Substituted 1,5-naphthyridine derivatives as novel antileishmanial agents. Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2018.04.033

Visceral leishmaniasis is a parasitic disease that affects, among other areas, both sides of the Mediterranean Basin. The drugs classically used in clinical practice are pentavalent antimonials (Sb^V) and amphotericin B, which are nephrotoxic, r

Full text of DOI:10.1016/j.ejmech.2018.04.033

Accepted Manuscript
Substituted 1,5-naphthyridine derivatives as novel antileishmanial agents. Synthesis
and biological evaluation
Ana Tejería, Yolanda Pérez-Pertejo, Rosa M. Reguera, Rafael Balaña-Fouce,
Concepción Alonso, María González, Gloria Rubiales, Francisco Palacios
PII:
S0223-5234(18)30367-2
10.1016/j.ejmech.2018.04.033
EJMECH 10382
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 February 2018
Revised Date: 11 April 2018
Accepted Date: 16 April 2018
Please cite this article as: A. Tejería, Y. Pérez-Pertejo, R.M. Reguera, R. Balaña-Fouce, Concepció.
Alonso, Marí. González, G. Rubiales, F. Palacios, Substituted 1,5-naphthyridine derivatives as novel
antileishmanial agents. Synthesis and biological evaluation, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.04.033.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Substituted 1,5-naphthyridine derivatives as novel
antileishmanial agents. Synthesis and biological
evaluation.
§
§
§
§,
Ana Tejería, Yolanda Pérez-Pertejo, Rosa M. Reguera, Rafael Balaña-Fouce, *
#
#
#
#,
Concepción Alonso, María González, Gloria Rubiales, and Francisco Palacios *
§
Departamento de Ciencias Biomédicas; Universidad de León, Campus de Vegazana s/n; 24071
León (SPAIN), Phone 34 987 291590.
#
Departamento de Química Orgánica I, Facultad de Farmacia and Centro de Investigación
Lascaray (Lascaray Research Center). Universidad del País Vasco/Euskal Herriko
Unibertsitatea (UPV/EHU). Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain.

ACCEPTED MANUSCRIPT
Substituted 1,5-naphthyridine derivatives as novel
antileishmanial agents. Synthesis and biological
evaluation.
§
§
§
§,
Ana Tejería, Yolanda Pérez-Pertejo, Rosa M. Reguera, Rafael Balaña-Fouce, *
#
#
#
#,
Concepción Alonso, María González, Gloria Rubiales, and Francisco Palacios *
§
Departamento de Ciencias Biomédicas; Universidad de León, Campus de Vegazana s/n; 24071
León (SPAIN), Phone 34 987 291590.
#
Departamento de Química Orgánica I, Facultad de Farmacia and Centro de Investigación
Lascaray (Lascaray Research Center). Universidad del País Vasco/Euskal Herriko
Unibertsitatea (UPV/EHU). Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain.
Keywords: Leishmania, naphthyridines, DNA-topoisomerase
Abbreviations: VL, Visceral Leishmaniasis; TopIB, Type IB DNA Topoisomerase; IRFP, Infra
Red Fluorescent Protein; FCS, Fetal Calf Serum; DMSO, Dimethyl sulfoxide; SI, Selectivity
Index; CPT, camptothecin; CPTs, camptothecin derivatives; HDAr, hetero-Diels-Alder reaction;
TLC, thin layer chromatography; ERC, electrocyclic ring closure.
1

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ABSTRACT. Visceral leishmaniasis is a parasitic disease that affects, among other areas, both
sides of the Mediterranean Basin. The drugs classically used in clinical practice are pentavalent
V
antimonials (Sb ) and amphotericin B, which are nephrotoxic, require parenteral administration,
and increasing drug resistance in visceral leishmaniasis has been observed. These circumstances
justify the search of new families of compounds to find effective drugs against the disease.
Eukaryotic type I DNA topoisomerase (TopIB) has been found essential for the viability of the
parasites, and therefore represents a promising target in the development of an antileishmanial
therapy. In this search, heterocyclic compounds, such as 1,5-naphthyridines, have been prepared
by cycloaddition reaction between N-(3-pyridyl)aldimines and acetylenes and their
antileishmanial activity on promastigotes and amastigote-infected splenocytes of Leishmania
infantum has been evaluated.
In addition, the cytotoxic effects of newly synthesized compounds were assessed on host murine
splenocytes in order to calculate the corresponding selective indexes (SI). Excellent
antileishmanial activity of 1,5-naphthyridine 19, 21, 22, 24 and 27 has been observed with
similar activity than the standard drug amphotericin B and higher selective index (SI >100)
towards L. infantum amastigotes than amphotericin B (SI > 62.5). Special interest shows the 1,5-
naphthyridine 22 with an IC50 value (0.58 ± 0.03 µM) similar to the standard drug amphotericin
B (0.32 ± 0.05 µM) and with the highest selective index (SI = 271.5). In addition, this compound
shows remarkable inhibition on leishmanial TopIB. However, despite these interesting results,
further studies are needed to disclose other potential targets involved in the antileishmanial effect
of these novel compounds.
2

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1
. Introduction
Similar to other Neglected Tropical Diseases, the treatment of the three forms of leishmaniasis
resides in a limited number of medicines that are either outdated or plenty of side effects [1].
V
Briefly, first line pentavalent antimony (Sb ) derivatives are metabolically-activated pro-drugs
III
that have to be reduced to toxic trivalent antimony (Sb ). The second line drugs include polyene
fungicides, such as amphotericin B (AMB) or the oral anticancer drug miltefosine. AMB,
®
administered either as sodium deoxycholate salt (Fungizone ) or as liposomal suspension
(AmBisome®), is nephrotoxic and needs patient hospitalization [2]. For its part, miltefosine is
the most remarkable antileishmanial compound introduced in the last decades, except for the
potential embriotoxicity reported in pregnant women [3]. Therefore, an efficient, safe,
economically affordable and self-administrable drug that accomplishes the Target Product
Profile requirements for leishmaniasis requested by Drug for Neglected Diseases initiative
(DNDi) is an urgent need (https://www.dndi.org). In addition, new and closer-to-reality disease
models are demanded in order to test under High Throughput Screening conditions the myriads
of existing and novel small molecules present in different libraries and repositories [4].
The role played by DNA topoisomerases (Top) in DNA physiology has inspired several
anticancer and antimicrobial drugs based on the irreversible inhibition of these enzymes [5].
Unlike the type II family, eukaryotic type I Top (TopI) have singular features in Leishmania (and
other trypanosomatids) that make them a selective target against these pathogens: i) the enzyme
has an increased expression during the rapidly invading stages of the parasite, and especially ii)
the pathogen’s enzyme has an atypical structure that can be exploited in targeted-driven
programs of drug design [6]. Several years ago, our research group found that L. infantum TopI
3

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(
LTopIB) had heterodimeric structure composed of two different protomers, which had to be
previously assembled to reconstitute a full active enzyme [7]. Furthermore, it was undoubtedly
demonstrated that the large protomer contained the four amino acids of the active site, whereas
the small protomer contained a catalytic tyrosine, the latter being a phylogenetically conserved
amino acid involved in the introduction of transient phosphodiester bonds to DNA substrate [8].
To date, two classes of TopIB inhibitors have been described: i) interfacial inhibitors or
poisons that stabilize the enzyme-DNA complex (cleavage complex), thus delaying the religation
step and facilitating the formation of single-strand breaks in DNA, and ii) catalytic inhibitors,
which interact either with the substrate DNA or with catalytic domains of the enzyme [9].
Camptothecin (CPT) their hydrosoluble derivatives topotecan and irinotecan [10], and other non-
CPT poisons such as indolocarbazoles and indenoisoquinolines [11] belong to the first group.
Catalytic inhibitors for their part, reversibly interact with the enzyme and do not induce DNA
lesions. Amongst them, many natural products and derivatives have been reported as potential
TopI catalytic inhibitors: plant terpenoids like betulinic acid, acetylenic fatty acids isolated from
sea animals or plant naphthoquinones. Many of these compounds have shown strong
antileishmanial activity [12].
In this study, we assess the antileishmanial effect of a series of 1,5-naphthyridines compounds
against both stages – free-living promastigotes and intracellular amastigotes – of L. infantum, the
aethiological agent responsible for VL in the Old World. For this purpose, an intracellular
screening of macrophages isolated from naturally infected BALB/c mice with an infrared-
emitting L. infantum strain was used [13]. Furthermore, we have studied the potential role played
by LTopIB as putative target of these compounds.
4

