added to a solution of the in situ

746
A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
Table 2
Comparative inhibitory activities of tetrahydroindeno-1,5-naphthyridines 5 and indeno-1,5-naphthyridines 6 on LTopIB and hTopIB.a
Entry
Compound
R
Leishmania TopIB
Time course (min)
Human TopIB
IC50
(m
M)
Time course (min)
IC50 (mM)
1
5
15
1
5
15
1
2
3
4
5
6
7
8
CPTb
5a
5b
5c
5d
5e
5f
5g
5h
5l
5n
5o
6a
6b
6d
6e
6f
6h
6i
6j
6k
6l
6m
6n
6o
þþ
n/a
n/a
e
e
e
no inhibition
45.22 0.10
48.97 0.03
no inhibition
60.59 0.06
41.76 5.26
no inhibition
no inhibition
21.78 0.73
>100
no inhibition
no inhibition
no inhibition
14.67 0.86
23.63 0.47
no inhibition
no inhibition
29.50 0.65
14.11 0.07
no inhibition
no inhibition
42.32 0.04
28.13 1.11
no inhibition
no inhibition
þþ
þþþ
e
e
e
no inhibition
46.45 0.83
no inhibition
no inhibition
22.35 0.19
no inhibition
24.37 3.72
no inhibition
no inhibition
10.73 7.23
no inhibition
no inhibition
no inhibition
87.69 1.21
27.45 3.26
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
no inhibition
31.56 0.59
83.39 2.57
no inhibition
no inhibition
C6H5
þþþ
þþþ
e
þþ
þþþ
e
þþ
e
e
4-F-C6H4
3-F-C6H4
4-CF3-C6H4
2,4-F2-C6H3
3,4-F2-C6H3
Me
3-MeO-C6H4
4-NO2-C6H4
4-pyr
e
e
e
e
n/a
þþþ
e
þþþ
þþ
e
þþþ
e
n/a
e
þþþ
e
þþþ
e
e
þþþ
e
þþ
e
e
e
e
e
e
9
þþþ
n/a
e
þþ
þþþ
e
e
e
e
e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
þþþ
e
þþþ
e
þþ
e
e
e
CO2Et
C6H5
e
e
e
e
e
e
e
e
e
e
e
e
4-F-C6H4
4-CF3-C6H4
2,4-F2-C6H3
3,4-F2-C6H3
3-MeO-C6H4
4-MeO-C6H4
4-MeS-C6H4
4-Me-C6H4
4-NO2-C6H4
2-naphthyl
4-pyr
þþþ
þþþ
e
þþþ
þþþ
e
þþ
þþþ
e
þþþ
þþþ
e
þþ
þþþ
e
e
þþþ
e
e
e
e
e
e
e
þþþ
þþþ
e
þþþ
þþþ
e
þþþ
þþþ
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
þþþ
þþþ
e
þþþ
þþþ
e
þþþ
þþþ
e
þþþ
þþþ
e
þþþ
þþþ
e
þþþ
þþþ
e
CO2Et
e
e
e
e
e
e
a
Compounds were preincubated with the enzyme for 15 min and then 0.5 mg supercoiled DNA was added. For time/course experiments, TopI inhibition was expressed
semiquantitatively according to the following criteria: þþþ total inhibition; þþ similar inhibition to CPT; - no inhibition. For dose/response experiments IC50 values are
expressed as X sd of three different experiments by triplicate.
b
Ref. [34].
prepared aldimine 3b (10 mmol) in CHCl3 (25 mL). The mixture was
stirred at reflux for 4 h. The reaction mixture was washed with 2 M
aqueous solution of NaOH (50 mL) and water (50 mL), extracted
with dichloromethane (2 ꢁ 25 mL), and dried over anhydrous
MgSO4. The removal of the solvent under vacuum afforded and oil
that was purified by silica gel flash column chromatography to
afford 2940 g (93%) of compound 5b as a white solid; mp
4JHH ¼ 1.7 Hz, 1H, Harom), 9.18 (d, 3JHH ¼ 7.8 Hz, 1H, Harom) ppm; 13
C
2
NMR (75 MHz, CDCl3)
d
: 38.0 (CH2), 115.9 (d, JCF ¼ 21.7 Hz,
2HCarom), 124.2 (HCarom), 124.7 (HCarom), 127.2 (HCarom), 127.8
3
(HCarom), 129.0 (HCarom), 131.0 (d, JCF ¼ 8.3 Hz, 2HCarom), 137.2
(Carom), 137.6 (HCarom), 140.2 (Carom), 140.7 (Carom), 143.6 (Carom),
144.5 (2Carom), 147.2 (Carom), 150.5 (HCarom), 155.9 (Carom), 163.6 (d,
1JCF ¼ 249.1 Hz, Carom) ppm; 19F RMN (282 MHz,CDCl3)
d: -
212e214 ꢀC (ethyl acetate/hexane). 1H NMR (300 MHz, CDCl3)
d:
112.6 ppm. HRMS (EI) for C21H13N2F [M]þ calcd 312.1062 found
312.1067.
