Antiparasitic activity and ultrastructural alterations provoked by organoruthenium complexes against: Leishmania amazonensis

Article abstract of DOI:10.1039/c8nj04657c

Four new organoruthenium complexes with formula [RuCl(η⁶-p-cymene)(μ-FCZ)]₂[Cl]₂ (1), [RuCl(FCZ)(η⁶-p-cymene)(PPh₃)]PF₆ (2), [RuCl(CTZ)(η⁶-p-cymene)(PPh₃)]PF₆ (3) and [RuCl(KTZ)(η⁶-p-cymene)(PPh₃)]PF₆ (4) (where FCZ: 2-(2,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)-2-propanol, CTZ: 1-[(2-chlorophenyl)-diphenylmethyl-1H-imidazole] and KTZ: cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) were synthesized, characterized and evaluated as potential inhibitors for Leishmania amazonensis growth by widely reported methods. Complexes 3 and 4 displayed effective IC₅₀ activities against Leishmania amazonensis promastigotes and intracellular amastigotes in the range of nanomolar concentration. Scanning and transmission electron microscopy analysis of Leishmania amazonensis promastigotes after treatment with 300 or 500 nM of complexes 3 and 4 for 48 h showed morphological alterations in the cell surface, a shortening of the flagellum, loss of mitochondrial matrix, disorganization of the kDNA and abnormal chromatin condensation. Thus, our strategy of incorporating a ruthenium atom into the structure of clinical drugs to improve their efficacy continues to demonstrate suitability for metallodrug discovery purposes.

Full text of DOI:10.1039/c8nj04657c

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Antiparasitic activity and ultrastructural alterations
provoked by organoruthenium complexes against
Leishmania amazonensis†
Cite this: New J. Chem., 2019,
43, 1431
a
Legna Colina-Vegas,
Joseane Lima Prado Godinho,bc Thallita Coutinho,bc
Rodrigo S. Correa,d Wanderley de Souza,bc Juliany Cola Fernandes Rodrigues,bce
Alzir Azevedo Batista*a and Maribel Navarro
*
fg
Four new organoruthenium complexes with formula [RuCl(Z6-p-cymene)(m-FCZ)]2[Cl]2 (1), [RuCl(FCZ)(Z6-
p-cymene)(PPh3)]PF6 (2), [RuCl(CTZ)(Z6-p-cymene)(PPh3)]PF6 (3) and [RuCl(KTZ)(Z6-p-cymene)(PPh3)]PF6
(4) (where FCZ: 2-(2,4-difluorophenyl)-1,3-di(1H-1,2,4-triazol-1-yl)-2-propanol, CTZ: 1-[(2-chlorophenyl)-
diphenylmethyl-1H-imidazole] and KTZ: cis-1-acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-
ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine) were synthesized, characterized and evaluated as
potential inhibitors for Leishmania amazonensis growth by widely reported methods. Complexes 3 and 4
displayed effective IC50 activities against Leishmania amazonensis promastigotes and intracellular
amastigotes in the range of nanomolar concentration. Scanning and transmission electron microscopy
analysis of Leishmania amazonensis promastigotes after treatment with 300 or 500 nM of complexes 3
and 4 for 48 h showed morphological alterations in the cell surface, a shortening of the flagellum, loss of
mitochondrial matrix, disorganization of the kDNA and abnormal chromatin condensation. Thus, our
strategy of incorporating a ruthenium atom into the structure of clinical drugs to improve their efficacy
continues to demonstrate suitability for metallodrug discovery purposes.
Received 12th September 2018,
Accepted 9th December 2018
DOI: 10.1039/c8nj04657c
rsc.li/njc
90 sandflies species. The three different clinical manifestations
of leishmaniasis lead to severe public health problems: (1) visceral
Introduction
Leishmaniasis is one of the most critical neglected diseases leishmaniasis is usually fatal when untreated and affects children
manifested worldwide affecting 101 countries with incidence mostly; (2) mucocutaneous leishmaniasis is a mutilating disease
estimated at 700 000 to 1 million new cases and 20 000 to 30 000 that leads to partial or total destruction of mucous membranes;
deaths, annually. This disease is caused by protozoan parasites and, (3) cutaneous leishmaniasis, the most common form of the
of the Leishmania genus that are transmitted to humans by over disease, is a disabling disease where several lesions can spread
throughout the body.1
The available treatments for leishmaniasis are far from
a
´
˜
˜
Departamento de Quımica, Universidade Federal de Sao Carlos, Sao Carlos, SP,
ideal. The classic first-line treatment for leishmaniasis relies
on pentavalent antimonials with sodium stibogluconate (pentostam)
and meglumine antimoniate (glucantime). The second-line of
treatment is based on using amphotericin B (deoxycholate or
liposomal) or pentamidine isethionate, which are toxic and
expensive. Also, all of these drugs have several limitations, such
as serious adverse effects, resistance cases, undesirable admin-
istration routes, long-term administration, and instability at
high temperatures.
In 2002, miltefosine was registered as the first oral treatment
for visceral leishmaniasis in India,2 and nowadays, it’s the first-
line chemotherapeutic agent in some countries in Asia, Africa
and Europe. Although miltefosine has 94% efficacy in Asia
(India) and it is administrated by the oral route, it is teratogenic,
hepatotoxic and nephrotoxic.3 Thus, there is an urgent need to
Brazil. E-mail: daab@ufscar.br
b
c
´
Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro,
Rio de Janeiro, Brazil
ˆ
Instituto Nacional de Ciencia e Tecnologia de Biologia Estrutural e Bioimagem
and Centro Nacional de Biologia Estrutural e Bioimagem-Cenabio,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
d
e
´
Departamento de Quımica, ICEB, Universidade Federal de Ouro Preto, Ouro Preto,
MG, Brazil
´
´
˜
Nucleo Multidisciplinar de Pesquisa UFRJ-Xerem, Divisao Biologia,
´
Campus UFRJ-Xerem, Duque de Caxias, Brazil
f Instituto Nacional de Metrologia, Qualidade e Tecnologia, INMETRO, Xerem, RJ,
Brazil
g
´
Departamento de Quımica, ICE, Universidade Federal de Juiz de Fora,
Juiz de Fora, MG, Brazil. E-mail: maribel.navarro@ufjf.edu.br
† Electronic supplementary information (ESI) available. CCDC 1830307 and
1830308. For ESI and crystallographic data in CIF or other electronic format
see DOI: 10.1039/c8nj04657c
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develop new therapeutic alternatives as the current treatment is
unsatisfactory due to toxicity, limited efficacy and high cost.
