Insights into the structural patterns of the antileishmanial activity of bi- and tricyclic N-heterocycles

Article abstract of DOI:10.1039/c6ob01149g

The influence of various structural patterns in a series of novel bi- and tricyclic N-heterocycles on the activity against Leishmania major and Leishmania panamensis has been studied and compounds that are active in the low micromolar region have been identified. Both quinolines and tetrahydrooxazinoindoles (TOI) proved to have significant antileishmanial activities, while substituted indoles were inactive. We have also showed that a chloroquine analogue induces Leishmania killing by modulating macrophage activation.

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Full Paper
Insights into the Structural Patterns of the Antileishmanial Activity of
Bi- and Tricyclic N-Heterocycles
Lizzi Herreraa,b, David E. Stephensc, Abigail D’Avilaa, Kathryn G. Georgea, Hadi Armanc, Yu Zhangc,
George Perryd, Ricardo Lleonarta, Oleg V. Larionov*c, Patricia L. Fernández*a
5
Received (in XXX, XXX) Xth XXXXXXXXX 2016, Accepted Xth XXXXXXXXX 2016
DOI: 10.1039/b000000x
Influence of various structural patterns in a series of novel bi- and tricyclic N-heterocycles on the activity
against Leishmania major and Leishmania panamensis has been studied and compounds that are active in
the low micromolar region have been identified. Both quinolines and tetrahydrooxazinoindoles (TOI)
10 proved to have significant antileishmanial activities, while substituted indoles were inactive. We have
also showed that a chloroquine analogue induces Leishmania killing by modulating macrophage
activation.
Several families of compounds have been tested for
45 antileishmania activity.8 Both, natural products and synthetic
compounds have been recently identified as promising leads
against leishmaniasis. Particularly promising scaffolds include
Introduction
Leishmaniasis is a tropical disease with a significant global health
15 burden that is caused by Leishmania flagellate protozoa.1 Twenty
Leishmania species that are pathogenic for humans have been
identified. They are transmitted by several sand flies species,
Phlebotomus in the Old World and Lutzomya in the New World.2
Leishmaniasis has a wide spectrum of clinical manifestations
20 depending on the Leishmania species and the immunological
status of the host. These include localized and diffused cutaneous
leishmaniasis, mucocutaneous form and visceral disease.3
quinolines
and
indoles.
Antiparasitic,9
antibacterial,10
antineoplastic,11 and antiviral12 activities have been reported for
50 quinoline derivatives. For instance, naphthylisoquinoline
alkaloids showed low micromolar activities against Leishmania
donovani,13 as well as against intracellular amastigote stage of
Leishmania major.14 Similarly, the naturally-occurring
hypocrellin A was found to be more active against L. donovani in
55 vitro than amphotericin
B
and pentamidine.15 Synthetic
antileishmanial 1,4-anthraquinones have also been described.16
Recently, abietane-type diterpenoids have emerged as potent
antileishmanial agents.17
Leishmania species differ in virulence, vectors preferences and
geographic distribution. However, all species have a similar life
25 cycle involving a motile, flagellated stage in the midgut of vector
(promastigote) and an intracellular non-motile stage (amastigote)
in host macrophages.4 Macrophages are the most important
effector cells in Leishmania infection, and their appropriate
activation is required to eliminate the parasite. The destruction of
30 the parasite by macrophages depends on the production of nitric
oxide (NO), tumor necrosis factor (TNF), interleukin (IL)-1
among other mediators, and is negatively affected by a variety of
factors including IL-10.5
60 Fig. 1. Structures of studied N-heterocycles.
In this study we evaluated the effects on intracellular amastigotes
Current treatments for leishmaniasis are based in drugs whose
35 specific mechanisms of action are poorly understood. The most
used drugs (e.g. pentavalent antimonials, pentamidine,
amphotericin B and miltefosine) require lengthy treatments and
have high toxicity and serious side effects.6 Furthermore,
resistance to some of these drugs has been reported for a diversity
40 of Leishmania strains.7 Consequently, the search for new drugs
for the treatment of the disease that carries a multitude of health
and socioeconomic problems in endemic countries is an enduring
challenge.
