Anti-leishmanial evaluation of C2-aryl quinolines: Mechanistic insight on bioenergetics and sterol biosynthetic pathway of Leishmania braziliensis

Article abstract of DOI:10.1016/j.bmc.2013.04.063

A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis based on the acid-catalyzed multicomponent imino Diels-Alder reactions. Seven selected quinolines were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2- phenylquinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mammalian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinization of acidocalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results validate the efficiency of quinoline molecules as leishmanicide compounds.

Full text of DOI:10.1016/j.bmc.2013.04.063

Bioorganic & Medicinal Chemistry 21 (2013) 4426–4431
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Anti-leishmanial evaluation of C2-aryl quinolines: Mechanistic
insight on bioenergetics and sterol biosynthetic pathway of
Leishmania braziliensis
Daznia Bompart a, , Jorge Núñez-Durán a, , Daniel Rodríguez b, Vladimir V. Kouznetsov d,
Carlos M. Meléndez Gómez d, Felipe Sojo c, Francisco Arvelo c,e, Gonzalo Visbal f, Alvaro Alvarez f,
Xenón Serrano-Martín a, , Yael García-Marchán a, ,
⇑
a Laboratorio de Señalización Celular y Bioquímica de Parásitos, Área de Salud, Instituto de Estudios Avanzados (IDEA), Caracas, Bolivarian Republic of Venezuela
b Laboratorio de Pesquisa Selectiva, Área de Salud, Instituto de Estudios Avanzados (IDEA), Caracas, Bolivarian Republic of Venezuela
c Área de Salud, Instituto de Estudios Avanzados (IDEA), Caracas, Bolivarian Republic of Venezuela
d Laboratorio de Química Orgánica y Biomolecular, Universidad Industrial de Santander, Bucaramanga, Colombia
e Laboratorio de Cultivo de Tejidos y Biología de Tumores, Instituto de Biología Experimental (IBE), Universidad Central de Venezuela, Caracas, Bolivarian Republic of Venezuela
f Laboratorio de Síntesis Orgánica y Compuestos Naturales, Centro de Química, Instituto Venezolano de Investigaciones Científicas (IVIC), Caracas, Bolivarian Republic of Venezuela
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of diverse simple C2-aryl quinolines was synthesized de novo via a straightforward synthesis
based on the acid-catalyzed multicomponent imino Diels–Alder reactions. Seven selected quinolines
were evaluated at different stages of Leishmania braziliensis parasite. Among them, the 6-ethyl-2-phenyl-
quinoline 5f was able to inhibit the growth of promastigotes of this parasite without affecting the mam-
malian cells viability and decreasing the number of intracellular L. braziliensis amastigotes on BMDM
macrophages. The mechanism of action studied for the selected compound consisted in: (1) alteration
of parasite bioenergetics, by disrupting mitochondrial electrochemical potential and alkalinization of aci-
docalcisomes, and (2) inhibition of ergosterol biosynthetic pathway in promastigote forms. These results
validate the efficiency of quinoline molecules as leishmanicide compounds.
Received 7 February 2013
Revised 11 April 2013
Accepted 18 April 2013
Available online 9 May 2013
Keywords:
Chemotherapy
L. braziliensis
Parasite bioenergetics
Sterol biosynthetic pathway
C2-aryl substituted quinolines
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
the treatment of visceral and cutaneous leishmaniasis, despite its
teratogenic characteristics, which limits its use during pregnancy.4
Leishmaniasis is one of the six more important parasitic dis-
eases worldwide, affecting 14 million people and 350 million on
risk of contracting the disease.1 Leishmaniasis includes three dif-
ferent clinical forms: cutaneous (CL), muco-cutaneous (ML) and
visceral (VL), being the last one, which causes more mortality in
humans. The first-line recommended therapy is based on pentava-
lent antimonials drugs like GlucantimeÒ and PentostamÒ. How-
ever, these drugs generate severe side-effects on heart, liver and
kidneys.2 Moreover, antimonial-resistant parasites have emerged
in endemic areas that have limited treatment and promotes the
used of second-line alternatives.3 At present, the alkyl-lysophos-
pholipid MiltefosineÒ appears to represent a major advance in
Cutaneous leishmaniasis is a disfiguring and stigmatizing dis-
ease with non-fatal consequences for the patient.5 In South Amer-
ica, 300,000 new cases of CL appear annually. Leishmania
braziliensis is responsible for nearly 90% of all CL cases, and absence
or incomplete treatment is associated with the subsequent devel-
opment of mucocutaneous leishmaniasis.6 The treatment of CL is
based on the same mentioned drugs, but it has been proved the
inconsistence in their effectiveness against different Leishmania
species.3
The use of natural and synthetic compounds is a major strategy
against parasitic diseases, and an example is antimalarial agent
chloroquine.7 Quinoline derivatives have also revealed a wide
spectrum of biological activities such as antibacterial,8 cytotoxic
and antineoplastic,9 antimycobacterial,10 and antiviral behavior.11
Quinolines have been widely used as leishmanicides since ancient
times. The Bolivian Indians have used extracts from plants of the
Rutacea family to treat the wounds of CL and a study of these plants
revealed the presence of diverse simple 2-substiuted quinolines
that may affect the viability of different Leishmania species.12 Since
⇑
Corresponding author. Tel.: +58 212 903 5095; fax: +58 212 903 5118.
