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A new ruthenium cyclopentadienyl azole compound with activity on tumor cell lines and trypanosomatid parasites
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Journal of Coordination Chemistry
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A new ruthenium cyclopentadienyl azole compound
with activity on tumor cell lines and trypanosomatid
parasites
Esteban Rodríguez Arcea, Cynthia Sarnigueta, Tania S. Moraesb, Marisol Vieitesa, A. Isabel
Tomazb, Andrea Medeirosc, Marcelo A. Cominic, Javier Varelad, Hugo Cerecettod, Mercedes
Gonzálezd, Fernanda Marquese, M. Helena Garcíab, Lucía Oteroa & Dinorah Gambinoa
a Departamento Estrella Campos, Cátedra de Química Inorgánica, Facultad de Química,
Universidad de la República, Gral. Flores 2124, 11800 Montevideo, Uruguay
b CCMM, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal
c Institut Pasteur de Montevideo, Group Redox Biology of Trypanosomes, Montevideo,
Uruguay
d Grupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias,
Click for updates
Universidad de la República, Uruguay
e Centro de Ciências e Tecnologias Nucleares (C2TN), Instituto Superior Técnico,
Universidade de Lisboa, Portugal
Accepted author version posted online: 18 Jun 2015.
To cite this article: Esteban Rodríguez Arce, Cynthia Sarniguet, Tania S. Moraes, Marisol Vieites, A. Isabel Tomaz, Andrea
Medeiros, Marcelo A. Comini, Javier Varela, Hugo Cerecetto, Mercedes González, Fernanda Marques, M. Helena García, Lucía
Otero & Dinorah Gambino (2015): A new ruthenium cyclopentadienyl azole compound with activity on tumor cell lines and
trypanosomatid parasites, Journal of Coordination Chemistry, DOI: 10.1080/00958972.2015.1062480
To link to this article: http://dx.doi.org/10.1080/00958972.2015.1062480
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Publisher: Taylor & Francis
Journal: Journal of Coordination Chemistry
DOI: http://dx.doi.org/10.1080/00958972.2015.1062480
A new ruthenium cyclopentadienyl azole compound with activity on tumor
cell lines and trypanosomatid parasites
ESTEBAN RODRÍGUEZ ARCEa, CYNTHIA SARNIGUETa, TANIA S. MORAESb, MARISOL VIEITESa,
A. ISABEL TOMAZb, ANDREA MEDEIROSc, MARCELO A. COMINIc, JAVIER VARELAd,
HUGO CERECETTOd, MERCEDES GONZÁLEZd, FERNANDA MARQUESe, M. HELENA GARCÍAb,
LUCÍA OTEROa and DINORAH GAMBINO*a
aCátedra de Química Inorgánica, Departamento Estrella Campos, Facultad de Química, Universidad de la República,
Gral. Flores 2124, 11800 Montevideo, Uruguay
bCCMM, Faculdade de Ciências da Universidade de Lisboa, Lisbon, Portugal
cInstitut Pasteur de Montevideo, Group Redox Biology of Trypanosomes, Montevideo, Uruguay
dGrupo de Química Medicinal, Laboratorio de Química Orgánica, Facultad de Ciencias,
Universidad de la República, Uruguay
eCentro de Ciências e Tecnologias Nucleares (C2TN), Instituto Superior Técnico, Universidade de Lisboa, Portugal
As part of our efforts to develop organometallic ruthenium compounds bearing activity on both
trypanosomatid parasites and tumor cells, a new Ru(II)-cyclopentadienyl clotrimazole complex,
[RuCp(PPh ) (CTZ)](CF SO ) where Cp = cyclopentadienyl, CTZ = clotrimazole, was
3 2 3 3
synthesized and characterized. The compound was evaluated in vitro on T. cruzi (Y strain), the
infective form of T. brucei brucei strain 427 (cell line 449), on three human tumor cell lines with
different sensitivity to cisplatin (A2780, ovary; MCF7, breast; HeLa, cervix) and on J774 murine
macrophages as mammalian cell model. The new compound is more cytotoxic on T. cruzi and on
the tumor cell lines than the reference drugs (Nifurtimox and cisplatin, respectively). In addition,
complexation of the bioactive CTZ to the {RuCp(PPh )} leads to significant increase of the
3
antiparasitic and antitumoral activity. To get insight into the potential “dual” mechanism of
antiparasitic action emerging from the presence of Ru(II) and CTZ in a single molecule, the
inhibitory effect of this new complex on the biosynthesis of T. cruzi sterols of membrane and the
interaction with DNA were studied. Although the tested complex does not affect DNA, it affects
the T. cruzi biosynthetic pathway of conversion of squalene to squalene oxide. According to the
results here reported, [RuCp(PPh ) (CTZ)][CF SO ] could be considered a prospective
3 2 3 3
antiparasitic and/or antitumoral agent that deserves further evaluation.