and promastigotes of L. panamensis and L. major of three
families
of
bi-
and
tricyclic
N-heterocycles:
tetrahydrooxazinoindoles (TOIs) 1, quinolines 2, and indoles 3
65 (Fig. 1). Since quinolines, e.g. amodiaquine, chloroquine,
mefloquine, and primaquine have been successfully used as
antimalarials, they may hold promise as a new class of
antileishmanial agents. Indeed, amodiaquine and its basic side
chain-modified analogues have been found to have a significant
70 antileishmanial activity.18 In addition, 7-chloro-4-quinolinyl
hydrazones have shown strong activity against the intracellular
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parasite.19 We have recently developed an efficient method of 50 products were obtained in good to excellent yields. The 4-chloro-
synthesis of 2-susbtituted quinolines from quinoline N-oxides that
allows for a simple access to various substituted quinolines,
including 2-substituted derivatives of amodiaquine and
chloroquine.20 Using this method, we have prepared a series of
substituted TOIs were isolated as single diastereomers, in line
with the previously observed results.21
X-ray crystal structures were elucidated for compounds 7-11
(Fig. 2), aiding in the confirmation of the structure of the TOI
5
quinolines bearing substituents in 2, 4, 5, 7 and 8 positions, 55 products and their precursors. Interestingly, although TOI
including novel amodiaquine and chloroquine analogues. The
1,2-oxazine moiety in tetrahydrooxazinoindoles (TOIs)
resembles pyridine in amodiaquine and chloroquine. In addition
10 to structural similarity, quinolines and 1,2-oxazines both contain
compounds 9 and 10 were prepared with >90% ee, only a small
amount of racemic crystals was obtained, indicating that the
racemate is less soluble than both enantiomers, as previously
observed for other scalemic mixtures.24 The indoles, quinolines
1
a weekly basic nitrogen atom that may be important for the 60 and TOIs were selected based on the combination of ready
antileishmanial activity. Hence, it was of interest to compare
these two classes of basic N-heterocycles. Our recently described
method of tetrahydrooxazinoindoles (TOIs) synthesis offered a
15 facile entry to the novel 1,2-oxazine-containing framework in
racemic and enantioselective fashion.21 Since TOI framework
contains an indole moiety, a series of substituted indoles were
also prepared, and their antileishmanial activities have been
compared with the quinolines and TOIs.
synthetic availability and structural diversity.
20 Our results show that most of the tested compounds are more
active against intracellular amastigotes than on promastigotes of
both Leishmania species assayed. L. panamensis amastigotes
appear to be more sensitive to our active compounds than L.
major amastigotes. Interestingly we also found that one of the
25 compounds inhibited the production of IL-10 by macrophages
infected with either L. panamensis or L. major.
Further, we describe herein that some TOIs are competent
antileishmanial agents, and their activity is related to the presence
of the 1,2-oxazine moiety. This result, along with the data on the
30 antileishmanial activity of several substituted quinolines, provide
important insights into the antileishmanial activity of N-
heterocycles and point to the potential of 1,2-oxazines22 as new
structural frameworks for biomolecular applications.23
35 Scheme 1. Structure and preparation of tetrahydrooxazinoindoles (TOIs).
Fig. 2. Single crystal X-ray crystallographic structures of compounds 7–
11.
Results and Discussion
Preparation of the compounds used in the antileishmanial
tests
65 Screening of compounds against L. major and L. panamensis
promastigotes.
