Coordination of Ru(II)-Arene Fragments to Dipyridophenazine Ligands Leads to the Modulation of Their In Vitro and In Vivo Anticancer Activity.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Coordination of Ru(II)-Arene Fragments to Dipyridophenazine\n Ligands Leads to the Modulation of Their In Vitro and In Vivo\n Anticancer Activity\n Stefan Nikolic\u0301, Jemma Arakelyan, Vladimir Kushnarev, Samah Mutasim Alfadul, Dalibor Stankovic\u0301,\n Yaroslav I. Kraynik, Sanja Grguric\u0301-S\u030c ipka,* and Maria V. Babak*\n Cite This: Inorg. Chem. 2023, 62, 8188\u22128199 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:23:23 (UTC).\n\n\n\n\n ABSTRACT: Despite extensive research on the anticancer properties of Ru\n complexes with dipyrido[3,2-a:2\u2032,3\u2032-c]phenazine (dppz) ligands, their in vivo\n efficacy is rarely investigated. Aiming to understand whether the coordination of\n certain half-sandwich Ru(II)-arene fragments might improve the therapeutic\n potential of dppz ligands, we prepared a series of Ru(II)-arene complexes with\n the general formula [(\u03b76-arene)Ru(dppz-R)Cl]PF6, where the arene fragment\n was benzene, toluene, or p-cymene and R was -NO2, -Me, or -COOMe. All\n compounds were fully characterized by 1H and 13C NMR spectroscopy and high-\n resolution ESI mass-spectrometry, and their purity was verified by elemental\n analysis. The electrochemical activity was investigated using cyclic voltammetry.\n The anticancer activity of dppz ligands and their respective Ru complexes was\n assessed against several cancer cell lines, and their selectivity toward cancer cells\n was assessed using healthy MRC5 lung fibroblasts. The substitution of benzene\n with a p-cymene fragment resulted in a more than 17-fold increase of anticancer\n activity and selectivity of Ru complexes and significantly enhanced DNA degradation in HCT116 cells. All Ru complexes were\n electrochemically active in the biologically accessible redox window and were shown to markedly induce the production of ROS in\n mitochondria. The lead Ru-dppz complex significantly reduced tumor burden in mice with colorectal cancers without inducing liver\n and kidney toxicity.\n\n\n \u25a0 INTRODUCTION\n Anticancer Ru complexes gained significant interest after two\n properties and excellent DNA binding affinity.9,10 Interest to\n the class of Ru(II) complexes with a dppz ligand was motivated\n iconic Ru(III) complexes\ufffdNAMI-A ((ImH)[trans- by the work of Barton et al., who demonstrated that the\n RuCl4(dmso-S)(Im)], Im = imidazole) and KP1339 (later [Ru(bipy)2(dppz)]2+ complex could enhance its photo-\n renamed as IT-139 and BOLD-100, Na[trans-RuCl4(Ind)2], luminescence by several orders of magnitude upon interactions\n Ind = indazole)\ufffdentered their first human clinical trials at the with DNA.11 Subsequent studies revealed that the structure of\n beginning of the 21st century.1 Half-sandwich Ru complexes the octahedral heteroleptic Ru-dppz complexes could be easily\n were suggested as alternatives to Ru(III) complexes since their fine-tuned depending on their potential applications. For\n structures could be easily modified according to the required example, the increase of the size of Ru complexes due to the\n design.2 Despite the excellent potential of (arene)Ru extension of the aromatic system in the dppz ligand12 or\n complexes as anticancer agents, none of them has entered introduction of several dppz ligands13 resulted in the\n clinical trials so far, probably due to their uncertain mechanism significant enhancement of their cytotoxicity. Moreover, slight\n of action.3 Interestingly, some half-sandwich Ru complexes variations in the structure of the complexes led to the\n were shown to exhibit anticancer properties due to their modulation of their intracellular accumulation and respective\n catalytic transfer hydrogenation activity in the reduction of anticancer mechanism of action. It is believed that the\n NAD+ to NADH.4\u22126 Other half-sandwich Ru complexes could\n bypass multidrug resistance and conventional programmed cell\n death pathways induced by commonly used chemotherapeutic Received: February 20, 2023\n agents.7,8 Therefore, this class of compounds still remains Published: May 18, 2023\n worthy of investigation.\n In the past decades, Ru complexes with dppz ligands have\n been extensively investigated as molecular imaging probes and\n phototherapeutic agents due to their unique photophysical\n\n \u00a9 2023 American Chemical Society https://doi.org/10.1021/acs.inorgchem.3c00570\n 8188 Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nanticancer activity of (polypyridyl)Ru-dppz complexes origi- ROS in the mitochondria of cancer cells. Importantly, this\nnates from their ability to intercalate into duplex DNA;14\u221216 work extended the limited in vivo data about the toxicity and\nhowever, it was also shown that some of these complexes could antitumor efficacy of Ru-dppz complexes and respective dppz\nreadily intercalate into DNA G-quadruplex17 and RNA ligands.\ntriplex18 or accumulate in mitochondria, thereby initiating\nmitochondria-mediated apoptosis.13,19\n In general, the coordination of dppz-derived ligands to the\nRu(polypyridyl) backbone yields highly stable Ru complexes\n \u25a0 EXPERIMENTAL SECTION\n Materials and Methods. Chemicals and Materials. All\n chemicals were of reagent-grade quality or higher, obtained from\nthat are inert under physiological conditions.16,20 On the other commercial suppliers, and used without further purification. Solvents\nhand, the use of organometallic fragments typically leads to the were used as received. RuCl3\u00b73H2O was purchased from Johnson\nformation of half-sandwich metal-dppz complexes with labile Matthey (London, United Kingdom). [(\u03b76-p-Cymene)RuCl2]2, [(\u03b76-\nchlorido ligands.21,22 It is believed that the hydrolysis of the toluene)RuCl2]2, and [(\u03b76-benzene)RuCl2]2 were prepared according\nlabile ligands results in the formation of highly reactive solvato to a published procedure.30 Thiazolyl blue tetrazolium bromide\ncomplexes that readily bind to the DNA,21 leading to DNA (MTT) was purchased from Alfa Aesar; Roswell Park Memorial\ndegradation. Therefore, various half-sandwich Ru complexes Institute (RPMI 1640) medium, Dulbecco\u2019s modified Eagle\u2019s medium\nwith dppz ligands and its analogues demonstrated promising (DMEM) medium, fetal bovine serum (FBS), Hank\u2019s balanced salt\nanticancer activity, which was dependent on their reactivity solution (HBSS), phosphate-buffered saline (PBS), and tris\u2212acetate\nand interactions with biomolecules. For example, Ru(II)-arene EDTA (TAE) were purchased from Thermo Fisher Scientific. Milli-\n Q-grade purified water was obtained from a Milli-Q UV purification\ncomplexes with diimine ligands, where diimine was 1,10- system (Sartorius Stedim Biotech S.A., Aubagne Cedex, France).\nphenanthroline, 5,6-dimethyl-1,10-phenanthroline, dppz, or 2\u2032,7\u2032-Dichlorodihydrofluorescein diacetate and Cremophor EL were\n11,12-dimethyl-dppz induced oxidative cleavage of the super- purchased from Sigma-Aldrich, Mitotracker Red CMXRos was\ncoiled DNA plasmid.23 The most pronounced effects were purchased from Molecular Probes, Eugene, OR, USA, and Hoechst\nrecorded for complexes with dppz-based ligands and the 33342 was purchased from B.D. Bioscience, San Jose, CA, USA,\nobserved DNA effects were in correlation with the cytotoxicity accordingly. DNA extraction and purification kits were obtained from\nof the complexes. Interestingly, the DNA-binding efficiency of QIAgen, Germany.\nRu(II)-arene complexes was not only defined by the chelating Instrumentation. 