Strong Influence of Ancillary Ligands Containing Benzothiazole or Benzimidazole Rings on Cytotoxicity and Photoactivation of Ru(II) Arene Complexes.

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/IC Strong Inﬂuence of Ancillary Ligands Containing Benzothiazole or Benzimidazole Rings on Cytotoxicity and Photoactivation of Ru(II) Arene Complexes Matteo Lari,†,¶ Marta Martínez-Alonso,†,¶ Natalia Busto,*,† Blanca R. Manzano,‡ Ana M. Rodríguez,§ M. Isabel Acuña,⊥ Fernando Domínguez,⊥ José L. Albasanz,‡ José M. Leal,† Gustavo Espino,*,† and Begoña García*,† † Departamento de Química, Facultad de Ciencias, Universidad de Burgos, Plaza Misael Bañuelos s/n, 09001 Burgos, Spain Departamento de Química Inorgánica, Orgánica y Bioquímica, Facultad de Ciencias y Tecnologías Químicas, IRICA and § Departamento de Química Inorgánica, Orgánica y Bioquímica, Escuela Técnica Superior de Ingenieros Industriales, Universidad de Castilla-La Mancha, 13071 Ciudad Real, Spain ⊥ CIMUS, Universidad de Santiago de Compostela, Avenida Barcelona s/n, 15782 Santiago de Compostela, Spain Inorg. Chem. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 11/01/18. For personal use only. ‡ S Supporting Information * ABSTRACT: A new family of neutral ruthenium(II) arene complexes of the type [Ru(η6-arene)X(κ2-O,N-L)] (η6-arene = p-cym, bz; X = Cl−, SCN−; HL1 = 2-(2′-hydroxyphenyl)benzimidazole, HL2 = 2-(2′hydroxyphenyl)benzothiazole) has been synthesized and characterized. The cytotoxic activity of the Ru(II) complexes was evaluated in several tumor cell lines (A549, HepG2 and SW480) both in the dark and after soft irradiation with UV and blue light. None of the complexes bearing benzimidazole (HL1) as a ligand displayed phototoxicity, whereas the complexes with a benzothiazole ligand (HL2) exhibited photoactivation; the sensitivity observed for UV was higher than for blue light irradiation. The interesting results displayed by HL2 and [Ru(η6-p-cym)(NCS)(κ2-O,N-L2)], [3a], in terms of photo cytotoxicity prompted us to analyze their interaction with DNA, both in the dark and under irradiation conditions, in an eﬀort to shed some light on their mechanism of action. The results of this study revealed that HL2 interacts with DNA by groove binding, whereas [3a] interacts by a dual mode of binding, an external groove binding, and covalent binding of the metal center to the guanine moiety. Interestingly, both HL2 and [3a] display a clear preference for AT base pairs, and this causes ﬂuorescence enhancement. Additionally, cleavage of the pUC18 plasmid DNA by the complex is observed upon irradiation. The study of the irradiated form demonstrates that the arene ligand is released to yield species such as [Ru(κ2-O,N-L2)(κ1-S-DMSO)2(μ-SCN)]2 [3c] and [Ru(κ2-O,N-L2)(κ1-S-DMSO)3(SCN)] [3d]. Such photo dissociation occurs even in the absence of oxygen and leads to cytotoxicity enhancement, an eﬀect attributed to the presence of [3d], thus revealing the potential of [3a] as a pro-drug for photoactivated anticancer chemotherapy (PACT). ■ INTRODUCTION However, Ru(III) pro-drugs are activated by reduction in physiological media,6 and thus, ruthenium(II) complexes (and especially half-sandwich derivatives) have subsequently aroused the attention of the organometallic chemistry community.7−9 In particular, the arene moiety stabilizes the lower oxidation state of ruthenium and improves lipophilicity; the chelate ligand confers additional stability by modulating the electronic properties of the metal center, and the monodentate ligand favors activation of the complex by generating a vacant coordination site that allows the binding of biomolecules.10 Interactions with DNA by forming covalent bonds with nitrogenous bases or reversible interactions (e.g., intercalation) are well-known mechanisms for ruthenium arene complexes and are thought to be responsible for the biological activity of these metal species.11,12 In recent years, the development of new drugs for cancer therapy has become one of the most important research ﬁelds. Nowadays, metal complexes with antiproliferative activity are regarded as a promising alternative to conventional organic drugs in anticancer chemotherapy. Platinum complexes, whose cytotoxic activity was discovered serendipitously in 1965 for cisplatin,1 is the ﬁrst type of metal compound to be applied in anticancer chemotherapy. Since then, several platinum drugs have been developed as antitumor drugs.2 However, platinum complexes generally have a number of undesired side eﬀects, a limited range of activity, and a certain degree of tumor resistance.3 In an eﬀort to overcome such side eﬀects, many research groups have devoted a great deal of eﬀort to the development of new complexes with metal cations other than Pt.4 This search has led to the rapid development of ruthenium(III) complexes such as KP1019, which has entered clinical trials.5 © XXXX American Chemical Society Received: August 25, 2018 A DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Inorganic Chemistry ■ Ruthenium arene derivatives can be easily tuned by changing their structural elements.13 Benzothiazole14,15 or benzimidazole16 derivatives have proved to exhibit antitumor activity. In addition, diﬀerent arene17−19 and nonarene20 ruthenium compounds with ligands containing a benzimidazole or benzothiazole core bound to a 2-pyridine unit or other organic moieties have been reported to exhibit antitumor activity and to have other biological applications.21−24 Our group has reported an SAR study on a new family of Ru(II) arene complexes of general formula [Ru(η6-arene)Cl(N∧N)] with aryldiazole ligands and these compounds showed anticancer activity.25 Moreover, a selective biological action toward the target cancer cells is highly desired for anticancer metal-drugs. Side eﬀects and systemic toxicity derived from nonselective drugs can preclude their clinical use. Photodynamic therapy (PDT)26 and photoactivated anticancer chemotherapy (PACT)27 are well-developed techniques to overcome these drawbacks; the latter technique targets the localized activation of a pro-drug by means of a source of light, with the aim of limiting side eﬀects. Interestingly, many ruthenium complexes exhibit photoactive properties prone to be exploited in anticancer strategies;28,29 among these, some arene complexes have shown promising photoreactivity.30,31 Bearing the above in mind, two diﬀerent 2-(2′-hydroxyphenyl)benzazole molecules (see Chart 1) have been used as pro-ligands Article RESULTS AND DISCUSSION Synthesis and Characterization of New Ruthenium Complexes. The synthesis of the new half-sandwich Ru(II) chlorido-derivatives of type [Ru(η6-arene)Cl(O∧N)], [1a], [1b], [2a], and [2b], was achieved by reacting overnight at room temperature the appropriate starting dimeric material [RuCl(μ-Cl)(η6-arene)]2 (arene = p-cymene or benzene) with the corresponding pro-ligand (HL1 = 2-(2′-hydroxyphenyl)-1Hbenzimidazole, HL2 = 2-(2′-hydroxyphenyl)benzothiazole), and triethylamine in methanol or methanol/acetonitrile (see Scheme 1). The Ru(II) isothiocyanato-analogs of general formula [Ru(η6-arene)(NCS)(L2)] ([3a] and [3b]) were synthesized by a related protocol, which includes an additional stirring period at 70 °C in the presence of excess KSCN to achieve the Cl−/SCN− metathesis reaction. All of the complexes are chiral at the metal site (C1 symmetry), and thus, they were obtained as racemates (RRu plus SRu) in moderateto-good yields in the form of yellow, orange, or brown powders. The chlorido-derivatives are soluble in common organic solvents, whereas the isothiocyanato derivatives are only sparingly soluble in these solvents (see details in the Synthesis of New Complexes subsection). Interestingly, crystals of complex [3c] were obtained from a solution of [3a] or [3b] in DMSO/acetone. The new derivatives were fully characterized by spectroscopic and analytical methods. In the cases of [1a], [1b], [2a], and [3c], the corresponding crystal structures were solved by X-ray diﬀraction. Comprehensive assignment of the signals in the NMR spectra was performed from 2D experiments, except for [3b] due to its extremely low solubility. In particular, the 1 H NMR spectra of all complexes contain signals for the protons of the O∧N ligands, L1 and L2, which, due to OH deprotonation and metal coordination, remain deshielded when compared to those of the free pro-ligands. The [1a], [2a], and [3a] derivatives gave rise to spectroscopic patterns that are compatible with the presence of two diastereotopic methyls and four inequivalent aromatic protons in the p-cymene ring, as one would expect for molecules with C1 symmetry. Moreover, complexes [1a] and [1b] each display