The tetrahydrooxazinoindole compounds 4 bearing substituents in
40 positions 3, 4, 4a, 5, 6, and 9 were accessed using the inverse
electron demand [4+2] cycloaddition reaction of indoles 5 with
transient nitrosoalkenes that were generated in situ from -
chlorooximes 6 (Scheme 1).21 The indoles 5 were prepared by
means of N-alkylation and a reductive C3-alkylation (See
45 Supporting Information for details).
The enantiomerically enriched TOIs were prepared using
Cu(DM-Binap)OTf as a catalyst, while racemic TOIs 4 were
prepared using Cu(rac-Binap)OTf as a catalyst, in the presence of
silver carbonate as a chloride scavenger and a base. The TOI
Libraries of TOIs, indoles and quinolines were tested against L.
major and L. panamensis extracellular promastigotes. To evaluate
the effect of compounds on promastigotes of both Leishmania
70 species we performed a first screening at a fixed concentration of
10 μM. Any compound inducing a growth inhibition of 50% or
more, was further tested using a four concentration points
including 1, 3, 10 and 30 µM. Viability of promastigotes was
assessed by using an ATP-bioluminescence assay after 24 hours
75 of incubation with the compound. 25 Our results showed that none
of the tested compounds were active for L. major promastigotes
(Tables 1 and 2), whereas four compounds from the TOI library
2
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(10% of the total number of the tested compounds) were effective
than on L. major (IC50= 12.27, 4.30, 3.84 M) (Table 2). It is
important to note that SI values are consistently higher for L.
panamensis than for L. major (Tables 1 and 2), suggesting that L.
panamensis is more sensitive to both families of compounds than
40 L. major. Compounds of the indole series showed no activity
against any Leishmania species or stage tested (Table S1).
Considering the specificity of the most active compounds for the
amastigote form, we evaluated the possible immunomodulatory
effect of compounds 9, 12, 13, 15, and 17-19. Our results showed
in the killing of L. panamensis promastigotes, with IC50 values
ranging from 8 to 12 μM (Table 2). Several compounds, e.g. 11-
14, showed some toxicity to uninfected macrophages.
5
Antileishmanial activity of compounds on intracellular
amastigotes.
The evaluation of the effect of compounds against L. major and
L. panamensis intracellular amastigotes was performed by using
10 the Giemsa staining method.26 As described above for 45 that compound 12 inhibited production of IL-10 by macrophages
promastigotes, a first screening was performed at a compound
concentration of 10 μM. Accordingly, active compounds were
subjected to a four-point dose response evaluation. Active
compounds and their IC50 values are presented in Table 1
15 (quinolines 12 and 15) and Table 2 (TOI compounds 9, 11, 13,
14, 16-20).
infected with L. panamensis and L. major (Fig. 3) in a dose
dependent manner. These results suggest that the compound-
induced parasite killing mechanism may include a regulation of
the macrophage activation.
Compounds from the quinoline family showed similar effect on
both Leishmania species. Only two quinoline derivatives (10 %
of the compounds tested) were active and exhibited similar IC50
20 values for L. major and L. panamensis (Table 1 and Fig S2).
Compound 12 exhibited cytotoxic effects on macrophages with a
50% cytotoxic concentration (CC50) of 14.03 M. However, that
cytotoxic concentration is still tenfold higher than the IC50
calculated for L. panamensis and L. major (1.07±0.51 and
25 1.65±0.3 respectively). Both quinoline derivatives, however, have
similar values of selectivity index (Table 1).
50
Fig. 3. Compound 12 inhibits the production of IL-10 from Leishmania-
infected macrophages. Peritoneal macrophages from Balb/c mice were
infected with L. panamensis (A) or L. major (B) and treated with
compound 12. Supernatants were collected after 24 hours of the stimulus
Compounds from TOIs family showed differential activity
against both Leishmania species. Compounds 17, 18 and 19 were
exclusively active against intracellular L. major whereas
30 compounds 11, 14, and 20 showed effect only for L. panamensis,
55 and levels of IL-10 were measured by Elisa. Data represent mean ± SEM
from stimuli performed in duplicates and are representative of two
independent experiments.