*Corresponding author. Email: dgambino@fq.edu.uy
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Keywords: Organometallic ruthenium compound; Clotrimazole; Cancer; Trypanosomes
1. Introduction
American Trypanosomiasis (Chagas disease) and Human African Trypanosomiasis (Sleeping
sickness), diseases caused by the genetically related trypanosomatid parasites Trypanosoma cruzi
and Trypanosoma brucei (Trypanosoma brucei gambiense and Trypanosoma brucei
rhodesiense), respectively, constitute major health concerns in the developing world [1-6],
although in the context of the current global society the impact of these diseases is not strictly
geographically confined [7, 8]. The available chemotherapeutics are decades old and suffer from
limited efficacy, undesirable collateral effects and development of resistance. Therefore, novel
strategies for the development of more efficient and less toxic agents, that could also circumvent
drug resistance, are urgently needed. Based on the recognition of common targets in both
parasites, drugs that are active against both protozoa could offer an innovative approach for drug
discovery [4].
In a not very different context, cancer is the second largest cause of death in developed
countries, and the number of worldwide deaths from this disease is projected by WHO to rise to
over 13.1 million people in 2030 [9]. Metabolic pathways of highly proliferative cells, like tumor
cells and parasites, are expected to have resemblances that could lead to a correlation between
antiparasitic and antitumor activities. In fact, several antitumoral drugs also show significant
antiparasitic activity and vice versa [10].
The development of bioactive metal-based compounds through inorganic medicinal
chemistry strategies has been recognized as a promising and attractive approach in the search for
antitumoral and antiparasitic drugs [11-27].
In particular, ruthenium complexes have been the most widely studied non-platinum,
metal-based anticancer candidates and hold potential as successful alternatives in cancer
treatment [10, 13, 16, 18, 28, 29]. In this perspective, ruthenium-cyclopentadienyl complexes
presenting N-heteroaromatic ligands have exhibited excellent antitumoral properties and are very
promising agents for cancer therapy [30-34]. In addition, ruthenium classical coordination
compounds and organometallic compounds have been recognized as prospective agents against
trypanosomatid parasites [35-42]. The design of antiparasitic compounds which combine ligands
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bearing antitrypanosomal activity and pharmacologically active metals in a single chemical
entity could provide drugs capable of modulating multiple targets simultaneously. Our group has
been working on the development of ruthenium complexes with different families of
trypanosomicidal ligands [10, 21, 38-42]. In particular, Ru-p-cymene compounds with this type
of ligands were developed and studied in depth [39-41].
Based on a similar approach, Sánchez-Delgado's group performed leading research in the
field by developing ruthenium and other metal complexes with the bioactive azole derivative
clotrimazole (CTZ, figure 1) as ligand [35, 37, 43-45]. CTZ is a well-known antifungal agent,
acting, like other azole derivatives, as a sterol biosynthesis inhibitor in fungi. In particular, it
inhibits the cytochrome P-450 dependent C(14) demethylation of lanosterol to ergosterol. The
sterol biosynthetic pathways of trypanosomatids are similar to those of pathogenic fungi and
therefore CTZ also displays moderate activity on T. cruzi [45, 46]. Five classical Ru-CTZ
coordination compounds and four organometallic Ru-p-cymene CTZ compounds were
developed by this group. Most of them displayed higher antiproliferative activity than free CTZ
on T. cruzi suggesting a synergistic effect [35, 37, 44, 45]. The biological profile clearly changed
when modifying the coordination sphere of the Ru(II), making further modification of the
Ru(II)-CTZ environment interesting [10, 37].
As part of our efforts to rationally develop organometallic ruthenium compounds bearing
activity on different trypanosomatid parasites but also on tumor cells, a new Ru(II)-
cyclopentadienyl CTZ complex, [RuCp(PPh ) (CTZ)](CF SO ) (abbreviated as RuCpCTZ), was
3 2 3 3
synthesized and characterized in this work. The compound was evaluated in vitro on the
epimastigote form of T. cruzi (Y strain), the infective form of T. brucei brucei strain 427 (cell
line 449) and the tumor cell lines A2780 (ovary), MCF7 (breast) and HeLa (cervix). To test
selectivity, the cytotoxicity on J774 murine macrophages as mammalian cell model was
evaluated. In order to get insight into the potential “dual” mechanism of antiparasitic action,
emerging from the presence of Ru(II) and CTZ in a single molecule, the inhibitory effect of the
complex on the biosynthesis of T. cruzi membrane sterols and the interaction with DNA were
studied.
2. Results and discussion
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Clotrimazole (CTZ, figure 1) is a classic imidazole ligand with a single nitrogen coordination
site (N1) capable of binding to a metal ion. It usually reacts readily under mild conditions
[37, 44, 45, 47-50]. A Ru-cyclopentadienyl complex (figure 2) with this bioactive ligand was
synthesized with high purity and good yield by the reaction of the precursor [RuCp(PPh ) Cl]
3 2
and CTZ (figure 1). Unfortunately, it was impossible to obtain adequate single crystals of the
complex for performing structural characterization by X-ray analysis. Nevertheless, the
compound was fully characterized in the solid state and in solution by using elemental analyses,
conductometric measurements, and FTIR and 1H-, 13C- and 31P-NMR spectroscopies.