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Some chemicals showing good human TopIB inhibition, such as CPT (Figure 1), its
derivatives and indeno[1,5]naphthyridines, have been studied as LTopIB inhibitors [14]. From a
chemical point of view, these derivatives present nitrogenated fused ring heterocycles. This
feature may be important towards their effectiveness as TopIB inhibitors. In addition, a wide
range of approved drugs [15] and antileishmanial lead compounds revealed the importance of the
skeleton of nitrogen-containing heterocycles [16]. Taking this into account, other quasi-flat
condensed heterocycles, such as naphthyridine derivatives (Figure 1) [17], might be also
adequate candidates as LTopIB inhibitors and overcome some of CPTs inherent limitations.
Figure 1. Structure of camptothecin (left), amphotericin B (middle) and newly synthesized 1,5-
naphthyridines (right).
A wide range of six-membered nitrogen-containing heterocyclic compounds play a major role in
organic chemistry through their widespread presence in nature and in their consequent biological
activity with applications in biochemistry, pharmacology and material science. [15, 18] Many
strategies are described in the literature for the synthesis of nitrogenated heterocycles, among
which one of the most straightforward is the hetero-Diels-Alder reaction (HDAr). This reaction
type is an atom-economic alternative for the carbon-carbon and carbon-heteroatom bond
construction [19] and represents an excellent tool for the generation of six-membered rings with
a high molecular complexity [20], which may have industrial applications.[21] Among those
strategies, the Povarov reaction [22, 23] allows the preparation of nitrogen-containing
5

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heterocyclic compounds in an excellent way. [24] This methodology also represents a direct
route to the naphthyridine core structure of interesting biologically active compounds as TopI
inhibitors and with antiproliferative activity against several cancer cell lines as reported in our
research group.
This manuscript describes the synthesis of 1,5-naphthyridines by cycloaddition reaction between
N-(3-pyridyl)aldimines and alkynes as well as the antileishmanial effect of new heterocyclic
compounds on L. infantum, the aethiological 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. [13] In addition, the inhibitory effect of these compounds has been studied on recombinant
TopIB from both Leishmania and human sources. These results point 1,5-naphthyridines as
promising antileishmanial drugs.
2
. Chemistry
First, the synthesis of target compounds, 1,5-naphthyridines, was accomplished. We started with
the preparation of the corresponding N-(3-pyridyl)aldimines 3-9 by using 3-aminopyridine 1 and
aromatic aldehydes 2 in chloroform as solvent and in the presence of molecular sieves (Scheme
1
).
Scheme 1. Preparation of novel 1,5-naphthyridines 17-32. Reagents and conditions: (A) 3-pyridylamine 1 (1
equiv), aldehyde 2 (1 equiv), molecular sieves 4 Å, chloroform, reflux, 15-48 h. (B) alkyne 10-15, chloroform,
reflux, 15-70h.
6

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Afterwards, the obtained aldimines 3-9 were reacted with acetylenes 10-15 (Chart 1). The
reaction without catalyst did not work and the starting products were recovered. However, the
use of trifluorboroetherate as Lewis acid catalyst gave us good results: the optimal ones were
when 2 equivalents of Lewis acid were used and only 2,4-disubstituted 1,5-naphthyridines 17-31
2
were regioselectively obtained (Chart 2) when terminal alkynes were used (R = H, Chart 1),
while the formation of the other regioisomers, namely 2,3-disubstituted 1,5-naphthyridines was
not observed. [27] It is noteworthy that not only terminal alkynes 10-14 (Chart 1) can be used for
the synthesis of 1,5-naphthyridines 17-31 but also internal alkyne 15 is appropriate for the
formation of corresponding heterocyclic compounds, such as nahthyiridine 32 (Chart 2).
Chart 1. Structures of aldimines 3-9 and acetylenes 10-15 used.
The structure of compounds 17-32 was assigned on basis of NMR spectra and mass
spectrometry. For example, when aldimine 5 (Ar = 4-CF C H ) and 4-methoxyphenyl acetylene
3
6
4
1
2
1
2 (R = 4-MeOC H , R = H) were used the 1,5-naphthyridine 25 (Chart 2) was obtained. Its
6 4
structure was assigned by means of NMR experiments and confirmed by HRMS. For instance, in
1
9
the F-NMR spectrum of compound 25 one signal was observed at δ = - 63.1 ppm and in the
F
1
H-NMR spectrum the corresponding two signals at low field corresponding to two protons of
3
the naphthyridine ring, one double doublet at δ
H
= 8.49 ppm with coupling constants JHH = 8.5
7

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4
3
Hz, JHH = 1.8 Hz and another double doublet at δ
H
= 9.01 with coupling constants JHH = 4.3 Hz
4
1
and J
= 1.8 Hz. In general for 1,5-naphthyridines 17-32, in the H-NMR spectra, a
HH
representatively characteristic signal appears as a singlet approximately at 8.1 ppm for the proton
1
3
at 3 position of naphthyridine ring. Moreover, in C NMR spectra signals for aromatic carbons
were observed for 1,5-naphthyridines 17-32. Among them two signals could be representatively
characteristic for these compounds, such as signal of CH aromatic carbon at 8 position which
appears at δ = 137.9 – 138.2 ppm range and signal at δ = 150.0 – 151.6 ppm range which
corresponds to the CH at 6 position of the naphthyridine ring. Moreover, its structure has been
unequivocally confirmed by X-ray analysis. [27]
a
Chart 2. Structures of 1,5-naphthyridines 17-32 obtained ( Reference 27).
8

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Formation of 1,5-naphthyridines 17-32 could be explained by a formal [4+2] process of imines
3
-9 with alkynes 10-15 and subsequent aromatization under the reaction conditions to afford
corresponding naphthyridines 17-32 (Scheme 1). In fact, our previous work based on a combined
experimental and computational study of the Povarov reaction between N-(3-pyridyl)aldimines
and acetylenes with a Lewis acid suggested a stepwise [4+2]-cycloaddition mechanism through a
3
-azatriene intermediate 16. [27] A wide scope of 1,5-naphthyridines 17-32 with electron-
donating and electron-withdrawing aromatic substituents, including fluorine substituents, [28]
has been achieved, and in order to increase the diversity of these compounds also derivatives
1
with heterocyclic substituents, such as thiophene (R = 3-thienyl), 30 and 31 were also obtained.
Biological activity against promastigotes and amastigotes-infected macrophages of L. infantum,
has been studied for all these 1,5-naphthyridine heterocyclic compounds 17-32 (Chart 2).
3
. Biological results and discussion
Activity of 1,5-naphthyridine against L. infantum. To assess the proficiency of new 1,5-
naphthyridines against Leishmania parasites, an improved protocol consisting in evaluating the
effect on both free-living promastigotes and intracellular amastigotes that infect natural host cells
were used. This method uses a specially engineered L. infantum strain that constitutively express
the infrared fluorescent protein (iRFP) called L. infantum-iRFP, which provides infrared
emission (708 nm) to both viable promastigotes and amastigotes. In this report we used infected
macrophages harboring amastigotes that were isolated from BALB/c mice infected 5 to 6 weeks
earlier with L. infantum-iRFP [13]. This method has several advantages: i) allows the rapid
assessment of viable pathogens under drug pressure; ii) the action of the drugs is performed
under natural ex vivo conditions, which includes the immunological environment that can help to
destroy the invading cells within spleen macrophages [29]. Finally, drug cytotoxicity, was
9

