Effect of 1,2,3-triazole salts, non-classical bioisosteres of miltefosine, on Leishmania amazonensis

Article abstract of DOI:10.1016/j.bmc.2017.03.051

Here, we report the effect of new non-classical bioisosteres of miltefosine on Leishmania amazonensis. Fifteen compounds were synthesized and the compound dhmtAc, containing an acetate anion, a side chain of 10 carbon atoms linked to N-1 and a methyl group linked to N-3, showed high and selective biological activity against L. amazonensis. On the intracellular amastigotes, stages of the parasite related to human disease, the IC₅₀ values were near or similar to the 1.0?μM (0.9, 0.8 and 1.0?μM on L. amazonensis-WT, and two transgenic L. amazonensis expressing GFP and RFP, respectively), being more active than miltefosine. Furthermore, dhmtAc did not show toxic effects on human erythrocytes and macrophages (CC₅₀?=?115.9?μM) being more destructive to the intracellular parasites (selectivity index?>?115). Promastigotes and intramacrophage amastigotes treated with dhmtAc showed low capacity for reversion of the effect of the compound. A study of the mechanism of action of this compound showed some features of metazoan apoptosis, including cell volume decreases, loss of mitochondrial membrane potential, ROS production, an increase in the intracellular lipid bodies, in situ labeling of DNA fragments by TUNEL labeling and phosphatidylserine exposure to the outerleaflet of the plasma membrane. In addition, the plasma membrane disruption, revealed by PI labeling, suggests cell death by necrosis. No increase in autophagic vacuoles formation in treated promastigotes was observed. Taken together, the data indicate that the bioisostere of miltefosine, dhmtAc, has promising antileishmanial activity that is mediated via apoptosis and necrosis.

Full text of DOI:10.1016/j.bmc.2017.03.051

Accepted Manuscript
Effect of 1,2,3-Triazole salts, non-classical bioisosteres of miltefosine, on
Leishmania amazonensis.
Pedro H.F. Stroppa, Luciana M.R. Antinarelli, Arturene M.L. Carmo, Jacy
Gameiro, Elaine S. Coimbra, Adilson D. da Silva
PII:
S0968-0896(17)30096-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.051
BMC 13649
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
16 January 2017
21 March 2017
23 March 2017
Please cite this article as: Stroppa, P.H.F., Antinarelli, L.M.R., Carmo, A.M.L., Gameiro, J., Coimbra, E.S., da Silva,
A.D., Effect of 1,2,3-Triazole salts, non-classical bioisosteres of miltefosine, on Leishmania amazonensis.,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.03.051
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Effect of 1,2,3-Triazole salts, non-classical bioisosteres of miltefosine, on Leishmania
amazonensis.
Pedro H. F. Stroppa1, Luciana M. R. Antinarelli2, Arturene M. L. Carmo1, Jacy
Gameiro2, Elaine S. Coimbra2, Adilson D. da Silva1*
1Departamento de Química, I.C.E., Universidade Federal de Juiz de Fora, Campus
Universitário, Juiz de Fora, Minas Gerais 36036-900, Brasil.
2Departamento de Parasitologia, Microbiologia e Imunologia, I.C.B., Universidade
Federal de Juiz de Fora, Campus Universitário, Juiz de Fora, Minas Gerais 36036-900,
Brasil.
*Corresponding author
E-mail address: david.silva@ufjf.edu.br (A.D da Silva).
Tel.: +55 32 2102 3310; fax: +55 32 2102 3310.
1

Abstract
Here, we report the effect of new non-classical bioisoteres of miltefosine on
Leishmania amazonensis. Fifteen compounds were synthesized and the compound
dhmtAc, containing an acetate anion, a side chain of 10 carbon atoms linked to N-1 and
a methyl group linked to N-3, showed high and selective biological activity against L.
amazonensis. On the intracellular amastigotes, stages of the parasite related to human
disease, the IC50 values were near or similar to the 1.0 µM (0.9, 0.8 and 1.0 µM on L.
amazonensis-WT, and two transgenic L. amazonensis expressing GFP and RFP,
respectively), being more active than miltefosine. Furthermore, dhmtAc did not show
toxic effects on human erythrocytes and macrophages (CC50 = 115.9 µM) being more
destructive to the intracellular parasites (selectivity index > 115). Promastigotes and
intramacrophage amastigotes treated with dhmtAc showed low capacity for reversion
of the effect of the compound. A study of the mechanism of action of this compound
showed some features of metazoan apoptosis, including cell volume decreases, loss of
mitochondrial membrane potential, ROS production, an increase in the intracellular
lipid bodies, in situ labeling of DNA fragments by TUNEL labeling and
phosphatidylserine exposure to the outerleaflet of the plasma membrane. In addition, the
plasma membrane disruption, revealed by PI labeling, suggests cell death by necrosis.
No increase in autophagic vacuoles formation in treated promastigotes was observed.
Taken together, the data indicate that the bioisotere of miltefosine, dhmtAc, has
promising antileishmanial activity that is mediated via apoptosis and necrosis.
Keywords: 1,2,3-triazoles; Leishmania; miltefosine; antileishmanial activity; apoptosis;
necrosis.
2

1. Introduction
Leishmaniasis is a complex vector-borne protozoan disease caused by over 20
species of the genus Leishmania that are pathogenic to humans 1. In South American
countries, L. amazonensis is one of the main etiological agents of leishmaniasis and
capable of producing a wide spectrum of manifestations including localized cutaneous,
mucocutaneous which is characterized by latency and chronicity, diffuse cutaneous
which is commonly refractory to available treatments and most severe and potentially
fatal if left untreated visceral leishmaniasis 2-4
.
The current chemotherapy options for leishmaniasis include pentavalent
antimonials, pentamidine, amphotericin B and its lipoidal formulations, but these are
associated with limited effectiveness, a long-term treatment, toxicity, and significant
side effects 5.
Miltefosine (Figure 1), a member of a class of alkyl-phosphocholines, is the first
orally antileishmanial drug which was originally developed as an anticancer drug, and
has been recently used to treat visceral leishmaniasis in India 5. Several similar
mechanisms of action have been described for the drug in both human tumor cells and
Leishmania parasites 6-8. However, its exact mechanism of action is not completely
understood. At present, the drug is reported to trigger apoptosis effects such as
mitochondrial dysfunction, externalization of phosphatidylserine, cell-cycle arrest at the
sub-G0/G1 phase and DNA fragmentation in Leishmania species 7, 8, impaired
phospholipid sterol metabolism in the parasite as well as acting as an immunomodulator
9, promoting macrophage activation 10, and lipid-dependent cell signaling in tumor cells
11. Miltefosine shows important drawbacks including gastrointestinal side effects,
occasional hepato- and nephrotoxicity and potential teratogenicity 12. Another limitation
of this drug is emerging drug resistance, the occurrence of relapses in
immunocompetent patients and variation in its sensitivity between American cutaneous
leishmaniasis species 13, 14
.
Non-classical bioisosteres include the replacement of entire functional groups,
exchanging cyclic and non-cyclic forms and the inversion of functional groups
(retroisosteres); this powerful concept has inspired rational drug design in medicinal
chemistry 15. Based on this concept, the aim of the present study was to investigate
whether the replacement of the phosphocholine functional group on miltefosine by the
3