Elemental analyses agree with the proposed formula. The molar conductivity value
obtained for the complex in DMSO solution demonstrates that it is a 1:1 electrolyte 51. No
conductivity changes were observed during at least 5 days at 25 °C, hence suggesting that the
complex is stable in air in DMSO.
Relevant solid FT-IR absorption bands were tentatively assigned to molecular vibration
modes of the coordinated CTZ ligand 37, the triflate counterion 34 and the {RuCp} moiety
52. Three new medium to strong bands were identified in the low wavenumber region (600-
400 cm-1) at 572, 533 and 522 cm-1 that could correspond to metal-ligand vibrations.
A detailed characterization of the new complex in solution was performed by NMR
spectroscopy. The resonances were assigned on the basis of 1D 1H-NMR, and 2D homonuclear
correlated spectroscopy (COSY) and heteronuclear correlation (HSQC) experiments as well as
on the comparison with those for CTZ and other previously reported Ru(II)–CTZ complexes
37, 44, 45. Table 1 shows the 1H-NMR chemical shifts () of the complex. Figure 2 shows the
corresponding numbering scheme. 1H-NMR integrations and signal multiplicities were in
agreement with the proposed formula and structure.
1H-NMR resonance of the 5-cyclopentadienyl ring is in the characteristic range of
cationic ruthenium(II) complexes [30]. The heteroaromatic ring protons of CTZ ligand are
shielded upon coordination to Ru(II) centre, with exception of the protons adjacent to the
coordinated N; these two protons, H2 and H4, are deshielded by 0.31 and 0.16 ppm, respectively.
This effect, perhaps due to the influence of the organometallic moiety on the ring current of the
heteroaromatic ligand, was also observed in other piano stool Ru(II) η5-cyclopentadienyl [34]
and Ru(II) η6-arene compounds with pyridylpyrazole, pyridylimidazole [53], and phenoxazine
ligands [54], coordinated by N of the heteroaromatic rings.
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13C-NMR spectra revealed the same general effect observed for the protons, although it
was not possible to unequivocally identify all the carbons, showing high complexity in the 127-
138 ppm region with signals corresponding to both PPh and CTZ ligands. However, analysis of
3
the HSQC spectra allowed clear identification of C2 (138.2 ppm) and C6 (130.0 ppm) of the
CTZ ligand. As previously reported, these are the most shifted CTZ carbon signals as a
consequence of complexation [44]. The signal at 83.2 ppm corresponds to equivalent carbons of
the Cp ligand [52]. Furthermore, a signal at 121.9 ppm corresponding to CF SO - counterion was
3 3
also observed [55].
31P-NMR spectra of the complex show a single sharp signal for the triphenylphosphane
coligand at ~41 ppm, and the expected deshielding upon coordination is in accord with the
σ-donor character of this ligand.
2.1. Biological results
2.1.1. Antiparasitic activity
The effect of RuCpCTZ on the epimastigote form of T. cruzi, Y strain, was evaluated and
compared with that of CTZ. The results are presented in table 2. RuCpCTZ showed high
cytotoxic activity on this parasite with an IC value in the submicromolar range. A six-fold
50
increase of activity was observed with respect to free CTZ. In addition, the complex showed an
activity about 30 times higher than the reference drug Nifurtimox (IC = 8.0 µM) [56].
50
For T. brucei brucei (strain 427), the new ruthenium compound showed growth inhibitory
activity, inducing a dose-dependent antiproliferative effect on parasites treated for 24 h (table 2).
CTZ displayed a lower cytotoxicity towards T. brucei (IC >25 μM) than against T. cruzi (IC
50 50
1.8 μM). This selectivity is in good agreement with the sterol content in each species and life
cycle stage of the parasites; T. cruzi epimastigotes contain 40% ergosterol whereas bloodstream
T. brucei contains predominantly cholesterol incorporated from the medium by a receptor-
mediated endocytic mechanism [57]. As a consequence of complex formation, the potency of
RuCpCTZ against the infective form of African trypanosomes is 40-fold higher (IC = 0.6 μM)
50
compared to the activity of the parental compound (IC >25 μM). This suggests that the RuCp
50
moiety of the CTZ complex is a major determinant for the biological activity of this compound
against T. brucei.
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In addition, RuCpCTZ showed a lower IC value on T. brucei brucei (strain 427) than
50
previously reported [Ru (p-cymene) (L) ]X complexes, where L = 5-nitrofuryl containing
2 2 2 2
thiosemicarbazones and X = Cl- or PF - [39].
