Pro-apoptotic activity of ruthenium 1-methylimidazole complex on non-small cell lung cancer.

Accepted Manuscript Pro-apoptotic activity of ruthenium 1-methylimidazole complex on non-small cell lung cancer Júlia Scaff Moreira Dias, Henrique Vieira Reis Silva, Guilherme Álvaro Ferreira da Silva, Marisa Ionta, Charlane Cimini Corrêa, Fernando Almeida, Legna Colina-Vegas, Marília Imaculada Frazão Barbosa, Antônio Carlos Doriguetto PII: DOI: Reference: S0162-0134(18)30189-2 doi:10.1016/j.jinorgbio.2018.06.008 JIB 10519 To appear in: Journal of Inorganic Biochemistry Received date: Revised date: Accepted date: 31 March 2018 12 June 2018 13 June 2018 Please cite this article as: Júlia Scaff Moreira Dias, Henrique Vieira Reis Silva, Guilherme Álvaro Ferreira da Silva, Marisa Ionta, Charlane Cimini Corrêa, Fernando Almeida, Legna Colina-Vegas, Marília Imaculada Frazão Barbosa, Antônio Carlos Doriguetto , Proapoptotic activity of ruthenium 1-methylimidazole complex on non-small cell lung cancer. Jib (2018), doi:10.1016/j.jinorgbio.2018.06.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT Pro-apoptotic activity of ruthenium 1-methylimidazole complex on non-small cell lung cancer Júlia Scaff Moreira Diasa, Henrique Vieira Reis Silvaa, Guilherme Álvaro Ferreira da Silvab, Marisa Iontab, Charlane Cimini Corrêac, Fernando Almeidad, Legna Colina- Instituto de Química, Universidade Federal de Alfenas, CEP 37130-000, Alfenas-MG, Brazil. Departamento de Ciências Biomédicas, Universidade Federal de Alfenas, CEP 37130- NU b SC a RI PT Vegase, Marília Imaculada Frazão Barbosaa* and Antônio Carlos Doriguettoa* 000, Alfenas-MG, Brazil. Departamento de Química - ICE Universidade Federal de Juiz de Fora Campus CEP MA c 36036-900, Juiz de Fora – MG, Brazil. D Instituto de Ciências Biomédicas – icb4, Universidade de São Paulo, CEP 05508-900, PT E d São Paulo-SP, Brazil. Departamento de Química, Universidade Federal de São Carlos, CEP 13565-905, São Carlos-SP, Brazil AC CE e * To whom correspondence should be addressed. e-mail: doriguetto@unifal-mg.edu.br (Antonio Carlos Doriguetto) and mariliaifrazaob@gmail.com (Marília I. F. Barbosa). Tel.: +55 35 3701-9712 1 ACCEPTED MANUSCRIPT Abstract Herein, novel ruthenium(II) complexes containing 1-methylimidazole as a ligand were obtained with the following formulas: [RuCl(1Meim)(dppb)(bpy)]Cl (1), [RuCl(1Meim)(dppb)(4,4’-DMbpy)]Cl (2), [RuCl(1Meim)(dppb)(5,5’-DMbpy)]Cl (3) and [RuCl(1Meim)(dppb)(phen)]Cl (4) where, 1Meim = 1-methylimidazole, dppb = PT 1,4-Bis(diphenylphosphino)butane, bpy = 2,2’-bipyridine, 4,4’-DMbpy = 4,4’-dimethyl= 1,10- RI 2,2’-bipyridine, 5,5’-DMbpy = 5,5’-dimethyl-2,2′-bipyridine and phen SC phenanthroline. Additionally, crystal structures containing the cations of (1) and (3) were obtained when the counter ion was exchanged, leading to the formation of (5) and [RuCl(1Meim)(dppb)(5,5’-DMbpy)]PF6 NU [RuCl(1Meim)(dppb)(bpy)]PF6 methanol solvate (6) where PF6 = hexafluorophosphate, showing one 1-methylimidazole MA molecule coordinated through the imidazole nitrogen, as expected. The complexes were characterized by elemental analysis, molar conductivity, infrared and UV-Vis 1 H, 13 C{1H} and 31 P{1H} NMR, mass spectrometry and cyclic D spectroscopy, PT E voltammetry. The interactions of complexes 1-4 with DNA and human serum albumin (HSA) were evaluated, and the cytotoxicity profiles of compounds 1-4 were determined CE using four different tumor cell lines derived from human cancers (melanoma: HT-144, colon: HCT-9, breast: MDA-MB-231 and lung: A549). A higher cytotoxic activity was AC observed for compound (3) against non-small cell lung cancer (A549). Complex (3) inhibited the clonogenic capacity and cell cycle progression of A549 cells and induced apoptosis involving mitochondrial pathway activation. Therefore, the data obtained in the present study support further investigations concerning molecular targets of complex (3) in non-small cell lung cancer. Keywords: 1-methylimidazole; lung cancer; antitumor activity; ruthenium(II) complexes. 2 ACCEPTED MANUSCRIPT 1. Introduction The strong therapeutic properties of imidazole-related drugs have encouraged medicinal chemists to synthesize novel chemotherapeutic agents with this molecule to test against different diseases, including cancers [1-4]. It is relevant to improve therapeutic treatments for cancer patients, considering that cancer is a complex disease PT and was responsible for 8.8 million deaths in 2015, representing the second leading RI cause of death globally [5]. SC Ruthenium compounds such as [im][trans-RuCl4(DMSO)(Him)], NAMI-A, [im][trans-RuCl4(Him)] (im = imidazolium cation, Him = imidazole) and [ind][trans- NU RuCl4(Hind)2], KP1019, (ind = indazolium cation, Hind = indazole) are already progressing through clinical trials [2-4]. However, the antitumor mechanism of these MA ruthenium compounds is not fully understood [6–9]. NAMI-A has been characterized as antimetastatic agent, in particular for lung cancer, due to its ability of inhibiting in vitro D cell migration and invasion [10-11]. The antimetastatic properties of KP1019 have also PT E already been described [12]; however, it acts preferentially as a cytotoxic drug on primary tumors, especially on colorectal cancer, due to its pro-apoptotic and pro-oxidant CE activities [12]. Based on the interesting antitumor properties of imidazole derivatives and AC related properties of ruthenium, the present report describes the synthesis and characterization of Ru(II) complexes containing 1-methylimidazole (1Meim). The obtained complexes were evaluated for the ability to interact with DNA and human serum albumin (HSA). In addition, their in vitro antitumor potential was evaluated. By means of the A549 cell line, we demonstrated that the complex [RuCl(1Meim)(dppb)(5,5’-DMbpy)]Cl (dppb = 1,4-Bis(diphenylphosphino)butane and 5,5’-DMbpy = 5,5’-dimethyl-2,2′-bipyridine) is a promising antitumor agent against 3 ACCEPTED MANUSCRIPT lung cancer due to its ability to inhibit clonogenic capacity and cell cycle progression. We also demonstrated that the potent cytotoxic activity of this complex was due at least 2. Experimental Section 2.1. Materials for synthesis PT in part to its capacity to promote apoptosis. RI Solvents were purified by standard methods. All chemicals used were of reagent grade or comparable purity. RuCl3∙xH2O and ligands 1-methylimidazole, 1,4- SC Bis(diphenylphosphino)butane, 2,2’-bipyridine (bpy), 4,4’-dimethyl-2,2’-bipyridine NU (4,4’-DMbpy), 5,5’-dimethyl-2,2’-bipyridine and 1,10-phenanthroline (phen) were used 2.2. MA as received from Sigma-Aldrich. Instrumentation D Elemental analyses were performed in a TruSpec CHNS-O model (Leco PT E Instruments LTDA). The IR spectra were recorded on KBr pellets in the 4000-200 cm-1 region in a Bomen–Michelson FT MB-102 instrument. The UV-vis spectra were CE recorded in CH2Cl2 solution, in a Hewlett Packard diode array – 8452A. Cyclic voltammetry (CV) experiments were carried out at room temperature in CH2Cl2 solution AC containing 0.10 M Bu4N+ClO4 (TBAP-Fluka Purum) using a BAS-100B/W Bioanalytical Systems instrument; the working and auxiliary electrodes were stationary Pt foils, a Luggin capillary probe was used, and the reference electrode was Ag/AgCl. All NMR experiments were recorded on a BRUKER, 300 MHz equipment, in a BBO 5 mm probe, at 298 K, and TMS for internal reference. For 1H, 13C{1H}, and 31P{1H} NMR spectra the CDCl3-d was used as solvent. The splitting of proton, carbon and phosphorus resonances was reported as s = singlet, d = doublet, t = triplet, and m = 4 ACCEPTED MANUSCRIPT multiplet. The triple quadrupole mass spectrometer TSQ Quantum Max (Thermo Scientific) was operated at positive polarity and the ionization conditions were 240 °C for capillary temperature, 35 °C for vaporizer temperature, 3500 