Discovery and Pharmacological Studies of 4-Hydroxyphenyl-Derived Phosphonium Salts Active in a Mouse Model of Visceral Leishmaniasis

Article abstract of DOI:10.1021/acs.jmedchem.9b00998

We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC₅₀ from 0.04 to 0.28 μM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of four daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant reactive oxygen species production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum, indicating its potential as antileishmanial lead.

Full text of DOI:10.1021/acs.jmedchem.9b00998

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Article
Discovery and pharmacological studies of 4-hydroxyphenyl-derived
phosphonium salts active in a mouse model of visceral leishmaniasis
José Ignacio Manzano, Eduardo jose Cueto diaz, Ana Isabel Olías-Molero, Ana Perea, Tomás
Herraiz, Juan Jose Torrado, José Maria Alunda, Francisco Gamarro, and Christophe Dardonville
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00998 • Publication Date (Web): 08 Nov 2019
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Discovery and pharmacological studies of 4-hydroxyphenyl-derived phosphonium
salts active in a mouse model of visceral leishmaniasis
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José Ignacio Manzanoa‡, Eduardo J. Cueto-Díazb‡, Ana Isabel Olías-Moleroc, Ana Pereaa,
Tomás Herraizd, Juan J. Torradoe, José María Alundac, Francisco Gamarroa*, Christophe
Dardonvilleb*
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a
Instituto de Parasitología y Biomedicina “López Neyra”, IPBLN–CSIC, Parque
Tecnológico de Ciencias de la Salud, 18016 Granada, Spain
b Instituto de Química Médica, IQM–CSIC, Juan de la Cierva 3, E–28006 Madrid, Spain.
c Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense
de Madrid, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain
d Instituto de Ciencia y Tecnología de Alimentos y Nutrición, ICTAN–CSIC, Juan de la
Cierva 3, E–28006 Madrid, Spain.
eDepartamento de Farmacia Galénica y Tecnología Alimentaria, Facultad de Farmacia,
Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain
KEYWORDS. Leishmania donovani, Leishmania infantum, phosphonium salt, in vitro
activity, in vivo activity, pharmacokinetics, mechanism of action.
Abstract
We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in
the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan
parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of
compound
1
[(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium
bromide] in a mouse model of visceral leishmaniasis were established. Compound 1
reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an
oral dosage of 4 daily doses of 1.5 mg/kg. Mode of action studies showed that compound
1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of
Leishmania parasites. Disruption of the energetic metabolism, with a decrease of
intracellular ATP levels as well as mitochondrial depolarization together with a
significant ROS production, contributes to the leishmanicidal effect of 1. Importantly,
this compound was equally effective against antimonials and miltefosine-resistant clinical
isolates of Leishmania infantum indicating its potential as antileishmanial lead.
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Introduction
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Leishmaniasis is a neglected parasitic disease caused by parasites of the genus
Leishmania. The clinical manifestations range from the (often self-healing) cutaneous
form to the deadly visceral form caused by Leishmania donovani and L.infantum. With
an estimated 700,000 to 1 million new cases and some 26,000 to 65,000 deaths occurring
every year, leishmaniasis continues to be a menace in countries across the globe.1 The
epidemiology and spreading of this devastating disease, which was mainly distributed in
tropical and subtropical areas, have been changing in the last years due to environmental,
demographic, human behavioural factors and co-infections.2 The lack of an anti-
Leishmania vaccine and the toxicity of current drugs, combined with the rapid emergence
of drug resistant Leishmania strains, represent major challenges for the control of this
parasitosis. Current antileishmanial drugs are expensive and nonspecific, and usually
require prolonged treatment regimens; as a result, high toxicity and low compliance are
commonly observed. The increasing drug resistance and therapeutic failure reported and
the reduced arsenal of drugs require the development of new compounds and treatment
regimens that are safer, more effective and less vulnerable to the development and
selection of drug resistant strains/isolates.
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The cidal activity of benzyltriphenylphosphonium salts against trypanosomatid parasites
was demonstrated 40 years ago by Kinnamon et al.3-5 However, recently the interest for
the antiparasitic activity of phosphonium salt derivatives rekindled.6, 7 In recent years, we
have synthesized several series of potent antiparasitic (bis)phosphonium salt derivatives
that target the mitochondrion of kinetoplastid parasites Trypanosoma brucei8 and
Leishmania.9 In T. brucei, the trypanocidal action of these compounds occurs by targeting
the mitochondrial FoF1 ATPase10 whereas in Leishmania, succinate dehydrogenase
(complex II) appears to be the main target.9 Lately, we have developed inhibitors of the
trypanosome alternative oxidase (TAO) as potential chemotherapeutic agents against
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African trypanosomes.11,
By applying a mitochondrion-targeting strategy using
lipophilic cations (e.g. triphenylphosphonium salts, TPP) we were able to dramatically
improve drug targeting and trypanocidal activity while retaining potency against the
target protein.13, 14
TAO is not a validated target of Leishmania because the amastigote stage causing the
disease in humans has a fully functional respiratory pathway, in contrast to bloodstream
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form (BSF) trypanosomes which lack the canonical oxidative phosphorylation processes
and rely exclusively on TAO for respiration.15 However, based on the literature reports
and our own experience, we envisaged that our mitochondrion-targeted (TAO inhibitor)
compounds may have chemotherapeutic potential against Leishmania parasites.
Accordingly, we carried out a focused screening of an in-house series of TAO inhibitors
against L. donovani parasites which resulted in the discovery of potent leishmanicides
(Table S1). As TAO is not a validated target in Leishmania, we set about to discover the
mechanism of action (MoA) of these mitochondrion-directed compounds. As the most
active and selective compounds presented potential metabolic instability, new derivatives
were synthesized and tested against promastigote and amastigote forms of L. donovani
(Chart 1).
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We report here the discovery of new phosphonium salts active against L. donovani in
vitro, pilot preclinical pharmacological studies and strong in vivo activity in a mouse
model of visceral leishmaniasis with an exploration of uptake and mechanism of action
of lead compound 1.
RESULTS
Preliminary screening and SAR studies. Thirty TAO inhibitors11, 12, 14 were screened
in vitro against promastigotes and the human-relevant intracellular amastigote forms of
L. donovani HU3, and their cytotoxicity against MRC-5 or THP-1 cells was determined
(Table S1). Eleven cationic compounds showed IC50 < 1 µM against intracellular
amastigotes, among which eight were phosphonium salts (15–18, 23–24, 26, 31) with
selectivity indices (SI) versus mammalian cells ranging from 2 to 27. In general, the
quinolinium salt derivatives were more cytotoxic and only three compounds (10, 11, 32)
presented a fair activity/selectivity profile (IC50 ≈ 0.8 µM, SI = 3.7–5). Remarkably,
compounds with a C14 methylene chain were slightly more selective towards Leishmania
than shorter analogues with C10 or C12 (compare 10/11, 15/16, 29/30). Three
phosphonium hit compounds displaying EC50 < 0.2 µM against promastigotes and
intracellular amastigotes of L. donovani and satisfactory selectivities (SI > 10) were
selected as possible leads for in vivo studies (Table S1 and Chart 1: 16, 17, and 18). Since
the selected compounds contain an ester bond that can be hydrolyzed by serum hydrolases
(e.g. the half-life of compound 16 is 1 h in mouse serum12), which may potentially limit
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their in vivo efficacy, we decided to change the ester bond for a more metabolically stable
keto link between the phenyl ring and the methylene chain (compounds 1–3, Chart 1).
The newly synthesized keto compounds were tested against promastigote and
intracellular amastigote forms of L. donovani (Table 1), and the most promising lead
compound (1) was selected for in vivo assays in a mouse model of visceral leishmaniasis.
Pharmacokinetics, MoA and uptake studies were carried out. Besides, the
trinaphthylphosphonium analogue (4) of compound 1 was synthesized as fluorescent
probe for MoA studies whereas the aliphatic derivative 9 was used as control for SAR
studies (Scheme 1).