1,2,3-triazole and the size of the carbon chain (Figure 1) could maintain or even
increase the biological activity of miltefosine. The triazole nucleus is a well known a
class of five-membered heterocyclic compounds of great importance for the design of
new drugs with broad and potent biological properties. In this work, fifteen compounds
were synthesized, being three as 1,4-disubstituted-1,2,3-triazole precursors molecules
(bht, hht and dht) and twelve salts of 1,4-disubstituted-1,2,3-triazole derivatives
(Figure 1). Preliminary biological studies were performed with all triazole derivatives
(data not shown). Serendipitously, only the compound dhmtAc, containing an acetate
anion and a side chain of 10 carbon atoms, showed high selective biological activity
against Leishmania parasites without any toxic effect on mammalian cells. These results
showed that the non-classical bioisosterism proved advantageous since the compound
dhmtAc was more effective on L. amazonensis than the miltefosine prototype. In
addition, this work also showed that the action of dhmtAc on the parasite involves
biochemical and cellular changes related to apoptosis and necrosis, similar to the
previously related to mechanism of action of miltefosine.
O
O
P
O
-
O
Miltefosine prototype
+
N
Non-classical
bioisosterism
-
-
n: 4, X: I , R: CH3
-
=
hhmtI
hhptBr
hhmtAc
hhptAc
n: 2, X:
I
,
R: CH3= bhmtI
n: 2, X: Br , R: C3H7 bhptBr
bhmtAc
bhptAc
-
n: 4, X: Br , R: C3H7
-
=
=
-
n: 4, X: Ac , R: CH3
-
=
n: 2, X: Ac , R: CH3
=
HO
N
n
-
n: 4, X: Ac , R: C3H7
=
n: 2, X: Ac , R: C3H7
=
X-
+
n: 8, X: I-, R: CH3 = dhmtI
n: 8, X: Br-, R: C3H7 = dhptBr
n: 8, X: Ac-, R: C3H7 = dhptAc
n: 8, X: Ac-, R: CH3 = dhmtAc
N
N
R
Best compound
HO
5
N
1
4
3
N
2
N
+
CH3COO-
H3C
dhmtAc
Figure 1: Chemical Structure of Miltefosine prototype and non-classical bioisosteres.
4

2. Results
2.1 Chemistry
The synthesis of the 1,4-disubstituted-1,2,3-triazole derivatives and their salts
were realized in two steps, and after the salt of acetate was obtained by metathesis
reaction. The synthesis of the starting material began by preparing alkyl azides
azidobutane, azidohexane and azidodecane via an azidation reaction of the alkyl halides
1-bromobutane, 1-bromohexane and 1-bromodecane in the presence of sodium azide
and a mixture 1:1 of ethanol:deionized water 16. Subsequently, the copper(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) provided regiospecifically the 1,4-disubstituted-
1,2,3-triazoles bht, hht and dht after 48 h reaction time. Addition reaction of 1,2,3-
triazoles with alkylhalide (iodomethane and 1-bromopropane), in acetonitrile at 80oC
during 24 h to give the salts 4-(hydroxymethyl)-3-alkyl-1-alkyl-1H-1,2,3-triazole-3-ium
halide. The acetate salt dhmtAc was previously reported in the patent literature 17. The
salts with the ion acetate were obtained passing the corresponding salts halide by anion
exchange resin and subsequently neutralizing species formed according to the literature
18 (Scheme 1). The 1,2,3-triazoles were properly purified, partially characterized and the
structure elucidation of the new compounds was obtained by the aid of 1H,13C NMR,
and Mass spectrometry (Supplementary Figure - S1).
HO
HO
CH3I or C3H7Br
N
n
N
CuSO4.5H2O
sodium ascorbat
HO
n
X-
+
N
N
N
N
MeCN
EtOH/H2O 1:1,
t.a., 48h,
yield 76-84%
R
80ºC, 24 h
yield 75-90%
N3
n
1 - Amberlite OH
2 - AcOH
yield 78-97%
n = 2, 4, 8
X = I, Br
R= CH3, CH2CH2CH3
HO
CH3COO-
N
N
n
+
N
R
Scheme 1. Synthesis of 1,4-disubstituted-1,2,3-triazole derivatives and their salts.
2.2 Biological essays
2.2.1 In vitro antileishmanial activity and mammalian cytotoxicity of dhmtAc
5

An initial screening with twelve non-classical bioisosteres of miltefosine on
murine macrophages and promastigotes of L. amazonensis was done. SAR studies
showed that among the salts 1,4-disubstituted-1,2,3-triazole derivatives, only
compounds containing then carbons atoms linked to N-1 (dhmtI, dhmtAc, dhptBr and
dhptAc) exhibited activity against promastigotes of L. amazonensis (IC50 values of 14.3
µM, 2.6 µM, 0.70 µM and 0.73 µM, respectively). All other compounds did not show
antileishmanial activity at maximum concentration tested (IC50 > 100.0 µM). Among
the four active compounds, two compounds dhptBr and dhptAc, containing a propyl
group linked to N-3, showed a strong toxic effect on macrophages (CC50 values of
0.0039 µM and 4.6 µM, respectively). Finally, the compound dhmtAc, containing the
acetate anion showed to be 5.5 times more active against promastigotes of L.
amazonensis than the compound dhmtI, possessing the iodide anion. So, this compound
was chosen for more studies about its antileishmanial activity and mechanism of action.
On promastigote forms, the proliferative insect vector-borne stage of
Leishmania, the compound dhmtAc showed high activity with IC50 values below 16.0
µM (2.6, 3.3 and 15.6 µM, against L. amazonensis-WT, L. amazonensis-GFP and L.
amazonensis-RFP, respectively) (Table 1). Next, we tested the effect of compound on
intracellular amastigotes, which are the pathogenic stage of the parasite in mammalian
host. Our results showed that sensitivity to the compound dhmtAc among various
Leishmania used in this work was uniform. In addition, the antileishmanial activity was
very expressive, with IC50 values near or similar to the 1.0 µM (0.9, 0.8 and 1.0 µM
against L. amazonensis-WT, L. amazonensis-GFP and L. amazonensis-RFP,
respectively), being more active than miltefosine, a control standard drug. The IC50
values for miltefosine were 4.2, 6.6 and 12.7 µM, for L. amazonensis-WT, L.
amazonensis-GFP and L. amazonensis-RFP, respectively. These are promising results,
given that the compound dhmtAc proved to be markedly more potent that miltefosine
against both proliferative stages of this parasite.
Table 1 also shows that the compound dhmtAc presented a low cytotoxic effect
on murine macrophages with CC50 of 115.9 µM. The selectivity index (SI) was also
calculated and the compound was much more destructive to the parasitic form than to
the macrophages, exhibiting an appreciable SI of 128.8, 144.9 and 115.9 for L.
amazonensis-WT, L. amazonensis-GFP and L. amazonensis-RFP, respectively.
6

Table 1: Effect of dhmtAc on L. amazonensis, murine macrophages and selectivity
index.
L. amazonensis (IC50 µM)a
Macroph-
ages
Selectivity index (SI)#
Promastigotes
WTc GFPd
Amastigotes
RFPe
15.6
0.1
WTc
0.9
GFPd
0.8
0.1
RFPe (CC50µM)b
1.0
WTc
128.8
GFPd
144.9
RFPe
115.9
dhmtAc
2.6
0.1
3.3
0.7
115.9
1.1
0.6
0.5
Miltefosi-
ne*
22.0 13.2 0. 18.9
0.5 0.1
4.2
0.2
6.6
1.8
12.7
0.9
143.5
9.1
34.2
21.7
11.3
8
aValues of inhibitory concentration of 50% of parasites (IC50). bValues of cytotoxic concentration of 50% of
macrophages (CC50). cWT (L. amazonensis-Wild type; IFLA/BR/1967/PH8 strain); dGFP (L. amazonensis
transfected with Green Fluorescent Protein; WHOM/BR/75/Josefa strain); eRFP (L. amazonensis transfected with
Red Fluorescent Protein; IFLA/BR/1967/PH8 strain). *Miltefosine was used as reference drug. The data are
expressed as the means from three experiments performed in duplicate. #Selectivity Index (SI) was calculated as the
ratio between the CC50 for macrophages and the IC50 against intracellular amastigotes.
To determine whether the inhibitory effect in multiplication on promastigotes
and intracellular amastigotes of L. amazonensis induced by dhmtAc was reversible, the
cells were treated with different concentration of this compound. Promastigotes were
treated with 20.0, 10.0, 5.0 or 2.5 µM compound (corresponding to 8, 4, 2 and 1 times
of IC50 value) for 72 h, and after this time the cells were washed and reincubated for
another 72 h without the compound dhmtAc. The results showed that promastigotes
treated with 20.0, 10.0 and 5.0 µM dhmtAc presented low proliferate capacity when the
compound was removed from the culture in the time analyzed, which reflected in their
interruption of cell proliferation and reduced re-adaptation to the medium conditions
(Figure 2A). For intramacrophage amastigotes, the infected macrophages were treated
for 72 h with compound dhmtAc at 3.6, 1.8 and 0.9 µM (corresponding to 4, 2 and 1
times of IC50 value) and after this time, the cells were washed, placed in a medium
without the compound for 72 h and we investigated the ability of these intracellular
stages to differentiate into promastigotes. As shown in the Figure 2B, the treatment with
dhmtAc at concentrations of 3.6; 1.8 and 0.9 µM reduced the capacity of the remaining
amastigotes to differentiate into promastigotes by 83.1, 69.0 and 61.9%, respectively, as
compared to the untreated parasites.
7