2.37 (dd, 2JHH ¼ 12.4 Hz, 3JHH ¼ 7.3 Hz, 1H, CH2), 3.19e3.26 (m, 2H,
3
HC6a and CH2), 3.73 (s, 1H, NH), 4.61 (d, JHH ¼ 6.9 Hz, 1H, HC11b),
4.68 (s, 1H, HC6), 6.86e7.16 (m, 7H, Harom), 7.35e7.42 (m, 2Harom),
3
4
7.74 (d, JHH ¼ 7.2 Hz, 1H, Harom), 7.98 (dd, JHH ¼ 1.7 Hz,
5.1.3. Synthesis of ethyl 7H-indeno[2,1-c][1,5]naphthyridine-6-
carboxylate (6ꢀ)
3JHH ¼ 4.1 Hz, 1H, Harom) ppm; 13C NMR (75 MHz, CDCl3)
d: 31.7
2
(CH2), 48.08 (HC), 49.3 (HC), 57.2 (HC), 115.5 (d, JCF ¼ 21.1 Hz, 2C,
To a solution of tetrahydro-5H-indeno[2,1-c][1,5]-naphthyridine
5o (2 mmol, 0.585 g) in acetic acid (7 mL) Mn(AcO)3 (10 mmol,
2.67 g) was added and the mixture was stirred at room temperature
for 24 h. The reaction mixture was poured onto water (20 mL),
neutralized with saturated sodium bicarbonate solution, extracted
with CH2Cl2 (2 ꢁ 20 mL) and dried with MgSO4. The solvent of the
resulting solution was removed under vacuum leading to an oil that
was purified by column chromatography on alumina (ethyl acetate/
hexane 1:20) to afford compound 6o 0.458 g (79%) as a yellow solid,
HCarom), 121.9 (HCarom), 122.5 (HCarom), 124.5 (HCarom), 126.6
3
(HCarom), 126.9 (HCarom), 127.3 (HCarom), 128.3 (d, JCF ¼ 8.1 Hz, 2C,
HCarom), 137.8 (d, 4JCF ¼ 3.0 Hz, Carom), 140.9 (HCarom), 141.4 (Carom),
142.4 (Carom), 144.7 (Carom), 145.1 (Carom), 162.4 (d, 1JCF ¼ 246.0 Hz,
C
arom) ppm; 19F NMR (282 MHz, CDCl3)
d: - 115.2 ppm. HRMS (EI)
for C21H17N2F [M]þ calcd 316.1376; found 316.1379.
5.1.2. Synthesis of 6-(4-fluorophenyl)-7H-indeno[2,1-c][1,5]
naphthyridine (6b) by dehydrogenation of compound 5b with DDQ
To a solution of tetrahydro-5H-indeno[2,1-c][1,5]-naphthyridine
5b (1 mmol, 0.316 g) in toluene (20 mL) was added DDQ (1 mmol,
0.227 g) and the mixture was irradiated with microwave at
150 W at 25 ꢀC for 2 h. The formed solid was filtered off, the solvent
of the resulting solution removed under vacuum leading to an oil
that was purified by column chromatography on alumina (ethyl
acetate/hexane 1:20) to afford 0.193 g (62%) of compound 6b as a
white solid, mp ¼ 190e192 ꢀC (ethyl acetate/hexane). 1H RMN
mp 153e154 ꢀC (ethyl acetate/hexane). 1H RMN (300 MHz, CDCl3):
3
d
: 1.47 (t, JHH ¼ 7.2 Hz, 3H, CH3), 4.39 (s, 2H, CH2), 4.54 (q,
3JHH ¼ 7.2 Hz, 2H, CH2), 7.41e7.50 (m, 2H, Harom), 7.61e7.66 (m, 2H,
3
3
H
arom), 8.56 (dd, JHH ¼ 8.7 Hz, JHH ¼ 1.7 Hz, 1H, Harom), 9.02 (dd,
4 3
3JHH ¼ 7.3 Hz, JHH ¼ 1.8 Hz, 1H, Harom), 9.07 (dd, JHH ¼ 4.1 Hz,
4JHH ¼ 1.7 Hz, 1H, Harom) ppm; 13C NMR (75 MHz, CDCl3)
d:14.4
(CH3), 38.5 (CH2), 62.2 (OCH2), 124.3 (HCarom), 124.6 (HCarom), 127.0
(HCarom), 127.4 (HCarom), 129.4 (HCarom), 138.5 (HCarom), 139.1
(Carom), 139.7 (Carom), 141.3 (Carom), 142.7 (Carom), 145.1 (Carom), 145.2
(Carom), 148.2 (Carom), 152.4 (HCarom), 165.3 (C¼O) ppm. HRMS (EI)
for C18H14N2O2 [M]þ calcd 290.1055 found 290.1059.