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assessed in free-parasite macrophages obtained under the same conditions from spleens dissected
-from uninfected BALB/c mice.
Table 1 shows the antileishmanial effect of the new series of compounds with the 1,5-
naphthyridine core rings by means of dose-response curves on L. infantum-iRFP promastigotes
and amastigotes, fitted by nonlinear analysis with the Sigma-Plot statistical package. For both
forms the drug effect on the pathogen was expressed as the 50%-reduction of infrared emission
(
IC ) with respect to negative control (that contains DMSO as drug vehicle). Selectivity indexes
50
of each compound were raised from the ratio between their cytotoxicity on uninfected explants
CC ) vs. the IC values obtained on infected ex vivo splenic explants. The leishmanicidal effect
(
50
50
of AMB (drug in clinical use) was included as positive control just for comparison purposes.
In the case of fluoro substituted 1,5-naphthyridines 20, 23, 26 and 31 (Ar = 4-F-C H , Table
6
4
1
, entries 5, 8, 11 and 16) a high degree of the macrophage destruction or low killing capability
of the parasite were observed. Interestingly, the values of antiparasitic activity obtained with 1,5-
naphthyridines 17-32 derivatives were better on intracellular amastigotes than on free-living
promastigotes (Table 1). It is noteworthy the remarkable effect observed for compounds 25, 29,
3
0 and 32 with trifluoromethyl substituents (Ar = 4-CF -C H ), where generally the cytotoxicity
3 6 4
on free-living promastigotes is higher than 100 µM (Table 1, entries 10, 14, 15 and 17). At the
same time the macrophage destruction is not observed for these mentioned compounds, showing
in murine splenocytes low cytotoxic values (over 100 µM in almost all these cases), which
means a selective killing effect on intracellular parasites.
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Table 1: Chemical structure and biological activity of 1,5-naphthyridine compounds (17-32) on L. infantum iRFP promastigotes and
amastigotes (naturally-infecting mouse macrophages). Each value is the mean ± sd of at least three different experiments made by
triplicate.
IC (µM)
CC (µM)
5
0
50
L. infantum
Promastigotes
0.77 ±0.15
L. infantum
Amastigotes
Murine
splenocytes
>20
1
2
entry
Compound
Ar
R
R
SI*
>62.5
6.5
1
2
3
4
5
6
7
8
9
Amphotericin B (AMB)
0.32 ±0.05
8.42±1.91
2.82±0.76
0.47±0.95
7.41±1.26
1.02±0.12
0.58±0.03
5.04±0.19
0.29±0.35
2.26±0.45
0.76±0.06
0.58±0.04
3.79±0.81
9.47±0.12
3.67±0.16
7.93±22.26
2.37±0.12
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
C
6
H
5
C
6
H
5
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
27.2 ± 12.3
>100
54,8 ± 6,1
21.7 ± 4.8
3,4-F -C H
C H5
6
7.7
2
6
3
4-CF -C H
C H5
6
37.3 ± 7.1
>100
65.7 ± 5.4
139.8
16.7
143.1
271.5
30.2
103.9
67.1
31.8
187.3
35.1
18.8
49.4
4.1
3
6
4
4-F-C
6
H
4
C
6
H
5
124 ± 44.3
3-NO -C H
C H5
6
79.24±2.28
>100
145.90±20.83
157.45±2.36
152.54±37.49
30.13±3.87
151.6 ± 89.7
24.16±2.92
108.65±0.35
132.71±1.74
178.4 ± 63.2
181.4 ± 51.5
32.51±12.30
126.1 ± 39.6
2
6
4
3-MeO-C H4
C H5
6
6
4-F-C
6
H
4
4-C
6
H
5
-C
6
H
4
4
45.17±5.26
15.09±0.42
>100
3-NO -C H
4-C H -C H
2
6
4
6
5
6
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
4-CF -C H
4-MeO-C H4
6
3
6
4
4-F-C
6
H
4
4-MeO-C
4-MeO-C H4
6
H
4
97.17±3.5
20.69±0.95
45.18±8.47
>100
4-NO -C H
2
6
4
4
6
3-NO -C H
4-MeO-C H4
2
6
6
3
4-CF -C
6
H
4
3 6 4
4-CF -C H
4-CF -C H
3-thienyl
3-thienyl
>100
3
6
4
4-F-C H4
66.95±1.51
>100
6
3
4-CF -C
6
H
4
C
6
H
5
3
53.2
a
Antileishmanial effects (IC50 ± SD) on promastigotes and amastigotes of L. infantum. Cytotoxicity effects (CC50 ± SD) on murine splenocytes. *SI: Selective
Index.
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In addition, when nitro substituted 1,5-naphthyridines 27 (Ar = 4-NO -C H ) and 21
2
6
4
and 24 (Ar = 3-NO -C H ) were tested high selectivity indexes (SI > 100) were
2
6
4
1
observed (Table 1, entries 6, 9 and 12). For example, when 27 (Ar = 4-NO -C H , R =
2
6
4
2
4
-MeOC H , R = H) was tested no macrophage destruction was observed with an IC
6 4 50
of murine splenocytes of 108.65±0.35 µM and a promastigotes killing capability of IC50
0.58±0.04, which results in a very high selectivity index (SI = 187.3). Even
=
1
2
trifluoromethyl substituted 1,5-naphthyridine 19 (Ar = 4-CF -C H , R = C H , R = H)
3
6
4
6
5
shows an excellent selectivity index (SI = 139.8). These results along with the previous
ones may suggest that the presence of electron-withdrawing substituents such as
trifluoromethyl or nitro groups in the aromatic ring of position 2 in 1,5-naphthyridines
1
9, 21, 24, 25, 27, 29, 30 and 32 increases the antileishmanial effect.
Interestingly, the inclusion of an electron-donating subtituent in the phenyl
substituent at position 2 of the 1,5-naphthyridine core, very much improved both the
antileishmanial effect (amastigote) and the corresponding SI values while macrophage
1
destruction was minimum. In such a way, the compound 22 (Ar = 3-MeO-C H , R =
6
4
2
C H , R = H) showed the highest SI value of this family (SI = 271.5; Table 1, entry 7).
6
5
Inhibition of leishmanial and human TopIB. Previous results reported by the
authors showed that the antileishmanial effect of a series of tetrahydroindeno-1,5-
naphthyridines and indeno-1,5-naphthyridines was due in part to their selective
inhibition on LTopIB [14]. With similar aims we performed conventional DNA
relaxation assays with the new series of compounds. Therefore, in order to study the
effect of 1,5-naphthyridines on TopIB from human and Leishmania sources,
conventional relaxation assays were carried out.
1
2

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These assays are based on the ability of these compounds to prevent the relaxation of
circular supercoiled DNA mediated by TopI. For this purpose, recombinant LTopIB and
hTopIB genes expressed in a TopIB-deficient Saccharomyces cerevisiae platform and
purified as described elsewhere [7] were used. The effect of these compounds was
compared with the inhibitory effect of CPT that was used as positive control in these
experiments [14,17].
To assess the effect of 1,5-naphthyridines on TopIB enzymes, we performed
previous studies with or without a 15-min preincubation step (Figure 2) of purified
TopIB with a fixed concentration of compound 17 (as representative compound of the
whole series) both at 4ºC and 37ºC. However, since CPT does not bind the enzyme in
the absence of DNA, the preincubation step was unnecessary [25]. Preincubation
temperature plays an important role in TopIB inhibition of compound 17 and in the rest
of the compounds, as well, since only when this compound was in contact with TopIB
at 37 ºC, the inhibition was effective. The concentration of the drug was set at 100 µM
and CPT was used as a positive control at the same concentration. Relaxation assay was
triggered by addition of 0.5 µg supercoiled pSK DNA substrate and subsequent
incubation for increasing time periods ranging from (1-16 min) (see Supplementary
Material, Fig. 1.SM). Reactions were stopped adding SDS and topoisomers were
resolved by agarose gel electrophoresis at the conditions established in Material and
Methods section.
Once established the optimal assay conditions, the IC50 of each compound was
calculated by means of dose-response curves. Table 2 shows the inhibition of both
LTopIB and hTopIB mediated by 1,5-naphthyridines calculated in terms of the
conversion of supercoiled DNA to its relaxed form, as determined by quantifying the
bands representing relaxed or supercoiled DNA in agarose gels according to the method
1
3