E-mail addresses: yaelgarciamarchan@gmail.com, ygarcia@idea.gob.ve (Y.
García-Marchán).
Present address: Grupo de Biología y Quimioterapia de Parasitosis Tropicales,
Area de Salud, Instituto de Estudios Avanzados (IDEA), Caracas, Bolivarian Republic of
Venezuela.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.063

D. Bompart et al. / Bioorg. Med. Chem. 21 (2013) 4426–4431
4427
this discovery, compounds with a quinoline skeleton became to be
considered attractive scaffolds to develop new anti-protozoal
agents.13,14
able to inhibit considerably the viability of promastigotes (IC50
650 M) while five quinoline molecules 5a-e with a 2-(3,4-meth-
ylenedioxyphenyl) moiety (dubamine analogs) were inactive. The
results also displayed that the quinoline 5g was highly cytotoxic
toward BMDM macrophages (Table 2).
l
Sitamaquine, an 8-aminoquinoline, has been proposed as oral
treatment alternative against VL, but its efficacy for CL was very
poor.15 The mechanism of action of this experimental drug on
Leishmania donovani promastigotes consists in the inhibition of
complex II of the respiratory chain. This effect produces a depletion
in mitochondrial electrochemical potential, a drop in ATP levels,
and finally an apoptosis-like lethal response.16 Although some bio-
logical details against the quinoline mechanism on Leishmania are
now available, further studies of the quinoline structure-activity
relationship, and mechanistic insight are urgently needed. Further-
more, there are few studies that evaluate this family compounds as
a possible alternative for the treatment of CL caused by L. brazilien-
sis.17–19
In the course of our ongoing screening program for new bio-
logically actives N-heterocycles compounds, we have previously
reported the antiparasitic and antifungal effects of different
substituted quinolines.20,21 Herein, we report the anti-leishman-
ial properties of diverse C2-aryl quinoline derivatives that can
modify bioenergetics of L. braziliensis through disrupting mito-
chondrial electrochemical potential and alkalinization of
acidocalcisomes.
Employing the Molinspiration software and software Sparc
v4.6,24 the selected quinolines 5a–e were subjected to the Lipin-
ski’s rule of five analysis (drug-likeness), which helps to predict
and explain biological behavior of small molecules such as quino-
line compounds. Calculated logP parameters (Table 2) indicated
that more lipophilic molecules 5c,e (inactive) and 5f,g (active) have
the different logP values from 3.87 to 5.19 that reflects nature R
and Ar substituents dependence. However, the latter active quino-
lines 5f,g, being formula isomers with similar high logP (5.12 and
5.19), have drastic behavior on BMDM macrophages. It should be
also noted that experimental drug sitamaquine, which was se-
lected as structural analog, is even less lipophilic than the studied
quinolines. Miltefosine, reference drug in this study, resulted be
also less lipophilic molecule. We also addressed to another quanti-
tative parameter for assessing lipophilicity,—logD depends on the
partitioning of the neutral portion of the molecule population plus
the partitioning of the ionized portion of the molecule population,
that is it is a pH dependent function. Calculated values demon-
strated good permeability, but low absorption for compounds
5f,g and experimental drug sitamaquine (3 < logD7.4 < 5), and mil-
tefosine (logD7.4 <1).25
2. Results and discussion
2.1. Chemistry
Having these results, we selected quinoline 5f by its adequate in
silico cell permeability and antiparasitic effect. We determined the
EC50 of the chosen compound on promastigotes and intracellular
amastigotes of L. braziliensis evaluating its effect on 5f cellular bio-
energetics and sterol biosynthetic pathway in promastigotes of this
parasite.