V for spray voltage and 5 bar for sheath gas pressure. The samples were analyzed by direct infusion using a 0.5 X-ray crystallography RI 2.3. PT mL syringe and the samples were prepared in methanol at 10 µg.mL-1 concentration. SC Well-shaped single crystals of (5) and (6) were chosen for the X-ray diffraction experiments that were performed at 298(2) K on an automatic diffractometer Agilent NU Technologies brand, Model SuperNova with graphite monochromated Mo-Kα radiation  = 0.71073 Å). The programs CrysAlis CCD and CrysAlis RED [13] were used for MA data collection, cell refinement, data reduction, and multi-scan method absorption correction. The structures were solved and refined using the software Sir2014 [14] and D SHELXL-2013 [15], respectively. All atoms, except hydrogen, were clearly identified PT E and refined by least squares full matrix F2 with anisotropic thermal parameters. The hydrogen atoms bonded to carbons were stereochemically positioned following a riding CE model with fixed C—H bond lengths of 0.93, 0.96, and 0.97 Å for the aromatic, methyl and methylene groups, respectively. The hydrogen atom bonded to oxygen atom of the AC methanol molecule solvating the crystal structure of (6) were also stereochemically positioned following a riding model with idealized OH group (C-O-H angle tetrahedral and O—H bond length of 0.82 Å). The isotropic thermal parameters (Uiso) of all hydrogens depended on the equivalent isotropic thermal displacements of the atoms bonded to them [Uiso(H) = 1.2Ueq (C-aromatic and C-methylene) or 1.5Ueq (C-methyl and O-methanol). The crystallographic tables were generated by WinGX [16] and the structure representations by Mercury software [17] and OLEX2 [18]. The main crystal 5 ACCEPTED MANUSCRIPT data collections and structure refinement parameters for (5) and (6) are summarized in Table 1. CCDC 1831213 and 1831214 contains the supplementary crystallographic data for (5) and (6). These data can be obtained free of charge from the Cambridge Crystallographic Data Centre (www.ccdc.cam.ac.uk/getstructures). Synthesis PT 2.4. RI The precursors cis-[RuCl2(dppb)(X-bpy)] or cis-[RuCl2(dppb)(phen)], where Xbpy = 2,2’-bipyridine (bpy), 4,4’-dimethyl-2,2’-bipyridine (4,4’-DMbpy), 5,5’- the ruthenium(II) 1-methylimidazole NU according to literature [19]. Posteriorly, SC dimethyl-2,2’-bipyridine (5,5’-DMbpy) and 1,10-phenanthroline (phen) were prepared complexes with N-N = bpy (1), 4,4’-DMbpy (2), 5,5’-DMbpy (3) and phen (4) were MA prepared by reacting (0.06 mmol;  50 mg) of cis-[RuCl2(dppb)(X-bpy)] with an excess of 1-methylimidazole ligand (0.30 mmol; 0.024 mL) in 20 mL of dichloromethane D previously degassed. The solution was kept under inert atmosphere and was stirred for PT E 24 h. The final solution was concentrated to ca. 2 mL, and 10 mL of hexane was added to precipitate an orange powder. The solids were filtered off, washed with hexane and [RuCl(1Meim)(dppb)(bpy)]Cl (1) Yield: 38 mg (68%). Anal. Calc. for AC 2.4.1 CE then dried under vacuum. C42H42Cl2N4P2Ru: exp.(calc) C, 60.50 (60.29); H, 5.08 (5.06); N, 6.73 (6.70). m/z exp.(calc): C42H42ClN4P2Ru - 801.10 (801.27) [M]+. 31 P{1H} NMR (121.50 MHz, CDCl3-d, 298 K): (ppm) 38.8 and 39.6 (d) Hz (2JP-P= 35.4 Hz). 1H NMR (300 MHz, CDCl3-d, 298 K): (ppm) 8.97 (s, CH-1Meim-H2), 8.37 (s, CH-1Meim-H1), 6.84 (s, CH-1Meim-H3), 7.75-6.50 overlapped signals, 29 H aromatic hydrogen for dppb, 3.15 (s, CH3-1Meim-H4), and (8H, CH2 of dppb) 4.00-1.0. 13C{1H} NMR (75.46 MHz, 6 ACCEPTED MANUSCRIPT CDCl3-d, 298 K): (ppm) 138.25 (C-1 of 1Meim), 138-120 overlapped signals and 34.37 (C-4 of 1Meim). UV-Vis (CH2Cl2, 5.18 x 10-5 M): /nm (/M-1 L cm-1) 288 (21.035), 332 (shoulder), 448 (3.668). 2.4.2 [RuCl(1Meim)(dppb)(4,4’-DMbpy)]Cl (2) Yield: 36 mg (65%). Anal. Calc. PT for C44H46Cl2N4P2Ru: exp. (calc) C, 61.33 (61.11); H, 5.34 (5.36); N, 6.46 (6.48). m/z RI exp.(calc): C44H46ClN4P2Ru 829.18 (829.33) [M]+. 31P{1H} NMR (121.50 MHz, SC CDCl3-d, 298 K): (ppm) 38.6 and 40.1 (d) MHz (2JP-P= 34.6 Hz). 1H NMR (300 MHz, CDCl3-d, 298 K): (ppm) 8.86 (s, CH-1Meim-H2), 8.56 (s, CH-1Meim-H1), 6.85 (s, NU CH-1Meim-H3), 7.75-6.50 overlapped signals, 29 H aromatic hydrogen for dppb, 3.15 (s, CH3-1Meim-H4) and (8H, CH2 of dppb) 4.00-1.0. 13C{1H} NMR (75.46 MHz, MA CDCl3-d, 298 K): (ppm) 138.32 (C-1 of 1Meim), 138-120 overlapped signals and 34.18 (C-4 of 1Meim). UV-Vis (CH2Cl2, 5.33 x 10-5 M): /nm (/M-1 L cm-1) 286 PT E D (19.137), 330 (shoulder), 440 (3.189). m/z 829.18 [M]+ 2.4.3 [RuCl(1Meim)(dppb)(5,5’-DMbpy)]Cl (3) Yield: 36 mg (65%). Anal. Calc. for CE C44H46Cl2N4P2Ru: exp. (calc) C, 61.28 (61.11); H, 5.38 (5.36); N, 6.50 (6.48). m/z AC exp.(calc): C44H46ClN4P2Ru - 829.14 (829.33) [M]+. 31P{1H} NMR (121.50 MHz, CDCl3-d, 298 K): (ppm) 38.6 and 40.1 (d) MHz (2JP-P= 35.4 Hz). 1H NMR (300 MHz, CDCl3-d, 298 K): (ppm) 8.97 (s, CH-1Meim-H2), 8.37 (s, CH-1Meim-H1), 6.82 (s, CH-1Meim-H3), 3.18 (s, CH3-1Meim-H4), 7.75-6.50 overlapped signals, 29 H aromatic hydrogen for dppb and (8H, CH2 of dppb) 4.00-1.0. 13C{1H} NMR (75.46 MHz, CDCl3d, 298 K): (ppm) 138.50 (C-1 of 1Meim), 138-120 overlapped signals and 34.44 (C-4 of 1Meim). UV-Vis (CH2Cl2, 4.92 x 10-5 M): /nm (/M-1 L cm-1) 294 (20.935), 304 (shoulder), 432 (3.862). m/z 829.14 [M]+ 7 ACCEPTED MANUSCRIPT 2.4.4 [RuCl(1Meim)(dppb)(phen)]Cl (4) Yield: 33 mg (60%). Anal. Calc. for C44H42Cl2N4P2Ru: exp.(calc) C, 61.59 (61.40); H, 4.94 (4.92); N, 6.49 (6.51). m/z exp.(calc): C44H42ClN4P2Ru - 825.14 (825.30) [M]+. 31P{1H} NMR (121.50 MHz, CDCl3-d, 298 K): (ppm) 38.5 and 39.1 (d) MHz (2JP-P= 34.6 Hz). 1H NMR (300 MHz, CDCl3-d, 298 K): (ppm) 9.41 (s, CH-1Meim-H2), 9.03 (s, CH-1Meim-H1), 6.88 (s, PT CH-1Meim-H3), 3.09 (s, CH3-1Meim-H4), 7.75-6.50 overlapped signals, 29 H aromatic RI hydrogen for dppb and (8H, CH2 of dppb) 4.00-1.0. 13C{1H} NMR (75.46 MHz, CDCl3- SC d, 298 K): (ppm) 138.18 (C-1 of 1Meim), 138-120 overlapped signals and 34.18 (C-4 of 1Meim). UV-Vis (CH2Cl2, 4.60 x 10-5 M): /nm (/M-1 L cm-1) 268 (23.913), 426 [RuCl(1Meim)(dppb)(bpy)]PF6 (5) and [RuCl(1Meim)(dppb)(5,5’-DMbpy)] MA 2.4.5 NU (652). PF6 (6) D The synthesis of complexes (5) and (6) was performed similarly to the others PT E however, NH4PF6 was added to counter ion exchange. In a schlenk flask 50 mg (0.06 mmol) of the precursor cis-[RuCl2(dppb)(X-bpy)], (X-bpy) = bpy (5) and 5,5’-DMbpy CE (6), with an excess of 1-methylimidazole ligand (0.30 mmol; 0.024 mL) were added AC in 20 mL of dichloromethane previously degassed, then 20.8 mg (0.128 mmol) of NH4PF6 were added and the solution was kept under inert atmosphere and stirred for 4 h. The final solution was concentrated to ca. 2 mL, and 10 mL of hexane was added to precipitate an orange powder. The solids were filtered off, washed with hexane and then dried under vacuum. Single-crystals of the complexes (5) and (6), which were used only in the X-ray diffraction experiment, were grown from the evaporation of their solutions in CH2Cl2/CH3OH (6:4 v/v). 