6
2.1.2. Cytotoxicity on J774 murine macrophages
The specificity of the antitrypanosomal activity of RuCpCTZ and CTZ was evaluated by
analyzing their cytotoxicity against a murine macrophage-like cell line (J774). The results are
depicted in table 2. These results enable comparison of selectivity indexes (SI) of RuCpCTZ and
CTZ calculated as SI = IC macrophages / IC parasites.
50 50
Although RuCpCTZ showed higher cytotoxicity than CTZ, RuCpCTZ showed fairly
good selectivities towards both parasites (T. cruzi and T. brucei) with respect to mammalian cells
(J774 murine macrophages). In the case of T. brucei, the comparison of the selectivity index
values reveals that RuCpCTZ (SI = 3) is not only more potent but also more selective than CTZ
(SI 2).
2.1.3. Cytotoxic activity in human tumor cells
The cytotoxic activities of RuCpCTZ and CTZ were evaluated on three human cancer cells,
ovarian A2780, breast MCF7 and cervical HeLa, representing common cancer diseases and also
presenting different degrees of resistance to metallodrugs. The cells were treated with the
compounds in the concentration range 100 nM - 100 µM during a 72 h incubation period. A
colorimetric MTT assay was used to evaluate the cytotoxic activity measured as the half-
inhibitory concentration (IC ). IC values found for RuCpCTZ ranged between 0.5 and 5 µM
50 50
while those obtained for CTZ spanned between 6 µM and 20 µM (table 3). The A2780 cells were
particularly much more sensitive to both compounds, in particular to RuCpCTZ, than the MCF7
and HeLa cells. Complex formation seems to be responsible for the observed higher sensitivity
of the cells to the Ru complex relative to the free ligand. Cisplatin was presented for comparison,
since it is the metallodrug in clinical application. In the cell lines studied, the cytotoxic activity
of RuCpCTZ is three to five times higher than that of the reference drug cisplatin. In addition,
RuCpCTZ is 4- to 63-fold more active on A2780 tumor cells and 2- to 12-fold more active on
MCF7 cells than a series of organometallic Ru compounds previously developed by our group
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[41] and shows a significantly lower IC value on HeLa and MCF7 cells than recently reported
50
Ru(II) cytotoxic compounds [59, 60].
2.2. Mechanism of antiparasitic action
RuCpCTZ was designed to include a bioactive ligand and the pharmacologically relevant
Ru-cyclopentadienyl center, searching for a dual mechanism of action by affecting different
targets or biologically significant processes in the parasite. Accordingly, studies were performed
in order to validate proposed targets and to get insight into the probable mechanism of
antiparasitic action of RuCpCTZ.
2.2.1. Inhibition of sterol membrane biosynthesis
CTZ mechanism of action involving -14 C demethylase inhibition from the sterol membrane
biosynthesis pathway is well known [45, 46]. Therefore, the effect of RuCpCTZ on this
biochemical pathway was studied [61]. The obtained results (figure 3) showed that, while
ergosterol is present in all the studied treatments, its concentration seemed to be lower for those
incubations treated with RuCpCTZ, CTZ or terbinafine than for the negative control. As
expected, treatment with CTZ produced accumulation of lanosterol due to inhibition of this
enzyme that catalyzes the conversion of lanosterol to ergosterol. On the other hand, the treatment
with RuCpCTZ led to squalene accumulation, effect observed for terbinafine but not for free
CTZ. The results indicate that RuCpCTZ affects sterol membrane biosynthesis at the same step
as terbinafine, inhibiting squalene-2,3-epoxidase enzyme [62].
2.2.2. Interaction with DNA
Metal compounds and particularly ruthenium ones are usually able to interact with DNA. In
addition, DNA has been proposed as a parasitic target for other Ru-CTZ complexes [45].
Therefore, this biomolecule was investigated as a potential target of RuCpCTZ. The well-
established fluorescent DNA probe ethidium bromide (EB) was used to detect RuCpCTZ
interaction with DNA. When EB is specifically intercalated into double stranded DNA, the
{DNA-EB} adduct shows a strong fluorescence emission (λ = 510 nm) with a maximum at
exc
594 nm in our experimental conditions [63, 64]. Results obtained for the titration of this
{DNA-EB} adduct with RuCpCTZ are summarized in figure 4. No quenching in the emission of
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{DNA-EB} was observed upon increasing Ru complex concentration. This result indicates no
strong interaction (through intercalation or covalent binding) between the Ru complex and DNA,
in agreement with what is observed in the direct quantification test for binding of RuCpCTZ to
DNA described in section 4.5.2. The DNA binding level obtained by this method (nmol Ru / mg
DNA ≈ 1) is negligible when compared to the values ranging from 40-300 nmol Ru / mg DNA
reported for other Ru compounds that efficiently interact with DNA and for antitumor metal
complexes having DNA as molecular target [39, 42, 65, 66].