–
both promastigotes and amastigotes (Table 2). These
Discussion
differences in sensitivity to some compounds were previously
described for Leishmania species.27 Compounds 9, 13 and 16
were active for amastigotes of both species but the effect on L.
35 panamensis was higher (IC50= 0.8, 1.22, 0.87 μM respectively)
60 Leishmaniasis is recognized as one of the most neglected
diseases, and the development of new drugs against leishmaniasis
is an important therapeutic goal.28
Table 1. Antileishmanial activity of select quinoline derivatives.a
IC50 (μM) ± SD
Intracellular Intracellular
L. major L. panamensispromastigotes promastigotes cytotoxicity
L. major L. panamensis Macrophage
SI (L. major/L.
panamensis)b
Compound
Structure
amastigotes amastigotes
CC50 (μM)
12
1.65±0.3
1.07±0.51
3.29±2.72
NA
NA
NA
NA
14.03±2.65
8.5/13.1
9.7/15.3
15
5.19±2.33
>30
65 a NA, non-active. IC50 and CC50 values are mean ± standard deviation (SD) of two independent experiments. The control drug was amphotericin B, with
an IC50 value for intracellular amastigotes of 0.103 μM for L. panamensis and 0.157 μM for L. major. The IC50 of amphotericin B for promastigotes was
0.1 μM for L. panamensis and 0.243 μM for L. major. b The selectivity index (SI) is calculated as the ratio between CC50 on peritoneal macrophages and
IC50 on intracellular L. major or L. panamensis amastigotes. SI for amphotericin B is 1723.3 for L. panamensis and 1130.5 for L. major.
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There is evidence of recent appearance of Leishmania resistance 20 higher activities than both larger benzylic and smaller (methyl)
to antimonials – the current first line of treatment.7b,d Molecular
and biochemical differences among species influence the
sensitivity of Leishmania species to different chemical agents,
complicating the search for antileishmanial drugs with broad
groups. On the other hand, both aromatic substituents and ester
groups in C3 were well tolerated. Displacement of the O1 with
TsN (S14, see Table S2) led to the loss of activity, further
highlighting the importance of substitution in the C ring of the
5
activity profile. Here, we studied synthetic compounds of three 25 TOI system.
classes and identified positive hits that inhibit the intracellular
amastigotes and promastigotes of L. panamensis and L. major.
We also showed that L. panamensis is more sensitive than L.
The C4 position in 1,2-oxazines is generally difficult to access
synthetically. Hence, influence of the substituents in C4 position
was tested with TOI compounds bearing a chlorine atom anti to
the C4a substituent. Interestingly, compounds 17 and 19 were
10 major to these compounds. Quinoline compounds were
previously identified as efficient antimalarial and antileishmanial 30 found to be active only against intracellular L. major amastigotes,
agents.18 Several quinoline derivatives showed inhibitory
capacity against different Leishmania species that is comparable
to reference drugs.8 In view of the lack of antileishmanial activity
15 of substituted indoles, we further focused on the study of the
indicating that significant selectivity can be achieved through
modulation of the 1,2-oxazine moiety.
In general, TOI framework has provided more hits than
quinolines. In the quinoline series, only two compounds (12 and
influence of substituents in the oxazine ring of the TOI 35 15) that are structurally related to amodiaquine and chloroquine
compounds. In the TOI series, presence of the bulky aromatic
rings in the N9 and C4a positions generally led to the loss of
antileishmanial activity. Allyl groups in N9 and C4a resulted in
exhibited significant activity against intracellular L. major and L.
panamensis amastigotes, with no activity against promastigotes
(Table 1).
Table 2. Antileishmanial activity of select tetrahydrooxazinoindoles (TOIs).a
40
IC50 (μM) ± SD
Compound R1
R2
R3
R4
R5
Intracellular Intracellular L.