a broad signal in CDCl3 at δ 10.86 and 10.67 ppm, respectively, and this is attributed to the NH group of L1. The 1H−1H NOESY spectra of complex [1a] showed exchange peaks between the arene hydrogen atoms H2 ↔ H6 and H3 ↔ H5, and the methyl groups of the isopropyl unit, which suggest a transient Cl− decoordination process that involves inversion of the conﬁguration at the Ru center (RRu ↔ SRu).36,37 This process cannot be observed for the benzene derivative [1b]. In addition, the 13C{1H} NMR spectra are fully consistent with the molecular structures established by 1H NMR spectroscopy. Interestingly, the chemical shift (δC) of the signal for the SCN− ligand of [3a] in DMSO-d6 is 135 ppm, which suggests the presence of an N-bonded SCN− ligand according to the literature data,38 (δ(M-SCN) < 131 ppm < δ(M-NCS)). Moreover, the IR spectrum of [3a] shows a strong peak at 2090 cm−1 for the C−N stretching frequency, which is also in agreement with a Ru−NCS coordination mode. Typically, wavenumbers higher than 2100 cm−1 are assumed to correspond to S-bonded isomers (ν(M−SCN) > 2100 cm−1 > ν(M−NCS)).39−41 For complex [3b], we assume the same coordination mode on the basis of its C−N stretching frequency in the IR spectrum, which appears at 2098 cm−1. However, the low solubility of [3b] prevented us from conﬁrming this structure by 13C{1H} NMR spectroscopy. Chart 1. Pro-ligands Used in This Work to obtain ruthenium arene complexes. Recently, semiempirical calculations on HL1 and HL2 have been reported32 and zinc and copper complexes were synthesized to analyze their luminescent properties33,34 or their activity toward Alzheimer’s disease.35 However, the antitumor activity of ruthenium complexes with these ligands has not been addressed to date. We report here on a new family of neutral Ru(II) halfsandwich complexes of formula [Ru(η6-arene)X(N∧O)] with the anionic ligands L1 and L2 (Chart 1). To analyze the cytotoxicity of these complexes against cancer cells and to establish useful structure−activity relationships, two diﬀerent arenes (benzene, bz, and p-cymene, cym), two leaving groups (Cl− and SCN−), and two ancillary ligands that diﬀer only in one group were selected. After deprotonation, the ligands adopt a bidentate chelate coordination mode through the N and O atoms, Ru(N∧O). The cytotoxicity of these complexes was successfully tested in the human tumor cell lines A549 (lung), HepG2 (liver), and SW480 (colon) in the dark and upon irradiation with both UV and blue light. The results obtained revealed the potential of [Ru(η6-pcym)(NCS)(κ2-O,N-L2)] as a photoactivated chemotherapy agent. DNA studies suggest that a dual mode of binding is feasible, namely covalent binding to guanine and groove binding. Moreover, photocleavage of plasmid DNA occurred when an aqueous solution of this complex was irradiated with light, thus transforming the initial complex into a more active derivative. B DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Scheme 1. Synthesis of Ruthenium Complexes (Ar = Arene) Figure 1. ORTEP diagrams for the molecular structures of (A) [1a], (B) [1b], (C) [2a], and (D) [3c] solved by X-ray diﬀraction. For the sake of clarity, some hydrogen atoms are omitted. Thermal ellipsoids are shown for 30% probability. The FAB+ mass spectra contain sets of peaks with m/z values and isotopic distributions that are consistent with the molecular ions, [M]+, as well as with the cationic fragments [M − Cl]+ or [M − NCS]+. Molar conductivity (ΛM) measurements were performed at room temperature (20−22 °C) in acetonitrile solution (10−3 M) for soluble complexes ([1a], [1b], [2a], and [3a]). The ΛM values are very low (1.9−13.0 S cm2 mol−1), and this is in agreement with the nonelectrolyte nature of the new complexes.42 Solid State Characterization. The molecular and crystal structures of [1a], [1b]·CH3OH, [2a]·0.5H2O, and [3c] were determined by X-ray diﬀraction. The crystallographic data are given in the Supporting Information (Tables S1−S5), and the corresponding ORTEPs are provided in Figure 1. A selection of bond distances, angles, and other geometric parameters is provided in Table 1 and Table S2. The corresponding unit cells of the mononuclear derivatives [1a] and [2a] contain the two enantiomers, RRu and SRu, stemming from the stereogenic nature of the metal center. By contrast, the unit cell of [1b] C DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Table 1. Selected Bond Distances (Å) and Angles (Deg) For Compounds [1a], [1b]·CH3OH, and [2a]·0.5H2O distance/angle [1a] [1b]·CH3OH Ru1−Cl1 Ru1−N1 Ru1−O1 O1−Ru1−N1 O1−Ru1−Cl1 N1−Ru1−Cl1 2.4312(8) 2.076(2) 2.068(2) 83.72(9) 87.15(6) 83.89(7) 2.4289(8) 2.086(3) 2.069(3) 82.86(9) 87.35(6) 86.03(6) [2a]·0.5H2Oa 2.408(12) 2.114(3) 2.090(2) 82.9(1) 84.8(8) 85.57(8) 2.399(1) 2.105(3) 2.073(2) 83.4(12) 85.51(8) 87.37(8) a Values for the two independent molecules found in the unit cell of [2a]·0.5H2O. The Ru−N bond distances are 2.035(3) Å (Ru−NCS) and 2.118(3) Å (Ru−N∧O). Lastly, the Ru−O bond distance is 2.047(3) Å. The geometry of the Ru−N−C−S units deviates slightly from linearity since the Ru−N−C angles are 164.3(3)°, that is, below 180°, whereas the SCN angles are 179.6(4)° and the Ru−S−C angles are 104.5(1)°. Moreover, in the eightmembered Ru2(SCN)2 metallacycle, all of the atoms essentially lie in the same plane. The angle formed between the benzothiazole and hydroxyphenyl planes is 7.5°. An analysis of the respective 3D crystal structures revealed the presence of π−π interactions in complexes [1a], [2a], and [3c] involving pairs of ligands L1 or L2 (Table S5). In the case of [1a], this interaction, which extends along the crystallographic b axis, is triple and involves the three rings of L1 arranged in a head-to-tail disposition (Figure S1). In the case of [2a], the interaction is established between the ligands of two independent molecules and involves the benzene ring of one ligand and the thiazole ring of the other (Figure S2). In the case of [3c], both L2 ligands of the dinuclear molecule are entangled in π−π interactions and, in this way, a chain that extends along the crystallographic a axis is formed (Figure S3). The interaction is double for every pair of ligands, which are arranged in a head-to-tail disposition, and involves the hydroxyphenyl ring of one ligand and the thiazole ring of the other. Additionally, for [1a], [1b], and [2a], hydrogen bonds involving the chloride anions or oxygen atoms of the complexes are formed. Likewise, the crystal structure of [1b] features one CH3OH molecule per asymmetric unit, which takes part in hydrogen bonds both as a donor and as an acceptor, with three diﬀerent ruthenium entities to provide additional stability to the 3D crystal network. Interestingly, in [3c] there is an S-π interaction between the sulfur atom of the thiocyanate bridge and the phenolate ring of the O∧N ligand (see Figure S4 and Table S4, S-centroid = 3.97 Å). This kind of interaction is very common in proteins (e.g., cysteine moieties) and the usual S-centroid distance is 3.9 Å.46,47 Cellular Uptake. The cellular uptake of the six synthesized complexes in SW480 cells was evaluated by means of ICP-MS (Figure 2). In light of the gathered results, it is clear that the leaving group has no eﬀect on the ability of these complexes to get into the cells. By contrast, the Ru accumulation inside the cells depends mainly on the ancillary ligand since complexes bearing L2 are more internalized than those with L1, and second, on the arene moiety being the complexes with p-cymene two-fold more accumulated than benzene derivatives. Cytotoxic Activity. The cytotoxicity of the ligands and their complexes, except the insoluble derivative [3b], was investigated toward diﬀerent human cancer cell lines: A549 (lung carcinoma), HepG2 (liver carcinoma), and SW480 (colon adenocarcinoma) at 24 h of incubation time, both in the dark and upon irradiation. The IC50 (μM) values (concentration required to inhibit 50% of the cell growth) obtained in the dark and after only contains the enantiomer SRu. Indeed, this complex crystallizes in the orthorhombic chiral space group P212121, suggesting that [1b] has undergone spontaneous chiral resolution upon crystallization from a racemic solution.43,44 The three metallic congeners adopt the expected half-sandwich three-legged piano-stool geometry, and the arene ring has a π-bonded η6-coordination mode, whereas the anionic 2-(2′hydroxyphenyl)benzimidazolate or 2-(2′-hydroxyphenyl)benzothiazolate ligands assume a bidentate-chelate coordination mode (κ2-N,O). A chloride anion occupies the last site in the coordination sphere of Ru(II). The unit cell of [2a] contains two independent molecules (diﬀerent enantiomers), which diﬀer slightly in their geometric features (Table 1). Moreover, the benzene ring of [1b] exhibits rotational disorder in such a way that two diﬀerent rotamers are detected in diﬀerent unit cells. In all of the structures, the O∧N chelate ring features an envelope conformation. This is a common feature of sixmembered metallacycles comprising at least one atom with sp3 hybridization, that is, the O atom in these complexes. In addition, the aromatic rings of the respective ligands (L1 or L2) did not exhibit coplanarity between each other, with angles between the benzimidazole (or benzothiazole) and hydroxyphenyl planes ranging from 16 to 20°. The Ru-centroid distances fall in a narrow range (1.66−1.67 Å). The Ru−Cl lengths (2.399(1)−2.4312(8) Å) are close to the upper limit of the characteristic range found in the Cambridge Structural Database (CSD), probably owing to the strong σ-donor nature of the chelate anionic ligand, O∧N. Indeed, the Ru−O distances (2.068(2)−2.090(2) Å) are slightly shorter than the Ru−N distances (2.076(2)−2.114(3) Å) in all cases. The unit cell of [3c] contains six neutral dinuclear units with the formula [Ru(L2)(κ1-S-DMSO)2(μ-SCN)]2 and all of these units have the ΛΔ conﬁguration, which is the meso form. The formation of these dinuclear units from a solution of [3b] in DMSO involves the replacement of the arene with DMSO molecules followed by dimerization through two bridging SCN− groups in a head-to-tail disposition (μ-SCN, Figure 1). Both of the Ru(II) centers have a distorted octahedral coordination environment. The coordination sphere for each ruthenium atom is completed with a 2-(2′-hydroxyphenyl) benzimidazolate ligand, which adopts a bidentate-chelate coordination mode (N∧O), and two S-bonded dimethyl sulfoxide molecules in a cis arrangement. The crystallization of [3c] from a solution of [3b] in DMSO demonstrates the easy loss of the arene moiety and its replacement by S-bonded DMSO molecules. Compound [3c] also crystallized from a DMSO solution of [3a]. A search in the CSD of related crystal structures containing the [Ru(SCN)]2 unit showed only one record for thiocyanato-bridged ruthenium complexes.45 The Ru−S distances for the thiocyanate (2.487(1) Å) are longer than those for the S-bonded DMSO molecules (2.258(1)−2.265(1) Å). D DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 3. Calculated photoindex (PI = IC50 dark/IC50 irr) values for the Ru(II) complexes in A549, HepG2, and SW480 cancer cells. (A) Complexes. (B) Ligands. Stuﬀed bars for UV (5 min, λ = 365 nm, 20 mW/cm2) and striped bars for blue (20 min, λ = 460 nm, 5.5 mW/cm2) light irradiation. Figure 2. Ruthenium accumulation in SW480 cells treated with 3 μM solutions of the synthesized complexes during 24 h. soft irradiation with UV (at 365 nm, 20 mW/cm2 for 5 min) and blue (at 460 nm, 5.5 mW/cm2 for 20 min) light are listed in Table 2. HL1 and its derivatives [1a] and [1b] display negligible cytotoxic activity against the studied tumor cell lines. Although the cytotoxicity displayed by HL2 is low, coordinated to ruthenium it increases sharply, [2b] being the most cytotoxic derivative in the dark. The results obtained in the dark point up the inﬂuence of the ancillary ligand (S substituent in HL2 versus NH in HL1) as a key factor for cytotoxicity with no signiﬁcant diﬀerences in terms of biological activity as a function of the leaving group. As to the photo cytotoxicity of the ligands and the metal complexes, the calculated photoindex values (PI) are plotted (Figure 3) for both UV and blue light irradiation in the studied tumor cell lines. Interestingly, none of the complexes bearing benzimidazole as a ligand (HL1) displayed phototoxicity. On the contrary, the complexes with HL2 ligand exhibit certain degree of photo activation under the soft irradiation conditions; the sensitivity observed for UV is higher than for blue light irradiation, [3a] being the most active complex against the irradiated tumor cell lines (Figure 3A). As for the ligands, there is no signiﬁcant irradiation eﬀect on the cytotoxicity of HL1, whereas HL2 is very sensitive to UV irradiation in all the cellular lines studied and its cytotoxicity remains unaltered with blue light irradiation. Figure 3B shows the calculated PI values for both ligands upon UV irradiation. This interesting result prompted us to analyze the physicochemical properties of complex [3a] and its HL2 pro-ligand in solution by studying more in detail this complex with an important DNA biological target. Physicochemical Properties of HL2 and [3a] in Water. A study of the physicochemical properties and the reactivity of 2-(2′-hydroxyphenyl) benzothiazole (HL2) and [3a] was conducted to determine the role played by the ligand in the reactivity of the metal complex. HL2 was only poorly soluble (μM concentrations) in buﬀer solution, I = 6.5 mM (NaClO4), pH = 7.0 and 2% DMSO/ H2O (v:v). The UV−vis absorption spectrum displays two maxima at 288 and 327 nm and a 460 nm band is visible in the emission spectra. Hence, HL2 displays a large Stokes shift of the emission peak (Figure S5). Interestingly, a kinetic process was recorded both in absorbance and ﬂuorescence modes (Figure 4) for concentrations CL > 3 μM, CL being the molar concentration of HL2. The well-deﬁned isosbestic points at 272 and 343 nm in the spectral curves denote two species in equilibrium (Figure 4A). Figure 4B shows the evolution with time undergone by the ﬂuorescence spectra; the band at 460 nm gradually vanishes, producing a shoulder, while a new band emerges at 508 nm. According to the literature,48 the ﬂuorescence spectrum of the keto-enamine tautomer shows a strong emission band at ∼535 nm and a weak shoulder at 456 nm. This ﬁnding demonstrates that the process illustrated in Figure 4B corresponds to the enol-imine to keto-enamine conversion (eq 1) induced by ESIPT (excited state intramolecular proton transfer) eﬀect. It follows that a dual emission occurs from mixtures, with the keto-enamine form emitting at longer wavelengths. The contribution of the isomers primarily depends on the DMSO content. Actually, the tracks at 385 nm in the UV−vis absorption spectra (Figure 4A, inset) and at 460 nm in the emission spectra (Figure 4B, inset) reveal that the reaction rate diminishes when the DMSO content increases and becomes very slow above 10% DMSO. The rate constants obtained from ﬁtting of a monoexponential function to the kinetic traces Table 2. IC50 (μM) Values for Ligands HL1 and HL2, Complexes [1a]−[3a], [1b]−[3b], and Cisplatin (CDDP) in Cell Lines A549, HepG2, and SW480 after 24 h Incubation Time, in the Dark and upon Irradiation with UV (5 min, λ = 365 nm, 20 mW/cm2) or Blue Light (20 min, λ = 460 nm, 5.5 mW/cm2) A549 Hep G2 SW480 compound dark UV blue dark UV blue dark UV blue HL1 HL2 [1a] [1b] [2a] [2b] [3a] CDDP >100 87.7 ± 4.2 >100 >100 74.9 ± 5.7 16.5 ± 1.2 83.1 ± 6.2 46.8 ± 2.3 >100 15.3 ± 1.3 >100 >100 41.8 ± 4.5 13.2 ± 1.1 41.2 ± 5.3 >100 90.2 ± 5.6 >100 >100 43.5 ± 2.9 12.3 ± 0.9 34.1 ± 2.4 71.5 ± 2.1 80.7 ± 3.1 >100 >100 24.0 ± 1.7 4.2 ± 0.2 23.8 ± 4.3 32.7 ± 1.5 57.8 ± 1.4 17.0 ± 1.3 >100 >100 13.7 ± 0.6 4.4 ± 0.5 13.9 ± 1.3 67.8 ± 1.4 79.0 ± 4.8 >100 >100 20.2 ± 1.0 4.8 ± 1.0 17.2 ± 3.6 >100 >100 >100 >100 10.4 ± 1.1 7.7 ± 1.1 8.7 ± 1.5 41.2 ± 1.8 >100 12.2 ± 0.7 >100 >100 8.3 ± 0.4 6.0 ± 0.7 5.4 ± 0.7 >100 >100 >100 >100 6.2 ± 1.0 4.9 ± 1.0 4.6 ± 0.6 E DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 4. Kinetics of HL2 in buﬀer solution. (A) Absorbance versus time for the experiment at 0.2% DMSO; inset: track at 385 nm for diﬀerent DMSO contents. (B) Fluorescence versus time example; inset: track at 460 nm for diﬀerent DMSO contents. CL = 10 μM and λexc = 325 nm. are 0.0068 s−1, 0.0043 s−1, and 0.00066 s−1 at 1, 2, and 10% DMSO, respectively. In aqueous solution (2% DMSO), NaClO4 and neutral pH, the UV−vis spectra recorded for [3a] as a function of time, show ﬁrst-order kinetic pattern (k = 4.0 × 10−4 s−1, Figure S6A) related to aquation process. In the same conditions, we have observed aquation also for [3b] (k = 6.5 × 10−4 s−1, Figure S6B), but not for the remainder complexes studied. These values resemble aquation processes of other ruthenium complexes.25 For [3a], substitution of SCN- by Cl- was observed in the presence of 4 mM NaCl, with rate constant 1.5 × 10−3 s−1, but aquation of Cl−Ru was not observed. A recent contribution by Sadler49 reveals that the aqua arene complexes with N∧O chelating ligands are very stable and the kinetics of aquation from the corresponding chlorido complexes are faster than from complexes bearing N∧N ligands. Actually, the observed reaction is put down to SCN− release and