Ru(II)/diclofenac-based complexes: DNA, BSA interaction and their anticancer evaluation against lung and breast tumor cells.

View Article Online Dalton View Journal Transactions An international journal of inorganic chemistry Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: K. M. Oliveira, J. Honorato, G. R. Gonçalves, M. R. Cominetti, A. A. Batista and R. S. Correa, Dalton Trans., 2020, DOI: 10.1039/D0DT01591A. This is an Accepted Manuscript, which has been through the Volume 46 Number 10 Dalton 1 P 4 a g M e a s r c 3 h 0 7 2 3 0 - 1 3 7 412 Royal Society of Chemistry peer review process and has been accepted for publication. Transactions Accepted Manuscripts are published online shortly after acceptance, An international journal of inorganic chemistry rsc.li/dalton before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. 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In no event ISSN 0306-0012 shall the Royal Society of Chemistry be held responsible for any errors CDNan o- O dHuM eg tetlM ea llrs u U o r Wc N eyn .I cy CSllit A ce c Tp act hI ai O o erbn n N e s en ( teX a sH l.t +a, bXil i=z eSd, Spea,r Teen)t sulfenyl, selenenyl, or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/dalton Page 1 of 11 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins ARTICLE Ru(II)/diclofenac-based complexes: DNA, BSA interaction and their anticancer evaluation against lung and breast tumor cells Received 00th January 20xx, Katia M. Oliveiraa,†,*, João Honoratob,†, Guilherme R. Gonçalvesa, Marcia R. Cominettic, Alzir A. Accepted 00th January 20xx Batistab, Rodrigo S. Correaa,* t p DOI: 10.1039/x0xx00000x Ruthenium(II) diclofenac-based complexes of the general formula [Ru(dicl)(P-P)(bpy)]PF [dicl = diclofenac, bpy = 2,2’- i 6 r bipyridine, and P-P = 1,4’-bis(diphenylphosphino)butane (dppb) (1), 1,2’-bis(diphenylphosphino)ethane (dppe) (2), 1,3’- c bis(diphenylphosphino)propane (dppp) (3) or 1,1’-bis(diphenylphosphino)ferrocene (dppf) (4)] are synthesized. The s complexes (1 - 4) are characterized by elemental analyses, infrared, NMR, and UV-Vis spectroscopy and (3) and (4) by single u crystal X-ray diffraction. The DNA binding of the complexes (1 - 4), studied by circular dichroism (CD) and Hoechst 33258 n staining assay, indicates their binding with the minor grooves. The complexes interact with BSA with binding constants (K) b in the range of 2.5 × 103 - 5.5 × 104 M1. The complexes exhibit high cytotoxicity against the tumors cell lines A549, MDA- a MB-231 and MCF-7 with IC values ranging from 0.56 to 15.28 μM. The complexes are more selective for the hormone M 50 dependent MCF-7 breast tumor cell line and the complex (1) is the most potent one. The study demonstrates the anticancer activity of ruthenium(II)/diclofenac-based complexes. d e ibuprofen5. These complexes displayed IC values in the range of 5 50 Introduction to 47 µM against hepatocellular and breast cancer cells, in which the t p complex with ibuprofen was the most active one4. The neutral Over the last couple of decades research towards the discovery organometallic complex [Ru(η6-p-cymene)(diclofenac)Cl] also e of metal complexes with antitumor properties has been increasing displays promising antiproliferative activity against three different c since the serendipitous discovery of the anticancer activity cisplatin. cell lines (lung cancer cell line A549, breast cancer cell line MCF-7 and A structure-activity relationship approach has led to the c cervix cancer cell line HeLa)6. Recent reports on this class of development of new platinum-based complexes such as carboplatin, A