1H and 13C NMR spectra were recorded using\nligand but also by the steric bulk of other participating co- Varian (Agilent, USA) 400 and 100 MHz NMR spectrometers\n equipped with a 5 mm ATB probe. The chemical shifts, \u03b4, were\nligands.24 It was shown that Ru(II)-dppz complexes with S-\n reported in ppm (parts per million), and coupling constants were\ncoordinated L-methionine or L-cysteine and bulky \u03b76- reported in Hertz. The residual solvent peaks were used as an internal\nhexamethylbenzene were characterized by the higher Kb values reference. The abbreviations for the peak multiplicities are as follows:\nthan their \u03b76-benzene analogues, suggesting stronger duplex s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q\nstabilization.24 Previously, some of us reported the synthesis, (quartet), m (multiplet), and br (broad). Elemental analysis was\ncharacterization, and preliminary biological assessment of carried out with the addition of V2O5 using an Elemental MicroCube\n(arene)Ru complexes with dppz-derived ligands.25,26 Several CHN analyzer. Infrared spectra were recorded on a Nicolet 6700 FT-\ncomplexes demonstrated enhanced cytotoxicity and selectivity IR spectrometer using the ATR technique. ESI mass spectra\nin comparison with the clinically used drug cisplatin. In measurements of complexes were carried out on a high-resolution\ncontrast, structurally similar half-sandwich Ru(II) complexes mass spectrometer (Sciex X500R Q-TOF). Cell viability assay was\n performed using the BioTek Synergy H1 microplate reader. Confocal\nwith bipyrimidine, tryptanthrin-6-oxime, or 11H-indeno[1,2-\n microscopy was performed using a Leica Confocal SP8 Microscope\nb]quinoxalin-11-one derivatives were devoid of cytotoxicity27 (Super Resolution). Agarose gel imaging was performed under UV\nor only moderately cytotoxic,28 indicating the importance of light using a ChemiDoc Touch Imaging System (BioRad). The DNA\ndppz-derived ligands in the biological activity of resulting concentrations were quantified using a NanoDrop One/OneC\ncomplexes. Microvolume UV\u2212Vis spectrophotometer.\n Surprisingly, despite continuous interest in the therapeutic Synthesis. General Procedure for Ligand Synthesis. All ligands\npotential of Ru-dppz complexes, the majority of the studies for were synthesized according to a literature procedure using a\nboth heteroleptic and half-sandwich Ru-dppz and structurally condensation reaction between 1,10-phenathroline-5,6-dione and\nsimilar complexes were mostly limited to in vitro experiments. diamine.31 The characterization of the ligands was in agreement\nThere are only few examples of in vivo studies with Ru-dppz with the literature data.32\u221234\n General Procedure for the Synthesis of Ru Complexes. The\ncomplexes or uncoordinated dppz-derived ligands. For\n solution of [(\u03b76-arene)RuCl2]2 (1 eq) in methanol (5 mL) was added\nexample, heteroleptic Ru(bipyridine)(dppz) complexes to a solution of its respective ligand (2 eq) in a methanol/CH2Cl2\nshowed high antiproliferative, antimetastatic, and antiangio- mixture (10 mL) 1/1 (v/v). The mixture was stirred at room\ngenic activity in the MDA-MB-231 zebrafish xenograft temperature for 5 h. NH4PF6 (3 eq) was then added to the solution\nmodel.29 Another heteroleptic Ru(tetra-imidazole)(dppz) and left to stir overnight. The formed yellow precipitate was filtered\ncomplex significantly inhibited tumor growth in mice and washed with cold MeOH and Et2O and dried in vacuo.\nxenografted with A549 lung cancer.19 Synthesis of [(\u03b76-Benzene)Ru(Medppz)Cl]PF6\u00b7H2O (1A). [(\u03b76-\n In this project, we prepared a series of dppz-derived ligands Benzene)RuCl2]2 (precursor 1) (50 mg, 0.099 mmol, 1 eq.), Medppz\nand respective piano-stool Ru-arene complexes with the (ligand A) (59 mg, 0.199 mmol, 2 eq.), and NH4PF6 (48 mg, 0.297\ngeneral formula [(\u03b76-arene)Ru(dppz-R)Cl]PF6. We discussed mmol, 3 eq.). Yield: 110 mg (82%). Calcd. for: C25H20ClF6N4PRuO\n C 44.55; H 2.99; N 8.31. Found: C 44.92; H 2.83; N 8.61. IR (cm\u22121):\nthe effects of the arene fragment and substituents in the dppz\n 3091, 1608 (C\ufffdN), 1501, 1414, 1357, 837, 557. 1H NMR (400\nligand on the electrochemical and anticancer properties of MHz, DMSO-d6): \u03b4 10.07 (d, J = 5.2 Hz, 2H), 9.39 (dd, J = 14.3, 8.1\nresulting complexes. We investigated the mechanism of action Hz, 2H), 8.26\u22128.16 (m, 2H), 8.00 (d, J = 8.6 Hz, 1H), 7.92 (s, 1H),\nof the lead drug candidates, including DNA degradation and 7.76 (d, J = 8.7 Hz, 1H), 6.35 (s, 6H), 2.56 (s, 3H). 13C{1H} NMR\nROS induction, and linked the electrochemical activity of these (101 MHz, DMSO-d6): \u03b4 157.96, 157.85, 150.58, 148.18, 148.01,\ncomplexes with their ability to induce the production of deadly 143.78, 142.18, 140.75, 138.86, 138.20, 135.47, 135.42, 135.34,\n\n 8189 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n129.62, 129.55, 129.12, 127.92, 127.75, 87.33, 22.06. ESI-HRMS: [M- 129.49, 128.04, 87.36, 53.46. ESI-HRMS: [M-PF6]+: 555.0353\nPF6]+: 511.0429 (calculated 511.02634). (calculated 555.01618).\n Synthesis of [(\u03b76-Toluene)Ru(Medppz)Cl]PF6 (2A). [(\u03b76-Toluene)- Synthesis of [(\u03b76-Toluene)Ru(CO2Medppz)Cl]PF6 (2C). [(\u03b76-\nRuCl2]2 (precursor 2) (50 mg, 0.095 mmol, 1 eq), Medppz (ligand Toluene)RuCl2]2 (precursor 2) (68,5 mg, 0.129 mmol, 1 eq),\nA) (56 mg, 0.189 mmol, 2 eq), and NH4PF6 (46 mg, 0.285 mmol, 3 CO2Medppz (ligand C) (88 mg, 0.258 mmol, 2 eq), NH4PF6 (63 mg,\neq). Yield: 124 mg (94%). Calcd. for: C26H20ClF6N4PRu C 46.61; H 0.387 mmol, 3 eq). Yield: 170 mg (92%). Calcd. for:\n3.01; N 8.36. Found: C 46.23; H 2.92; N 8.60. IR (cm\u22121): 3073, 1578 C27H20ClF6N4O2PRu C 45.42; H 2.82; N 7.85. Found: C 45.28; H\n(C\ufffdN), 1500, 1413, 1357, 840, 558. 1H NMR (400 MHz, DMSO- 2.85; N 7.57. IR (cm\u22121): 3078, 1731 (C\ufffdO), 1626 (C\ufffdN), 1448,\nd6): \u03b4 9.96 (d, J = 5.1 Hz, 4H), 9.35 (dd, J = 15.8, 8.2 Hz, 4H), 8.25\u2212 1254, 1091, 837, 730, 558. 1H NMR (400 MHz, DMSO-d6): \u03b4\n8.14 (m, 4H), 7.94 (d, J = 8.7 Hz, 2H), 7.87 (s, 2H), 7.72 (d, J = 8.7 10.09\u22129.98 (m, 2H), 9.53 (dd, J = 8.0, 4.0 Hz, 2H), 8.77 (s, 1H),\nHz, 2H), 6.45 (t, J = 5.7 Hz, 4H), 6.09 (d, J = 6.0 Hz, 4H), 5.90 (t, J = 8.36 (dt, J = 18.4, 5.2 Hz, 2H), 8.28\u22128.22 (m, 2H), 6.47 (t, J = 5.9\n5.6 Hz, 2H), 2.53 (s, 5H), 2.29 (s, 5H). 13C{1H} NMR (101 MHz, Hz, 2H), 6.11 (d, J = 6.1 Hz, 2H), 5.92 (t, J = 5.7 Hz, 1H), 3.99 (s,\nDMSO-d6): \u03b4 162.27, 157.74, 157.63, 150.58, 148.24, 148.07, 143.76, 3H), 2.29 (s, 3H). 13C{1H} NMR (101 MHz, DMSO-d6): \u03b4 165.56,\n142.10, 140.66, 138.82, 138.17, 135.37, 135.32, 129.51, 129.44, 161.69, 158.40, 158.25, 150.51, 148.91, 148.69, 143.66, 141.30,\n129.06, 127.86, 127.81, 127.76, 106.24, 90.54, 83.40, 80.64, 22.04, 141.00, 140.49, 135.95, 135.74, 132.77, 131.70, 131.06, 130.52,\n19.32. ESI-HRMS: [M-PF6]+: 525.0560 (calculated 525.04199). 129.39, 128.06, 106.26, 90.56, 83.47, 80.80, 53.45, 19.33. ESI-HRMS:\n Synthesis of [(\u03b76-Cymene)Ru(Medppz)Cl]PF6 (3A). [(\u03b76-Cymene)- [M-PF6]+: 569.0451 (calculated 569.03183).\nRuCl2]2 (precursor 3) (50 mg, 0.079 mmol, 1 eq), Medppz (ligand Synthesis of [(\u03b76-Cymene)Ru(CO2Medppz)Cl]PF6 \u00b7 H2O (3C). [(\u03b76-\nA) (40 mg, 0.158 mmol, 2 eq), NH4PF6 (38 mg, 0.237 mmol, 3 eq). cymene)RuCl2]2 (precursor 3) (74 mg, 0.12 mmol, 1 eq),\nYield: 81 mg (72%). Calcd. for: C29H26ClF6N4PRu C 48.92; H 3.68; CO2Medppz (ligand C) (82 mg, 0.241 mmol, 2 eq), NH4PF6 (59\nN 7.87. Found: C 48.75; H 3.59; N 8.29. IR (cm\u22121): 3132, 3047, mg, 0.361 mmol, 3 eq). Yield: 165 mg (88%). Calcd. for:\n2877, 1607 (C\ufffdN), 1498, 1411, 1357, 845, 558. 1H NMR (400 C30H28ClF6N4O3PRu C 46.55; H 3.65; N 7.24. Found: C 45.99; H\nMHz, DMSO-d6): \u03b4 9.96 (s, 2H), 9.54 (t, J = 7.8 Hz, 2H), 8.29\u22128.21 3.68; N 7.84. IR (cm\u22121): 3056, 2973, 1727 (C\ufffdO), 1624 (C\ufffdN),\n(m, 2H), 8.15 (d, J = 8.6 Hz, 1H), 8.06 (s, 1H), 7.86 (d, J = 8.8 Hz, 1415, 1320, 840, 773, 558. 1H NMR (400 MHz, DMSO-d6): \u03b4 10.02\n1H), 6.35 (d, J = 6.1 Hz, 2H), 6.12 (d, J = 6.1 Hz, 2H), 2.74\u22122.57 (d, J = 4.7 Hz, 2H), 9.60 (d, J = 8.1 Hz, 2H), 8.83 (s, 1H), 8.47\u22128.36\n(m, 4H), 2.18 (s, 3H), 0.96 (d, J = 6.8 Hz, 6H). 13C{1H} NMR (101 (m, 2H), 8.33\u22128.21 (m, 2H), 7.16 (s, 2H), 6.38 (d, J = 6.4 Hz, 2H),\nMHz, DMSO-d6): \u03b4 162.22, 157.63, 150.58, 148.25, 148.08, 143.80, 6.15 (d, J = 6.4 Hz, 2H), 3.98 (s, 3H), 2.67 (dt, J = 13.8, 6.9 Hz, 1H),\n142.35, 140.94, 139.31, 138.65, 135.48, 129.84, 129.79, 129.23, 2.19 (s, 3H), 0.97 (d, J = 6.9 Hz, 5H). 13C{1H} NMR (101 MHz,\n128.03, 127.96, 105.23, 103.13, 86.34, 84.65, 30.88, 22.18, 22.09, DMSO-d6): \u03b4 165.62, 148.84, 148.61, 143.73, 141.35, 141.28, 140.77,\n18.66. ESI-HRMS: [M-PF6]+: 567.1046 (calculated 567.08894). 