80
60
40
20
0
A
B
72 h of treatment
72 h subculture
40
30
20
10
0
**
**
*
*
**
**
**
¨
**
**
***
dhmtAc
dhmtAc
Figure 2: Reversibility of the effect of dhmtAc on proliferation of promastigotes and intracellular
amastigotes of L. amazonensis. (A) Promastigote forms of L. amazonensis were treated with the
indicated concentrations of dhmtAc for 72 h at 25 ºC. Parasite were counted, washed and incubated for
more 72 h at 25 ºC without the compound. (B) After 72 h of the treatment, the infected macrophages were
washed twice and incubated with Warren’s medium and 10% fetal bovine serum at 25 ºC for more 72 h
and the promastigotes were counted. P<0.001 (**), significant difference compared with the negative
control (NC) The data were expressed as the means of three different experiments performed in duplicate.
To test the safety of the compound on human erythrocytes, the ability of
dhmtAc to lyse erythrocytes was also assessed. The treatment did not cause significant
damage to human erythrocytes, exhibiting hemolysis levels of less than 5% at all
concentrations tested (Supplementary Figure - S2).
100
80
60
40
20
***
0
Figure S2: Cytotoxicity of dhmtAc against human erythrocytes. To evaluate the cytotoxicity to
human erythrocytes, cells were exposed to the compound dhmtAc as described in materials and methods.
NC (negative control). Saponine (1%) was used as positive control. P< 0.0001 (***), significant
difference compared with the negative control. The data were expressed as the means of three different
experiments performed in triplicate.
Since a significant effect of the non-classical bioisotere of miltefosine on L.
amazonensis was observed, our next question was whether dhmtAc could also trigger
8

the programmed cell death of parasites, similar to that observed for miltefosine 7, 8, 12
As compound dhmtAc is a new drug, preliminary studies were performed by using
promastigote forms of L. amazonensis-WT.
.
2.2.2 DhmtAc induces collapses of mitochondrial membrane potential
The mitochondrial function of the parasites was first analyzed by the
determination of mitochondrial membrane potential (∆ψm) using the fluorescent marker
JC-1 19. Fluorometric data presented in Figure 3 illustrate that the treatment of parasites
with the compound for 24 h resulted in a marked reduction in the ∆ψm at all
concentrations evaluated. In this sense, promastigotes treated with dhmtAc at 20.0,
10.0, 5.0 or 2.5 µM was capable of reducing the ∆ψm at all concentrations (41.5, 32.5,
20.0 and 37.5%, respectively). Promastigotes treated with FCCP, a classic protonophore
uncoupler, exhibited a strong decrease of the ∆ψm (87.2%).
100
***
80
***
***
***
60
40
20
***
0
dhmtAc
Figure 3: Changes in the ∆ψm of L. amazonensis promastigotes following treatment with dhmtAc.
Parasites were untreated (negative control) or treated with compound at 2.5, 5.0, 10.0 or 20.0 µM and
after 24 h were probed with JC-1. Fluorescence was measured fluorometrically. Negative control was
regarded as 100% and the results expressed as a percentage in relation of the control. FCCP (20.0 µM)
was used as positive control. P< 0.0001 (***), significant difference compared with the negative control.
The data were expressed as the means of three different experiments performed in triplicate.
2.2.3 DhmtAc increases total reactive oxygen species (ROS) in promastigotes of L.
amazonensis
The alterations of ∆ψm induced in Leishmania by a variety of molecules has
been associated with the generation of ROS, which triggers damage to the components
of the electron transport chain, collapses mitochondrial functionality and culminates in
9

apoptosis-like cell death 20. Figure 4 shows a significant increase in the ROS production
at all of the concentrations tested compared with untreated parasites (negative control).
The treatment with 20.0, 10.0, 5.0 and 2.5 µM caused significant increases in the ROS
levels of 75.5, 54.0, 40.7 and 38.4%, respectively. Promastigotes treated with
miltefosine (22.0 µM), used as a positive control, also increased the ROS production of
66.7%.
100
***
80
***
***
60
**
***
40
20
0
dhmtAc
Figure 4: ROS production in L. amazonensis promastigote forms treated with dhmtAc. Parasites
were untreated (negative control-NC) or treated with the compound at 2.5, 5.0, 10.0 and 20.0 µM for 72
h, and after this time the cells were probed with H2DCFDA. Miltefosine (22.0 µM) was used as positive
control. Fluorescence was measured fluorometrically and the data were expressed as the fluorescence
intensity in arbitrary units (A.U.) of means of three independent experiments. P< 0.0001 (***) and 0.001
(**), significant difference compared with the negative control.
2.2.4 Analysis of lipid bodies accumulation in promastigotes of L. amazonensis treated
with dhmtAc
Considering the effect of compound dhmtAc on the induction of mitochondrial
stress in promastigotes by (a) reduction of membrane potential and, (b) increase of ROS
generation, we determined whether this compound also induces the formation of lipid
inclusions, which is strongly related to mitochondrial dysfunction 21, 22. To evaluate the
effect of dhmtAc on the accumulation of neutral lipid bodies, we performed
fluorometric assay by using the Nile Red stain. Nile red is a fluorescent compound used
to localize and quantify lipids, particularly neutral lipid droplets within cells 23.
Fluorimetry analysis indicated that treatment with the compound in the highest
concentrations tested (20.0 and 10.0 µM) for 24 h was able to induce a significant
increase in the accumulation of lipid bodies (2.5 and 1.9 times, respectively), compared
with the untreated parasites. On the other hand, the treatment with 5.0 and 2.5 µM
10

compound did not show significant alteration in the formation of lipid inclusions
(Figure 5).
15000
***
**
10000
5000
0
dhmtAc
Figure 5: Lipid inclusions in L. amazonensis promastigote forms treated with compound dhmtAc.
Parasites were untreated (negative control-NC) or treated with dhmtAc at 2.5, 5.0, 10.0 or 20.0 µM and
after 24 h were probed with Nile Red. Fluorescence was measured fluorometrically and the data were
expressed as the fluorescence intensity in arbitrary units (A.U.) of three independent experiments. P<
0.0001 (***) and 0.001 (**) significant difference compared with the negative control. Data were
expressed as the means of three different experiments performed in triplicate.
2.2.5 Effect of dhmtAc on plasma membrane permeability of L. amazonensis
Plasma membrane rupture is a key event of necrotic cell death 24. In order to
investigate whether the strong mitochondrial collapse marked by loss of ∆Ψm could be
associated with the ability of compound to alter the plasma membrane permeability of
parasite, we evaluated the internalization of the fluorescent dye PI into the cytoplasm of
promastigotes previously treated with dhmtAc (20.0, 10.0, 5.0 and 2.5 µM) for 24 h.
Figure 6 shows that the intensity of PI fluorescence was higher at the maximum
concentration used (20.0 µM) than in the untreated cells, indicating permeability on the
parasite plasma membrane. All others concentrations tested caused no interference on
the percentage of PI-labelled parasites and probably did not undergo a necrotic process.
The positive control (parasites heated) induced strong permeabilization of the parasite
membrane (Figure 6).
11