compounds demonstrated that the ruthenium/arene/diclofenac oxaliplatin, and nedaplatin which are used in clinic as complex displays cytotoxicity against MCF-7 cells with an IC value 50 chemotherapeutics for the treatment of different types of cancer. of 25 µM7. Besides ruthenium, diclofenac complexes of Zn(II),8,9 s Despite the benefits of platinum compounds they have caused a Ag(I),10,11 Cu(II),12 Cd(II),13 Co(II), and Mn(II)14,15 have been studied for n number of side effects limiting their clinical applications1,2. their antibacterial/antioxidant properties. Cobalt-diclofenac o An interesting methodology used for designing new bioactive d- complexes also act as catechol oxidase and carbonic anhydrase block metal compounds with anticancer properties is based on i inhibitors16,17. t preparing coordination compounds bearing ligands that already In the last years, we also have used the strategy of combining c present some biological activity3. In this way, non-steroidal anti- active ligands with metal ions, based on their potential synergistic a inflammatory drugs (NSAIDs) which are largely used in clinic due to effects. Four classes of ligands have been used by us, in order to their antipyretic, antiarthritic, analgesic, and anti-inflammatory s obtain active ruthenium complexes containing nucleobases activities4 have been frequently used as ligands to synthesize active n derivatives18, natural products19, amino acids20, and organic drugs21. complexes. For example, promising antitumor results were obtained Metal complexes containing NSAIDs have shown antitumor a for [Ru(L)(dppm) ]PF complexes, where dppm = 1,1’- 2 6 properties, leading to cell death by apoptosis22. Therefore, as part of r bis(diphenylphosphine) methane and L is diclofenac (dicl) or our ongoing effort to obtain new active complex with interesting T structural/biological properties, here, we use diclofenac (dicl) as main ligand to obtain several ruthenium complexes exhibiting potent n a.Departamento de Química, ICEB, Universidade Federal de Ouro Preto (UFOP), CEP anticancer activities. The synthesis, characterization, DNA, BSA o 35400-000, Ouro Preto-MG, Brazil.*kmoliveiraq@gmail.com (K. M. Oliveira); interaction and cytotoxicity are reported herein. *rodrigocorrea@ufop.edu.br (R. S. Correa). Tel.: +55 31 35591229. t b.Departamento de Química, Universidade Federal de São Carlos (UFSCar), Rodovia l Washington Luiz, KM 235 CP 676, CEP 13561-901, São Carlos – SP, Brazil. a c.Departamento de Gerontologia, Universidade Federal de São Carlos (UFSCar), Material and Methods D Rodovia Washington Luiz, KM 235 CP 676, CEP 13561-901, São Carlos – SP, Brazil. † K. M. O and J.H contributed equally. Materials Electronic Supplementary Information (ESI) available: Fig. S1 (UV-Vis spectra of complexes 1 – 4). Fig. S2 (31P{1H} spectra of complexes 1 – 4). Fig. S3 – S6 (1H spectra All the reactions were carried out under argon atmosphere. The of complexes 1 – 4). Fig. S7 – S10 (13C{1H} spectra of complexes 1 – 4). Fig. 11 – 14 reagents and chemicals were of analytical grade and were used as (Cyclic voltammetry and differential pulse voltammetry of ferrocene and complexes received from commercial suppliers. The RuCl .nH O, KPF , dppe 1 – 4). Fig. S15 – 16 (Crystal structures of complexes 4 and 3a). Fig. 17 (Overlap of 3 2 6 the structures of complexes 3, 3a and 4). Fig. 18 (Crystal packing of complexes 3, 3a (1,2-bis-(diphenylphosphino)ethane), dppp (1,3- and 4). Table S1 (Infrared assignment for complexes 1 - 4). Table S2 (X-ray data bis(diphenylphosphino)propane), dppb (1,4- collections and refinement details of