Article pubs.acs.org/Organometallics
Synthesis, Structures, and Biological Studies of Heterobimetallic Au(I)−Ru(II) Complexes Involving N‑Heterocyclic Carbene-Based Multidentate Ligands Luca Boselli,†,‡ Mael̈ le Carraz,§,∥ Serge Mazères,⊥,# Lucie Paloque,§,∥ Germán González,§,∥ Françoise Benoit-Vical,†,‡ Alexis Valentin,§,∥ Catherine Hemmert,*,†,‡ and Heinz Gornitzka*,†,‡ †
CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, BP 44099, F-31077 Toulouse Cedex 4, France Université de Toulouse, UPS, INPT, F-31077 Toulouse Cedex 4, France § Université de Toulouse; UPS; UMR 152 Pharma-DEV; Université Toulouse 3; Faculté des sciences pharmaceutiques; 35 Chemin des maraîchers, F-31062 Toulouse Cedex 9, France ∥ Institut de Recherche pour le Développement; IRD; UMR 152 Pharma-DEV, F-31062 Toulouse Cedex 9, France ⊥ CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France # Université de Toulouse, UPS, IPBS, Toulouse, France ‡
S Supporting Information *
ABSTRACT: Three heterobimetallic gold(I)−ruthenium(II) complexes containing heteroditopic bipyridine−N-heterocyclic carbene (NHC) ligands were synthesized and fully characterized by spectroscopic methods and in one case by single-crystal Xray diffraction. In addition, the in vitro cytotoxic, antileishmanial, and antimalarial activities of these new heterobimetallic complexes were assessed. Moreover, the photophysical properties of two compounds have been used to localize them in tumor cells by confocal microscopy.
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rhodium,2 palladium,3 or ruthenium4 to the other site of the same triazolediylidene. These mixed-metal complexes were successfully employed in tandem reactions in which each metal carries out one catalytic transformation in a one-pot procedure. The use of poly-NHC ligands, in which two or more NHC groups are linked by variable spacers, for the formation of heterobimetallic compounds is rare because of the difficulty in prohibiting a chelating mode instead of the desired bridging coordination mode and in deprotonating the usually equal carbene precursors selectively to introduce the desired metals stepwise. Braunstein and co-workers prepared a stable free dicarbene by deprotonation of the bis(imidazolium) precursor salt and reacted it with [IrCl(cod)]2 in ethanol to give a mixture of the dinuclear Ir(I) complex and the mononuclear Ir(I)
INTRODUCTION Since the discovery of transition-metal complexes with Nheterocyclic carbenes (NHCs) by Wanzlick and Ö fele in 1968,1 NHCs have become established ligands in organometallic chemistry and homogeneous catalysis. NHCs can be easily modified by attaching functional groups at their nitrogen atoms that can act as additional donor ligands. NHC ligands with nitrogen, oxygen, phosphorus, or sulfur as donor atoms have been described. Such functionalized ligands or polycarbene systems give access to bimetallic complexes. In contrast to homobimetallic complexes, only a limited number of heterobimetallic transition-metal complexes with NHC ligands have been reported. Concerning polycarbene systems, Peris and co-workers used a dicarbene ligand to connect two different metal centers by stepwise deprotonation of one 1,2,4-trimethyl1,2,4-triazolium precursor salt. In this way, it was possible first to bind iridium to one binding site of the ligand and then © XXXX American Chemical Society
Received: November 17, 2014
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DOI: 10.1021/om501158m Organometallics XXXX, XXX, XXX−XXX
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properties, and biological activities of the here-presented complexes are discussed.
complex with its second NHC unit reprotonated. After isolation of the monometalated species, it was reacted with [RhCl(cod)]2 and Cs2CO3 to generate a heterodinuclear Ir(I)− Rh(I) complex.5 Cowie and co-workers also used mono-NHC metal complexes with a pendant imidazolium unit as precursors for bridged bis-NHC Ir(I)−Rh(I),6,7 Pd(II)−Ir(I),6 and Pd(II)−Rh(I)6,8 mixed-metal complexes. Hahn and co-workers designed tris-NHC-type ligands that could be metalated step by step, giving rise to Pd(II)−Rh(III),9 Pd(II)−Ir(III),9 Rh(III)− Rh(III)−Ir(III),10 and Rh(III)−Rh(III)−Au(I)10 complexes. Another approach for preparing heterobimetallic NHCcontaining complexes is based on the combination of NHCs with ancillary ligands. This strategy was applied by Straub and co-workers, who synthesized Pd−Au and Pd−Cu complexes using a thiol-functionalized asymmetrical bis(triazolium) precursor.11 In 2013, Peters presented a salen-bridged bisNHC system permitting the preparation of Pd−Ag, Pd−Ni, and Ag−Ni complexes.12 In 2005, Arduengo reported a Ru−Pd complex that incorporates a cyclopentadienyl-annulated imidazol-2-ylidene moiety.13 Catalano used pyridyl- and quinolyl-containing substituents on an NHC for the synthesis of Ag−Au,14 Ag−Cu,14 and Cu−Au15 compounds. Chen achieved binding of two different metal centers (Pd−Cu and Pd−Ag) using an asymmetrical 3-(1,10-phenanthrolin-2-yl)-1pyridin-2-ylmethyl)imidazolylidene.16 Most of the targeted applications of these polynuclear heterobimetallic complexes are in materials research and catalysis. Research on metal−metal interactions plays a marginal role in this case in comparison to homopolynuclear complexes. For several years we have been working on the design of functionalized mono- and bis-NHC ligands for the stabilization of Au(I) and Au(III) centers in order to study aurophilic interactions and luminescent properties of such complexes.17 Recently we focused our research on the biological activities of mononuclear gold complexes. Gold−NHC compounds have received increased attention in recent years as potential anticancer agents.18 Studies concerning the cellular uptake, intracellular distribution, and mode of action of Au(I) compounds have been done, and the major mode of action discussed for such compounds targets thioredoxin reductase.19 In some cases, luminescent ligands have been attached to the metal center in order to study mechanistic aspects using confocal microscopy techniques.20 Some examples of monoand heterobimetallic complexes in the context of cell imaging for mechanistic studies, where the metals play an essential role in the luminescence properties of the complexes, are described in the literature.21 Furthermore, some studies have reported the design and synthesis of heterometallic systems for biomedical applications.22 In our case, the most active complexes involved heteroditopic NHC ligands containing aromatic23 or aliphatic amino-functionalized side arms. These side arms can also be used to introduce a secondary transition metal in order to synthesize heterobimetallic complexes. We focused on the bipyridine (bipy)-containing side arm, a motif well-known as a ligand for transition metals. Moreover, the corresponding gold−bis(NHC) complex shows interesting biological activities. 23 Herein we present a series of Au(I)−Ru(II) heterobimetallic complexes combining a gold−NHC unit and a Ru(bipy)3 building block. Both involved metals are known to have biological activities, and the RuII(bipy)3 unit is known for its luminescence properties. Such properties could be helpful to study the mode of action of drugs. The synthesis, luminescence