135.81, 132.81, 131.72, 130.57, 129.60, 128.20, 105.31, 86.37, 84.67,\n Synthesis of [(\u03b7 6 -Benzene)Ru(NO 2dppz)Cl]PF 6 (1B). [(\u03b76 - 53.44, 30.88, 22.20, 18.69. ESI-HRMS: [M-PF6]+: 611.0967\nBenzene)RuCl2]2 (precursor 1) (46 mg, 0.092 mmol, 1 eq); (calculated 611.07878).\nNO2dppz (ligand B) (60 mg, 0.183 mmol, 2 eq), NH4PF6 (45 mg, Cyclic Voltammetry. Electrochemical measurements were\n0.275 mmol, 3 eq). Yield: 114 mg (90%). Calcd. for: conducted in a three-electrode cell (volume of 5 mL), where glassy\nC24H15ClF6N5O2PRu C 41.97; H 2.20; N 10.20. Found: C 42.30; carbon electrode (3 mm diameter) was used as a working electrode,\nH 2.26; N 10.11. IR (cm\u22121): 3092, 1732, 1611 (C\ufffdN), 1526 (NO2), Ag/AgCl (3 M KCl) was used as a reference electrode, and platinum\n1352 (NO2), 1053, 833, 729, 556. 1H NMR (400 MHz, DMSO-d6): \u03b4 wire was used as a counter electrode. The concentration of the Ru\n10.12 (s, 2H), 9.66 (d, J = 5.5 Hz, 2H), 9.18 (s, 1H), 8.73 (d, J = 8.7 complexes was 1 mg/mL in DMSO containing 0.1 M tetrabuty-\nHz, 1H), 8.61 (d, J = 9.2 Hz, 1H), 8.29 (s, 2H), 6.35 (s, 6H). lammonium perchlorate as the supporting electrolyte. Measurements\n13\n C{1H} NMR (101 MHz, DMSO-d6): \u03b4 160.91, 158.90, 158.74, were carried out in a potential range from \u22120.8 to 1.5 V at a scan rate\n149.21, 149.15, 148.94, 144.12, 142.06, 141.58, 140.96, 136.37, of 50 mV/s using a CHI instrument model 760b (Austin, Texas,\n136.10, 131.92, 129.47, 129.42, 128.25, 125.92, 125.66, 87.44. ESI- USA).\nHRMS: [M-PF6]+: 542.0118 (calculated 541.99578). Stability Studies. To check the stability of 1A\u22123A, 1B, 2B, and\n Synthesis of [(\u03b7 6 -Toluene)Ru(NO 2 dppz)Cl]PF 6 (2B). [(\u03b7 6 - 1C\u22123C in DMSO, these compounds were dissolved in DMSO-d6,\nToluene)RuCl2]2 (precursor 2) (50 mg, 0.094 mmol, 1 eq); NO2dppz and 1H NMR spectra were obtained at 0, 24, 48 and 72 h timepoints.\n(ligand B) (62 mg, 0.188 mmol, 2 eq); NH4PF6 (46 mg, 0.282 mmol, For stability studies, all compounds were dissolved in 1% DMSO in\n3 eq) Yield: 128 mg (97%). Calcd. for: C25H17ClF6N5O2PRu C 42.84; PBS or complete colorless cell culture media (DMEM containing 10%\nH 2.44; N 9.99. Found: C 42.42; H 2.66; N 9.79. IR (cm\u22121): 3093, FBS and 1% penicillin\u2212streptomycin)/H2O (1:9) solvent system. All\n1610 (C\ufffdN), 1529 (NO2), 1354 (NO2), 1499, 1419, 1354, 839, solutions were kept at 37 \u00b0C throughout the experiment. The UV\u2212\n729, 558. 1H NMR (400 MHz, DMSO-d6): \u03b4 10.04 (d, J = 5.3 Hz, Vis profiles of the samples were recorded at 0, 24, and 72 h using a\n2H), 9.64 (dd, J = 8.1, 3.0 Hz, 2H), 9.18 (d, J = 2.2 Hz, 1H), 8.72 Cary 50 spectrophotometer (Varian) with the following parameters:\n(dd, J = 9.3, 2.3 Hz, 1H), 8.60 (d, J = 9.3 Hz, 1H), 8.29 (dt, J = 8.3, start wavelength = 200 nm, end wavelength = 800 nm, scan speed =\n5.1 Hz, 2H), 6.47 (t, J = 5.9 Hz, 2H), 6.10 (d, J = 6.1 Hz, 2H), 5.90 400 nm/min, sampling interval = 0.5, slit width = 1.5, and path length\n(t, J = 5.6 Hz, 1H), 2.28 (s, 3H). 13C{1H} NMR (101 MHz, DMSO- = 10. Blank calibration and baseline correction were performed before\nd6): \u03b4 158.66, 158.51, 149.27, 149.14, 149.00, 144.10, 142.10, 141.63, every measurement. Quartz cuvettes have been used for the\n140.94, 136.23, 135.94, 131.88, 129.38, 129.33, 128.23, 128.18, measurement of original samples.\n125.90, 125.62, 106.26, 90.56, 83.47, 80.84, 19.31. ESI-HRMS: [M- Cell Lines and Culture Conditions. Human cancer cell lines\nPF6]+: 556.0288 (calculated 556.01143). HCT116 (human colorectal carcinoma), CT26 (murine colorectal\n Synthesis of [(\u03b76-Benzene)Ru(CO2Medppz)Cl]PF6 (1C). [(\u03b76- adenocarcinoma), MDA-MB-231 (human breast adenocarcinoma),\nBenzene)RuCl2]2 (precursor 1) (65 mg, 0.129 mmol, 1 eq), and MRC5 (human lung fibroblasts, non-cancerous) were obtained\nCO2Medppz (ligand C) (88 mg, 0.259 mmol, 2 eq), NH4PF6 (64 from ATCC. HCT116, MDA-MB-231, CT26, and MRC5 were\nmg, 0.389 mmol, 3 eq). Yield: 157 mg (86%). Elemental analysis: cultured in DMEM containing 10% FBS and 1% penicillin\u2212\nCalcd. for: C26H18ClF6N4O2PRu C 44.62; H 2.59; N 8.00. Found: C streptomycin (10,000 U/mL). Cells were grown in tissue culture\n44.58; H 2.66; N 7.83. IR (cm\u22121): 3091, 1731 (C\ufffdO), 1627 (C\ufffd flasks (75 cm2 and 25 cm2, SPL Life Sciences). All cell lines were\nN), 1256, 1092, 836, 558. 1H NMR (400 MHz, DMSO-d6): \u03b4 10.10 grown at 37 \u00b0C in a humidified atmosphere of 95% air and 5% CO2.\n(t, J = 4.1 Hz, 4H), 9.55 (dd, J = 7.8, 3.6 Hz, 4H), 8.79 (d, J = 1.0 Hz, All drug stock solutions were prepared in DMSO, and the final\n2H), 8.38 (dt, J = 16.5, 5.2 Hz, 4H), 8.30\u22128.23 (m, 4H), 6.36 (s, concentration in the medium did not exceed 1%, at which cell viability\n11H), 3.99 (s, 6H). 13C{1H} NMR (101 MHz, DMSO-d6): \u03b4 165.57, was not inhibited.\n158.54, 158.40, 150.51, 150.50, 148.85, 148.62, 143.70, 141.34, Cell Viability Assay. The cytotoxicity of compounds was\n140.98, 140.47, 136.06, 135.85, 132.79, 131.72, 131.09, 130.54, determined using an MTT colorimetric test. The cells were harvested\n\n 8190 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nfrom culture flasks by trypsinization and seeded into Cellstar 96-well samples were loaded onto 1% agarose gel and subjected to\nmicroculture plates at the seeding density of 6000 cells per well (6 \u00d7 electrophoresis for 2 h at 80 V in a TAE running buffer (tris\u2212acetate\n104 cells/mL). After the cells were allowed to resume exponential EDTA). The gels were photographed under UV light using a\ngrowth for 24 h, they were exposed to drugs at different ChemiDoc Touch Imaging System (BioRad). The digital images were\nconcentrations in media for 72 h. The drugs were diluted in complete analyzed using BioRad Image Lab Software.\nmedium at the desired concentration and added to each well (100 In Vivo Studies. All animal procedures were performed under the\n\u03bcL) and serially diluted to other wells. After exposure for 72 h, the Guidelines for Care and Use of Laboratory Animals of the City\nmedia was replaced with MTT in media (5 mg/mL, 100 \u03bcL/well) and University of Hong Kong and approved by the Animal Ethics\nincubated for an additional 50 min. Subsequently, the medium was Committee of the City University of Hong Kong. All mice were\naspirated, and the purple formazan crystals formed in viable cells were maintained in the laboratory animal research unit (LARU) of the City\ndissolved in DMSO (100 \u03bcL/well). Optical densities were measured University of Hong Kong in specific pathogen-free conditions. All\nat 570 nm using a BioTek Synergy H1 microplate reader. The mice experiments were performed in compliance with the guidelines\nquantity of viable cells was expressed in terms of treated/control (T/ and protocols approved by the institutional and local ethics\nC) values in comparison to untreated control cells, and 50% committee of HKSAR, Department of Health. Six- to eight-week-\ninhibitory concentrations (IC50) were calculated from concentra- old BALB/c mice were purchased from the Laboratory Animal\ntion\u2212effect curves by interpolation. Evaluation was based on means Research Unit (LARU) of the City University of Hong Kong. Animals\nfrom at least three independent experiments, each comprising three were randomly assigned to different groups. Prior to assignment to\nreplicates per concentration level. groups, the weight variation of the animals did not exceed 20% of the\n ROS Detection via Confocal Microscopy. HCT116 cells were mean weight. Animals were grouped and housed in solid bottom\nseeded onto 4-well chamber slides with removable wells (Nunc Lab- polycarbonate cages (5 mice per cage) and provided with pelleted\nTek II Chamber Slide System) at a 1 \u00d7 105/mL density (1 mL per food and water ad libitum. Environmental controls for the animal\nwell). Cells were allowed to resume exponential growth for 24 h. The room were set to maintain 22\u221227 \u00b0C, a relative humidity of 55\u221275%,\ncell culture medium was aspirated and washed with PBS (2\u00d7). In a a minimum of 10 air changes/h, and a 12 h light/12 h dark cycle. No\nlow-light environment, the H2DCFDA (2\u2032,7\u2032-dichlorodihydrofluor- known