(300 MHz, CDCl3) d: 4.24 (s, 2H, CH2), 7.25e8.04 (m, 8H, Harom), 8.53
(dd, 3JHH ¼ 8.5 Hz, 4JHH ¼ 1.7 Hz, 1H, Harom), 9.11 (dd, 3JHH ¼ 4.1 Hz,

A. Tejería et al. / European Journal of Medicinal Chemistry 124 (2016) 740e749
747
5.2. Biology
The animal research described in this manuscript complies with
Spanish Act (RD 53/2013) and European Union Legislation (2010/
63/UE). The used protocols were approved by the Animal Care
5.2.4. Recombinant expression of L. infantum and human TopIB
Cloning of LTopIB (encoding large and small subunits) and
hTopIB ORFs, expression and purification of functional enzymes
were carried out as previously described [16]. Briefly, S. cerevisiae
EKY3 strain deficient in TopIB activity [MAT
a ura3-52 his3D200
ꢀ
Committee of the University of Leon (Spain), project license num-
leu2 1 trp1 63 top1 :TRP1], was transformed with the bicis-
D
D
D
ber (PI12/00104).
tronic pESC-URA vector, which carries both LTopIB or hTopIB ORFs.
Single colonies were incubated overnight in SC-uracil media sup-
plemented with 2% dextrose (w/v). Induction of GAL1/GAL10 pro-
moters was performed with 2% galactose (w/v) for 6 h. Yeasts were
harvested, washed with cold 1 ꢁ TEEG buffer (50 mM TriseHCl pH
7.4, 1 mM EDTA, 1 mM EGTA, 10% glycerol) and resuspended in
15 mL of the same buffer supplemented with 0.2 M KCl and pro-
tease inhibitors cocktail (Roche Farma SA, Spain). Yeasts were dis-
rupted by vortexing in a glass bead-beater at 4 ꢀC. Protein extracts
were obtained by centrifugation at 15,000g for 45 min in cold and
precipitated with ammonium sulphate added to 75% saturation at
5.2.1. Leishmania strain for in vitro assays
Leishmania infantum BCN150 iRFP (from now on L. infantum-
iRFP) is a genetically modified strain that constitutively in-
corporates the iRFP infrared gen for infrared detection [34]. Pro-
mastigotes are routinely cultured in M199 medium supplemented
with 25 mM HEPES (pH 6.9), 10 mM glutamine, 7.6 mM hemin,
0.1 mM adenosine, 0.01 mM folic acid, 1 ꢁ RPMI 1640 vitamin mix
(Sigma), 10% (v/v) heat-inactivated foetal calf serum (FCS), and
antibiotic cocktail (50 U/mL penicillin and 50 mg/mL streptomycin).
Naphthyridine screening is performed in black 96-well plates with
clear bottom. Stock solutions of each compound were prepared in
DMSO and stepwise diluted in M199 media. Growing concentra-
4
ꢀC. Enzymes were further purified by ion-exchange (P-11 phos-
phocellulose) and hydrophobic interaction (phenyl-sepharose)
FPLC (Akta, GE Healthcare).
tions of each compound (0.07e300 mM) were added to each well.
