Ruthenium(II)-Polypyridyl Compounds with π-Extended Nitrogen Donor Ligands Induce Apoptosis in Human Lung Adenocarcinoma (A549) Cells by Triggering Caspase-3/7 Pathway.

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX pubs.acs.org/IC Ruthenium(II)-Polypyridyl Compounds with π‑Extended Nitrogen Donor Ligands Induce Apoptosis in Human Lung Adenocarcinoma (A549) Cells by Triggering Caspase-3/7 Pathway Bruno Peña,† Sayan Saha,† Rola Barhoumi,‡ Robert C. Burghardt,‡ and Kim R. Dunbar*,† † Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, Texas 77843, United States ‡ Inorg. Chem. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 09/21/18. For personal use only. S Supporting Information * ABSTRACT: Ru(II)-polypyridyl complexes exhibit antitumor properties that can be systematically tailored by means of adjusting the ligand environment. In this work, the eﬀect of incorporating πextended moieties into anionic N∧O− based chelating ligands on the cytotoxic properties of Ru compounds is explored. Four new Ru(II) complexes, [Ru(bpy)2(dphol)][PF6] (1; bpy = 2,2′-bipyridine, dphol = dibenzo[a,c]phenazin-10-olate), [Ru(phen)2(dphol)][PF6] (2; phen = 1,10-phenanthroline), [Ru(bpy)2(hbtz)][PF6] (3; hbtz = 2(benzo[d]thiazol-2-yl)phenolate), and [Ru(phen)2(hbtz)][PF6] (4) were synthesized and thoroughly characterized. In vitro cytotoxicity was investigated in human lung adenocarcinoma (A549) cells, which revealed that 4 is the most cytotoxic compound (IC50 = 0.8 μM) in the series including a control compound [Ru(bpy)2(quo)][PF6] (5; quo = 8-hydroxyquinolinate) and is nearly 8-fold more cytotoxic than cisplatin. An investigation of the mechanism of cell death led to the ﬁnding that compounds 1−4 disrupt the mitochondrial transmembrane potential (ΔΨm) in a concentration-dependent fashion, which is an event associated with the intrinsic pathway of apoptosis. Moreover, compound 4 triggers the activity of caspase-3/7, which eventually induces the apoptotic cellular death of A549 cells. Thus, increasing the overall lipophilicity of the Ru compounds by introducing π-extended moieties in the anionic N∧O− ligand is a successful strategy for realizing a new family of pro-apoptotic compounds with a [RuIIN5O]+ coordination environment. ■ INTRODUCTION Cancer is a disease characterized by uncontrolled cellular growth and has been one of the leading causes of death globally for the past few decades. In fact it is the second leading cause of death in the United States after heart related disease. Since the fortuitous discovery of cisplatin, cis-Pt(NH3)2Cl2 in the late 1960s, inorganic cancer chemotherapy is still largely dependent on the administration of Pt-based chemotherapeutic drugs. Despite its success and high cure rates, speciﬁcally against metastatic testicular and ovarian cancer, cisplatin suﬀers from detrimental dose-limiting side eﬀects and tumor resistance. 1,2 To circumvent the undesired limitations associated with Pt drugs, researchers are actively searching for alternative therapies involving diﬀerent transition metals to produce drugs with lower systemic toxicity and higher selectivity. Over the past two decades, ruthenium compounds have emerged as promising anticancer agents due to their lower toxicities and eﬀectiveness against Pt resistant tumors. The initial success of NAMI-A and KP1019, both currently in advanced phases of clinical trials, sparked intense investigation of Ru compounds in cancer chemotherapy.3−8 Ruthenium compounds are a suitable choice for medicinal applications © XXXX American Chemical Society owing to several factors: (a) they form thermodynamically stable coordination compounds with slow ligand exchange rates enabling them to reach biological targets without being modiﬁed; (b) they exhibit multiple oxidation states that are stable under physiological conditions; and (c) they are capable of mimicking iron binding for transportation, resulting in lower toxicities.9,10 Within the realm of anticancer compounds, Ru(II) polypyridyl complexes are well established for their antitumor properties and are known to inhibit tumor growth by triggering apoptotic/necrotic pathways of cellular death when interacting with mitochondria, the nucleus, and other cell organelles.11−15 They interact with key cellular targets, including DNA and proteins,16−19 and can be engineered to release biologically active molecules inside the cell using light. It is also possible to tune their cytotoxicity and cellular uptake simply by modifying the coordination environment through proper choice of ancillary ligands, thereby providing a rich platform for the development of a variety of new drugs. Although the choice of ligand environment provides an ample ﬁeld to explore, it is Received: July 15, 2018 A DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry recorded on a Shimadzu UVPC-3001 spectrophotometer. Electrochemical measurements were performed under a N2 atmosphere in dry acetonitrile and 0.1 M tetra-n-butylammonium hexaﬂuorophosphate ([nBu4N][PF6]) as the supporting electrolyte with a HCH Electrochemical Analyzer model CH 1620A using a BAS Pt disk working electrode, Pt wire auxiliary electrode, and Ag/AgCl (3 M KCl(aq)) reference electrode at a 100 mV/s scan rate. The concentration of the Ru complexes for the electrochemical experiments was ∼1 mM. Ferrocene was used as an internal standard and exhibited an E1/2 = 0.44 V vs Ag/AgCl for the Fc+/Fc couple under the same experimental conditions. The E1/2 values of the Ru complexes were referenced vs NHE using the following expression: E1/2 vs NHE = [(E1/2 vs Ag/AgCl of Ru complex) + (0.64−0.44)] V, where 0.64 V = E1/2 [Fc+/Fc] vs NHE and 0.44 V = E1/2 [Fc+/Fc] vs Ag/AgCl. Synthetic Details. Dibenzo[a,c]phenazin-10-ol (dpholH). A mixture of 2,3-diaminophenol (210 mg, 1.69 mmol) and 9,10phenanthrenequinone (336 mg, 1.61 mmol) in 20 mL of ethanol/ acetic acid (1:1) was heated to reﬂux for 2 h. A yellow precipitate was collected by ﬁltration, dissolved in hot dichloromethane (100 mL), and ﬁltered through a short plug of SiO2. A yellow band was eluted with dichloromethane, and the combined fractions were reduced to ca. 25 mL. After the yellow solution was kept in an ice bath for 30 min, a light yellow precipitate was collected and washed with cold dichloromethane. Yield: 208 mg (44%). 