ACCEPTED MANUSCRIPT
described in material and methods. Equal concentrations of DMSO were added to each
reaction in order to assess the potential negative effect of the vehicle in the experiments.
Figure 2. Representative agarose gels showing the role of preincubation time in the inhibition of the
relaxation activity of TopIB. Compound 17 was assayed on LdTopIB (top panel) and on hTopIB (bottom
panel) without (figures a and d) and with 10 min preincubation at 4°C (figures b and e) and at 37 °C
(
figures c and f), respectively. The relative position of the negatively supercoiled DNA substrate is
indicated by SC, N is the nicked DNA. Reactions were stopped with 1% (w/v) SDS. Lanes (C) contain
.5 µg of pSK plasmid DNA. Lanes (DMSO) show the activity of the enzymes in absence of drugs but
0
with 2.5% (v/v) DMSO. Lanes (CPT) show the inhibitory effect of 100 µM CPT. Lanes (6a) show the
effect of 100 µM of compound 17.
Table 2 gathers the IC values of a total of 16 compounds tested on both LTopIB
5
0
1
and hTopIB enzymes. According to the results only the compound 17 (Ar = R = C H ,
6
5
2
R = H) showed significant ability to prevent the relaxation of supercoiled DNA by
conventional TopIB assay (IC50 = 24.42 µM). However, this compound was not
selective inhibiting the leishmanial enzyme since it prevented hTopIB-mediated DNA
relaxation at even lower concentrations (IC = 11.24 µM).
5
0
1
4

ACCEPTED MANUSCRIPT
Table 2. Inhibition of relaxation activity of LTopIB and hTopIB by 1,5-naphthyridines
1
7-32. Each value is the mean ± sd of at least three different experiments made by
triplicate.
Leishmania TopIB Human TopIB
1
2
entry Compound
Ar
R
R
IC (µM)
IC (µM)
5
0
50
1
2
3
4
5
6
7
8
9
CPT
no inhibition
24.42 ± 1.69
76.57 ± 1.82
69.46 ± 0.38
81.94 ± 4.70
No inhibition
53.04 ± 0.91
58.05 ± 0.94
78.73 ± 0.94
No inhibition
No inhibition
No inhibition
No inhibition
79.24 ± 0.73
62.81 ± 2.46
49.26 ± 0.02
75.69 ± 3.17
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
C H
C H
H
H
11.24±0.95
73.11±1.11
72.68±0.94
72.77±1.08
No inhibition
82.03±0.97
83.25±6.50
67.53±10.34
No inhibition
No inhibition
No inhibition
No inhibition
50.93±3.85
82.08±1.30
43.85±3.72
70.61±4.09
6
5
6
5
5
5
5
5
5
3,4-F -C H
C H
6
2
6
3
4-CF -C H
C H
6
H
3
6
4
4-F-C H
C H
6
H
6
4
3-NO -C H
C H
6
H
2
6
4
3-MeO-C H
C H
6
H
6
4
4-F-C H
4-C H -C H
H
6
4
6
5
6
4
3-NO -C H
4-C H -C H
H
2
6
4
6
5
6
4
1
0
1
2
3
4
5
6
7
4-CF -C H
4-MeO-C H
H
3
6
4
6
4
4
4
4
1
1
1
1
1
1
1
4-F-C H
4-MeO-C H
H
6
4
6
4-NO -C H
4-MeO-C H
H
2
6
4
6
3-NO -C H
4-MeO-C H
H
2
6
4
6
4-CF -C H
4-CF -C H
4
H
3
6
4
3
6
4-CF -C H
3-thienyl
3-thienyl
C H
H
3
6
4
4-F-C H
H
6
4
4-CF -C H
CH3
3
6
4
6
5
a
Compounds were preincubated with the enzyme for 15 min and then 0.5 µg supercoiled DNA was added.
For time/course experiments and for dose/response experiments IC values are expressed as mean ± sd of
50
three different experiments by triplicate.
1
2
A similar picture was found with compounds 20 (Ar = 4-F-C H , R = C H , R =
6
4
6
5
1
2
1
2
H), 18 (Ar = 3,4-F -C H , R = C H , R = H) and 19 (Ar = 4-CF -C H , R = C H , R
2
6
4
6
5
3
6
4
6
5
=
H), which displayed from one to two fluorine heteroatoms and a trifluoromethyl
group [28] in the 2-aryl substituent, respectively. In these cases, the introduction of
fluorine atoms to the phenyl substituent did not improve the inhibitory effect on any
1
2
TopIB, despite compound 19 (Ar = 4-CF -C H , R = C H , R = H) had an interesting
3
6
4
6
5
1
2
1
39.8 SI over the parasites. Only compounds 22 (Ar = 3-MeO-C H , R = C H , R =
6 4 6 5
1
2
H) and 23 (Ar = 4-F-C H , R = 4-C H -C H , R = H) showed relative selectivity over
6
4
6
5
6
4
LTopIB, but their IC50 values are far from the antileishmanial effect showed on
intracellular amastigotes and their corresponding SIs.
1
5

ACCEPTED MANUSCRIPT
1
When an electron-donating methoxy group was introduced in R in position 4 of the
bicyclic ring those compounds were frankly inactive on both TopIB (compounds 25, 26,
2
7 and 28), despite some of them displayed interesting Selective Indexes on the
bioassays. However, compound 22 containing a methoxy group in the 2 position of the
naphthyridine ring resulted more selective towards LTopIB. Finally, the 1,5-
1
naphthyridine derivatives with a thienyl group in R (compounds 30 and 31) recovered
1
in part the inhibitory effect on TopIB. Interestingly, compound 30 (Ar = 4- CF C H , R
3
6
4
2
=
3-thienyl, R = H) was more selective in inhibiting LTopIB than other compounds of
the series and retained an interesting SI of 49.4 in killing leishmania amastigotes.
4
. Conclusions
In conclusion, a wide range of substituted 1,5-naphthyridines has been prepared, by
using the Povarov reaction, and their antileishmanial activity on promastigotes and
amastigote-infected splenocytes of L. infantum has been evaluated as well as their
inhibition of LTopIB and hTopIB enzymatic activity. The synthesis of these
nitrogenated heterocycles 17-32 involves the Aza-Diels-Alder reaction of functionalized
imines 3-9, obtained from 3-aminopyridine 1 with aldehydes 2, and acetylenes 10-15.
In general, 1,5-naphthyridines 17-32 have shown higher antiparasitic activity on
intracellular amastigotes than on free-living promastigotes. The presence of electron-
withdrawing substituents increases the antileishmanial effect. For example, high
selectivity indexes (SI > 100) were observed for nitroaryl substituted 1,5-naphthyridines
2
7 (Ar = 4-NO -C H ) and 21 and 24 (Ar = 3-NO -C H ). In this sense, it is noteworthy
2 6 4 2 6 4
1
2
that when compound 27 (Ar = 4-NO -C H , R = 4-MeOC H , R = H) was tested no
2
6
4
6
4
macrophage destruction was observed with an IC50 of murine splenocytes of
08.65±0.35 µM and a promastigotes killing capability of IC = 0.58±0.04, resulting in
1
5
0
1
6

ACCEPTED MANUSCRIPT
a very high selectivity index (SI = 187.3). Also trifluoromethyl substituted 1,5-
1
2
naphthyridine 19 (Ar = 4-CF -C H , R = C H , R = H) showed an excellent selectivity
3
6
4
6
5
index (SI = 139.8). However, among all new derivatives, 1,5-naphthyridine 22 (Ar = 3-
1
2
MeO-C H , R = C H , R = H) containing a methoxy group in the aryl in position 2 of
6
4
6
5
naphthyridine resulted the most interesting compound to emerge from these derivatives,
due to its high in vitro antileishmanial activity and low toxicity showing the highest SI
value of this family (SI = 271.5).
Few of these compounds had selective inhibitory effects on LTopIB-mediated
relaxation assays compatible to their strong biological in vivo and ex vivo effects. This
is the case of 22, the only compound of the series containing an electron-donating
methoxy group in the aryl group of position 2 of naphthyridines that combines certain
selective effect over the leishmanial enzyme and has the highest SI of the series (SI =
2
71.5). Despite the good biological activity of 22 is hardly attributable to LTopIB
inhibition, this compound can be considered a good candidate and do offer possibilities
1
for further studies. Whereas, the presence of the 4-MeO-C H group in R in the
6
5
position 4 of naphthyridines 25-28 originated a series of 1,5-naphthyridines with no
TopIB activity.
We can conclude, in the light of the current data, that the significant antileishmanial
effect displayed by these compounds is poorly related to LTopIB inhibition, which just
might play a secondary role in their biological efficacy. Nevertheless, the strong
antileishmanial activity and the relative safety of many of the current compounds
provide a promising basis for further development of biologically active naphthyridines.
1
7