complexes 3, 3a and 4). Table S3 (Torsion bis(diphenylphosphino)butane), dppf (1,1’- angles of complexes 3, 3a and 4). CCDC 2000312 (3), 2000313 (3a) and 2000314 (4). bis(diphenylphosphino)ferrocene), bpy (2,2’-bipyridine), sodium See DOI: 10.1039/x0xx00000x Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 2 of 11 ARTICLE Journal Name diclofenac (dicl), Calf-Thymus DNA (CT-DNA) and Bovine Serum 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) (d, 51.57; 32.08 /2J = 2 2 Albumin (BSA) were purchased by Sigma-Aldrich. All precursor 56.70 Hz). 1H NMR (400 MHz, DMSO-d , 298 K): (ppm) 8.41 – 5.76 6 complexes were synthesized as described in the literature23,24. (m, an overlap of aromatic protons of phenyl groups of dppp (20H), bpy(8H) and dicl(8H)), 3.07 – 1.97 (m, 6H, chain aliphatic of dppp and 2H of dicl). Physical measurements [Ru(dicl)(dppf)(bpy)]PF (4): Elemental analysis for 6 [C H Cl F FeN O P Ru]: exp. (calc) (%) C, 55.96 (55.65), H, 3.83 58 46 2 6 3 2 3 The NMR experiments (31P{1H}, 1H and 13C{1H}) were recorded on (3.70), N, 3.59 (3.36). IR (ATR) v/cm: 3333, 1523, 1432, 1092 and Bruker Avance III spectrometer with a 5 mm internal diameter 828. 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) (d, 54.38; 2 2 indirect probe with ATMATM (Automatic Tuning Matching). Elemental 47.41/2J = 30.78 Hz). 1H NMR (400 MHz, DMSO-d , 298 K): (ppm) 6 analyses were carried out on an CHNS Element Analyzer (Thermo 8.52 – 6.06 (m, 36 H, an overlap of aromatic protons of phenyl groups t p Scientific, Fisons EA-1108 model). UV-vis spectra were recorded of dppf (20H), bpy(8H) and dicl(8H), 4.88 – 4.43 (m, 8H, aromatic C- using a Genesys 10S UV-Vis Spectrophotometer (Thermo Scientific). H ferrocene of dppf), 2.76 – 2.51 (m, 2H of CH of dicl). i 2 r Fluorescence spectra were measured on a SpectraMax i3 (Molecular c Devices). The electrochemical studies were recorded using a BAS X-ray structures determination s model 100B electrochemical analyzer employing a three-electrode u configuration consisting of an Ag/AgCl (0.10 M TBAP in CH 2 Cl 2 ) Single-crystals of the complex (3) were obtained by slow reference electrode and Pt disc working and auxiliary electrodes. The n evaporation of the acetone-methanol (1:1 v/v) solution with one voltammograms were recorded using solutions of the complexes in drop of water. In addition, crystals of this complex (named 3a) were a CH Cl containing 0.10 M tetrabutylammonium perchlorate (TBPA) 2 2 also obtained from DMSO solution used in the study that evaluated M (Fluka Purum) as supporting electrolyte at room temperature at the the stability of the complex. Crystals of complex (4) were obtained scan rate of 50 mV s. Differential pulse voltammetry by the diffusion of ethyl ether into a dichloromethane solution. X-ray measurements: scan rate = 20 mV s–1, amplitude = 50 mV and d diffraction data were collected in a Mach3 diffractometer with an modulation time = 50 ms. Under the conditions mentioned ferrocene APEX II CCD area detector and graphite-monochromatized radiation e electrochemical standard undergoes oxidation at +0.56 V (Fig. S11, (MoK = 0.71073 Å) from an Incoatec MicroFocus IS 1.0 microsource t Supplementary Material). equipped with a Helios MX Incoatec focusing optics. The structure of p the complexes were solved by direct methods and the models were e Synthesis of complexes refined by full-matrix least-squares on F2 by means of SHELXL 201825. c Anisotropic displacement parameters were applied for ruthenium, [Ru(dicl)(dppb)(bpy)]PF (1). To a solution of cis-[RuCl (dppb)(bpy)] c 6 2 nitrogen, phosphorus, carbon and oxygen