1H NMR (500 MHz, CDCl3): δ 9.39 (dd, 1H, 3J = 8.0, 4J = 1.5), 9.29 (dd, 1H, 3J = 8.0, 4J = 1.0), 8.57 (m, 2H), 8.15 (s, 1H), 7.87 (dd, 1H, 3J = 8.5, 4J = 1.0), 7.84−7.72 (m, 5H), 7.31 (dd, 1H, 3J = 7.5, 4J = 1.5). HRMS (ESI+): Calcd for [C20H13N2O]+ ([M + H]+), 297.1028. Found: 297.1026. [Ru(bpy)2(dphol)][PF6] (1). Samples of cis-RuCl2(bpy)2·2H2O (150 mg, 0.29 mmol), dpholH (95 mg, 0.32 mmol), and NaHCO3 (74 mg, 0.88 mmol) were suspended in 70 mL of ethanol and heated to reﬂux for 8 h. The resulting dark red solution was cooled to room temperature and ﬁltered. A quantity of NH4PF6(aq) (5 equiv dissolved in 2 mL of water) was added to the ﬁltrate, and a dark red solid precipitated. The solid was dissolved in dichloromethane (50 mL) and washed with water (3 × 30 mL). The organic layer was dried with MgSO4 and reduced to dryness. The residue was dissolved in 10 mL of acetone, and diethyl ether was added slowly until a solid began precipitating. The mixture was stored at 0 °C overnight, during which time a dark red microcrystalline solid formed, which was collected by ﬁltration and washed with diethyl ether (25 mL). Yield: 195 mg (79%). 1H NMR (500 MHz, (CD3)2CO): δ 9.01 (m, 2H), 8.94 (d, 1H, 3J = 5.0), 8.86 (d, 1H, 3J = 8.5), 8.75 (d, 1H, 3J = 8.5), 8.51 (d, 1H, 3J = 5.5), 8.47 (d, 1H, 3J = 8.0), 8.33 (m, 3H), 8.25 (ddd, 1H, 3J = 8.0, 3J = 8.0, 4J = 1.5), 8.16 (d, 1H, 3J = 8.0), 8.03 (ddd, 1H, 3J = 8.0, 3J = 8.0, 4J = 1.5), 7.87 (ddd, 1H, 3J = 8.0, 3J = 8.0, 4J = 1.5), 7.77 (ddd, 1H, 3J = 8.5, 3J = 7.0, 4J = 1.5), 7.70 (ddd, 1H, 3J = 8.0, 3J = 7.0, 4 J = 1.0), 7.66 (m, 2H), 7.61 (ddd, 1H, 3J = 7.5, 3J = 5.5, 4J = 1.5), 7.59 (d, 1H, 3J = 5.5), 7.41−7.35 (m, 2H), 7.29 (ddd, 1H, 3J = 7.5, 3J = 5.5, 4J = 1.5), 7.24 (dd, 1H, 3J = 8.0, 4J = 1.0), 7.18 (ddd, 1H, 3J = 7.5, 3J = 5.5, 4J = 1.5), 7.13 (ddd, 1H, 3J = 8.5, 3J = 7.5, 4J = 1.0), 6.94 (dd, 1H, 3 J = 8.0, 4 J = 1.5). HRMS (ESI+): Calcd for [C40H27N6ORu]+ ([M−PF6]+), 709.1290. Found: 709.1287. Anal. Calcd for C40H27N6OF6PRu·1.05(CH3)2CO: C, 56.66; H, 3.67; N, 9.19. Found: C, 56.84; H, 3.82; N, 9.36. [Ru(phen)2(dphol)][PF6] (2). This compound was prepared in a fashion similar to that described for 1 with cis-RuCl2(phen)2·2H2O (151 mg, 0.27 mmol), dpholH (87 mg, 0.19 mmol), and NaHCO3 (69 mg, 0.82 mmol) in ethanol (70 mL). A dark red microcrystalline solid was obtained. Yield: 185 mg (77%). 1H NMR (500 MHz, (CD3)2CO): δ 9.18 (dd, 1H, 3J = 5.5, 4J = 1.0), 9.05 (dd, 1H, 3J = 8.0, 4 J = 1.0), 8.75 (m, 2H), 8.70 (dd, 1H, 3J = 8.0, 4J = 1.0), 8.66 (d, 1H, 3 J = 8.0), 8.49 (dd, 1H, 3J = 8.0, 4J = 1.0), 8.41 (dd, 1H, 3J = 5.5, 4J = 1.0), 8.36 (d, 2H, 3J = 8.0), 8.28−8.19 (m, 3H), 8.18 (d, 1H, 3J = 8.0), 8.11 (d, 1H, 3J = 9.0), 8.01 (dd, 1H, 3J = 8.0, 3J = 5.5), 7.88 (dd, 1H, 3J = 8.0, 3J = 5.5), 7.75−7.66 (m, 2H), 7.63 (t, 1H, 3J = 8.0), 7.56 (dd, 1H, 3J = 5.5, 4J = 1.0), 7.46 (dd, 1H, 3J = 8.0, 3J = 8.0), 7.29− 7.22 (m, 2H), 7.21 (ddd, 1H, 3J = 8.0, 3J = 7.0, 4J = 1.0), 6.87 (dd, 1H, 3J = 8.0, 4J = 1.0), 6.44 (t, 1H, 3J = 7.5). HRMS (ESI+): Calcd for surprising that most of the reports on the biological activity of octahedral Ru(II) polypyridyl molecules have focused mainly on substitutionally inert complexes that possess neutral Ndonor ligands ([RuIIN6]2+),20−22 with much less focus on other donor atoms. One advantage of using anionic donor ligands is that the lipophilicity and thus the cellular uptake of the compound increases due to a decrease of the overall charge on the complex. Only a few studies on the cytotoxicity of Ru complexes possessing anionic N∧O− bidentate ligands and a [RuIIN5O]+ coordination environment are reported in the literature. Meggers et al. used a combinatorial, high throughput screening approach that led to the discovery of [Ru(tBu2bpy)2(phox)][PF6] (t-Bu2bpy = 4,4′-di-tert-butyl-2,2′bipyridine; phox = deprotonated 2-(2′-hydroxyphenyl)oxazoline) which exhibits low micromolar IC50 values in HeLa cervical cancer cells and was found to decrease the mitochondrial membrane potential of Burkitt-like lymphoma (BJAB) cells, suggesting the involvement of the intrinsic pathway of apoptosis.23 Glazer et al. reported that coordination of hydroxyquinolines to Ru-centers leads to a marked increase in their cytotoxicity through rapid processes which induces apoptosis,24 whereas Liu and coworkers have shown 8hydroxyquinoline Ru(II)-complexes can inhibit angiogenesis and in vivo tumor growth of HepG2 (human hepatocellular liver carcinoma cells) xenografted on mouse through ERK and AKT signaling pathways.25 On a diﬀerent note, Wang et al. explored the non-innocent behavior of Ru-hydroxyquinoline complexes which exhibited DNA photocleavage upon visible light irradiation through a radical mediated pathway that can be utilized in photodynamic therapy (PDT).26,27 To expand the library of [RuN5O]+ cytotoxic compounds, we decided to incorporate π-extended moieties into the N∧O− ligands. Four new Ru(II) complexes (Figure S1), [Ru(bpy)2(dphol)][PF6] (1), [Ru(phen)2(dphol)][PF6] (2), [Ru(bpy)2(hbtz)][PF6] (3), and [Ru(phen)2(hbtz)][PF6] (4) were synthesized and thoroughly characterized. The eﬀect of adding π-extended systems on their cytotoxic properties was investigated in human lung adenocarcinoma (A549) cells, and the mechanism of cytotoxicity was probed using two diﬀerent biological assays. ■ EXPERIMENTAL SECTION General Methods. Standard Schlenk-line techniques under a N2 atmosphere were used during the preparation of the compounds. Solvents were of reagent grade quality. Ethanol (KOPTEC 200 proof), acetone (EMD Chemicals), dichloromethane (EMD Chemicals), diethyl ether (EMD Chemicals), and glacial acetic acid (EMD Chemicals) were used as received without further puriﬁcation. RuCl3· xH2O (Pressure Chemicals Co.), 2,2′-bipyridine (bpy, Alfa Aesar), 1,10-phenanthroline (phen, Alfa Aesar), 8-hydroxyquinoline (quoH, Acros Organics), 2-(2-hydroxyphenyl)benzothiazole (hbtzH, SigmaAldrich), 2,3-diaminophenol (Sigma-Aldrich), 9,10-phenanthrenequinone (Sigma-Aldrich), NH4PF6 (Sigma-Aldrich), NaHCO3 (Mallinckrodt), and K2CO3 (Spectrum Chemicals) were purchased and used as received. The compounds cis-RuCl2(N∧N)2·2H2O (N∧N = bpy, phen)28 were prepared following reported procedures. Instrumentation. The 1H NMR spectra were recorded on an Inova 500 MHz spectrometer. Chemical shifts are reported in δ (ppm) and coupling constants (J) in hertz (Hz). The residual solvent peak was used as an internal reference (δ 7.26 for CDCl3, δ 1.94 for CD3CN, 2.05 for (CD3)2CO). Electrospray ionization (ESI) mass spectra were acquired on an Applied Biosystems PE SCIEX QSTAR mass spectrometer (MDS Sciex). Elemental analyses were performed by Atlantic Microlab, Inc. (Norcross, GA). Absorption spectra were B DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry [C44H27N6ORu]+ ([M−PF6]+), 757.1290. Found: 757.1284. Anal. Calcd for C44H27N6OF6PRu: C, 58.61; H, 3.02; N, 9.32. Found: C, 58.60; H, 3.06; N, 9.30. [Ru(bpy)2(hbtz)][PF6] (3). This compound was prepared in a fashion similar to that described for 1 with cis-RuCl2(bpy)2·2H2O (158 mg, 0.30 mmol), hbtzH (77 mg, 0.34 mmol), and K2CO3 (88 mg, 0.64 mmol) in ethanol/water 1:1 (30 mL) as solvent. A dark red microcrystalline solid was obtained. Yield: 175 mg (73%). 