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Chart 1. Benzoate hit compounds active in vitro against L. donovani (16–18) and new
keto analogues being studied (1–3)
O
R
Br-
P (CH2)14
Br-
O
R
P
(CH2)14
O
OH
OH
1
2
16
17
: R = OH
: R = CH3
: R = OH
: R = CH3
O
CH3
O
Br-
(CH2)14
CH3
Br-
P
P
(CH2)14
O
N
N
OH
OH
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Phosphonium
Lipophilic
Cation
Methylene
Linker
Benzoate
linkage
4-OH-phenyl
head
Chemistry. The phosphonium salt derivatives 1–4 were synthesized by nucleophilic
substitution of the bromoalkyl precursors 6 with triphenylphosphine (1, 2),
diphenyl‐2‐pyridylphosphine (3), and tris(naphthalen‐1‐yl)phosphane (4), respectively
(Scheme 1).
Compounds 6a and 6b were obtained by acylation of resorcinol and m-cresol with 16-
bromohexadecanoic acid 5, respectively. Compound 9 was synthesized by reaction of 14-
bromotetradecanol 8 with Ph3P in CH3CN at 80 ºC. The 14-bromotetradecanol precursor
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8 was synthesized in 3 steps from tetradecanedioic acid following reported procedures.16,
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Scheme 1. Synthesis of compounds 1–4 and 9
HO
R1
R1
O
HO
(CH2)15 Br
HO
(CH2)15 OH
(CH2)15 Br
i
ii
O
O
HO
5
6a
6b
: R1 = OH
: R1 = CH3
iii or iv
Cmpd
R1
R2
R3
1
2
3
4
OH
CH3
CH3
OH
R1
O
R2
. Br-
(CH2)15
P
R2
N
R3
HO
1
4
-
O
vi
RO
HO
OR
R
O
R = H
R = Me
7
: R = OH
v
vii
iii
8
: R = Br
+
-
9
: R = PPh3Br
aReagents and conditions: (i) HBr 48%, 110 ºC; (ii) BF3-Et2O, 110 ºC; (iii)
triphenylphosphine (for 1 and 2) or diphenyl‐2‐pyridylphosphine (for 3), CH3CN, 80 ºC,
5-18 days; (iv) tris(naphthalen‐1‐yl)phosphane (for 4), DMF, 110 ºC, 5 days; (v) SOCl2,
MeOH, rt; (vi) LiAlH4, THF, reflux; (vii) HBr 48%, cyclohexane, reflux.
Biology
In vitro activity against promastigotes and amastigotes of L. donovani. New keto
derivatives 1–3 inhibited the growth of L. donovani promastigotes and intracellular
amastigotes with submicromolar EC50 values similar to those of the ester counterparts
16–18 (Table 1). Remarkably, the leishmanicidal activity of 1–3 against intracellular
amastigote forms (EC50 = 0.1 µM) was twice as good as the control drug amphotericin B
(AmB) (EC50 = 0.24 µM). The cytotoxicity of 1–3 against THP-1 cells was comparable
or slightly lower than the ester analogues 16–18, resulting in higher selectivity for the
new compounds (SI >20).
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Based on the promising in vitro activity/selectivity profile of the new keto derivatives
against intracellular amastigotes of L. donovani, which is the human-relevant form of the
parasite, compound 1 (EC50 = 90 nM, SI = 31.2) was selected as lead for further
pharmacological studies.
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Table 1. Activity of 4-hydroxyphenyl keto analogues 1–3 and ester counterparts 16–18
in promastigote and intracellular amastigote forms of L. donovani HU3 and cytotoxicity
against THP-1 cellsa
Cmpd
EC50(µM)
SIb
L. donovani
intracellular
amastigotes
L. donovani
promastigotes
THP-1 cells
1
2
0.07 ± 0.02
0.09 ± 0.01
0.28 ± 0.05
3.54 ± 0.36c
0.04 ± 0.01
0.09 ± 0.02
0.18 ± 0.02
0.33 ± 0.04
0.13 ± 0.02
6.51 ± 0.35
0.09 ± 0.02
0.10 ± 0.01
0.10 ± 0.02
1.25 ± 0.22c
0.11 ± 0.02
0.15 ± 0.01
0.12 ± 0.03
0.35 ± 0.05
0.24 ± 0.06
0.91 ± 0.05
2.81 ± 0.35
2.45 ± 0.41
2.11 ± 0.12
12.85 ± 2.30
3.02 ± 0.47
1.84 ± 0.22
2.35 ± 0.38
3.05 ± 0.61
27.85 ± 3.50
26.25 ± 2.58
31.2
24.5
21.1
10.3
27.6
12.3
19.6
8.7
3
4
16
17
18
9
AmBd
MILe
116
28.8
a Parasites and THP-1 cells were grown in the presence of increasing concentrations of compounds. Data
are means ± standard deviations from three independent experiments. b SI= Selectivity Index (EC50 THP-
c
d
1 / EC50 parasite (amastigote forms)). Compound tested in L. infantum. AmB: amphotericin B. e MIL:
Miltefosine.
Activity of compound 1 against resistant clinical isolates of L. infantum. The
increasing therapeutic failure and appearance of drug resistance represents a serious
limitation of current antileishmanial chemotherapy ,18-22 hence we assessed the sensitivity
of compound 1 in both promastigote and intracellular amastigote forms of resistant lines
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of L. infantum obtained from clinical cases.23 No significant differences in sensitivity to
compound 1 were found between the drug sensitive reference line (LEM3049) and L.
infantum LEM 3323 (resistant to SbIII)24, L. infantum LEM 5159 (resistant to
miltefosine)24 and L. infantum LEM 2126 (resistant to miltefosine and paramomycin)25
(Table 2).
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Table 2. Leishmanicidal activity of compound 1 on promastigote and intracellular
amastigote forms of clinical isolates of L. infantuma
EC50(µM)
Promastigotes
Intracellular amastigotes
L. infantum
Cmpd 1
AmB
Cmpd 1
AmB
LEM3049
LEM3323
LEM5159
LLM2126
0.08 ± 0.03
0.10 ± 0.02
0.11 ± 0.02
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0.10 ± 0.02
0.12 ± 0.06
0.15 ± 0.07
0.14 ± 0.02
0.14 ± 0.01
0.17 ± 0.02
0.18 ± 0.02
0.16 ± 0.03
0.16 ± 0.01
a
Parasites were grown as described in Experimental section, in the presence of increasing concentrations
of compounds. L. infantum clinical isolates used: LEM 3049 line as a reference line; LEM 3323 a clinical
line resistant to SbIII; LEM 5159 a clinical line resistant to miltefosine (MIL) and LEM 2126 a clinical line
resistant to MIL and paromomycin. Amphotericin B (AmB) was used as a reference drug for Leishmania.
Data are means ± standard deviation from three independent experiments.
Preliminary pharmacokinetic studies of compound 1. A snapshot pharmacokinetic
study was performed with male BALB/c mice to determine the oral bioavailability of
compound 1. Since the molecule is hardly soluble in water, it was formulated in 8%
DMSO in water and a single dosage of 20 mg/kg, corresponding to 500 µg of compound
1, was given orally (n = 3 mice). The maximum plasma concentration (Cmax = 182 µM)
was reached rapidly 1 h after oral dosage (Figure 1, Table S2). The compound showed an
apparent multi compartmental behavior with a rapid elimination from the plasma 6 h after
treatment (t1/2 = 1 h) and slower elimination in the 6–72 h post-treatment (t1/2 apparent = 23
h). Taking into account the PK data and the fact that signs of acute toxicity were observed
at the 20 mg/kg dosage, a 16-fold lower dose (1.5 mg/kg) and different vehicle (i.e. γ-
cyclodextrin) was selected for in vivo efficacy studies.
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Figure 1. Plasma concentration of compound 1 obtained after oral administration of 20
mg/kg of compound 1 in 5-8% DMSO to BALB/c mice. Values correspond to the
determination in pooled samples (n=3).