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RESULTS AND DISCUSSION Synthesis and Characterizations. The carbene precursors 1 to 3 (see Scheme 1) were synthesized as described for 1 in the literature.23 In the case of methyl- and butylimidazole, an excess of imidazole was heated with 5-(bromomethyl)-2,2′bipyridine in toluene. In the case of benzylimidazole, only 1 equiv was used. The most notable features in the 1H and 13C NMR spectra of the imidazolium salts are the resonances for the imidazolium protons (H2) located between 9.37 and 10.76 ppm and the corresponding carbon atoms (C2) in the range of 134.9−139.0 ppm. The high-resolution mass spectra of all of these compounds exhibit the classical peak corresponding to [M − Br]+ cations. 2 and 3 were crystallized from CH2Cl2/ pentane and analyzed by X-ray diffraction (see the Supporting Information). The syntheses of the ruthenium complexes and the heterobimetallic Ru−Ag and Ru−Au complexes are summarized in Scheme 1. In order to obtain these heterobimetallic complexes, two synthetic strategies are possible: (i) formation of the carbene−gold unit first, followed by complexation of Ru(bipy)2 through functionalization of the gold−NHC Scheme 1. Syntheses of the Ruthenium Complexes 1Ru to 3Ru, the Ruthenium−Silver Complexes 1RuAg to 3RuAg, and the Ruthenium−Gold Complexes 1RuAu to 3RuAu
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Organometallics complex; (ii) complexation of the ruthenium by the substituted imidazolium salts first, followed by transformation of the imidazolium into the NHC−gold(I) unit. The gold−NHC can be formed under mild conditions via the silver oxide route, while the complexation reaction of ruthenium demands relatively drastic conditions (EtOH/H2O under reflux). Taking into account the fact that the gold−NHC complex is less stable than the RuII(bipy)3 complex (transmetalation or formation of gold nanoparticles can occur), we decided to use the second strategy starting with the complexation of ruthenium. 1Ru and 3Ru were formed by heating 1 and 3, respectively, in EtOH/H2O followed by addition of Ru(bipy)2Cl2. The solutions were concentrated, and anion exchange with NH4PF6 led to orange precipitates. In the case of 2Ru, the complex was obtained without anion exchange directly by evaporation of the solvents, addition of acetonitrile, and precipitation at low temperature. The 1H NMR spectra are in agreement with the expected compounds. The imidazolium protons H2 at δ = 8.90 ppm (1Ru), 10.14 ppm (2Ru), and 9.10 ppm (3Ru), respectively, show small shifts in comparison with those in compounds 1 to 3, indicating the formation of the desired complexes. This was confirmed by the peaks corresponding to [M − X]+ (X = PF6 for 1Ru and 3Ru and X = Cl for 2Ru) found by high-resolution mass spectrometry (HRMS). The silver complexes 1RuAg and 3RuAg were obtained by adding 1 equiv of Ag2O and KCl to solutions of the corresponding ruthenium complexes in acetonitrile; in the case of 2RuAg the addition of KCl was not necessary. The lack of the H2 proton signal in the 1H NMR spectra indicated the formation of the carbene complexes. The three gold complexes were synthesized by classical 1:1 transmetalation reactions between 1RuAg to 3RuAg, respectively, and Au(SMe2)Cl. All of these reactions were carried out in acetonitrile. NMR spectroscopy unequivocally demonstrates the formation of the gold(I) complexes: the 13C spectra show the resonances for the carbenic carbon atoms at 172.3, 170.2, and 171.1 ppm for 1RuAu, 2RuAu, and 3RuAu, respectively. These values are in the range of reported values for Au(I)− NHC complexes having C−Au−X (X = halide) motifs.24 X-ray Structures. The results of the X-ray structure analysis of proligands 2 and 3 are presented in the Supporting Information. Very small crystals of complex 1Ru were obtained by slow evaporation of a methanol solution. The analysis clearly shows the complexation of the ruthenium cation by three bipy units in a classical distorted octahedral geometry (Figure 1). The benzyl substituted complex 3Ru was crystallized by slow vapor diffusion of diethyl ether in a saturated solution of the compound in methanol. As in 1Ru, the ruthenium atom is coordinated by three bipy units in a classical way (Figure 2). The gold complex 3RuAu was crystallized at low temperature from a concentrated acetonitrile solution. To the best of our knowledge, this structure (Figure 3) represents the first example of a heterobimetallic Ru(II)−Au(I) complex involving an NHC ligand. The coordination geometries for the two metals are both classical: an octahedral geometry for the ruthenium(II) coordinated by three bipy units and a nearly linear coordination of the gold(I) by one NHC and one chloride anion. The gold−carbene (1.95(2) Å) and gold− chlorine (2.27(1) Å) distances are in good agreement with literature values (Au−C between 1.91 and 2.07 Å; Au−Cl between 2.26 and 2.33 Å).25 The two parts of the complex, the NHC−Au−Cl unit and the Ru(bipy)3 unit, seem to be
Figure 1. Cationic part of the molecular structure of 1Ru depicted at the 30% level for the ellipsoids. Hydrogen atoms and disordered anions have been omitted for clarity. Selected bond lengths (Å) and angles (deg): Ru−N between 2.04 and 2.07, N−Ru−N between 78 and 96.
Figure 2. Cationic part of the molecular structure of 3Ru depicted at the 30% level for the ellipsoids. Hydrogen atoms, anions, and disordered noncoordinating solvent molecules have been omitted for clarity. Selected bond lengths (Å) and angles (deg): Ru−N between 2.05 and 2.07, N−Ru−N between 78 and 97.
