Communication pubs.acs.org/Organometallics
Cytotoxicity Studies of Cyclometallated Ruthenium(II) Compounds: New Applications for Ruthenium Dyes Bruno Peña,†,⊥ Amanda David,†,⊥ Christiane Pavani,‡ Mauricio S. Baptista,‡ Jean-Philippe Pellois,§ Claudia Turro,*,∥ and Kim R. Dunbar*,† †
Department Department § Department ∥ Department ‡
of of of of
Chemistry, Texas A&M University, P.O. Box 30012, College Station, Texas 77842-3012, United States Biochemistry, University of São Paulo, São Paulo 05508-070, Brazil Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, United States Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio 43210-1340, United States
S Supporting Information *
ABSTRACT: The antiproliferative activity of four Ru(II) dyes incorporating the cyclometallated ligand phpy− (deprotonated 2-phenylpyridine) have been tested against HeLa cells. All of the compounds exhibit cytotoxic activity similar to that of cisplatin. The most active compound, [Ru(phpy)(bpy)(dppn)]+ (4; bpy = 2,2′-bipyridine, dppn = benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine), is 6 times more active than the platinum drug, and it is able to disrupt the mitochondria membrane potential. In addition, [Ru(phpy)(biq)2]+ (3; biq = 2,2′-biqinoline), with strong absorption at 640 nm, exhibits enhanced activity upon irradiation with 633 nm light. These findings demonstrate that coordinatively saturated cyclometallated Ru dyes have the potential to emerge as a new family of organometallic anticancer compounds, both in the dark and upon irradiation with low-energy light. The compound [Ru(phpy)(pap)(NCCH3)2]+ (5; pap = 2-(phenylazo)pyridine) was also synthesized and structurally characterized as a new precursor for the preparation of tris-heteroleptic dyes.
T
cisplatin in a number of cancer cell lines,9 and the RAPTA complexes developed by Dyson and co-workers, [(η6-arene)RuCl2(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane), are known to have selective activity on metastatic tumors in vitro and in vivo similar to that of NAMI-A.10 A structurally different series of compounds with the anionic ligand phpy− (deprotonated 2-phenylpyridine) was reported by Pfeffer,11 where [Ru(phpy)(phen)(NCCH3)2]+ (RDC11; phen = 1,10-phenanthroline) was the most active compound against A-172 glioblastoma cells11 and was more efficient than cisplatin at inhibiting the growth of various tumors implanted in mice. Interestingly, toxicity to the liver or kidneys was not observed when mice were treated with RDC11.12 Cyclometallated Os(II) complexes with good to excellent anticancer activities against A172 glioblastoma cells were also reported by the same group.13 More recently, a cyclometallated piano-stool Ru(II) compound of the type [(η6-p-cymene)RuCl(L)], where L is a phpy−benzimidazole derivative, was reported to have cytotoxicities higher than those of cisplatin against epithelial ovarian carcinoma, breast, and colon cancer cells and to induce early apoptosis.14 In light of the promising cytotoxic properties of cyclometallated Ru(II) compounds, we have studied some
he discovery of the antitumor properties of cisplatin by Barnett Rosenberg in the 1960s1 and its subsequent approval by the FDA for the treatment of testicular and ovarian cancers in 1978 had a tremendous impact on the field of inorganic chemistry. In the ensuing decades since this seminal discovery, there has been considerable progress in improving Pt-based drugs (e.g., carboplatin, oxalipatin, satraplatin, picoplatin)2 and developing other metal-based compounds that do not cause the severe side effects of cisplatin and/or do not suffer from the drawback of acquired tumor resistance associated with this drug.3 Among the many early- and latetransition-metal-based anticancer compounds,4 ruthenium (Ru) complexes have garnered a great deal of interest.5 In fact, the Ru(III) compounds NAMI-A ([H2im][trans-RuCl4(S-dmso)(Him)]; Him = imidazole, dmso = dimethyl sulfoxide) and KP1019 ([H2ind][trans-RuCl4(Hind)2]; Hind = indazole) are in phase II clinical trials. The former exhibits an impressive antimetastatic activity,6 and the latter has been selected for further development against colorectal cancer.7 In the past 10 years, the growth of bioorganometallic chemistry has led the discovery of new families of organometallic Ru(II) compounds with carcinostatic activities. Complexes with the Ru(η6-arene) scaffold are some of the most well studied.8 For example, the piano-stool complexes [(η6-arene)RuCl(en)]+ (en = 1,2-ethylenediamine) reported by Sadler exhibit anticancer activities in vitro comparable to that of © 2014 American Chemical Society
Received: January 2, 2014 Published: February 18, 2014 1100
dx.doi.org/10.1021/om500001h | Organometallics 2014, 33, 1100−1103
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derivatives and herein communicate the syntheses, characterization, and cytotoxicity data against HeLa cells of the complexes [Ru(phpy)(bpy)2]+ (1), [Ru(phpy)(phendione)2]+ (2), [Ru(phpy)(biq)2]+ (3), and [Ru(phpy)(bpy)(dppn)]+ (4) (Figure 1; bpy = 2,2′-bipyridine, phendione = 1,10-phenanthro-
Figure 1. Molecular structures of cyclometallated Ru compounds.