Another group of antileishmanial compounds are the sterol
biosynthesis inhibitors, including terbinafine, imidazole derivatives
(ketoconazole KTZ, clotrimazole CTZ, triazoles: fluconazole
FCZ, itraconazole ITZ, and azasterols), however they are still
in an experimental phase and there is not enough evidence for
their use in clinical treatments.4
Over the past two decades, our research group has demon-
strated that metal complexes with some imidazole derivatives
and another type of ligand are active against several parasites
responsible for neglected diseases; thus, according to this
strategy, combining a well-known bioactive agent and a metal
fragment into a single molecule results in a synergy that can
translate into improved activity and/or selectivity against
parasites.5–8 More recently, various organoruthenium and
manganese complexes with different azole antifungal agents
were reported with significant antiparasitic activity at low
micromolar concentrations against Leishmania major, Tripanosoma
cruzi and Schistosoma mansoni.9–11 Taking into account all of the
above, in this work four new organoruthenium complexes with CTZ,
Scheme 1 Synthesis of the organometallic azole–Ru(II) complexes and
ligands with numbering for NMR data. a-PA: a-phellandrene.
The UV-Vis spectra of the compounds are essentially analogous
KTZ and FCZ molecules, using triphenylphosphine and chlorido as to the ruthenium starting material complexes and characterized by
auxiliary ligands, were synthesized and characterized looking for intense absorption bands in the UV region, at B250 nm, char-
compounds with good biological properties. The complexes were acteristic of p–p* transitions of the aromatic ligands. This band
evaluated against Leishmania amazonensis promastigotes and intra- was followed by one or two broad and less intense bands, which
cellular amastigotes to determine the IC50 values by MTS/PMS are placed between 300 and 350 nm. The solid state FT-IR spectra
assay and characterize the morphological and ultrastructural of the complexes presented the characteristic bands in the range
alterations induced by the treatment of promastigotes with of 3142–3155 n(C–H); 1557–1586 n(CQN); 1491–1512 n(CQC);
ruthenium(II) complexes.
840–856 n(P–F); 557 d(P–F); 510–527 n(Ru–P) and 298–299 n(Ru–
Cl). The molar conductivities found, in acetone, were in the
range of 2 : 1 electrolytes for complex 1 and in the range of 1 : 1
electrolytes for complexes 2–4. Electrospray ionization mass
spectra were obtained in acetone (Fig. S1, ESI†). The spectra
for 1 present the fragment at a low intensity corresponding to
Results and discussion
Synthesis and characterization
The azoles here selected are well-established ligands for transition [M–2Cl–H]+ m/z 1153.1753 and also exhibited peaks of high
metal complexes; in particular, CTZ and KTZ have been more intensity corresponding to the cations [M–2Cl]2+ m/z 577.0665
reported when compared to the FCZ ligand, which can adopt two and [Ru(p-cymene)(FCZ)]+ m/z 541.0880. The compounds pro-
different monodentate coordination modes of the 1,2,4-triazole vide parent peaks corresponding to the cations [M–PF6]+ m/z
ring via N1 or N4.12,13 The desired ruthenium complexes were 839.1696 (2), 877.1675 (3), 1065.1963 (4), [M–PPh3–PF6]+ m/z
obtained in good yields by similar procedures (Scheme 1), in which 615.0872 (3), 533.0614 (4) and [M–XTZ–PF6]+ m/z 533.0444 (2),
the reaction was carried out in methanol.
533.0698 (3), 497.0935 (4), where XTZ: FCZ, CTZ or KTZ.
The binuclear compound [RuCl(Z6-p-cymene)(m-FCZ)]2Cl2
All NMR signals were assigned on the basis of 1D (1H and
(1) was obtained by reaction of FCZ ligand with the chloro- 13C{1H} NMR) and 2D experiments such as Correlation Spectro-
bridged arene ruthenium complex [RuCl2(Z6-p-cymene)]2; the scopy (1H–1H gCOSY), Heteronuclear Single Quantum Coherence
reaction of FCZ with [RuCl2(Z6-p-cymene)(PPh3)], in boiling (1H–13C gHSQC) and Heteronuclear Multiple Bond Coherence
methanol, lead to the mononuclear compound [RuCl(FCZ)- (1H–13C gHMBC). The H NMR of binuclear complex 1 presents
1
(Z6-p-cymene)(PPh3)]PF6 (2). A similar procedure was followed characteristic resonances of p-cymene and the fluconazole ligand
to obtain complexes [RuCl(CTZ)(Z6-p-cymene)(PPh3)]PF6 (3) as well as widening and splitting of the signals. A possible
and [RuCl(KTZ)(Z6-p-cymene)(PPh3)]PF6 (4). The complexes explanation for these results can be attributed to the different
under investigation are stable at room temperature, and soluble conformations namely anti–gauche (A–G) and anti–anti (A–A) of a
in common organic solvents chloroform, dichloromethane, fluconazole ligand (Fig. 1), which generate a fluxional molecule.14
acetone, and dimethylsulfoxide, and insoluble in water, hexane
The 1H NMR spectra of complexes 2, 3 and 4 show the
and diethyl ether. Half-sandwich complexes 2, 3 and 4 with characteristic resonances of p-cymene, fluconazole and triphenyl-
three-legged piano stool geometry with four different substituents phosphine ligands with a relative integral ratio of 1: 1 : 1 and the
[Mabcd] are examples of organometallic compounds with a stereo- signals typical of N-coordinated azole binds to the metal through
genic metal center.
the unsubstituted N1 atom, a good donor site of these molecules.