Selected diverse quinoline compounds 5 were prepared de novo
using a straightforward synthesis based on acid-catalyzed imino
Diels–Alder (imino DA) reactions22 starting with commercial and
inexpensive reagents. Performed imino DA cycloaddition of substi-
tuted anilines 1, N-vinylpyrrolidin-2-one 2 and diverse benzalde-
hydes 3 in refluxing MeCN in the presence of 20% mol BiCl3
conducted to the formation of the tetrahydroquinolines 4, which
were used as crude, without further purification, to obtain the final
quinoline products 5a-g (Table 1) via an oxidation-aromatization
process. These quinolines were obtained as stable solids in good
yields (45–70%) after their column chromatography purification21
(Fig. 1).
The quinoline 5f affected the viability of L. braziliensis prom-
astigotes, with an EC50 value of 6.0 lM (Fig. 2). This value is similar
to sitamaquine, evaluated on this and other parasite specie.17 It
was noted that the substituents nature on the quinoline ring are
essential for leishmanicidal activity of quinoline derivatives.12,26,27
Paloque et al.27 demonstrated that a series of quinoline deriva-
tives with substitutions at C2 position, affected the viability of
promastigotes of L. donovani with an EC50 of 6.6 lM. This effect
is also similar to that obtained in this study with compound 5f.
2.2.2. Intracellular amastigotes evaluation
2.2. Biology
Leishmanicidal effect of quinoline 5f on the clinically relevant
stage was realized thought the design of an in vitro infection model
with macrophages BMDM and L. braziliensis amastigotes. Using this
model, a 40% maximum infection was obtained, similar to previ-
ously results.28 The effect of 5f was evaluated at 96 hours post-
treatment. Figure 3 shows that as the molecule 5f concentration
is increased, a concomitant decrease in the number of intracellular
2.2.1. Antiparasitic and cytotoxicity activity
Seven C2-aryl substituted quinolines 5a-g were selected for
evaluation on L. braziliensis promastigotes viability, and cytotoxic-
ity toward mouse bone marrow-derived macrophages (BMDM).
Macrophages BMDM are commonly used as an in vitro infection
model for anti-leishmanial drugs assays on the intracellular
amastigotes stage.23 In the first screening we found that among
quinoline compounds there were two molecules, 6-ethyl-2-phen-
ylquinoline 5f and 5,7-dimethyl-2-phenylqunoline 5g, that were
amastigotes was detected with an EC50 value of 20 lM. Similar val-
ues were reported for intracellular amastigotes of Leishmania major
exposed to other quinoline derivatives.29 Furthermore, we found
Table 1
Physico-chemical data of 2-aryl quinolines 5a–g
Compd 5
R1
R2
R3
Ar
MF
MW
Mp (°C)
Yield (%)
a
b
c
d
e
f
H
H
H
H
Me
H
Me
H
Me
Et
MeO
Me
Et
H
H
H
H
Me
H
Me
3,4-(OCH2O)C6H3
3,4-(OCH2O)C6H3
3,4-(OCH2O)C6H3
3,4-(OCH2O)C6H3
3,4-(OCH2O)C6H3
C6H5
C16H11NO2
C17H13NO2
C18H15NO2
C17H13NO3
C18H15NO2
249.26
263.29
277.32
279.29
277.11
233.31
233.31
90–92
166–168
175–177
139–141
170–173
63–66
50
45
45
60
62
55
70
C17H15
N
N
g
H
C6H5
C17H15
71–74
MF: molecular formula; MW: molecular weight; Mp: melting point.

4428
D. Bompart et al. / Bioorg. Med. Chem. 21 (2013) 4426–4431
Figure 1. Scheme of synthesis.