ACCEPTED MANUSCRIPT
5
5
5
. Experimental protocols
.1 Chemistry
.1.1. General experimental information
All reagents from commercial suppliers were used without further purification. All
solvents were freshly distilled before use from appropriate drying agents. All other
reagents were recrystallized or distilled when necessary. Reactions were performed
under a dry nitrogen atmosphere. Analytical TLCs were performed with silica gel 60
F254 plates. Visualization was accomplished by UV light. Column chromatography was
carried out using silica gel 60 (230-400 mesh ASTM). Melting points were determined
with a digital melting point apparatus without correction. NMR spectra were obtained
on a 300 MHz and on a 400 MHz spectrometers and recorded at 25 ºC. Chemical shifts
1
13
for H NMR spectra are reported in ppm downfield from TMS, chemical shifts for C
NMR spectra are recorded in ppm relative to internal chloroform (δ = 77.2 ppm for
1
3
19
C), chemical shifts for
F NMR are reported in ppm downfield from
fluorotrichloromethane (CFCl ). Coupling constants (J) are reported in Hertz. The terms
3
1
3
19
m, s, d, t, q refer to multiplet, singlet, doublet, triplet, quartet. C NMR, and F NMR
were broadband decoupled from hydrogen nuclei. High resolution mass spectra
(HRMS) was measured by positive-ion electrospray ionization (EI) method using a
mass spectrometer Q-TOF. Aldimines 3-5 and 9 [25] and aldimines 6, 8 [14] were
prepared as previously described. Compounds 17, 19, 25, 27 and 29 were prepared as
previously described. [27]
1
8

ACCEPTED MANUSCRIPT
5
.1.2. Compounds Purity Analysis
All synthesized compounds were analyzed by HPLC to determine their purity. The
analyses were performed on Agilent 1260 infinity HPLC system (C-18 column,
Hypersil, BDS, 5µm, 0.4 mm×25 mm) at room temperature. All the tested compounds
were dissolved in dichloromethane, and 5µL of the sample was loaded onto the column.
Ethanol and heptane were used as mobile phase, and the flow rate was set at 1.0
mL/min. The maximal absorbance at the range of 190−400 nm was used as the
detection wavelength. The purity of all the tested compounds (compounds 17-32) is
>
95%, which meets the purity requirement by the Journal.
5
.1.2.1. N-[(3-Nitrophenyl)methylene]-3-pyridinamine (7). To a solution of 3-
aminopyridine 1 (10 mmol, 0.941 g) in CHCl (30 mL) 3-nitrobenzaldehyde (10 mmol,
3
1
.412 g) was added. The mixture was stirred under nitrogen at room temperature for 15
h. The reaction product is unstable during distillation and/or chromatography and was
1
used in situ for further reactions. H RMN of crude reaction mixture (300 MHz, CDCl )
3
5
3
3
4
δ: 7.35 (ddd, J = 0.7 Hz, J = 4.8 Hz, J = 8.0 Hz, 1 H), 7.56 (ddd, J = 1.5
HH
HH
HH
HH
4
3
3
3
Hz, J = 2.5 Hz, J = 8.0 Hz, 1 H), 7.68 (dd, J = 7 Hz, J = 8.1 Hz, 1 H), 8.25
HH
HH
HH
HH
4
3
3
4
4
(
ddd, J = 2.4 Hz, J = 8.2 Hz, J = 8.3 Hz, 1 H), 8.34 (ddd, J = 1.1 Hz, J
=
=
HH
HH
HH
HH
HH
3
4
2
1
.2 Hz, J = 8.2 Hz, 1 H), 8.51-8.53 (m, 2 H), 8.56 (s, 1 H, HC=N), 8.75 (dd, JHH
HH
3
13
1
.9 Hz, J = 3.9 Hz, 1 H) ppm; C { H} NMR of crude reaction mixture (75 MHz,
HH
CDCl ) δ: 123.7 (HC), 123.9 (HC), 126.2 (HC), 127.4 (HC), 127.9 (HC), 130.1 (HC),
3
1
1
33.9 (HC), 134.4 (HC), 137.4 (C), 142.7 (HC), 146.8 (C), 148.1 (HC), 148.8 (C),
59.1 (HC=N) ppm.
5
.1.2.2. General procedure for the synthesis of naphthyridines 17-32. To a solution
of of the in situ prepared aldimines 3-9 (5 mmol) in chloroform the corresponding
1
9

ACCEPTED MANUSCRIPT
acetylenes 10-15 (7 mmol) and BF ·Et O (10 mmol, 1.230 mL) were added and the
3
2
1
mixture was stirred at the opportune temperature until TLC and H NMR spectroscopy
indicated the disappearance of aldimine. The reaction mixture was washed with 2M
aqueous solution of NaOH (25 mL) and water (25 mL), extracted with dichloromethane
(
2 x 25 mL), and dried over anhydrous MgSO The removal of the solvent under
4.
vacuum afforded and oil that was purified by silica gel flash column chromatography
using a gradient elution of 10-40% ethyl acetate in hexane to afford products 17-32.
5
.1.2.2.1. 2,4-Diphenyl-1,5-naphthyridine (17). The compound was prepared and
characterized as previously described. [27] Purity 99.95 % (EtOH/Heptane = 10/90, Rt
4.741 min).
=
5
.1.2.2.2. 2-(3,4-Difluorophenyl)-4-phenyl-1,5-naphthyridine (18). The general
procedure was followed using imine 4 prepared in situ and phenylacetylene 10 (7 mmol,
.768 mL) and the reaction mixture was stirred at refluxing chloroform for 24 h.
0
Compound 18 was obtained (0.954 g, 60 %) as a white solid; mp 221-222 ºC (ethyl
1
acetate/hexane). H NMR (400 MHz, CDCl ) δ: 7.20-7.29 (m, 1 H), 7.41-7.52 (m, 3 H),
3
3
3
7
.61 (dd, J = 8.5 Hz, J = 4.1 Hz, 1 H), 7.72-7.75 (m, 2 H,), 7.84-7.89 (m, 1 H),
HH HH
3
3
3
7
.96 (s, 1 H), 8.1 (ddd, JHH = 11.3 Hz, J = 7.7 Hz, J = 2.2 Hz, 1 H), 8.41 (dd,
HH HH
3
4
3
4
JHH = 8.5 Hz, J = 1.9 Hz, 1 H), 8.92 (dd, J = 4.1 Hz, J = 1.9 Hz, 1H) ppm.
HH
HH
HH
1
3
1
2
2
C { H} NMR (100 MHz, CDCl ) δ: 117.3 (d, J = 67.8 Hz, HC), 117.5 (d, J
=
3
CF
CF
3
4
6
1
1
2
7.8 Hz, HC), 121.7 (HC), 123.8 (dd, JCF = 6.8 Hz, J = 3.7 Hz, HC), 124.8 (HC),
CF
3
4
28.6 (2 HC), 129.2 (HC), 130.7 (2 HC), 136.2 (dd, JCF = 5.6 Hz, J = 3.7 Hz, C),
CF
1
37.0 (C), 137.9 (HC), 141.4 (C), 144.5 (C), 149.6 (C), 151.0 ( HC), 151.1 (dd, J CF
=
2
1
2
47.4 Hz, J = 12.1 Hz, FC), 151.8 (dd, J = 250.4 Hz, J = 10.1 Hz, FC), 155.4
CF
CF
CF
1
9
(
C) ppm. F NMR (282 MHz, CDCl ) δ: - 137.3 to -137.1 (m), -136.6 to -136.5 (m)
3
2
0