3. Conclusion
A new organometallic ruthenium clotrimazole compound, [RuCp(PPh ) (CTZ)](CF SO ), was
3 2 3 3
synthesized and fully characterized. This compound showed high cytotoxic activity on
trypanosomatid parasites (T. cruzi and T. brucei), validating our proposal of the development of
broad spectrum metal-based drugs active against multiple trypanosomatid protozoa as a
potentially innovative approach for antiparasitic drug discovery. In addition, the compound
shows high cytotoxicity on three tumor cell lines with different sensitivity to that of cisplatin.
The compound is more cytotoxic on the parasites and on the tumor cell lines than the reference
antitrypanosomal and antitumoral drugs. In all cases [RuCp(PPh ) (CTZ)](CF SO ) is more
3 2 3 3
active than free CTZ. The experiments performed gave insight into the probable mechanism of
antitrypanosomatid action of the ruthenium compound. It affects the T. cruzi biosynthetic
pathway responsible for the conversion of squalene to squalene oxide. According to the results
obtained, [RuCp(PPh ) (CTZ)](CF SO ) could be considered a prospective antiparasitic and/or
3 2 3 3
antitumor agent that deserves further development.
4. Materials and methods
4.1. Materials
All common laboratory chemicals were purchased from commercial sources and used without
purification. All syntheses were carried out under dinitrogen using Schlenk techniques and the
solvents used were dried by standard methods [67]. The precursor [Ru(η5-C H )(PPh ) Cl] was
5 5 3 2
synthesized from RuCl , triphenylphosphane and cyclopentadienyl in ethanol according to a
3
previously reported procedure [68].
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4.2. Synthesis of [RuCp(PPh ) (CTZ)](CF SO )
3 2 3 3
[RuCp(PPh ) Cl] (0.5 mmol, 0.320 g) and AgCF SO (0.45 mmol, 0.116 g) were dissolved in
3 2 3 3
dried distilled dichloromethane. The reaction mixture was kept at room temperature for 1.5 h.
CTZ (0.5 mmol, 0.172 g) was added and allowed to react at room temperature for about 2 h. The
reaction was followed by TLC (stationary phase silica gel GF254; mobile phase petroleum
ether/acetone 1:1v/v) until no reactants were detected. AgCl was filtered off on line under
dinitrogen pressure and the solvent was evaporated in vacuo. The yellow complex was obtained
by separation through silica column under N using petroleum ether/acetone (1:1v/v) as mobile
2
phase.
[RuCp(PPh ) (CTZ)](CF SO ). Yield: 190 mg, 32%. Yellow solid. Anal (%) calc. for
3 2 3 3
C H ClF N O P RuS (%): C, 64.89; H, 4.42; N, 2.36; S, 2.71. Found: C, 64.75; H, 4.44; N,
64 52 3 2 3 2
2.38; S, 2.67. FT-IR (KBr, cm-1, m = medium, w = weak, s = strong): 3055 (mw), (C-H from
arene), (C H ); 1479 (mw), 1446 (ms); 1430 (m), (C=N and/or C=C from arene); 696 (s),
5 5
520(s); 1273 (s), (CF SO ). (DMSO) 380 nm ( = 2.8×103 M-1cm-1). (DMSO) =
3 3 M
34.6 Scm2mol-1.
4.3. Physicochemical characterization
C, H and N analyses were carried out with a Thermo Scientific Flash 2000 elemental analyzer.
Conductimetric measurements were done over time (5 days) at 25 °C in 10−3 M DMSO solutions
using a Conductivity Meter 4310 Jenway to determine the type of electrolyte and to assess the
stability of the complex in such medium [51]. The FTIR absorption spectra (4000-300 cm–1) of
the complex, the precursor and CTZ were measured as KBr pellets with a Shimadzu
IRPrestige-21 instrument. UV-vis spectrum was measured in DMSO from 300-800 nm with a
spectrophotometer Shimadzu UV 1603 instrument. 1H-NMR and 13C-NMR spectra were
recorded in DMSO-d at 30 °C on a Bruker DPX-400 instrument (at 400 MHz and 100 MHz,
6
respectively). Homonuclear (COSY) and heteronuclear correlation experiments (2D-HETCOR),
HSQC (heteronuclear single quantum correlation), were carried out with the same instrument.
Tetramethylsilane was used as the internal standard. 31P{1H}-NMR spectrum was recorded in
DMSO-d on a Bruker AVANCE III 400 instrument (at 161.98 MHz). Chemical shifts are
6
reported in ppm.
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4.4. Biological activity
4.4.1. Anti-T. cruzi activity
Trypanosoma cruzi epimastigotes (Y strain) were grown at 28 °C in an axenic milieu
(BHI-Tryptose) supplemented with 5% fetal bovine serum (FBS) as previously described
[69, 70]. Cells from a 10-day-old culture (stationary phase) were inoculated into 50 mL of fresh
culture milieu to give an initial concentration of 106 cells/mL. Cell growth was followed every
day by measuring the absorbance of the culture at 600 nm. Before inoculation, the milieu was
supplemented with the indicated amount of the studied compound from a freshly prepared stock
solution in DMSO. Nifurtimox (Nfx) was used as the reference trypanosomicidal drug. The final
concentration of DMSO in the culture milieu never exceeded 0.4%, and the control was run in
the presence of 0.4% DMSO and in the absence of the studied compounds. No effect on
epimastigote growth was observed due to the presence of up to 1% DMSO in the culture milieu.