contaminants were present in the diet or water at levels that\nescein diacetate) solution prepared in 1\u00d7 HBSS (20 \u03bcM, 1 mL) was might interfere with this study. To identify the appropriate dose of the\nadded to each well and incubated for 30 min (37 \u00b0C, 5% CO2). experimental drugs for subsequent in vivo efficacy studies, BALB/c\nDCFDA solution was removed and washed with HBSS (2 \u00d7 500 \u03bcL). mice were injected every second day via the i.p. route with 20 mg/kg\nThe drug solutions at desired concentrations were prepared in of Ru complex 3A or 0.8 mg/kg of ligand A for 6 days (3 injections in\ncolorless, serum-free cell culture media and added to the cells, and total). Subsequently, the mice were monitored for 5 more days. The\nthen the chamber slides were incubated for 4 h (37 \u00b0C, 5% CO2). control group received only the respective vehicle (DMSO/\nAfter that, the drug-containing media was removed and the wells were Cremophor EL/PBS). The location of the i.p. injection was the\nwashed with PBS (2 \u00d7 500 \u03bcL). Then, the cells were incubated with a lower left abdominal quadrant. Animals were controlled for distress\nculture medium containing 1 \u03bcM Mitotracker Red CMXRos and 10 development. Their weight changes were monitored every second day\n\u03bcg/mL Hoechst 33342 for 15 min. After that, the cells were washed for 2 weeks. On day 6, two mice receiving 20 mg/kg of 3A died;\nwith PBS (2 \u00d7 500 \u03bcL) and mounted on a slide with mounting media therefore, another group of mice was treated with 8 mg/kg of 3A\nglycerol:PBS (9:1). The samples were protected from photo- using the similar regimen. Mice receiving 8 mg/kg of 3A, 0.8 mg/kg\ndegradation by covering them with aluminum foil before imaging. of A, or a vehicle did not show any signs of toxicity. For in vivo\nImages were acquired using a Leica confocal laser scanning efficacy studies, BALB/c mice were subcutaneously injected with 0.1\nmicroscope and analyzed via Microscope Software Platform LAS X mL of CT26 cells (5 \u00d7 106 cells/L) on the right flank. After the\nLife Science. tumors became palpable (day 10 after implantation), treatment was\n Quantitative ROS Detection via Flow Cytometry. HCT116 initiated. Animals were randomly divided into three groups (n = 5),\ncells were seeded onto 6-well plates at a 3 \u00d7 105 cells/mL density (2 and the tumor dimensions were measured using a caliper. The volume\nmL per well). Cells were allowed to resume exponential growth for 24 (mm3) of the tumor was calculated according to the formula tumor\nh. The cell culture medium was aspirated and washed with PBS (1\u00d7). volume = (longest diameter) \u00d7 (shortest diameter)2 \u00d7 0.5. Drug-\nThe drug solutions at desired concentrations were added to the cells treated groups were injected with 8 mg/kg 3A or 0.8 mg/kg A in\nand incubated for 4 h (37 \u00b0C, 5% CO2). Subsequently, the drug- sterile PBS with DMSO/Cremophor EL, whereas the control group\ncontaining media was removed, and the wells were washed with PBS was injected with sterile PBS with DMSO/Cremophor EL as a\n(2 \u00d7 500 \u03bcL). Afterward, the cells were trypsinized, normalized to 1 \u00d7 vehicle. Treatment was performed on days 10, 12, 14, 16, and 18.\n106 cells/mL, transferred to microcentrifuge tubes, washed, and After the end of treatment, animals were sacrificed on day 21. Animals\ncentrifuged to eliminate the supernatant. After centrifugation, the cells were controlled for distress development. Their weight changes were\nwere resuspended in a colorless medium (FBS and phenol red-free) monitored every day for 3 weeks. All mice were bright, alert, and\ncontaining H2DCFDA (20 \u03bcM) and incubated for 30 min (37 \u00b0C, 5% responsive during the whole study. After sacrificing the mice, the\nCO2). Next, the H2DCFDA solution was removed, and the cells were tumors were collected, weighed, and together with the organs (liver\nwashed and resuspended in PBS. Immediately before analyzing by and kidney) were harvested for further histopathological examination.\nflow cytometry, the cells were stained with propidium iodide (PI, 100 Histopathological Analysis. Tumor, liver, and kidney tissues\n\u03bcg/mL) to exclude necrotic cells and subsequently protected from were fixed in 10% buffered formalin overnight and washed twice with\nphotodegradation with aluminum foil. A cytometer by Beckman PBS. The tissues were then embedded in paraffin. Sections with\nCoulter (CytoFLEX) was used to evaluate the distribution of the thicknesses of 4 \u03bcm were prepared, mounted on slides, and\nDCF-stained cells. FlowJo software (ver. 10.8.1) was used for the deparaffinized in xylene (twice). Then, the sections were rehydrated\nanalysis of flow cytometry data. in a graded series of alcohol (2 \u00d7 100% alcohol and 2 \u00d7 75% alcohol)\n DNA Degradation Assay. HCT116 cells were seeded onto 6-well and distilled water. Later, the sections were stained with a\nplates (Greiner Bio-One) at a density of 3 \u00d7 105 cells/mL (2 mL per hematoxylin solution, rinsed in water, passed through a 70% ethanol\nwell). The cells were allowed to resume exponential growth for 24 h. solution containing 1% HCl, and rinsed again with tap water. The\nThe cell culture medium was aspirated and washed with PBS (2 \u00d7 1 sections were stained with eosin for 5 min and rinsed with absolute\nmL) and replaced with compounds at the respective IC50 (72h) alcohol and xylene for 5 min.35 The slides were analyzed using a Leica\nconcentrations in DMEM for 24 h at 37 \u00b0C and 5% CO2. DNA DM2700 microscope and a Panoramic 250 Flash III (3D Histech,\nextraction and purification were done based on the manufacturer\u2019s Hungary). Necrosis, hemorrhages, steatosis in hepatocytes, cell\ninstructions (QIAgen DNA Extraction and Purification Kit). The shrinking, nuclear enlargement or pyknosis, chromatin condensation,\nDNA concentrations were normalized using a NanoDrop One/OneC ruptured cell membranes, and apoptotic bodies were evaluated as\nMicrovolume UV\u2212Vis spectrophotometer. The normalized DNA potential signs of liver and kidney toxicity.36 The extent of tumor\n\n 8191 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 1. Synthetic Route toward Complexes 1A\u22123A, 1B, 2B, and 1C\u22123C with the Numbering Scheme Used for the NMR\nAssignment\n\n\n\n\nnecrosis was quantified on H&E-stained slides by evaluating the protons in complexes, which were located in the ortho and\npercent of tumor necrotic area by the visual assessment of an meta positions to the nitrogen in the phen fragment. On the\nexperienced pathologist. other hand, the protons in the para position as well as the\n Statistical Analysis. Quantitative data are presented as means \u00b1\n phenazine protons remained largely unaffected. Similar\nSD from at least three independent experiments. For the in vivo data,\nthe significance of the differences was evaluated with two-way observations were previously reported for other Ru-dppz\nANOVA followed by Tukey\u2019s multiple comparison post-test. complexes.21 For example, in the uncoordinated ligand A, the\nStatistical analysis was executed with GraphPad Prism Software signals of H2(H2\u2032) and H4(H4\u2032) protons appeared at 9.47 and\n(San Diego, CA, USA) using p < 0.05 as the critical significance level. 9.16 ppm, respectively. Upon coordination to the (arene)Ru\n moiety, the signals of H2(H2\u2032) and H4(H4\u2032) protons shifted\n\u25a0 RESULTS AND DISCUSSION\n Synthesis and Characterization. Ru complexes 1A\u22123A,\n to 10.07 and 9.39 ppm, respectively. A similar downfield shift\n occurred for the signal of H3(H3\u2032) protons, which shifted from\n 7.90 ppm in the uncoordinated ligand A to 8.20 ppm. Atoms\n1B, 2B, and 1C\u22123C were synthesized following the synthetic H10, H11, and H11\u2032 were less affected by the coordination\nroute described in Scheme 1. 11-Methyldipyrido[3,2-a:2\u2032,3\u2032- and displayed a slight upfield shift in comparison with\nc]phenazine (A), 11-nitrodipyrido[3,2-a:2\u2032,3\u2032-c]phenazine (B), uncoordinated ligand A. Ligand B showed less pronounced\nand methyl dipyrido[3,2-a:2\u2032,3\u2032-c]phenazine-11-carboxylate proton shifts upon coordination to the Ru center. Protons\n(C) were used as ligands. They were prepared using a H2(H2\u2032) displayed an upfield shift from 10.15 ppm in the\nstandard method based on the Schiff base condensation of\n uncoordinated ligand B to 10.11 and 10.04 ppm in complexes\n1,10-phenanthroline-5,6-dione with the respective diamine.