ACCEPTED MANUSCRIPT
+
ppm. HRMS (EI): calculated for C H N F [M] 318.0989 found 318.0992. Purity
2
0
12
2 2
9
9.91 % (EtOH/Heptane = 10/90, Rt = 5.146 min).
5
.1.2.2.3. 4-Phenyl-2-(4-(trifluoromethyl)phenyl)-1,5-naphthyridine (19). The
compound was prepared and characterized as previously described [27]. Purity 98.84 %
EtOH/Heptane = 10/90, Rt = 5.228 min).
(
5
.1.2.2.4. 2-(4-Fluorophenyl)-4-phenyl-1,5-naphthyridine (20). The general procedure
was followed using imine 6 (5 mmol, 1.006 g) and phenylacetylene 10 (7 5 mmol, 0.768
ml) and the reaction mixture was stirred at refluxing chloroform for 36 h. Compound 20
1
was obtained (1.125 g, 75%) as a white solid ; mp 164-165 ºC (ethyl acetate/hexane). H
3
NMR (300 MHz, CDCl ) δ: 7.16 (t, J = 7.8 Hz, 2 H), 7.42-7.52 (m, 3 H), 7.59 (dd,
3
HH
3
3
JHH = 8.5 Hz, J = 4.5 Hz, 1 H), 7.73-7.76 (m, 2 H,), 8.08 (s, 1 H), 8.12-8.17 (m, 2
HH
3
4
3
4
H), 8.44 (dd, J = 7.9 Hz, J = 1.8 Hz, 1 H), 8.92 (dd, J = 4.1 Hz, J = 1.8 Hz,
HH
HH
HH
HH
1
3
1
2
1
H) ppm. C { H} NMR(75 MHz, CDCl ) δ: 116.1 (d, J = 22.0 Hz, 2 HC), 122.1
3
CF
3
(
HC), 124.7 (HC), 128.5 (2 HC), 129.0 (HC), 129.7 (d, J = 8.5 Hz, 2 HC), 130.6 (2
CF
HC), 135.1 (C), 137.1 (C), 137.7 (HC), 141.3 (C), 144.4 (C), 149.4 (C), 150.7 (HC),
1
19
1
56.7 (C), 164.2 (d, J = 251.0 Hz, FC) ppm. F NMR (CDCl ) δ: - 112.0 ppm.
CF 3
+
HRMS (EI): calculated for C H FN [M] 300.1096 found 300.1075. Purity 96.62 %
2
0
13
2
(EtOH/Heptane = 10/90, Rt = 5.792 min).
5
.1.2.2.5. 2-(3-Nitrophenyl)-4-phenyl-1,5-naphthyridine (21). The general procedure
was followed using imine 7 (5 mmol, 1.136 g) and phenylacetylene 10 (7 mmol, 0.768
ml) and the reaction mixture was stirred at chloroform reflux for 15 h. Compound 21
1
was obtained (0.572 g, 35%) as a white solid; m.p. 170-171 (ethyl acetate/hexane). H
NMR (400 MHz, CDCl ) δ: 7.58-7.61 (m, 3 H), 7.70-7.76 (m, 2 H), 7.82-7.85 (m, 2 H),
3
3
4
8
.16 (s, 1 H), 8.34-8.37 (m, 1 H), 8.54 (dd, JHH = 8.7 Hz, J = 1.8 Hz, 1 H) 8.58-8.60
HH
2
1

ACCEPTED MANUSCRIPT
3
4
3
4
(
m, 1H), 9.04 (dd, J = 4.2 Hz, J = 1.8 Hz, 1 H), 9.11 (dd, J = 4.3 Hz, J
=
HH
HH
HH
HH
1
3
1
1
.8 Hz, 1 H) ppm. C { H} NMR (75 MHz, CDCl ) δ: 121.8 (HC), 122.7 (HC), 124.4
3
(HC), 125.0 (HC), 128.6 (2 HC), 129.2 (HC), 130.1 (HC), 130.6 (2 HC), 133.5 (HC),
1
36.8 (C), 138.1 (HC), 140.8 (C), 141.6 (C), 144.6 (C), 150.0 (C), 151.4 (HC), 155.0
+
(
C) ppm. HRMS (EI): calculated for C H N O [M] 327.1008 found 327.1018. Purity
2
0
13
3
2
9
9.95 % (EtOH/Heptane = 10/90, Rt = 7.840 min).
5
.1.2.2.6. 2-(3-Methoxyphenyl)- 4-phenyl-1,5-naphthyridine (22). The general
procedure was followed using imine 8 (5 mmol, 1.062 g) and phenylacetylene 10 (7
mmol, 0.768 ml) and the reaction mixture was stirred at refluxing chloroform for 20 h.
Compound 22 was obtained (0.783 g, 50%) as yellowish oil after purification by flash
1
chromatography; Rf: 0.67 (50:50 ethyl acetate/hexane). H NMR (400 MHz, CDCl ) δ:
3
3
4
4
3
7
1
.95 (s, 3 H, OCH ), 7.06 (ddd, J = 8.1 Hz, J = 2.6 Hz, J = 0.7 Hz, 1 H), 7.44-
3 HH HH HH
3
3
.59 (m, 4 H), 7.66 (dd, JHH = 8.5 Hz, J = 4.3 Hz, 1 H), 7.75-7.86 (m, 4 H), 8.11 (s,
HH
3
4
3
4
H), 8.52 (dd, J = 8.4 Hz, J = 1.7 Hz, 1 H), 8.99 (dd, J = 4.2 Hz, J = 1.7
HH
HH
HH
HH
1
3
1
Hz, 1 H) ppm. C { H} NMR (100 MHz, CDCl ) δ: 55.5 (OCH ), 112.9 (HC), 115.8
3
3
(HC), 120.2 (HC), 122.4 (HC), 124.6 (HC), 128.4 (2 HC), 128.8 (HC), 130.0 (HC),
1
30.5 (2 HC), 137.2 (C), 137.8 (HC), 140.5 (C), 141.5 (C), 144.4 (C), 149.0 (C), 150.5
+
(
HC), 157.6 (C), 160.3 (C) ppm. HRMS (EI): calculated for C H N O [M] 312.1263
2
1
16
2
found 312.1269. Purity 98.85 % (EtOH/Heptane = 10/90, Rt = 7.741 min).
5
.1.2.2.7. 4-[(1,1'-Biphenyl)-4-yl]-2-(4-fluorophenyl)-1,5-naphthyridine (23). The
general procedure was followed using imine 6 (5 mmol, 1.006 g) and 4-ethynylbiphenyl
1
1 (7 mmol, 1.248 g) and the reaction mixture was heated at refluxing chloroform for
3
6 h. Compound 23 was obtained (1.034 g, 55%) as a white solid; m.p. 206-207 (ethyl
1
acetate/hexane). H NMR (400 MHz, CDCl ) δ: 7.22-7.27 (m, 2 H), 7.38-7.42 (m, 1 H),
3
3
3
7
.47-7.52 (m, 2 H), 7.67-7.71 (m, 3 H), 7.79 (d, J = 8.6 Hz, 2 H), 7.92 (d, J = 8.4
HH HH
2
2