L. major panamensis promastigotes promastigotes cytotoxicity (L. major/L.
amastigotes amastigotes
CC50 (μM) panamensis)b
L. major
L. panamensis Macrophage
SI
9
H
Me
All
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
H
H
Cl
H
12.2±0.19
NA
0.8±0.49
1.95±0.99
0.87±0.14
0.35±0.18
1.22±0.63
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11.60±0.20
NA
>30
28.48±2.19
9.15±1.24
30
5.96/90.9
NA/14.6
2.38/10.5
NA/85.7
9.57/33.7
29.3/NA
12.2/NA
13.3/NA
NA/47.0
11
13
H
H
H
H
H
H
H
H
All
Bn
iPr
3.84±2.5
NA
iPr
14
16
8.28±0.83
12.53±0.54
NA
All
All
All
All
cPent
All
4.30±1.73
1.92±1.81
6.13±0.05
5.57±0.53
NA
>30
17
18
>30
All 2-Naphth
NA
NA
>30
19
20
All p-MeOPh Cl
NA
NA
>30
iPr
Ph
H
1.13±0.21
11.51±0.32
>30
a NA, non-active. IC50 and CC50 values are mean ± standard deviation (SD) of two independent experiments. The control drug was amphotericin B, with
an IC50 value for intracellular amastigotes of 0.103 μM for L. panamensis and 0.157 μM for L. major. The IC50 of amphotericin B for promastigotes was
0.1 μM for L. panamensis and 0.243 μM for L. major. b The selectivity index (SI) is calculated by the ratio between CC50 on peritoneal macrophages and
45 IC50 on intracellular L. major or L. panamensis amastigotes. SI for amphotericin B is 1723.3 for L. panamensis and 1130.5 for L. major.
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It has been known that there are important differences in the
novel antileishmanial agents and for further elucidation of their
mechanism of action.
sensitivity towards chemical agents between both parasite stages,
and between different Leishmania species.25,29 We showed herein
that, of the 39 assayed compounds, none was active against L.
major promastigotes, while 20% of the compounds were active
against L. major intracellular amastigotes. Promastigotes differ
biologically from amastigotes in metabolism, morphology and
60
Experimental
5
Materials and methods - Dichloromethane was dried and purified
under an argon atmosphere using an LC technology Solutions’
SP-1 Solvent Purifier All oximes were synthesized according to
surface composition and these differences have an impact on the 65 the literature procedure.21 All heterocyclic N-oxides were
sensitivity of parasites to chemical agents.30 Moreover, to be
synthesized according to reported procedures.20b N'-(2-chloro-1-
10 active against amastigotes, compounds must cross the cellular
membrane and maintain its stability in the intracellular
environment. Additionally, some compounds may be toxic to the
phenylethylidene)-4-methylbenzenesulfonohydrazide
was
synthesized according to literature procedure.34 All other reagents
were purchased and used without further purification. Column
parasite only when metabolized inside the macrophage, then 70 chromatography was performed using CombiFlash Rf-200
showing the behavior of being inactive in promastigotes and
15 active in the amastigote. Another interesting possibility may be
that some of the compounds, instead of being toxic directly to the
parasite, may be able to activate the macrophage to fully develop
(Teledyne-Isco) automated flash chromatography system. 1H,
13C, 19F NMR spectra were recorded at 500 (1H), 125 (13C), and
282 MHz (19F) on Varian Mercury VX 300 and Agilent
Inova 500 instruments in CDCl3 solutions. Chemical shifts (δ) are
their antiparasitic activity. We also showed here that compound 75 reported in parts per million (ppm) from the residual solvent peak
12 inhibits the production of IL-10 by macrophages infected with
20 L. panamensis and L. major. It is well-known that IL-10 is an
antiinflammatory mediator that inhibits a variety of macrophage
functions including phagocytosis, expression of co-stimulatory
and coupling constants (J) in Hz. Proton multiplicity is assigned
using the following abbreviations: singlet (s), doublet (d), triplet
(t), quartet (quart.), quintet (quint.), septet (sept.), multiplet (m),
broad (br). Infrared measurements were carried out neat on a
molecules and production of pro-inflammatory cytokines, with 80 Brüker Vector 22 FT-IR spectrometer fitted with a Specac
important consequences in macrophage activation.31 Interleukin
25 10 has been implicated as a key factor in the survival of
Leishmania infection both in vitro and in vivo. High levels of IL-
10 have been linked to leishmaniasis progression and parasite
diamond attenuated total reflectance (ATR) module.