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Pharmacokinetic parameters
200
Tmax
1 h
150
100
50
Cmax
182 µM
t1/2
First phase (< 6 h)
Second phase (6–72 h)
1 h
23 h
0
13 6
24
48
Time (h)
72
Microsomal stability. The microsomal stability of 1 toward metabolism by cytochrome
P450 (Phase-I metabolism) and Uridine Glucuronosyl-Transferase (UGT) (Phase-II
metabolism) was studied in presence of NADPH and UDPGA. Compound 1 was rapidly
metabolized by mouse (CD-1) liver microsomes (high intrinsic clearance) with half-life
< 30 min, confirming the PK data observed in vivo. In contrast, 1 was metabolized slowly
by human liver microsomes (low to moderate intrinsic clearance) with t1/2 > 2 h, and very
slowly by human liver S9 fraction within 2 h of reaction (Table S3) suggesting species-
related differences. In comparison, high intrinsic clearance of the control drug diclofenac
by human fractions was observed under the same conditions (t1/2 < 30 min).
In vivo efficacy assay in BALB/c mouse model of visceral leishmaniasis. Compound
1 was formulated as a colloidal suspension in 5% γ-cyclodextrin in water (w/w); the
thermodynamic solubility of 1 in this vehicle was measured as 520 ± 10 mg/L. Based on
the pharmacokinetics, stability and absorption data, compound 1 was administered orally
in the L. infantum BALB/c mouse model at 1.5 mg/kg for 4 days. We established three
groups of mice (five mice per group): (i) G1, non-infected mice + compound 1; (ii) G2,
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infected mice + vehicle, and (iii) G3, infected mice + compound 1. The results are
expressed as mean number of amastigotes per mg of tissue. Amastigote burdens in the
liver and spleen were determined after 48 h of the end of treatment with compound 1. The
vehicle-treated controls showed high amastigote burdens in the spleen (607.3 ± 177.0)
and lower burdens in the liver (4.92 ± 1.21) and we observed a reduction of 98.9% (6.47
± 1.77) and 95.3% (0.18 ± 0.07), respectively in the infected mice treated with compound
1 (Table 3).
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Table 3. Activity of compound 1 in the L. infantum BALB/c mouse model with respect
to organ burden and efficacy (percentage of reduction)
Amastigote burden
Liver
Spleen
Mean + SD
Dosing group
Meana + SD % reduction
% reduction
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G1 (Non-infected + Cmpd 1 at
-
-
-
-
-
-
1.5 mg/kg p.o.b for 4 days )
G2 (Infected + vehicle [5% γ-
cyclodextrin])
4.92 ± 1.21
0.18 ± 0.07
607.25 ± 176.96
6.47 ± 1.77
G3 (Infected + Cmpd 1 at 1.5
mg/kg p.o. for 4 days)
95.3
98.9
aMean number of amastigotes per mg of tissue ± SD. bp.o., orally.
Pharmacokinetics and biodistribution. Compound 1 was only detected in plasma
samples corresponding to the first sampling (after the 4 days of treatment) (Table S4).
Notably, all the plasma concentrations were well over the EC50 value measured for L.
donovani (0.09 µM). In all other cases (solid organs and plasma 2 days after the end of
treatment) the concentration of 1 was below the detection limit of the HPLC technique.
Absence of detection of 1 in the samples obtained 2 days after the end of the treatment is
consistent with the rapid clearance observed in the snapshot experiments and with
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microsomal fractions in vitro. Hence, the absence of detection of 1 in the solid organs
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analyzed (liver, spleen) could be due to the elimination of the molecule in target organs.
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Mode of action studies: uptake and effect of compound 1 on mitochondrial
membrane potential (∆Ψm), ATP levels, reactive oxygen species (ROS), plasma
membrane integrity and DNA fragmentation of L. infantum parasites.
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Triphenylphosphonium salt derivatives are cationic molecules with a delocalized charge
and enhanced hydrophobic character; these properties allow their diffusion across
biological membranes driven by the electrochemical potential, resulting in their
accumulation in organelles with high membrane potential such as mitochondria.8-10, 26
To directly measure the cytotoxicity of compound 1, we studied if the mitochondrial
function of Leishmania was affected by the molecule. The effect of compound 1 on the
mitochondrial membrane potential (ΔΨm) of L. infantum promastigotes was determined
using the JC-1 fluorescence marker. We observed a fast depolarization of ∆Ψm from the
first concentration (5 times under EC50) and time-point tested (0.02 µM, 30 min) with a
decrease of 86%, showing a level of ∆Ψm similar to the classical uncoupling reagent
FCCP, used at 10 µM for 30 min (Figure 2A). The maximum decrease in ∆Ψm was
observed after 24 h at 1 µM of compound 1 (97%) with visible signs of cellular death in
the culture of L. infantum parasites. These results strongly suggest that compound 1
affects the mitochondrion.
Bearing in mind that ∆Ψm is essential for mitochondrial ATP synthesis,27 we analyzed the
levels of ATP in parasites treated with 0.02, 0.1 and 1 µM of 1 after 30, 60, 120 min and
24 h using the bioluminescence assay CellTiter-Glo. Compound 1 elicited a rapid time
and concentration dependent depletion of ATP in the treated parasites (Figure 2B). In the
first 30 min, a dose-dependent decrease of 50–74% of ATP levels was observed upon
treatment with 1. The maximum decrease of ATP levels (91, 89 and 87% after incubation
with 0.02, 0.1 and 1 µM of 1, respectively) was reached after 120 min (Figure 2B).
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Figure 2. Altered mitochondrial membrane potential and ATP levels in L. infantum
lines exposed to compound 1. L. infantum line was preincubated with 0.02, 0.1 and 1
µM of 1 for 30 min, 60 min, 120 min, and 24 h. (A) To determine ∆Ψm, parasites were
incubated with 5 µM JC-1 for 10 min, as described in the Experimental section. The
FL2/FL1 fluorescence ratio was measured by flow cytometry analysis. Parasites were
pretreated with 10 µM FCCP for depolarization control. The fluorescence intensity was
determined by flow cytometry analysis and expressed as Relative Fluorescence Units
(RFU). (B) To analyze ATP levels, a 25 µL aliquot of parasites was transferred to a 96-
well plate and mixed with the same volume of CellTiter-Glo (Promega) for measurement
of the sample bioluminescence. Data are the means ± SD of at least three independent
experiments. Significant differences versus the control line were determined by Student's
t-test (*: p< 0.01).
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Several antileishmanial drugs have been described to induce cell death mediated by the
accumulation of ROS (Reactive Oxygen Species) in the mitochondria of parasites.28
Taking into account the relationship between the decrease of ∆Ψm induced in Leishmania
by different drug treatments29 and ROS production, we studied ROS levels in parasites
treated with 1 µM of compound 1 for 2 h using the intracellular probe H2DCFDA. A
significant increase (8-fold) of ROS levels was observed compared with the untreated
control parasites. This effect was even higher than that of H2O2 (100 µM, 15 min) used
as positive control for the generation of ROS (Figure 3). Consequently, the generation of
ROS after treatment with 1 could contribute to the leishmanicidal effect of the compound.
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Figure 3. ROS levels in L. infantum lines exposed to compound 1. Intracellular ROS
levels were measured using the fluorescent probe H2DCFDA. L. infantum line was
incubated with 1 µM of compound 1 for 2 h, as described in the Experimental section.
After treatment, parasites were incubated with 40 nM H2DCFDA for 30 min at 28 ºC. The
fluorescence intensity was determined by flow cytometry analysis and expressed as
Relative Fluorescence Units (RFU). We used H2O2 (100 µM, 15 min) as positive control
of generation of ROS. Data are the means ± SD of at least three independent experiments.
Significant differences versus the control line were determined by Student's t-test (*: p<
0.01).
One of the mechanisms involved in cell death which usually acts together with the loss
of ∆Ψm, ATP depletion and generation of ROS, is the alteration of plasma membrane
permeability. Hence, we studied this parameter by the increase in fluorescence of SYTOX
green, a membrane impermeable intercalating agent. Permeabilized promastigotes with
1% Triton-X100 (TX100) were used as a positive control. Incubation of Leishmania
parasites with 0.02, 0.1 and 1 μM of compound 1 did not affect the plasma membrane
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permeability significantly (Figure 4). Additionally, depolarization of the plasma
membrane was measured using DiBAC4(3) as a fluorescence probe. After treatment with
0.02, 0.1 and 1 μM of compound 1 for 2 h, we observed significant changes in the plasma
membrane potential in a concentration-dependent manner (Fig. 4B). Thus, compound 1
does not induce osmolysis as it enters the parasite by depolarizing the plasma membrane
without inducing pore formation.