80000
60000
40000
20000
0
***
**
dhmtAc
Figure 6: Cell membrane integrity of L. amazonensis promastigote forms treated with dhmtAc.
Parasites were untreated (negative control) or treated with compound dhmtAc at 2.5, 5.0, 10.0 or 20.0
µM for 24 h and stained with PI. Fluorescence was measured fluorometrically and the data were
expressed as the fluorescence intensity in arbitrary units (A.U.) of means of three independent
experiments. P< 0.0001 (***) and P< 0.001 (**), significant difference compared with the negative
control. Positive control: cells heated at 65 ºC for 10 min.
2.2.6 DhmtAc induces phosphatidylserine exposure in promastigotes of L. amazonensis
Apoptosis is marked by biochemical alterations, including phosphatidylserine
exposure to the outerleaflet of the plasma membrane. Annexin V, a Ca+2-dependent
phospholipid-binding protein with an affinity for phosphatidylserine, is routinely used
to label the externalization of phosphatidylserine. Since annexin V can also label
necrotic cells, a PI stain was used to differentiate among apoptotic cells (annexin
V+/PI), necrotic cells (annexin V-/PI+) and normal cells (annexin V-/PI-) 25. As shown in
Figure 7, a significant percentage (> 28%) of promastigote forms that were treated for
24 h with dhmtAc were annexin-V positive, compared with only 5.3% in the untreated
parasites (negative control), indicating phosphatidylserine exposure. The histogram
analysis also showed that promastigotes treated at 20.0 and 10.0 µM were double-
stained with annexin V and PI (16.9 and 11.2%, respectively). Parasites treated with
miltefosine (44.0 µM), used as a positive control, exhibited similar behavior:
promastigotes annexin V-positive (53.2%); double-stained with annexin V and PI
(12.2%) and only PI-positive (7.1%).
12

Figure 7: Phosphatidylserine exposure on the cell membrane in promastigote forms of L.
amazonensis treated with dhmtAc. Promastigotes were incubated with 2.5, 5.0, 10.0 or 20 µM dhmtAc
for 24 h. After, the cells were stained with the fluorescent probes annexin V-FITC and PI and analyzed by
flow cytometer. Promastigotes treated with miltefosine (44.0 µM), were used as a positive control.
Histograms are representative of three independent experiments, with 10,000 parasites analyzed. The
percentage of annexin V-positive cells are shown in the upper and lower right quadrants. The percentage
of PI-positive cells are shown in the upper and lower left quadrants.
2.2.7 DhmtAc decreases cell volume of parasite
Morphological changes were evaluated by optical microscopy. The untreated
parasites exhibited typical features of the promastigote forms, with an elongated cell
body and a free flagellum. In contrast, parasites that were treated with 2.5, 5.0, 10.0 and
20.0 µM dhmtAc exhibited an altered shape, a size reduction, and a rounded cell body
(Supplementary Figure - S3).
13

Figure S3: Morphological analysis of promastigotes of L. amazonensis treated with dhmtAc.
Promastigotes were untreated or treated with 5.0, 10.0 and 20.0 µM dhmtAc for 24 h and were stained
with Giemsa. (A) Untreated parasites; (B) Promastigotes treated with 20 µM dhmtAc; (C) Promastigotes
treated with 10.0 µM dhmtAc; (D) Promastigotes treated with 5.0 µM dhmtAc. Scale bars=10 µm.
2.2.8 DhmtAc induces DNA fragmentation in promastigotes of L. amazonensis
DNA fragmentation is a typical hallmark of cells undergoing the final outcome
of the apoptosis-like death and is strongly related to the oxidative imbalance 25. Thus,
the TUNEL assay was performed in order to verify whether the compound was able to
induce nuclear DNA and/or mitochondrial DNA fragmentation in promastigotes of L.
amazonensis. Promastigotes that were treated with the compound dhmtAc at 5.0 and
10.0 µM for 24 h exhibited bright fluorescence, indicating DNA double-strand ruptures
compared with untreated parasites (Figure 8). Bright fluorescence was also observed
with the promastigote treated with the reference compound, miltefosine (22.0 µM).
DAPI fluorescence revealed nuclei and kinetoplasts in both untreated control and
treated promastigotes with dhmtAc and miltefosine.
14

Figure 8: DNA fragmentation in promastigote forms of L. amazonensis. Parasites were treated with the
compound dhmtAc (5.0 and 10.0 µM) for 24 h and stained with TUNEL kit and examined by
fluorescence microscopy at 100X. Control group (untreated parasites). Promastigotes incubated with
Miltefosine at 22.0 µM were utilized as positive control. Green fluorescence indicates DNA
fragmentation. Blue fluorescence of DAPI show nuclei and kinetoplasts in both control and treated
promastigotes. Merged images revealed the localization of the nuclei and kinetoplasts inside the parasites.
Scale bars=10 µm.
15

2.2.9 Promastigotes treated with dhmtAc did not exhibited increase of the autophagic
vacuoles
Finally, to evaluate the hypothesis that treatment with compound can also induce
autophagy death of the parasite, promastigotes treated with the compound dhmtAc
were labeled with MDC, a selective marker for the presence of autophagosomes 26.
Promastigotes that were treated with dhmtAc (2.5, 5.0 and 10.0 µM) displayed a non-
significant increase in MDC fluorescence in relation to the untreated parasites.
Interestingly, the fluorescence by parasites treated with compound at 20.0 µM was
significantly lower (49.8%) than that observed in untreated parasites (Supplementary
Figure - S4). These results show that treatment with compound did not result in the
formation of autophagic vacuoles in L. amazonensis promastigotes.
15
10
*
5
0
dhmtAc
Figure S4: Evaluation of autophagic vacuoles by MDC labeling of L. amazonensis promastigotes
treated with compound dhmtAc. Parasites were untreated (negative control-NC) or treated with
compound dhmtAc at 2.5, 5.0, 10.0 or 20.0 µM for 24 h and stained with MDC. Reading was performed
in a microplate reader using wavelengths of 335 and 460 nm for excitation and emission, respectively.
The data were expressed as the fluorescence intensity in arbitrary units (A.U.) of means of three
independent experiments. P< 0.01 (*) significant difference compared with the negative control.
3. Discussion
In this work, we show that dhmtAc, a non-classical bioisotere of miltefosine,
showed a strong effect on promastigote and amastigote forms of L. amazonensis, being
more effective than the alkylphosphocholine miltefosine prototype. Miltefosine exhibits
variation in activity against several Leishmania species, laboratory strains, and also
between the same species from different endemic areas 13, 27. Furthermore, the New
World Leishmania species are considered less susceptible to miltefosine action,
16

suggesting that higher doses of miltefosine would be needed to treat patients infected
with Leishmania species prevalent in Brazil 27. Therefore, it was interesting to note that
the compound dhmtAc showed IC50 values near the 1.0 µM against all L. amazonensis
amastigotes tested in this work. As can be noted, amastigotes of L. amazonensis-GFP
(Josefa strain) and L. amazonensis-RFP (PH8 strain) presented IC50 values very close to
the IC50 value of L. amazonensis-WT (PH8 strain), highlighting the
antileishmanialeffect of dhmtAc. Furthermore, dhmtAc exhibited low cytotoxicity for
murine macrophages (CC50 value of 115.9 µM), being much more destructive to the
intracellular parasites of all Leishmania strains analyzed (SI = 115.0) than to the host
cell. These are promising results, given that this compound proved to be more selective
to the amastigotes than miltefosine. For Leishmania sp, an SI > 20 is considered
adequate for subsequent in vivo studies 28. In addition, the ability of dhmtAc to cause
red blood cell lysis was also assessed and this compound did not present a significant
effect on the erythrocyte viability at the highest concentration tested. It is known that
miltefosine has high hemolytic activity and could not be administered intravenously 29.
Preliminary studies about the mechanism of action of the compound dhmtAc
were performed using promastigote forms of L. amazonensis and the mitochondria
organelle was initially the target chosen for the studies. The single mitochondria is vital
for the survival of Leishmania parasite and alterations in this organelle can to induce the
loss of cell viability and represents an important target for drugs 20, 30. In this sense, our
results strongly suggest that dhmtAc induces mitochondrial deregulation with marked
reduction of the ∆ψm and increase in the ROS production. Also, promastigotes of L.
amazonensis treated with this compound exhibited an increase in the intracellular lipid
bodies. Lipid body (LP) formation can be caused by different perturbations to the
parasite cellular functions and LPs are a hallmark of cellular stress can be caused by
mitochondrial dysfunction and constitute a characteristic of apoptotic cell death 21, 22, 31
.
Since we observe in this work that the miltefosine bioisostere affected the
mitochondria, and several works show that miltefosine induces most of the features
associated with metazoan apoptosis in Leishmania sp 7, 8, 32, 33, our next question was
whether the treatment of L. amazonensis with dhmtAc could also be associated with
biochemical features related to apoptosis. We found that dhmtAc was able to induce
nuclear DNA and/or mitochondrial DNA fragmentation using TUNEL staining in
promastigotes, and findings suggest that this compound might also interact with DNA
17