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Table 1 Selected bond lengths around the metal of the complexes 1
and 5
Fragment
1
5
Ru(1)–N(1)
Ru(1)–N(11)
Ru(1)–Cl(1)
Ru(1)–Ct
Ru(1)–O(1W)
Ru(1)–P(1)
2.114(4)
2.139(3)
2.4056(10)
1.668(1)
—
—
—
2.411(2)
1.734(3)
2.144(10)
2.373(3)
Fig. 1 Conformational isomers of fluconazole: (a) anti–gauche, (b) anti–anti.
—
The resonances of the p-cymene ring are in the characteristic
range of mono-cationic Ru(II). The most remarkable characteristics
of these complexes compared with the precursor [RuCl2(Z6-p-
cymene)(PPh3)] is the chemical shift of the aromatic protons of
p-cymene that exhibit four pairs of doublets at 6.0–5.0 ppm.
This behavior was reported for other complexes such as [Ru(Z6-
p-cymene)(O-S)Cl], [Ru2(Z6-p-cymene)2(EPh3)2(m-4,40-bipy)(Cl)2]
and [Ru(Z6-p-cymene)(PPh3)(L)Cl]PF6 with O-S: acylthioureas,
E: As or P and L: S-donor systems based on heterocyclic thiourea
derivatives.15–17 The NMR 31P{1H} spectra of the precursor
[Ru(Z6-p-cymene)(PPh3)Cl2] present a singlet signal at 24.0 ppm
corresponding to the PPh3 ligand while the NMR 31P{1H} spectra
of the complexes, after azole coordination to the ruthenium
metal center, showed a significant chemical shift around
36.0 ppm and an additional heptet signal at ꢀ114.1 ppm
Fig. 3 Crystal structure of the complex [RuCl(H2O)(Z6-p-cymene)-
(PPh3)]PF6 (5), showing the ellipsoids at 30% probability. The PF6 was
ꢀ
omitted.
ꢀ
the p-cymene ring, is in agreement with Ru(II)–p-cymene with
diamine ligands.18
attributed to the PF6 anion.
Crystal structure determination of 1 by single-crystal X-ray
diffraction confirmed the formation of a binuclear complex
containing two FCZ ligands making a bridge between the two-
It is interesting to observe that the precursor [RuCl2(Z6-p-
cymene)(PPh3)] used to obtain the complexes 2, 3 and 4 showed
aquation in a methanol/water solution mixture, resulting in the
substitution of one chlorido by a water molecule, forming the
complex [RuCl(H2O)(Z6-p-cymene)(PPh3)]PF6 (5) that also pre-
sents a stereogenic ruthenium center. In the structure of 5, the
Ru(II)–Ct distance [1.735 Å] is slightly longer compared with
complex 1, probably due to the influence of the PPh3 ligand
(Table 1). Based on the analysis of the crystal structure of the
complex 5, the presence of two molecules was established in
the asymmetric unit. Both molecules are very similar. For the
sake of clarity, Fig. 3, presenting an Ortep-3 type, shows the
structure of just one molecule. The separation between these
ligands and Ru(II) is represented in Table S2 (ESI†). Analyzing
the supramolecular behavior in complex 5, it can be observed
that while the coordinated water molecule is involved in
O1–Hwꢁ ꢁ ꢁF–P intermolecular interactions, the Cl– ligand does
not display any interaction with it.
%
ruthenium centers. The complex crystallizes in a P1 centrosym-
metric space group, in which the molecule is located in a
special position (inversion center). In the structure, there are
two chlorides as counter-ions, per complex. The chloride anion
is hydrogen bonded to the hydroxyl group of the FCZ ligand,
presenting an O–Hꢁ ꢁ ꢁCl separation of 2.259 Å. This interaction
displays an important role for crystal structure stabilization.
Moreover, disordered methanol and water located in the cavity
along the b axis can be observed.
As can be seen in Fig. 2, the complex presents a 20-membered
macrocyclic ring with the RuꢁꢁꢁRu atoms at a separation of 10.103 Å.
The Ru(II)–Ct distance [1.666 Å], where Ct means the centroid of
Antiproliferative effects on Leishmania amazonensis
promastigotes and intracellular amastigotes
The ruthenium complexes synthesized here were tested against
Leishmania amazonensis promastigotes and intracellular amastigotes.
First of all, they were analyzed against promastigotes using two
different methods: (1) MTS/PMS assay for measuring cell pro-
liferation and viability using a sensitive quantification method
based on the activity of NAD(P)H-dependent dehydrogenase
enzymes in metabolically active cells; (2) cell count every 24 h in
a phase contrast light microscope using the Neubauer chamber
during 96 h of growth.
Fig. 2 Crystal structure of complex 1, showing the ellipsoids at 30%
probability. The Cl– and disordered solvent were omitted.
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Table 2 IC50 values from MTS/PMS assay after treatment of Leishmania
amazonensis promastigotes
Compound
IC50 (mM) 24 h
IC50 (mM) 48 h
1
2
Z10
Z10
Z10
Z10
3
4
0.24 ꢂ 1.65
0.08 ꢂ 2.62
0.82 ꢂ 1.12
Z3.00
0.57 ꢂ 5.30
0.15 ꢂ 3.35
0.87 ꢂ 1.17
Z3.00
Clotrimazole
Ketoconazole
Fluconazole
Z100
Z100
The MTS/PMS assay revealed significantly lower IC50 values
(Table 2) when compared with the compounds without ruthenium.
The fluconazole ligand free did not present significant activity
against promastigotes, resulting in an IC50 higher than 100, and
also complexes 1 and 2 displayed IC50 values higher than 10 mM.
On the other hand, complex 4 was the most effective complex
with an IC50 value of 80 nM and 150 nM for 24 h and 48 h of
treatment, respectively. Complex 3 was also active against
promastigotes, however it was less potent than 4. It is interesting
to compare the CTZ and KTZ ligand free complexes; the IC50 values
were 0.87 mM and 3.00 mM after 48 h of treatment, respectively.