ACCEPTED MANUSCRIPT
3
3
3
Hz, 2 H), 8.12 (s, 1 H), 8.23 (dd, JHH = 8.8 Hz, J = 5.3 Hz, 2 H), 8.51 (dd, J
=
HH
HH
4
3
4
13
1
8
.2 Hz, J = 1.7 Hz, 1 H), 9.01 (dd, J = 4.1 Hz, J = 1.7 Hz, 1 H) ppm. C { H}
HH
HH
HH
2
NMR (100 MHz, CDCl ) δ: 116.1 (d, J = 21.9 Hz, 2 HC), 121.9 (HC), 124.7 (HC),
3
CF
3
1
1
27.3 (2 HC), 127.4 (2 HC), 127.7 (HC), 129.0 (2 HC), 129.7 (d, J = 8.5 Hz, 2 HC),
CF
31.0 (2 HC), 135.4 (C), 136.1 (C), 137.9 (HC), 140.9 (C), 141.4 (C), 142.0 (C), 144.5
1
19
(
C). 148.9 (C), 150.7 (HC), 156.8 (C), 164.2 (d, JCF = 250.1 Hz, FC) ppm. F NMR
+
(
CDCl ) δ: - 112.1 to -112.3 (m) ppm. HRMS (EI): calculated for C H FN [M]
3
26 17
2
3
76.1376 found 376.1383. Purity 99.98 % (EtOH/Heptane = 10/90, Rt = 6.735 min).
5
.1.2.2.8. 4-[(1,1'-Biphenyl)-4-yl]-2-(3-nitrophenyl)-1,5-naphthyridine (24). The
general procedure was followed using imine 7 (5 mmol, 1.136 g) and 4-ethynylbiphenyl
1 (7 mmol, 1.248 g) and the reaction mixture was stirred at chloroform reflux for 24 h.
1
Compound 24 was obtained (0.604 g, 30%) as a yellowish solid; m.p. 197-198 (ethyl
1
acetate/hexane) . H NMR (400 MHz, CDCl ) δ: 7.38-7.43 (m, 1 H), 7.48-7.52 (m, 2 H),
3
7
1
.60-7.64 (m, 2 H), 7.68-7.77 (m, 3 H), 7.80-7.83 (m, 2H), 7.93-7.95 (m, 2 H), 8.22 (s,
3
4
3
4
H), 8.37 (dd, J = 8.4 Hz, J = 2.3 Hz, 1 H), 8.57 (dd, J = 8.4 Hz, J = 1.8
HH
HH
HH
HH
3
4
4
Hz, 1 H), 8.60-8.62 (m, 1 H), 9.07 (dd, JHH = 4.1 Hz, J = 1.8 Hz, 1 H), 9.13 (t, J
HH
HH
1
3
1
=
1.8 Hz, 1H) ppm. C { H} NMR (100 MHz, CDCl ) δ: 121.6 (HC), 122.8 (HC),
3
1
24.5 (HC), 125.0 (HC), 127.4 (4 HC), 127.8 (HC), 129.1 (2 HC), 130.1 (HC), 131.1 (2
HC), 133.5 (HC), 135.7 (C), 138.0 (HC), 140.7 (C), 140.8 (C), 141.6 (C), 142.2 (C),
1
44.6 (C), 149.1 (C), 149.7 (C), 151.5 (HC), 155.1 (C) ppm. HRMS (CI): calculated for
+
C H N O [M] 403.1321 found 403.1334. Purity 97.64 % (EtOH/Heptane = 10/90,
26
17
3
2
Rt = 8.657 min).
2
3

ACCEPTED MANUSCRIPT
5
.1.2.2.9. 4-(4-Methoxyphenyl)-2-(4-(trifluoromethyl)phenyl)-1,5-naphthyridine (25).
The compound was prepared and characterized as previously described [27]. Purity
9
9.92 % (EtOH/Heptane = 10/90, Rt = 6.601 min).
5
.1.2.2.10. 2-(4-Fluorophenyl)-4-(4-methoxyphenyl)-1,5-naphthyridine (26). The
general procedure was followed using imine 6 (5 mmol, 1.006 g) and 4-
methoxyphenylacetylene 12 (7 mmol, 0.908 ml) and the reaction mixture was heated at
refluxing chloroform for 24 h. Compound 26 was obtained (0.495 g, 30%) as a white;
1
m.p. 174-175 (ethyl acetate/hexane). H NMR (400 MHz, CDCl ) δ: 3.90 (s, 3 H,
3
3
3
OCH ), 7.08-7.12 (m, 2 H), 7.20-7.25 (m, 2 H), 7.65 (dd, J = 8.6 Hz, J = 4.3 Hz,
3
HH
HH
3
4
1
H), 7.74-7.83 (m, 2 H), 8.04 (s, 1H), 8.18-8.22 (m, 2 H), 8.47 (dd, J = 8.6 Hz, J
HH HH
3
4
13
1
=
1.7 Hz, 1 H), 8.98 (dd, JHH = 4.3 Hz, J = 1.7 Hz, 1 H) ppm. C { H} NMR (75
HH
2
MHz, CDCl ) δ = 55.6 (OCH ), 114.1 (2 HC), 116.0 (d, J = 22.0 Hz, 2 HC), 121.6
3
3
CF
3
(
HC), 124.5 (HC), 129.4 (C), 129.7 (q, J = 8.4 Hz, 2 HC), 132.0 (2 HC), 135.5 (C),
CF
1
37.8 (HC), 141.5 (C), 144.6 (C), 148.8 (C), 150.4 (HC), 156.8 (C), 160.4 (C), 164.3 (d,
1
19
JCF = 248.4 Hz, CF) ppm. F NMR (CDCl ) δ: - 112.0 to - 112.1 (m) ppm. HRMS
3
+
(
EI): calculated for C H FN O [M] 330.1168 found 330.1176. Purity 97.25 %
2
1
15
2
(EtOH/Heptane = 10/90, Rt = 6.566 min).
5
.1.2.2.11. 2-(4-Nitrophenyl)-4-(4-methoxyphenyl)-1,5-naphthyridine (27). The
compound was prepared and characterized as previously described [27]. Purity 96.92 %
EtOH/Heptane = 10/90, Rt = 15.835 min).
(
5
.1.2.2.12. 2-[(3-(Nitrophenyl)-4-(4-methoxyphenyl)]-1,5-naphthyridine (28). The
general procedure was followed using imine 7 (5 mmol, 1.136 g) and 4-
methoxyphenylacetylene 12 (7mmol, 0.908 ml) and the reaction mixture was stirred at
refluxing chloroform for 70 h. Compound 28 was obtained (0.714 g, 40%) as a white
2
4

ACCEPTED MANUSCRIPT
1
solid; m.p. 193-194 (ethyl acetate/hexane). H NMR (400 MHz, CDCl ) δ: 3.92 (s, 3 H,
3
3
3
OCH ), 7.12 (d, J = 8.8 Hz, 2 H), 7.69-7.76 (m, 2 H), 7.83 (d, J = 8.8 Hz, 2 H),
3
HH
HH
3
4
4
3
8
.14 (s, 1H), 8.35 (ddd, JHH = 8.1 Hz, J = 2.2 Hz, J = 1.1 Hz, 1 H), 8.53 (dd, J
HH
HH
HH
4
3
4
4
=
8.5 Hz, J = 1.8 Hz, 1 H), 8.59 (ddd, J = 8.0 Hz, J = 1.7 Hz, J = 1.0 Hz, 1
HH
HH
HH
HH
3
3
4
13
H), 9.03 (dd, J = 8.6 Hz, J = 4.0 Hz, 1H), 9.10 (t, J = 1.7 Hz, 1H) ppm. C
HH
HH
HH
1
{
1
1
H} NMR (75 MHz, CDCl ) δ: 55.6 (OCH ), 114.2 (2 HC), 121.3 (HC), 122.7 (HC),
3 3
24.4 (HC), 124.9 (HC), 128.9 (C), 130.1(HC), 132.9 (2 HC), 133.5 (HC), 138.0 (HC),
40.8 (C), 141.7 (C), 144.6 (C), 149.1(C), 149.6 (C), 151.2 (HC), 155.0 (C), 160.6 (C)
+
ppm. HRMS (EI): calculated for C H F N O [M] 342.4005 found 342.4009. Purity
2
2
18
3
2
2
9
6.99 % (EtOH/Heptane = 10/90, Rt = 11.464 min).
5
.1.2.2.13. 2,4-Bis-(4-Trifluoromethyl)phenyl-1,5-naphthyridine (29). The compound
was prepared and characterized as previously described [27]. Purity 99.85 %
EtOH/Heptane = 10/90, Rt = 5.316 min).
(
5
.1.2.2.14. 4-(Thiophen-3-yl)-2-(4-(trifluoromethyl)phenyl)-1,5-naphthyridine (30).
The general procedure was followed using imine 5 (5 mmol, 1.250 g) and 3-
ethynyltiophene 14 (7 mmol, 0.693 ml) and the reaction mixture was stirred at refluxing
chloroform for 40 h. Compound 30 was obtained (0.890 g, 50%) as a yellowish solid;
1
3
m.p.195-196 (ethyl acetate/hexane). H NMR (300 MHz, CDCl ) δ: 7.43 (dd, J = 5.0
3
HH
3
3
3
Hz, J = 3.0 Hz, 1 H), 7.61 (dd, J = 8.5 Hz, J = 4.1 Hz, 1 H), 7.71-7.74 (m, 3
HH
HH
HH
3
4
H), 8.14 (s, 1H,), 8.24-8.25 (m, 3 H), 8.41 (dd, JHH = 8.5 Hz, J = 1.7 Hz, 1H), 8.95
HH
3
4
13
1
(
dd, J = 4.1 Hz, J = 1.7 Hz, 1H) ppm. C { H} NMR (75 MHz, CDCl ) δ: 120.7
HH HH 3
1
3
(
HC), 123.9 (q, J = 271.8 Hz, CF ), 124.7 (HC), 125.7 (HC), 126.0 (q, J = 3.8 Hz,
CF 3 CF
2
2
HC), 128.1 (2 HC), 128.3 (HC), 129.1 (HC), 131.6 (q, JCF = 32.9 Hz C-CF ), 137.0
3
(C), 138.1 (HC), 141.2 (C), 142.5 (C), 143.2 (C), 144.7 (C), 150.8 (HC), 156.5 (C) ppm.
2
5