parasite (i.e., by binding to DNA or intercalating DNA), thus leading to structural
changes in DNA, reflected by an increase in the population of cells in the sub-G0/G1
phase (data not shown). DNA fragmentation can disturb the parasite duplication,
leading to cell cycle arrest, culminating in apoptosis 34.
In a parallel step, we studied the effect of dhmtAc on the plasma membrane of
promastigotes of L. amazonensis. The study of the alterations of the plasma membrane
provides us with important data to distinguish between apoptosis and necrosis,
considering the rupture of the plasma membrane as a morphological feature of the
necrosis and the exposure of phosphatidylserine residues on the external leaflet of the
parasite membrane has been associated with apoptosis 24. By using Annexin V/FITC
staining, we demonstrated the exposure of phosphatidylserine residues on the external
leaflet of the parasite membrane after treatment with dhmtAc. Also, parasites treated
with dhmtAc at the maximum concentrations tested (20.0 and 10.0 µM) caused
disruption of the plasma membrane, revealed by PI labeling. Altogether, these results
indicate that the target of this molecule is partially related to the plasma membrane of
the parasites and not exclude the possibility that death occurred through classic necrosis.
De Macedo-Silva et al. 23 related the alterations of lipid composition resulting
from treatment with drugs, which were also revealed here by Nile red accumulation in
treated promastigotes, and showed that they could interfere with plasma membrane
integrity, culminating in parasite death by necrosis. Interestingly, in this work, the
treatment of parasites with miltefosine (44.0 µM) also altered the percentage of cells
labeled with PI, indicating the possibility of necrosis-like death in the later stages. This
result is not surprising and previous studies showed that miltefosine induces necrosis in
Leishmania 8.
Autophagy is another form of programmed cell pathway which has been well
described for trypanosomatids, and involves degradation of unnecessary cellular
components through the actions of lysosomes 35. Although apoptosis and autophagy
should be considered as two distinct processes, they are closely related to mitochondrial
dysfunction associated with loss of membrane potential and an increase in ROS
formation in treated promastigotes 34. Besides these effects observed on the
promastigotes mitochondria, our data clearly demonstrated that dhmtAc did not induce
the formation of autophagic vacuoles in L. amazonensis promastigotes at the time and
concentrations tested, thus ruling out the possibility of cell death by autophagy.
18

Our results also showed that this compound had a strong effect on
intramacrophage amastigotes and the intracellular parasites showed a low capacity
towards reversion of the treatment, reflecting the efficacy of the treated and infected-
macrophages in eliminate these stages of parasite. It has been suggested that miltefosine
presents direct effect on the intracellular parasite since this drug retain their
antileishmanialeffect on immunodeficient mice 10. Since the compound dhmtAc is an
amphiphilic molecule and is positively charged, we hypothesized that it could interact
with both negatively charged proteins and membrane phospholipids and be able to cross
membranes, reaching the parasite within the parasitophorous vacuole 36.
It has been reported in literature that the miltefosine unresponsive strain
Leishmania resists formation ROS 37, 38. Regarding this, the new compound dhmtAc
may be seen as an alternative against miltefosine unresponsive strain.
Finally, our findings provide clear evidence that, like miltefosine, dhmtAc also
induces promastigote cell death involving a cascade of biochemical events which
display major hallmarks of apoptosis-like such as mitochondrial depolarization, ROS
production, lipid body accumulation, phosphatidylserine externalization in plasma
membrane and DNA fragmentation. However, necrosis-like death in later stages can
also be involved in the mechanism of action of this miltefosine bioisostere on L.
amazonensis.
4. Conclusions
In this work, we showed the successful rational drug design using the
bioisosterism concept. A new miltefosine bioisostere showed superior activity over the
prototype on L. amazonensis along with an appreciable selectivity index and therefore
makes it a strong candidate for further investigations towards the development of new
antileishmanial drugs. Furthermore, the present data provide insights into the
mechanism of action of this compound. In vivo activity in murine models, as well in
vitro studies with other Leishmania species are now being conducted in order to better
establish the antileishmanial effect of this compound.
5. Experimental
19

5.1 Chemicals
Infrared (FT-IR) spectra were recorded with an Alpha Bruker FT-IR Eco-ATR
spectrometer, in the region 4000 - 600 cm−1. It was used a 4 cm−1 spectral resolution and
an average of 24 scans. 1H NMR and 13C NMR spectra were recorded on Bruker Avance
III HD 500 at 500.13 MHz. The chemical shifts (δ) are given in parts per million (ppm)
relative to tetramethylsilane. Spectra were acquired in DMSO-d6, CD3OD and CDCl3.
High resolution mass spectra were obtained with a Bruker MicrOTOF II ESI mass
spectrometer. The mass of the molecular ion was identified through analysis by
(MALDI-TOF MS) using a pulsed nitrogen ultraviolet laser (λ = 337 nm) of an AXIMA
Performance MALDI-TOF MS (Shimadzu Biotech). All reagents of analytical grade
were obtained from commercial suppliers and were used without previous purification.
.
5.1.1 General procedures for the preparation of alkyl azides
In a flask, sodium azide (15.8 g, 0.243 mol) in 40 mL of (EtOH) and 40 mL of
deionized water was added. Alkyl halide (1-bromobutane, 1-bromohexane and 1-
bromodecane) (0.243 mol) was added to sodium azide at room temperature with
stirring. The reaction was allowed to proceed at 60°C and monitored by 1H NMR
analysis of aliquots. The alkyl azides were not isolated, following cycloaddition reaction
in situ.
5.1.2 General procedure for the preparation of triazols (bht 39, hht and dht)
Propargyl alcohol (78 mmol), CuSO4.5H2O (3 mmol), and sodium ascorbate (24
mmol) were added in the solution of EtOH/H2O with the alkyl azides (60 mmol). The
reactions were stirred at room temperature for 48 h with monitoring by 1H NMR
analysis of aliquots. Filtration and washing with EtOH afforded 84% of (bht), 76% of
(hht) and 81% of (dht). A brown oil was formed from the reactions (bht and hht) and a
brown solid was formed to the dht.
bht
Yield 84%; 1H NMR (500 MHz, DMSO-d6) δ 0.88 (t, 3H, J= 7.3, CH3), 1.23 (sex, 2H,
J= 7.5, CH3), 1.77 (qui, 2H, J= 7.2, CH2), 4.32 (t, 2H, J= 7.0, CH2), 5.16 (s, 2H,
20