Figure 3. Cationic part of the molecular structure of 3RuAu depicted at the 50% level for the ellipsoids. Hydrogen atoms and anions have been omitted for clarity. Selected bond lengths (Å) and angles (deg): Ru−N between 2.05 and 2.07, C−Au 1.949(13), Au−Cl 2.272(4), C− Au−Cl 177.1(4), N−Ru−N between 78 and 96.
independent of each other from an electronic point of view, excluding electronic interactions. Luminescence Studies. All of the complexes presented in this study were investigated photophysically at standard pressure and room temperature in solution and in the solid C
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Organometallics state. The energies involved in the electronic transitions in the methyl-, butyl-, and benzyl-substituted systems did not show significant differences. Some remarkable points will be discussed for the butylsubstituted system in water (see Figure 4). The absorption
Table 1. In Vitro Antileishmanial, Antiplasmodial, and Cytotoxic Activities (IC50) of Proligands 1 to 3, Ruthenium Complexes 1Ru to 3Ru, and Heterobimetallic Ru−Au Complexes 1RuAu to 3RuAu compound
L. infantum (promastigotes) IC50 [μM]
1 2 3 1Ru 2Ru 3Ru 1RuAu 2RuAu 3RuAu reference
>50 >50 >50 >50 >50 >50 >50 >25 >50 0.01 (amphotericin B)
P. falciparum IC50 (esd) [μM] >50 17.6 (4.0) 39.3 (3.5) >50 >50 >50 25.5 (5.6) 16.1 (2.2) 15.8 (4.1) 0.19 (0.02) (chloroquine)
Hep3B IC50 [μM] − − − >100 >100 >100 30.4 17.1 18.9 7.2 (Sorafenib)
for the tests on the cancer cell line Hep3B, only the gold complexes show activities. The moderate activities (IC50 > 10 μM) of gold complexes 1RuAu to 3RuAu could only be explained by the presence of the Ru(bipy)3 unit. This phenomenon could be due to steric hindrance induced by addition of the bulky sphere of the Ru(bipy)3 unit or to the fact that we are in the presence of a neutral NHC−Au−Cl unit instead of a cationic NHC−Au−NHC one. For gold−NHC complexes, various antiproliferative effects have been evidenced as increased formation of reactive oxygen species (ROS)27 or apoptosis induction. For some complexes, a mitochondrial pathway has been demonstrated,28 and for most Au(I)−NHC compounds, thioredoxin reductase has been identified as a central target.18b In order to gain deeper insight into mechanistic aspects, the most active complex 2RuAu and the related precursor 2Ru were selected for imaging studies in Hep3B cells using confocal microscopy. The important points are cellular uptake and intracellular distribution of the complexes. For this, cells were treated with 10 μM solutions of the complexes, and the luminescence was studied at t = 0, 24, 48, and 72 h. At t = 0, a series of images showed cell-membrane labeling followed by fast cell uptake of both compounds (see the Supporting Information), excluding a steric problem for cell penetration. The images at t = 24, 48, and 72 h gave very similar information concerning the inner-cell distributions of both complexes (see Figure 5), although we showed that only 2RuAu was cytotoxic on these cells. Both compounds were localized in the cytoplasm, mostly in the peripherical area of the nuclei, but no trace could be observed in the nuclei themselves (Figure 6). In order to verify possible antimitochondrial activity of the complexes, MitoTracker (green) was used (Figure 7). This experiment showed no colocalization of the compounds in the mitochondria, most probably excluding this often-discussed mechanism for gold compounds in the present case. The emission spectra obtained for 2Ru and 2RuAu in Hep3B cells at t = 0 present a maximum at 640 nm for 2Ru and at 620 nm for 2RuAu. This difference provided the possibility to follow the evolution of the active gold complex in cells in time (see the Supporting Information). At t = 0 and 24 h, the spectra correspond to the complex 2RuAu with maxima at 620 nm. At t = 48 and 72 h, the spectra become larger and the maxima shift to 640 nm. These spectra correspond to a mixture of 2RuAu
Figure 4. Absorption and emission spectra of the butyl-substituted complexes 2Ru (green) and 2RuAu (red) and of Ru(bipy)3 (black) in water at room temperature.
spectra of the ruthenium complex 2Ru (green curve) and the heterobimetallic complex 2RuAu (red curve) exhibit the characteristic bands found (and well-known) for the Ru(bipy)3 complex (black curve). The intense band at 285 nm encompasses the ligand-centered (LC) transition, and the other two bands at 240 and 450 nm are attributed to metal-toligand charge transfer (MLCT) transitions. The emission spectra of the complexes display a broad and structureless emission band centered around 625 nm that is attributed to 3 MLCT transitions.26 Even though all of the complexes show characteristics very similar to those of Ru(bipy)3 concerning luminescence properties, it should be noted that the luminescence quantum yield in water increases by about 30% in going from the ruthenium complex 2Ru (ϕem = 0.020) to the corresponding ruthenium−gold complex (ϕem = 0.026). The lifetime calculated for the Ru−Au complex (τ = 432 ns) is about 30% longer than the one for the ruthenium complex without gold (τ = 335 ns), in good agreement with the observed quantum yield difference. Moreover, the emission maximum of 2Ru (630 nm) is slightly red-shifted in comparison with that of 2RuAu (615 nm). This shift could be due to solvatochromic effects, as a charged imidazolium unit in 2Ru is changed to a neutral carbene unit in 2RuAu. On the other side, a gold chlorine is coordinated by the carbene, leading also to strong changes in the molecule. Biological Studies. All of the compounds presented herein except for the silver complexes were tested in vitro against Leishmania infantum promastigotes, Plasmodium falciparum FcB1-Colombia strain, and the human hepatocellular carcinoma (Hep3B) cell line. The tested compounds show moderate activities or absence of activity (Table 1). Earlier studies22 have shown good antiplasmodial activity of the cationic Au(NHC)2 complex with NHC corresponding to the derivative of 1 (IC50 = 0.33 μM against P. falciparum strain FcM29-Cameroon). In these series, the proligands show low activities against Plasmodium, and the addition of Ru(bipy)2 seems to deactivate the molecules. Moreover, the corresponding gold−ruthenium complexes show some activity in the case of P. falciparum. Also, D
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Figure 6. Images of a cell treated with 2RuAu (8.5 μM, 24 h) and DAPI. (top left) Fluorescence image of DAPI (in cyan). (top right) Fluorescence image of 2RuAu (in red). (bottom left) Superposition of the two top panels. (bottom right) Transmission image of the cell.