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Finally, the DNA fragmentation, typical of apoptotic processes, was tested after treatment
with compound 1 using the TUNEL assay. Parasites incubated with different
concentrations of compound 1 (0.02, 0.1, 1 and 5 µM) for 4 h were TUNEL negative (Fig.
5A). Additionally, necrotic cell death was analyzed by parasite staining with propidium
iodide (PI). We observed PI positive parasites in all concentrations tested (Fig. 5B).
Consequently, the cell death produced by the treatment with compound 1 is not related
with the apoptotic process.
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Figure 4. Plasma membrane permeability and membrane potential in L. infantum
lines exposed to compound 1. L. infantum line was incubated with 0.02, 0.1 and 1 µM
of 1 for 2 h, as described in the Experimental section. (A) After treatment, parasites were
incubated with SYTOX Green for 15 min at 28 ºC. The fluorescence intensity was
determined by flow cytometry analysis and expressed as Relative Fluorescence Units
(RFU). Permeabilized promastigotes with 1% Triton-X100 (TX100) were used as a
positive control. (B) After treatment, parasites were incubated with 2 μM DiBAC4(3) for
10 min at 28 °C. Parasites treated with a 10 μM concentration of the depolarizing agent
carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 15 min were used as controls.
Data are the means ± SD of at least three independent experiments. Significant differences
versus the control line were determined by Student's t-test (*: p< 0.01).
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Figure 5. DNA fragmentation in L. infantum lines exposed to compound 1.
Representative histogram of (A) TUNEL analysis and (B) propidium iodide (PI) labeling,
respectively, of L. infantum promastigotes treated with compound 1. Parasites were
treated with different concentrations of compound 1: 0.01 (b), 0.2 (c), 1 (d) and 5 (e) μM
for 4 h at 28 °C, using untreated parasites (a) as controls. Fluorescein-dUTP and PI nucleic
acid labeling were analyzed by flow cytometry as described in Experimental part.
Histograms are representative of three independent experiments with 10,000 parasites
analyzed per group.
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Uptake of compound 4. To expand the knowledge about the mechanism of action of
compound 1, we used a fluorescent analogue (compound 4) to study the uptake of this
kind of TPP molecules. Considering the EC50 and the limit of the fluorescence signal
obtained, we used 20 µM of compound 4 (Exc. 340 nm; Em. 510 nm). The uptake of
compound 4 at 28 ºC was fast and reached saturation in about 10 min (Figure 6A). No
differences in compound 4 uptake were observed in the ATP-depleted parasites or at 4 ºC
(Figure 6B). Compound 4 showed fast entry kinetics into Leishmania parasites in an
energy- and temperature-independent process, this suggesting plasma membrane
diffusion process as expected for this class of lipophilic cations.
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Figure 6. Uptake of compound 4 by L. infantum parasites. (A) Kinetics of compound
4 uptake at 28 ºC in promastigotes at different time-points (1, 5, 10, 30 and 60 min). (B)
Energy and temperature dependence of compound 4 uptake. We used non-treated
parasites (control, incubated at 28 ºC), treated with 2-deoxy-D-glucose plus NaN3
(2DDG+SA) or parasites incubated at 4 ºC. Data are the means ± SD of three independent
experiments.
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DISCUSSION AND CONCLUSIONS
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The mitochondrion of trypanosomatid parasites is a well-known target of cationic
compounds.9, 10, 14, 30 This is the case, for instance, for miltefosine and paromomycin, two
of the currently used drugs against Leishmania.31, 32 The TPP salt derivatives reported here
are lipophilic cations that can accumulate in negatively charged organelles driven by the
electrochemical potential, so their profound effect on Leishmania mitochondria was
expected. These compounds are weak acids with pKa values close to 7.4 (the measured
pKa value of compound 1 is 7.62 at 25 ºC in H2O, see experimental section), meaning that
approximately 50% of the molecule will exist as phenolate ion at physiological pH.
Hence, 50% of molecule 1 will behave as electrically neutral zwitterion (logP = 0.38)
whereas another 50% will behave as cation (log P = 1.17). These physicochemical
properties of 1 may have an impact on the drug solubility and absorption, and possibly
on its MoA. In fact, the logP value of cation 1, that falls within the logP range of 1–3 for
compounds considered to be highly permeable,33 agrees with the observed oral
bioavailability of compound 1 in vivo.
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Our data show that compound 1 is taken up by Leishmania parasites by diffusion through
the plasma membrane; in addition, we observed a depolarization of the plasma membrane
that did not affect the integrity of parasites. Then, the compound acts on the
mitochondrion affecting the energy metabolism with a depolarization of ∆Ψm that
influences the ATP synthesis together with a significant ROS production. Cortes et al6
have shown that related gallic acid–TPP conjugates with linkers of 10 and 12 methylene
units, which are trypanocidal in vitro against T. cruzi, also exert an uncoupling effect (i.e.
the positive charge of the TPP cation counteracts the negative charge at the level of the
mitochondrial matrix) in T. cruzi trypomastigotes, although to a lower extent than the
classical uncoupler FCCP. In contrast, compound 1 displayed a fast uncoupling effect as
strong as the control drug FCCP in promastigotes of L. infantum at low concentration (5
times under EC50). This collapse of ∆Ψm could be explained by the combined action of
an uncoupling effect and respiratory chain inhibition, although plasma membrane
permeabilization does not appear to be involved.34
To determine whether compound 1 produced apoptosis-like cell-death, which is
characterized by DNA nicking and fragmentation as the final outcome, its effect on the
DNA fragmentation was studied; our results were consistent with a non-programmed cell-
death or necrosis.
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The preliminary SAR studies obtained from the screening of compounds 10–40 show that
the antileishmanial activity/selectivity profile depends on the substitution pattern of the
4-hydroxyphenyl head. Indeed, the lower efficacy (3- to 8-fold higher EC50 value)
observed for the alkyl compound 9 lacking the (2-Me/2-OH)-4-hydroxy aromatic head,
compared with 1, 2, 16, and 17 agrees with these findings and warrants future SAR studies
on this scaffold. Advantageously, the new keto analogues 1–4 synthesized in this work
are metabolically more stable than the benzoate counterparts 16–1812 and they also
proved to be slightly more selective (2- to 4-fold) towards Leishmania. Thus, lead
compound 1 was relatively stable when challenged by phase I and phase II metabolism
with human fractions, indicating favorable pharmacokinetic properties for future studies.
At present, miltefosine is the only effective oral drug available among the limited arsenal
of leishmanicidal treatments. The increasing appearance of drug resistance and
therapeutic failure, together with the high cost of the current treatments, underscores the
need to find new orally active drugs for this disease. The activity (EC50) of compound 1
against promastigotes and intracellular amastigotes of L. donovani (0.07 and 0.09 µM,
respectively) with a SI=31.2 compared well with those obtained for miltefosine (6.51 and
0.91 µM, respectively) with a SI=28.8. Furthermore, the results from the in vivo acute
murine model showed a strong reduction of parasite burden in spleen and liver (98.9 and
95.3%, respectively) with an oral administration of 1.5 mg/kg of 1 for 4 days. Similar
results in the reduction of parasite burden and efficacy have been published recently with
a high dose of miltefosine (i.e. 40 mg/kg once a day for 5 days) in a similar mouse model
of leishmaniasis,35 this reinforcing the relevance of the efficacy results obtained with
compound 1 at much lower dosage. The potential of compound 1 for the treatment of
chronic leishmaniasis using hamster or dog models will be the subject of future studies.