atoms. Hydrogen atoms (0.05 g, 0.066 mmol) in CH 2 Cl 2 (20 mL) and CH 3 OH (10 mL) was added were located by Fourier maps and constrained at geometrically A sodium diclofenac (0.033 g, 0.099 mmol) followed by KPF (0.024 g, 6 calculated positions and refined with the appropriate riding model 0.133 mmol) as solid and the solution was kept under argon considering Uiso(H) = 1.2Ueq (Caromatic/Cmethylene). Figures were s atmosphere and reflux overnight. The volume of the solution was prepared using Mercury 4.3.126. n reduced to about 3 mL in a rotary evaporator and the yellow solid that precipitated upon the addition of water (~ 1 mL) was filtered, o Cell culture and MTT assay washed with distilled water and ethyl ether, and dried under i vacuum. Similar procedure was employed for the synthesis of the t The cell lines used in this study were the human lung A549 (ATCC: c complexes (2 – 4) using the precursor complexes cis- CCL-185), non-tumor lung MRC-5 (ATCC: CCL-171), human breast [RuCl (dppe)(bpy)], cis-[RuCl (dppp)(bpy)] and cis- a 2 2 MDA-MB-231 (ATCC: HTB-26) and MCF-7 (ATCC: HTB-22) and non- [RuCl (dppf)(bpy)], respectively. s 2 tumor breast cells, MCF-10A (ATCC: CRL-10317). The cell lines A549, MRC-5 and MDA-MB-231 were maintained in DMEM medium and n [Ru(dicl)(dppb)(bpy)]PF (1): Elemental analysis for MCF-7 cells in RPMI medium. Both media were supplemented with a 6 streptomycin and penicillin (100 g mL), L-glutamine (2 mM) and [C H Cl F N O P Ru].H O: exp. (calc) (%) C, 54.35 (54.70), H, 4.15 r 52 46 2 6 3 2 3 2 (4.24), N, 3.53 (3.68). IR (ATR) v/cm: 3256, 1502, 1448, 1090 and fetal bovine serum (10%). MCF-10A cells were cultivated in T DMEM/F12 medium containing horse serum (HS) 5%, EGF (0.02 mg 838. 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) (d, 49.03; 47.76 2 2 mL), hydrocortisone (0.05 mg mL) and insulin (0.01 mg mL). All n /2J = 34.02 Hz). 1H NMR (400 MHz, DMSO-d , 298 K): (ppm) 8.21 – 6 the cells were kept at 37 C, in humidified incubator with 5% CO 5.85 (overlapped signals, 36H aromatic hydrogen of dppb (20H), bpy 2 o atmosphere. (8H) and dicl (8H)), 2.87 – 1.48 (m, 8H chain aliphatic of dppb and 2H t of dicl). The cytotoxic activity of ruthenium complexes was analyzed by l a MTT assay. For that, the cells (1.5 × 104 cell/well) were seeded in 200 [Ru(dicl)(dppe)(bpy)]PF (2): Elemental analysis for 6 L of medium in 96-well plates and incubated at 37 C, in 5% CO D [C H Cl F N O P Ru]1/2C H : exp. (calc) (%) C, 55.84 (55.89), H, 2 50 42 2 6 3 2 3 6 14 4.61 (4.34), N, 3.69 (3.69). IR (ATR) v/cm: 3381, 1520, 1434, 1105 atmosphere for 24 h. Next, the ruthenium complexes (50 to 0.012 M) were added and the plates were incubated again for 48 h. The and 833. 31P{1H} NMR (162 MHz, CH Cl , 298 K): (ppm) (d, 84.06; 2 2 complexes were solubilized in DMSO, and the maximum percentage 76.98 /2J = 29.16 Hz). 1H NMR (400 MHz, DMSO-d , 298 K): (ppm) 6 of DMSO used in the assay was 0.5%. Thereafter, the medium was 8.35 – 5.33 (m, overlapped signals, 20H of dppe, 8H of bpy and 8H of removed, MTT solution (0.5 mg mL, 50 L) was added and after 3 h dicl), 3.14 – 1.66 (m, 4H of dppe and 2H of dicl). of incubation, the formazan crystals were solubilized by isopropanol. [Ru(dicl)(dppp)(bpy)]PF (3): Elemental analysis for 6 The absorbance was registered using an automated microplate [C H Cl F N O P Ru]: exp. (calc) (%) C, 55.58 (55.19), H, 4.04 (4.00), 51 45 2 6 3 2 3 reader at 570 nm. N, 4.08 (3.79). IR (ATR) v/cm: 3256, 1506, 1434, 1094 and 835. 