Table 2
Evaluation of C2-aryl substituted quinolines 5a–e on viability of L. braziliensis promastigotes and BMDM macrophages
Compd
Structure
Physico-chemical properties in silico
IC50 (lM)
logP
logD5.2
3.40
logD7.4
L. braziliensis
BMDM macrophages
O
O
5a
4.00
3.46
3.94
4.42
3.95
P50
NT
N
Me
5b
4.48
4.90
3.87
3.73
4.28
3.78
P50
P50
P50
NT
NT
NT*
O
O
N
Me
O
5c
N
O
MeO
O
5d
N
O
Me
5e
4.97
4.05
4.38
P50
NT
O
Me
Me
N
O
5f
5.12
5.19
4.85
3.65
5.04
5.10
650
650
P200
6100
N
Me
5g
Me
N
Me
N
MeO
Structural analog
4.04
1.78
3.52
65024
610017a
HN
N
Me
Me
Sitamaquine
CH3
N+
CH3
O
-O
P
O
Reference drug
CH3
( )16
À0.212
0.12
0.12
650
P200
O
CH3
Miltefosine
*
NT—not tested on macrophages BMDM, very low activity against promastigotes of L. braziliensis.
that this effect is specific for intracellular amastigotes without
affecting the viability of host cells (BMDM macrophages) in the
concentration ranges tested.
2.2.3. Mechanisms of action studies
2.2.3.1. Parasite bioenergetics.
the antiparasitic activity generated by quinoline 5f, its effect on the
To probe the molecular basis of

D. Bompart et al. / Bioorg. Med. Chem. 21 (2013) 4426–4431
4429
ure of this drug generates an activation of an electrophoretic mech-
anism causing the failure of mitochondrial potential, compromising
viability of parasites.30 Moreover, the tafenoquine generates a loss
of mitochondrial potential on L. donovani by inhibition of cyto-
chrome C reductase of the mitochondrial respiratory chain,31 while
the sitamaquine inhibits electron transport chain, at succinate
deshydrogenase level.16
When the promastigotes were loaded with acridine orange
(Fig. 5), it was observed that quinoline 5f generated a rapid alkalin-
ization of the parasite acidocalcisomes. These results were corrob-
orated with the addition of nigericin, a K+/H+ exchanger capable to
alkalinize these compartments.32 The same effect was reported in
L. donovani exposed to sitamaquine and chloroquine.33
Figure 2. Evaluation of 5f on L. braziliensis promastigotes. Growth curve of L.
braziliensis promastigotes, exposed to crescent concentrations of 5f. Each condition
2.2.3.2. Sterol biosynthetic pathway.
The sterol biosynthetic
was made for triplicate. EC50
6 lM ( 2 lM).
pathway has been extensively validated as an important chemo-
therapeutic target.34 In this study, we demonstrated that quinoline
5f interfere this pathway in promastigotes of L. braziliensis. Indeed,
we were able to show an accumulation of squalene and a depletion
of 5-dehydroepisterol in treated parasites (Table 3). A similar effect
was observed in promastigotes of Leishmania mexicana exposed to
miltefosine at the same period of time, affecting the viability of this
species of parasites.35 This is a first report that demonstrated the
effect of aryl-quinoline compounds on the viability of L. braziliensis,
through a blockade of sterol biosynthesis pathway.
3. Conclusion
This study demonstrates the effect of the 2-phenylquinoline 5f
on L. braziliensis promastigotes and intracellular amastigotes, with-
out affecting the viability of the host cells. This confirms the effec-
tiveness of aryl quinolinic structures against this parasite species,
especially if we consider that the mechanisms of action demon-
strated in this study have been previously validated with other
compounds.30,33,34,35
Figure 3. Quinoline 5f evaluation on intracellular amastigotes of L. braziliensis.
Dose–response curve, BMDM macrophages were infected with promastigotes of L.
braziliensis (1:10) and incubated with 5f for 96 h. d % macrophages total, j %
infected macrophages and N N° amastigotes  macrophages. EC50: 20
lM ( 3 lM)
Given the proven effectiveness of quinolines on VL,9 in addition
to the studies shown in this paper, we suggest that 2-phenylquin-
oline compounds (such as the compd 5f) could represent an alter-
native for conducting more advanced studies, in the search of
alternative treatments for patients with CL by Leishmania
braziliensis.
calculated based on N° de amastigotes  macrophages. Each experiment was made
by triplicate.
mitochondrial electrochemical potential and the accumulation of H+
in acidocalcisomes was analyzed. Parasites loaded with rhodamine
123 were exposed to the quinoline 5f (Fig. 4), showing a rapid in-
crease in fluorescence similar to that obtained with trif-
4. Experimental
4.1. Chemistry
luorocarbonylcyanide phenylhydrazone (FCCP),
a
classical
protonophore uncoupler that dissipates the mitochondrial H+ gradi-
ent. The uncoupling of mitochondrial H+ gradient was previously re-
ported with the 8-aminoquinoline sitamaquine on promastigotes of
L. donovani parasites.30 The authors suggested that the cationic nat-
The melting points (uncorrected) were determined on a Fisher–
Johns melting point equipment. The IR spectra were recorded on a
Figure 4. Action of 5f on mitochondrial electrochemical potential in L. braziliensis. Promastigotes were loaded with rhodamine 123 for 45 min. Letf panel: compound 5f at
M (arrow), followed by the addition of FCCP at 1 M (arrow). Right panel: FCCP at 1 M (arrow), followed by the addition of the compound 5f at 6 M (arrow). The
experiment shown is typical of at least three repeats.