8 ACCEPTED MANUSCRIPT 2.5. Biological assays 2.5.1 Cell lines and treatment schedule The following cell lines derived from human cancers were used in the present study: A549 (lung), HCT-9 (colon), HT-144 (melanoma), and MDA-MB-231 (breast). Fibroblasts (CCD-1059Sk) derived from normal skin were also examined. The cell PT cultures were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium, Sigma, RI CA, USA) supplemented with 10% fetal bovine serum (Vitrocell, Campinas, Brazil). SC Cells were grown in a 37 C humidified incubator containing 5% CO2. The Ru(II) complexes were solubilized in DMSO immediately before use, and the amount of NU DMSO in the culture medium did not exceed 0.4% (v/v). Cells were seeded into 96-well plates (cell viability assay), 12-well plates (annexin V assay), or 35 mm Petri plates MA (cell cycle analysis). After attachment (24 h), the cultures were treated with different PT E 2.5.2 Cell viability analysis D complexes over 24 h. Cell viability was measured by MTS (dimethylthiazol carboxymethoxyphenyl CE sulfophenyl tetrazolium) assay using the CellTiter 96® Aqueous Non-Radiative Cell Proliferation assay (Promega) according to the manufacturer’s instructions. The MTS AC tetrazolium compound is bioreduced by metabolically active cells to give a colored formazan product that absorbs light at 490 nm. Viable cells rate is directly proportional to the amount of formazan produced by dehydrogenase enzymes. The experiments were conducted in triplicate wells and repeated twice. The data are presented as the mean ± standard deviation (SD). The cells were seeded into 96-well plates at a density of 5 x 103 cells/well (A549) or 1 x 104 cells/well (HCT-9, MDA-MB-231, and HT-144). The complexes (1 – 4) were used at 20 µM (A549 cell line) or 40 µM (A549, HCT-9, MDA- 9 ACCEPTED MANUSCRIPT MB-231, and HT-144) over 24 h for evidencing the most promising complexes. In next step, complexes (3) was evaluated in different concentrations (0 - 80 µM) for 24 h. IC50 values were determined from non-linear regression using GraphPad Prism® (GraphPad Software, Inc., San Diego, CA, USA). PT 2.5.3 Cell cycle analysis RI Cell cycle analysis was performed according to literature [20]. Briefly, cells were treated with complex (3) for 24h at 5, 10 or 20 µM. Cells were fixed with 75% SC ethanol at 4 C overnight and rinsed twice with cold phosphate-buffered saline (PBS). NU Afterwards, the cells were homogenized in a dye solution [PBS containing 30 μg.mL-1 propidium iodide (PI) and 3 mg.mL-1 RNAase]. DNA was quantified 1h after staining. MA The analysis was performed by flow cytometry (Guava easyCyte 8HT, Hayward, CA, PT E 2.5.4 Clonogenic assay D USA). Results are presented as the mean ± SD of three independent experiments. The clonogenic assay was performed according to [21] with some modifications. CE Briefly, 100 cells were seeded into 35mm Petri plates. The cells were treated for 24h with complex (3) at a concentration of 5, 10, or 20 µM and recovered in a drug-free AC medium for an additional 15 days. Afterwards, the colonies were fixed and stained with crystal violet. Only the colonies with > 50 cells were counted by direct visual inspection with a stereomicroscope at 20 × magnification. Assays were performed in triplicate, and the data are presented as the mean ± SD of three independent experiments. 2.5.5 Apoptosis evaluation using annexin V assay Cells were seed into 12-well plates at 8 × 104 cells/well. After 24 h of treatment with complex (3) at a concentration of 10 or 20 µM, we evaluated the 10 ACCEPTED MANUSCRIPT phosphatidylserine externalization using Guava Nexin® Kit (Merck Millipore, Massachusetts, USA) according to manufacturer’s instructions. Briefly, cells were collected by enzymatic digestion (Trypsin/EDTA, Sigma), centrifuged at 1,000 rpm for 5 min at 4 °C, and washed with ice-cold PBS, and then 2 × 104 cells were resuspended in 100 μL of DMEM. In the next step, 100 μL of a mixed solution containing buffered PT Annexin V-PE and 7-AAD was added. The samples were read after 20 min of RI incubation at room temperature in a dark chamber. The analysis was performed by flow SC cytometry using GuavaSoft 2.7 software. The experiments were conducted in triplicate NU and repeated twice. The data are presented as the mean ± SD. 2.5.6 Mitochondrial membrane potential (ΔΨm) analysis by JC-1 fluorescence MA The alteration of ΔΨm in A549 cells were analyzed through a JC-1 staining using Guava®MitoPotential Kit (Merck/millipore) according to manufacturer’s D instructions. Briefly, the cells were trypsinized and washed twice with PBS; after that, PT E the cells were labeled with the fluorescent dye JC-1/7-AAD in the dark chamber for 30 min at 37 °C. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was used as a CE positive control to reduce ΔΨm. The analysis was performed by flow cytometry using GuavaSoft 2.7 software. The data are shown as mean ± SD from three independent AC experiments. 2.5.7 ATM activation profile Ataxia telangiectasia-mutated (ATM) activation was measured by flow cytometry (software GuavaSoft 2.7) using Cell Cycle Checkpoint ATM DNA Damage Kit (Merck-millipore) according to manufacturer’s instructions. Briefly, the cells were trypsinized, washed twice and fixed. After permeabilization process, anti-p-ATM 11 ACCEPTED MANUSCRIPT (Ser1981) was incubated por 1 h (4 °C in dark chamber). The nuclei were stained with Propidium iodide for 30 min. The data are shown as mean ± SD from three independent experiments. 2.5.8 Statistical Analysis PT The results of the biological assays were tested for significance using one-way RI analysis of variance (ANOVA) followed by Tukey’s post-test using GraphPad Prism®. HSA fluorescence NU 2.5.9 SC The values are expressed as the mean ± SD. Fluorescence spectroscopy is an effective method for exploring the interactions MA between small molecules and biomacromolecules. The fluorescence of HSA comes from its tryptophan, tyrosine and phenylalanine residues, where the latter two contribute D to its fluorescence to only a minor extent [22]. The protein interaction was examined in PT E 96-well plates used for fluorescence assays. HSA (~2.5 x 10-6 mol.L1) was prepared by dissolving the protein in Tris–HCl at pH = 7.4, and the complexes were dissolved in CE sterile DMSO. For the fluorescence measurements, the HSA concentration in the buffer Tris–HCl was kept constant in all samples, while the complex concentration was AC increased from 0.78 to 100 μM, and quenching of the emission intensity of the HSA tryptophan residues at 305 nm (excitation wavelength of 270 nm) was monitored at different temperatures (298 and 310 K). A standard solution was prepared with 180 μL of albumin and 20 μL of DMSO. The experiments were carried out in triplicate and analyzed using the classical Stern–Volmer equation as follows: F0/F = 1 + Kqτo[Q] = 1 + Ksv[Q] Eq. (1) 12 ACCEPTED MANUSCRIPT where F0 and F are the fluorescence intensities in the absence and presence of quencher, respectively, [Q] is the quencher concentration, and Ksv is the Stern–Volmer quenching constant, which can be written as Kq= Ksv/τo, where Kq is the bimolecular quenching rate constant and τo is the average lifetime of the fluorophore in the absence PT of quencher (6.2 x 10-9 s) [23]. Therefore, Eq. (1) was applied to determine Ksv by linear regression of a plot of Fo/F vs. [Q]. RI The binding constant (Kb) and the number of complexes bound to HSA (n) were SC determined by plotting the double logarithmic graph of the fluorescence data using Eq. NU (2) as follows: MA log [(F0-F)/F] = log Kb + nlog [Q] Eq. (2) The thermodynamic parameters were calculated from the eq. (3): Eq. (3), PT E D ln (K2/K1) = [(1/T1)-(1/T2)] ΔH/R where K1 and K2 are the binding constants at temperatures T1 and T2, respectively, and Eq. (4): CE R is the gas constant. Furthermore, the change in free energy (ΔG) was calculated from AC ΔG = -RT ln K = ΔH – TΔS Eq. (4) The inner filter effect on the intensity of fluorescence of the protein and complexes were corrected according to the Eq (5) where Fcorr and Fobs are the corrected and observed fluorescence intensities and Aex and Aem are the absorbance values at excitation and emission wavelengths. Eq. (5) 2.5.10 DNA interaction studies 13 ACCEPTED MANUSCRIPT The interactions of the complexes with ctDNA (calf thymus DNA) were analyzed by absorption spectrophotometric analysis at room temperature using a Hewlett Packard diode array – 8452 UV–vis spectrophotometer. A standard solution of calf thymus DNA (ctDNA) from Sigma-Aldrich was prepared in Tris-HCl buffer (5 mM Tris–HCl and 50 mM NaCl, pH 7.4). The concentration of this ctDNA solution was PT determined from its absorption intensity