line-5,6-dione, biq = 2,2′-biquinoline, dppn = benzo[i]dipyrido[3,2-a:2′,3′-c]phenazine). These compounds resemble the structure of several cyclometallated Ru dyes used in dyesensitized solar cells,15 and our findings of their cytotoxicity will increase the diversity of metal-based anticancer compounds, since many of the reported Ru dyes in the literature may also display antitumor properties. In addition, the synthesis and structural characterization of [Ru(phpy)(pap)(NCCH3)2][PF6] (5; pap = 2-(phenylazo)pyridine) is presented as a new precursor for the synthesis of tris-heteroleptic cyclometallated compounds. The syntheses of compounds 1, 3, and 4 were previously reported by us16,17 (an improved synthesis of 3 is detailed in the Supporting Information). Compound 2 was prepared in a similar fashion by reacting [Ru(phpy)(NCCH3)4][PF6] with 2 equiv of phendione in refluxing EtOH. The ESI(+) mass spectrum of 2 shows only one peak at m/z 676.05 for [Ru(phpy)(phendione)2]+, and its C1-symmetric structure was confirmed by 1H NMR spectroscopy, which verified that the protons of the two phendione ligands are not magnetically equivalent. The most shielded proton resonance of 2 appears at 6.60 ppm (Figure S1, Supporting Information) and corresponds to the proton ortho to the Ru−C bond, as also observed in 1 (6.41 ppm),18 3 (6.25 ppm),17 and 4 (6.61 ppm).16 Compound 5 was prepared by reacting [Ru(phpy)(NCCH3)4][PF6] with 1 equiv of pap in CH2Cl2 at room temperature under reduced light conditions. The substitution of the acetonitrile ligands in 5 was also explored to demonstrate its suitability as precursor for expanding the structural diversity of Ru cyclometallated compounds. Reaction of 5 with 1 equiv of dcmb (4,4′-dicarboxymethyl-2,2′-bipyridine) in refluxing MeOH afforded the tris-heteroleptic compound 6. Both compounds were obtained as single geometric isomers, and their structures were unambiguously confirmed by X-ray diffraction (Figure 2, CCDC 978317 and 978318; see Tables S3−S5 and Figure S15−S16 in the Supporting Information for more details) and NMR spectroscopy (Figures S3−S7, Supporting Information). The metal centers in 5 and 6 are surrounded by five N atoms and one C atom in a distorted-octahedral environment. Their Ru1−C1 bond lengths (2.039(4) and 2.047(4) Å, respectively) are similar to those observed for related cyclometallated compounds.11,17 The Ru−N bond in the position trans to the Ru1−C1 bond is the longest in both complexes (Ru1−N4
Figure 2. Molecular structures of 5, 5a, and 6 (top) and thermal ellipsoid plots at the 50% probability level of the X-ray structures of 5 (middle) and 6 (bottom). The [PF6]− anions and H atoms have been omitted for the sake of clarity.