6
l
l
l
l

4430
D. Bompart et al. / Bioorg. Med. Chem. 21 (2013) 4426–4431
Figure 5. Evaluation of 5f on acidocalcisomes alkalinization in L. braziliensis. Promastigotes were loaded with acridine orange for 5 min. Left panel: compound 5f at 6 lM
(arrow), followed by the addition of nigericine (Nig) at 2
lM (arrow). Right panel: Nig at 2
l
M (arrow), followed by the addition of the compound 5f at 6
lM (arrow). The
experiment shown is typical of at least three repeats.
Table 3
Effect of quinoline 5f on the biosynthesis of free sterol in L. braziliensis promastigotes
Sterols
Retention time (min)
Mass percent after treatment with
p-Value
Control
Compd 5f at 6 lM
Exogenous cholesterol
Endogenous 14-methyl squalene
Endogenous 14-desmethyl 5-dehydroepisterol
24.2
20.5
28.9
13.6 0.51
22.7 0.12
63.7 1.02
12.3 1.87
64.3 0.89
23.4 0.22
0.0232
<0.0001
<0.0001
Mass percent of squalene and 5-dehydroepisterol, in this biosynthetic pathway. Parameters determined by Gas Chromatography and High Resolution Mass Spectrometry.
Was made a ‘t-test’ for statistics analysis, using InfoStat 2012 version,36 (p significance 6 0.05).
Lumex infralum FT-02 spectrophotometer in KBr. 1H NMR and 13
C
4.2.2. Anti-leishamial activity and cytotoxicity
NMR spectra were recorded on Bruker AC-200 or Bruker AC-400
spectrometers. Chemical shifts are reported in ppm (d) relative to
the solvent peak (CHCl3 in CDCl3 at 7.24 ppm for protons). Signals
are designated as follows: s, singlet; d, doublet; dd, doublet of dou-
blets; ddd, doublet of doublets of doublets; t, triplet; td, triplet of
doublets; q, quartet; m, multiplet; br., broad. A Hewlett–Packard
5890a series II Gas Chromatograph interfaced to an HP 5972 mass
selective detector (MSD) with an HP MS Chemstation Data system
was used for MS identification at 70 eV using a 60 m capillary col-
umn coated with HP-5 [5%-phenyl-poly(dimethyl-siloxane)]. Ele-
mental analyses were performed on a Perkin–Elmer 2400 Series
II analyzer, and were within 0.4 of theoretical values. The reaction
progress was monitored using thin layer chromatography on a
silufol UV254 TLC aluminum sheet.
In order to evaluate the effect of seven 2-aryl quinoline derivatives
on the promastigotes viability, a colorimetric test, as reported by
Saint–Pierrre–Chazalet,38 was performed with modifications. Briefly,
200,000 parasites/well were seeded in a 96-well plate, adding a un-
ique concentration of 50
lM of each compound, and it was incubated
for 96 h at 29 °C. After the incubation, 1
lg/mL 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was added and
than it was incubated in darkness for 4 h. After this time, the parasites
were lysed with acidic isopropanol and the plate was read at 570 nm
in a spectrophotometer Synergy HT (Biotek) with the software KC4
3.4 Rev. 21. MiltefosineÒ was used as reference drug.
Compounds that generated a 50% or more inhibition of viability
(compared to untreated parasites) were selected for further evalu-
ation. The citotoxicity on mammalian host cells (macrophages
BMDM) was evaluated by the same assay, with few modifications.
For this case, 5000 cells/well were seeded and the concentrations
The chemical purity of obtained 2-aryl quinolines 5 was con-
firmed using elemental analyses, performed on a Perkin Elmer
2400 Series II analyzer, that were within 0.4 of theoretical values.
Their spectral and physical properties were in agreement with
those reported in literature.21
of the compounds 100, 200 and 300 lM were tested.