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In summary, the results disclosed here provide the evidence of significant antileishmanial
activity of 4-hydroxyphenyl phosphonium derivatives in vitro and in vivo in a BALB/c
mouse model. The highest activity and the best selectivity index were presented by
compound 1. In fact, the reduction observed in the parasite burden in target organs after
treatment with compound 1 in our murine model is similar to that obtained with
miltefosine with much higher doses. Exact MoA is unknown although the data obtained
suggest favorable PK characteristics, a fast entry of these compounds by diffusion across
the plasma membrane, alteration of the energy metabolism (∆Ψm, ATP), production of
ROS and a necrosis. The determination of the specific target protein (complex) of
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compound 1 in the mitochondrial electron transport chain will be approached in future
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EXPERIMENTAL PART
Chemistry. Anhydrous solvents were purchased to ACROS Organics in AcroSeal®
bottles and used as received. Thin Layer chromatography (TLC) was performed on silica
gel 60 F254 aluminum TLC plates (MERCK). Chromatography was performed on silica
gel 60 (0.040–0.063 mm, 230–400 mesh ASTM, MERCK). LC-MS spectra were
recorded on a WATERS apparatus integrated with a HPLC separation module (2695),
PDA detector (2996) and Micromass ZQ spectrometer using electrospray ionization
(ES+). Analytical HPLC was performed with a SunFire C18-3.5 μm column (4.6 mm ×
50 mm). Mobile phase A: CH3CN + 0.08% formic acid and B: H2O + 0.05% formic acid.
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UV detection was carried over 190 to 440 nm. H NMR and C NMR spectra were
registered on a Bruker Avance-300, Varian Inova-400, Varian-Mercury-400, and Varian-
1
500. Chemical shifts of the H NMR spectra were referenced to tetramethylsilane (δ 0
ppm) for Chloroform-d and the residual peak of the deuterated solvent (δ 3.31 ppm for
Methanol-d4) Chemical shifts of the 13C NMR spectra were referenced to CDCl3 (δ 77.16
ppm) and CD3OD (δ 49.0 ppm). Coupling constants J are expressed in hertz (Hz).
Accurate mass was measured with an Agilent Technologies Q-TOF 6520 spectrometer
using electrospray ionization. All of the biologically tested compounds were ≥ 95% pure
by HPLC.
(15-(2,4-Dihydroxyphenyl)-15-oxopentadecyl)triphenylphosphonium bromide (1).
A Kimax tube was charged with 6a (75 mg, 0.18 mmol) and Ph3P (51 mg, 0.19 mmol),
and flushed with argon. Anhydrous acetonitrile (1.5 mL) was added and the tube was
screw-capped. The reaction mixture was stirred 18 days at 80 ºC. The tube was allowed
to stand at 4 ºC for several days whereupon a precipitate settled at the bottom of the tube.
The supernatant was discarded and a small volume of CH3CN was added to the tube to
rinse the precipitate. The tube was centrifuged and the supernatant was discarded. Rinsing
of the precipitate was repeated with Et2O following the same protocol. The pure product
1 was isolated as a film-like colorless solid (85 mg, 100%). HPLC >95%, 1H NMR (400
MHz, Chloroform-d) δ 12.71 (s, 1H), 9.70 (s, 1H), 7.78 – 7.56 (m, 15H), 7.48 (d, J = 8.9
Hz, 1H), 6.55 (dd, J = 8.9, 2.4 Hz, 1H), 6.33 (d, J = 2.4 Hz, 1H), 3.41 (td, J = 13.1, 12.6,
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7.3 Hz, 2H), 2.74 (t, J = 7.5 Hz, 2H), 1.60 (q, J = 7.4 Hz, 2H), 1.56 – 1.42 (m, 4H), 1.30
– 1.05 (m, 20H). 13C NMR (101 MHz, Chloroform-d) δ 205.2, 165.3, 165.0, 135.3 (d, J
= 3.0 Hz), 133.6 (d, J = 9.9 Hz), 132.2, 130.7 (d, J = 12.5 Hz), 118.2 (d, J = 86.0 Hz),
112.8, 108.7, 103.4, 37.9, 30.6 (d, J = 15.5 Hz), 29.5, 29.4, 29.3, 29.2, 29.14, 29.11, 29.08,
25.2 (d, J = 1.8 Hz), 22.8 (d, J = 50.3 Hz), 22.7 (d, J = 4.5 Hz). HRMS (ESI+) m/z
609.3482 (C40H50PO3 requires: 609.3498).
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(16-(4-Hydroxy-2-methylphenyl)-16-oxohexadecyl)triphenylphosphonium bromide
(2). A Kimax tube was charged with 6b (6.9 mg, 0.016 mmol) and Ph3P (4.2 mg, 0.016
mmol), and flushed with argon. Anhydrous acetonitrile (0.6 mL) was added and the tube
was screw-capped. The reaction mixture was stirred 5 days at 80 ºC. The solvent was
removed under vacuum and the product was isolated by precipitation from
MeOH/Et2O/hexane at 4 ºC overnight. The supernatant was removed and the precipitate
was rinsed successively with Et2O and hexane. Off-white gummy solid (7.6 mg, 69%).
HPLC ≥ 95%. 1H NMR (400 MHz, Chloroform-d) δ 12.36 (s, 1H), 7.84 – 7.74 (m, 6H),
7.72 (td, J = 7.3, 1.8 Hz, 3H), 7.63 (td, J = 7.7, 3.5 Hz, 6H), 7.57 (d, J = 8.2 Hz, 1H), 6.71
(d, J = 1.7 Hz, 1H), 6.63 (dd, J = 8.2, 1.7 Hz, 1H), 3.76 (dt, J = 13.2, 7.4 Hz, 2H), 2.87
(t, J = 7.5 Hz, 2H), 2.27 (s, 3H), 1.66 (p, J = 7.4 Hz, 2H), 1.60 – 1.50 (m, 2H), 1.38 – 1.07
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(m, 22H). C NMR (101 MHz, Chloroform-d) δ 206.6, 162.8, 147.9, 135.1 (d, J = 3.0
Hz), 133.9 (d, J = 10.0 Hz), 130.6 (d, J = 12.5 Hz), 130.1, 120.3, 118.7 (d, J = 85.6 Hz),
118.6, 117.3, 38.4, 30.6 (d, J = 15.5 Hz), 29.71, 29.69, 29.67, 29.64, 29.57, 29.54, 29.45,
29.41, 29.3, 24.8, 23.0 (d, J = 49.3 Hz), 22.8 (d, J = 4.6 Hz), 22.1. HRMS (ESI+) m/z
607.3709 (C41H52PO2 requires: 607.3705).
(16-(4-Hydroxy-2-methylphenyl)-16-oxohexadecyl)diphenyl(pyridin-2-
yl)phosphonium bromide (3). Using the same procedure as above for compound 2, a
mixture of 6b (5.7 mg, 0.014 mmol) and Ph3P (3.7 mg, 0.014 mmol) in dry acetonitrile
(0.6 mL) was stirred at 80 ºC for 5 days under argon atmosphere. The solvent was
removed under vacuum and the product was isolated by precipitation from
MeOH/Et2O/hexane at 4 ºC overnight. The supernatant was removed and the precipitate
was rinsed successively with Et2O and hexane. Brownish gum (4.6 mg, 48%). HPLC ≥
95%. 1H NMR (400 MHz, Chloroform-d) δ 12.36 (s, 1H), 8.81 (dt, J = 4.7, 1.5 Hz, 1H),
8.42 (ddt, J = 7.9, 5.9, 1.1 Hz, 1H), 8.09 (tdd, J = 7.9, 5.1, 1.8 Hz, 1H), 7.87 – 7.78 (m,
4H), 7.77 – 7.70 (m, 2H), 7.62 (tdd, J = 9.1, 4.5, 3.2 Hz, 5H), 7.57 (d, J = 8.0 Hz, 1H),
6.71 (dd, J = 1.6, 0.9 Hz, 1H), 6.63 (dd, J = 8.0, 1.6 Hz, 1H), 3.75 – 3.62 (m, 2H), 2.92 –
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2.82 (m, 2H), 1.72 – 1.46 (m, 4H), 1.37 – 1.07 (m, 22H). 13C NMR (101 MHz,
Chloroform-d) δ 206.6, 162.8, 151.9 (d, J = 19.1 Hz), 147.9, 145.4, 144.2, 138.8 (d, J =
10.3 Hz), 135.1 (d, J = 3.0 Hz), 134.1 (d, J = 9.6 Hz), 132.5 (d, J = 24.5 Hz), 130.5 (d, J
= 12.6 Hz), 130.1, 128.1 (d, J = 3.5 Hz), 120.3, 118.6, 117.9 (d, J = 85.4 Hz), 117.3, 38.4,
30.6 (d, J = 15.2 Hz), 29.72, 29.70, 29.68, 29.63, 29.58, 29.56, 29.46, 29.32, 29.28, 24.8,
22.62, 22.59 (d, J = 13.6 Hz), 22.1, 22.0. HRMS (ESI+) m/z 608.3666 (C40H51PNO2
requires: 608.3657).