\n 1B and 2B, respectively. The signal of H4(H4\u2032) shifted\nThe subsequent complexation of 2 eq of dppz ligands A\u2212C\n downfield from 9.55 ppm in the uncoordinated ligand B to\nwith 1 eq of the corresponding (arene)Ru dimers in methanol\n 9.64 ppm in 1B and 2B. The most significant shift was\nyielded complexes 1A\u22123A, 1B, 2B, and 1C\u22123C in moderate\n observed for ligand C and complex 1C. Ligand protons\nto almost quantitative yields. They were found to be soluble in\n H2(H2\u2032) and H4(H4\u2032) shifted from 9.29 and 9.15 ppm in the\nDMSO, methanol, and acetonitrile. All complexes were\ncharacterized by high-resolution mass-spectrometry (Figures uncoordinated ligand C to 10.10 and 9.55 ppm in complex 1C,\nS1\u2212S8), 1H NMR, and 13C NMR spectroscopy (Figures S9\u2212 respectively. In complexes 2C and 3C, these protons were\nS24), and their purity was confirmed by elemental analysis. located at 10.02 and 9.55 ppm, respectively. Proton signals\n The identity of the complexes was verified by high- originated from H3(H3\u2032) atoms were also shifted downfield\nresolution mass spectrometry, and their purity was confirmed from 7.64 ppm in the uncoordinated ligand C to 8.27 ppm in\nby elemental analysis. The mass spectra of all the complexes 1C\u22123C. The downfield shift for the protons that were located\nrevealed high-intensity signals corresponding to M+ ions. The further from the Ru center, such as H10, H11, and H11\u2032, was\nobserved isotopic distribution pattern was in good agreement less significant (around 0.2\u22120.3 ppm).\nwith the calculated mass distribution. Analysis of the NMR In 13C{1H} NMR spectra, the signals from the arene\nspectra revealed that the chemical shifts of the protons and moieties appeared at usual values for this type of piano-stool\ncarbons of the Ru complexes were in agreement with the complexes.21,25 For example, 13C signals from the benzene\npreviously published structurally similar compounds.21,25 As moiety were located at 87.4 ppm in complexes 1A, 1B, and 1C.\n 13\nexpected, the arene-to-ligand ratio was found to be 1:1 as C signals from the toluene moiety in complexes 2A, 2B, and\ndetermined by the integration of protons in the 1H NMR 2C appeared at 106.2, 90.5, 83.4, and 80.6 ppm, while a methyl\nspectra. The six protons originated from the benzene moiety in group was located at 19.3 ppm. 13C signals from the cymene\ncomplexes 1A\u22121C were located at 6.35 ppm. The signals from moiety were present at 105.2, 103.1, 86.3, and 84.6 ppm, while\nthe toluene moiety in complexes 2A\u22122C were present at ca. a methyl group was located at 18.6 ppm. 13C signals from the\n6.46, 6.10, and 5.91 ppm, whereas the methyl group was isopropyl group were located at 30.8 ppm (CH) and 22.2 ppm\ndetected at 2.45 ppm. The signals from the cymene moiety in (CH3). 13C signals of the C2 and C3 atoms of the dppz ligands\ncomplexes 3A and 3C were located at ca. 6.36 and 6.13 ppm; were mostly affected by the coordination to the Ru center.\nthe signals from one methyl group and two other methyl Whereas in the uncoordinated ligands, C2 signals were located\ngroups were at 2.18 and 0.96 ppm, respectively, while the at ca. 152 ppm, in 1A\u22123A, these signals were shifted downfield\nsignal from the isopropyl group was located at 2.65 ppm. to ca. 157.5 ppm, in 1B and 2B to ca. 158.6 ppm, and in 1C\u2212\n The coordination of ligands A\u2212C to the (arene)Ru 3C to ca. 158.2 ppm. The C3 signals in the uncoordinated\nfragments resulted in the significant downfield shift of the ligands were present at 133.6 ppm, and in all complexes, they\n 8192 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 1. Cyclic voltammograms of dppz ligands A\u2212C and Ru-dppz complexes 1A\u22123A, 1B, 2B, and 1C\u22123C in DMSO/n-Bu4NClO4 with a glassy\ncarbon working electrode at a scan rate of 50 mV s\u22121.\n\nwere shifted downfield to 136.2\u2212135.3 ppm. The 13C signals of the methyl group with -NO2 or -COOMe groups resulted in\nother atoms in the coordinated dppz ligands were less affected less negative redox potentials. Thus, ligands B and C are easier\nby the coordination. to reduce, as expected upon the introduction of the electron-\n The IR spectrum of the ligand A exhibited a very strong withdrawing substituents, leading to the decrease of the\nband at 1586 cm\u22121 due to the C\ufffdN stretching. In complexes electron density within the dppz ligand. Similar observations\n1A, 2A, and 3A characteristic bands at 1608, 1578, and 1607 were reported in the literature for structurally similar dppz\ncm\u22121 from pyridine \u03bd(C\ufffdN) showed a downward and upward ligands.37\nshift in comparison with the uncoordinated ligand. The C\ufffdN The electrochemical behavior of the corresponding Ru\nstretching in the ligand B was observed at 1618 cm\u22121, and it complexes was more complex. Briefly, in the positive region of\nwas shifted to 1611 and 1610 cm\u22121 in 1B and 2B, respectively. the cyclic voltammograms, all complexes with the exception of\nAn asymmetric stretch from the nitro group in the ligand B 2B displayed one irreversible redox peak between 1.12 and\nwas observed at 1521 cm\u22121, and in complexes 1B and 2B, it 1.29 V, corresponding to Ru2+/Ru3+ oxidation. In contrast, the\nwas observed at 1526 and 1529 cm\u22121, respectively. The negative region of the cyclic voltammograms of the Ru-dppz\nsymmetric stretch of the nitro group also showed a downward complexes was less well-defined. Complexes 1A\u22123A with a\nshift from 1343 cm\u22121 in the uncoordinated ligand B to 1352 Me-substituted dppz ligand displayed one irreversible reduc-\nand 1354 cm\u22121 in complexes 1B and 2B, respectively. The tion (E1red = \u22120.45, \u22120.38, and \u2212 0.47 V for 1A, 2A, and 3A,\npyridine \u03bd(C\ufffdN) stretch band was shifted from 1621 cm\u22121 in respectively, with respect to the Ag/AgCl reference electrode).\nthe uncoordinated ligand C to 1627, 1626, and 1624 cm\u22121 in On the contrary, the cyclic voltammograms of complexes 1B\ncomplexes 1C, 2C, and 3C, respectively. The \u03bd(C\ufffdO) stretch and 2B with a NO2-substituted dppz ligand displayed the first\nin the uncoordinated ligand C was observed at 1737 cm\u22121 and irreversible reduction peak at E1red = \u22120.33 and \u22120.29 V for 1B\nwas shifted to 1731, 1731, and 1727 cm\u22121 in the corresponding and 2B, respectively, and the second reversible reduction peak\ncomplexes 1C, 2C, and 3C, respectively. at E1red = \u22120.55 V for 1B and 2B with respect to the Ag/AgCl\n Electrochemistry. Electrochemical measurements of the reference electrode. The reduction processes in complexes\nligands and their respective Ru complexes were carried out in 1C\u22123C seemed to be irreversible with E1red = \u22120.35, \u22120.36,\ntetrabutylammonium phosphate supporting electrolyte at scan and \u22120.43 V for 1C, 2C, and 3C, respectively, with respect to\nrate of 50 mV s\u22121 (Figure 1), and the electrochemical the Ag/AgCl reference electrode. Additionally, the cyclic\npotentials are summarized in Table S1. In the case of the voltammogram of 3C was characterized by the very defined\nmethyl-substituted dppz ligand A, only one reduction can be second irreversible reduction peak at E2red = \u22120.61 V. The\nobserved (E1red = \u22120.47 V with respect to Ag/AgCl reference observed electrochemical behavior of the dppz ligands and\nelectrode). The nitro-substituted dppz ligand B and methox- (arene)Ru-dppz complexes indicates that all tested compounds\nycarbonyl-substituted dppz ligand C showed two reduction might be electrochemically active in the cells, leading to the\nprocesses (E1red = \u22120.30 V and E2 red = \u22120.37 V for B and E1red induction of ROS; however, they are not expected to undergo\n= \u22120.28 V and E2 red = \u22120.33 V for C with respect to the Ag/ reversible redox cycling unlike other metal complexes, which\nAgCl reference electrode). As can be seen, the substitution of generate ROS via Fenton-like chemistry.38\n 8193 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. Cytotoxicity of dppz Ligands and Corresponding Half-Sandwich Ru Complexes Determined by the MTT Assay after\n72 h of Exposure\n IC50 (\u03bcM)a\n type compound MDA-MB-231 HCT116 CT26 MRC5 SFMDAb SFHCT116c\n ligand A (-Me) 2.0 \u00b1 0.3 1.3 \u00b1 0.2 2.5 \u00b1 1.0 13 \u00b1 4 6 10\n B (-NO2) 1.8 \u00b1 0.6 1.1 \u00b1 0.2 1.1 \u00b1 0.4 2.2 \u00b1 0.9 1 2\n C (-CO2Me) >2d >2 >2 >2\n Ru complex 1A 35 \u00b1 7 81 \u00b1 15 166 \u00b1 57 178 \u00b1 24 5 2\n 2A 23 \u00b1 8 27 \u00b1 5 168 \u00b1 19 121 \u00b1 40 5 5\n 3A 2.0 \u00b1 0.6 2.1 \u00b1 0.2 12 \u00b1 2.0 12 \u00b1 5 6 6\n 1B 82 \u00b1 17 84 \u00b1 10 >125d >125 >2 >1\n 2B 64 \u00b1 5 85 \u00b1 18 >125 >125 >2 >1\n 1C 47 \u00b1 14 >83d >83 >83 >2\n 2C 31 \u00b1 1 78 \u00b1 23 >62d >62 >2 <1\n 3C 2.9 \u00b1 0.8 1.9 \u00b1 0.4 >33d >33 >11 >17\n cisplatin 4.7 \u00b1 1.8 1.1 \u00b1 0.1 n.d.e 7.0 \u00b1 0.8 1 6\n doxorubicin n.d. 1.0 \u00b1 0.1 n.d. n.d.