CH2OH), 7.95 (s, 1H, H-triazole); 13C NMR (125 MHz, DMSO- d6) δ 13.3, 19.1, 31.8,
48.9, 55.0, 122.6, 147.9; IR νmax (cm-1): 3322, 2955, 1638, 1460.
hht
Yield 76%; 1H NMR (500 MHz, CDCl3) δ 0.88 (t, 3H, J= 6.9, CH3), 1.23 (m, 6H), 1.90
(qui, 2H, J= 7.0, CH2), 4.34 (t, 2H, J= 7.2, CH2), 4.78 (s, 2H, CH2OH), 7,62 (s, 1H, H-
triazole); 13C NMR (125 MHz, CDCl3) δ 14.1, 22.6, 26.3, 30.4, 31.3, 50.7, 56.5, 122.9,
149.6; IR νmax (cm-1): 3307, 3137, 2925, 1661, 1459.
dht
Yield 81%; 1H NMR (500 MHz, CDCl3) δ 0.89 (t, 3H, J= 7.2, CH3), 1.38 (m, 14H),
1.90 (m, 2H, CH2), 4.41 (t, 2H, J= 7.2, CH2), 4.67 (s, 3H, CH3), 8.14 (s, 1H, H-
triazole); 13C NMR (125 MHz, CDCl3) δ 14.6, 23.8, 27.5, 30.2, 30.5, 30.7, 31.3, 31.8,
33.1, 37.1, 51.8, 56.9, 140.4, 165.0; IR νmax (cm-1): 3326, 2917, 1653, 1463.
5.1.3 General procedure for the preparation halide salts (bhmtI, hhmtI, dhmtI, bhptBr,
hhptBr and dhptBr)
In a flask, a mixture of the 1,4-dissubstituted-1,2,3-triazole (bht, hht and dht)
(50 mmol) and alkyl halide (iodomethane and 1-bromopropane) (200 mmol) was stirred
at 80°C for 24 h in 10 mL of acetonitrile. The reaction mixture was concentrated in
vacuo to afford bhmtI (75%), hhmtI (81%), dhmtI (90%), bhptBr (87%), hhptBr
(88%) and dhptBr (82%). A brown oil was formed from the reactions, except for the
dhmtI (brown solid).
bhmtI
Yield 75%; 1H NMR (500 MHz, CD3OD) δ 1.00 (t, 3H, J= 7.30, CH3), 1,43 (sextet, 2H,
J= 7.30, CH2), 2.01 (quintet, 2H, J= 7.3, CH2), 4.32 (s, 3H, CH3), 4.64 (t, 2H, J= 7.20,
CH2), 4.87 (s, 2H, CH2OH), 8.70 (s, 1H, H-triazole); 13C NMR (125 MHz, CD3OD) δ
13.8, 20.5, 32.4, 39.0, 53.9, 54.9, 129.9, 145.4; MALDI-TOF MS m/z, calcd for [M]+
170.1293, found 170.1280; IR νmax (cm-1): 3282, 3084, 2955, 1581, 1454.
hhmtI
21

Yield 81%; 1H NMR (500 MHz, CDCl3) δ 0.89 (t, 3H, J= 7.20, CH3), 1.34 (m, 6H),
2.01 (quintet, 2H, J= 7.30, CH2), 4.39 (s, 3H, CH3), 4.64 (t, 2H, J= 7.30, CH2), 5.04 (s,
2H, CH2OH), 8.95 (s, 1H, H-triazole); 13C NMR (125 MHz, CDCl3) δ 14.1, 22.5, 26.0,
29.5, 31.1, 39.7, 53.0, 54.6, 130.0, 143.9; MALDI-TOF MS m/z, calcd for [M]+
198.1606, found 198.1613; IR νmax (cm-1): 3281, 3078, 2924, 1582, 1452.
dhmtI
Yield 90%; 1H NMR (300 MHz, CDCl3) δ 0.82 (t, 3H, J= 7.25, CH3), 1.27 (m, 14H),
1.97 (m, 2H, CH2), 4.34 (s, 3H, CH3), 4.58 (t, 2H, J= 7.2, CH2), 4.98 (s, 2H, CH2OH),
8.91 (s, 1H, H-triazole); 13C NMR (75 MHz, CDCl3) δ 14.2, 22.7, 26.3, 28.9, 29.3, 29.4,
29.5, 29.6, 31.9, 39.7, 52.9, 54.5, 129.9, 143.8; MALDI-TOF MS m/z, calcd for [M]+
254.2232, found 254.2228; IR νmax (cm-1): 3302, 2918, 1592, 1465.
bhptBr
1
Yield 87%; H NMR (500 MHz, CDCl3) δ 0.98 (t, 3H, J= 7.30, CH3), 1.03 (t, 3H, J=
7.40, CH3), 1.42 (sex, 2H, J= 7.50, CH2), 2.02 (qui, 2H, J= 7.50, CH2), 2.07 (sex, 2H,
J= 7.30, CH2), 4.64 (t, 2H, J= 7.30, CH2), 4.70 (t, 2H, J= 7.30, CH2), 4.97 (s, 2H,
CH2OH), 9.18 (s, 1H, H-triazole); 13C NMR (125 MHz, CDCl3) δ 11.0, 13.4, 19.5, 22.7,
31.3, 52.4, 53.8, 54.0, 130.0, 143.7; MALDI-TOF MS m/z, calcd for [M]+ 198.1606,
found 198.1600; IR νmax (cm-1): 3236, 2958, 1579, 1460.
hhptBr
Yield 88%; 1H NMR (500 MHz, CDCl3) δ 0.88 (t, 3H, J= 7.0, CH3), 1.03 (t, 3H, J=
7.3, CH3), 1.35 (m, 6H, 3CH2), 2.05 (m, 4H, 2CH2), 4.62 (t, 2H, J= 7.3, CH2), 4.67 (t,
2H, J= 7.3, CH2), 4.98 (s, 2H, CH2OH), 9.15 (s, 1H, H-triazole); 13C NMR (125 MHz,
CDCl3) δ 11.0, 14.1, 22.5, 22.7, 26.0, 29.5, 31.1, 52.6, 53.9, 54.4, 130.1, 143.9;
MALDI-TOF MS m/z, calcd for [M]+ 226.1918, found 226.1910; IR νmax (cm-1):
3297, 2928, 1630, 1459.
dhptBr
Yield 82%; 1H NMR (500 MHz, CDCl3) δ 0.88 (t, 3H, J= 7.5, CH3), 1.03 (t, 3H, J=
7.5, CH3), 1.25 (m, 16H), 2.03 (m, 2H, CH2), 4.63 (m, 4H, 2CH2), 4.97 (s, 2H,
CH2OH), 9.12 (s, 1H, H-triazole); 13C NMR (125 MHz, CDCl3) δ 10.9, 14.1, 22.7, 22.9,
22

26.2, 28.9, 29.1, 29.3, 29.4, 29.5, 29.6, 31.9, 52.5, 53.8, 54.3, 130.0, 143.7; MALDI-
TOF MS m/z, calcd for [M]+ 282.2545, found 282.2541; IR νmax (cm-1): 3335, 2924,
1626, 1465.
5.1.4 General procedure for the preparation acetate salts (bhmtAc, hhmtAc, dhmtAc,
bhptAc, hhptAc and dhptAc)
Ethanolic solution of the salts of 1,2,3-triazolium cations containing hydroxide
as anion were prepared from 1,2,3-triazolium halide (bhmtI, hhmtI, dhmtI, bhptBr,
hhptBr and dhptBr) using anion exchange resin AMBERLITE IRA400OH (Sigma).
To these solutions were added equimolar amounts of acetic acid in ice bath. The
mixture was stirred for 16 h at room temperature. Then solvent was evaporated to give
the product as yellow oil. bhmtAc (78%), hhmtAc (96%), dhmtAc (85%), bhptAc
(97%), hhptAc (91%) and dhptAc (86%).
bhmtAc
1
Yield 78%; H NMR (500 MHz, CDCl3) δ 0.97 (t, 3H, J= 7.30, CH3), 1.40 (sextet, 2H,
J= 7.50, CH2), 1.87 (s, 3H, CH3COO-), 1.98 (qui, 2H, J= 7,30, CH2), 4.34 (s, 3H, CH3),
4.60 (t, 2H, J= 7.40, CH2), 4.93 (s, 2H, CH2OH), 9.23 (s, 1H, H-triazole); 13C NMR
(125 MHz, CDCl3) δ 13.5, 19.6, 24.9, 31.4, 38.4, 52.5, 53.8, 130.2, 145.2, 178.1;
MALDI-TOF MS m/z, calcd for [M]+ 170.1293, found 170.1288; IR νmax (cm-1):
3375; 3080; 2957; 1566; 1387.
hhmtAc
1
Yield 96%; H NMR (500 MHz, CDCl3) δ 0.88 (t, 3H, J= 7.2, CH3), 1.32 (m, 6H,
3CH2), 1.86 (s, 3H, CH3COO-), 1.97 (qui, 2H, J= 7,2, CH2), 4.31 (s, 3H, CH3), 4.60 (t,
2H, J= 7.3, CH2), 4.90 (s, 2H, CH2OH), 9.03 (s, 1H, H-triazole); 13C NMR (125 MHz,
CDCl3) δ 14.1, 22.5, 25.1, 26.0, 29.4, 31.4, 38.2, 52.4, 54,0, 130.0, 145.0, 177.7;
MALDI-TOF MS m/z, calcd for [M]+ 198.1606, found 198.1605; IR νmax (cm-1):
3358; 3095; 2925; 1565; 1391.
dhmtAc
Yield 85%; 1H NMR (500 MHz, CDCl3) δ 0.88 (t, 3H, J= 7.0, CH3), 1.31 (m, 14H),
1.90 (s, 3H, CH3COO-), 1.98 (qui, 2H, J= 7.4, CH2), 4.33 (s, 3H, CH3), 4.56 (t, 2H, J=
23