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(16-(2,4-Dihydroxyphenyl)-16-oxohexadecyl)tri(naphthalen-1-yl)phosphonium
bromide (4). Compound 6a (53.6 mg, 0.13 mmol) and tris(naphthalen‐1‐yl)phosphane
(67.0 mg, 0.16 mmol) were suspended in 2.0 mL of anhydrous DMF in a Kimax tube
under inert atmosphere. The tube was screw-capped and the mixture was kept under
stirring at 110 ºC for 5 days. The solvent was evaporated under high vacuum and the
crude solid was purified by flash chromatography on silica gel (Hexane/EtOAc: 0→20%)
leading to a mixture of 2 and tri(naphthalen-1-yl)phosphine oxide. The latter was removed
by preparative TLC (EtOAc/MeOH: 8/2) and 4 was obtained as pale white solid (7 mg,
6.4%). HPLC > 95%. 1H NMR (500 MHz, Methanol-d4) δ 8.43 (dd, J = 8.5, 1.5 Hz, 3H),
8.20 – 8.11 (m, 6H), 7.97 (d, J = 8.6 Hz, 3H), 7.78 – 7.61 (m, 7H), 7.54 – 7.47 (m, 3H),
6.33 (ddd, J = 8.8, 2.4, 0.6 Hz, 1H), 6.20 (d, J = 2.4 Hz, 1H), 4.00 (br, 2H), 2.88 (t, J =
7.4 Hz, 2H), 1.68 (p, J = 7.4 Hz, 2H), 1.63 – 0.95 (m, 22H). HRMS (ESI+) m/z 759.3981
(C52H56O3P requires 759.3967).
16-Bromohexadecanoic acid (5). A solution of 16-hydroxyhexadecanoic acid (1.0 g,
3.67 mmol) in acetic acid (4 mL) and HBr 48% aq (w/w) (4 mL) was stirred at 110 ºC for
41 h in a screw-capped Kimax tube. When cooled, a precipitate formed at the top of the
solution. The precipitate was filtered, washed with water, and dried under high vacuum,
obtaining 1.671 g of crude product. Recrystallization from hexane yielded 5 as white
crystals (1.074 g, 87%). 1H NMR (400 MHz, Chloroform-d) δ 3.34 (t, J = 6.9 Hz, 2H),
2.28 (t, J = 7.5 Hz, 2H), 1.79 (p, J = 7.2 Hz, 2H), 1.57 (p, J = 7.5 Hz, 2H), 1.40 – 1.12
(m, 22H). 13C NMR (101 MHz, Chloroform-d) δ 179.9, 34.03, 34.02, 32.9, 29.62, 29.60,
29.58, 29.54, 29.44, 29.42, 29.2, 29.1, 28.8, 28.2, 24.7.
16-Bromo-1-(2,4-dihydroxyphenyl)hexadecan-1-one (6a). An oven-dried Kimax tube
was charged with resorcinol (33 mg, 0.3 mmol) and 16-bromohexadecanoic acid 5 (104
mg, 0.3 mmol). The tube was flushed with argon and boron trifluoride diethyl etherate
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(1.5 mL) was added at once. The tube was screw-capped and stirred at 110 ºC for 2 h.
The cold reaction mixture was poured into crushed ice and the product was extracted with
EtOAc (25 mL). The organic phase was washed with brine (5 mL), dried (MgSO4), and
evaporated to give a crude brown oil. Silica chromatography (10 g) eluting with
hexane/EtOAc: 100/0→90/10 yielded the product as colorless glassy solid (58 mg, 45%).
HPLC (UV) >90%. 1H NMR (300 MHz, Chloroform-d) δ 12.81 (s, 1H), 7.59 (d, J = 8.9
Hz, 1H), 6.40–6.20 (m, 2H), 6.11 (brs, 1H), 3.34 (t, J = 7.0 Hz, 2H), 2.82 (t, J = 7.5 Hz,
2H), 1.78 (p, J = 7.1 Hz, 2H), 1.65 (p, J = 7.3 Hz, 2H), 1.45 – 1.06 (m, 20H). 13C NMR
(75 MHz, Chloroform-d) δ 205.6, 165.4, 162.7, 132.6, 114.0, 107.9, 103.7, 38.2, 34.3,
33.0, 29.76, 29.73, 29.67, 29.61, 29.57, 29.52, 28.9, 28.3, 25.1.
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16-Bromo-1-(4-hydroxy-2-methylphenyl)hexadecan-1-one (6b). Same procedure as
6a starting from m-cresol (30.6 mg, 0.28 mmol). Silica chromatography with
hexane/EtOAc: 100/0 →30/1 yielded an impure product as colorless paraffin-like solid
(75 mg). The product was purified further by silica chromatography using hexane 100%
to remove the impurity ethyl 16-bromohexadecanoate. Off-white paraffin-like solid (15
mg, 13%). HPLC (UV) > 95%. 1H NMR (400 MHz, Chloroform-d) δ 12.36 (s, 1H), 7.57
(d, J = 8.3 Hz, 1H), 6.72 (d, J = 1.5 Hz, 1H), 6.63 (dd, J = 8.3, 1.5 Hz, 1H), 3.34 (t, J =
6.9 Hz, 2H), 2.89 – 2.84 (m, 2H), 2.28 (s, 3H), 2.25 – 2.18 (m, 2H), 1.83 – 1.74 (m, 2H),
1.66 (p, J = 7.4 Hz, 2H), 1.59 – 1.50 (m, 2H), 1.39 – 1.17 (m, 18H). 13C NMR (101 MHz,
Chloroform-d) δ 206.5, 162.8, 147.9, 130.0, 120.2, 118.7, 117.3, 38.4, 34.6, 34.2, 33.0,
29.74, 29.73, 29.68, 29.61, 29.59, 29.58, 29.49, 29.41, 29.29, 28.91, 28.33, 25.14, 24.86.
(14-Hydroxytetradecyl)triphenylphosphonium bromide (9). Using the same
procedure as above for compound 2, a mixture of 14-bromotetradecan-1-ol 811 (37.6 mg,
0.13 mmol) and Ph3P (33.6 mg, 0.13 mmol) in dry acetonitrile (1 mL) was stirred at 80
ºC for 4 days under argon atmosphere. The solvent was removed under vacuum and the
product was isolated by precipitation from MeOH/Et2O. The precipitate was rinsed with
1
Et2O to yield 9 as colorless solid (44 mg, 61%). HPLC > 95%. H NMR (300 MHz,
Chloroform-d) δ 7.85 – 7.69 (m, 9H), 7.64 (ddd, J = 8.5, 6.6, 3.5 Hz, 6H), 3.82 – 3.66 (m,
2H), 3.56 (t, J = 6.7 Hz, 2H), 1.61 – 1.43 (m, 6H), 1.31 – 1.07 (m, 18H). 13C NMR (101
MHz, Chloroform-d) δ 135.1 (d, J = 3.0 Hz), 133.8 (d, J = 10.0 Hz), 130.6 (d, J = 12.5
Hz), 118.5 (d, J = 85.8 Hz), 62.9, 32.9, 30.5 (d, J = 15.6 Hz), 29.57, 29.53, 29.51, 29.48,
29.44, 29.3, 29.2, 25.8, 22.9 (d, J = 49.7 Hz), 22.7 (d, J = 4.6 Hz). HRMS (ESI+) m/z
475.3127 (C32H44OP requires: 475.3130).