Thus, the combination of ruthenium with azoles in L. amazonensis
promastigotes significantly increased the activity of the com-
pounds. To confirm the effect induced by the treatment with 3
or 4 obtained by MTS/PMS assay, we carried out experiments of
growth curves counting the cells in the Neubauer chamber to
follow the parasites over several days. Growth curves revealed a
concentration- and time-dependent effect of 3 and 4 against
promastigotes (Fig. 4), where the concentrations of 500 and
800 nM were very potent inducing around 100% growth inhibition.
Complexes 3 and 4 were also tested against L. amazonensis
intracellular amastigotes cultivated in murine macrophages.
The IC50 values in Table 3 were significantly lower than those
found in promastigotes. For both complexes, they were in
the nanomolar range. After 48 h of treatment with different
concentrations, the IC50 values obtained were 25.8 nM and
15.5 nM for complexes 3 and 4, respectively.
Fig. 4 Growth curves of Leishmania amazonensis promastigotes treated
with different concentrations of (A) 3 and (B) 4 over 96 h.
Table 3 IC50 values obtained after treatment of Leishmania amazonensis
intracellular amastigotes with compounds 3 and 4 for 48 h
Cell viability of the macrophages was also investigated to
evaluate possible cytotoxicity effects of the treatment in the
mammalian host cells. The results obtained indicated that the
cells began to suffer in concentrations higher than 1 mM of 4,
which was not observed for the treatment with 3 (Fig. 5A and B).
Finally, we also followed the treatment of infected-macrophages
with intracellular amastigotes over 48 h using a range of con-
centrations to describe the effect of the ruthenium complexes
better and the results demonstrated that they act in a
concentration-dependent manner, with activity starting at a
low concentration of 10 nM (Fig. 5C and D).
Compound
IC50 (nM)
IC90 (nM)
3
4
25.80 ꢂ 1.09
15.53 ꢂ 1.30
52.65 ꢂ 1.25
108.60 ꢂ 2.60
Fig. 6A–F shows different images of untreated promastigotes
(Fig. 6A), and treated with complexes 3 and 4 (Fig. 6B–F).
The effects observed appeared to be concentration-dependent
as we treated the parasites with 300 and 500 nM. For both
ruthenium(II) complexes, promastigotes became rounded and
swollen; some of them presented protrusions in the cell surface
(arrows) and in the flagellum (arrowhead). After treatment with
300 nM complex 3 (Fig. 6B) and complex 4, we also observed a
shortening of the flagellum, however cells without a flagellum
Ultrastructural effects of ruthenium(II)–azole complexes on
Leishmania amazonensis promastigotes
Electron microscopy was used to determine the morphological also appeared (Fig. 6D and E).
and ultrastructural alterations induced by ruthenium(II) complexes
The treatment with these Ru(II) complexes was also evaluated by
on promastigotes. For the analysis of the morphology of the cell transmission electron microscopy trying to characterize the cellular
body and cell surface, scanning electron microscopy was used. target of the inhibitors. Fig. 7A shows a control promastigote,
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Fig. 5 Effects on cell viability of macrophages (A and B) and L. amazonensis
intracellular amastigotes (C and D). (A and B) MTS/PMS assay was used to
measure the cell viability of murine macrophages during treatment with 3
and 4 for 24 h and 48 h. Cytotoxic effects were only observed with 1 mM of 4.
(C and D) Compounds 3 and 4 were tested against L. amazonensis
intracellular amastigotes when cultivated in murine macrophages.
Fig. 7 Ultrathin sections of Leishmania amazonensis promastigotes.
(A) Control and (B and C) parasites treated with complex 4 for 48 h.
(A) General overview of a control parasite presenting a ramified mitochon-
drion (M) and nucleus (N) without any alteration in its ultrastructure.
Images (B) and (C) revealed important alterations in the mitochondrion,
such as: swelling and loss of the matrix content, and disorganization of the
kDNA structure. We also observed abnormal chromatin condensation
(B) and the presence of cytoplasmic vacuoles containing cellular debris
(C, arrows). F, flagellum; N, nucleus; M, mitochondrion; K, kinetoplast.
without treatment, showing the mitochondrion and nucleus
displaying a typical ultrastructure. Three kinds of alterations
predominate for both complexes 4 and 3: (1) mitochondrial
swelling followed by a loss of mitochondrial matrix (Fig. 7B, C
and 8A–D); (2) disorganization of the kDNA structure (Fig. 7C
and 8A, B), which could be related to arrest of the cell cycle, as
some cells appear to have more than one kinetoplast (arrow-
heads); (3) abnormal chromatin condensation (Fig. 7B and 8D),
which could be related to apoptosis-like cell death. Moreover,
in some cells we also observed the presence of cytoplasmic
vacuoles containing membrane profiles, small vesicles and
parts of the cytoplasm (Fig. 7B and C, arrows).
For other ergosterol biosynthesis inhibitors, including the
group of azoles, the mitochondrion was one of the organelles
most affected by the treatments.19–22 In the Trypanosomatidae
family, mitochondria have a special lipid composition, where
ergosterol, episterol and 5-dehydroepisterol are essential ster-
ols to maintain the mitochondrial membranes as shown for
Trypanosoma cruzi epimastigotes.23
Fig. 6 Scanning electron microscopy (SEM) of Leishmania amazonensis
This special requirement could explain why mitochondria
promastigotes. (A) Control and (B–F) parasites treated with ruthenium
complexes for 48 h. (B) 300 nM complex 3; (C and D) 500 nM complex 3; are dramatically affected after treatment with this class of
(E and F) 300 nM complex 4. (A) General overview of a control parasite
without any alteration in its morphology and cell surface. SEM images
compounds. An interesting alteration observed here was the
effect on kDNA structure; in some of the images the kinetoplast
appeared as a structure that did not divide correctly, once more
suggest a significant reduction in the size of the cell body, where some
cells appeared rounded and swollen (B–F). We also observed the presence
than one kDNA network was observed. This should be related to
a possible effect of the ruthenium(II) complexes in the DNA
of membrane protrusions on the plasma membrane that covers the cell
body (arrows) and the flagellum (arrowheads).