at 260 nm using a molar absorption coefficient RI value of 6600 M−1 cm−1. The ctDNA solution was protein-free given that the ratio of the SC UV absorbances at 260 and 280 nm was approximately 1.8-2.0. The absorption titrations were recorded in the range of 200–450 nm while keeping the concentration of NU the Ru(II) complexes constant (1.0 mM) and increasing the amount of ctDNA after each addition. The intrinsic equilibrium binding constant (Kb) of the complexes to ctDNA MA was obtained by monitoring the changes in the absorption intensity with increasing D concentration of ctDNA and analyzed by regression analysis. Results and discussion 3.1. Synthesis reaction of 1-methylimidazole with ruthenium precursors cis- CE The PT E 3. [RuCl2(dppb)(X-bpy)] or cis-[RuCl2(dppb)(phen)] was employed to obtain the AC complexes [RuCl(1Meim)(dppb)(bpy)]Cl (1), [RuCl(1Meim)(dppb)(4,4’-DMbpy)]Cl (2), [RuCl(1Meim)(dppb)(5,5’-DMbpy)]Cl (3) and [RuCl(1Meim)(dppb)(phen)]Cl (4) by single chloride exchange under mild conditions (Scheme 1). The elemental analyses are described in the experimental section, and they agreed well with the proposed formulations. The MS spectra were acquired in the positive mode, and the charged complex ions resulting from loss of the respective chloride ions were observed (Supporting material: Figures 1S-4S). The molar 14 ACCEPTED MANUSCRIPT conductance values measured for complexes (1-4) in acetone at room temperature ranged from 93.3 to 113.9 S.cm2.mol-1, revealing 1:1-type compounds [24]. Furthermore, the complexes were characterized by 31P{1H}, 13C{1H} and 1H NMR spectroscopy, UV-Vis and IR spectroscopy, and cyclic voltammetry. Compounds (5) and (6), which contained the cations present in (1) and (3), respectively, had their PT structures determined by X-ray crystallography, confirming the coordination sphere RI around the Ru(II) cation containing the imidazole molecule (details in section 3.2). SC The 31P{1H} NMR spectra of complexes (1-4) in CDCl3 presented a typical AX spin system, indicating the magnetic nonequivalence of the two phosphorus atoms, in NU which one is trans to the N (X-bpy or phen) ligand and the other is trans to the N of the 1-methylimidazole ligand (Supporting material: Figure 5S-8S). The chemical shifts and MA coupling constants (2JP-P) are shown in the experimental section. For all complexes, the 31 P{1H} NMR chemical shifts were different from those of the cis-[RuCl2(dppb)(X- D bpy)] or cis-[RuCl2(dppb)(phen)] starting material, suggesting that the presence of a 1- PT E methylimidazole ligand coordinated to the metal shifted the electron density of the phosphorus atoms of the dppb ligand. CE In the 1H NMR spectrum of the free 1-methylimidazole, a singlet at 3.64 ppm assigned to the methyl group and signals at 7.39, 7.06 and 6.86 ppm assigned to H1, H2 AC and H3 were observed (Scheme 1). For all complexes, H1 and H2 characteristic deshielded signals at 9.41-8.37 and 9.03-8.37 ppm, respectively, were observed, as expected, due to imidazolic nitrogen coordination. Other aromatic hydrogen atom resonances were in the range 5.00–8.00 ppm and were attributed to the protons present in the aromatic phosphine and X-bpy or phen ligands. Additionally, 8 hydrogens of the CH2 groups of dppb were observed at 4.00-1.0 ppm. All complex spectra exhibited a 15 ACCEPTED MANUSCRIPT singlet at 3.18-3.09 ppm assigned to the methyl group of 1-methylimidazole (Supporting material: Figure 9S-12S). The chemical shifts of the 13C{1H} NMR spectra for the free 1-methylimidazole and their respective complexes are summarized in Table S1 (Supporting material). All of the complex signals ranging from 33.05-34.44 ppm were attributed to the methyl PT group C4, and signals at 138.18-138.66 ppm were relative to C1, whereas signals RI referent to C4 and C1 in the free ligand were observed at 33.14 ppm and 137.79. SC Aromatic carbon atoms of X-bpy, phen and phosphine were also identified in the range of 170–120 ppm (Supporting material: Figure 13S-16S). NU Time-dependent 31P{1H} NMR experiments in solution were carried out to evaluate the stability of the complexes. Thus, the complexes were diluted in DMSO and MA analyzed from 0 to 48 h (Supporting material: Figures 17S-20S). The 31P{1H} NMR spectra reveal that all complexes (1-4) were stable over this length of time. D Infrared spectra of the complexes presented characteristic bands due to 1- PT E methylimidazole, X-bpy or phen and dppb ligand vibrations (Supporting material: Figures 21-25S). The stretching modes of the aromatic imidazole C–H bonds displayed CE maxima ranging from 3126 to 3132 cm-1. The strong band at 1521 cm-1 was assigned to ν(C=C). The methyl groups of the 1Meim ligand were indicated by bands near 2920, AC 2922, 2924 and 2920 cm-1, respectively, for complexes (1-4). The ν(C=N) of the imidazole ring appeared close to 1629, 1622, 1624 and 1625 cm-1 for compounds (1-4), respectively, which was different from that of the free ligand (1649 cm-1) and indicated the coordination of the ligand by the imidazolic nitrogen atom. The low-intensity band ranging from 485 to 489 cm-1 was assigned to the Ru-N stretch [25]. Table 2 summarizes the main IR frequencies (cm-1) of 1-methylimidazole and complexes (1-4). 16 ACCEPTED MANUSCRIPT The electronic spectra of complexes (1-4) (Supporting material: Figures 26S29S) showed two bands in the UV region (268–304 nm) assigned to π → π* transitions, which were also present in the spectra of the free dppb and diimine ligands [26, 27]. The bands at approximately 440 nm were attributed to metal-to-ligand charge transfer, probably from ruthenium to the diimine, pyridine and dppb ligands. Similar assignments PT have been proposed for other Ru(II) complexes [28]. RI The electrochemical behavior of complexes (1-4) containing the 1- SC methylimidazole ligand was similar to that found for other Ru(II) complexes presenting diimine and dppb ligands [29] (Supporting material: Figures 30S-33S). These NU experiments were performed under the same conditions, and it was observed that complexes (1-4) exhibited a quasi-reversible process assigned to the redox pair MA Ru(II)/Ru(III), with Epa ranging from 1010 to 1080 mV. The spectral difference observed among the complexes may be due to their stereochemistry differences. The D E½ values found for the complexes were considerably more anodic than those observed PT E for both precursors [RuCl2(dppb)(X-bpy)] and [RuCl2(dppb)(phen)] [19], indicating that the ruthenium center was more stable after coordination with 1-methylimidazole than CE was the precursor. The metal center stabilization occurred due to replacement of a chloride by an imidazolic nitrogen of 1-methylimidazole. A linear correlation of AC the pKa values of the X-bpy ligands with the redox potential of the complexes were observed (Supporting material: Table S1). The lowest redox potential of complex (3) could be correlated with the highest activity against A549 cells. In general, the redox activity of Ru complexes is associated with the formation of ROS in cells and might lead to activation in the reductive environment of tumors (30). 