Twenty microliters of each compound are added per well to com-
plete 180 mL, final volume with the culture of promastigotes. The
final concentration of DMSO was never higher than 0.1%. DMSO and
AMB were included as negative and positive controls, respectively.
The viability of free-living promastigotes was assessed recording
the fluorescence emission at 708 nm in an Odyssey (Li-Cor) infrared
imaging system. All compounds and controls were assayed by
triplicate.
5.2.5. DNA relaxation assays
TopIB activity was assayed by the relaxation of negatively
supercoiled plasmid DNA. One unit of recombinant LTopIB was
incubated with the corresponding drug for 15 min at 4 ꢀC. Then, the
reaction mixture containing 0.5
Tris-HCl buffer pH 7.5, 5 mM MgCl2, 0.1 mM EDTA, 15
serum albumin, 50 mM KCl, in a total volume of 20
m
g of supercoiled pSK DNA, 10 mM
g/mL bovine
L was added.
m
m
Reaction mixtures were incubated for 5 min at 25 ꢀC. Enzyme re-
actions were stopped by the addition of up to 1% SDS (w/v) (final
concentration) and digested with 1 mg/mL proteinase K at 37 ꢀC
during one extra hour to remove the protein that remained linked
to DNA fragments. The extent of plasmid DNA relaxation was
assessed in 1% agarose gels by electrophoresis in 0.1 M Tris borate
EDTA buffer (pH 8.0) at 2 V/cm for 16 h. Gels were visualized with
UV illumination after ethidium bromide (0.5 mg/mL) staining. The
gel was run for 16 h at 4 V/cm and the images of gels were acquired
with a G-BOX (Syngene UK).
5.2.2. Ex vivo splenic explant cultures
To obtain primary infected splenic explants, BALB/c mice are
inoculated intraperitoneally with 108 L. infantum iRFP metacyclic
promastigotes isolated from stationary-phase culture by negative
selection with peanut agglutinin [35]. Five weeks post-infection,
mice are slaughtered and spleens aseptically dissected, washed in
cold phosphate-buffered saline (PBS), and placed in petri dishes.
Small pieces were obtained by using a scalpel. In order to obtain a
single-cell suspension, tissue was incubated with 5 mL of 2 mg/mL
collagenase D (Roche) prepared in buffer (10 mM HEPES [pH 7.4],
150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) for
20 min at 37 ꢀC. A cell suspension containing traces of spleen mass
Acknowledgement
This collaborative research was carried out under the auspices of
was gently passed through a 100-mm cell strainer to remove tissue
Ministerio de Economía
y Competitividad (MINECO; CYTED
fragments [20b]. Splenocytes were washed twice with PBS by
centrifugation (500 ꢁ g for 7 min at 4 ꢀC) and resuspended in RPMI
medium supplemented with 10% FCS, 1 mM sodium pyruvate,
214RT0482, CTQ2015-67871-R), Instituto de Salud Carlos III-Feder
ꢀ
(PI12/00104), Junta de Castilla
y
Leon (Grants to: Gr238,
LE182U13) and by Gobierno Vasco, Universidad del País Vasco (GV,
IT 992-16; UPV). UPV/EHU-SGIker technical support (MINECO, GV/
EJ, European Social Fund) is also gratefully acknowledged. M. G.
thanks Basque Government for a formation contract.
1 ꢁ RPMI vitamins, 10 mM HEPES, and 50 U/mL penicillin and 50
mg/
mL streptomycin at 37 ꢀC with 5% CO2. Cells were counted and
diluted at different cell densities. Cells were seeded until confluence,
and different concentrations of the studied compounds were
administered to the explants for 48 h. Amastigote viability was
assessed by recording the fluorescence emission of infected sple-
nocytes at 708 nm in an Odyssey (Li-Cor) infrared imaging system.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.09.017.
5.2.3. Cytotoxicity assay and selectivity index (SI) determination
The cytotoxicity of both series of indeno-1,5-naphthyridines was
assessed on uninfected splenocytes, obtained and cultured as
described elsewhere. Splenocytes were seeded in 96-well plates in
the presence of different concentrations of the assayed compounds
for 48 h at 37 ꢀC. Viability of the cultures was determined using the
Alamar Blue staining method, according to manufacturer's rec-
ommendations (Invitrogen). Selectivity Indexes (SI) for the com-
pounds were determined as the ratio between the CC50 values for
non-infected mouse splenocytes and the IC50 values for amasti-
gotes in infected splenocytes.
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