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through elemental analyses, electrospray ionization mass, UV-Vis,
NMR, molar conductivity, IR spectroscopy and X-ray techniques.
Single-crystal X-ray diffraction confirmed the formation of a binuc-
lear complex [RuCl(Z6-p-cymene)(m-FCZ)]2Cl2 containing two FCZ
ligands making a bridge between the two-ruthenium centers. Half-
sandwich complexes [RuCl(L)(Z6-p-cymene)(PPh3)]PF6 (L: FCZ, CTZ,
KTZ, H2O) are examples of chiral-at-metal organometallic com-
pounds. The complexes [RuCl(CTZ)(Z6-p-cymene)(PPh3)]PF6 and
[RuCl(KTZ)(Z6-p-cymene)(PPh3)]PF6 are significantly active in
the inhibition of the proliferation of the Leishmania parasite at
a very low level of concentration and both complexes increased the
cytotoxic effect on promastigotes when compared to the free ligand
activity. Electron microscopy images of Leishmania amazonensis
promastigotes after treatment with the complexes showed
morphological alterations such as reduction in the size of the
cell body, membrane protrusions, alterations in the mitochondria
including swelling and loss of the matrix content, disorganization of
the kDNA structure, abnormal chromatin condensation and the
presence of cytoplasmic vacuoles containing cellular debris. Further
studies will be conducted to establish a better structure–activity
correlation. The results obtained from this study have so far
demonstrated the feasibility of the strategy to coordinate
an organic molecule with the desirable biological property to a
transition metal to develop an effective and alternative treatment
for the Leishmania parasite.
Fig. 8 Ultrathin sections of Leishmania amazonensis promastigotes
treated with complex 3 for 48 h. (A and B) 300 nM complex 3; (C and D)
500 nM complex 3. As observed in complex 4, parasites treated with
complex 3 also presented significant alterations in the mitochondrion and
kDNA structure. Cells appeared to present more than one kinetoplast,
which may be due to cell cycle arrest. It is important to note that the
nucleus also presented alterations in the chromatin condensation, as
observed in D. F, flagellum; K, kinetoplast; M, mitochondrion; N, nucleus.
Experimental section
Materials and methods
All the syntheses of the complexes were performed under an
argon atmosphere. Solvents were purified by standard methods.
All chemicals used were of reagent grade or comparable purity.
The RuCl3ꢁ3H2O and a-phellandrene were purchased from
Sigma-Aldrich. The ligands CTZ, KTZ and FCZ were supplied
structure. It is important to mention that some images also
suggested alterations in the chromatin condensation that could
be a typical feature found in dead cells.
Thus, our results with L. amazonensis indicate that the coordina-
tion between azoles and the ruthenium increases the efficacy of the
activity of the compound, producing a new hybrid molecule, which
is much more potent against the parasite inhibiting the proliferation
and inducing significant ultrastructural alterations in a nanomolar
range of concentrations.
These interesting results demonstrate once more that our
strategy using a synergistic effect concept can significantly increase
the activity of the free ergosterol biosynthesis inhibitors (CTZ and
KTZ) activity against trypanosomatid parasites when coordinated to
transition metals to provide new metal–drug complexes.12,13,24–27
This approach is an alternative way to search for new candidates to
treat neglected diseases such as leishmaniasis.
˜
by the Calendula Pharmacy (Sao Paulo, Brazil). Starting materials
[RuCl2(Z6-p-cymene)]2 and [RuCl2(Z6-p-cymene)(PPh3)] were pre-
pared following the method described in the literature.28,29 The
microanalyses were performed using a FISIONS CHNS, mod. EA
1108 microanalyzer. The IR spectra were recorded on an FT-IR
Bomem-Michelson 102 spectrometer in the range of 4000–250 cmꢀ1
using KBr pellets. Conductivity data were obtained using a Meter
Lab CDM2300 instrument; measurements were taken at room
temperature using 1.0 mM solutions. The UV-Vis spectra were
recorded on a Hewlett Packard diode array-8452A. The electrospray
mass (ESI-MS) spectra were recorded on Water Synapt HDMS TOF
with a hybrid quadrupole analyzer, using acetone as a solvent. All
the 1D and 2D NMR experiments (1H, 13C{1H}, 31P{1H}, 1H–1H
gCOSY, 1H–13C gHSQC, 1H–13C gHMBC) were recorded at 298 K
on a 9.4 T Bruker Avance III spectrometer with a 5 mm internal
diameter indirect probe with ATMATM (Automatic Tuning
Matching).
Conclusions
Synthesis of the metal complexes
Four ruthenium(II)–azole complexes were prepared and their
[RuCl(g6-p-cymene)(l-FCZ)]2Cl2 (1). A solution of [RuCl2(Z6-
characterization was achieved in both the solid state and in solution, p-cymene)]2 (0.20 mmol) with an excess of FCZ (0.45 mmol) was
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stirred for 30 min in methanol (20 mL). The precipitated solid H5-CTZ), 6.51 (s, 1H, H4-CTZ), 5.89 (d, J = 5.7 Hz, 1H, Ph-p-cy),
was filtered off and washed with methanol and diethyl ether 5.77 (d, J = 6.0 Hz, 1H from Ph-p-cy), 5.30 (d, J = 5.9 Hz, 1H, Ph-p-
and dried under vacuum. Yield 73%; elemental analysis (%) for cy), 5.07 (d, J = 6.1 Hz, 1H, Ph-p-cy), 2.30–2.16 (m, 1H, isopropyl-
C
46H52Cl4F4N12O2Ru2ꢁ3CH3OH: exp. (calc.) C 44.54 (44.55); H p-cy), 1.14 (d, J = 6.9 Hz, 3H, p-cy), 1.02 (d, J = 6.8 Hz, 3H, p-cy).