3.2. Single-Crystal X-ray Analysis 17 ACCEPTED MANUSCRIPT Attempts to obtain a single crystal of (1-4) for X-ray diffraction experiments were all unsuccessful. Therefore, an attempt was also made to obtain single crystals containing the large cations of (1-4) by replacing chloride with hexafluorophosphate (PF6). These experiments were successful for crystal growth of (5) and (6). When the small chloride counter-ion was exchanged, single crystals containing the large cations of PT (1) and (3) were obtained containing the large PF6- counter-ion, leading to formation of RI (5) and (6), respectively. This provides examples of the general principle that solid salts SC separate from aqueous solutions easiest in combinations of either small cation-small anion or large cation-large anion (as here), preferably with systems having the same but NU opposite charges on the counter-ions [31]. The entrance of solvent into the crystalline lattice also helped in the crystallization of (6). MA The X-ray structures shown in Figure 1 confirm that (5) and (6) were sixcoordinate complexes of a ruthenium cation bound to the P1 and P2 phosphorous atoms D from the dppb ligand, the N1 and N2 nitrogen atoms from the bipyridines bpy (5) and PT E 5,5’-DMbpy in (6), the N3 nitrogen atom from the 1-methylimidazole ligand, and the chlorido anion. The 1Meim ligand was positioned cis to the coordinated chlorido ligand CE and trans to the P2 atom from the dppb ligand. Considering the precursors cis[RuCl2(dppb)(bpy)] [19] and cis-[RuCl2(dppb)(5,5’-DMbpy)] [32], it was confirmed AC that the 1Meim ligand replaced the chlorido ligand trans to the dppb phosphorous atom, in agreement with analogous complexes [25, 33-38]. A molecular superposition of (5) and (6) (overlaid using the Ru atoms and the six atoms coordinated to them as homologous atom pairs) showed that the two complexes differed significantly in the orientation of the phenyl groups linked to the dppb ligand P2 atom (Figure 1c). Analysis of the octahedral geometry around the Ru cation in (5) and (6) showed that for both structures, as expected, the smallest metal-ligand distances involved the 18 ACCEPTED MANUSCRIPT nitrogen atoms (N1 and N2) from the bipyridines (bpy or 5,5`-DMbpy ligands), whereas the largest distances were between the Ru metal and the chlorido ligand (Table 3). The bond lengths of the Ru-P2 were comparable to those of Ru-P1 in either (5) or (6). This structural feature was also expected in (5) and (6), taking into account that their respective P1 and P2 phosphorus atoms were both trans to the nitrogen atoms (N1 and PT N2) from the bipyridines. Therefore, the two phosphorous atoms were not susceptible to RI different trans competitions, as observed in the analogous complex having a P2 trans to SC the CO ligand [28, 37]. The Ru-N3 distances in (5) and (6) did not present a significant difference (Table 3) and were very similar to those observed for the analogous NU complexes having pyridines instead of 1Meim groups completing their coordination sphere, including pyridine (Ru-N3 = 2.215(3) Å) [25], 4-methylpyridine (Ru-N3 = MA 2.175(7) Å) [34], 4-amine-pyridine (Ru-N3 = 2.203(4) Å) [25], 4-phenylpyridine (RuN3 = 2.170(3) Å) [38] or 4-vinylpyridine (Ru-N3 = 2.213(3) Å) [39]. Finally, upon D comparing the bond angle values around the octahedral Ru coordination sphere (Table PT E 3), it was observed that the largest deviation from the orthonormality involved the two nitrogen atoms (N1-Ru-N2 angle) from the bipyridinic rigid bidentate ligand, which CE was also in agreement with other analogous complexes [25, 28, 33-39]. As emphasized in our previous work [37], the overall geometries of the AC compounds with the general formula ct-[RuCl(L)(dppb)(bipyridine)] having L cis to the chlorido ligand and trans to dppb were very similar. Therefore, confirming our expectation, the intramolecular geometry of (5) was similar to those observed for the analogous complexes containing one bpy and one dppb as bidentate ligands and one chlorido and one pyridine-derivative (pyridine [25], 4-methylpyridine [34], 4-aminepyridine [25] or 4-phenylpyridine [38]) as monodentate ligands (Supporting material: Figure 35S). 19 ACCEPTED MANUSCRIPT A very interesting aspect of the crystallographic part of the present work is the finding that (6) and its analogous cis-[RuCl(4-vinylpyridine)(dppb)(5,5`-Mebipy)] [39] (5,5`-Mebipy = 5,5’-dimethyl-2,2′-bipyridine), were crystals with isotype features [40]. That means, in spite of the different chemical compositions (the crystal structure of (6) had the 1Meim ligand instead 4-vinylpyridine in addition to being solvated by PT methanol), they crystallized in the same space group (Pbca) with very similar unit cell RI metrics (a = 15.0023(3), b = 20.3898(3), and c = 29.2586(6) Å in (6) vs. a = 14.669(3), SC b = 20.499(3), and c = 29.401(6) Å in the 4-vinylpyridine analogue [39] and had comparable fractional atomic coordinates for the homologous atoms (Ru, Cl, dppb and NU 5,5’-DMbpy) (Figure 2) and equivalent intermolecular arrays (packing) (Figure 3). MA Even though the term “very similar” has not been defined in quantitative terms, [41] the unit cell dimensions of these isotype crystals are expected to be slightly different as a consequence of their different chemical compositions. Moreover, since PT E D 1Meim is smaller than 4-vinylpyridine, it would be expected that the unit cell of (6) was smaller than that of the 4-vinylpyridine analogue. However, this was only observed with respect to their respective b and c-axes. The a-axis of (6) was 0.333 Å longer than that CE of its previously reported analogue. Closer scrutiny showed that the a-axis increase in AC (6) was a consequence of solvent insertion and not ligand replacement (Figure 3). In the specific case discussed here, these two variables are, of course, correlated, since to retain the same structure, the void left by replacing a larger ligand (4-vinylpyridine) with a smaller one (1Meim) had to be filled by the solvent (methanol) to stabilize the packing of (6). It is important to emphasize that the void volume occupied by the methanol molecule in (6) was 3% (272.50 Å3) of its unit cell volume (calculated by MERCURY [17] using a grid spacing of 0.7 Å and probing-sphere radius of 1.1 Å). It is also important to note that the difference in volume between the two unit cells was only 20 ACCEPTED MANUSCRIPT 1.2% (8950 Å3 in (6) vs. 8841 Å3 in its analogue). The contact surfaces of the calculated voids in (6) occupied by methanol are shown in (Supporting material: Figure 36S). It is relevant to mention that the differentiation of isotype crystals by PXRD analysis could be a difficult task since their PXRD patterns are expected to be almost identical, which was confirmed here by comparing the calculated PXRD patterns to the crystal structures Biological targets: DNA and HSA binding studies SC 3.3. RI PT of (6) and its analogue (CCDC code BUZYIF [39]) (Supporting material: Figure 37S). 3.3.1 Fluorescence spectra of HSA–Ru complexes NU HSA solutions exhibit a strong fluorescence emission with a peak at 338 nm, which is provided mainly by a single residue of tryptophan located at position 214 along MA the chain in subdomain IIA [42, 43]. To understand the mechanism of interaction between complexes (1-4) and HSA, fluorescence quenching experiments were D performed. The experiments were carried out by holding the concentration of the HSA PT E solution constant and adding increasing concentrations of complexes (1-4) at temperatures of 298 and 310 K while monitoring the fluorescence intensity suppression. CE The effects of the complexes on the protein fluorescence intensity are presented in (Supporting material: Figure 38S). The constants obtained for complexes (1-4) are AC listed in Table 4. The KSV values displayed an inverse correlation with the temperature, which suggested that this quenching mechanism was static and initiated by adduct formation [44]. The binding constants acquired have been ranked as 4 > 3~2 > 1, indicating a more potent interaction between HSA and complex (4). This conduct may be related to the size and electronic density of the phenantroline ligand, which is a larger substituent and a better activator than bipyridine and methyl-bipyridine. The quantity of the binding 21 ACCEPTED MANUSCRIPT sites (n) was approximately 1, indicating at least one binding site with HSA. The thermodynamic parameters (ΔHº, ΔSº and ΔGº) were obtained to evaluate the intermolecular forces between the complexes and the protein. As observed in Table 4, for compounds (1, 2 and 3), the negative ΔHº and ΔSº values reflect van der Waals forces or hydrogen bond formation, whereas for complex (4), the positive ΔHº and ΔSº PT values indicate the predominance of hydrophobic interactions [22,45-47]. Furthermore, RI the negative ΔGº values observed for all complexes reveal that the interaction was SC spontaneous. Thus, complexes (1-4) can be stored in the protein and released at targets. The results are in agreement with those for other ruthenium compounds such as NU [RuCl(CTZ)(bipy)(P-P)]PF6 (CTZ = clotrimazole, bipy = 2,2’-bipyridine and P-P = 1,2bis(diphenylphosphino)ethane, 1,4-bis(diphenylphosphino)butane and 1,1’- MA bis(diphenylphosphino)ferrocene). CTZ, a kind of imidazole, binds to BSA with moderate affinity through a static quenching mechanism, and the thermodynamic PT E D parameters reveal the predominance of hydrophobic interactions with the protein [48]. 