7.4, CH2), 4.91 (s, 2H, CH2OH), 9.20 (s, 1H, H-triazole); 13C NMR (125 MHz, CDCl3)
δ 14.1, 22.6, 24.4, 26.2, 28.8, 29.2, 29.3, 29.4, 29.4, 31.8, 38.1, 52.2, 53.8, 130.1, 145.2,
177.5; MALDI-TOF MS m/z, calcd for [M]+ 254.2232, found 254.2241; IR νmax (cm-
1): 3363, 3090,2923, 1566, 1390.
bhptAc
1
Yield 97%; H NMR (500 MHz, CDCl3) δ 0.97 (t, 3H, J= 7.3, CH3), 1.01 (t, 3H, J=
7.5, CH3), 1.38 (sex, 2H, J= 7.4, CH2), 1.90 (s, 3H, CH3COO-), 1.99 (qui, 2H, J= 7.4,
CH2), 2.03 (sex, 2H, J= 7.3, CH2), 4.57 (t, 2H, J= 7.4, CH2), 4.60 (t, 2H, J= 7.3, CH2),
4.92 (s, 2H, CH2OH), 9.36 (s, 1H, H-triazole); 13C NMR (125 MHz, CDCl3) δ 10.8,
13.3, 19.4, 22.6, 24.7, 31.2, 52.2, 53.1, 53.6, 130.4, 145.1, 177,7; MALDI-TOF MS
m/z, calcd for [M]+ 198.1606, found 198.1615; IR νmax (cm-1): 3372, 3088, 2961,
1566, 1388.
hhptAc
Yield 91%; 1H NMR (500 MHz, CDCl3) δ 0.88 (t, 3H, J= 7.0, CH3), 1.01 (t, 3H, J=
7.2, CH3), 1.31 (m, 6H, 3CH2), 1.89 (s, 3H, CH3COO-), 2.03 (m, 4H, 2CH2), 4.57 (t,
2H, J= 7.5, CH2), 4.59 (t, 2H, J= 7.2, CH2), 4.91 (s, 2H, CH2OH), 9.24 (s, 1H, H-
triazole); 13C NMR (125 MHz, CDCl3) δ 10.9, 14.0, 22.5, 22.7, 24.8, 25.9, 29.4, 31.1,
52.4, 53.3, 54.0, 130.4, 145.0, 177.8; MALDI-TOF MS m/z, calcd for [M]+ 226.1918,
found 226.1928; IR νmax (cm-1): 3350, 3111, 2927, 1565, 1391.
dhptAc
Yield 86%; 1H NMR (500 MHz, CDCl3) δ 0.86 (t, 3H, J= 7.1, CH3), 0.99 (t, 3H, J=
7.3, CH3), 1.23 (m, 14H, 7CH2), 1.86 (s, 3H, CH3COO-), 1.99 (m, 4H, 2CH2), 4.55 (m,
4H, 2CH2), 4.88 (s, 2H, CH2OH), 9.09 (s, 1H, H-triazole); 13C NMR (125 MHz, CDCl3)
δ 11.0, 14.3, 22.7, 22.8, 24.5, 26.3, 29.0, 29.4, 29.5, 29.5, 29.6, 32.0, 52.4, 53.3, 54.1,
130.3, 144.8, 178,0; MALDI-TOF MS m/z, calcd for [M]+ 282.2545, found 282.2559.
5.2 Biological Assays
5.2.1 Reagents
24

Miltefosine was supplied by Cayman Chemical Company (Michigan, USA).
Propidium Iodide (PI), 9-diethylamino-5H-benzo[alpha] phenoxazine-5-one (Nile Red),
monodansylcadaverin (MDC), poly-L-lysine, Hank´s Balanced Sal Solution (HBSS),
carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), geneticin (G418),
dimethylsulfoxide (DMSO), hemin, saponin, folic acid and 3-[4,5-dimethylthiazol-2-
yl]-2,5-100 diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St.
Louis, MO, USA). Fetal bovine serum (FBS), RPMI 1640 medium, penicillin G and
streptomycin were purchased from Cultilab (Campinas, São Paulo, Brazil); brain heart
infusion (BHI) was purchased from Himedia (Mumbai, Indian). The terminal
deoxyribonucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) kit
was supplied by Promega, Madison, WI, USA. Annexin V-FITC was obtained from
Invitrogen (Eugene, OR, USA). 4',6-Diamidino-2-Phenylindole, Dihydrochloride (Dapi)
was acquired from Santa Cruz Biotechnology (Dallas, TX, USA).
5.2.2 Parasite culture
Promastigotes of L. amazonensis wild-type (IFLA/BR/1967/PH8), L.
amazonensis (IFLA/BR/1967/PH8) transfected with the gene of red fluorescent protein
(RFP) 40 and L. amazonensis (WHOM/BR/75/Josefa) transfected with the gene of green
fluorescent protein (GFP) 41 were used. Leishmania amazonensis-WT, L. amazonensis-
GFP and L. amazonensis-RFP were used in antipromastigote and antiamastigote assays,
and L. amazonensis-WT was used in the study of action mechanism. The promastigotes
of L. amazonensis-WT, L. amazonensis-GFP and L. amazonensis-RFP were cultured as
previously reported 42,43. The parasites were routinely isolated from cutaneous lesions of
infected BALB/c mice and passaged as promastigote forms in culture. All protocols
were approved by the Ethical Committee for Animal Research of Federal University of
Juiz de Fora (#055/2013 -CEUA).
5.2.3 Antipromastigote assay
The promastigotes of L. amazonensis-WT, L. amazonensis-GFP and L.
amazonensis-RFP (2x106 cells/mL) from logarithmic phase culture were not exposed
(negative control) or exposed to an increasing concentration of the compound (1.6-
100.0 µM) for 72 h at 25 °C in a BOD incubator. The viability of the promastigotes was
determined by using MTT colorimetric assay 42,43. The results were expressed as the
25