substitution by a H2O molecule, according to eq 2. From now on, the species formed are denoted as aqua-[3a]. No kinetics of exchange of SCN− with DMSO was observed: for formation of some ruthenium hydroxo-compounds.25 Note that, even though the pKa values for HL2 and the complex are similar, the acid−base equilibria concern diﬀerent functional groups, namely the hydroxo group and the leaving water group for HL2 and aqua-[3a], respectively: [(p − cym)RuL2(H 2O)]+ + H 2O Ka′ XooY [(p − cym)RuL2(OH)] + H3O+ Below pH = 11, kinetic eﬀects were absent. At pH = 11, a slow kinetic eﬀect (data not shown) was observed; as the new peaks formed are the same as those of the anion ligand form (Figure S7B), we assumed that highly basic conditions can disrupt the binding between the metal and the L2 ancillary ligand. The prevailing forms for HL2 and aqua-[3a] were characterized under the buﬀer conditions used. The cytotoxicity of some organometallic Ru(II) complexes can be ascribed to DNA binding; therefore, to shed some light into the underlying mechanism of the aqua-[3a]/DNA interaction upon irradiation, we undertook the study of the DNA binding of the HL2 proligand and the aqua-[3a] complex in the dark. Interaction of HL2 and Aqua-[3a] with DNA. First, we conducted ﬂuorescence titrations of polynucleotides with HL2. The presence of CT-DNA and poly(dGdC)2 gave rise to a strong red shift in the emission of HL2 (Δλem = 55 nm) and, in the presence of poly(dAdT)2, a sudden ﬂuorescence enhancement was also observed (Figure 5A). Since the emission bands centered at 460 and 508 nm are related to the enol-imine and keto-enamine tautomers, respectively, Figure 5A shows that in fresh HL2 solution only the enol tautomer is present. With the addition of DNA, a new band, which corresponds with keto tautomer emission, appears. Actually, Figure 5B shows that the ﬂuorescence enhancement of HL2 is much higher in the presence of the p(dAdT)2 sequence than in the presence of p(dGdC)2 or CT-DNA. The results provided by ﬂuorometric titrations are endorsed by other complementary techniques. Actually, the HL2/CTDNA system exhibited a slight decrease both in the circular dichroism bands (Figure S9A) and viscosity measurements (Figure S9B). Moreover, HL2 did not alter the melting temperature of the DNA double-stranded conformation (Figure S9C). These results point out that keto-HL2 tautomer displays selectivity toward AT base-pairs, concurrent with binding to the minor groove.53,54 Most minor groove binders (netropsin, distamycin, Hoechst 33258, berenil, 40,6-diamidino-2-phenylindole, and SN-6999) are prone to interact with [(p − cym)RuL2(NCS)] + H 2O k V [(p − cym)RuL2(OH 2)]+ + SCN− (2) Since aqua-complexes are species generally more reactive than their chlorido precursors,50 the experiments were carried out after completing the aquation of [3a]. Acid−Base Behavior of HL2 and Aqua-[3a]. Regarding pH, ﬂuorescence enhancement of HL2 was observed for increasing pH values (Figure S7A). In addition, a notable variation of the absorbance spectra was recorded for diﬀerent pH values (Figure S7B). The pKa value obtained by means of the Henderson−Hasselbalch equation,51 9.4 ± 0.1, refers to the deprotonation of the phenol group (eq 3). This value concurs with the pKa obtained for other (hydroxyphenyl) benzothiazoles (for example, for 2-(3′-hydroxyphenyl)benzothiazole, pKa = 9.5 and, for 2-(4′-hydroxyphenyl)benzothiazole pKa = 8.8).52 Ka HL2 + H 2O XooY L2− + H3O+ (4) (3) As for aqua-[3a], the absorbance spectra recorded for increasing pH values show changes and two isosbestic points at 290 and 330 nm (Figure S8), ascribable to formation of the hydroxo-compound [(p-cym)Ru(L2)(OH)], according to eq 4. The pKa′ value 9.2 ± 0.1 obtained from the track at 303 nm (Figure S8, inset) indicates that the complex is present in the aqua form at neutral pH. Similar pKa values have been reported F DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 5. (A) Comparison of the ﬂuorescence spectra of the titration of HL2 with diﬀerent polynucleotides at CP/CL = 10. (B) Titration track at λem = 500 nm for the HL2/p(dAdT)2, HL2/CT-DNA, and HL2/p(dGdC)2 systems; the ﬂuorescence intensity F is divided by F0, that is, the ﬂuorescence intensity of the free ligand at λem = 500 nm. CL = 2.5 μM, CP = 0−180 μM, and λex = 355 nm. Figure 6. (A) Example of kinetic spectra for the interaction between the aqua-[3a] complex (D) and dGMP (P), CD = 14 μM, CP/CD = 70; inset: track at 345 nm and ﬁtting (red line) by monoexponential function. (B) Fitting of the rate constants by the 1/τ = k1CP equation; 2% DMSO, I = 6.5 mM (NaClO4), pH = 7.0 (NaCac), and T = 25.0 °C. Figure 7. (A) Example of kinetic spectrogram for the interaction between the aqua-[3a] complex (D) and CT-DNA (P), CD = 14 μM, CP/CD = 45; inset: track at 340 nm. (B) Fitting of eq 6 to the data pairs (red line); 2% DMSO, I = 6.5 mM (NaClO4), pH = 7.0 (NaCac) and T = 25.0 °C. plot displays close-to-zero intercept, indicating that an irreversible reaction between aqua-[3a] and dGMP is at work, yielding the [3a]/dGMP complex (eq 5). The slope value provides the reaction rate constant k1 = (0.20 ± 0.01) M−1 s−1: AT-rich sequences, bringing about only a slight distortion of the double helix.55 Concerning the aqua-[3a] complex, prior to the study of its interaction with DNA, kinetic studies by absorbance measurements were performed in the presence of deoxyadenosine-5′monophosphate (dAMP) and deoxyguanosine-5′-monophosphate (dGMP) in excess of nucleotides. No signal variation was detected for aqua-[3a] with dAMP, whereas for the aqua[3a]/dGMP system a slow kinetic eﬀect was perceptible. The reaction caused hypochromism at 383 nm and a hyperchromic eﬀect at 340 nm in the aqua-[3a] spectra, with an isosbestic point at 353 nm (Figure 6A). Actually, various ruthenium arene complexes have been reported to bind guanine through the N7 site by substitution of their leaving groups.56,57 The time constants (1/τ) increased linearly with the increase in the dGMP concentration (CP) (Figure 6B). In addition, the k1 aquo‐[3a] + dGMP → [3a] /dGMP (5) We also studied the reaction between aqua-[3a] and dGMP by means of 1H NMR and 31P{1H}-NMR spectroscopy. New sets of peaks emerged in the presence of dGMP. In particular, for the [3a]/dGMP system, a new set of proton peaks was recorded after 3 days, suggesting a slow reaction between the complex and dGMP (Figure S10). In addition, a small signal in the 31P NMR spectrum appeared for [3a]/dGMP (Figure S11). Hence, we can hypothesize the presence of a minor species in which ruthenium is bound to the phosphate group. Moreover, G DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 8. (A) Fluorescence titration of aqua-[3a] by addition of increasing amounts of CT-DNA (P). (B) Titration track at λem = 500 nm for the aqua-[3a]/p(dAdT)2, aqua-[3a]/CT-DNA, and aqua-[3a]/p(dGdC)2; the ﬂuorescence intensity F is normalized for the value at λem = 500 nm of the free complex, F0. CD = 2.5 μM, CP = 0−150 μM, I = 6.5 mM, pH = 7.0, λex = 355 nm, 2% DMSO, and T = 25.0 °C. Figure 9. (A) Irradiated aqua-[3a]/pUC18 samples, I. Lane 1: Molecular weight marker. Lane 2: pUC18 Irradiated control (CI), tirr = 250 min. Lanes 3−8: aqua-[3a]/pUC18 (I), tirr = 0(D), 50, 100, 150, 200, 250 min. Lane 9: pUC18 Dark Control (CD). CD = 20 μM, CP = 20 μM, [NaClO4] = 4.0 mM, [NaCac] = 2.5 mM, pH = 7.0 and λirr = 325 nm. Incubation at T = 37 °C, overnight. (B) Quantiﬁcation of the cleavage. K1CP 1 = k2 τ 1 + K1CP a second more intense peak is ascribed to the major Ru−N species. Regarding the interaction aqua-[3a]/CT-DNA, certain variation of the absorbance spectra with time was also observed. Figure 7A shows the spectral kinetic curves, with an isosbestic point at 359 nm. The inset shows the absorbance versus time track at 340 nm. The rate constants (1/τ) were obtained from ﬁtting of a monoexponential function to the data pairs (inset red line). The spectral changes are similar to those observed for the aqua-[3a]/dGMP system (Figure 6A); hence, a similar interaction with the guanine moiety can be surmised. However, in this case, the increase in the rate constant versus CP (Figure 7B) is nonlinear, diﬀerently from that observed with dGMP (Figure 6B). This kinetic behavior agrees well with a fast preassociation mechanism of the metal complex to the nucleic acid via noncovalent binding, which modulates the kinetic rate of the covalent binding to the guanine.58 Noncovalent interactions are generally fast, whereas covalent binding to the nitrogenous bases is slower. Therefore, we can assume: (i) the aqua-[3a] species reacts quickly with CT-DNA to form the noncovalent PD complex, K1 being the equilibrium