ACCEPTED MANUSCRIPT
1
9
+
F NMR (CDCl ) δ: - 63.0 ppm. HRMS (EI): calculated for C H F N S [M]
3
19 11
3
2
3
56.0595 found 356.0592. Purity 99.67 % (EtOH/Heptane = 10/90, Rt = 5.695 min).
5
.1.2.2.15. 2-(4-Fluorophenyl)-4-(thiophen-3-yl)-1,5-naphthyridine (31). The general
procedure was followed using imine 6 (5 mmol, 1.006 g) and 3-ethynyltiophene 14 (7
mmol, 0.693 ml) and the reaction mixture was heated at refluxing chloroform for 48 h.
Compound 31 was obtained (0.459 g, 30%) as a white solid; m.p. 106-107 (ethyl
1
acetate/hexane). H NMR(300 MHz, CDCl ) δ: 7.21-7.25 (m, 2 H), 7.49-7.52 (m, 1 H),
3
7
.64-7.69 (m, 1 H), 7.78-7.80 (m, 1 H), 8.16-8.22 (m, 3 H), 8.29-8.31 (m, 1 H), 8.46
3
4
3
4
(
dd, J = 8.6 Hz, J = 1.8 Hz, 1H, H), 9.00 (dd, J = 4.0 Hz, J = 1.8 Hz, 1H,
HH HH HH HH
1
3
1
2
H) ppm. C { H} NMR (75 MHz, CDCl ) δ: 116.0 (d, J = 21.7 Hz, 2 HC), 120.6
3
CF
3
(
HC), 124.6 (HC), 125.6 (HC), 128.0 (HC), 129.1 (HC), 129.6 (d, J = 8.9 Hz, 2 HC),
CF
1
35.5 (C), 137.1 (C), 137.9 (HC), 141.1 (C), 143.0 (C), 144.6 (C), 150.3 (HC), 157.0
1
19
(
C), 164.0 d, J = 252.3 Hz, C) ppm. F NMR (CDCl ) δ: -112.1 to -112.0 ppm.
CF 3
+
HRMS (EI): calculated for C H FN S [M] 306.0627 found 306.0633. Purity 99.97 %
1
8
11
2
(EtOH/Heptane = 10/90, Rt = 5.679 min).
5
.1.2.2.16. 4-Phenyl-3-methyl-2(4-trifluoromethylphenyl)-1,5-naphthyridine (32). The
general procedure was followed using imine 5 (5 mmol, 1.250 g) and propyl-1-yl-
benzene 15 (7 mmol, 0.802 ml) and the reaction mixture was heated at refluxing
chloroform for 58 h. Compound 32 was obtained (0.910 g, 50%) as a yellowish solid;
1
m.p. 189-190 (ethyl acetate/hexane). H NMR (400 MHz, CDCl ) δ: 2.25 (CH ), 7.38-
3
3
3
7
.40 (m, 2 H), 7.48-7.52 (m, 1 H), 7.55-7.61 (m, 3 H), 7.79 (q, JHH = 8.5 Hz, 4 H), 8.45
3
4
3
4
(
dd, J = 8.5 Hz, J = 1.8 Hz, 1 H), 8.95 (dd, J = 4.1 Hz, J = 1.8 Hz, 1 H)
HH HH HH HH
1
3
1
1
ppm. C { H} NMR (100 MHz, CDCl ) δ: 19.1 (CH ), 123.8 (HC), 124.7 (q, J =
CF
3
3
3
2
75.0 Hz, CF ), 125.5 (q, J = 3.7 Hz, 2 HC), 128.1 (HC), 128.4 (2 HC), 129.5 (2
3 CF
2
HC), 130.1 (2 HC), 130.5 (C), 130.6 (q, JCF = 32.5 Hz, C-CF ), 136.6 (C), 137.3 (HC),
3
2
6

ACCEPTED MANUSCRIPT
1
9
1
41.8 (C), 142.2 (C), 144.6 (C), 149.3 (C), 151.3 (HC), 160.3 (C) ppm. F NMR
+
(
CDCl ) δ: - 63.0 ppm. HRMS (EI): calculated for C H F N [M] 364.1187 found
3
22 15
3
2
3
64.1189. Purity 97.43 % (EtOH/Heptane = 10/90, Rt = 5.507 min).
5
.2. Biology
All used protocols described in this work were approved by the Animal Care Committee
of University of Leon, project license PI12/00104. It complies with European Union
Legislation (2010/63/UE) and Spanish Act (RD 53/2013).
5
.2.1. L. infantum strain and culture conditions. A genetically modified L. infantum
BCN150 strain that constitutively produces the near infrared fluorescent protein iRFP
L. infantum-iRFP) as readable reporter [13] was used in the experiments. Promastigotes
(
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, Aldrich), 10% (v/v) heat-inactivated foetal calf serum (FCS) and
antibiotic cocktail (50 U/mL penicillin and 50 µg/mL streptomycin). To test the
6
leishmanicidal effect of compounds on promastigotes, 10 cells/mL were seeded in
black 96-well plates with clear bottom. Stock solutions of each naphthyridine were
prepared in DMSO and stepwise (three-fold) diluted in M199 media and growing
concentrations of each compound (0.07 to 300 mM) were added to each well. Final
concentration of DMSO in each well was never higher than 0.1% (v/v). The viability of
promastigotes was monitored recording the infrared emitted fluorescence of living
promastigotes in an Odyssey facility (λ exc. 684 nm; λ em. 708 nm) (LI-COR, USA).
AMB deoxycholate was used as positive control, whereas 0.1 (v/v) DMSO – the
maximum concentration of solvent used in the experiments – was included as negative
control. All compounds and controls were assayed by triplicate.
2
7

ACCEPTED MANUSCRIPT
5
.2.2. Intracellular ex vivo infections. The effect of 1,5-naphthyridines on intracellular
amastigotes of L. infantum was performed in ex vivo splenic explants prepared from
infected mice according to [13]. Female BALB/c mice were infected intravenous with
1
08 metacyclic promastigotes of L. infantum iRFP. Briefly, after 5 weeks post-infection,
animals were euthanized, and their spleens were aseptically recovered to obtain a
primary culture of infected splenic explant. Spleens were washed in cold phosphate-
buffered saline (PBS) and cut in small pieces. To obtain a single cell suspension, the
tissue was incubated with 5 mL of collagenase D (Roche®) at 2 mg/mL prepared in
buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM
CaCl2) for 20 min at 37 °C. The cell suspension and remaining tissue fragments were
gently passed through 100 mm mesh to remove the tissue fragments. After washing 3x
by centrifugation (500 × g for 7 min at 4 °C) with PBS, splenocytes were seeded in
black 384-wells clear bottom plates in RPMI supplemented with 20% (v/v) FCS, 1 mM
sodium pyruvate, 1x RPMI vitamins, 10 mM HEPES and 100 U/mL penicillin and 100
mg/mL streptomycin. Cells were counted and diluted at different cell densities in order
to fix the fluorescence level per well. Different concentrations (in a concentration range
between 10 and 0.07 µM) of naphthyrydines were added to the cultures for an extended
period of 72 h. Amastigote viability was assessed by recording the fluorescence
emission of infected splenocytes at 708 nm in an Odyssey (Li-Cor) infrared imaging
system.
5
.2.3. Cytotoxicity assessment and selectivity index (SI) determination. The
cytotoxicity of substituted 1,5–naphthyridines was assessed on mouse uninfected
splenocytes. The splenocytes used to determine cytotoxicity were obtained and cultured
as described above. Cells were seeded in 96-well plates in the presence of different
concentrations (in a concentration range of 100 to 0.8 µM) of the assayed compounds
2
8