concentration inhibiting parasite growth by 50% (IC50). Miltefosine (6.2-100.0 µM) was
used as an antileishmanial reference drug. Results were obtained from three independent
experiments performed in duplicate.
5.2.4 Reversibility effect of dhmtAc on promastigote proliferation
To determine whether the antipromastigote effect of dhmtAc was reversible,
promastigotes of L. amazonensis-WT were incubated with this compound (2.5-20.0
µM) for 72 h at 25 ºC in BOD incubator, and the number of parasites was then counted.
The parasites were centrifuged, washed twice in PBS and the number of cells was
adjusted (10x106 cells/mL) and incubated with Warren’s medium plus 10% SBF
without the compound dhmtAc. After 72 h at 25 ºC in BOD incubator, the number of
viable promastigotes was counted in a Neubauer chamber 44. Results were obtained
from three independent experiments performed in duplicate.
5.2.5 Viability assay against murine macrophages
Peritoneal macrophages (2x106 cells/mL) were obtained from mice in a protocol
already described 45. Adherent macrophages were incubated with increasing
concentrations of dhmtAc (9.4-150.0 µM) or with RPMI 1640 medium for 72 h at 33
ºC and 5% CO2. Viability of the macrophages was determined by MTT assay. The
cytotoxic concentration of the compound to reduce 50% of viable macrophages (CC50)
was obtained from three independent experiments performed in duplicate. The
Selectivity Index (SI) of this compound was calculated as the ratio between the CC50 for
macrophages and the IC50 against intracellular amastigotes 44. All protocols were
approved by the Ethical Committee for Animal Research (#054/2013-CEUA).
5.2.6 Antiamastigote activity
Murine macrophages obtained according to protocol previously described 45,
were infected with promastigotes of L. amazonensis in the stationary growth phase and
L. amazonensis-macrophages were treated with different concentrations of the
compound dhmtAc (0.78-100.0 µM) diluted in sterile deionized H2O. The parasite
burden was evaluated by counting the intracellular amastigotes, uninfected and infected
macrophages (at minimum 200 macrophages) in treated and untreated cultures 45.
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The antiamastigote assays using L. amazonensis-GFP and L. amazonensis-RFP
were performed as previously described 42, 43. The infected macrophages were treated
with different concentrations of the compound dhmtAc (0.78-100.0 µM) and after 72 h
of treatment, the fluorescence intensity of the cultures was measured using a plate-
reader fluorometer (FLx800, BioTek Instruments, Inc., Winooski, VT, USA) set at 540
nm excitation/600 nm emission for L. amazonensis-RFP 43 and at 485 nm excitation/528
nm emission for L. amazonensis-GFP 42. Miltefosine (1.6-25.0 µM) was used as the
reference drug. The results were obtained from three independent experiments
performed in duplicate.
5.2.7 Analysis of the reversibility of the effect of dhmtAc on the intracellular parasite
proliferation
The macrophages infected with L. amazonensis-WT were treated with dhmtAc
(0.45; 0.9; 1.8 and 3.6 µM) for 72 h at 33 ºC in 5% CO2. After this time, the infected
macrophages were washed twice in PBS and incubated with Warren’s medium
supplemented with 10% SBF, without the compound, at 25 ºC in a BOD incubator for
more 72 h. After, the parasite viability was quantified by counting the viable
promastigotes under the microscope 44. Results were obtained from three independent
experiments performed in duplicate. All protocols were approved by the Ethical
Committee for Animal Research of Federal University of Juiz de Fora (#0056/2013-
CEUA).
5.2.8 Erythrocytes lysis assay
The hemolytic activity was determined by incubation of a suspension of fresh
human red blood cells in PBS with different concentrations of dhmtAc (9.4-150.0 µM)
as previously described 46.
5.2.9 Determination of mitochondrial membrane potential
The promastigotes of L. amazonensis-WT (1x107 cells/mL) in the exponential
phase were untreated or treated with different concentrations starting from IC50 of the
compound dhmtAc (2.5, 5.0, 10.0 or 20.0 µM) for 24 h at 25 °C in a BOD incubator.
Cells were harvested, washed, incubated with JC-1 and the fluorescence was
spectrofluorometrically measured (FLx800, BioTek Instruments, Inc., Winooski, VT,
27

USA) at both 485/528 nm and 540/600 nm wavelength 42. The promastigotes treated
with FCCP (20.0 µM) for 30 minutes were utilized as positive control. Relative ∆ψm
value was quantified using the ratio between the reading at 600 nm and the reading at
528 nm 42.
5.2.10 Detection of reactive oxygen species (ROS) production
The detection of ROS production was done by using the fluorescent probe
H2DCFDA 42. Briefy, L. amazonensis-WT promastigotes in the exponential phase were
untreated or treated with compound dhmtAc (2.5, 5.0, 10.0 or 20.0 µM) for 24 h at 25
°C in a BOD incubator. Parasite concentration was adjusted to 2x107 promastigotes in
PBS and H2DCFDA was added, incubated for 30 min in dark at room temperature and
the ROS generation were measured using an excitation and emission wavelengths of
485 and 528 nm, respectively, in a fluorometer microplate reader 42. Promastigotes
incubated with miltefosine (22.0 µM) were used as positive control.
5.2.11 Detection of formation of lipid droplets
Exponential phase L. amazonensis-WT promastigotes (1x107 cells/mL) were
cultured for 24 h in the absence or presence of the compound dhmtAc (2.5, 5.0, 10.0 or
20.0 µM), at 25 ºC in a BOD incubator (Fanem, São Paulo, SP, Brazil). For detection of
lipids droplets, the parasites were incubated with Nile Red and the fluorescence was
47
.
spectrofluorometrically measured
5.2.12 Cell membrane integrity
Promastigotes of L. amazonensis-WT (1x107 cells/mL) in the exponential phase
were untreated or treated with 2.5, 5.0, 10.0 or 20.0 µM dhmtAc for 24 h at 25 °C in
BOD incubator. After washed, the parasites were incubated with PI at 1.0 µg/mL for 15
min in dark at room temperature 42. Fluorescence was spectrofluorometrically measured
(excitation wavelength of 540 nm and an emission wavelength of 600 nm). As positive
control, cells heated at 65°C for 10 min were used45. Alterations in the PI fluorescence
were quantified as the percentage of increase in the fluorescence compared with the
untreated promastigotes.
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5.2.13 Detection of phosphatidylserine exposure by annexin-V-FITC labeling
Exponential phase L. amazonensis-WT promastigotes (1x107 cells/mL) were
cultured for 24 h in the absence or presence of 2.5, 5.0, 10.0 or 20.0 µM dhmtAc , at 25
ºC in a BOD incubator (Fanem, SP, Brazil). Phosphatidylserine exposure was evidenced
using annexin V-FITC and propidium iodide (PI) assay as already described 44.
Promastigotes treated with miltefosine (44.0 µM) were used as positive control. Data
acquisition and analysis were performed using a FACsCanto II flow cytometer (Becton
Dickinson, Rutherford, NJ, USA) equipped with DIVA software (Joseph Trotter,
Scripps Research Institute, La Jolla, CA, USA). A total of 10,000 events were analysed
per sample in the region that was previously established as the one that corresponded to
the parasites.
5.2.14 Evaluation of morphology of promastigotes
Promastigotes of L. amazonensis (1x107 cells/mL) in the exponential phase were
treated with 2.5, 5.0, 10.0 or 20.0 µM dhmtAc for 24 h at 25 °C in BOD incubator.
After, the parasites were harvested, resuspended in PBS, smeared on glass slides, fixed
with absolute ethanol and stained with Giemsa and visualized using an Olympus BX53
microscopy.
5.2.15 DNA Fragmentation Assay
Exponential phase L. amazonensis-WT promastigotes (1x107 cells/mL) were
cultured for 24 h in the absence or presence of 5.0 or 10.0 µM dhmtAc at 25 ºC in a
BOD incubator (Fanem, São Paulo, SP, Brazil). DNA fragmentation was analysed
using a terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling
(TUNEL) apoptosis detection system, as previously reported 48. Promastigotes treated
with miltefosine (22.0 µM) were used as positive control. The slides were observed
using an Olympus BX53 fluorescence microscope, and images were captured with an
Olympus camera DP73.
5.2.16 Evaluation of autophagic vacuoles
Leishmania amazonensis-WT promastigotes (1x107 cells/mL) in the exponential
phase were treated with the compound dhmtAc (2.5, 5.0, 10.0 and 20.0 µM) or
untreated (negative control) for 24 h, at 25 ºC in a BOD incubator (Fanem, SP, Brazil).
29