Figure 5. (left) Fluorescence, (middle) transmission, and (right) superposed images of cells treated with 2Ru (cyan) and 2RuAu (red) at 10 μM for 24, 48, and 72 h.
and 2Ru. This suggests that the gold−ruthenium complexes are slowly transformed into the corresponding ruthenium complexes by release of gold cations to the cells, leading to cell death. These results are in good agreement with the fact that the presence of gold is essential for the cytotoxic activity. It is notable that at high 2RuAu concentrations (>50 μM), all parts of the cell (cytoplasm, organelles, nuclei, and nucleoli) are rapidly occupied by the complex, leading immediately to cell death. Such behavior has been observed by Parker and coworkers for lanthanide complexes, where concentrations beyond their IC50 led to penetration of the nuclei and lower concentrations showed a different pattern.21a−d
Figure 7. Fluorescence and transmission images of the cells treated with 2RuAu (in red, 10 μM, 24 h) and labeled with MitoTracker (in green).
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CONCLUSIONS A series of new mononuclear ruthenium(II) complexes and the corresponding heterobimetallic Ru(II)−Au(I) complexes have been synthesized. These mixed-metal complexes have been designed for two distinguished properties: photophysical properties concerning the ruthenium part and biological activities concerning the gold unit. All of the complexes show photophysical properties very similar to those of Ru(bipy)3. All of the compounds were tested for their biological activities E
DOI: 10.1021/om501158m Organometallics XXXX, XXX, XXX−XXX
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121.2 (1C, C9), 121.2 (1C, C12), 50.1 (1C, C17), 49.9 (1C, C6), 31.9 (1C, C18), 19.4 (1C, C19), 13.4 (1C, C20). MS (FAB+): m/z 293 [M − Br−]+. HRMS (ES+): calcd for C18H21N4 293.1766, found 293.1770. Anal. Calcd for C18H21BrN4: C, 57.92; H, 5.67; N, 15.01. Found: C, 58.23; H, 5.59; N, 14.98. 3-Benzyl-1-{[6-(pyridin-2-yl)pyridin-3-yl]methyl}-1H-imidazol-3ium Bromide (3). 1-Benzylimidazole (0.562 g, 3.55 mmol) and 5(bromomethyl)-2,2′-bipyridine (0.884 g, 3.55 mmol) were stirred in toluene (40 mL) at 130 °C for 24 h. After the mixture was cooled to r.t., the solvent was removed, and the solid was dried under vacuum to give a beige paste. The paste was dissolved in a small amount of CH2Cl2, and Et2O was added, leading to the formation of a fine yellow suspension and a brown oily film on the glass wall. The suspension was transferred to a round-bottom flask, and the solvent was removed. The solid was dried under vacuum to give the pure product as a yellow powder (0.986 g, 68%). The proligand was crystallized from CH2Cl2/ pentane. 1H NMR (300 MHz, CDCl3): δ 10.67 (s, 1H, H2), 8.84 (d, 1H, J = 1.8 Hz, H16), 8.55 (d, 1H, J = 4.6 Hz, H15), 8.26 (d, 1H, J = 8.2 Hz, H12), 8.18 (d, 1H, J = 8.0 Hz, H9), 8.07 (dd, 1H, J = 8.2, 2.2 Hz, H13), 7.74 (s, 1H, H5), 7.72−7.63 (m, 1H, H8), 7.38 (m, 4H, H4, H14, H20), 7.30−7.17 (m, 3H, H19, H21), 5.76 (s, 2H, H17), 5.48 (s, 2H, H6). 13C NMR (75 MHz, CDCl3): δ 156.9 (1C, C10), 155.0 (1C, C11), 149.5 (1C, C15), 149.2 (1C, C16), 137.8 (1C, C8), 136.9 (1C, C13), 136.7 (1C, C2), 132.7 (1C, C7), 129.5 (1C, C21), 129.4 (2C, C19), 129.2 (1C, C18), 128.9 (2C, C20), 124,1 (1C, C14), 122.6 (1C, C5), 122.2 (1C, C4), 121.3 (1C, C9), 121.2 (1C, C12), 53.4 (1C, C17), 50.3 (1C, C6). MS (FAB+): m/z 327 [M − PF6−]+. HRMS (ES +): calcd for C21H19N4 327.1610, found 327.1612. Anal. Calcd for C21H19BrN4: C, 61.93; H, 4.70; N, 13.76. Found: C, 61.78; H, 4.53; N, 13.77. Preparation of Ruthenium(II) Complexes. (3-Methyl-1-{[6(pyridin-2-yl)pyridin-3-yl]methyl}-1H-imidazol-3-ium)bis(2,2′bipyridine)ruthenium(II) Tris(hexafluorophosphate) (1Ru). 1 (0.300 g, 0.9 mmol) was dissolved in EtOH(aq) (95/5) (45 mL) under N2, and the solution was heated to reflux. Ru(bpy)2Cl2 (0.437 g, 0.9 mmol) was added, and the mixture was heated to reflux for 3 h. After cooling to r.t., the solution was concentrated and cooled in an ice bath. A saturated aqueous solution of NH4PF6 (3−5 mL) was added, and the formed precipitate was filtered, washed with Et2O, and dried under vacuum to give the pure product 1Ru as a bright-orange solid (0.88 g, 88%). 1H NMR (400 MHz, acetone-d6): δ 8.90 (s, 1H, H2), 8.87− 8.74 (m, 6H, Harom), 8.29−8.15 (m, 6H, Harom), 8.10−7.97 (m, 6H, Harom), 7.73 (s, 1H, H16), 7.64−7.52 (m, 6H, Harom), 5.68−5.53 (m, 2H, H6), 4.05 (s, 3H, H17). 