constant of this step, and (ii) the PD complex converts into covalent PD* in a second unimolecular, irreversible step, k2 being the rate-determining constant (eq 6). Bearing this in mind, eq 7 was ﬁtted to the 1/τ versus CP data-pairs, obtaining the parameters K1 = (4 ± 1) × 103 M−1 and k2 = (5.8 ± 0.5) × 10−5 s−1: K1 k2 fast slow P + D XoooY PD ⎯⎯⎯→ PD* (7) To determine the type of interaction that governs the formation of PD, sets of experiments with freshly prepared solutions were carried out. The ﬂuorometric titration of aqua-[3a] with CT-DNA (Figure 8) caused a similar ﬂuorescence light-switch and shifted the maximum emission to a wavelength longer than that observed for the HL2/CT-DNA system (Figure 5A). The increase in the emission (or light-switch) in the presence of DNA has been veriﬁed for other ruthenium complexes.59,60 The respective titration experiments of [3a] with poly(dAdT)2 and poly(dGdC)2 exhibit preference for AT base-pairs, as observed for the HL2 ligand. Similarly to HL2, few modiﬁcations were observed by circular dichroism, viscometry, and melting temperature, agreeing with the minor groove binding hypothesis (Figures S12A−C). These results indicate that the binding of HL2 to the groove promotes the fast interaction observed for aqua-[3a] to form PD. Groove binding has been veriﬁed for other ruthenium arene complexes.18 In conclusion, a dual mode of binding of aqua-[3a] to CTDNA was observed: fast binding to the minor groove, governed by the ancillary chelating ligand (HL2) to give PD. The groove binding would promote the association of the metal complex to the nucleic acid and then covalent interaction with guanine occurs to give PD* in a two-step mechanism (eq 6). Recently, our group reported a stable bifunctional interaction (covalent and partially intercalated) between the [(η6-p-cymene)Ru(κ2N,N-2-pydaT)]2+ fragment and CT-DNA, where both interactions are present concurrently.61 The diﬀerence in the type of the complexes formed with DNA, groove binding for (6) H DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 10. UV−vis absorption kinetics of photorelease of p-cymene from the [3a] complex in (A) DMSO (Inset: track at 412 nm ﬁtted by a monoexponential curve, 1/τ = 5.7 × 10−4 s−1) and (B) buﬀer solution (2% DMSO, aqua-[3a]), I = 6.5 mM (NaClO4), and pH = 7.0 (Inset: Track at 383 nm). CD = 20 μM, λirr = 325 nm, and T = 25.0 °C. Figure 11. 1H NMR spectra of an irradiated solution of [3a] in DMSO-d6. Blue circle, [3a] complex; red circle, [3d] complex; black square, free p-cymene. HL2 ligand, and bifunctional binding for aqua-[3a], has provided a body of evidence about the key role played by the metal regarding DNA interaction due to its ability to bind covalently with the guanine N7 site. Photoreactivity of Aqua-[3a] Complex. Photo reactivity of the aqua-[3a] ruthenium complex was evaluated by cleavage of the pUC18 plasmid DNA. The photo cleavage was evaluated by conversion of the plasmid native supercoiled form (Form I) to the circular form (Form II), by means of eq 9 (see Methods subsection). Separation of the diﬀerent forms was performed by electrophoresis (Figure 9A). Figure 9B shows certain degree of cleavage, which increased with enhancement of the irradiation time at λ = 325 nm. To assess the oxygen dependence of the DNA cleavage, new photo cleavage experiments were conducted in the presence of scavengers of reactive oxygen species (ROS). Actually, no cleavage decrease was found in the samples containing L-histidine (for 1O2), DMSO (for OH•), and superoxide dismutase (SOD, for O2−) (Figure S13). Magennis et al.62 had reported similar nondependence on oxygen of photo cleavage of the plasmid DNA in the presence of a ruthenium complex (which releases its arene under nitrogen atmosphere). To properly elucidate if the DNA cleavage is due only to DNA binding of the aqua-[3a] complex or to changes in the complex upon irradiation, deoxygenated solutions of aqua-[3a] were irradiated, observing important changes in its absorbance spectra (Figure 10), which suggests alterations in its structure. Furthermore, under the same experimental conditions the kinetic rate is faster in DMSO (Figure 10A) respect to buﬀer solution (Figure 10B). The stability of [3a] in DMSO-d6 was monitored by 1H NMR for 3 months under exposure to ambient light and N2 atmosphere at room temperature. No signiﬁcant changes were observed after 24 h. However, after 3 months, signals of free p-cymene (see Figure S14) and a new set of peaks for a new species, denoted as [3d], were detected along with the resonances of [3a], which reveals a slow decomposition process that involves arene loss. Moreover, irradiation of a [3a] sample in DMSO-d6 with an arc lamp source of 325 nm speeds up the process in such a way that degradation was completed after 29 h (Figure 11). Again, resonances for free p-cymene (labeled with black squares) and a new Ru(II) complex bearing L2 ([3d], orange circles) were observed by 1H NMR in the ﬁnal mixture. The molecular structure of [3d] (Scheme 2) was elucidated by FAB-MS experiments. In particular, the resulting peaks indicate that p-cymene was replaced by three DMSO molecules: 470 ([3d-2DMSO-d6]+), 496 ([3d-NCS-DMSO-d6]+), 554 ([3d-DMSO-d6]+), 580 ([3d-NCS]+), 638 ([3d]+) Da (see spectra in Figures S15−S17). Release of p-cymene from I DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Scheme 2. Proposed Photoreaction Found in DMSO Involving Release of p-Cymene Ligand that aqua-[3a] is able to interact with DNA also in the dark suggests a complex mechanism for its biological activity, bearing in mind that the observed arene photodissociation can be easily related to the plasmid DNA photo cleavage. Interestingly, DNA cleavage occurs even in the presence of ROS scavengers, conﬁrming an oxygen-independent mechanism. Therefore, these results add interest toward such type of complexes for photoactivation chemotherapy strategies. The possibility of tuning the properties of the complex with arylbenzazoles or adding new ancillary ligands that can promote further interactions could be of interest for new syntheses of PACT (photoactivated chemotherapy) molecules. other Ru(II) arene complexes has been reported in previous studies.63,64 The photodissociation of the arene and its replacement by solvent molecules leads to labile positions in the coordination sphere of the metal center, which are prone to interact with DNA nucleobases; therefore, we postulate that these interactions could be responsible for the observed photocleavage. Hence, [3a] is a potential O2-independent PACT agent that accomplishes its biological activity through photoinduced dissociation of the arene from the metal center to give [3d], which, in turn, would be the actual species responsible for the observed photoactivity. Arene photodissociation has been observed for all the complexes bearing L2 as ancillary ligand (Figure S18), regardless of the arene moiety or leaving group; the metallic products that stem from irradiation are able to cleave plasmid DNA (Figure S19). However, complexes bearing L1 are unable to release the arene moiety after irradiation and cannot cleave DNA. These features conﬁrm arene− photodissociation as the underlying mechanism for the observed photoreactivity. The high cytotoxicity of HL1 in the dark indicates that our future strategy should be based on designing PACT (photo activated chemotherapy) molecules able to release this ligand, that is, chemotherapeutic agents with potential dual activity when both the free ligand (HL1) and the metal fragment exhibit cytotoxic action.27,65 On the other hand, the high photo cytotoxicity of HL2 can be of interest in the design of photodynamic therapeutic agents. ■ MATERIALS AND METHODS Materials. Starting Materials. RuCl3·xH2O was purchased from Apollo Scientiﬁc Ltd. and used as received. [(η6-Arene)Ru(μ-Cl)Cl]2 (arene = p-cym or bz,) were prepared according to literature procedures.66 The ligands 2-(2′-hydroxyphenyl)-1H-benzimidazole (HL1) and 2-(2′-hydroxyphenyl)benzothiazole (HL2) were purchased from Aldrich and used without further puriﬁcation. Deuterated solvents were obtained from SDS and Euriso-top and deaerated by freezing-vacuum cycles and in dry nitrogen atmosphere. Occasionally, some of them were also dried with molecular sieves (MS). Lyophilized calf thymus (CT) DNA sodium salt was purchased from Sigma-Aldrich. It was dissolved in bidistilled water and sonicated to obtain a mean length of 1000 bp, conﬁrmed by electrophoresis assay. Standardization of stock solutions was performed spectrophotometrically (ε = 13 200 cm−1 M−1 in bp at λ = 260 nm, I = 0.1 M (NaCl), pH = 7.0, and T = 25.0 °C). Poly(deoxyadenylicdeoxythymidylic) acid sodium salt (p(dAdT) 2 ), and poly(deoxyguanylic-deoxycytidylic) acid sodium salt (p(dGdC)2) were purchased from Sigma and the concentration was checked by UV−vis spectra, using ε = 13 400 cm−1 M−1 in base pairs (bp) at λ = 260 nm for p(dAdT)2 and ε = 16 600 cm−1 M−1 in bp at λ = 254 nm for p(dGdC)2, I = 0.1 M (NaCl), pH = 7.0, and T = 25.0 °C.67 Plasmid pUC18 (2686 bp) for the photocleavage study was extracted from bacteria and puriﬁed by means of a HP Plasmid Midi Kit (OMEGA Biotek, VWR). The concentration of the polynucleotides is always expressed in molarity base pairs, and denoted as CP. 2′-Deoxyguanosine-5′-monophosphate (5′-dGMP) was purchased from Sigma-Aldrich (purity of 99%) and used without further puriﬁcation. Sodium cacodylate trihydrate ((CH3)2AsOONa·3H2O, NaCac, purity 98%) and sodium perchlorate monohydrate (NaClO4· H2O, purity 98%) were purchased from Fluka, and they were just dissolved in water to obtain stock solution. All the experiments between aqua-[3a] and mono- and polynucleotides were carried out in double distilled water, at a ﬁxed ionic strength (I) and pH. Working solutions had I = 6.5 mM and pH = 7.0. Methods. All synthetic manipulations were carried out under an atmosphere of dry, oxygen-free nitrogen using standard Schlenk techniques. The solvents were distilled from the appropriate drying agents and degassed before use. Elemental analyses were performed ■ CONCLUSIONS We have synthesized and fully characterized a series of new neutral complexes of type [Ru(η6-arene)X(κ2N,O)] using arylbenzazoles as N,O ligands (HL1 = 2-(2′-hydroxyphenyl)benzimidazole, Z = NH, HL2 = 2-(2′-hydroxyphenyl)benzothiazole, Z = S). The cytotoxic activity was tested for all the complexes in various cell lines in the dark and after soft irradiation with UV and blue light, revealing that the ancillary ligand (only substituent Z is changed) is a key factor for the observed cytotoxicity. Complexes bearing L1 (Z = NH) are not cytotoxic in the dark or under UV and blue light irradiation. However, complexes bearing L2 as the ancillary ligand (Z = S) are cytotoxic in the dark and can be photoactivated both with UV and blue light. The most interesting results in terms of photoindex, corresponding to [3a] complex and HL2 ligand, were studied in depth. The stability study of complex [3a] in DMSO and water revealed the arene loss after irradiation with UV light, even in an oxygen-free atmosphere. This fact conﬁrmed that the process is only and exclusively light-dependent. The observation J DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry with a LECO CHNS-932 microanalyzer. IR spectra were recorded on a Nicolet Impact 410 (within the frequency range 4000−400 cm−1), and in a Jasco FT/IR-4200 spectrophotometers as KBr pellets. FAB mass spectra (position of the peaks in DA) were recorded with an Autospec spectrometer. The isotopic distribution of the heaviest set of peaks matched very closely that calculated for the formulation of the complex cation in all cases. Conductivity measurements were carried out with a CRISON 522 conductometer, connected to a conductivity cell CRISON 52 92 with platinum electrodes. The solutions of the complexes (10−3 M) in acetonitrile (dielectric constant = 36.2 S cm2 mol−1) were prepared in 5 mL volumetric ﬂasks and measured in test tubes. NMR samples were prepared under nitrogen atmosphere by dissolving the suitable amount of compound in 0.5 mL of the respective oxygen-free deuterated solvent, and the spectra were recorded at 298 K (unless otherwise stated) on a Varian Unity Inova-400 (399.94 MHz for 1 H; 161.9 MHz for 31P; 100.6 MHz for 13C). Typically, 1D 1H NMR spectra were acquired with 32 scans into 32K data points over a spectral width of 16 ppm. 1H and 13C{1H} chemical shifts were internally referenced to tetramethylsilane via 1,4-dioxane in D2O (δ = 3.75 and 67.19 ppm, respectively) or via the residual 1H and 13C signals of the corresponding solvents according to the values reported by Fulmer et al.68 Chemical shift values are reported in ppm and coupling constants (J) in hertz. The splitting of proton resonances in the reported 1H NMR data is deﬁned as s = singlet, d = doublet, t = triplet, st = pseudotriplet, q = quartet, sept = septet, m = multiplet, bs = broad singlet. 2D NMR spectra such as 1H−1H gCOSY, 1H−1H NOESY, 1H−13C gHSQC, and 1H−13C gHMBC were recorded using standard pulse sequences. A standard unit calibrated with methanol as a reference controlled the probe temperature (±1 K). All NMR data processing were carried out using MestReNova v10.0.2−15465. pH Measurements. Desired pH of working solutions was reached by adding small aliquots of concentrated solution of HClO4 and NaOH, using a Metrohm 16 DMS Titrin pH meter, with a glass electrode containing a 3 M KCl solution. The calibration of the instrument was attained through nine buﬀer solutions. Spectrophotometric Experiments. UV−vis spectra were recorded by means of a Hewlett-Packard 8453A spectrophotometer (Agilent Technologies), with diode-array detector and coupled with a computer-assisted temperature-control system. Kinetic absorbance study of the interaction between aqua-[3a] with 5′-dGMP and CTDNA was performed recording the spectra at a deﬁned time interval and the kinetic curve was treated at a speciﬁc wavelength. Circular Dichroism Equipment. Circular dichroic spectra were recorded with a MOS-450 Bio-Logic dichrograph (Claix, France), at diﬀerent CD/CP (CP = 5.0 × 10−5 M), CD, CP, and L being the analytical concentrations of the complex, ligand, and polynucleotide, respectively. The spectra were recorded from 200 to 600 nm, with an acquisition rate of 0.5 nm s−1. Viscosity. The elapsed time of 3 mL of DNA solution passing through a capillar was determined in an Ubbelhode microviscometer. Viscosity measurements allows one to determine the elongation of the DNA using eq 8,69 where t0, t1, and t2 are the elapsed time of the solvent, DNA and complex/DNA solutions (CP = 2.0 × 10−4 M), respectively. The temperature was kept at 25.0 °C by an external water thermostat: jij η zyz jj zz jj η zz k 0{ 1/3 ji t − t 0 zyz z = jjj 2 j t1 − t 0 zz k { a double beam spectrophotometer Evolution 300 UV−vis (ThermoScientiﬁc). The connection allows irradiation of samples at a deﬁned wavelength (λirr) for a deﬁned time period (tirr) and, then, recording the spectrum. Thus, the kinetics of a photoreaction can be monitored. Moreover, the system was used to irradiate the samples for the photocleavage proofs at diﬀerent irradiation times and wavelengths. Cleavage Assay. The separation and detection of the plasmid forms was achieved with an electrophoretic apparatus, coupled with a UV lamp. The solvent was the 1% TBE buﬀer and the gel 1% TBE containing 1% agarose. After irradiation, the samples were incubated for 14−16 h at 37.0 °C in a KS 4000 I control incubator (IKA) and, then, 10 μL of the solution (Plasmid concentration: 20 μM) was mixed with 2 μL of loading buﬀer (Bio-Rad, Glycerol 25%) and loaded in the hole. Runs were performed at 5 V/cm for 90 min. To visualize the DNA with the lamp, ethidium bromide was previously added to the gel. Densitometry data were obtained with ImageJ program, and a quantitative value of the cleavage parameter (Cleavage) was obtained through eq 9,70 where DI, DII, and DIII are the areas of the supercoiled, circular, and linear forms, respectively. For DI, a correction factor of 1.4 was applied for the decreased ability of the dye to intercalate inside the supercoiled form:71 Cleavage = DII + 2DIII DI + DII + 2DIII (9) X-ray Crystallography. Single crystals were obtained by slow evaporation from a [1b] solution in methanol and from a [2a] solution in methanol/water. As for [1a], single crystals were grown by slow diﬀusion of n-hexane into a dichloromethane solution of this complex. In the case of dinuclear complex [3c], a monocrystalline sample was achieved from a solution of the parent mononuclear compound [3b] in DMSO/acetone as a result of arene/DMSO exchange. A summary of the crystal data collection and reﬁnement parameters for all compounds is given in Table S1 in the Supporting Information. Single crystals of [1a], [1b]·CH3OH, [2a]·0.5H2O, and [3c] were mounted on a glass ﬁber and transferred to a Bruker X8 APEX II CCD-based diﬀractometer equipped with a graphite-monochromated Mo Kα radiation source (λ = 0.71073 Å). The highly redundant data sets were integrated using SAINT72 and corrected for Lorentz and polarization eﬀects. The absorption correction was based on the function ﬁtting to the empirical transmission surface as sampled by multiple equivalent measurements with the SADABS program.73 The software package