The percentage of inhibition (PGI) was calculated as follows: PGI (%) = {1 - [(Ap - A0p)/(Ac -
A0c)]} 100, where Ap = A of the culture containing the studied compound at day 5;
600nm
A0p = A of the culture containing the studied compound just after addition of the inocula
600nm
(day 0); Ac = A of the culture in the absence of the studied compound (control) at day 5;
600nm
A0c = A in the absence of the studied compound at day 0. To determine IC values (50%
600nm 50
inhibitory concentrations) parasite growth was followed in the absence (control) and presence of
increasing concentrations of the corresponding compound. At day 5, the absorbance of the
culture was measured and related to the control. The IC value was taken as the concentration of 50
compound under study necessary to reduce the absorbance ratio to 50%.
4.4.2. Activity on T. brucei brucei strain 427
The infective form of T. brucei brucei strain 427, cell line 449 (encoding one copy of the
tet-represor protein: PleoR [71]), was aerobically cultivated in a humidified incubator at 37 °C
with 5% CO in HMI-9 medium [72] supplemented with 10% (v/v) fetal calf serum (FCS),
2
10 U/mL penicillin, 10 µg/mL streptomycin and 0.2 g/mL phleomycin. The assay was
performed as previously described [73]. Ten mM stock solutions of the compounds were
prepared using DMSO as solvent and then diluted in culture medium to obtain seven
experimental concentrations (from 25 to 0.3 µM). The maximum DMSO concentration used in
the cytotoxicity assays did not exceed 1%, which is known to be nontoxic to T. brucei parasites.
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Compounds, DMSO (up to 1%) or medium were immediately added at the concentrations
described above and the culture plate was incubated at 37 °C with 5% CO . After 24 h, living
2
parasites were counted with a Neubauer chamber under the light microscope. Each condition was
tested in triplicate.
For each compound concentration, cytotoxicity was calculated according to the following
equation: Cytotoxicity (%) = {(experimental value - DMSO control)/(growth control - DMSO
control)} × 100. The data were plotted as percentage cytotoxicity versus drug concentration. IC
50
values were obtained from dose response curves fitted to a sigmoidal equation (Boltzmann
model) or extrapolated from non-linear fitting plots.
4.4.3. Cellular viability assays in human tumor cells
Three human tumor cell lines: A2780 ovarian, MCF7 breast and HeLa cervical (ATCC) were
cultured in medium (Gibco, Invitrogen) RPMI 1640 (A2780) or DMEM containing GlutaMax I
(MCF7, HeLa) supplemented with 10% fetal bovine serum and 1% antibiotics at 37 °C, 5% CO
2
in a humidified atmosphere (Heraeus, Germany). The cells were adherent in monolayers and
upon confluency were harvested by digestion with trypsin–EDTA (Gibco). Cell viability was
evaluated by using a colorimetric assay based on the tetrazolium salt MTT [74]. For this purpose,
cells were seeded in 200 µL aliquots in complete media RPMI or DMEM into 96-well plates.
Adequate cellular densities were chosen to ensure exponential growth of untreated control
samples throughout the experiments (2-5 × 104 cells/well). For 24 h cells were allowed to adhere
followed by the addition of dilution series of the test compounds in fresh medium (200 µL/well).
Ligand and complex were first solubilized in DMSO and then in medium, and added to final
concentrations in the range 100 nM - 100 µM. DMSO in cell culture medium did not exceed 1%
(final concentration). DMSO does not show cytotoxic effect by itself at this concentration. After
continuous treatment of the cells with the compounds for 72 h at 37 °C with 5% CO , the
2
medium was replaced by 200 µL of MTT solution in PBS (0.5 mg/mL). After 3-4 h incubation,
MTT solutions were removed and the formazan product formed was dissolved in DMSO
(200 µL/well). The cellular viability was evaluated by measuring the absorbance at 570 nm using
a plate spectrophotometer (PowerWave Xs, Bio-tek Instruments, VT, USA). The cytotoxic
effects of compounds were quantified by calculating the IC value based on nonlinear regression
50
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analysis of dose-response curves (GraphPad Prism version 4.0). Evaluation was based on at least
two independent experiments, each comprising six replicates per concentration.