General procedure for the synthesis of 1,2-oxazines - To an oven
dried flask was added 3Å MS (5 scoops), CuOTf ½ PhMe (10–20
mol%), rac-BINAP or (S)-DM-BINAP (10–20 mol%) and
persistence.32 The addition of IL-10 to L. major-infected 85 dichloromethane (0.1–0.2M). The reaction was stirred for 15 min
macrophages results in uncontrolled parasite replication.32a Our
30 results suggest that the antileishmanial activity of compound 12
might be, at least in part, mediated by the modulation of the
macrophage activation. The selectivity index of this chloroquine
and then cooled to –78 C under argon. Indole (1 equiv.), oxime
(1 equiv.) and silver carbonate (3 equiv.) were added
sequentially. The reaction was allowed to warm to either 20 or
15 C and was stirred for the specified time. The reaction
analogue was 3 to 4 times higher (8.5/13.1 L. major/L. 90 mixtures were then filtered, concentrated under reduced pressure,
panamensis) than the SI of chloroquine (3.1) previously reported
35 under similar experimental conditions33 Since chloroquine is an
approved drug, these SI values suggest that compound 12 is a
promising candidate for further therapeutic investigation.
and purified by column chromatography [hexanes/EtOAc, silica
gel] to yield the desired products.
Mice – Female and male Balb/c mice, 8 weeks of age, were
provided by INDICASAT’s animal facility. Animals were
We have shown here that novel synthetic derivatives of several 95 maintained with 12 hours light/dark cycle, at a constant
families of compounds are promising antileishmanial hits,
40 opening new possibilities for further development of 1,2-oxazine-
based antileishmanial agents as a way towards new and effective
drugs against this neglected disease. Due to the differences in
temperature of 24 °C with free access to food and water. All
experimental procedures were approved by the Institutional
Animal Care and Use Committee of INDICASAT (IACUC-14-
002) and were based in the strict observance of the ethic
Leishmania species sensitivity to drugs, the search of species- 100 guidelines related to the handling of lab animals in accordance
specific antileishmanial drugs has been encouraged. The higher
45 values of SI for L. panamensis than for L. major for the active
compounds described herein indicate that they are good leads in
the development of L. panamensis-specific drugs.
with international regulations and those established by
INDICASAT.
Parasites
–
Promastigotes
of
L.
panamensis
(MHOM/PA/94/PSCI-1) and L. major (Restrepo et al., 2013)
105 were cultured at 25°C, in Schneider medium (Sigma)
supplemented with 20% FBS (Gibco). Parasite virulence for both
strains was maintained by inoculating them previously in
hamster.
Promastigote Inhibition Assay – L. panamensis and L. major
110 parasites from stationary phase culture were washed with PBS 1X
and centrifuged at 1700xg for 10 minutes. Parasites were diluted
in Schneider media supplemented with 20 % FBS and seeded in
96 well white opaque plate (Thermo Scientific, Nunc) at a density
of 2x106 parasites per well in a volume of 99 µL. Each well was
115 treated with 1 µL of compound at a concentration of 10 µM in
Conclusions
In conclusion, a targeted library of tetrahydrooxazinoindoles
50 (TOIs) was synthesized, and antileishmanial activities were
discovered for a number of the TOI compounds. The activity was
compared with indoles that were found to be inactive, and with
quinolines. For quinolines, only amodiaquine and chloroquine
analogues were found to be active. We have identified that the
55 antileishmanial activity of the chloroquine analogue 12 may also
be due to a modulation of macrophage activation. The activity of
TOI compounds opens an avenue for the search of structurally
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screening assays and later at 1, 3, 10 and 30 µM in dose-response
assays. The parasites were incubated at 25°C during 24 hours.