WINGX, version 2014.1,74 was used for space group determination, structure solution, and reﬁnement by full-matrix leastsquares methods based on F2. A successful solution by direct methods provided most non-H atoms from the E map. The remaining non-H atoms were located in an alternating series of least-squares cycles and diﬀerence Fourier maps. All non-H atoms were reﬁned with anisotropic displacement coeﬃcients. Except the N−H for [1a] and [1b]·CH3OH, all hydrogen atoms were placed using a “riding model” and included in the reﬁnement at calculated positions. CCDC reference numbers for [1a], [1b]·CH3OH, [2a]·0.5H2O, and [3c] are 1815683, 1815684, 1815685, and 1815686, respectively. Cellular Uptake. SW480 cells were seeded in 12-well plates (1.5 × 105 cells/well) with DMEM medium supplemented with 10% newborn calf serum and 1% amphotericin−penicillin−streptomycin solution and incubated at 37 °C under 5% CO2 atmosphere for 24 h. Then cells were exposed to 3 μM of the studied metal complexes during 24 h. Before analysis, cells were washed three times with DPBS (Dulbecco’s phosphate buﬀered saline), then they were harvested. The pellets were resuspended in 1 mL of DPBS and 10 μL was used to count cells by means of an automated cell counter (TC20, BioRad). Then cells were digested for ICP-MS with 65% HNO3 during 24 h. Finally, solutions were analyzed in a 7700 ICP-MS (Agilent Technologies). Data are reported as the mean ± the standard deviation (n = 3). MTT Assay. Approximately 1 × 104 of A549, HepG2 or SW480 cells were cultured in 200 μL of culture medium per well (DMEM medium), supplemented with 10% newborn calf serum and 1% amphotericin−penicillin−streptomycin solution in 96-well plates, and 1/3 (8) Diﬀerential Scanning Calorimetry (DSC). Thermal denaturation study was carried out by means of a Nano-DSC (TA, Waters LLC, New Castle, USA). The working solutions (solutions at diﬀerent CD/CP, CP = 4.0 × 10−4 M) were degassed before injection in the equipment. The system was pressurized at 3 atm and the solutions were heated from 20 to 110 °C at 1 °C min−1 scan rate. Illumination System. The study of photodissociation was carried out by means of an illuminator. Brieﬂy, it consists of a Xenon short arc lamp (Ushio) coupled with a monochromator. The intensity of the radiation is regulated by means of the power lamp (ﬁxed to 150 W) and the slits of the monochromator. This system was connected with K DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Synthesis of [(η6-Benzene)RuCl(κ2-O,N-L1)], [1b]. The synthesis was performed as with [1a] in the presence of the ligand 2-(2′hydroxyphenyl)benzimidazole (0.0843 g, 0.400 mmol), [RuCl2(bz)]2 (0.0998 g, 0.200 mmol), and Et3N (58 μL, 0.420 mmol) in methanol/ acetonitrile (10:2 mL). Red−brown powder. Yield: 69.1 mg (0.163 mmol, 49%). Mr (C19H15ClN2ORu) = 423.8630 g/mol. Anal. Calc. for C19H15ClN2ORu·(H2O)1.5: C 50.61; H 4.02; N 6.21. Found: C 50.14; H 3.61; N 6,40 1H NMR (400 MHz, CDCl3, 25 °C) δ 10.86 (s, 1H, HN−H), 7.56 (d, J = 8.4 Hz, 1H, Hf), 7.18 (m, 2H, Hc, H3′), 7.04−6.98 (m, 1H, He, or H5′), 6.94 (m, 2H, H6′, He, or H5′), 6.78 (t, J = 7.3 Hz, 1H, Hd), 6.46 (t, J = 6.8 Hz, 1H, H4′), 5.56 (s, 6H, Hbz) ppm. FT-IR (KBr, cm‑1) selected bands: 3450(w, νN−H), 3053 (w, νCH), 2961 (w, νCH), 1621 (m, νC−N), 1600 (s, νCC), 1538 (m), 1479 (vs, νCN), 1460 (m), 1445 (m), 1310 (m), 1265 (s, νC−O), 1139 (m, δN‑Hip), 1038 (w), 1006 (w), 856 (w), 765 (s, δN‑Hoop), 750 (s, δC‑Hoop). MS (FAB+): m/z (%) = 424 (5) ([M]+), 389 (90) ([M-Cl]+). Molar Conductivity (CH3CN): 13.0 S cm2 mol−1. Solubility: soluble in methanol, dichloromethane, chloroform, dimethyl sulfoxide, and acetone. Slightly soluble in water. Synthesis of [(η6-p-Cymene)RuCl(κ2-O,N-L2)], [2a]. The synthesis was performed as with [1a] in the presence of the ligand 2-(2′hydroxyphenyl)benzothiazole (0.0743 g, 0.327 mmol), [RuCl2(pcym)]2 (0.0999 g, 0.163 mmol), and Et3N (48 μL, 0.347 mmol) in methanol (10 mL). Yellow−orange powder. Yield: 85 mg (0.171 mmol, 52%). Mr (C23H22ClNORuS) = 497.0216 g/mol. Anal. Calc. for C23H22ClNORuS·(H2O)0.5: C 54.59; H 4.58; N 2.77; S 6.34 Found: C 54.79; H 4.42; N 3.19; S 5.79. 1H NMR (400 MHz, CDCl3, 25 °C) δ 8.47 (d, J = 8.4 Hz, 1H, Hf), 7.78 (dd, J = 8.0, 0.6 Hz, 1H, Hc), 7.54 (ddd, J = 8.4, 7.3, 1.2 Hz, 1H, He), 7.49 (dd, J = 7.9, 1.7 Hz, 1H, H3′), 7.39 (m, 1H, Hd), 7.26 (m, 1H, H5′), 7.18 (dd, J = 8.5, 1.2 Hz, 1H, H6′), 6.60 (ddd, J = 8.0, 6.9, 1.2 Hz, 1H, H4′), 5.49 (d, J = 6.0 Hz, 1H, H2, or H6), 5.33 (d, J = 5.9 Hz, 1H, H2, or H6), 5.30 (d, J = 5.8 Hz, 1H, H3, or H5), 5.23 (d, J = 5.6 Hz, 1H, H3, or H5), 2.68 (sept, J = 6.9 Hz, 1H, H7), 2.04 (s, 3H, H10), 1.14 (d, J = 6.9 Hz, 3H, H8, or H9), 1.10 (d, J = 6.9 Hz, 3H, H8, or H9) ppm. 13 C{1H} NMR (101 MHz, CDCl3, 25 °C) δ 169.5 (s, 1C, C1′), 166.9 (s, 1C, Ca), 153.1 (s1C, Cg), 134.0 (s, 1C, C5′), 131.7 (s, 1C, Cb), 129.6 (s, 1C, C3′), 127.1 (s, 1C, Ce), 125.5 (s, 2C, Cf, Cd), 124.4 (s, 1C, C6′), 121.5 (s, 1C, Cc), 121.3 (s, 1C, C2′), 116.1 (s, 1C, C4′), 103.3 (s, 1C, C1 o C4), 97.5 (s, 1C, C1 o C4), 83.0 (s), 81.24 (s), 81.1 (s), 80.60 (s), 30.9 (s, 1C, C7), 23.1 (s, 1C, C8 or C9), 21.6 (s, 1C, C8 or C9), 18.8 (s, 1C, C10) ppm. FT-IR (KBr, cm‑1) selected bands: 3033 (m, νCH), 2958 (w, ν−CH), 1597 (s, νC−N), 1540 (s, νCC), 1491 (vs, νCN), 1463 (s), 1441 (s), 1420 (m), 1332 (s), 1213 (s, νC−O), 1153 (s, νCS), 1128 (w), 1033 (w), 875 (w), 832 (w), 753 (s, δC‑Hoop). MS (FAB+): m/z (%) = 497 (14) ([M]+), 462 (61) ([M-Cl]+). Molar Conductivity (CH3CN): 3.4 S cm2 mol−1. Solubility: soluble in acetone, dichloromethane, chloroform, methanol, and acetonitrile and partially soluble in ethanol; slightly soluble in water. Synthesis of [(η6-Benzene)RuCl(κ2-O,N-L2)], [2b]. The synthesis was performed as with [1a] in the presence of the ligand 2-(2′hydroxyphenyl)benzothiazole (0.0913 g, 0.402 mmol), [RuCl2(bz)]2 (0.1001 g, 0.200 mmol), and Et3N (58 μL, 0.420 mmol) in methanol/ acetonitrile (8:2 mL). Yellow powder. Yield: 134.6 mg (0.305 mmol, 77%). Mr (C19H14ClNORuS) = 440.9144 g/mol. Anal. Calc. for C19H14ClNORuS·H2O: C 50.17; H 3.37; N 3.52; S 6.57 Found: C 49.73; H 3.51; N 3.05; S 6.99. 1H NMR (400 MHz, CDCl3, 25 °C) δ 8.53 (d, J = 8.3 Hz, 1H, Hf), 7.80 (d, J = 8.0 Hz, 1H, Hc), 7.57 (t, J = 7.7 Hz, 1H, He), 7.50 (d, J = 7.8 Hz, 1H, H3′), 7.42 (t, J = 7.6 Hz, 1H, Hd), 7.33−7.27 (m, 1H, H5′), 7.24 (d, J = 8.2 Hz, 1H, H6′), 6.64 (t, J = 7.2 Hz, 1H, H4′), 5.63 (s, 6H, Hbz) ppm. 13C{1H} NMR (101 MHz, CDCl3, 25 °C) δ 169.9 (s, 1C, C1′), 167.4 (s, 1C, Ca), 152.9 (s, 1C, Cg), 134.3 (s, 1C, C5′), 131.8 (s, 1C, Cb), 129.6 (s, 1C, C3′), 127.4 (s, 1C, Ce), 125.6 (s, 1C, Cd), 125.2 (s, 1C, Cf), 123.7 (s, 1C, C6′), 121.9 (s, 1C, C2′), 121.6 (s, 1C, Cc), 116.5 (s, 1C, C4′), 83.7 (s, 6C, Cbz) ppm. FT-IR (KBr, cm‑1) selected bands: 3056 (w), 3038 (w, νCH), 3006 (w, νCH), 1599 (s, νC−N), 1545 (m, νCC), 1489 (vs, νCN), 1467 (s), 1440 (s), 1420 (m), 1336 (m), 1217 (s, νC−O), 1155 (m, νCS), 1125 (w), 1036 (w), 832 (w), 753−748 incubated at 37 °C under 5% CO2 atmosphere. Cells were treated with diﬀerent concentrations of the tested drugs and incubated for 1 h. Then cells were irradiated or not with UV light (at 365 nm, 20 mW/cm2 for 5 min) and with blue light (at 460 nm, 5.5 mW/cm2 for 20 min). After 24 h overall incubation, the treatment was retired and cells were incubated with 100 μL of MTT (3-(4,5-dimethyltiazol2-yl)-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich) dissolved in culture medium (500 μg/mL) for a 3 h further period. At the end of the incubation, the formazan was dissolved by adding 100 μL of solubilizing solution (10%SDS and 0.01 M HCl) to each well. Then plates were incubated at 37 °C with soft agitation. After 18 h, absorbance was read at 590 nm in a microplate reader (Cytation 5 Cell Imaging Multi-Mode Reader, Biotek Instruments, USA). Four replicates per dose were included. The concentrations that produced 50% inhibition of cell viability (IC50) were calculated from MTT data using nonlinear regression of the GraphPadPrism Software Inc. (version 6.01) (USA). Synthesis of New Complexes. The atom numbering for the ligands and the p-cymene ring is reﬂected in Chart 2. Chart 2. Atom Numbering Synthesis of [(η6-p-Cymene)RuCl(κ2-O,N-L1)], [1a]. In a 100 mL Schlenk ﬂask, the ligand 2-(2′-hydroxyphenyl)benzimidazole (0.086 g, 0.409 mmol) was added to a solution of [RuCl2(p-cym)]2 (0.1251 g, 0.204 mmol) in degassed methanol (10 mL). Et3N (58 μL, 0.416 mmol) was then added, and the mixture was stirred at room temperature for 20 h and under nitrogen atmosphere. The solution was concentrated, and water was then added to precipitate the product and remove Et3NHCl. The solid was washed with cold diethyl ether (3 mL). The resulting red−brown powder was dried under vacuum. Yield: 142.8 mg (0.298 mmol, 73%). M r (C 2 3 H 2 3 ClN 2 ORu) = 479.9702 g/mol. Anal. Calc. for C23H23ClN2ORu·H2O: C 55.47; H 5.06; N 5.63 Found: C 55.13; H 4.99; N 5.98. 