2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A Page 3 of 11 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE Results and discussion Morphology assay MCF-7 cells were seeded (1  105 cells/well) in 12-well plates and Synthesis and characterization after 24 h of incubation at 37 C in 5% CO atmosphere, to allow cell 2 adhesion, the cells were treated with complex (1) at different The synthetic route employed for the preparation of new series concentrations (1 to 15 M). Alterations in the morphology were of Ru(II)/diclofenac-based complexes (1  4) is described in Scheme registered using an inverted microscope coupled with a camera 1. The novel complexes were obtained by reacting precursors (Motican 1000 – 1.3MP Live Resolution). complexes cis-[RuCl (P-P)(bpy)] and diclofenac ligand in 2 methanolic/dichloromethane solution (1:2 v/v), in which P-P means Evaluation of cell apoptosis by flow cytometry t 1,4’-bis(diphenylphosphino)butane (dppb) [for complex 1], 1,2’- p bis(diphenylphosphino)ethane (dppe) [for complex 2], 1,3’- The mechanism of cell death induced by complex (1) in MCF-7 i cells was investigated using 7-AAD annexin V and PE (BD Biosciences) bis(diphenylphosphino)propane (dppp) [for complex 3], and 1,1’- r bis(diphenylphosphino)ferrocene (dppf) [for complex 4]. The c staining. For that, the MCF-7 cells (0.7  105 cells/well) were seeded in 24-well plate and after 24 h treated with complex (1) in different coordination between the diclofenac ligand and the precursors cis- s concentrations (0.67 to 5.7 M) for 24 h. The plates were [RuCl 2 (P-P)(bpy)] occurs by an exchange reaction with substitution of u centrifuged, medium collected, cells washed with ice-cold PBS and two chloride ligands by one monoanionic carboxylate of diclofenac n stained with 7-AAD annexin V and PE, according to the as bidentate (Scheme 1). a manufacturer’s instructions. The cells were analyzed using a flow cytometer BD Accuri C6 Plus. N Cl N +PF6 - M DNA binding assays P P Ru N Cl + Cl NH O O- CH2C K l2 P / F C 6 H3OH P P Ru O N d Cl O e The interaction of Ru(II)-diclofenac complexes with DNA was studied by circular dichroism (CD) and competitive displacement WherePP: t p assay using Hoechst 33258 as probe. Firstly, the CT-DNA solution was prepared by dissolution in Tris-HCl buffer (4.5 mM Tris-HCl, 0.5 mM P P P P e Fe Tris-base, 50 mM NaCl, pH 7.4). CT-DNA concentrations were P P P P c determined by UV-Vis spectrophotometry using a molar absorption c coef F f o ic r i e t n h t e o C f D D N ex A p a e t r i 2 m 6 e 0 n n t m s t ( h 6 e 6 0 sa 0 m m p o le l s 1 o d f m C 3 T c - m DN 1 A ). 2 ( 7 50 M) in the d ( p 1 p ) b d ( p 2 p ) e d ( p 3 p ) p d ( p 4 p ) f A presence or absence of ruthenium complexes at different Scheme 1. Synthetic route used to obtain the Ru(II)-diclofenac s concentrations (0 – 25 M) were prepared and incubated for 18 h at complexes. 37 C. The measurements were recorded using a spectropolarimeter n JASCO J720 in the range of 240 to 350 nm at 298 K. The nitrogen flush Complexes (1  4) are air-stable yellow solids, soluble in common o was kept constant during the analysis. organic solvents (methanol, ethanol, dimethylsulfoxide, acetone), i The competitive displacement assay was performed by and insoluble in diethyl ether, hexane and water. The formation and t monitoring the fluorescence quenching of CT-DNA-Hoechst 33258 by composition of complexes were confirmed by IR, UV-Vis, 31P{1H}, 1H, c adding the ruthenium complex. For that, the CT-DNA (125 M) was 13C{1H} NMR, cyclic voltammetry and X-ray experiments (For 3 and a incubated with Hoechst (2.5 M), and the ruthenium complexes in 4). s different concentrations (0 – 125 M) were added to the wells of an The IR