13C NMR (75 MHz, acetone-d6): δ 157.4 (1C), 157.2 (1C), 157.1 (1C), 157.1 (1C), 157.0 (1C), 156.7 (1C), 152.1 (1C), 151.9 (1C), 151.8 (1C), 151.7 (1C), 151.7 (1C), 150.0 (1C), 138.2 (1C), 138.1 (1C), 138.1 (2C), 138.0 (2C), 137.5 (1C), 137.3 (1C), 134.9 (1C, C2), 128.1 (1C), 128.0 (1C), 127.9 (1C), 127.9 (1C), 127.8 (1C), 124.8 (1C), 124.6 (1C), 124.5 (1C), 124.4 (1C), 124.3 (2C), 122.7 (1C), 49.4 (1C, C6), 36.0 (1C, C17). MS (FAB+): m/z 955 [M − PF6−]+ (100%). HRMS (ES+): calcd for C35H31F12N8P2Ru 955.1008, found 955.1023. Anal. Calcd for C35H31F18N8P3Ru: C, 38.23; H, 2.84; N, 10.19. Found: C, 38.41; H, 3.10; N, 10.01. (3-Butyl-1-{[6-(pyridin-2-yl)pyridin-3-yl]methyl}-1H-imidazol-3ium)bis(2,2′-bipyridine)ruthenium(II) Bromide Dichloride (2Ru). 2 (0.300 g, 0.8 mmol) was dissolved in 45 mL of EtOHaq (95/5) under N2, and the solution was heated to reflux. Ru(bpy)2Cl2 (0.389 g, 0.8 mmol) was added, and the mixture was heated to reflux for 3 h. After the mixture was cooled to r.t., the solvent was removed, and the solid was washed with Et2O to give a red powder. The crude product was dissolved in a minimum volume of acetonitrile and precipitated at low temperature (one night in the freezer). This precipitation was done several times, giving pure 2Ru (0.570 g, 82%). 1H NMR (300 MHz, CD3CN): δ 10.14 (s, 1H, H2), 8.76−8.64 (m, 4H, Harom), 8.63−8.56 (m, 2H, Harom), 8.25−8.16 (m, 1H, Harom), 8.15−8.04 (m, 6H, Harom), 7.85−7.78 (m, 2H, Harom), 7.75−7.65 (m, 4H, Harom), 7.61−7.46 (m, 2H, Harom), 7.45−7.33 (m, 4H, Harom), 5.70−5.49 (m, 2H, H6), 4.23 (t, 2H, J = 7.4 Hz, H17), 1.83 (tt, 2H, J = 7.3, 7.4 Hz, H18), 1.42−1.29 (tq, 2H, J = 7.3, 7.4 Hz, H19), 0.94 (t, 3H, J = 7.4 Hz, H20). 13C NMR
against Leishmania infantum, Plasmodium falciparum, and a human hepatocellular carcinoma cell line. The compounds show low activities in comparison to the gold complex of 1. Exploring the luminescence properties of the complexes, we have shown that this is not due to steric reasons. Furthermore, we have shown that probably neither a DNA-concerning nor an antimitochondrial mechanism is at the origin of the cytotoxic effects. Moreover, the difference of 20 nm between the emission spectra of the gold−ruthenium complex and the gold-free ruthenium complex has been used to evidence the slow degradation of the gold complex in the cells. Identification of the main targets of this family of compounds and further modifications of the compounds in order to increase their biological activities are under investigation. Modifications must also be done in order to obtain bigger shift differences between the emission maxima of the gold−ruthenium compound and the gold-free complex, allowing more information to be obtained concerning the mechanisms and kinetics of the complex transformations and correlated biological activities.
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EXPERIMENTAL SECTION
Chemistry. All manipulations were performed under an inert atmosphere of dry nitrogen using standard vacuum line and Schlenk tube techniques. Glassware was dried at 120 °C in an oven for at least 3 h. Dichloromethane and acetonitrile were dried over CaH2 and subsequently distilled. All other reagents were used as received from commercial suppliers. 5-(Bromomethyl)-2,2′-bipyridine,29 3-Methyl-1{[6-(pyridin-2-yl)pyridin-3-yl]methyl}-1H-imidazol-3-ium bromide,23 1-mesitylimidazole,30 and Ru(bpy)2Cl231 were prepared as described in the literature. All reactions involving silver compounds were performed with exclusion of light. 1H (300 or 400 MHz) and 13C NMR spectra (75 or 101 MHz) were recorded at 298 K on Bruker DPX300, Bruker AV300, or Bruker AV400 spectrometers in CDCl3, CD3CN, CD3OD, acetone-d6, or DMSO-d6 as the solvent. The atom numbering for NMR assignments is shown in Scheme 2. Fast atom
Scheme 2. Numbering for NMR
bombardment (FAB) was performed with a NERMAG R10-10 spectrometer, and HRMS was performed with a Thermo Finnigan MAT 95 XL spectrometer using electrospray ionization (ESI) by the Service de Spectrométrie de Masse de ChImidie UPS-CNRS (Toulouse). Elemental analyses were carried out by the Service de Microanalyse du Laboratoire de Chimie de Coordination (Toulouse). Preparation of Imidazolium Salts. 3-Butyl-1-{[6-(pyridin-2yl)pyridin-3-yl]methyl}-1H-imidazol-3-ium Bromide (2). 1-Butylimidazole (1.6 mL, 12.4 mmol) and 5-(bromomethyl)-2,2′-bipyridine (0.884 g, 3.55 mmol) were stirred in toluene (40 mL) at 130 °C for 24 h. After cooling to r.t., the desired white/yellow precipitate was filtered off and dried under vacuum. Yield 1.02 g (77%). 1H NMR (300 MHz, CDCl3) δ 10.54 (s, 1H, H2), 8.85 (s, 1H, H16), 8.53 (d, 1H, J = 4.1 Hz, H15), 8.25 (d, 1H, J = 8.2 Hz, H12), 8.18 (d, 1H, J = 7.9 Hz, H9), 8.09 (dd, 1H, J = 8.2, 1.5 Hz, H13), 7.80 (s, 1H, H5), 7.74−7.63 (m, 1H, H8), 7.49 (s, 1H, H4), 7.25−7.14 (m, 1H, H14), 5.80 (s, 2H, H6), 4.20 (t, 2H, J = 7.3 Hz, H17), 1.88−1.68 (m, 2H, J = 7.5 Hz, H18), 1.34−1.12 (m, 2H, H19), 0.81 (t, 3H, J = 7.3 Hz, H20). 13C NMR (75 MHz, CDCl3): δ 156.7 (1C, C10), 155.0 (1C, C11), 149.5 (1C, C15), 149.2 (1C, C16), 137.8 (1C, C2), 137.0 (2C, C13), 136.6 (1C, C8), 129.5 (1C, C7), 124.1 (1C, C14), 122.6 (1C, C5), 122.5 (1C, C4), F