5.20 (4.88); N 12.51 (12.72). IR (KBr, cmꢀ1): n(O–H) 3421, n(C–H) 31P{1H} NMR (162 MHz, CDCl3): d 36.24 (s, PPh3), ꢀ144.15 (h,
3118, n(CQN) 1535; n(CQC) 1501; n(Ru–Cl) 290. H NMR (400 PF6). 13C{1H} NMR (100.62 MHz, CDCl3): d ppm (attribution)
1
MHz, DMSO-d6): d (integral, attribution) 8.40 and 7.80 (4H, 141.64 (C2-CTZ), 139.29–124.01 (Cq and CH-Ph CTZ and PPh3),
0
0
H
2,2 ,5,5 -FCZ), 7.35–6.48 (3H, H9,10,12-FCZ), 6.10–5.40 (4H, Ph-p-cy), 113.65 (Cq-p-cy), 103.55 (Cq-p-cy), 94.19, 90.07, 88.15 and 84.14
0
4,60 (4H, H6,6 -FCZ), 2.83 (1H, isopropyl-p-cy), 1.80 (3H, p-cy), 1.13 (CH-Ph-p-cy), 76.90 (C24-CTZ, superposition with solvent signal),
(6H, p-cy). 13C{1H} NMR (100 MHz, CDCl3): d ppm (attribution) 30.59 (cisopropyl-p-cy), 23.27 and 20.93 ((CH3)2-p-cy), 18.08
0
150.3–149.6 (C5,5 -FCZ), 110.9–103.7 (C9,10,12-FCZ), 85.8 and 82.13 (CH3-p-cy). High resolution ESI(+)-MS (m/z; %) (acetone):
(CH-Ph-p-cy), 56.2 (C6,6 -FCZ), 30.9 (cisopropyl-p-cy), 22.2: 20.4 [M–PF6]+ (877.1675; 92.20), [M–PPh3–PF6]+ (615.0872; 71.42),
0
((CH3)2-p-cy), 18.0 (CH3-p-cy). High resolution ESI(+)-MS (m/z; %) [M–CTZ–PF6]+ (533.0698; 38.96), [CTZ-Imidazol]+ (277.0724; 100.00).
(acetone): [M–2Cl–H]+ (1153.1753; 19.0), [M–3Cl–Ru–(p-cymene)–
[RuCl(KTZ)(g6-p-cymene)(PPh3)]PF6 (4). Yield 85%; elemental
H]+ (833.1898; 9.52), [M–2Cl]2+ (577.0665; 95.23), [M–4Cl–Ru– analysis (%) for C54H57Cl3F6N4O4P2Ruꢁ2H2O: exp. (calc.) C 52.48
( p-cymene)–(FCZ)–H]+ (541.0808; 100).
(52.37), H 5.35 (5.19), N 4.86 (4.44). IR (KBr, cmꢀ1): n(C–H) 3142,
General procedure for 2–4. A solution of [RuCl2(Z6-p- n(CQN) 1557–1586; n(CQC) 1512; n(P–F) 851; d(P–F) 557;
cymene)(PPh3)] (0.20 mmol) in methanol (30 mL) was stirred n(Ru–P) 510–527; n(Ru–Cl) 298. 1H NMR (400 MHz, CDCl3) d
until complete dissolution was achieved, an excess of KPF6 and 7.58 (s, 1H, H2-KTZ), 7.49–7.17 (m, 19H, Ph from KTZ and PPh3),
the ligand (FCZ, CTZ or KTZ) dissolved in methanol (0.25 mmol) 7.03 (d, J = 9.0 Hz, H5-KTZ), 6.94 (d, J = 9.0 Hz, H4-KTZ),
was added. The resultant mixture was stirred and refluxed for 6.87–6.76 : 6.66 (m, 3H, H8,9,11-KTZ), 5.98, 5.91, 5.86 and 5.78
8 h, and then the final yellow solutions were concentrated to (d, J = 6.0 Hz, 2H, Ph from p-cy), 5.28, 5.22, 5.18 and 5.05 (d,
ca. 2 mL and the solid was precipitated by addition of water. J = 6.0 Hz, 2H, Ph from p-cy), 4.37–3.60 (m, 11H, H6,15,16,18,27,31-KTZ),
The solid obtained was filtrated off, washed with diethyl ether 3.06 (m, 4H, H28,30-KTZ), 2.31 (m, 1H, isopropyl p-cy), 2.12 : 211 (s,
and water to remove the excess of the ligand or salts, and dried 3H, H33-KTZ), 1.61 : 1.60 (s, 3H, p-cy), 0.97 (m, 6H, p-cy). 31P{1H}
under vacuum. Before the biological evaluation, the stability NMR (162 MHz, CDCl3): d 36.70 : 36.61 (s, PPh3), ꢀ144.15 (h, PF6).
of the complexes was tested using the 31P{1H} NMR technique 13C{1H} NMR (100 MHz, CDCl3): d ppm (attribution) 169.10
in DMSO or Tris–HCl solution containing 70% DMSO. After (C32-KTZ), 153.02–115.06 (CH-Ph and Cq of KTZ, PPh3),
seven days, the spectra of these complexes were the same, 107.31 : 101.01 (Cq-p-cy), 103.52 : 103.03 (Cq-p-cy), 94.80 : 94.33,
when compared with those recorded using fresh solutions 90.29 : 90.25, 87.21 : 87.03 and 83.74 : 83.37 (CH-Ph-p-cy),
(Fig. S6, ESI†).
74.96 : 74.46, 67.67, 52.24 : 51.75 (C6,15,16,18,27,31-KTZ), 50.97-41.37
[RuCl(FCZ)(g6-p-cymene)(PPh3)]PF6 (2). Yield 78%; elemental (C28,30-KTZ), 31.12 : 30.73 (cisopropyl-p-cy), 21.33 (C33-KTZ),
analysis (%) C41H41ClF8N6OP2RuꢁH2O: exp. (calc.) C 46.47 23.21 : 22.71 and 20.58 (–(CH3)2-p-cy), 18.19 : 18.11 (CH3-p-cy). High
(46.54); H 4.26 (4.35); N 10.94 (11.23). IR (KBr, cmꢀ1): n(O–H) resolution ESI(+)-MS (m/z; %) (acetone): [M–PF6]+ (1065.1963;
3432, n(C–H) 3145, n(CQN) 1599; n(CQC) 1502; n(P–F) 847; 71.42), [M–PPh3–PF6]+ (803.1275; 37.66), [M–KTZ–PF6]+ (533.0614;
1
d(P–F) 558; n(Ru–P) 512–527; n(Ru–Cl) 305. H NMR (400 MHz, 100.00), [M–KTZ–PF6–Cl]+ (497.0935; 71.42).