spectra of the complexes show bands in the region of n opaque 96-well plate. Fluorescence spectra were recorded on a 1502–1523 cm1 and 1432–1448 cm1 for v (COO) and v(COO) of as s a SpectraMax i3 (Molecular Devices), from 300 to 500 nm by exciting diclofenac carboxylate, suggesting the bidentate coordination to the at 343 nm at 37 C. ruthenium6,28 (Table S1). According to literature, the r T variation between the asymmetrical and symmetrical frequencies of Albumin binding studies carboxylate groups [Δv = v (COO) - v(COO)] shorter than 165 cm1 as s n can be attributed to bidentate coordination of carboxylate28. On the The investigation of the interaction between ruthenium(II) other hand, when Δv for the carboxylate stretching vibrations are up o complexes (1 - 4) and bovine serum albumin (BSA) was carried out by to 230 cm1, this may confirm the coordination of the O-carboxylate t monitoring the fluorescence quenching of the protein in the group to the metal in a unidentate or bridge coordination mode28. l a presence of different concentrations of the complexes. For that, the Thus, since all complexes present Δv lower than 90 cm1, the ruthenium complexes in different concentrations (0–80 M) were bidentate coordination is suggested. In addition, the spectra of all D added to a BSA (2.5 M) solution in Tris-HCl buffer (pH = 7.4). Using complexes show a characteristic vibrational band of the counterion the molar absorption coefficient of BSA at 279 nm (43824 mol L PF  at the region of 828 – 838 cm, assigned to the vPF stretching 6 cm1), its concentration was determined. The samples were added to vibration. an opaque 96 wells plate and the fluorescence was monitored on a The electronic spectra for complexes (1 – 4) show strong SpectraMax i3 (Molecular Devices) instrument at 298 and 310 K by absorption bands in the UV region around 300 nm assigned to exciting at 270 nm. intraligand transitions IL (π→π*) that involve the phosphine, diclofenac and bipyridine ligands23. Also, the band in the visible region, ranging from 420 to 500 nm, can be attributed to a metal-to- This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3 Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 4 of 11 ARTICLE Journal Name ligand charge transfer (MLCT) transitions, occurring due to transition In order to investigate the redox behavior of the complexes 1 – 4 from Ru(dπ) to the ligand (π*). In the complex (4), the band in this the cyclic voltammograms were recorded in dichloromethane at region is partially overlapped due to the d-d transition of the Fe atom room temperature in the potential range of 0 to 1600 mV on a Pt disc of the ferrocene group29. working electrode and an Ag/AgCl reference electrode (Fig. S11 – Given that one of the goals of the present report was to evaluate S14). The complexes exhibit anodic and cathodic waves with E½ the therapeutic potential of the Ru(II)/diclofenac-based complexes values in the range of 1295 to 1350 mV for the Ru(II)/Ru(III) redox as antitumor drugs, it is important to monitor their hydrolysis in couple33 (Figure 2; Table 1). The precursor complexes cis-[RuCl (P- 2 DMSO. Thus, the stabilities of complexes (1 – 4) in DMSO solutions at P)(bpy)] present E½ values in the range of 640 to 700 mV (Table 1). room temperature were monitored by recording the UV-Vis spectra The chloride ligands are good -and -donors and the oxygen donors at 0, 24, 48, and 72 h time points (DMSO). As a result, no changes of the carboxylate group of diclofenac have a weak electronic donor were observed in the spectra, indicating that all complexes are stable character leading