1H NMR (500 MHz, CD3CN): δ 8.94 (d, 1H, 3J = 5.5), 8.61 (d, 1H, 3J = 5.5), 8.43 (d, 1H, 3J = 8.0), 8.40 (d, 1H, 3J = 8.0), 8.34 (m, 2H), 8.06 (d, 1H, 3J = 5.5), 8.01−7.94 (m, 2H), 7.85 (ddd, 1H, 3J = 8.0, 3J = 7.5, 4J = 1.5), 7.82 (d, 1H, 3J = 8.0), 7.76 (ddd, 1H, 3J = 8.0, 3J = 7.5, 4J = 1.5), 7.57 (dd, 1H, 3J = 8.0, 4J = 1.5), 7.47 (ddd, 1H, 3J = 7.5, 3J = 5.5, 4 J = 1.5), 7.40 (d, 1H, 3J = 5.5), 7.34 (ddd, 1H, 3J = 7.5, 3J = 5.5, 4J = 1.5), 7.20−7.11 (m, 3H), 7.02 (ddd, 1H, 3J = 8.5, 3J = 7.0, 4J = 1.5), 6.90 (ddd, 1H, 3J = 8.5, 3J = 7.0, 4J = 1.5), 6.45 (ddd, 1H, 3J = 8.5, 3J = 7.0, 4J = 1.5), 6.40 (d, 1H, 3J = 8.5), 6.37 (dd, 1H, 3J = 8.5, 4J = 1.0). HRMS (ESI+): Calcd for [C33H24N5OSRu]+ ([M−PF6]+), 640.0748. Found: 640.0752. Anal. Calcd for C 33 H 24 N 5 OF 6 PSRu·0.95(CH3)2CO: C, 51.27; H, 3.56; N, 8.34. Found: C, 51.36; H, 3.55; N, 8.42. [Ru(phen)2(hbtz)][PF6] (4). This compound was prepared in a fashion similar to that described for 1 using cis-RuCl2(phen)2·2H2O (177 mg, 0.31 mmol), hbtzH (81 mg, 0.36 mmol), and K2CO3 (86 mg, 0.62 mmol) in ethanol/water 1:1 (30 mL). A dark red-green microcrystalline solid was obtained. Yield: 156 mg (60%). 1H NMR (500 MHz, CD3CN): δ 9.27 (dd, 1H, 3J = 5.0, 4J = 1.0), 9.03 (dd, 1H, 3 J = 5.0, 4J = 1.0), 8.56 (m, 2H), 8.32 (dd, 1H, 3J = 8.0, 4J = 1.0), 8.25−8.18 (m, 3H), 8.15 (d, 1H, 3J = 9.0), 8.11 (d, 1H, 3J = 9.0), 8.06 (d, 1H, 3J = 8.5), 7.88 (dd, 1H, 3J = 8.5, 3J = 5.0), 7.78−7.73 (m, 2H), 7.62 (dd, 1H, 3J = 8.0, 4J = 1.5), 7.46 (dd, 1H, 3J = 5.5, 3J = 1.5), 7.34−7.28 (m, 2H), 7.08 (ddd, 1H, 3J = 8.0, 3J = 7.0, 4J = 1.0), 7.00 (ddd, 1H, 3J = 8.5, 3J = 7.0, 4J = 1.5), 6.72 (ddd, 1H, 3J = 8.5, 3J = 7.5, 4 J = 1.5), 6.50−6.42 (m, 2H), 6.28 (dd, 1H, 3J = 8.5, 4J = 1.0). HRMS (ESI+): Calcd for [C37H24N5OSRu]+ ([M−PF6]+), 688.0745. Found: 688.0714. Calcd for C37H24N5OF6PSRu·(CH3)2CO: C, 53.30; H, 3.44; N, 7.97. Found: C, 53.51; H, 3.58; N, 7.89. [Ru(bpy)2(quo)][PF6] (5). This compound was prepared in a fashion similar to that described for 1 with cis-RuCl2(bpy)2·2H2O (121 mg, 0.23 mmol), quoH (41 mg, 0.28 mmol), and K2CO3 (65 mg, 0.47 mmol) in ethanol/water (1:1, 30 mL). A dark green-red microcrystalline solid was collected by ﬁltration and was washed with diethyl ether (25 mL). Yield: 138 mg (85%). 1H NMR (500 MHz, (CD3)2CO): δ 8.89 (d, 1H, 3J = 5.5), 8.74−8.65 (m, 4H), 8.17 (d, 1H, 3J = 6.0), 8.13−8.04 (m, 4H), 8.00 (m, 2H), 7.89 (d, 1H, 3J = 5.0), 7.65 (ddd, 1H, 3J = 7.5, 3J = 6.0, 4J = 1.5), 7.55 (dd, 1H, 3J = 5.0, 4 J = 1.0), 7.46 (m, 1H), 7.40 (m, 2H), 7.32 (t, 1H, 3J = 8.0), 7.20 (dd, 1H, 3J = 8.5, 3J = 5.0), 6.87 (dd, 1H, 3J = 8.0, 4J = 1.0), 6.79 (dd, 1H, 3 J = 8.0, 4J = 1.0). HRMS (ESI+): Calcd for [C29H22N5ORu]+ ([M− PF6]+), 558.0868. Found: 558.0866. Calcd for C29H22N5OF6PRu· 0.5(CH3CH2)2O: C, 50.34; H, 3.68; N, 9.47. Found: C, 50.29; H, 3.79; N, 9.23. X-ray Crystallography. Single crystals of compounds 1, 3, and 4 were obtained by slow diﬀusion of diethyl ether into acetone solutions of the compounds at room temperature. X-ray data were collected at 110 K on a Bruker APEX II CCD X-ray diﬀractometer equipped with a graphite monochromated Mo Kα radiation source (λ = 0.71073 Å). The data sets were integrated with the Bruker SAINT software package.29 The absorption correction (SADABS)30 was based on ﬁtting a function to the empirical transmission surface as sampled by multiple equivalent measurements. Solution and reﬁnement of the crystal structures were carried out using the SHELX31 (2013) suite of programs and the graphical interface ShelXle32 was used during the reﬁnement. The structures were solved by the Patterson method; all non-hydrogen atoms were reﬁned with anisotropic displacement parameters using a full-matrix least-squares technique on F2. Hydrogen atoms were ﬁxed to parent atoms and reﬁned using the riding model.33,34 PLATON/SQUEEZE was employed in the case of 4 after attempts to model a disordered diethyl ether solvent molecule failed. The solvent molecules in the unit cell (one solvent molecule per asymmetric unit, Z = 4) were determined to occupy 642.9 Å3. The number of electron counts in voids per unit cell was 159, which is close to that expected for four diethyl ether molecules (168 electrons). Cell Culture Experiments. The human lung adenocarcinoma A549 cell line, derived from type II pneumocytes (CCL 185), was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM/F-12 medium (Dulbecco’s Modiﬁed Eagle Medium: Nutrient Mixture F-12) with 10% FBS. Cell cultures were incubated in a humidiﬁed atmosphere containing 5% CO2 at 37 °C and were approximately 80% conﬂuent at the time of analysis. In vitro Cytotoxicity. A549 cells were plated in a 96 well plate and preincubated in a humidiﬁed atmosphere containing 5% CO2 at 37 °C for 24 h. Solutions of the metal complexes in DMEM/F-12 medium were added at diﬀerent concentrations (ﬁnal concentrations of compounds: 0−50 μM range, 0.1% DMSO) and the cells were incubated for another 48 h. Cells were then washed twice with PBS and ﬁxed with methanol for 30 min. After ﬁxation, Janus green B (1 mg/mL, Alfa Aesar) was added to each well and incubated at room temperature for 5 min. Cells were washed again twice with PBS and 100 μL of methanol was added to each well to extract the dye. Janus Green B signal was then measured using a BioTek Synergy 4 plate reader set to an absorbance of 630 nm. Two experiments were conducted on diﬀerent days with each experiment having 8 replicates per concentration. The absorbance of Janus Green B is directly proportional to the number of living cells. JC-1 Assay. Live cell imaging studies were performed using a Zeiss 510 META NLO multiphoton system consisting of an Axiovert 200 MOT inverted laser scanning confocal microscope (Carl Zeiss Microimaging, Thornwood, NY). A Zeiss Plan-Neoﬂuar 40×/NA = 1.3 oil immersion objective was used to acquire the images. Cells were plated in Nunc Lab-Tek II chambered coverglass slides (Thermo Scientiﬁc) for 24 h prior to treatment with compounds 1−4 and cisplatin for 48 h. Cells were then washed with PBS and labeled with the mitochondrial membrane potential probe, JC-1 (Invitrogen) at a ﬁnal concentration of 5 μg/mL for 30 min at 37 °C. Excitation of JC-1 was performed using an argon ion laser at 488 nm and emission data were collected using a dichroic 545 nm SP in combination with 2 ﬁlters 500−550 BP (green signal) and 565−615 BP (red signal). At least eight areas per well were scanned. Two wells were analyzed per treatment. Two experiments were conducted on diﬀerent days. Ratio (red signal/green signal) was used as an indicator of cellular mitochondria membrane potential. Calcein AM Assay. Cells were plated in chambered coverglass slides for 24 h prior to treatment with compound 4 or cisplatin for 48 h. Cells were then washed with PBS and incubated with 1 μg/mL Hoeschst 33258 (Invitrogen) and 10 μM acetoxymethyl ester of the membrane-permeable live-cell labeling dye Calcein (calcein AM, Invitrogen) for 30 min, and with 50 nM Mitotracker Deep Red FM (Invitrogen) for 15 min. Following loading, cells were washed and 1 mM cobalt(II) chloride hexahydrate was added to the cells and images were acquired. To collect Hoechst 33258 (Invitrogen) ﬂuorescence, cells were irradiated with the Chameleon tunable Ti:sapphire laser (Coherent Inc., Santa Clara, CA) at an excitation wavelength of 740 nm (which is roughly equivalent to 370 nm in single photon excitation with a continuous wavelength laser system) and emission was collected at 430−480 nm. Calcein was excited with an argon ion laser at 488 nm and emission was monitored using a band-pass 500−530 ﬁlter. Mitotracker Deep Red FM was excited with a He−Ne laser at 633 nm and emission was collected using a BP 650−710 ﬁlter. Image acquisition was performed sequentially to reduce the possibility of bleed-through between channels. At least eight areas per well were scanned. Two wells were analyzed per treatment. Two experiments were conducted on diﬀerent days. Caspase Glo Assay. Cells were cultured for 24 h prior to addition of compound 4 or cisplatin for 48 h. Cells were then washed twice with PBS and 100 μL of the Caspase-Glo 3/7 reagent solution (Promega) was added to each well. Cells were then scanned every 10 min for 30 min and luminescence readings were recorded with C DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry BioTek Synergy 4 plate reader. Four wells per concentration were recorded. ■ RESULTS AND DISCUSSION Synthesis and Characterization. The precursors for the syntheses of 1−5, namely cis-RuCl2(N∧N)2 (N∧N = bpy, phen), were prepared by reacting RuCl3,xH2O with 2 equiv of N∧N in reﬂuxing DMF and in the presence of LiCl (Figure S1).28 The crude products [Ru(N∧N)2(N∧O−)][PF6] were obtained by reacting cis-RuCl2(N∧N)2 with 1.1 equiv of N∧OH (dpholH, hbtzH, and quoH) in reﬂuxing ethanol (or aqueous ethanol) and in the presence of a base (NaHCO3 or K2CO3) to deprotonate the phenol moiety of N∧OH, followed by precipitation with NH4PF6(aq). The ﬁve complexes were obtained as dark red microcrystalline solids after recrystallization from acetone/diethyl ether. Although hbtzH is commercially available, surprisingly, there is only one previous report of a Ru(II) complex of the ligand that is similar to 3 but there were no synthetic details or further characterization provided.35 Due to the C1 symmetry of 1−5, there are no magnetically equivalent protons in their 1H NMR spectra (Figures S2 and S3); the integration of the signals matches the expected number of protons for each complex (1, 27H; 2, 27H; 3, 24H; 4, 24H; 5, 22H). The identity and purity of the compounds were conﬁrmed by elemental analyses and ESIMS, where a single peak corresponding to the [M−PF6]+ cations was observed for all of the complexes (1, m/z = 709.1287; 2, m/z = 757.1284; 3, m/z = 640.0752; 4, m/z = 688.0714; 5, m/z = 558.0866). X-ray Structures of Compounds 1, 3, and 4. Single crystals of 1, 3, and 4 were obtained by slow diﬀusion of diethyl ether into acetone solutions of the compounds at room temperature. Their X-ray structures are shown in Figures 1 and 2 and the crystallographic data are compiled in Tables S1−S4. Compounds 1 and 3 crystallize in the triclinic space group P1̅, whereas 4 crystallizes in the monoclinic space group P2/n. There is an interstitial acetone molecule in the asymmetric unit of 1 and 3. A similar structure of 3 was previously reported in the literature which crystallizes in monoclinic P21/n space group with interstitial acetonitrile molecule in the asymmetric unit.35 The coordination sphere of the metal center in the structures of the three ruthenium molecules consists of ﬁve N atoms and one O atom in a distorted octahedral environment. The Ru−O bond distances are ∼2.060 Å in the three compounds and are similar to the one reported for [Ru(bpy)2(quo)][PF6] (5; 2.063(6) Å).36 Their Ru−N bond distances to bpy and phen fall in the 2.019−2.079 Å range and are also in agreement with the respective distances in [Ru(bpy)2(quo)][PF6] and Ru(II) polypyridyl compounds in a [RuN6] coordination environment. In contrast, the Ru−N bond distances to dphol in 1 (Ru−N1, 2.151(3) Å) and to hbtz in 3 (Ru−N5, 2.110(2) Å) and 4 (Ru−N5, 2.110(3) Å) are longer. The elongation of these Ru−N bonds is likely to be caused by steric repulsions between the bulky phenanthrene moiety of dphol and adjacent bpy ligand in the case of 1, and between the benzothiazolyl moiety of hbtz and adjacent bpy and phen ligands in the case of 3 and 4, respectively. The dphol ligand in 1 is twisted as depicted in Figure 1, displaying dihedral angles of −30.7(5)° for Ru1−N1−C19−C18 and −19.3(6)° for N1−C19−C18−C17. The hbtz ligands in 3 and 4 are twisted around the C−C bond that connects the phenoxido and benzothiazolyl moieties, exhibiting dihedral Figure 1. (Top) Thermal ellipsoid plots at the 50% probability level of the X-ray structure of [Ru(bpy)2(dphol)][PF6] (1). The [PF6]− anion and H atoms were omitted for the sake of clarity. (Bottom) Distortion of the dphol ligand in compound 1. angles of 8.5(3)° (N5−C27−C26−C21) and −16.3(5)° (N5− C31−C30−C25), respectively. Electrochemical Properties. The redox properties of complexes 1−5 were studied by cyclic voltammetry in acetonitrile. The half-wave potential values (E1/2) vs Ag/ AgCl were obtained from the cyclic voltammograms of 1−5 (Figures S4−S5). The E1/2 values were referenced vs NHE for the discussion of the results (NHE = normal hydrogen electrode) as described in the Experimental Section; the values are presented in Table 1. The redox events observed in the cyclic voltammograms of 1−5 are quasi-reversible (ipa/ipc ≈ 1), except for the metal-based oxidation processes for 1 and 2 which are irreversible. The Ru3+/2+ redox couples of 1−5 (E1/2 [Ru3+/2+] = 0.86− 0.74 V range) occur at less positive potentials with respect to [Ru(bpy)3]2+ (E1/2 [Ru]3+/2+ = 1.54 V37). The anionic character and π-donating ability of the O atom donor of the N∧O− ligands destabilize the Ru(dπ) “t2g-type” orbitals (Figure S6) and facilitates metal oxidation. This destabilization eﬀect was veriﬁed by DFT calculations reported for the known compound 5,38 where the HOMO has contributions from both the Ru(dπ) and O(pπ) orbitals and the HOMO−1 and HOMO−2 are mainly metal-based. The redox events observed for 5 are in agreement with previous reports of related compounds.38−40 The metal-based redox couple at E1/2 = 0.76 V corresponds to the Ru3+/2+ oxidation couple, whereas those at E1/2 = −1.28 and −1.53 V D DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 2. Thermal ellipsoid plots at the 50% probability level of the X-ray structures of [Ru(bpy)2(hbtz)][PF6] (3) and [Ru(phen)2(hbtz)][PF6] (4). The [PF6]− anion and H atoms were omitted for the sake of clarity. Table 1. Half Wave Redox Potentials (E1/2) of 1−5 Recorded in Acetonitrile E1/2 (V) vs NHE (ΔE = Epa − Epc in mV) compound