3.3.2 DNA-binding: UV-Vis spectrophotometric titration CE UV–visible absorption spectroscopy is a useful direct method for determining the DNA binding constants of metal complexes that can interact at distinct binding sites AC (groove binding outside of the DNA helix along the major or minor groove, electrostatic binding to a phosphate group and intercalation). To investigate DNA as a potential target for the complexes, spectroscopic studies were carried out. Upon adding the solution of ctDNA to each complex (1–4), a decrease in the absorption intensity (hypochromism) was observed, which suggests interaction between the complexes and ctDNA. A representative absorption spectrum of compound (3) is provided in Figure 4, and those for compounds (1, 2 and 4) are in (Supporting material: Figure 39S). 22 ACCEPTED MANUSCRIPT The magnitude of such interaction was indicated by the DNA binding constant, Kb, (Table 5) which was calculated according to Eq. 4 [34] as follows: [ctDNA]/(εa-εf) = [ctDNA]/(εb-εf) + 1/Kb(εb-εf) Eq. (4) where [ctDNA] is the concentration of ctDNA in base pairs, εa is the ratio of the PT absorbance/[Ru], εf is the extinction coefficient of the free Ru(II) complex, and εo is the RI extinction coefficient of the complex in the fully bound form. The ratio of the slope to SC the intercept in the plot of [DNA]/(εa-εf) vs. [DNA] gives the value of Kb, which was calculated from the metal-to-ligand charge transfer (MLCT) absorption band (λmax) at NU 424 nm for complexes (1-3) and 400 nm for complex (4). The values obtained for the binding constant and the percentage of hypochromism for each complex are MA summarized in Table 5. Compounds (1-4) interacted with DNA with binding constants, Kb, on the order PT E D of 104 M-1. The magnitude of Kb found here is comparable with those of metal complexes that bind ctDNA through noncovalent (electrostatic or hydrogen bonding) interactions reported in the literature [33, 34], and such interactions that occur with 3.4. AC CE other Ru(II)/phosphinic/diimine complexes, as reported elsewhere [33, 34, 48]. Biological assays The complexes (1-4) were evaluated against 4 different tumor cell lines at 40 µM for 24 h. We observed significant cytotoxic activity by complex (3) on HT-144 and MDA-MB-231 when compared to control samples (Figure 5A). Interestingly, in these experimental conditions, all complexes displayed potent cytotoxicity against A549 cells, reducing almost 100% of cell viability (data not shown). Thus, the complexes were again evaluated on A549 cells at 20 µM, and results showed high cytotoxicity of 23 ACCEPTED MANUSCRIPT complex (3) (Figure 5A). Some studies have reported a great responsiveness of A549 cells to ruthenium complexes treatment when compared to other tumor cell lines [2, 20, 49-50]. This finding is very relevant, considering that A549 cells are derived from nonsmall cell lung cancer, a type of tumor that represents 75-80% of all lung cancers diagnosed [51]. PT We selected complex (3) to perform dose-response curves. We included in the RI analysis both the ruthenium complex precursor [RuCl2(dppb)(5,5’-DMbpy)] and free 1- SC methylimidazole (ligand). We provided evidence that complex (3) was much more cytotoxic when compared to the precursor (Figure 5B). The IC50 value (Table 6) of the NU complex (3) was approximately 4-fold higher than that of cisplatin, and the ligand did not display cytotoxic activity on A549 cells, at least not in the concentration range MA tested. These findings evidence that the coordinated structure of the complex was critical for its cytotoxic activity, highlighting the importance of the positions of the D metal [20] and methyl groups [52]. We also examined the cytotoxic profile of complex PT E (3) on human normal fibroblasts, which was lower than that toward A549 cells (Table 6), indicating a certain selectivity of the tested compound toward tumor cells. CE The morphological features of the A549 cells treated with complex (3) or the vehicle are shown in Figure 6A. A reduction in cell density was observed in all treated AC samples compared to untreated cultures. In addition, cell morphology was profoundly altered by treatment with complex (3) at 10 µM and 20 µM. Rounded cells were frequently observed in cultures treated with complex (3) at 20 µM, indicating cell detachment from the substrate, similarly to the process observed in cell death (Figure 6A). In the next step, to better understand the biological mechanisms underlying complex (3) activity toward A549 cells, we verified whether complex (3) reduced 24 ACCEPTED MANUSCRIPT clonogenic capacity and/or promoted apoptosis in A549 cells. The data showed that the complex (3) was very cytotoxic at 10 and 20 µM once colonies were not observed in treated samples. Interestingly, we observed a significant reduction of the number of colonies in samples treated with complex (3) at 5 µM compared to the control group. In addition, there was a reduction in the diameter of the colonies, indicating that the PT proliferation rate of the A549 cells was affected by the treatment, even after drug RI removal (Figure 6B and C). These findings are very important, considering that the SC sustained proliferative behavior of tumor cells is critical for tumor progression and metastasis [53]. The negative influence of complex (3) on the proliferative behavior of NU A549 cells over a prolonged period demonstrates its promising antitumor potential. There are a few reports concerning the influence of ruthenium complexes on the MA clonogenic capacity of A549 cells. It has been reported that cis-[RuCl2(NH3)4]Cl [54] and [Ru(pipe)(dppb)(bipy)]PF6 (pipe = piperonylic acid and bipy = 2,2’-bipyridine) [20] D inhibited the colony-formation ability of A549 cells. However, these studies did not PT E report reductions in colony diameters observed in the present study. To investigate whether the antiproliferative activity of complex (3) on A549 CE cells was triggered by cell cycle arrest, we performed DNA quantification by flow cytometry using propidium iodide (PI) staining. We observed a significant increase of AC the G0/G1 population and reduction of the cell population at G2/M in cultures treated with complex (3) at sub-toxic concentrations (5 µM), indicating cell cycle arrest at the G1/S transition. We also observed an elevated subdiploid peak (sub-G1 phase) in samples treated with complex (3) at 10 and 20 µM compared to the control group, indicating complex (3) promoted an increase of the dead cell population. These findings corroborated the results obtained in the clonogenic assay and observations concerning cell morphology features. Accumulation of cells in the G0/G1 phase is often the result 25 ACCEPTED MANUSCRIPT of cell cycle checkpoint activation [55]. Studies have shown that some ruthenium compounds induce the arrest of cells in the G0/G1 phase by modulating the activity of