to a decrease in the electron density on ruthenium t in DMSO, as showed by UV-Vis spectra (Fig. S1). upon coordination with diclofenac. Consequently, the metal center p The 31P{1H} NMR spectrum of the precursor complex cis- becomes electron deficient, shifting the oxidation and reduction i [RuCl (dppb)(bpy)] in CH Cl exhibits two doublets at 42.19 and 30.43 potentials to more positive region. The same behavior was observed r 2 2 2 ppm (2J = 32.64 Hz) typical of AB spin system. The complex for bioactive ruthenium complexes containing naphthoquinones and c P–P [Ru(dicl)(dppb)(bpy)]PF (1) also shows the AB spin system with two cinnamic acid19,34. Further, Ru(II) complexes with promising s 6 doublets at 49.03 and 47.76 ppm (Figure 1, Table 1). This indicates anticancer properties are found to display E 1/2 values for the RuII/RuIII u the non-equivalence of the phosphorus donors of the phosphinic redox couple at > 1000 mV35,36. n ligands and the formation of an unsymmetrical complex (see Table 1). This behavior is expected for phosphorus trans to the oxygen a donors of the carboxylate group and phosphorus trans to the M nitrogen donors of the bpy ligand30. The complexes 2 – 4 also display two doublets for the P-donors with higher chemical shift values than d that of the respective precursor complex as a result of the replacement of the two chloride ligands (σ- and π-donor) by a e carboxylate group of the diclofenac ligand. Complex (2) with the t dppe ligand presents chemical shifts higher than that of the p complexes 1, 3, and 4 (Fig. S2). The deshielding of the P-donors of the e dppe ligand in (2) is attributed to the formation of the stable planar c five-membered ring around the metal centre which provide good c directionality to electron density distribution from P to Ru thereby deshielding the P donor atoms31,32. A Figure 2. (A) Cyclic voltammogram and (B, C) differential pulse s voltammogram of complex (1). Electrolyte: 0.10 M TBAP in CH Cl ; 2 2 n Electrodes: Pt disc as working and auxiliary and Ag/AgCl, as a reference; Scan rate: 50 mV s. o i t c a s n Figure 1. 31P{1H} NMR spectrum of the complex (1), in DMSO-d . 6 a r In all compounds, the 1H NMR integrations and signal T multiplicities are in agreement with the proposed structures. The new Ru(II)/diclofenac complexes (1 – 4) show similar {1H}-chemical n shift pattern. Some aromatic H-atoms of phosphines, bpy, and diclofenac (dicl) are partly overlapped in the 1H NMR spectra (Fig. S3 o – S6). In the case of complex (1), for example, multiplets referring to t aromatic hydrogens of dppb, bpy and dicl (8.21 – 5.58 ppm), and CH l 2 a of dppb and dicl (2.78 – 1.48 ppm) can be observed. Also, the 13C D NMR spectra of complexes (1–4) are in agreement with the proposed structures (Figs. S7 – S10). The chemical shift (δ) of the C of carboxylate group occurs at around 187 ppm in the complexes, while to free diclofenac the value is 176 ppm. This can be related to the decrease in the electronic density of carbon atoms after the coordination of the carboxylate group to the ruthenium, indicating the formation of Ru(II)-diclofenac complexes. 