E1/2 [M(n+1)+/n+]a E1/2,red1 E1/2,red2 E1/2,red3 [Ru(bpy)2(dphol)][PF6] (1) [Ru(phen)2(dphol)][PF6] (2) [Ru(bpy)2(hbtz)][PF6] (3) [Ru(phen)2(hbtz)][PF6] (4) [Ru(bpy)2(quo)][PF6] (5) 0.86b 0.85b 0.74 (80) 0.74 (90) 0.76 (95) −0.94 (66) −0.93 (64) −1.25 (60) −1.26 (65) −1.28 (68) −1.35 (66) −1.36 (85) −1.52 (78) −1.54 (86) −1.53 (94) −1.61 (88) −1.59 (80) a Ru3+/2+ couple for 1−5. bIrreversible; the anodic peak potential (Ep,a) is reported. correspond to consecutive 1e− reductions of the bpy ligands. Reduction of quo is not observed in the 1.8 to −1.8 V range of potentials. The Ru3+/2+ redox events for 1 and 2 are irreversible and are shifted anodically by ∼100 mV with respect to 5 (Figure S4); i.e., compounds 1 and 2 are more diﬃcult to oxidize than 5. The ﬁrst reduction waves of 1 and 2 (E1/2,red1 ∼ − 0.94 V) occur at less negative potentials than that of 5 (E1/2,red1 = −1.28 V, bpy-based reduction) and are assigned as a reduction of the dphol ligand: dphol + e− → dphol−. The dphol ligand is easier to reduce than bpy (or quo) due to its more delocalized π-system and it is likely that the electron is delocalized in the phenanthrene moiety. The other two ligand-based reduction waves at ∼ −1.35 and ∼ −1.60 V are assigned as consecutive 1e− reductions of bpy in the case of 1, and phen in the case of 2. Compounds 3 and 4 display metal-based and ligand-based redox processes at E1/2 values very similar to those of 5 (Figure S5). Therefore, it can be concluded that the E1/2 [Ru3+/2+] couples of 3 and 4 occur at E1/2 = 0.74 V and that the ligandbased redox events at E1/2 − 1.25 and −1.52 V for 3, and E1/2 − 1.26 and −1.54 V for 4 correspond to consecutive 1e− reductions of bpy and phen ligands, respectively. The reduction of the hbtz ligand is likely to occur at more negative potentials and is not observed in the potential window of the cyclic voltammogram experiments. Electronic Properties. The absorption maxima (λabs) and molar extinction coeﬃcients (ε) for 1−5 in acetonitrile are listed in Table 2, and their electronic absorption spectra in acetonitrile are shown in Figures S7−S9. The Ru compounds Table 2. Electronic Absorption Data for 1−5 Recorded in Acetonitrile compound λmax, nm (ε × 104 M−1 cm−1) [Ru(bpy)2(dphol)] [PF6] (1) [Ru(phen)2(dphol)] [PF6] (2) [Ru(bpy)2(hbtz)] [PF6] (3) [Ru(phen)2(hbtz)] [PF6] (4) [Ru(bpy)2(quo)] [PF6] (5) 710 (0.14), 500 (0.83), 363 (1.92), 292 (4.67), 246 (5.65) 710 (0.23), 525a (1.10), 485 (1.26), 380 (1.80), 355 (2.07), 263 (9.30), 226 (8.26) 652 (0.16), 496 (0.81), 351 (1.35), 292 (4.29) a 650 (0.20), 480 (1.28), 351 (1.00), 267 (7.14), 225 (7.53) 506 (0.89), 460 (0.65), 394 (0.74) 368 (0.80), 295 (3.47), 252 (3.52) Shoulder. with phen as an ancillary ligand (2 and 4) exhibit slightly higher molar absorptivity coeﬃcients in the visible region than those with bpy ligands (1, 3, and 5). The absorption maxima at ∼500 nm for 1, 3, and 5 are assigned as singlet metal-to-ligand charge transfer (1MLCT) transitions [Ru(dπ) → bpy(π*)]. Similarly, broad 1MLCT bands centered in the 480−500 nm interval are observed for 2 and 4, which arise from Ru(dπ) → phen(π*) transitions. Such 1MLCT bands for 1−5 are redshifted with respect to the prototype Ru(II) complex [Ru(bpy)3]2+ (1MLCT at 450 nm37) due to the destabilization of the Ru-HOMOs (occupied t2g-type orbitals), which in turn decreases the HOMO−LUMO gap and the energy of the MLCT transition, as illustrated in Figure S6. Compounds 1 and 2 exhibit additional absorption features in the 650−800 nm region that are assigned as [Ru(dπ) → dphol(π*)] 1 MLCT transitions. These low energy transitions are in E DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry indicated by a decrease in the red/green emission intensity ratio (R) of JC-1.45 A549 cells were incubated with 1−4 at diﬀerent concentrations for 48 h. Following this period of time, the cells were incubated with JC-1, and R values were calculated. The results are shown in Figure 3. In general, an increase of the agreement with the less negative reduction potential of coordinated dphol with respect to quo or hbtz, indicating that the π* MOs of dphol are lower in energy. The transitions in the 300−400 nm range arise from 1ππ* ligand-centered (LC) transitions of the N∧O− ligands because the free ligands dpholH (374 and 393 nm), hbtzH (332 nm), and quoH (312 nm) display absorption maxima in this region (Figure S9). The maxima at higher energies (λ < 300 nm) correspond to overlapping L(π) → L(π*) and bpy(π) → bpy(π*) LC transitions in the case of 1 (L = dphol), 3 (L = hbtz), and 5 (L = quo), and overlapping L(π) → L(π*) and phen(π) → phen(π*) LC transitions for 2 (L = dphol) and 4 (L = hbtz). Cytotoxic Properties. Compounds 1−5 exhibit IC50 values in the low micromolar range (Table 3). Cisplatin was Table 3. Cytotoxicity Data for Complexes 1−5 against A549 Cells (Using Janus Green B Assay)a compound IC50, μM (95% CIb) [Ru(bpy)2(dphol)][PF6] (1) [Ru(phen)2(dphol)][PF6] (2) [Ru(bpy)2(hbtz)][PF6] (3) [Ru(phen)2(hbtz)][PF6] (4) [Ru(bpy)2(quo)][PF6] (5) cisplatin 6.6 (3.2 to 13.9) 1.3 (0.8 to 2.1) 1.1 (0.7 to 1.9) 0.8 (0.5 to 1.2) >50 6.2 (2.9 to 13.5) a Compounds 1−5 were dissolved in DMEM/F-12 medium (with 0.1% DMSO). Incubation time = 48 h. bValues in parentheses represent the 95% conﬁdence interval. used as a positive control (IC50 = 6.2 μM). Compound 1 exhibits activity comparable to that of cisplatin, whereas compounds 2−4 are more active than the platinum drug. Compound 4 is the most cytotoxic of the Ru series (IC50 = 0.8 μM) and is 8-fold more cytotoxic than cisplatin. In sharp contrast, compound 5 is not active in the range of concentrations that were tested (0−50 μM). It is likely that the higher cytotoxicities of 1−4 with respect to 5 is due to increased lipophilicities because they feature extended aromatic systems on the ligands dphol (1 and 2) and hbtz (3 and 4), in support of our initial hypothesis. The higher lipophilicity of dphol and hbtz with respect to quo may increase the cellular uptake of 1−4, as also shown in the work of Meggers9 and in several studies on the anticancer properties of other Ru23,4,24−26 complexes. Investigation of the Mechanism of Cancer Cell Death. Disruption and permanent dissipation of the inner mitochondrial transmembrane potential (ΔΨm) is an event that is associated with the intrinsic pathway of apoptosis.41,42 To determine the eﬀect of 1−4 on mitochondria, mitochondrial dysfunction was assessed by measuring the changes in ΔΨm using JC-1 (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl carbocyanine iodide).22,43 JC-1 is a lipophilic cationic dye that accumulates in mitochondria due to the negative potential of the inner membrane on these organelles (−120 to −160 mV41,44), and its ﬂuorescence wavelength is potentialdependent: regions within the cell with high mitochondrial polarization (high