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Physicochemical properties. pKa and logP were measured by potentiometry using the
SIRIUS T3 apparatus. As compound 1 is poorly soluble in water, its pKa was measured
in H2O/MeOH mixture and extrapolated to 0% co-solvent using the Yasuda-Shedlovsky
procedure as implemented in the SIRIUS T3 Refine software. The thermodynamic
solubility measurements were performed by a miniaturized shake-flask method and
analyzed by UV-spectrophotometry.
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Biology
Chemical compounds
For the biological assays, stock solutions of synthesized compounds in DMSO at 10 mM
were
prepared.
Triton
X-100
(TX100),
carbonylcyanide
p-
trifluoromethoxyphenylhydrazone (FCCP), m-chlorophenylhydrazone (CCCP),
amphotericin B (AmB), 4',6-diamidino-2-phenylindole dilactate (DAPI), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI)
and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich (St.
Louis, MO). Miltefosine (MIL) was purchased from Zentaris GmbH (Frankfurt,
Germany). L-glutamine and penicillin/streptomycin were obtained from Gibco. Cell-Titer
Glo was purchased from Promega. JC-1, H2DCFDA, bis-(1,3-dibutylbarbituric
acid)trimethine oxonol [DiBAC4(3)] and Sytox Green were purchased from Invitrogen
(Carlsbad, CA). All chemicals were of the highest quality available.
Leishmania cell lines and culture
Leishmania donovani (MHOM/ET/67/HU3) line was employed in the screening of
synthesized compounds. Leishmania infantum LEM 3049 line24 was employed as the
reference line to perform the experiments related to mechanism of action, uptake and in
vivo sensitivity. Three resistant lines obtained from clinical cases, namely L. infantum
LEM 3323 (a line clinical resistant to SbIII)24, L. infantum LEM 5159 (a line clinical
resistant to MIL)24 and L. infantum LEM 2126 (a line clinical resistant to MIL and
paromomycin)25 were used for sensitivity assays. All these lines were grown at 28 °C in
RPMI 1640-modified medium (Invitrogen) supplemented with 10% hiFBS (Invitrogen),
as described.36
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Sensitivity analysis and in vitro infection of THP-1 derived macrophages with
Leishmania lines
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The sensitivity of Leishmania promastigote lines to the different compounds was
determined after incubation for 72 h at 28 °C in presence of increasing concentrations of
the compounds. The concentration of compound required to inhibit 50 % of parasite
growth (EC50) was calculated using the MTT colorimetric assay, as described
previously.37 For the intracellular Leishmania amastigotes' susceptibility to compounds,
stationary-phase promastigotes were used to infect macrophage-differentiated THP-1
cells at a macrophage/parasite ratio of 1:10, as described previously.37 After overnight
infection at 35 ºC with 5% CO2 in RPMI 1640 medium plus 5% hiFBS, extracellular
parasites were removed by washing with serum-free medium. Infected macrophages were
incubated with different concentrations of compounds in RPMI 1640 medium plus 10%
hiFBS at 37 ºC and a 5% CO2 atmosphere, for 96 h. Following incubation, samples were
fixed for 20 min at 4 ºC with 2.5% paraformaldehyde in PBS buffer and then
permeabilized with 0.1% TX100 in PBS for 10 min. Infection level (54.3 ± 5.6%; control)
and mean number of intracellular parasites/cell (3.6 ± 0.7; control) were detected by
nuclear staining with Prolong-Gold antifade agent with DAPI (Invitrogen). The
percentage of infection and the mean number of amastigotes per infected macrophage
were determined in 200 macrophages/well.
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Human cell lines culture and determination of cellular toxicity
Human myelomonocytic cell line THP-1 was grown in RPMI-1640 supplemented with
10% heat-inactivated fetal bovine serum (hiFBS), 2 mM glutamate, 100 U/mL penicillin
and 100 mg/mL streptomycin at 37 °C and 5% CO2. 5 × 105 THP-1 cells per well in 24-
well plates were differentiated to macrophages with 20 ng/mL of PMA treatment for 48
h followed by 24 h of culture in complete fresh medium.
MRC-5 cells, a SV-40 transformed human fetal lung fibroblast cell line, were maintained
at 37 °C and 5% CO2 in DMEM supplemented with 10% hiFBS, 100 U/mL penicillin and
100 μg/mL streptomycin.
The cellular toxicity of all compounds was determined using the colorimetric MTT-based
assay,38 as described for Leishmania promastigotes, except for the incubation
temperature, which was 37 °C in this case.
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ATP measurement in Leishmania parasites
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ATP was measured using a CellTiter-Glo® luminescence assay as described39 with
modifications. Briefly, parasites (4 × 106/mL promastigotes) were incubated with 0.02,
0.1 and 1 µM compound 1 for different time-points (0, 30, 60, 120 min and 24 h) before
being mixed with the same volume of CellTiter-Glo® (Promega) and incubated in the dark
for 10 min following the manufacturer instructions. The bioluminescence was measured
using an Infinite® F200 Luminescence System (Tecan Austria GmbH).
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Measurement of mitochondrial membrane potential in promastigote forms of
Leishmania
The mitochondrial membrane potential (∆Ψm) was measured using the JC-1 fluorescent
marker as previously described.39 JC-1 is a cationic fluorescent compound (green colour
as monomer, FL1 emission at 530 nm after excitation at 490 nm) which exhibits potential-
dependent accumulation in mitochondria, as indicated by a shift in its fluorescence
emission from green to red (FL2 emission at 590 nm) due to the formation of red
fluorescent aggregates. Depolarization of ∆Ψm is indicated by a reduced accumulation of
the dye and a shift from red to green with a decrease in the red/green fluorescence ratio.
A cell suspension (4 × 106 parasites per mL) was treated with 0.02, 0.1 and 1 µM of
compound 1 for 30, 60, 120 min and 24 h, and then further incubated at 28 ºC with 5 µM
JC-1 in HBS buffer for 10 min. Cellular fluorescence was quantified by calculating the
ratio between FL2 and FL1 in a FACScan flow cytometer (Becton-Dickinson, San Jose,
CA). The uncoupling agent FCCP (10 μM), was used as a control for mitochondrial
depolarization.
Determination of plasma membrane integrity in Leishmania parasites
Cell membrane permeability was determined using the Sytox Green nucleic acid stain as
described with modifications.40 L. infantum promastigotes (2×106) were incubated in
HBS buffer with 0.02, 0.1 and 1 µM compound 1 for 2 h. As a positive control we used
parasites permeabilized with 1% TX100. After compound 1 treatment, parasites were
incubated in the presence of 0.5 µM SYTOX Green for 30 min at 28 ºC. The parasites
where then washed and analyzed by flow cytometry using a FACScan flow cytometer
(Becton-Dickinson, San Jose, CA). Fluorescence emission was quantified at 523 nm
wavelength upon excitation at 490 nm by using Cell Quest software.
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Determination of plasma membrane depolarization in Leishmania parasites
The membrane potential-sensitive probe DiBAC4(3) was used to measure potential
changes as previously described previously.38 Thus, parasites (1 × 107 promastigotes)
were incubated without or with different concentrations of compound 1 (0.02, 0.1 and 1
µM) for 2 h in culture medium at 28 °C and then treated with 2 μM DiBAC4(3) for 10
min at 28 °C. Parasites treated with a 10 μM concentration of the depolarizing agent
carbonyl cyanide m-chlorophenylhydrazone (CCCP), for 15 min were used as controls.
DiBAC4(3) fluorescence was analyzed by flow cytometry using a FACScan flow
cytometer (Becton-Dickinson, San Jose, CA).
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Measurement of ROS accumulation in Leishmania parasites
Intracellular ROS accumulation was measured using the fluorescent probe H2DCFDA as
described with some modifications.28 Briefly, 1 × 107 Leishmania promastigotes were
incubated in HBS buffer (21 mM HEPES, 0.7 mM Na2HPO4, 137 mM NaCl, 5 mM KCl,
and 6 mM glucose, pH 7) containing 1 µM of compound 1 for 2 h at 28 ºC. As a positive
control we used parasites incubated with 100 µM H2O2. After this treatment, parasites
were washed and incubated in the presence of 0.1 mM H2DCFDA for 30 min at 28 ºC.
After incubation, the parasites were washed again with HBS buffer and analyzed by flow
cytometry using a FACScan flow cytometer (Becton-Dickinson, San Jose, CA).