\na\n Effective concentrations of 50% in MDA-MB-231 (human breast adenocarcinoma), HCT116 (human colorectal carcinoma), CT26 (murine\ncolon carcinoma), and MRC5 (human lung fibroblasts) determined by means of the MTT assay with the exposure time of 72 h. Values are means\n\u00b1 standard deviations obtained from at least three independent experiments. bThe selectivity factor (SF) was determined as IC50(MRC-5)/\nIC50(MDA-MB-231). cThe selectivity factor (SF) was determined as IC50(MRC-5)/IC50(HCT116) dThe maximum tested concentrations were\nlimited by the solubility of the compounds in DMSO. eNot determined.\n\n Stability Studies. All Ru complexes were found to be intensity, while small peaks at 396 and 376 nm were replaced\npoorly soluble in aqueous solutions and adequately soluble in by one broad peak centered at 375 nm (Figure S33B).\nDMSO; therefore, their subsequent biological analysis was Comparable changes were observed for all other tested\nperformed in 1% DMSO in cell culture media. To ensure that complexes. Complex 3A demonstrated the highest stability in\ninvestigated complexes are stable in DMSO and do not PBS and complete cell culture media/H2O mixture (Figures\nundergo decomposition or exchange of chlorido ligand, we S35).\ninvestigated the stability of all complexes in DMSO-d6 solvent Anticancer Activity. The in vitro anticancer activity of the\nusing 1H NMR spectroscopy for 72 h. The 1H NMR signals Ru complexes and their corresponding ligands was determined\ndid not undergo any changes within the tested period, by the colorimetric MTT assay in the breast cancer cell line\nindicating that complexes were stable in DMSO (Figures (MDA-MB-231) and colorectal cancer cell lines (HCT116 and\nS25\u2212S32). To assess the stability of compounds in aqueous CT26) with an exposure time of 72 h. The selectivity of the\nmedia, we also performed UV\u2212Vis spectrophotometric drug candidates toward the cancer cell lines was assessed in\nexperiments in 1% DMSO diluted with either PBS or complete comparison with a non-cancerous human lung fibroblast cell\ncell culture media (DMEM containing 10% FBS and 1% line (MRC5). The maximum tested concentration was limited\npenicillin\u2212streptomycin) diluted with H2O in a ratio of 1:9 by the solubility of the compounds in DMSO. The IC50 values\n(Figures S33\u2212S40). Complexes 1B, 2A, 3A, and 2C are listed in Table 1, and the concentration\u2212effect curves are\ndemonstrated high stability in PBS, reflected by only minor shown in Figure S41.\nchanges of intensity (Figures S34A\u2212S36A and S39A). On the The activity of the ligands decreased in the following order:\ncontrary, the UV\u2212Vis spectra of 1A, 2B, and 1C recorded after A \u2248 B \u226b C. While A and B showed similar cytotoxicity in the\n72 h of incubation were characterized by the reduced low-micromolar concentration range, ligand C was non-\nintensities of the peaks at 285, 300, or 283 nm, respectively, cytotoxic, possibly due to its poor solubility in DMSO (\u22640.2\nand the appearance of isosbestic points at 405, 414, or 402 nm, mM). It should be noted that it was previously reported that\nrespectively (Figures S33A, S37A, and S38A). The decrease of dppz and its analogues were devoid of any anticancer activity\nintensity might indicate the potential aggregation or precip- against different cancer cell lines,25,39,40 with the exception of\nitation of the complexes from the aqueous media. At the same ligand A, which was moderately cytotoxic against the HeLa cell\ntime, the formation of isosbestic points is typically associated line (IC50(24 h) \u2248 60 \u03bcM).39 The majority of the tested Ru\nwith the formation of new species. The UV\u2212Vis spectrum of complexes exhibited improved aqueous solubility but modest\ncomplex 3C in PBS was characterized by the most significant anticancer activity, similar to previously published Ru-dppz\nchanges, reflected by the marked increase of intensity of the complexes.17,19,21,25,26,41,42 Only 3A and 3C were cytotoxic in\npeak centered at 275 nm and its slight shift to 287 nm after 72 the low micromolar range in MDA-MB-231 and HCT116 cell\nh. Moreover, we observed the increase in intensity of a small lines in agreement with their higher aqueous stability.\npeak at 395 nm and the formation of several isosbestic points Interestingly, most of the tested compounds did not show\n(Figure S40A). Dissolution of compounds in complete cell any effects in the murine colon cancer CT26 cell line,\nculture media resulted in the quick formation of new species, indicating their selectivity toward human cancer cell lines.\nas indicated by the appearance of several isosbestic points and Overall, the cytotoxicity of the complexes showed the\nother changes in the UV\u2212Vis absorption bands after 24 h following trend in human cancer cell lines: 3 \u226b 2 \u2248 1,\n(Figures S33B\u2212S40B). For example, in the case of complex suggesting the beneficial role of the cymene arene fragment. In\n1A, the strongest band centered at 285 nm demonstrated a agreement, previously published (cymene)Ru complexes with\ntime-dependent shift toward 280 nm and reduction of dppz ligands showed high cytotoxicity in the MDA-MB-231\n 8194 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\ncell line, reflected by IC50 values between ca. 5 and 9 \u03bcM.21,26 reflected by smearing band patterns. The remaining band\nIn general, the substitution of the arene had more profound intensities for 2A, doxorubicin, and 3A were ca. 79, 31, and\neffect on the cytotoxicity of the complexes than the variation of 15% respectively, in comparison with untreated cells. The\nsubstituents in the dppz backbone. Additionally, we assessed improved DNA degradation of 3A in comparison with equally\nthe selectivity of all tested compounds toward cancer cell lines cytotoxic ligand A might be related to the interactions of the\nby comparing their IC50 values in MDA-MB-231 or HCT116 DNA nucleobases with an Ru center.46 On the other hand,\nwith IC50 values in the non-cancerous human lung fibroblast DNA extracted from cells treated with 1A and A remained\nMRC5 cell line. As illustrated in Table 1, all drug candidates relatively intact, although ligand A caused some degree of\nwere more selective toward non-cancerous cells; in particular, DNA smearing, indicating initial fragmentation of high-\n3C was at least 17 times less toxic against MRC5 than molecular weight DNA. Since all compounds were used at\nHCT116. In comparison with clinically used anticancer drug equipotent concentrations, the most prominent DNA degra-\ncisplatin, 3A and 3C were more active against MDA-MB-231 dation caused by (cymene)Ru-dppz complex 3A might be one\nbreast cancer cells (ca. 10 and 7 times, respectively). Moreover, of the underlying causes of its highest cytotoxicity among the\nthey were markedly more selective than cisplatin toward tested complexes.