CDCl3) d (multiplicity, integral, attribution) 8.82–7.78 (m, 4H,
[RuCl(H2O)(g6-p-cymene)(PPh3)]PF6 (5). A solution of [RuCl2(Z6-
0
0
H
H
H
2,2 ,5,5 -FCZ), 7.40 (m, 15H, Ph from PPh3), 6.80–6.62 (m, 3H, p-cymene)(PPh3)] (0.20 mmol) in methanol/water (2 : 1) was stirred
9,10,12-FCZ), 5.93–5.16 (m, 4H, Ph-p-cy), 4.82–4.30 (m, 4H, until complete dissolution was achieved, and then an excess of
0
6,6 -FCZ), 2.40 (m, 1H, isopropyl-p-cy), 1,85 (m, 1H, OH-FCZ), KPF6 was added, and red crystals were obtained. Yield 73%;
1.70 (m, 3H, p-cy), 1.19 (m, 6H, p-cy). 31P{1H} NMR (162 MHz, elemental analysis (%) for RuC28H31ClF6OP2: exp. (calc.) C 48.63
CDCl3): d 36.65 : 36.21 (s, PPh3), ꢀ144.15 (h, PF6). 13C{1H} (48.32), H 4.32 (4.49). IR (KBr, cmꢀ1): n(C–H) 3142, n(CQC) 1523;
1
NMR (100 MHz, CDCl3): d ppm (attribution) 151.8–144.7 n(P–F) 850; d(P–F) 556; n(Ru–P) 527–510; n(Ru–Cl) 295. H NMR
0
0
(C2,2 ,5,5 -FCZ), 134.2–128.9 (CH–Ph–PPh3), 129.7, 112.3 and (400 MHz, DMSO-d6) d 7.56 (m, 15H, Ph form PPh3), 5.87 (d, J =
104.4 (C9,10,11-FCZ), 95.0, 89.1, 87.7 and 84.8 (CH-Ph-p-cy), 6.0 Hz, 2H, Ph from p-cy), 5.59 (d, J = 6.0 Hz, 2H, Ph from p-cy),
0
56.1 : 55.2 (C6,6 -FCZ), 31.0 : 29.6 (cisopropyl-p-cy), 22.4 : 19.6 5.34 (d, J = 6.4 Hz, 2H, Ph from p-cy), 5.10 (d, J = 6.0 Hz, 2H, Ph
((CH3)2-p-cy), 18.27 : 18.18 (CH3-p-cy). High resolution ESI(+)-MS from p-cy), 2.61 (m, 1H from isopropyl p-cy), 1.88 (s, 3H, p-cy),
(m/z; %) (acetone): [M–PF6]+ (839.1696; 20.00), [M–Cl–FCZ–PF6]+ 1.15 (t, 6H, p-cy). 31P{1H} NMR (162 MHz, DMSO-d6): d ppm
(497.0852; 60.00), [M–FCZ–PF6]+ (533.0444; 100.00).
(multiplicity, attribution) 33.07 (s, PPh3), ꢀ144.50 (h, PF6).
[RuCl(CTZ)(g6-p-cymene)(PPh3)]PF6 (3). Yield 90%; elemental
Single crystal X-ray structure data analysis
1
analysis (%) C50H48Cl2F6N2OP2Ruꢁ2H2O: exp. (calc.) C 57.50
(57.70), H 4.66 (4.65), N 2.78 (2.69). IR (KBr, cmꢀ1): n(C–H) Data collection and processing of the X-ray diffraction studies
3155, n(CQN) 1570–1586; n(CQC) 1491; n(P–F) 840; d(P–F) 557; were performed in a Nonius Kappa-CCD diffractometer, Mo Ka
n(Ru–P) 511–526; n(Ru–Cl) 299. 1H NMR (400 MHz,CDCl3) d 7.88 (l = 0.71073 Å), graphite monochromator, T = 298 K. The
(s, 1H, H2-CTZ), 7.56–7.27 (m, 25H, Ph from CTZ and PPh3), 6.89 COLLECT30 and SCALEPACK31 programs were used to refine
(dd, J = 22.5, 7.5 Hz, 4H, H6,7,8,9-CTZ), 6.68 (d, J = 7.9 Hz, 1H, the cells and in all cases, all the reflections were used to obtain
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the final parameters of the cell. The data were reduced using for 24 h, 48 h and 72 h. The MTS/PMS assay reaction was
the DENZO-SMN and SCALEPACK programs. A Gaussian quantified by optical density measurement at 490 nm in a
method implemented in WinGX was used for the absorption microplate reader and SpectraMax M2/M2e spectrofluorometer
correction.32,33 Using Olex2,34 the structure was solved with the (Molecular Devices, United States). As a negative control, para-
SIR200435 structure solution program using Direct Methods sites were fixed with 0.4% nascent formaldehyde for 10 min at
and refined with the ShelXL-2018/336 refinement package using room temperature before incubation. Data were plotted and
Least Squares minimisation. All non-hydrogen atoms were subjected to statistical analysis using Prism 4 (GraphPad software).
refined with anisotropic displacement parameters. The hydrogen For intracellular amastigote assays, murine macrophages and para-
atoms were located from the difference synthesis of electron sites were obtained as published previously.38 After 24 h of initial
density and refined using the riding model on their parent atoms infection, different concentrations of the compounds were added,
with Uiso(H) = 1.5Ueq for water and methyl H atoms or 1.2Ueq for the and the medium with the drug was changed every day for 3 days.