Fluorescence was measured at 517–527 nm wavelengths upon excitation at 490 nm by
using Cell Quest software.
DNA fragmentation analysis
DNA fragmentation was analyzed by terminal deoxynucleotidyltransferase (TdT)-
mediated dUTP end labeling (TUNEL) using the Roche in situ cell death detection kit as
described previously.41 Parasites (107 promastigotes) were incubated with 0.02, 0.1, 1,
and 5 μM of compound 1 for 4 h at 28°C in culture medium, fixed with 4% formaldehyde
in PBS for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 for 2
min at 4°C. Then, the parasites were labeled with TUNEL reaction mixture following the
manufacturer's instructions. Fluorescence was measured by flow cytometry, in a
FACScan flow cytometer (Becton-Dickinson, San Jose, CA). Unfixed parasites were
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labeled with 0.4 μg/ml PI for 5 min at 4°C and measured by flow cytometry to detect
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necrotic cells.
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Uptake of compound 4 and effect of temperature and energy
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Promastigotes (1 × 108 cells/mL) were treated with 20 µM of compound 4 for 1, 5, 10, 30
and 60 min in culture medium at 28 ºC. Afterwards, parasites were washed with the same
medium followed by phosphate-buffered saline (PBS; 1.2 mM KH2PO4, 8.1 mM
NaH2PO4, 130 mM NaCl and 2.6 mM KCl, pH 7), and re-suspended in 1% TX-100.
Compound 4 accumulation was determined fluorimetrically by recording an emission
spectrum in the range 510 ± 20 nm upon excitation at 340 nm an Infinite® F200
Luminescence System (Tecan Austria GmbH).
For energy-depletion studies, parasites were pre-incubated for 10 min with 5 mM 2-
deoxy-D-glucose and 20 mM NaN3 at 28 °C in HBS buffer without glucose. To analyze
the effect of the temperature in the uptake, we treated the parasites with compound 4 and
determined the uptake at 28 ºC (control) and 4 ºC.
Statistical analysis
Statistical comparisons between groups were performed using the Student’s t-test.
Differences were considered significant at a level of p< 0.05.
Pharmacokinetics
A pharmacokinetic study was performed with male BALB/c mice to determine the oral
bioavailability of compound 1. In the snapshot experiment, the compound was suspended
in 8% DMSO in water and a single dosage of 20 mg/kg was administered orally (n = 3
mice). Serum samples were obtained by facial vein puncture at 0, 1, 3, 6, 24, 48 and 72 h
post administration. Serum samples were deproteinized by adding 1.5 mL of acetonitrile,
mixed in a vortex and centrifuged at 1370 × g for 10 min. Supernatant was filtered through
0.45 μm mesh (Millex® HV PVDF, Merck Millipore) and assayed by modular HPLC
(Jasco, Japan). Liver and spleen samples were weighed and mixed with diluted acetic
acid. After homogenization, the samples were subjected to three freezing/thawing cycles
to extract the compound. Acetonitrile (0.75 mL) was added, mixed and centrifuged at
1370 × g for 10 min. Supernatant was filtered and assayed by HPLC as above. The flow
rate was 1.5 mL/min of a mobile phase containing a mixture 80:20 of acetonitrile:purified
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water containing 0.05% orthophosphoric acid. The stationary phase was a BDS
Hypersil® C18 column (Thermo Scientific, USA) 5 µm, 200 × 4.6 mm. The injection
volume was 100 µL and the detection wavelength was 275 nm. Apparent half-lives were
calculated based on a first order elimination rate.
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Microsomal stability
Microsomal stability of compound 1 towards metabolism by cytochrome P450 (Phase-I
metabolism) and Uridine Glucuronosyl-Transferase (UGT) (Phase-II metabolism) was
studied in presence of NADPH and UDPGA, respectively. Incubation media (0.6 mL,
final volume) containing human liver microsomes (HLM) (Gentest; Corning) (0.66 mg
prot./mL), mouse (CD-1) liver microsomes (Sigma-Aldrich) (0.66 mg prot./mL) or
human liver S9 fraction (Sigma-Aldrich) (0.83 mg prot./mL) in 80 mM potassium
phosphate buffer (pH 7.4) were added with compound 1 (5–10 µM) or diclofenac (20–40
µM) and NADPH (1 mM), and incubated in a water bath at 37 ºC for 2 h. On the other
hand, incubation media (0.6 mL, final volume) containing human or mouse liver
microsomes (0.66 mg prot./mL) or human liver S9 mix fraction (0.83 mg prot./mL) in 80
mM potassium phosphate buffer (pH 7.4), and 4 mM MgCl2 were sonicated for 3 min
with tubes in ice bath and added with compound 1 (10–20 µM), UDPGA (2 mM), and
incubated at 37 ºC for 2 h. Aliquots (100 µL) from incubation media were withdrawn at
0, 15, 30, 60 and 120 min and added to 100 µL of acetonitrile, vortexed and centrifuged
at 10000 rpm. An aliquot of supernatant (20 µL) was analyzed by RP-HPLC (Agilent
1200 apparatus with a 1200 diode array detector (DAD) using a reversed phase 3.9×150
mm, 4 µm, Nova-pak C18 column (Waters, Milford, MA, USA) under the following
chromatographic conditions: eluent A: 50 mM ammonium phosphate buffer (pH 3) and
eluent B: 20 % A in acetonitrile. A linear gradient was used from 0 to 100% B in 8 min,
and then 100% B for 10 min. Under these conditions 1 eluted at t = 11.7 min. Compound
1 was detected at 215 and 275 nm and diclofenac at 280 nm. Assays were carried out at
least in duplicate and the maximum amount of DMSO or CH3CN was 0.25% and 0.5%,
respectively.
In vivo assays
Animals and ethics statement. BALB/c mice (six-week-old, female) from Charles River
Breeding Laboratories were housed and fed under pathogen-free conditions in the Animal
Facility Service of the Instituto de Parasitología y Biomedicina “López-Neyra”, CSIC.
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The use of laboratory rodents was carried out in strict accordance to all mandatory
guidelines (EU directives, including the Revised Directive 2010/63/EU on the Protection
of Animals used for Scientific Purposes that came into force on 1 January 2013 and the
declaration of Helsinki in its latest version) and was approved by the ethical committee
of the Instituto de Parasitología y Biomedicina “López-Neyra” – CSIC (file CEA-03-11-
2018). Pharmacological studies (experimental design and housing conditions) were
approved by the Committee of Animal Experimentation (Universidad Complutense de
Madrid) and regional authorities (Community of Madrid) (Ref. PROEX 169/15).
Experiments were carried out at the animal house ES280790001164 following the 3Rs
principles. Animal handling and sampling were performed by trained and officially
qualified personnel.
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Analysis of in vivo infection
Animals (5 mice/group) were injected intraperitoneally with 5×107 stationary-phase L.
infantum promastigotes resuspended in PBS (following the technical recommendation of
our Animal Facility in order to establish the most homogeneous infections rate). Two
weeks after infection, the parasite burden with this Leishmania line reached the maximum
as routinely determined in our Animal Facility under their experimental conditions. Two
weeks after infection, animals were treated orally for 4 days with 1.5 mg/kg of compound
1 suspended in 5% (w/w) γ-cyclodextrin (Ashland Industries Europe, Cavamax® W8,
Switzerland) in distilled water. Two days after the end of the treatment, spleen and liver
were removed for parasite quantification using the limiting dilution technique as
described previously.42, 43 Briefly, target tissues were homogenised in 5 mL of RPMI
1640 medium supplemented with 20 % hiFBS, 100 U/mL penicillin and 100 μg/mL
streptomycin using a tissue grinder. After centrifuging at 100 × g for 10 min at 4 °C, the
supernatant was collected and centrifuged at 2000 × g for 20 min at 4 °C. The pellet was
resuspended in 5 mL of RPMI 1640 medium and then dispensed in 96-well plates using
progressive 1:10 dilutions. Parasite burden was determined using light microscopy to
examine the dilution which yielded promastigotes after 7 days of incubation at 28 °C.
ASSOCIATED CONTENT
Supporting Information Available.
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