4.4.4. Cytotoxicity on murine macrophages
The J774 mouse macrophage cell line was cultivated in a humidified 5% CO /95 % air
2
atmosphere at 37 °C in DMEM medium supplemented with 10% (v/v) FCS, 10 U/mL penicillin
and 10 µg/mL streptomycin. Stock solutions of the compounds to be tested were prepared as
described for anti-T. brucei activity tests and diluted in culture medium to obtain seven
experimental concentrations (from 100 to 0.5 µM). The maximum DMSO concentration used did
not exceed 1%, which is known to be tolerated by murine macrophages from J774 cell line. Each
condition was tested in triplicate. The cytotoxic effect of the compounds towards macrophages
was evaluated by colorimetric assay of cell viability with a tetrazolium salt (WST-1 reagent).
The absorbance of the formazan dye produced by metabolically active cells was measured at 450
nm (reference wavelength at 630 nm) with an EL 800 microplate reader (Biotek). The
methodological procedure is essentially as described previously [39]. Cytotoxicity was
calculated and IC values were obtained as described for cytotoxicity on T. brucei brucei.
50
4.5. Mechanism of action
4.5.1. Inhibition of the biosynthesis of membrane sterols
Epimastigote form of T. cruzi (Tulahuen 2 strain) was maintained in an axenic medium
(BHI-Tryptose). The experiments were carried out in cultures at 28 °C and with strong aeration.
The studied compound was added to the culture (10×106 cells/mL) as a DMSO solution in a
concentration equivalent to IC . Compound and parasites were incubated 72 h at 28 °C. Final
50
concentration of DMSO in the culture never exceeded 0.4%. A control including 0.4% DMSO
was prepared. Positive controls with terbinafine were also incubated. After incubation the control
and drug-treated parasites were centrifugated at 3000 rpm during 10 min and then the
supernatants were discarded and the pellets were collected and suspended in 1 mL phosphate
buffer (0.05 M, pH 7.4) and centrifugated at 3000 rpm during 10 min. The pellets were treated
with 1 mL chloroform/methanol (2:1) during 12 h at 4 °C. After addition of saturated NaCl
aqueous solution a new extraction with 600 μL chloroform and an extraction with 600 μL hexane
were performed. The total volume of extraction was seeded on a silica TLC plate. Squalene was
12
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identified by two chromatographic runs with hexane as mobile phase and ergosterol by one run
with n-hexane:AcOEt (8:2). The TLC was developed by UV light and with iodine vapors. The
compound was seeded together with a negative control, a positive control (terbinafine) and
standards of squalene, lanosterol, cholesterol and ergosterol [75].
4.5.2. Interaction with DNA
Fluorescence studies. Experiments for competitive binding to calf-thymus DNA (ct-DNA,
SIGMA, Type I, No. D-1501) with ethidium bromide (EB, SIGMA) were carried out in 10 mM
Tris-HCl buffer at pH 7.4. Millipore® water was used for the preparation of all aqueous
solutions. Fluorescence measurements were carried out on individually prepared samples to
ensure the same pre-incubation time for all samples in each assay. Due to the low solubility of
the complex in aqueous media, DMSO was used to prepare concentrated stock solutions
followed by appropriate dilution to obtain the targeted concentration and the same content of
DMSO (5% v/v) in the final samples. DNA stock solutions were prepared by hydrating ct-DNA
in Tris-HCl buffer (1 mg/mL, ~2 mM.nuc-1) during 3-4 days at 4 °C, swirling the solution about
4-5 times a day until full dissolution was attained, and a clear solution was obtained. This
solution was kept at 4 °C (in the refrigerator) between measurements and discarded after 4 days.
The concentration of each stock solution was determined by UV spectrophotometry using the
molar extinction coefficient ε = 6600 M−1cm−1 [76]. A 5 mM EB solution was prepared in
(260nm)
Tris-HCl buffer. ct-DNA was pre-incubated with EB at 4 °C for 24 h. Samples were prepared
with a total concentration of DNA and of EB of 20 µM and 10 µM, respectively, varying the
total complex concentration from 1-110 µM. They were incubated at 37 °C for 30 min. Samples
with complex alone and samples with complex and EB but no DNA were used as blanks.
Fluorescence spectra were recorded from 520 nm to 650 nm at an excitation wavelength
of 510 nm on a Shimadzu RF-5301PC spectrofluorimeter. Fluorescence emission intensity was
corrected for the absorption and emission inner filter effect at the maximum emission
wavelength (594 nm) using the UV-Visible absorption data recorded for each sample according
to the following equation [77].
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ct-DNA interaction experiments. The complex was tested for their global DNA interaction
ability using native ct-DNA by a modification of a previously reported procedure [78, 79].
ct-DNA (50 mg) was dissolved in water (30 mL) overnight. Solutions of the complex in DMSO
(spectroscopy grade) (1 mL, 10-3 M) were incubated at 37 °C with solution of ct-DNA (1 mL)
during 96 h. DNA/complex mixtures were exhaustively washed to eliminate the un-reacted
complex. Quantification of bound metal was done by atomic absorption spectroscopy on a Perkin
Elmer 5000 spectrometer. Standards were prepared by diluting a metal standard solution for
atomic absorption spectroscopy. Final DNA concentration per nucleotide was determined by UV
absorption spectroscopy using molar absorption coefficient of 6600 M-1cm-1 at 260 nm [76].