ΔΨm) are indicated by red ﬂuorescence (590 nm) due to the formation of dye aggregates (so-called Jaggregates). In contrast, green ﬂuorescence (527 nm) of dye monomers is observed when the mitochondria are depolarized (low ΔΨm).41,45 Therefore, mitochondrial depolarization is Figure 3. R values from JC-1 assay in A549 cells after 48 h of incubation when exposed to diﬀerent concentrations of compounds (a) 1, (b) 2, (c) 3, and (d) 4. The graphs represent means with standard deviation. Control experiments (no metal complex) are depicted in gray. concentration of the compounds is accompanied by a progressive decrease of R, indicating that 1−4 induce mitochondria depolarization in a dose-dependant fashion, a result that signiﬁes cell death occurs via the intrinsic pathway of apoptosis. To compare the changes of R among 1−4, the change of R (ΔR) with respect to a control (no complex added) was calculated at 5 μM concentration of Ru complex (ΔR = [(R5 μM − Rcontrol)/Rcontrol] × 100%). The observed trend is as follows: 4 (−97%) > 2 (−66%) ≈ 3 (−63%) > 1 (−23%), where the values in parentheses represent ΔR. Interestingly, ΔR was directly proportional to the cytotoxicity, where the most cytotoxic compound, 4, decreases R to the greatest extent. Moreover, 4 induces a greater ΔΨ m depolarization as compared to cisplatin (Figure 4), with ΔR = −70% and −15% for 4 and cisplatin at 1.5 μM concentration, respectively. The typical ﬁbrillar mitochondrial structure in A549 cells is shown in Figure S10, where cells were incubated only with JC1. Green (Figure S10a) and red (Figure S10b) ﬂuorescence images are shown to highlight the dual emission of JC-1 in mitochondria. Dramatic morphological changes of mitochondria occur when A549 cells are incubated with 4 for 48 h at concentrations as low as 0.5 μM, as shown in Figure 5. Complex 4 induces swelling and fragmentation of mitochondria, producing small and rounded organelles.41,46 Such morphological changes indicate cell death by the intrinsic F DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry 6a−c). In contrast, incubation of A549 cells with 1 μM 4 for 48 h leads to calcein ﬂuorescence quenching inside mitochondria, and dim green ﬂuorescence is observed throughout the cells (Figure 7). These results indicate long-lasting opening of the mitochondrial permeability transition pore (MPTP) complex, which allows CoII ions to enter the mitochondrial matrix and quench calcein ﬂuorescence, thus supporting the intrinsic pathway of apoptosis as the mechanism of cancer cell death. Caspases are cysteine proteases that are involved in both the initiation and execution phases of apoptosis and, indeed, the activation of these proteolytic enzymes is often taken as a hallmark of apoptosis.47,48 In particular, caspase-3/7, known as the executioner caspases, are activated after cytochrome c leaks out of mitochondria when a cell dies via the intrinsic pathway of apoptosis.41 The eﬀect of a chemotherapy drug on the activity of caspase-3/7 can be examined using the Caspase-Glo assay kit, which includes (i) the caspase-3/7 substrate DEVDNH-luciferin and (ii) the Luciferase enzyme. Caspase-3/7, which are activated during apoptosis, cleave the amino acid sequence DEVD (Asp-Glu-Val-Asp), releasing 6-aminoluciferin (Figure S11). The latter undergoes oxidative decarboxylation catalyzed by the Luciferase enzyme, producing oxyluciferin in an electronically excited state (oxyluciferin*), which emits light (hv) when it is deactivated and returns to the ground state (oxyluciferin* → oxyluciferin + hv). The luminescence intensity is proportional to the amount of caspase-3/7 activity. A549 cells were treated with compound 4 at three diﬀerent concentrations (0.75, 1.5, and 2.25 μM) for 48 h, and caspase3/7 activity was determined after this period of time. Compound 4 induced caspase-3/7 activity in a concentration-dependent fashion as depicted in Figure 8, in which an increase in the oxyluciferin luminescence intensity is observed upon increasing the concentration of 4, results that conﬁrm that this Ru compound induces cell suicide via the intrinsic pathway. The data in Figure 8 show that a lower concentration of 4 is needed to induce caspase-3/7 activity with respect to cisplatin, in which 2.25 μM 4 reaches activity comparable to that of 6.25 μM cisplatin, indicating higher apoptosis inducing ability of 4. Figure 4. R values from JC-1assay in A549 cells after 48 h of incubation when exposed to diﬀerent concentrations of cisplatin and compound 4. The graphs represent means with standard deviation. apoptosis pathway. A progressive decrease of the red ﬂuorescence intensity from J-aggregates can be also seen when the concentration of 4 increases from 0 to 1 μM (Figures 5a−c), indicating loss of ΔΨm as described previously. Induction of outer mitochondrial membrane permeabilization (MMP) is a crucial event during cell death via the intrinsic pathway of apoptosis and is often considered as the “point of no return”.41 Such an event is accompanied by a dissipation of ΔΨm and permeabilization of the inner mitochondrial membrane (IMM). To further support that the intrinsic pathway of apoptosis is triggered by these series of compounds, a biological assay that uses the calcein AM dye was carried out for compound 4. In this experiment, A549 cells were loaded with calcein AM and CoCl2. Calcein AM undergoes removal of the ester groups by intracellular esterases to form calcein which gets trapped in cytosolic compartments, including mitochondria.41 The CoII ions quench the ﬂuorescence of calcein in all subcellular compartments except in mitochondria because the IMM is impermeable to CoII ions and water. When the IMM barriers are functional, a distinct bright green ﬂuorescence signal from calcein identiﬁes mitochondria,41 as can be observed in Figure 6a. Dim green ﬂuorescence from calcein is observed elsewhere because CoII ions quench calcein ﬂuorescence. The signal from calcein overlays very well with the red ﬂuorescence from Mitotracker, a mitochondria-speciﬁc ﬂuorescent dye, thus conﬁrming the localization of calcein in mitochondria (Figures ■ CONCLUSIONS The lack of studies in the literature of cytotoxic properties of Ru(II) polypyridyl complexes in a [RuIIN5O]+ coordination Figure 5. Confocal red ﬂuorescence images of JC-1 in A549 cells incubated with (a) 0, (b) 0.5, and (c) 1 μM of compound 4. Only red ﬂuorescence images were shown to emphasize the progressive decrease of red ﬂuorescence intensity with concentration. Field of view = 75 × 75 μm. Images were collected after 48 h of incubation with the corresponding concentration of the compound. G DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry Figure 6. Confocal ﬂuorescence images of A549 cells coincubated with Calcein AM and Mitotracker in the absence of compound 4. (a) Green ﬂuorescence from Calcein (+CoCl2), (b) red ﬂuorescence from Mitotracker, and (c) overlay of (a, b) images. Field of view = 75 × 75 μm. Images were collected after 48 h of incubation. Figure 7. Confocal ﬂuorescence images of A549 cells coincubated with compound 4 (1 μM), Calcein AM, and Mitotracker. (a) Green ﬂuorescence from Calcein AM (+CoCl2), (b) red ﬂuorescence from Mitotracker, and (c) overlay of (a, b) images. Field of view = 75 × 75 μm. Images were collected after 48 h of incubation with the compound. the less positive oxidation potential for this series of compounds with respect to the prototype complex [Ru(bpy)3]2+. The four compounds are cytotoxic against human lung adenocarcinoma (A549) cells with IC50 values in the low micromolar range. Compound 4, the most active of the series, is ∼8 times more cytotoxic than cisplatin in this cell line. The four Ru complexes induce loss of ΔΨm in a concentrationdependent fashion, and the cytotoxicity of 1−4 is directly proportional to the loss of ΔΨm, indicating that 1−4 induce apoptosis via the intrinsic pathway in A549 cells. Moreover, compound 4 promotes the long-lasting opening of the MPTP complex and induces the activity of caspase-3/7, conﬁrming that A549 cells die by the intrinsic pathway of apoptosis when incubated with this type of Ru complex. These results support the hypothesis that increasing the lipophilicity of the N∧O− ligand is a successful strategy for accessing a new family of proapoptotic Ru complexes with a [RuIIN5O]+ coordination environment. This work complements the strategy23 to increase cellular uptake and anticancer activity. These results are expected to encourage the exploration of the anticancer activities of other octahedral Ru(II) polypyridyl complexes possessing coordination environments diﬀerent than [RuIIN6]2+. Figure 8. Caspase-3/7 activity measurements when A549 cells are exposed for 48 h to compound 4 or cisplatin. The graphs represent means with standard deviation. environment prompted us to explore the biological activities of a new series of Ru polypyridyl compounds with the N∧O− bidentate ligands dphol and hbtz. Four new Ru complexes (1− 4) were prepared and characterized by 1H NMR spectroscopy, mass spectrometry, elemental analysis, and X-ray crystallography. The 1MLCT transitions for these complexes are bathochromically shifted with respect to that of [Ru(bpy)3]2+ due to the π-donating ability of the O−-donor of N∧O−, which destabilizes the Ru(dπ) HOMOs and decreases the energy of the Ru(dπ) → L(π*) transition. This eﬀect is also reﬂected in H DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry ■ 116 colorectal cancer to the ruthenium-based drug NAMI-A. J. Inorg. Biochem. 2017, 168, 90−97. (9) Havrylyuk, D.; Heidary, D. K.; Nease, L.; Parkin, S.; Glazer, E. C. Photochemical Properties and Structure−Activity Relationships of RuII Complexes with Pyridylbenzazole Ligands as Promising Anticancer Agents. Eur. J. Inorg. Chem. 2017, 2017 (12), 1687−1694. (10) Lenis-Rojas, O. A.; Roma-Rodrigues, C.; Fernandes, A. R.; Marques, F.; Pérez-Fernández, D.; Guerra-Varela, J.; Sánchez, L.; Vázquez-García, D.; López-Torres, M.; Fernández, A.; Fernández, J. J. Dinuclear RuII(bipy)2 Derivatives: Structural, Biological, and in Vivo Zebrafish Toxicity Evaluation. Inorg. Chem. 2017, 56 (12), 7127− 7144. (11) Schatzschneider, U.; Niyhesel, J.; Ott, I.; Gust, R.; Alborzinia, H.; Wölfl, S. Cellular Uptake, Cytotoxicity, and Metabolic Profiling of Human Cancer Cells Treated with Ruthenium(II) Polypyridyl Complexes [Ru(bpy)2(N-N)]Cl2 with N-N = bpy, phen, dpq, dppz, and dppn. ChemMedChem 2008, 3 (7), 1104−1109. (12) Schäfer, S.; Ott, I.; Gust, R.; Sheldrick, W. S. Influence of the Polypyridyl (pp) Ligand Size on the DNA Binding Properties, Cytotoxicity and Cellular Uptake of Organoruthenium(II) Complexes of the Type [(η6-C6Me6)Ru(L)(pp)]n+ [L = Cl, n = 1; L = (NH2)2CS, n = 2]. Eur. J. Inorg. Chem. 2007, 2007, 3034−3046. (13) Tan, C.; Lai, S.; Wu, S.; Hu, S.; Zhou, L.; Chen, Y.; Wang, M.; Zhu, Y.; Lian, W.; Peng, W.; Ji, L.; Xu, A. Nuclear Permeable Ruthenium(II) β-Carboline Complexes Induce Autophagy To Antagonize Mitochondrial-Mediated Apoptosis. J. Med. Chem. 2010, 53 (21), 7613−7624. (14) Tan, C.; Wu, S.; Lai, S.; Wang, M.; Chen, Y.; Zhou, L.; Zhu, Y.; Lian, W.; Peng, W.; Ji, L.; Xu, A. Synthesis, structures, cellular uptake and apoptosis-inducing properties of highly cytotoxic rutheniumNorharman complexes. Dalton Trans. 2011, 40 (34), 8611−8621. (15) Qian, C.; Wang, J.-Q.; Song, C.-L.; Wang, L.-L.; Ji, L.-N.; Chao, H. The induction of mitochondria-mediated apoptosis in cancer cells by ruthenium(ii) asymmetric complexes. Metallomics 2013, 5 (7), 844−854. (16) Garner, R. N.; Gallucci, J. C.; Dunbar, K. R.; Turro, C. [Ru(bpy)2(5-cyanouracil)2]2+ as a Potential Light-Activated DualAction Therapeutic Agent. Inorg. Chem. 2011, 50, 9213−9215. (17) Respondek, T.; Garner, R. N.; Herroon, M. K.; Podgorski, I.; Turro, C.; Kodanko, J. J. Light Activation of a Cysteine Protease Inhibitor: Caging of a Peptidomimetic Nitrile with RuII(bpy)2. J. Am. Chem. Soc. 2011, 133, 17164−17167. (18) Sears, R. B.; Joyce, L. E.; Ojaimi, M.; Gallucci, J. C.; Thummel, R. P.; Turro, C. Photoinduced ligand exchange and DNA binding of cis-[Ru(phpy)(phen)(CH3CN)2]+ with long wavelength visible light. J. Inorg. Biochem. 2013, 121, 77−87. (19) Sgambellone, M. A.; David, A.; Garner, R. N.; Dunbar, K. R.; Turro, C. Cellular Toxicity Induced by the Photorelease of a Caged Bioactive Molecule: Design of a Potential Dual-Action Ru(II) Complex. J. Am. Chem. Soc. 2013, 135, 11274−11282. (20) Komor, A. C.; Barton, J. K. The path for metal complexes to a DNA target. Chem. Commun. 2013, 49, 3617−3630. (21) Gill, M. R.; Thomas, J. A. Ruthenium(II) polypyridyl complexes and DNA-from structural probes to cellular imaging and therapeutics. Chem. Soc. Rev. 2012, 41, 3179−3192. (22) Pierroz, V.; Joshi, T.; Leonidova, A.; Mari, C.; Schur, J.; Ott, I.; Spiccia, L.; Ferrari, S.; Gasser, G. Molecular and Cellular Characterization of the Biological Effects of Ruthenium(II) Complexes Incorporating 2-Pyridyl-2-pyrimidine-4-carboxylic Acid. J. Am. Chem. Soc. 2012, 134, 20376−20387. (23) Mulcahy, S. P.; Grundler, K.; Frias, C.; Wagner, L.; Prokop, A.; Meggers, E. Discovery of a strongly apoptotic ruthenium complex through combinatorial coordination chemistry. Dalton Trans. 