DOI: 10.1021/om501158m Organometallics XXXX, XXX, XXX−XXX
Article
Organometallics (75 MHz, CD3CN): δ 157.2 (1C), 157.1 (1C), 156.9 (1C), 156.8 (1C), 156.7 (2C), 152.9 (1C), 152.0 (1C), 151.6 (2C), 151.4 (1C), 151.3 (1C), 137.7−137.9 (6C), 137.6 (1C), 134.9 (1C, C2), 127.9 (1C), 127.7 (2C), 127.5 (2C), 125.0 (1C), 124.8 (2C), 124.5 (1C), 124.4 (2C), 122.7 (1C), 122.6 (1C), 49.4 (1C, C6), 48.8 (1C, C17), 31.6 (1C, C18), 19.0 (1C, C19), 12.8 (1C, C20). MS (FAB+): m/z 823 [M − Cl−]+ (20%); m/z 583 [Ru(bpy)2(Me-bpy)]+ (100%). HRMS (ES+): calcd for C38H36N8Ru(2+) 353.1058, found 353.1058 (65%); calcd for C34H28N8Ru(2+) [M − Bu]2+ 325.0745, found 325.0747. Anal. Calcd for C38H37BrCl2N8Ru: C, 53.22; H, 4.35; N, 13.07. Found: C, 52.94; H, 4.54; N, 12.95. (3-Benzyl-1-{[6-(pyridin-2-yl)pyridin-3-yl]methyl}-1H-imidazol-3ium)bis(2,2′-bipyridine)ruthenium(II) Tris(hexafluorophosphate) (3Ru). 3 (0.300 g, 0.7 mmol) was dissolved in 45 mL of EtOH(aq) (95/5) under N2, and the solution was heated to reflux. Ru(bpy)2Cl2 (0.339 g, 0.7 mmol) was added, and the mixture was heated to reflux for 3 h. After cooling to r.t., the solution was concentrated and cooled in an ice bath. A saturated aqueous solution of NH4PF6 (3−5 mL) was added, and the formed precipitate was filtered, washed with Et2O, and dried under vacuum to give the pure product 3Ru as a bright-orange solid (0.711 g, 86%). 1H NMR (300 MHz, acetone-d6): δ 9.10 (s, 1H, H2), 8.88−8.80 (m, 4H, Harom), 8.79−8.72 (m, 2H, Harom), 8.28−8.14 (m, 6H, Harom), 8.13−7.99 (m, 5H, Harom), 7.99−7.94 (m, 1H, Harom), 7.82 (s, 1H, H16), 7.63−7.44 (m, 11H, Harom), 5.66−5.60 (m, 2H, H17), 5.59−5.54 (m, 2H, H6). 13C NMR (75 MHz, CD3CN): δ 157.4 (1C), 157.0 (2C), 156.9 (3C), 156.4 (1C), 151.9 (1C), 151.8 (1C), 151.7 (1C), 151.6 (2C), 151.5 (1C), 150.6 (1C), 137.9 (3C), 137.8 (1C), 137.2 (1C), 136.3 (1C), 133.8 (1C), 133.4 (1C), 129.4 (1C), 129.3 (2C), 128.8 (2C), 127.9 (1C), 127.6 (3C), 124.8 (1C), 124.4 (1C), 124.3 (2C), 124.2 (2C), 123.0 (2C), 53.1 (1C, C6), 49.4 (1C, C17). MS (FAB+): m/z 1117 [M − PF6−]+ (100%). HRMS (ES+): calcd for C41H35F12N8P2Ru 1031.1323, found 1031.1337. Anal. Calcd for C41H35F18N8P3Ru: C, 41.88; H, 3.00; N, 9.53. Found: C, 41.56; H, 2.78; N, 9.33. Preparation of Ruthenium(II)−Silver(I) Complexes. Synthesis of [Ag(MeIm)(CH2bipy)Ru(bipy)2][Cl][PF6]2 (1RuAg). 1Ru (0.440 g, 0.40 mmol) was dissolved in 20 mL of dry acetonitrile under N2. To the solution were added 0.030 g of KCl and, after 5 min, Ag2O (0.093 g, 0.40 mmol). The solution was stirred at 40 °C for 24 h. The mixture was cooled to r.t. and filtered on Celite, and the solvent was removed under reduced pressure to give the pure product 1RuAg (0.390 g, 99%). 1H NMR (300 MHz, acetone-d6): δ 8.85−8.67 (m, 6H, Harom), 8.29−8.05 (m, 6H, Harom), 8.05−7.93 (m, 5H, Harom), 7.62−7.47 (m, 5H, Harom), 7.43 (s, 1H, H16), 7.34 (d, 1H, Harom), 7.24 (d, 1H, Harom), 5.55−5.33 (m, 2H, H6), 3.79 (s, 3H, H17). Synthesis of [Ag(BuIm)(CH2bipy)Ru(bipy)2][Cl][Br] (2RuAg). 2Ru (0.356 g, 0.415 mmol) was dissolved in 20 mL of dry acetonitrile under N2. Ag2O (0.096 g, 0.415 mmol) was added, and the solution was stirred at 40 °C for 24 h. After that, the mixture was cooled to r.t. and filtered on Celite, and the solvent was removed under reduced pressure to give the pure product 2RuAg (400 mg, 100%). 1H NMR (300 MHz, CD3CN) δ 8.87−8.60 (m, 6H, Harom), 8.25−7.94 (m, 6H, Harom), 7.93−7.65 (m, 5H, Harom), 7.58−7.32 (m, 6H, Harom), 7.09− 6.85 (m, 2H, Harom), 3.97 (t, 2H, J = 7.1 Hz, H17), 2.68 (s, 2H, H6), 1.74 (tt, 2H, J = 7.5, 7.1 Hz, H18), 1.29 (tq, 2H, J = 8.1, 7.5 Hz, H19), 1.02−0.85 (t, 3H, J = 8.1 Hz, H20). Synthesis of [Ag(BnIm)(CH2bipy)Ru(bipy)2][Cl][PF6]2 (3RuAg). 3Ru (0.446 g, 0.38 mmol) was dissolved in 20 mL of dry acetonitrile under N2. To the solution were added 0.040 g of KCl and, after 5 min, Ag2O (0.088 g, 0.38 mmol). The solution was stirred at 40 °C for 24 h. The mixture was cooled to r.t. and filtered on Celite. The solvent was removed under reduced pressure, and the solid was dried under vacuum to give the pure product 3RuAg (0.357 g, 80%). 1H NMR (300 MHz, acetone-d6): δ 8.90−8.55 (m, 6H, Harom), 8.28−8.09 (m, 5H, Harom), 8.06−7.89 (m, 6H, Harom), 7.64−7.43 (m, 7H, Harom), 7.24 (s, 6H, H16), 5.43 (s, 2H, H17), 5.34 (s, 2H, H6). 13C NMR (75 MHz, acetone-d6) δ 157.1 (1C), 157.0 (2C), 156.9 (1C), 156.7 (1C), 156.6 (1C), 151.8 (2C), 151.7 (2C), 149.0 (1C), 138.1 (3C), 137.6 (1C), 136.8 (1C), 136.5 (1C), 128.9 (3C), 128.3 (2C), 128.0 (2C), 127.9 (2C), 127.8 (1C), 127.5 (2C), 124.7 (1C), 124.4 (3C), 124.3