\nMRC5 (SFMDA = 6, >11, and 0.3 for 3A, 3C, and cisplatin, ROS Detection. It is known that superoxide anionic\nrespectively), as well as previously published structurally radicals, hydroxyl free radicals, and other ROS are produced as\nsimilar (arene)Ru-dppz complexes.25,26 On the contrary, in a result of one or more oxygen electrons being reduced by\nHCT116 cells, cisplatin and another clinically used drug cellular enzymes or in the mitochondrial respiratory system.47\ndoxorubicin were more active than the majority of Ru These molecules play an important function in cellular\ncomplexes and comparably active to 3C and ligands A and signaling for various biological processes; however, an excess\nB. To understand whether the differences in the cytotoxicity of free radicals might lead to DNA damage and cell death as\nbetween Ru complexes with different arene fragments were well as DNA mutations and carcinogenesis.48 In cancer cells,\nlinked to their mechanism of action, we performed subsequent ROS production is elevated due to their higher metabolic rate\ninvestigations of their DNA-degrading properties. compared to normal cells.49 Various structurally different Ru\n DNA Degradation. Previously, various metal complexes complexes were shown to induce ROS-mediated cancer cell\nwith dppz-based ligands, including half-sandwich complex death.50,51 However, typically ROS experiments were carried\n[(\u03b75-C5Me5)Ru(dppz)(NO)](OTf), were shown to affect the out after prolonged drug exposure (12\u221248 h);21,50 thus, the\n ROS observed in drug-treated cancer cells might be the\nDNA mobility in gel electrophoresis experiments with plasmid\n consequence of cell damage caused by other mechanisms.\nDNA, indicating DNA cleavage.43\u221245 Herein, we assessed the\n Based on the results of cyclic voltammetry experiments, 1A\u2212\nability of Ru complexes 1A\u22123A to affect cancer cell DNA\n 3A, 1B, 2B, and 1C\u22123C were electrochemically active, and the\nusing agarose gel-based DNA degradation assay in comparison\n biologically accessible reduction was dppz ligand-based.\nwith uncoordinated ligand A and clinically used DNA\n Therefore, we expected that these complexes could initiate\ndamaging agent doxorubicin. HCT116 colorectal cancer cells ROS insult after a short timepoint, leading to DNA damage\nwere incubated with the tested compounds at concentrations and cancer cell death. We used the triple-staining protocol with\ncorresponding to their IC50(72 h) values (and 3\u00d7 IC50 value for nuclear staining Hoechst dye, mitochondria-specific Mito-\ndoxorubicin) for 24 h. Subsequently, DNA was extracted, tracker Red CMXRos dye, and ROS-sensitive DCFDA dye.\nnormalized, and assessed by 1% agarose gel electrophoresis. HCT116 cells were treated with complex 3A and respective\nQuantification of the bands using BioRad Image Lab Software ligand A for 4 h, and live cell images were obtained using\nrevealed the following trend in the efficacy of DNA confocal microscopy. As expected, 3A caused an increase in\ndegradation: 1A \u2248 A < 2A < doxorubicin < 3A. As can be mitochondrial ROS production reflected by enhanced green\nseen in Figure 2, DNA extracted from the cells treated with 2A, DCF fluorescence colocalized with mitochondrial staining. On\ndoxorubicin, and 3A underwent considerable degradation as the other hand, A did not induce marked ROS insult (Figure\n 3). These results suggest that mitochondrial ROS production\n might be one of the underlying causes of 3A-induced cancer\n cell death. Additionally, to quantify the amount of ROS\n generated by the compounds of interest, we performed flow\n cytometric analysis of drug-treated HCT116 cells (Figure\n S42). In agreement with the microscopic studies, ligand A was\n characterized by an insignificant increase of DCF fluorescence,\n while 3A caused more than 8-fold increase in DCF\n fluorescence similar to H2O2 (100 \u03bcM, cell-free, positive\n control). Inspired by promising in vitro activity of 3A, we\n subsequently performed an in vivo efficacy study using Balb/C\nFigure 2. Detection of DNA degradation using 1% agarose gel mice.\nelectrophoresis. HCT116 cells were treated with complexes 1A\u22123A In Vivo Experiments. Initially, we performed a pilot acute\nand A at their respective 1 \u00d7 IC50 values and doxorubicin as a positive toxicity study on BALB/c mice to determine the appropriate\ncontrol at a 3 \u00d7 IC50 value for 24 h. DNA was normalized using a\n dose of tested compounds for the subsequent efficacy study.\nNanoDrop One/OneC Microvolume UV\u2212Vis spectrophotometer,\nand 200 ng per sample was loaded into each well (lane 1, 1 kb DNA Animals (n = 12) were randomly distributed into four groups\nladder; lane 2, untreated cells (NT); lane 3, 1A; lane 4, 2A; lane 5, and were intraperitoneally injected with drug candidates or\n3A; lane 6, A; lane 7, doxorubicin (DOXO)). Gel imaging was respective vehicle (three injections within 1 week; Group 1: 10\nperformed under UV light using a ChemiDoc Touch Imaging System mg/kg of 3A, Group 2: 20 mg/kg of 3A, Group 3: 0.8 mg/kg\n(BioRad). of A, and Group 4: vehicle). The injected dose of the ligand\n 8195 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 3. ROS detection using live cell confocal microscopy imaging. HCT116 cells were stained with H2DCFDA (excitation/emission\nwavelengths of 504 nm/525 nm) for 15 min, incubated with complex 3A (1 \u00d7 IC50) and A (1 \u00d7 IC50) for 4 h. Hoechst 33342 dye (excitation/\nemission wavelength of 350/461) and Mitotracker Red CMXRos dye (excitation/emission wavelengths of 579 nm/599 nm) were used for nuclear\nand mitochondrial staining, respectively. Images were acquired using a Leica confocal laser scanning microscope and analyzed via Microscope\nSoftware Platform LAS X Life Science. The scale bar represents 50 \u03bcm. NT, untreated cells.\n\n\n\n\nFigure 4. Effects of A and 3A on CT26 tumor growth in vivo. (A) Representative tumor images at the endpoint (day 20) after five i.p. injections of\nA, 3A, or vehicle. (B) Time-dependent tumor growth in CT26 tumor-bearing Balb/C mice. Starting from day 8, the tumors became palpable and\ntheir volume was measured by a caliper weekly. Mice (n = 5) were treated with A at 0.8 mg kg\u22121, 3A at 8 mg kg\u22121, or respective vehicle (DMSO/\nCremophor EL in sterile saline) via the i.p. route every other day (arrows represent the injection days). (C) Weights of the isolated tumors shown\nin Figure 4A. (D) Body weight changes of Balb/C mice during the treatment. (E) Representative H&E-stained kidney tissue of a 3A-treated mouse,\ndemonstrating normal kidney structure. (F) Representative H&E-stained liver tissue of a 3A-treated mouse, demonstrating preserved liver tissue\narchitecture. The scale bar represents 50 \u03bcm. Statistical analysis was performed by (B) two-way ANOVA test with Dunnett\u2019s post hoc analysis or\n(C) unpaired t test using GraphPad Prism 9 software (GraphPad Software Inc., CA) with p < 0.05 considered as significant (*p < 0.05, ****p <\n0.0001, ns: not significant).\n\nwas severely limited by its precipitation from aqueous observed \u223c10% weight loss in one mouse after the second\nsolutions. However, the addition of Cremophor EL (final injection. Therefore, we chose 8 mg/kg of 3A for subsequent\nconcentration not exceeding 1\u22123%) ensured sufficient in vivo efficacy studies. Next, we established a mouse colorectal\nsolubility of drug candidates in physiological saline. Impor- cancer CT26 xenograft model. Animals (n = 15) were\ntantly, the solubility of Ru complex 3A allowed for testing at randomly divided into three groups as follows: Group 1, 3A\nsignificantly higher doses (up to 20 mg/kg), which indicates (8 mg/kg); Group 2, A (0.8 mg/kg); Group 3, vehicle (1%\none of the advantages of Ru coordination. The weight changes DMSO/Cremophor EL in sterile physiological saline). As soon\nand clinical signs of toxicity were monitored daily. Mice in the as the tumors became palpable (day 10), the mice were\nligand and control groups did not reveal any signs of toxicity intraperitoneally injected with drug candidates or the vehicle\nduring the whole observation period. In Group 2, two mice every second day for 10 consecutive days (overall five\nwere found dead after the first injection, while in Group 1, we injections). When the tumor volume in the control group\n 8196 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nreached 1500 mm3, the animals were humanely sacrificed