2010, 39, 8177−8182. (24) Heidary, D. K.; Howerton, B. S.; Glazer, E. C. Coordination of Hydroxyquinolines to a Ruthenium Bis-dimethyl-phenanthroline Scaffold Radically Improves Potency for Potential as Antineoplastic Agents. J. Med. Chem. 2014, 57 (21), 8936−8946. ASSOCIATED CONTENT * Supporting Information S The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01988. Reaction schemes, NMR spectra, crystallographic data and reﬁnement parameters, bond distances and bond angles, cyclic voltammograms, electronic absorption spectra, and confocal ﬂuorescence microscopy images (PDF) Accession Codes CCDC 997833−997835 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, 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 Author *E-mail: dunbar@chem.tamu.edu. ORCID Sayan Saha: 0000-0002-6724-6430 Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS Confocal microscopy studies were performed in the Texas A&M University College of Veterinary Medicine & Biomedical Sciences Image Analysis Laboratory, supported by NIH-NCRR (Grant 1S10RR22532-01). K.R.D. thanks the National Science Foundation (Grant CHE-12465067) for support of this work. ■ REFERENCES (1) Kelland, L. The resurgence of platinum-based cancer chemotherapy. Nat. Rev. Cancer 2007, 7 (8), 573−584. (2) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The status of platinum anticancer drugs in the clinic and in clinical trials. Dalton Trans. 2010, 39 (35), 8113−8127. (3) Groessl, M.; Reisner, E.; Hartinger, C. G.; Eichinger, R.; Semenova, O.; Timerbaev, A. R.; Jakupec, M. A.; Arion, V. B.; Keppler, B. K. Structure−Activity Relationships for NAMI-A-type Complexes (HL)[trans-RuCl4L(S-dmso)ruthenate(III)] (L = Imidazole, Indazole, 1,2,4-Triazole, 4-Amino-1,2,4-triazole, and 1-Methyl1,2,4-triazole): Aquation, Redox Properties, Protein Binding, and Antiproliferative Activity. J. Med. Chem. 2007, 50 (9), 2185−2193. (4) Trondl, R.; Heffeter, P.; Kowol, C. R.; Jakupec, M. A.; Berger, W.; Keppler, B. K. NKP-1339, the first ruthenium-based anticancer drug on the edge to clinical application. Chem. Sci. 2014, 5 (8), 2925− 2932. (5) Leijen, S.; Burgers, S. A.; Baas, P.; Pluim, D.; Tibben, M.; van Werkhoven, E.; Alessio, E.; Sava, G.; Beijnen, J. H.; Schellens, J. H. M. Phase I/II study with ruthenium compound NAMI-A and gemcitabine in patients with non-small cell lung cancer after first line therapy. Invest. New Drugs 2015, 33 (1), 201−214. (6) Bergamo, A.; Sava, G. Linking the future of anticancer metalcomplexes to the therapy of tumour metastases. Chem. Soc. Rev. 2015, 44 (24), 8818−8835. (7) Gransbury, G. K.; Kappen, P.; Glover, C. J.; Hughes, J. N.; Levina, A.; Lay, P. A.; Musgrave, I. F.; Harris, H. H. Comparison of KP1019 and NAMI-A in tumour-mimetic environments. Metallomics 2016, 8 (8), 762−773. (8) Bergamo, A.; Pelillo, C.; Chambery, A.; Sava, G. Influence of components of tumour microenvironment on the response of HCTI DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX Article Inorganic Chemistry (25) Yang, L.; Zhang, J.; Wang, C.; Qin, X.; Yu, Q.; Zhou, Y.; Liu, J. Interaction between 8-hydroxyquinoline ruthenium(ii) complexes and basic fibroblast growth factors (bFGF): inhibiting angiogenesis and tumor growth through ERK and AKT signaling pathways. Metallomics 2014, 6 (3), 518−531. (26) Zhang, Y.; Zhou, Q.; Zheng, Y.; Li, K.; Jiang, G.; Hou, Y.; Zhang, B.; Wang, X. DNA Photocleavage by Non-innocent LigandBased Ru(II) Complexes. Inorg. Chem. 2016, 55 (9), 4296−4300. (27) Zhang, Y.; Zhou, Q.; Tian, N.; Li, C.; Wang, X. Ru(II)Complex-Based DNA Photocleaver Having Intense Absorption in the Phototherapeutic Window. Inorg. Chem. 2017, 56 (4), 1865−1873. (28) Sullivan, B. P.; Salmon, D. J.; Meyer, T. Mixed Phosphine 2,2′Bipyridine Complexes of Ruthenium. Inorg. Chem. 1978, 17, 3334− 3341. (29) SMART and SAINT; Siemens Analytical X-ray Instruments Inc.: Madison, WI, 1996. (30) Sheldrick, G. M. SADABS; University of Gottinggen: Gottingen, Germany, 1996. (31) Sheldrick, G. A short history of SHELX. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, 64 (1), 112−122. (32) Hubschle, C. B.; Sheldrick, G. M.; Dittrich, B. ShelXle: a Qt graphical user interface for SHELXL. J. Appl. Crystallogr. 2011, 44 (6), 1281−1284. (33) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. OLEX2: a complete structure solution, refinement and analysis program. J. Appl. Crystallogr. 2009, 42 (2), 339−341. (34) Sheldrick, G. Crystal structure refinement with SHELXL. Acta Crystallogr., Sect. C: Struct. Chem. 2015, 71 (1), 3−8. (35) Keyes, T. E.; Leane, D.; Forster, R. J.; Coates, C. G.; McGarvey, J. J.; Nieuwenhuyzen, M. N.; Figgemeier, E.; Vos, J. G. Redox and Spectroscopic Orbitals in Ru(II) and Os(II) Phenolate Complexes. Inorg. Chem. 2002, 41 (22), 5721−5732. (36) Brissard, M.; Convert, O.; Gruselle, M.; Guyard-Duhayon, C.; Thouvenot, R. Enantiospecific Synthesis of Δ and Λ [Ru(bpy)2ppy]+ and [Ru(bpy)2quo]+ (bpy = 2,2′-Bipyridine, ppy = Phenylpyridine−H+, quo = 8-Hydroxyquinolate): 1H and 13C NMR Studies and X-ray Structure Determination of rac-[Ru(bpy)2quo]PF6. Inorg. Chem. 2003, 42, 1378−1385. (37) Sun, Y.; Joyce, L. E.; Dickson, N. M.; Turro, C. Efficient DNA photocleavage by [Ru(bpy)2(dppn)]2+ with visible light. Chem. Commun. 2010, 46, 2426−2428. (38) Zhao, H. C.; Harney, J. P.; Huang, Y.-T.; Yum, J.-H.; Nazeeruddin, M. K.; Grätzel, M.; Tsai, M.-K.; Rochford, J. Evaluation of a Ruthenium Oxyquinolate Architecture for Dye-Sensitized Solar Cells. Inorg. Chem. 2012, 51, 1−3. (39) Bhattacharya, S. 8-quinolinolate Complexes of Ruthenium(II). Synthesis, Characterization and Electron Transfer Properties. Polyhedron 1993, 12 (2), 235−239. (40) Pramanik, N. C.; Bhattacharya, S. Bis(quinolin-8-olato) Complexes of Ruthenium. Synthesis,Characterization and Cyclic Voltammetric Studies. J. Chem. Res., Synop. 1997, 98−99. (41) Kroemer, G.; Galluzzi, L.; Brenner, C. Mitochondrial Membrane Permeabilization in Cell Death. Physiol. Rev. 2007, 87, 99−163. (42) Boehning, D.; Patterson, R. L.; Sedaghat, L.; Glebova, N. O.; Kurosaki, T.; Snyder, S. H. Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat. Cell Biol. 2003, 5 (12), 1051−1061. (43) Qian, C.; Wang, J.-Q.; Song, C.-L.; Wang, L.-L.; Ji, L.-N.; Chao, H. The induction of mitochondria-mediated apoptosis in cancer cells by ruthenium(II) asymmetric complexes. Metallomics 2013, 5, 844− 854. (44) Lemasters, J. J.; Ramshesh, V. K. Imaging of mitochondrial polarization and depolarization with cationic fluorophores. Methods Cell Biol. 2007, 80, 283−295. (45) Johnson, I.; Spence, M. T. Z. The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies. http:// www.lifetechnologies.com/ipac/en/home/references/molecularprobes-the-handbook.html (accessed 17 Jan 2014). (46) Cao, R.; Jia, J.; Ma, X.; Zhou, M.; Fei, H. Membrane Localized Iridium(III) Complex Induces Endoplasmic Reticulum Stress and Mitochondria-Mediated Apoptosis in Human Cancer Cells. J. Med. Chem. 2013, 56, 3636−3644. (47) Li, J.; Yuan, J. Caspases in apoptosis and beyond. Oncogene 2008, 27, 6194−6206. (48) Tan, C.; Wu, S.; Lai, S.; Wang, M.; Chen, Y.; Zhou, L.; Zhu, Y.; Lian, W.; Peng, W.; Ji, L.; Xu, A. Synthesis, structures, cellular uptake and apoptosis-inducing properties of highly cytotoxic rutheniumNorharman complexes. Dalton Trans. 2011, 40, 8611−8621. J DOI: 10.1021/acs.inorgchem.8b01988 Inorg. Chem. XXXX, XXX, XXX−XXX