remaining aromatic and methine H atoms. The voids (188.4 Å3) in After each time, cultures were fixed in Bouin’s solution (70% picric
the structure 1 contain disordered solvent at partial occupancy. acid, 5% acetic acid and 25% formaldehyde in aqueous solution),
A satisfactory disorder model for the solvent was not found, and washed with 70% ethanol, followed by washing in distilled water,
therefore the OLEX2 Solvent Mask routine (similar to PLATON/ and then stained with Giemsa solution for 1 h. The number of
SQUEEZE) was used to mask out the disordered density. The intracellular amastigotes and macrophages (infected or not) was
ORTEP-337 was used to generate the molecular graphics. The counted via light microscopy. Association indices (the mean
X-ray crystallographic data for the structures of complexes 1 and number of parasites internalized multiplied by the percentage
5 reported in this article have been deposited at the Cambridge of infected macrophages divided by the total number of macro-
Crystallographic Data Centre (CCDC) under deposition numbers phages) were determined and used as a parameter to calculate
1830307 and 1830308, respectively.†
the percentage of infection for each condition used in this
study. The concentration that inhibited 50% of growth (IC50)
was calculated. At least three independent experiments were
Anti-Leishmania activities
Parasite. Leishmania amazonensis WHOM/BR/75/JOSEFA performed for each treatment.
strain was used in this study. It was isolated in 1975 from a
Electron microscopy. Control and treated promastigotes
patient with diffuse cutaneous leishmaniasis by Dr Cesar A. were fixed for at least 2 h in 2.5% glutaraldehyde (Sigma
Cuba–Cuba (Brasilia University, Brazil) and kindly provided by Chemical Co.) in a 0.1 M cacodylate buffer (pH 7.2), and post-
the Leishmania Collection of the Instituto Oswaldo Cruz (Code fixed in a solution containing 1% OsO4, 1.25% potassium
IOCL 0071 – FIOCRUZ). Parasites were maintained after inoculation ferrocyanide, 5 mM CaCl2, and 0.1 M cacodylate buffer (pH
of metacyclic infective promastigotes at the base of the tail of Balb/C 7.2) for 30 min. For scanning electron microscopy (SEM), cells
mice. Intracellular amastigotes were isolated from the lesions and were dehydrated in an ethanol series (50%, 70%, 90% and
then differentiated into promastigotes, which were maintained in 100%; last one for 3 times), critical point dried in CO2, mounted
Warren’s medium (Brain Heart Infusion plus 20 mg mLꢀ1 hemin on stubs, sputtered with a thin gold layer, and observed under a
and 10 mg mLꢀ1 folic acid) supplemented with 10% of fetal bovine ZEISS EVO scanning electron microscope. For transmission
serum (Cultlabs) at 25 1C. Infective metacyclic promastigotes electron microscopy (TEM), cells were dehydrated in an acetone
were used to infect murine macrophages to obtain intracellular series (50%, 70%, 90%, and 100%; last one for 3 times) and
amastigotes.
embedded in epoxy resin. Ultrathin sections were stained with
Antiproliferative effects against Leishmania amazonensis uranyl acetate and lead citrate and then observed under an FEI
promastigotes and intracellular amastigotes. For promastigote Tecnai T20 electron microscope.
assays, two different analyses were carried out: (1) growth curve
counting of the parasite cell number in a Neubauer chamber by
contrast phase light microscopy; (2) MTS/PMS assay to determine
Conflicts of interest
the cell viability. Growth curves of L. amazonensis promastigotes
There are no conflicts to declare.
were initiated with an inoculum of 1.0 ꢃ 106 cells per mL in a
Warren culture medium supplemented with 10% fetal bovine
serum. After 24 h of growth, different concentrations of the
compounds were added and the cells were cultured for 96 h,
Acknowledgements
˜
`
with the cell density calculated every 24 h by counting the The authors are grateful to Fundaçao de Amparo a Pesquisa do
˜
˜
number of cells in a Neubauer chamber using contrast phase Estado de Sao Paulo (FAPESP), Fundaçao Carlos Chagas Filho
`
light microscopy. Cell viability of promastigotes was evaluated by de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ)
CellTiter 96s Aqueous MTS Assay (Promega, United States) (1). and Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico
Regarding this, promastigotes were cultured in Warren’s (CNPq) for the financial suport. This study was financed in part
´
´
medium starting from a cell density of 1.0 ꢃ 106 cells per mL by Coordenaçao de Aperfeiçoamento de Pessoal de Nıvel
in 24-well plates; after 24 h of growth, different concentrations Superior-Brasil (CAPES)-Finance code 001. Legna Colina-Vegas
of the compounds were added to the cultures. Cell viability was thanks to FAPESP for her postdoctoral fellowship (grant
measured in triplicate, in a 96-well plate, after their incubation #2016/23130-5 and #2017/23254-9). R. S. Correa would like to
˜
`
1438 | New J. Chem., 2019, 43, 1431--1439
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18 L. Colina-Vegas, W. Villarreal, M. Navarro, C. R. de Oliveira,
A. E. Graminha, P. I. S. Maia, V. M. Deflon, A. G. Ferreira,
M. R. Cominetti and A. A. Batista, J. Inorg. Biochem., 2015,
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19 S. T. de Macedo-Silva, W. de Souza and J. C. F. Rodrigues,
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20 J. C. F. Rodrigues, C. F. Bernardes, G. Visbal, J. A. Urbina,
A. E. Vercesi and W. de Souza, Protist, 2007, 158, 447–456.
21 S. T. de Macedo-Silva, T. L. A. de Oliveira-Silva, J. A. Urbina,
W. de Souza and J. C. F. Rodrigues, Mol. Biol. Int., 2011,
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22 S. T. de Macedo-Silva, J. A. Urbina, W. de Souza and
J. C. F. Rodrigues, PLoS One, 2013, 8, e83247.
23 C. O. Rodrigues, R. Catisti, S. A. Uyemura, A. E. Vercesi, R. Lira,
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thank CNPq for financial support (project 403588/2016-2 and
308370/2017-1).
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