important regulators of the G1/S transition including p53, p21, CDK4/6 and cyclin D1 [20, 56]. Further investigations will be performed to identify the molecular targets of complex (3) in A549 cells. PT The pro-apoptotic activity of complex (3) was evaluated by a PE-Annexin assay RI based on its high cytotoxicity on A549 cells. We observed a frequency increase of SC annexin V-positive cells in cultures treated for 24 h at 10 and 20 µM (Figure 6D), indicating that apoptosis was effectively induced in treated samples in a concentration- NU dependent manner. Our data corroborate those reported by other authors demonstrating the pro-apoptotic activity of ruthenium complexes such as Ru(II)/phosphinic/diiminic MA complexes, cis-[RuCl2(NH3)4]Cl and [Ru(pipe)(dppb)(bipy)]PF6 [20, 53]. Evasion of apoptosis is one of the central features of tumor progression and drug resistance [53]. PT E antineoplastic agents. D Therefore, compounds that effectively induce apoptosis represent good candidates as We sought to investigate whether the pro-apoptotic activity of complex (3) CE toward A549 cells was related to activation of the intrinsic apoptotic pathway, also called the mitochondrial pathway. Thus, we verified the influence of this complex on AC the mitochondrial membrane potential (ΔΨm) using the fluorescent probe JC-1, which exhibits ΔΨm-dependent accumulation and emits strong orange fluorescence in normal mitochondria, while in unhealthy mitochondria, JC-1 emits a strong green fluorescence. We observed a strong reduction in the orange/green ratio in samples treated with complex (3) at 20 µM (Figure 6E), indicating a significant decrease of ΔΨm in A549 cells. Our data showed, therefore, that the pro-apoptotic activity of complex (3) is associated with intrinsic apoptotic pathway activation. Recent studies have 26 ACCEPTED MANUSCRIPT demonstrated that ruthenium complexes are effective in inducing apoptosis by mitochondrial pathway activation [20, 57]; however, the molecular target underlying this specific cellular response still remains unclear. The intrinsic apoptotic pathway may be activated by various intracellular stress signals including DNA damage. Thus, we also evaluated the activation profile of ATM PT by flow cytometry in A549 cells treated with complex (3), considering that ATM RI (ataxia telangiectasia mutated) is a kinase protein critically involved in the DNA SC damage response induced by radiation or chemotherapeutic drugs. ATM is a member of the phospho inositide 3-kinase (PI3K)-related Ser/Thr protein kinase family. Inactive NU ATM exists as a dimer but quickly dissociates and becomes phosphorylated on serine 1981 in response to ionizing radiation. Activated ATM phosphorylates several key MA proteins that act as DNA damage checkpoints, leading to cell cycle arrest, DNA repair, or apoptosis [58]. We did not evidence a significant increase in ATM activation after D treatment with complex (3) (Figure 6F), indicating that its pro-apoptotic activity on PT E A549 cells was not directly associated with primary damage to DNA. The findings obtained in the present study are very promising and support CE further molecular studies to identify the probable molecular targets for complex (3) in 4. AC non-small cell lung cancer. Conclusions This report presents the synthesis and characterization of four new cationic ruthenium complexes with X-bpy or phen and 1-methylimidazole. Crystal structures containing the cations of (1) and (3) were obtained when the counter ion was exchanged. A linear correlation of the pKa values of the X-bpy ligands with the redox 27 ACCEPTED MANUSCRIPT potential of the complexes was observed. The lowest redox potential of complex (3) could be correlated with the higher active against A549 cells. Interactions with DNA and human serum albumin (HSA) were performed with (1-4), and it was found that Ru(II)-complexes exhibit moderate DNA-binding affinity. The HSA binding experiments suggested predominance of Van der Waals forces for PT compounds (1, 2 and 3) and Van der Waals forces or hydrogen bond formation for RI complex (4). Thus, complexes (1-4) can be stored in the protein and released at targets, SC in agreement with the behavior reported for other Ru(II)/phosphinic/diiminic complexes. NU Evaluation of the antitumor activities showed that the A549 cell line was the most responsive, especially to complex (3). Also, complex (3) inhibited clonogenic MA capacity and cell cycle progression of A549 cells and induced apoptosis involving mitochondrial pathway activation. These findings demonstrate that complex (3) is a D very promising antitumor agent and support further investigations concerning its Acknowledgements CE 5. PT E molecular targets in non-small cell lung cancer. The authors thank FINEP, CNPq (448723/2014-0; 308162/2015-3), CAPES, and AC FAPEMIG (APQ-00273-14; APQ-02486-14; PPM-00533-16) for financial support. We also thank CNPq and CAPES for research fellowships (J.S.M.D.; H.V.R.S.; M.I.F.B.; A.C.D). This work is a collaborative research project by members of the Rede Mineira de Química (RQ-MG), supported by FAPEMIG (RED-00010-14). We also thank Dr. Alzir Azevedo Batista for collaboration in this work. 6. Appendix A. Supplementary data 28 ACCEPTED MANUSCRIPT Coordinates and other crystallographic data have been deposited with CCDC numbers 1831213 and 1831214 for the complex (5) and (6), respectively. Copies of this information may be obtained from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK, Fax: +44 1233 336033, E-mail: deposit@ccdc.cam.ac.uk or References RI 7. PT www.ccdc.cam.ac.uk. SC [1] G. V. M. Sharma, A. Ramesh, A. Singh, G. Srikanth, V. Jayaram, D. Duscharla, J. H. Jun, R. Ummanni, S. V. Malhotra, Imidazole derivatives show anticancer potential NU by inducing apoptosis and cellular senescence, Med. Chem. Commun. 5 (2014) 17511760. MA [2] L. Chen, G. Li, F. Peng, X. Jie, G. Dongye, K. Cai, R. Feng, B. Li, Q. Zeng, K. Lun, J. Chen, B. Xu, The induction of autophagy against mitochondria-mediated apoptosis in D lung cancer cells by a ruthenium (II) imidazole complex, Oncotarget. 7 (2016) 80716- PT E 80734. [3] W. M. Motswainyana, P. A. 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Lacerda, Induction of Cell Cycle Arrest and Apoptosis by Ruthenium Complex cis-(Dichloro)tetramineruthenium(III) Chloride in Human Lung Carcinoma Cells A549, Biol. Trace Elem. Res. 147 (2012) 8-15. [55] E. K. Cassimere, C. Mauvais, C. Denicourt, p27Kip1 Is Required to Mediate a G1 Cell Cycle Arrest Downstream of ATM following Genotoxic Stress, PLOS ONE. 11 (2016) 1-20. 36 ACCEPTED MANUSCRIPT [56] J. Chen, Y. Zhang, G. Li, F. Peng, X. Jie, J. She, G. Dongye, Z. Zou, S. Rong, L. Chen, Cytotoxicity in vitro, cellular uptake, localization and apoptotic mechanism studies induced by ruthenium(II) complex, J. Biol. Inorg. Chem. 23 (2017) 261-275. [57] C. O. D. S. Costa, J. H. A. Neto, I. R. S. Baliza, R. B. Dias, L. F. Valverde, M. T. A. Vidal, C. B. S. Sales, C. A. G. Rocha, D. R. M. Moreira, M. B. P. Soares, A. A. PT Batista, D. P. Bezerra, Novel piplartine-containing ruthenium complexes: synthesis, cell RI growth inhibition, apoptosis induction and ROS production on HCT116 cells SC Oncotarget. 8 (2017) 104367-104392. [58] D. Vecchio, G. Frosina, Targeting the Ataxia Telangiectasia Mutated Protein in AC CE PT E D MA NU Cancer Therapy, Curr. Drug. Targets. 17 (2016) 139-153. 