2.125(4) Å and Ru1−N2 2.127(3) Å, respectively), reflecting the strong trans influence of phpy−. The Ru−N bond with the azo moiety is the shortest in both compounds (Ru1−N6 1.960(3) and 1.944(3) Å, respectively) because of the greater π back-bonding into the azo moiety to stabilize the additional electron density on the metal center donated by phpy−, which is also accompanied by an elongation of the N−N bond of the azo group (1.304(5) and 1.306(5) Å, respectively) with respect to uncoordinated pap (1.25 Å).19 It is interesting to note that the coordinating moiety that is trans to the pyridyl group of the pap ligand in 5 is the ortho-metalated phenyl ring of phpy− but changes to the pyridyl ring of the phpy− ligand in 6. Such a difference in the arrangement of the ligands suggests that 5 isomerizes prior to coordination of dcmb, as was reported for the formation of the tris-heteroleptic compounds [Ru(phpy)(phen)(L)]+ (L = bpy and phen derivatives) from [Ru(phpy)(phen)(NCCH3)2]+.20 The thermal stability of 5 was investigated by 1H NMR spectroscopy in CD3CN, which revealed that one acetonitrile ligand exchanges with CD3CN at 21 °C (Figure S8, Supporting Information) but that isomerization does not occur at this temperature. The labile acetonitrile ligand is likely to be trans to the azo group, since the Ru1−N3 bond (2.056(4) Å) is longer 1101
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than the Ru1−N2 bond (2.047(4) Å). Dissociation of the second acetonitrile ligand is observed upon heating (Figure S9, Supporting Information), and it is accompanied by isomerization to a new species (5a, Figure 2). Compound 5a exhibits a doublet resonance at 5.64 ppm that corresponds to the proton ortho to the Ru−C bond (H1; Figures S10 and S11, Supporting Information). The higher field chemical shift of H1 in 5a with respect to 5 (7.76 ppm) indicates that H1 is directed toward the pyridyl ring of pap, and it appears at a higher field because of ring current effects. In addition, the proton ortho to the N atom (H8) in phpy− is deshielded (8.65 ppm) with respect to 5 (6.92 ppm), indicating that it is directed toward an acetonitrile ligand. Therefore, we propose that 5a is formed by dissociation of the pyridyl donor moiety of pap in 5 (facilitated by the strong donating properties of the ortho-metalated phenyl ring),20 followed by recoordination to the metal center in a position trans to the pyridyl group of phpy−. The viability of HeLa cells treated with compounds 1−4 was tested using the colorimetric cell viability MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the results were compared to those for cisplatin. The IC50 values (concentration of compound required to inhibit 50% of cell survival) were calculated after 2 h of incubation and are given in Table 1. The four compounds inhibited cell viability in
Figure 3. (a−c) JC-1 fluorescence (overlay of green and red channels) images of HeLa cells treated with 4 (7 μM) at (a) 0 h, (b) 1 h, and (c) 2 h. (d) Overlay of phase contrast and SYTOX green fluorescence images of HeLa cells treated with 4 (7 μM) for 2 h.
Table 1. Ru3+/2+ Redox Potentials and Cytotoxicity Data of 1−4 against HeLa Cells compd
E1/2(Ru3+/2+), V
1 2 3 4 cisplatin
+0.72 +0.93 +0.89 +0.76
1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide). The accumulation of this cationic dye in mitochondria is potential-dependent, and it is indicated by a shift in its fluorescence from green (∼525 nm) to red (∼590 nm),27 as can be observed in Figure 3a. The time lapse experiment indicated that treatment with compound 4 induces a timedependent depletion of Δψ (Figure 3a−c) which is indicated by an opposite fluorescence emission shift from red to green.27,28 These findings suggest that mitochondria are possible cellular targets of compound 4 in HeLa cells. Because of the strong absorption of 3 with a maximum at 640 nm (ε = 10500 M−1 cm−1), the complex was investigated as a potential agent for photochemotherapy (PCT). PCT has emerged as a noninvasive treatment with low systemic toxicity for the treatment and cure of early-stage lesions of endoscopically accessible tumors,29 but excitation in the 600−850 nm range is desirable for deeper tissue penetration of light.30 Figure S14 (Supporting Information) shows that cell death is not observed in the presence of 1 or 5 μM of 3, but