(2C), 124.2 (1C), 123.3 (1C), 122.7 (1C), 55.0 (1C, C6), 51.6 (1C, C17). Preparation of Ruthenium(II)−Gold(I) Complexes. Synthesis of [Au(MeIm)(CH2bipy)Ru(bipy)2][Cl][PF6]2 (1RuAu). 1RuAg (0.395 g, 0.36 mmol) was dissolved in 20 mL of dry acetonitrile under N2 and under protection from light. After the addition of Au(SMe2)Cl (0.106 g, 0.36 mmol), the mixture was stirred at r.t. overnight. The AgCl formed was filtered off on Celite and with a PTFE filter membrane (pore size 0.2 μm). The solvent was removed under reduced pressure. giving the pure product 1RuAu as an orange/red powder (0.365 g, 92%). 1H NMR (300 MHz, acetone-d6): δ 8.88−8.76 (m, 6H, Harom), 8.34−8.14 (m, 6H, Harom), 8.10−7.92 (m, 5H, Harom), 7.65−7.49 (m, 6H, Harom), 7.37 (d, J = 1.9 Hz, 1H, Harom), 7.34 (d, J = 1.9 Hz, 1H, Harom), 5.56−5.38 (m, 2H, H6), 3.88 (s, 3H, H17). 13C NMR (75 MHz, acetone-d6): δ 171.3 (1C, C2), 157.1 (1C), 157.0 (1C), 156.9 (1C), 156.8 (1C), 156.7 (1C), 156.6 (1C), 151.8 (2C), 151.7 (1C), 151.6 (2C), 148.7 (1C), 138.2 (1C), 138.1 (3C), 138.0 (1C), 137.6 (1C), 136.7 (1C), 128.2 (1C), 128.0 (2C), 127.9 (1C), 127.8 (1C), 124.7 (1C), 124.6 (1C), 124.5 (1C), 124.4 (1C), 124.3 (1C), 124.1 (1C), 123.1 (1C), 122.1 (1C), 50.8 (1C, C6), 37.9 (1C, C17). MS (FAB+): m/z 1041 [M − PF6−]+ (100%). HRMS (ES+): calcd for C35H30AuClF6N8PRu 1041.0638, found 1041.0658. Anal. Calcd for C35H30AuClF12N8P2Ru: C, 35.44; H, 2.55; N, 9.49. Found: C, 35.23; H, 2.83; N, 9.52. Synthesis of [Au(BuIm)(CH2bipy)Ru(bipy)2][Cl][PF6]2 (2RuAu). 2Ru (0.403 g, 0.47 mmol) was dissolved in 20 mL of dry acetonitrile under N2 and under protection from light. After the addition of Ag2O (0.120 g, 0.47 mmol), the mixture was stirred at 40 °C for 12 h. After the mixture was cooled to r.t., Au(SMe2)Cl (0.139 g, 0.47 mmol) was added, and the resulting mixture was stirred overnight. The AgCl formed was filtered off on Celite. The solvent was evaporated, and the solid was dried under vacuum to give the pure product as a dark-red powder (0.403 g, 81%). 1H NMR (300 MHz, CD3OD): δ 8.85−8.60 (m, 6H, Harom), 8.25−8.05 (m, 6H, Harom), 7.95−7.75 (m, 5H, Harom), 7.62−7.45 (m, 6H, Harom), 7.41−7.30 (m, 2H, Harom), 5.45−5.27 (m, 2H, H6), 4.19 (t, 2H, J = 7.0 Hz, H17), 1.84 (tt, 2H, J = 7.1 Hz, J = 7.0 Hz, H18), 1.45−1.25 (m, 2H, H19), 0.98 (t, 3H, J = 7.3 Hz, H20). 13C NMR (75 MHz, CD3OD): δ 168.7 (1C, C2), 155.6 (1C), 155.5 (1C), 155.4 (1C), 155.3 (1C), 155.2 (2C), 150.1 (1C), 149.9 (1C), 149.8 (2C), 147.4 (1C), 136.6 (2C), 136.5 (2C), 136.4 (1C), 136.2 (1C), 135.3 (1C), 126.6 (1C), 126.4 (1C), 126.3 (2C), 126.1 (1C), 123.2 (1C), 123.1 (2C), 122.9 (2C), 122.7 (1C), 120.8 (1C), 120.1 (1C), 115.4 (1C), 49.7 (1C, C6), 49.4 (1C, C17), 31.5 (1C, C18), 17.8 (1C, C19), 11.22 (1C, C20). MS (FAB+): m/z 937 [M − Cl− − Br−]2+ (100%). HRMS (ES+): calcd for C38H36AuClN8Ru(2+) 469.0734, found 469.0738. Anal. Calcd for C38H36AuBrCl2N8Ru: C, 43.32; H, 3.44; N, 10.64. Found: C, 43.69; H, 3.76; N, 10.98. Synthesis of [Au(BnIm)(CH2bipy)Ru(bipy)2][Cl][PF6]2 (3RuAu). 3RuAg (0.434 g, 0.37 mmol) was dissolved in 20 mL of dry acetonitrile under N2 and under protection from light. After addition of Au(SMe2)Cl (0.109 g, 0.37 mmol), the mixture was stirred at r.t. overnight. The AgCl formed was filtered off on Celite and with a PTFE membrane (0.2 μm). The solvent was removed under reduced pressure to give the pure product 3RuAu as an orange-red powder (0.460 g, 98%). 1H NMR (300 MHz, acetone-d6): δ 8.80 (m, 6H, Harom), 8.38−8.14 (m, 6H, Harom), 8.13−7.93 (m, 5H, Harom), 7.73− 7.34 (m, 13H, Harom), 5.65−5.33 (m, 4H, H6, H17). 13C NMR (75 MHz, CD3CN): δ 171.1 (1C, C2), 156.9 (3C), 156.8 (1C), 156.6 (1C), 156.5 (1C), 151.8 (1C), 151.7 (2C), 151.6 (1C), 151.5 (1C), 149.2 (1C), 138.0 (1C), 137.9 (1C), 137.8 (3C), 137.2 (1C), 136.4 (1C), 136.3 (1C), 129.0 (2C), 128.5 (1C), 127.9 (1C), 127.8 (2C), 127.7 (2C), 127.6 (2C), 124.5 (2C), 124.4 (1C), 124.3 (2C), 124.0 (1C), 122.7 (1C), 121.8 (1C), 54.7 (1C, C6), 51.0 (1C, C17). MS (FAB+): m/z 1041 [M − PF6−]+ (100%). HRMS (ES+): calcd for C41H34AuClN8RuPF6 1117.0953, found 1117.0977. Anal. Calcd for C41H34AuClF12N8P2Ru: C, 39.02; H, 2.72; N, 8.88. Found: C, 39.41; H, 2.98; N, 9.15. Crystallographic Data for 1Ru, 3Ru, and 3RuAu. All data were collected at low temperature using oil-coated shock-cooled crystals on a Bruker AXS APEX II diffractometer with Mo Kα radiation (λ = G
DOI: 10.1021/om501158m Organometallics XXXX, XXX, XXX−XXX