37 ACCEPTED MANUSCRIPT Figure legend Scheme 1: Synthetic route of complexes (1-4). Figure 1: Plots with partial atom labeling showing the cationic part of the asymmetric PT unit of (5) (a) and (6) (b) and the superposition of the compound backbones created by RI selecting the Ru atoms and the six atoms coordinated to them as homologous atom pairs SC (c). The H atoms, hexafluorophosphate anion and solvating methanol (present in (6)) were omitted for the sake of clarity. Ellipsoid plots with complete atom labeling for (5) NU and (6) are given in (Supporting material: Figure 34S). MA Figure 2: A molecular superposition of (6) (in orange) and its cis-[RuCl(4vinylpyridine)(dppb)(5,5`-Mebipy)] analogue (CCDC code BUZYIF [38] in light green) D created by selecting the Ru atoms and the six atoms coordinated to them as homologous PT E atom pairs. Hydrogen atoms are omitted for clarity. CE Figure 3: Partial packing of (6) (a) and its cis-[RuCl(4-vinylpyridine)(dppb)(5,5`Mebipy)] analogue (CCDC code BUZYIF [38]) (b) projected onto their respective ab AC planes highlighting the equivalence of their intermolecular arrays. The cationic complex and the hexafluorophosphate anion are depicted in green and blue, respectively. The black dotted circles highlight the region occupied by the 1Meim + methanol (in red) in (6) or by the 4-vinylpyridine in the analogous structure depicted in (b). Hydrogen atoms (excepted to those bind to methanol) were omitted for the sake of clarity. 38 ACCEPTED MANUSCRIPT Figure 4: Changes in the electronic absorption spectra of (3) with increasing concentrations of ctDNA. [3] = 1.00 × 10-3 M and [ctDNA] = 0 – 3.78 × 10-5 M in TrisHCl buffer (5 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 298 K. Figure 5: (A) A549 cell viability determined by MTS assay after 24h of treatment with PT different compounds at 20 µM or 40 µM. (B) Concentration-response curves of the RI complex (3), their precursor, and free 1-methylimidazole. *p < 0.05, **p < 0.01 and SC ***p < 0.001 determined using ANOVA followed by Tukey's post-test from three NU independent experiments. Figure 6: (A) Illustrative image showing the morphological features of A549 cultures MA obtained by phase contrast microscopy (60× magnification). (B) Illustrative images and quantitative data from the clonogenic assay. (C) Representative histograms obtained by D flow cytometry showing cell populations in different cell cycle phases. Brown (Sub-G1 PT E phase), pink (G0/G1 phases), green (S phase), blue (G2/M phase). (D) Dot plots from the annexin V/7-AAD assays. Viable cells (lower left quadrants), early apoptosis (lower CE right quadrants), late apoptosis (upper right quadrants), and nonviable cells (necrotic cells) (upper left quadrants). (E) Mitochondrial membrane potential assay using JC-1 as AC a fluorescent probe. CCCP was used as a positive control to reduce ΔΨm. (F) ATM activation was determined by measuring ATM phosphorylation levels at Ser1981. *p < 0.05, and ***p < 0.001 determined using ANOVA followed by Tukey's post-test from three independent experiments. 39 PT ACCEPTED MANUSCRIPT AC CE PT E D MA NU SC RI Scheme 1. 40 (a) PT ACCEPTED MANUSCRIPT (b) (c) AC CE PT E D MA NU SC RI Figure 1. 41 SC RI PT ACCEPTED MANUSCRIPT AC CE PT E D MA NU Figure 2. 42 AC CE PT E D MA NU SC RI PT ACCEPTED MANUSCRIPT Figure 3. 43 AC CE PT E D MA NU SC RI PT ACCEPTED MANUSCRIPT Figure 4. 44 AC Figure 5. CE PT E D MA NU SC RI PT ACCEPTED MANUSCRIPT 45 AC CE PT E D MA NU SC RI PT ACCEPTED MANUSCRIPT Figure 6. 46 ACCEPTED MANUSCRIPT Table 1. Crystal data and structure refinement for (5) and (6). R indices (all data) PT AC CE Largest diff. peak and hole (e.Å-3) (6) (C44H46Cl1N4P2Ru1)+ (P1F6)-.(C1H4O1) 1006.32 298(2) 0.71073 Orthorhombic Pbca a = 15.0023(3) b = 20.3898(3) c = 29.2586(6) 8950.0(3) 8 1.494 00.582 4128 3.256 to 29.787 20<=h<=20, -25<=k<=28, -40<=l<=40 191069 12270 [R(int) = 0.0532] 99.8 12270 / 0 / 552 1.129 R1 = 0.0583, wR2 = 0.1250 R1 = 0.0832, wR2 = 0.1376 0.780 and -0.642 RI SC PT E Reflections collected Independent reflections Completeness to  max. (%) Data/restraints/parameters Goodness-of-fit on F2 Final R indices [I>2(I)] NU Volume (Å3) Z Calc. Density (Mg/m3) Absorption coef. (mm-1) F(000)  range for data collection (°) Index ranges MA Formula weight Temperature (K) Wavelength (Å) Crystal system Space group Unit cell dimensions (Å) D Empirical formula (5) (C42H42Cl1N4O1P2Ru1)+ (P1F6)946.22 298(2) 0.71073 Orthorhombic Pca21 a = 17.0144(3) b = 15.0566(3) c = 16.6356(3) 4261.69(14) 4 1.475 0.605 1928 3.426 to 29.771 -23<=h<=23, -20<=k<=21, -22<=l<=21 99970 11162 [R(int) = 0.0553] 99.7 11162 / 1 / 514 1.026 R1 = 0.0409, wR2 = 0.0863 R1 = 0.0587, wR2 = 0.0944 0.524 and -0.315 47 ACCEPTED MANUSCRIPT Table 2. Main IR frequencies (cm-1) of 1-methylimidazole and complexes (1-4). 1Meim 2 3 4 3112 3126 3128 3132 3126 1649 1629 1622 1624 1625 1521 1535 1533 1535 1535 1286 1280 1282 1284 1282 1232 1230 1236 1232 1230 827-748 816-741 827-741 816-740 814-741 520-507 519-507 518-507 520-507 489 485 489 487 295 Abbreviations: s, strong; m, medium; w, weak; b, broad. AC CE PT E D MA NU SC RI ν (C-H) (w) ν (C=N) (bs) ν (C=C) (s) ν (C-N)arom (s) ν (C-N)alif (s) δ (C-H) (s) (Ru-P) (m) (Ru-N) (w) (Ru-Cl) (w) 1 PT Assignation 48 ACCEPTED MANUSCRIPT Table 3. Selected bond lengths and angles for (5) and (6). PT RI SC (6) 83.0(1) 84.0(1) 93.94(8) 170.14(8) 87.39(8) 92.19(7) 167.68(8) 88.22(3) 86.05(3) 176.91(8) 101.15(8) 93.20(3) 89.88(8) 101.23(8) 78.0(1) AC CE PT E D N3-Ru-N1 N3-Ru-N2 N3-Ru-P1 N3-Ru-P2 N3-Ru-Cl Cl-Ru-N1 Cl-Ru-N2 Cl-Ru-P1 Cl-Ru-P2 P1-Ru-N1 P1-Ru-N2 P1-Ru-P2 P2-Ru-N1 P2-Ru-N2 N1-Ru-N2 (6) 2.125(3) 2.069(3) 2.170(3) 2.338(1) 2.315(1) 2.4347(9) NU Ru-N1 Ru-N2 Ru-N3 Ru-P1 Ru-P2 Ru-Cl MA Bond (Å) (5) 2.112(4) 2.074(4) 2.176(4) 2.355(1) 2.323(1) 2.419(1) Angles (°) (5) 84.9(2) 82.5(1) 92.5(1) 175.5(1) 85.2(1) 91.2(1) 164.2(1) 86.11(4) 95.37(4) 176.4(1) 104.2(1) 92.06(4) 90.6(1) 96.2(1) 77.9(2) 49 ACCEPTED MANUSCRIPT Table 4. Stern–Volmer quenching constant (Ksv, M-1), biomolecular quenching rate constant (Kq, M−1 s−1), binding constant (Kb, M−1), number of binding sites (n), ΔG° (KJ·mol−1), ΔH° (KJ·mol−1) and ΔS° (J·mol−1·K) for the complex–HSA systems at different temperatures. ΔGº (mol∙L-1) 298 2.31 ± 0.002 5.16 ∙104 ± 1.99 1.09 ± 0.08 -26.89 310 1.73 ± 0.09 4 1.75 ∙10 ± 1.68 1.00 ± 0.05 -25.18 298 4.39 ± 0.08 1.97 ∙105 ± 1.72 1.16 ± 0.05 -30.21 310 3.41 ± 0.03 5 1.77 ∙10 ± 2.13 1.17 ± 0.07 -31.14 298 9.15 ± 0.001 1.77 ∙106 ± 1.50 1.32 ± 0.04 -35.65 310 5.78 ± 0.07 5 3.75∙10 ± 1.36 1.19 ± 0.03 -33.08 298 21.50 ± 0.003 1.71∙108 ± 1.20 1.70 ± 0.02 -46.96 17.00 ± 0.09 9 1.98 ± 0.01 -56.32 RI SC 3.09 ∙10 ± 1.14 ΔHº ΔSº -69.26 -142.18 -7.12 -77.47 -99.50 -214.27 185.48 780.00 MA 310 PT (10 mol∙L ) D 4 n (K) PT E 3 Kb -1 CE 2 Ksv 4 AC 1 Tem. NU Complexes 50 ACCEPTED MANUSCRIPT Table 5. Binding constants (Kb) and hypochromism percentage (%H) of compounds (1-4). Kb (105 M-1) %H 1 3.10 ± 0.37 7.23 2 3.21 ± 0,05 4.32 3 1.83 ± 0.04 12.02 4 0.20 ± 0.06 3.03 AC CE PT E D MA NU SC RI PT Compounds 51 ACCEPTED MANUSCRIPT Table 6. IC50 values (µM) determined from MTS data after 24 h of treatment. Compounds A549 CCD-1059Sk SI** 1Meim n.d. n.d - Complex (3) 14.65 ± 0.90 35.68 ± 0.79 2.39 Precursor n.d. n.d - Cisplatin* 59.54 ± 5.45 74.25 ± 5.26 1.25 AC CE PT E D MA NU SC RI PT * Cisplatin was used as a positive control; n.d: not determined because the reduction in cell viability was not enough for determining IC50 values. ** Selectivity index represents the ratio between the IC50 values obtained for the normal cell line and the tumor cell line. 52 ACCEPTED MANUSCRIPT D MA NU SC RI PT Graphical abstract PT E Ruthenium(II) complexes with 1-methylimidazole as ligand were obtained. DNA and human serum albumin interactions were evaluated. The complex (3) presented higher AC CE cytotoxic activity that was correlated with pro-apoptotic potential. 53 ACCEPTED MANUSCRIPT Highlights  Novel ruthenium(II) complexes containing 1-methylimidazole as ligand were obtained.  Interactions with DNA and human serum albumin (HSA) were performed.  Cytotoxicity profiles were determined for different tumor cell lines. AC CE PT E D MA NU SC RI PT  Complex (3) presented higher cytotoxic activity in non-small cell lung cancer. 54