Acknowledgements
E.R.A. and J.V. wish to thank ANII for a postgraduate grant. Authors thank CSIC-UdelaR
(project 800), PEDECIBA and ANII-SNI, Uruguay. M.A.C. acknowledges the support of
FOCEM (MERCOSUR Structural Convergence Fund, COF 03/11). A.I.T. and T.M. thank the
Portuguese Foundation for Science and Technology (FCT) for financial support (the IF Initiative
IF/01179/2013 and post-doctoral grant SFRH/BPD/93513/2013). Portuguese coauthors also
thank FCT for projects PTDC/QUI-QUI/118077/2010 and PEst-OE/QUI/UI0536.
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Table 1. 1H-NMR chemical shift values () in ppm for [RuCp(PPh ) (CTZ)](CF SO )
3 2 3 3
and CTZ in DMSO-d at 30 °C.
6
1H-NMR/ (integration) (multiplicity)
H
Proton number [RuCp(PPh ) (CTZ)](CF SO ) CTZ
3 2 3 3
H2 7.75 (1, s) 7.44 (1, t)
H4 7.07 (1, dd) 6.91 (1, t)
H5 6.72 (1, dd) 6.73 (1, t)
H8 6.79 (1, m) 6.80 (1, d)
H11 7.38* (1, m) 7.40 (1, t)
H9, H10, H14, H16, H18, H20, 6.79 (8, m) 7.32 (8, m)
H22, H24
H15, H17, H21, H23 6.66 (4, m) 7.01 (4, m)
Cp 4.50 (5, s) -
PPh 7.38 (12, m), 7.27 (6, m), 7.21 (12, m) - 3
*overlapped with PPh
3
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Table 2. In vitro activity (measured as the IC value, the half inhibitory concentration) against
50
T. cruzi, T. brucei brucei, cytotoxicity on murine macrophages and selectivity index (SI) values
of RuCpCTZ and CTZ.
T. cruzi T. brucei macrophages
Compound SI (fold)a SI (fold)b
IC / M IC / M IC / M
50 50 50
RuCpCTZ 0.25 ± 0.08 0.6 ± 0.1 1.9 ± 0.1 8 3
CTZ 1.8 ± 0.4 25 55.1 ± 0.5 31 2
a SI: IC macrophages / IC T. cruzi. b SI: IC macrophages / IC T. brucei brucei. Results are
50 50 50 50
the mean value of three different independent experiments.
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Table 3. IC values found for RuCpCTZ and CTZ in three different human
50
cancer cells lines after 72 h incubation at 37 °C. Cisplatin is included for
comparison.
IC (µM)
Compound 50
A2780 MCF7 HeLa
CTZ 5.7 ± 0.9 20.5 ± 8.1 18.4 ± 7.9
RuCpCTZ 0.54 ± 0.1 5.4 ± 2.0 3.6 ± 1.1
Cisplatin 1.9 ± 0.1 a 28 ± 6.0 a 7.00 ± 2.70 b
a From reference [33]. b From reference [58].
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Figure 1. Scheme for the synthesis of [RuCp(PPh ) (CTZ)](CF SO ).
3 2 3 3
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CF SO
10 3 3
9
11
4 5 8
7 12 Cl 20
Ru N N 6 21
19
Ph P
3 2
14
13 22
Ph 3 P 18 24
23
15
17
16
Figure 2. Proposed chemical structure for [RuCp(PPh ) (CTZ)](CF SO ). 3 2 3 3
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Figure 3. TLC of the lipidic extract of T. cruzi after treatment of the parasite with the compounds under study and terbinafine. The
sterols used as standards are included. The TLC was revealed with vapors of iodine. Lane 1: RuCpCTZ, Lane 2: CTZ, Lane 3:
terbinafine, Lane 4: negative control, Lane 5: ergosterol, Lane 6: lanosterol, Lane 7: cholesterol, Lane 8: squalene. Relevant pathways
of the sterol biosynthesis are shown in the attached scheme (right).
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0 M
1 M
250 5 M
7.5 M 140
15 M
m n
0 1
) 200 2 3
3
5 0
5
M M
M
m
n
120
5 = 150 4 4 0 5 M M 4 9 100
c 5
x 50 M @
).u
(
e
100
60 M
F
rro
c
80
.a
(
F 50
I
%
60
I
0 40
540 560 580 600 620 640 0 10 20 30 40 50 60
/nm [RuCpCTZ]/M
Figure 4. Left: Fluorescence data obtained for the competitive binding study of RuCpCTZ to
{EB-DNA}. Right: Relative fluorescence intensity (%) at = 594 nm with increasing
em
RuCpCTZ concentration. (C = 20 µM, C = 10 µM, samples prepared in 2% DMSO/Tris
DNA EB
HCl medium, 30 min incubation at 37 °C).
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