1H NMR (400 MHz, CDCl3, 25 °C) δ 10.67 (s, 1H, HN−H), 7.43 (d, J = 8.1 Hz, 1H, Hf), 7.13 (d, J = 7.8 Hz, 1H, Hc), 7.01 (d, J = 7.6 Hz, 1H, H3′), 6.91−6.81 (m, 3H, He, H6′, H5′), 6.63 (t, J = 7.5 Hz, 1H, Hd), 6.32 (t, J = 6.8 Hz, 1H, H4′), 5.38 (d, J = 5.8 Hz, 1H, H3, or H5), 5.35 (d, J = 5.8 Hz, 1H, H2, or H6), 5.29 (d, J = 5.7 Hz, 1H, H6, or H2), 5.20 (d, J = 5.7 Hz, 1H, H5 or H3), 2.34 (sept, J = 6.9 Hz, 1H, H7), 2.07 (s, 3H, H10), 0.91 (d, J = 6.9 Hz, 3H, H8 or H9), 0.80 (d, J = 6.8 Hz, 3H, H9 or H8) ppm. 13C{1H} NMR (101 MHz, CDCl3, 25 °C) δ 167.3 (s, 1C, C1′), 148.2 (s, 1C, Ca), 142.0 (s, 1C, Cg), 133.6 (s, 1C, Cb), 132.0 (s, 1C, C5′), 127.3 (s, 1C, C3′), 122.8 (s, 1C, Cd), 122.7 (s, 1C, C6′), 122.5 (s, 1C, Ce), 117.5 (s, 1C, Cf), 116.0 (s, 1C, C4′), 115.9 (s, 1C, C2′), 113.3 (s, 1C, Cc), 101.5 (s, 1C, C1), 97.7 (s, 1C, C4), 83.0 (s, 1C, C2 or C6), 81.8 (s, 1C, C3 or C5), 79.9 (s, 1C, C5 or C3), 79.8 (s, 1C, C6 or C2), 30.5 (s, 1C, C7), 22.8 (s, 1C, C8 or C9), 21.4 (s, 1C, C9 or C8), 18.8 (s, 1C, C10) ppm. FT-IR (KBr, cm‑1) selected bands: 3053 (w, νCH), 2961 (w, ν−CH), 1621 (m, νC−N), 1600 (s, νCC), 1532 (m), 1479 (vs, νCN), 1459 (s), 1445 (s), 1316 (m), 1261 (s, νC−O), 1136 (m, δN‑Hip), 1035 (w), 860 (m), 743 (s, δN‑Hoop). MS (FAB+): m/z (%) = 480 (5) ([M]+), 445 (100) ([M-Cl] + ). Molar Conductivity (CH 3 CN): 11.3 S cm2 mol−1. Solubility: soluble in methanol, dichloromethane, chloroform acetone, and acetonitrile. Partially soluble in water. L DOI: 10.1021/acs.inorgchem.8b02299 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry (w, νCH), 2099 (vs, νC−N(SCN)), 1597 (s, νC−N), 1543 (m, νCC), 1494 (s, νCN), 1456 (s), 1439 (s), 1420 (m), 1332 (m), 1238 (m), 1223−1210 (s, νC−O), 1149 (m, νCS), 1125 (w), 1036−1016 (w), 982 (w), 834 (m), 813 (w), 750 (s, νC−S(SCN)), 724 (w). MS (FAB+): m/z (%) = 406 (7) ([M-NCS]+) Molar Conductivity: It could not be measured due to their poor solubility. Solubility: partially soluble in dichloromethane. Slightly soluble in chloroform, methanol, dimethyl sulfoxide, acetonitrile, benzene, and acetone; insoluble in water. (s, δC‑Hoop), 723 (m). MS (FAB+): m/z (%) = 406 (10) ([M-Cl]+). Molar Conductivity: It could not be measured due to their poor solubility both water and acetonitrile. Solubility: soluble in methanol, dichloromethane, chloroform; partially soluble in acetone and slightly soluble in water and acetonitrile. Synthesis of [(η6-p-Cymene)Ru(NCS)(κ2-O,N-L2)], [3a]. In a 100 mL Schlenk ﬂask, the ligand 2-(2′-hydroxyphenyl)benzothiazole (0.0744 g, 0.327 mmol) was added to a solution of [RuCl2(cym)]2 (0.1000 g, 0.163 mmol) in degassed methanol (8 mL). Et3N (48 μL, 0.345 mmol) was then added, and the mixture was stirred at room temperature for 20 h and under nitrogen atmosphere. KSCN (0.0513 g, 0.528 mmol) was added to the mixture and heated at 70 °C for 4 h. The precipitate was ﬁltered and water was added to remove salts. After ﬁltering, the residue was washed with diethyl ether (3 mL). The resulting orange powder was dried under vacuum. Yield: 133.8 mg (0.257 mmol, 79%). Mr (C24H22N2ORuS2) = 519.6526 g/mol. Anal. Calc. for C24H22N2ORuS2·H2O: C 53.79; H 4.01; N 5.68; S 12.16 Found: C 53.61; H 4.50; N 5.21; S 11.93. 1H NMR (400 MHz, CD3CN, 25 °C) δ 8.25 (d, J = 8.4, 1.1, 0.7 Hz, 1H, Hf), 8.01 (ddd, J = 8.0, 1.2, 0.6 Hz, 1H, Hc), 7.69 (ddd, J = 8.5, 7.2, 1.3 Hz, 1H, He), 7.63−7.56 (m, 1H, H3′), 7.54 (ddd, J = 8.1, 7.2, 1.1 Hz, 1H, Hd), 7.33 (ddd, J = 8.4, 7.0, 1.7 Hz, 1H, H5′), 7.11−7.00 (m, 1H, H6′), 6.67 (ddd, J = 7.9, 7.0, 1.2 Hz, 1H, H4′), 5.66 (d, J = 5.9 Hz, 1H, H2, or H6), 5.54 (d, J = 5.3 Hz, 1H, H2, or H6), 5.43 (d, J = 6.1 Hz, 1H, H3, or H5), 5.37 (d, J = 6.0 Hz, 1H, H3, or H5), 2.69−2.56 (m, 1H, H7), 1.85 (s, 3H, H10), 1.16 (s, 3H, H8, or H9), 1.14 (s, 3H, H8, or H9). 1H NMR (400 MHz, DMSO-d6, 25 °C) δ 8.21 (d, J = 8.1 Hz, 1H, Hf), 8.17 (d, J = 8.0 Hz, 1H, Hc), 7.70 (t, J = 7.8 Hz, 1H, He), 7.58 (d, J = 6.7 Hz, 1H, H3′), 7.55 (t, J = 6.3 Hz, 1H, Hd), 7.31 (t, J = 7.7 Hz, 1H, H5′), 6.99 (d, J = 8.4 Hz, 1H, H6′), 6.65 (t, J = 7.4 Hz, 1H, H4′), 5.80 (d, J = 5.9 Hz, 1H, H2, or H6), 5.65 (t, J = 6.1 Hz, 2H, H6 or H2, H3, or H5), 5.58 (d, J = 6.0 Hz, 1H, H5, or H3), 1.89 (s, 3H, H10), 1.09 (d, J = 3.5 Hz, 3H, H8, or H9), 1.07 (d, J = 3.5 Hz, 3H, H9, or H8) ppm. H7 is overlapped into the residual DMSO signal. 13C{1H} NMR (101 MHz, CD3CN, 25 °C) δ 147.0 (s, 1C, C1′ or Ca), 145.6 (s, 1C, C1′ or Ca), 141.5 (s, 1C), 140.6 (s, 1C), 135.1 (s, 1C), 130.8 (s, 1C), 128.5 (s, 1C), 126.8 (s, 1C), 125.3 (s, 1C), 124.3 (s, 1C), 123.2 (s, 1C), 117.2 (s, 1C), 105.4 (s, 1C), 99.6 (s, 1C), 84.4 (s, 1C), 83.7 (s, 1C), 83.4 (s, 1C), 82.6 (s, 1C), 31.9 (s, 1C, C7), 22.8 (s, 1C, C8 or C9), 21.8 (s, 1C, C8 or C9), 18.8 (s, 1C, C10). 13C{1H} NMR (101 MHz, DMSO-d6, 25 °C) δ 168.4 (s, 1C, C1′), 166.4 (s, 1C, Ca), 152.1 (s, 1C, Cg), 135.4 (s, 1C, CSCN), 134.1 (s, 1C, C5′), 131.0 (s, 1C, Cb), 129.6 (s, 1C, C3′), 127.7 (s, 1C, Ce), 125.7 (s, 1C, Cd), 124.2 (s, 1C, Cf), 123.2 (s, 1C, C6′), 122.5 (s, 1C, Cc), 120.6 (s, 1C, C2′), 116.0 (s, 1C, C4′), 103.3 (s, 1C, C1), 98.5 (s, 1C, C4), 83.6 (s, 1C, C2 or C6), 82.3 (s, 1C, C6 or C2), 82.1 (s, 1C, C3 or C5), 81.5 (s, 1C, C5 or C3), 30.5 (s, 1C, C7), 22.2 (s, 1C, C8 or C9), 21.2 (s, 1C, C9 or C8), 18.1 (s, 1C, C10) ppm. FT-IR (KBr, cm‑1) selected bands: 3036 (w, νCH), 2962 (w, ν−CH), 2090 (vs, νC−N(SCN)), 1598 (s, νC−N), 1543 (s, νCC), 1489 (vs, νCN), 1455 (s), 1442 (s), 1419 (m), 1336 (s), 1212 (s, νC−O), 1152 (s, νCS), 1130 (w), 1032 (w), 877 (w), 836 (w), 819 (w), 751 (vs, νC−S(SCN)), 726 (w). MS (FAB+): m/z (%) = 982 (2) ([2M-NCS]+), 462 (100) ([M-NCS]+). Molar Conductivity (CH3CN): 1.9 S cm2 mol−1. Solubility: soluble in chloroform, acetonitrile, and dimethyl sulfoxide; partially soluble in methanol and insoluble in water and acetone. Synthesis of [(η6-Benzene)Ru(NCS)(κ2-O,N-L2)], [3b]. The synthesis was performed as for [3a] in the presence of the ligand 2-(2′hydroxyphenyl)benzothiazole (0.0911 g, 0.400 mmol), [RuCl2(bz)]2 (0.1002 g, 0.200 mmol), Et3N (59 μL, 0.424 mmol), and KSCN (0.053 g, 0.545 mmol) in methanol (8 mL). Orange powder. Yield: 113.7 mg (0.245 mmol, 62%). Mr (C20H14N2ORuS2) = 463.5454 g/mol. Anal. Calc. for C20H14N2ORuS2·(CH3CN)0.25·(H2O): C 50.06; H 3.43; N 6.41; S 13.04 Found: C 50.20; H 3.48; N 6.08; S 12.61. 1H NMR (400 MHz, CD2Cl2, 25 °C) δ 8.24 (d, J = 8.4 Hz, 1H, Hf), 7.91 (d, J = 8.0 Hz, 1H, Hc), 7.68 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H, He), 7.56 (dd, J = 7.9, 1.7 Hz, 1H, H3′), 7.51 (ddd, J = 8.4, 7.3, 1.1 Hz, 1H, Hd), 7.33 (ddd, J = 8.7, 7.0, 1.7 Hz, 1H, H5′), 7.17 (dd, J = 8.4, 1.2 Hz, 1H, H6′), 6.69 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H, H4′), 5.64 (s, 6H, Hbz) ppm. FT-IR (KBr, cm‑1) selected bands: 3055 (w, νCH), 3000 ■ ASSOCIATED CONTENT S Supporting Information * The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b02299. X-ray crystallographic ﬁles in CIF format; tables and ﬁgures with information on molecular and crystalline structures, 1H NMR spectra, photo physical studies of compounds in solution and in presence of DNA, FABMS spectra, cleavage electrophoresis (PDF) Accession Codes CCDC 1815683−1815686 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033. ■ AUTHOR INFORMATION Corresponding Authors *E-mail: nbusto@ubu.es. *E-mail: gespino@ubu.es. *E-mail: begar@ubu.es. ORCID Marta Martínez-Alonso: 0000-0002-0931-5274 Natalia Busto: 0000-0001-9637-1209 Blanca R. Manzano: 0000-0002-4908-4503 José L. Albasanz: 0000-0002-9927-5076 Gustavo Espino: 0000-0001-5617-5705 Begoña García: 0000-0002-0817-1651 Author Contributions ¶ M.L. and M.M.-A. have contributed equally to the realization of this work. Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS The ﬁnancial support by “la Caixa” Foundation ((LCF/PR/ PR12/11070003), MINECO (CTQ2014-58812-C2-2-R and CTQ2014-58812-C2-1-R FEDER Funds), and Junta de Castilla y León and Fondo Social Europeo (BU042U16), Spain, is gratefully acknowledged. ■ REFERENCES (1) Rosenberg, B.; Van Camp, L.; Krigas, T. Inhibition of Cell Division in Escherichia coli by Electrolysis Products from a Platinum Electrode. Nature 1965, 205, 698−699. (2) Ho, Y.-P.; Au-Yeung, S. C. F.; To, K. K. W. Platinum-based anticancer agents: innovative design strategies and biological perspectives. Med. Res. Rev. 2003, 23, 633−655. (3) Levina, A.; Mitra, A.; Lay, P. A. Recent developments in ruthenium anticancer drugs. Metallomics 2009, 1, 458−470. 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