The EC50 calculation of selected compounds was performed
using growth curves in LIT medium, as previously reported.39
Briefly, the cultures were established at 1 Â 106 promastigotes/
mL, and were added increasing concentration of quinoline 5f after
24 h at 29 °C. Parasite proliferation was monitored daily by direct
counting in a Neubauer chamber, three independent experiments
were performed.
4.2. Biology
4.2.1. General
Promastigotes of Leishmania (V.) braziliensis strain MHOM/CO/
87/UA301 (provided by Dr. Carlos Muskus. Programa de Estudio y
Control de Enfermedades Tropicales PECET, Universidad de Antio-
quia, Colombia) were isolated from footpad lesions in BalbC mice
previously infected. For differentiation and culture maintenance
was used medium LIT (tryptose 15 g/L, yeast extract 5 g/L, liver ex-
tract 2 g/L, hemin–NaOH 0.02 g/L, glucose 4 g/L, NaCl 9 g/L, KCl
0.4 g/L Na2HPO4, 7.5 g/L, pH 7.4) supplemented with 10% fetal calf
serum and maintained at 29 °C. Macrophages BMDM were ob-
tained from mouse bone marrow and differentiated in a condi-
tioned medium of mouse lung fibroblasts (medium L-929), as
previously reported methodologies.23,37
4.2.3. Intracellular amastigotes infections
The effect of selected compound on intracellular amastigotes
was performed by the protocol previously described.40 Briefly, a
mixture of macrophages BMDM and promastigotes of L. brazilien-
sis, in a proportion 1:10 diluted in DMEM +10% FBS, was prepared.
The mixture were placed on glass coverslips and keeping in a moist
chamber for 18 h at 37 °C and 5% CO2. After, the medium was re-
moved, adding new medium with the tested condition and incu-
bated for 96 h in same condition. Then, macrophages were
stained with Giemsa and counted.

D. Bompart et al. / Bioorg. Med. Chem. 21 (2013) 4426–4431
4431
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The mitochondrial membrane potential estimations were car-
ried out with the fluorescent dye rhodamine 123, that presents
maximum peaks in its excitation and emission spectra at 488
and 530 nm, respectively. Rhodamine 123, is a mitochondrion-
specific cationic dye that allows visualization of electrochemical
potential on this organelle. As described by Serrano-Martín and
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lg/mL of rho-
damine 123 in buffer 130 mM KCl, 1 mM MgCl2, 2 mM KH2PO4,
20 mM Tris–HCl, pH 7.4 for 30 min at 29 °C. All measurements
were performed in a Hitachi 7000 spectrofluorimeter at 29 °C, un-
der continuous agitation.
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spectrofluorimeter under continuous agitation in a stirred cuvette
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4.2.5. Free sterol contents
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raphy coupled with high-resolution mass spectrometry, as de-
scribed previously.40,41 Briefly, for the extraction and the
separation of neutral lipids, L. braziliensis was cultured in the pres-
ence of quinoline 5f, and total lipids were extracted with chloro-
form-methanol (2:1, vol/vol). The extract was dried and
resuspended in a minimum volume of chloroform. The suspension
was applied to a silicic acid column (1.5 by 4 cm) and washed with
chloroform to separate neutral lipids from other lipid fractions.
For the quantitative analysis of free sterols and structural
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high-resolution column (25 m by 0.20 mm [inner diameter]; Ul-
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Agilent Technologies 7890A gas chromatograph equipped with
mass-sensitive detector Agilent Technologies 5975C. The lipids
were dissolved in chloroform and injected into the column at an
initial temperature of 50 °C (1 min), followed by a temperature in-
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detector was kept at 280 °C.
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Acknowledgments
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Y.G.-M. and X.S.-M. thank Jacques Leañez for critical reading of
the manuscript, Jose Alvarez and Gustavo Benaim for laboratory
supplements and financial support from Fondo Nacional de Ciencia
Tecnología e Innovación, Project 2021002132. Financial support
from Patrimonio Autónomo Fondo Nacional de Financiamiento
para la Ciencia, la Tecnología y la Innovación, Francisco José de Cal-
das, contract RC-0572-2012, is gratefully acknowledged (V.V.K.).
C.M.M.G. acknowledges COLCIENCIAS for the fellowship.
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35. Serrano-Martín, X.; Payares, G.; De Lucca, M.; Martinez, J. C.; Mendoza-León,
A.; Benaim, G. Antimicrob. Agents Chemother. 2009, 53, 5108.
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