and mg/kg for A vs 8 mg/kg for 3A). The vehicle-treated and\nthe tumors, kidneys, and livers were harvested for further ligand A-treated mice were characterized by rapid tumor\nhistological examination. We observed disease stabilization in growth and the occurrence of necrosis, characteristic of rapidly\nthe group of 3A-treated mice since there was a significant proliferating tumors, as confirmed by the histopathological\ntumor growth inhibition after the beginning of treatment analysis. On the contrary, 3A significantly reduced the tumor\n(Figures 4A\u2212C). The histological examination of tumor tissues size and tumor weight in CT26-bearing mice without inducing\nfrom 3A-treated mice revealed solid sheets of high-grade any toxic side effects. While the cytotoxicity of 3A might not be\ncarcinoma with low levels of necrosis (Figure S43). Mean- sufficient for the successful preclinical development of this\nwhile, the vehicle-treated and ligand-treated groups were drug candidate, the results of this work justify the subsequent\ncharacterized by disease progression, reflected by the ca. 7\u22128- studies on other series of anticancer half-sandwich Ru(II)-dppz\nfold increase of tumor size after the beginning of the treatment, complexes and further elucidation of their structure\u2212activity\nas well as high levels of necrosis, characteristic for late stages of relationships.\nadvanced solid tumors and highly proliferative tumors (Figures\nS44 and S45). At the endpoint, the 3A-treated tumors were ca.\n3 and 4 times smaller than in the vehicle- and ligand-treated\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\ngroups, respectively (Figures 4A\u2212C). Importantly, no statisti- The Supporting Information is available free of charge at\ncally significant differences were observed between the vehicle- https://pubs.acs.org/doi/10.1021/acs.inorgchem.3c00570.\nand ligand-treated groups, suggesting that the presence of the\n(arene)Ru fragment is crucial for its therapeutic efficacy. Electrochemical data for dppz ligands A\u2212C and their\nDuring the whole length of the experiment, all mice were respective Ru(II) complexes 1A\u22123A, 1B, 2B, and 1C\u2212\nbright, alert, and responsive and did not exhibit any clinical 3C; high-resolution ESI-MS spectra of 1A\u22123A, 1B, 2B,\nsigns of toxicity (body weight changes not more than 7%; and 1C\u22123C; 1H and 13C NMR spectra of 1A\u22123A, 1B,\nFigure 4D). In agreement, the kidneys harvested from all three 2B, and 1C\u22123C; 1H NMR study on the stability of 1A\u2212\ngroups demonstrated normal structures without significant 3A, 1B, 2B, and 1C\u22123C in DMSO-d6 for 24 h; UV\u2212Vis\nmicroscopic structural changes (Figure 4E and Figure S46). In study on the stability of 1A\u22123A, 1B, 2B, and 1C\u22123C in\nlivers from all three groups, several areas of hemorrhages were 1% DMSO in PBS or complete cell culture media/H2O\ndetected as well as nuclei polymorphism, sinusoidal dilatation, in a ratio of 1:9 for 24 h; concentration\u2212effect curves for\nand cytoplasm vacuolization; however, the liver tissue 1A\u22123A, 1B, 2B, and 1C\u22123C in MDA-MB-231 and\narchitecture was preserved in all groups (Figure 4F and Figure HCT116 cell lines upon 72 h of exposure; flow\nS46), indicating that the observed effects were not caused by cytometry analysis of drug-induced intracellular ROS\nthe drug treatment. generation; H&E-stained tumor sections in vehicle-,\n ligand A- and Ru complex 3A-treated groups; H&E-\n\u25a0 CONCLUSIONS\nIn this work, we prepared eight Ru(II)-arene complexes with\n stained kidney and liver sections in vehicle- and ligand\n A-treated groups (PDF)\nthree different arene fragments and three dppz-based ligands.\nThe cytotoxicity of the complexes against the MDA-MB-231\nand HCT116 breast cancer cell lines varied from low\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\nmicromolar to high micromolar concentrations. We demon- Sanja Grguric\u0301-S\u030cipka \u2212 Faculty of Chemistry, University of\nstrated that the cytotoxicity of the complexes was more Belgrade, 11000 Belgrade, Serbia; Email: sanjag@\nstrongly dependent on the Ru(II)-arene fragment than chem.bg.ac.rs\nsubstituents in the dppz backbone. The cytotoxicity of the Maria V. Babak \u2212 Drug Discovery Lab, Department of\ncomplexes decreased in the following order: 3 \u226b 2 \u2248 1, Chemistry, City University of Hong Kong, Hong Kong SAR\nsuggesting the beneficial role of the p-cymene fragment. To the 999077, People\u2019s Republic of China; orcid.org/0000-\nbest of our knowledge, (p-cymene)Ru(II) complexes 3A and 0002-2009-7837; Email: mbabak@cityu.edu.hk\n3C represent one of the most cytotoxic half-sandwich Ru-dppz\ncomplexes reported in the literature (IC50 values \u2248 2\u22123 \u03bcM). Authors\nThe variation of the arene fragment drastically affected the Stefan Nikolic\u0301 \u2212 Innovative Centre of the Faculty of Chemistry\nstability of the complexes and their ability to degrade DNA in Belgrade, University of Belgrade, 11000 Belgrade, Serbia\ncolorectal cancer cells. Only 3A, but not 1A and 2A, degraded Jemma Arakelyan \u2212 Drug Discovery Lab, Department of\nDNA to a similar extent as a clinically used anticancer agent Chemistry, City University of Hong Kong, Hong Kong SAR\ndoxorubicin. Further mechanistic investigations revealed that 999077, People\u2019s Republic of China\nuncoordinated dppz ligand A and its respective Ru(II) Vladimir Kushnarev \u2212 Drug Discovery Lab, Department of\ncomplex 3A induced deadly ROS production in the Chemistry, City University of Hong Kong, Hong Kong SAR\nmitochondria only after 4 h, which was in agreement with 999077, People\u2019s Republic of China\ntheir electrochemical activity. The ROS production induced by Samah Mutasim Alfadul \u2212 Drug Discovery Lab, Department\n3A was considerably stronger in the case if the dppz ligand. of Chemistry, City University of Hong Kong, Hong Kong SAR\nAdditionally, all tested Ru complexes demonstrated some 999077, People\u2019s Republic of China\ndegree of selectivity toward healthy MRC5 lung fibroblasts; Dalibor Stankovic\u0301 \u2212 Faculty of Chemistry, University of\nhowever, the origin of this selectivity is yet to be determined. Belgrade, 11000 Belgrade, Serbia\nThe in vivo efficacy of a dppz ligand was hindered by its poor Yaroslav I. Kraynik \u2212 Drug Discovery Lab, Department of\nsolubility in aqueous buffers even in the presence of Chemistry, City University of Hong Kong, Hong Kong SAR\nCremophor EL, while the coordination of the Ru(II)-arene 999077, People\u2019s Republic of China\nfragment allowed for a 10-fold increase of the tested dose (0.8 Complete contact information is available at:\n 8197 https://doi.org/10.1021/acs.inorgchem.3c00570\n Inorg. Chem. 2023, 62, 8188\u22128199\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nhttps://pubs.acs.org/10.1021/acs.inorgchem.3c00570 [Ru(2,9-dmp)2(tbtc)]2+.Acta A Mol. Biomol. Spectrosc. 2008, 71,\n 1181\u22121187.\nNotes (15) Di Pietro, M. L.; La Ganga, G.; Nastasi, F.; Puntoriero, F.\n Ru(II)-Dppz Derivatives and Their Interactions with DNA: Thirty\nThe authors declare no competing financial interest.\n Years and Counting. Appl. Sci. 2021, 11, 3038.\n\n\u25a0 ACKNOWLEDGMENTS\nThis work was supported by the City University of Hong Kong\n (16) Huang, H.; Zhang, P.; Yu, B.; Chen, Y.; Wang, J.; Ji, L.; Chao,\n H. Targeting nucleus DNA with a cyclometalated\n dipyridophenazineruthenium(II) complex. J. Med. Chem. 2014, 57,\n(projects 9610518 and 7005614) and the Ministry of 8971\u22128983.\n (17) Wachter, E.; Moy\u00e1, D.; Parkin, S.; Glazer, E. C. Ruthenium\nEducation, Science and Technological Development of the\n Complex \u201cLight Switches\u201d that are Selective for Different G-\nRepublic of Serbia (contract numbers: 451-03-68/2023-14/ Quadruplex Structures. Chem. \u2212 Eur. J. 2016, 22, 550\u2212559.\n200288 and 451-03-47/2023-01/200168). M.V.B. acknowl- (18) Ni, W.; Liu, X.; Tan, L. Binding properties of chiral\nedges Ho-Jung Choe for generating the TOC artwork. ruthenium(II) complexes \u039b- and \u0394-[Ru(bpy)(2)dppz-11-CO(2)-\n\n\u25a0 REFERENCES\n (1) Alessio, E.; Messori, L. 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