After incubation period, 50 µL of CellTiter-Glo® reagent 60 Investigacion, Republic of Panama (SNI-34-2014) is gratefully
University of Texas at San Antonio, the National Science
Foundation (CHE-1455061), and the Sistema Nacional de
(Promega) was added to each well for lysing the parasites and the
plate was incubated at room temperature for 10 minutes to
stabilize the luminescent signal. The resulting ATP was recorded
in relative-light units (RLU) in a multi-detection microplate
reader (Synergy HT-Biotek).
acknowledged. Mass spectroscopic analysis was supported by a
grant from the National Institute on Minority Health and Health
Disparities (G12MD007591). We thank Hector Cruz and Kissy
de Gracias (INDICASAT) for their help with the intracellular
65 amastigotes experiments.
5
Amastigote Inhibition Assay – Peritoneal resident macrophages
10 from Balb/c mice were collected by peritoneal lavage with cold
PBS 1X (AppliChem). Cells were seeded in RPMI (Gibco) with
10% FBS (Gibco) at a density of 1x106 cells per well in 24 well
plates with a round glass coverslip in each well and cultured for 2
h at 37oC in an atmosphere of 5% CO2. Non-adherent cells were
15 removed by washing and adherent macrophages were infected
with late stationary phase promastigotes at 1:30 ratio
(cell:parasite) for L. panamensis and 1:10 for L. major during 1
hour at 37oC, 5% CO2. Non-internalized promastigotes were
removed by washing with RPMI media. Infected macrophages
20 were treated with the compounds at a final concentration of 10
µM. Dose-response curves were produced for active compounds
using concentrations of 1, 3, 10 and 30 µM. Amphotericin B
(Sigma) was used as positive control. Infected macrophages were
incubated for 24 hours at 37°C in 5% CO2. All negative controls
25 and stimulus were performed in the presence of 0.1% DMSO
(Sigma) since compounds are solubilized in this solvent. After
incubation, supernatants were collected for evaluation of the
presence of IL-10 and coverslips were washed once with PBS,
fixed with Methanol (Merck), and stained with Giemsa (Sigma).
30 The infection rate was calculated by counting the number of
amastigotes per cell in a total of 250 cells. The percentage of
parasite inhibition was calculated as
Notes and references
a Centro de Biología Molecular y Celular de Enfermedades, Instituto de
Investigaciones científicas y de alta tecnología (INDICASAT-AIP),
Edificio 219, Ciudad del Saber, Apartado 0843-01103, Panamá
70 República de Panamá; Fax: +507 507 0020; Tel: +507 517 0739; E-
mail: pllanes@indicasat.org.pa
b Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra
Pradesh, 522510, India
c Department of Chemistry, The University of Texas at San Antonio, San
75 Antonio, Texas, United States of America. Fax: +1 210 458 7428; Tel: +1
210 458 6050; E-mail: oleg.larionov@utsa.edu
d Department of Biology, The University of Texas at San Antonio, San
Antonio, Texas, United States of America.
*Corresponding authors: Email: pllanes@indicasat.org.pa;
80 oleg.larionov@utsa.edu
† Electronic Supplementary Information (ESI) available: Experimental
procedures and spectroscopic data. See DOI: 10.1039/b000000x/
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Financial support by the Welch Foundation (AX-1788), National
Institute of the General Medical Sciences (SC3GM105579), the
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