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Organometallics 0.71073 Å). The structures were solved by direct methods,32 and all non-hydrogen atoms were refined anisotropically using the leastsquares method on F2.33 1Ru: C35H31BrCl2N8Ru, Mr = 815.56, crystal size = 0.20 mm × 0.10 mm × 0.02 mm, monoclinic, space group P21/n, a = 15.238(4) Å, b = 13.917(3) Å, c = 17.217(4) Å, β = 92.49(1)°, V = 3647.8(14) Å3, Z = 4, T = 173(2) K, 19916 reflections collected, 5142 unique reflections (Rint = 0.2187), R1 = 0.0886, wR2 = 0.1929 [I > 2σ(I)], R1 = 0.2254, wR2 = 0.2560 (all data), residual electron density = 0.867 e Å−3. 3Ru: C45H49F18N8O3P3Ru, Mr = 1285.90, crystal size = 0.50 mm × 0.20 mm × 0.20 mm, monoclinic, space group P21/n, a = 12.179(2) Å, b = 26.720(5) Å, c = 17.866(4) Å, β = 109.07(3)°, V = 5495(2) Å3, Z = 4, T = 173(2) K, 29646 reflections collected, 7440 unique reflections (Rint = 0.0317), R1 = 0.0468, wR2 = 0.1220 [I > 2σ(I)], R1 = 0.0720, wR2 = 0.1350 (all data), residual electron density = 0.481 e Å−3. 3RuAu: C41H34AuClF12N8P2Ru, Mr = 1262.19, crystal size = 0.20 mm × 0.02 mm × 0.01 mm, orthorhombic, space group Pbca, a = 12.260(6) Å, b = 21.800(13) Å, c = 32.613(18) Å, V = 8717(8) Å3, Z = 8, T = 173(2) K, 111442 reflections collected, 7098 unique reflections (Rint = 0.0926), R1 = 0.0364, wR2 = 0.0646 [I > 2σ(I)], R1 = 0.0665, wR2 = 0.0728 (all data), residual electron density = 1.216 e Å−3. Photophysical Measurements. The experiments were carried out at 298 K in water solution contained in quartz cuvettes (optical path length 1 cm, Hellma). UV/vis absorption spectra were recorded with a PerkinElmer λ40 spectrophotometer. Fluorescence spectra were obtained with a PerkinElmer LS-50 spectrofluorimeter equipped with a Hamamatsu R928 phototube. Fluorescence quantum yields were measured according to the method of Crosby and Demas34 (standard used: Ru(bpy)3 in aerated water solution). Fluorescence lifetime measurements were performed using an Edinburgh FLS920 spectrofluorimeter equipped with a TCC900 card for data acquisition in time-correlated single-photon counting experiments (0.5 ns time resolution) with a D2 lamp and an LDH-P-C-405 pulsed diode laser. The estimated experimental errors are 2 nm on the band maximum, 5% on the lifetime, and 10% on the fluorescence quantum yield. Microscopy Imaging. Cell imaging was performed on an LSM 710 NLO-Meta confocal microscope with spectral detection (Zeiss, Göttingen, Germany). Images were taken through a 40×/1.2W objective. Excitation was provided by a 458 nm argon laser line, and images were recorded from 501 to 725 nm either in channel mode integrating the emitted fluorescence or in spectral mode with 9.8 nm steps. Microscopy imaging experiments were performed with Hep3B cells that were cultured in six-well plates and on glass slides (180000 cells per well) during 18 h before incubation with complexes at different concentrations for various times. For the nuclei- and mitochondriastaining experiments, Hep3B cells were incubated with 2RuAu (at concentrations of 8.5 μM (IC50/2), 17.1 μM (IC50), and 34.2 μM (2 × IC50) for 24 h. For nuclei-staining experiments, cells were washed three times in PBS, fixed for 10 min in 4% PFA, and incubated for 15 min with 2 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) at 37 °C. Excitation was provided by both a 720 nm two-photon line (DAPI) and a 458 nm argon laser line (2RuAu), and images were recorded in one pass in two channels (394−462 nm for DAPI and 516−670 nm for 2RuAu). For mitochondria-staining experiments, the MitoTracker Green probe (Invitrogen Molecular Probes) was incubated in live cells at a final concentration of 200 nM for 30 min at 37 °C in a 5% CO2 humidified incubator and then observed in one pass in two channels (478−541 nm for MitoTracker and 619−735 nm for 2RuAu) with excitation by a 458 nm laser line for both dyes. In both cases, merged images were generated to evaluate colocalization of nuclei and mitochondrial probes with the 2RuAu complex. Biology. Cell Line and Parasite Strains. The Leishmania species used in this study was L. infantum MHOM/MA/67/ITMAP-263 strain (CNR Leishmania, Montpellier, France) expressing luciferase activity. The human hepatocellular carcinoma (Hep3B) cell line was obtained from ATCC HB-8064. Cells were cultured in high-glucose DMEM containing also 10% fetal bovine serum, nonessential amino acids, and sodium pyruvate at 37 °C in a 5% CO2 humidified
incubator. Cells were routinely verified by the following tests: morphology microscopic examination, growth curve analysis, and mycoplasma detection (MycoAlert, Lonza, Basel, Switzerland). All experiments were started with low-passaged cells (