4 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A Page 5 of 11 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE Table 1. Cyclic voltammetry and 31P{1H} NMR data for Ru(II)-diclofenac complexes and precursors Complexes ΔE p (mV) E 1/2 (mV) Ipa/Ipc  (ppm) 2J P-P (Hz) Reference [Ru(dicl)(dppb)(bpy)]PF 110 1335 4.46 49.03; 47.76 34.02 6 [Ru(dicl)(dppe)(bpy)]PF 110 1295 5.22 84.06; 76.98 29.16 6 This work [Ru(dicl)(dppp)(bpy)]PF 100 1320 4.72 51.57; 32.08 56.70 6 [Ru(dicl)(dppf)(bpy)]PF 100/70* 1350/925* 5.37/1.07* 54.38; 47.41 30.78 6 cis-[RuCl(bpy)(dppb)] 91 641.5 1.04 42.19; 30.43 32.64 2 cis-[RuCl(bpy)(dppe)] 65 693.5 0.98 67.38; 60.34 21.87 2 cis-[RuCl(bpy)(dppp)] 42 669 0.92 38.31; 30.33 42.12 37 2 cis-[RuCl(bpy)(dppf)] 64/50 653/970* 1.35/0.99* 41.35; 35.70 29.16 2 *Redox potential FeII/FeIII of dppf. t is delocalized over the -O2-Ru1-O1-C1- moiety, through the p Crystal structure determination conjugated double bonds system. The C-O bond distances found for the two symmetry-independent molecules of complex (3) are in the i r Crystals of complexes (3) and (4) were evaluated by X-ray range of 1.266-1.274 Å. Comparing the C-O distances of the complex c crystallography (Table S2, Fig. 3 and S15), confirming the molecular (3) with those ones found to diclofenac acid values of 1.308-1.316 Å s structures proposed for these compounds. In addition, crystals for C-O and 1.227-1.229 Å for C=O are observed38. This aspect is u obtained from a DMSO solution of complex (3) prepared in order to consistent with the resonant bond given that the values found to study the stability of the complex, were also analyzed by X-ray complex (3) are between the C-O and C=O bond distances. As can be n diffraction. This crystal structure, which is a DMSO solvate form, is seen, the C1-O1 bond length (1.270-1.274 Å) is slightly longer than a identified here as (3a) (Fig. S16). The crystal structure of the complex C1-O2 distance (1.266-1.267 Å) probably due to the influence of the (3) is presented in Figure 3. Due to the poor quality of the crystals of N1-H1…O1 intramolecular hydrogen bond. M complex (4), it was not possible to achieve total refinement of the The Ru-P, Ru-N and Ru-O bond lengths are in the expected range structure. Thus, determining bond lengths and angles were not observed in similar diphosphinic Ru(II)-compounds reported recently d accurate enough for meaningful discussion. However, a lot of useful by us39. The Ru1-O1 bond [2.204 Å] is slightly longer than Ru1-O2 e information was still obtained from the diffraction study, especially bond [2.159 Å], as consequence of stronger trans influence of P1 t the complex stereochemistry and connectivity with Ru(II) metal phosphorus of dppp related to N2 nitrogen of bpy. This trend is also p center. Moreover, the X-ray structure of complex (4) agree with the observed for [Ru(O-O)(PP)(NN)]+ complexes with O-O bidentate e composition suggested by other characterization techniques [31P(1H) carboxylate previously reported39–41. Intra and intermolecular NMR and IR spectroscopies]. differences among (3), (3a) and (4) are depicted in Fig S17 and S18. c c In vitro cytotoxic activity A The cytotoxic activities of Ru(II)-diclofenac complexes (1  4), s cisplatin, and diclofenac free of metal were investigated against n three cancerous cell lines from lung (A549) and breast (MDA-MB-231 and MCF-7) and two non-tumor cell lines, also from lung (MRC-5) and o breast (MCF-10A) as controls, using the colorimetric MTT assay. i According to the IC results and selectivity indexes (SI), displayed t 50 c in Table 2, complexes (1 – 4) are more cytotoxic than the free diclofenac and cisplatin drugs, used as reference. The free diclofenac a is not cytotoxic at the maximum concentration tested (100 M). The s low IC 50 values and high SI against tumor cells encouraged detailed n studies on the biological properties of this class of compounds. a Specifically, complex (1) is five times more cytotoxic against MCF-7 tumor cells, when compared to the non-tumor MCF-10A. r T Comparing the cytotoxicity of the precursor complex cis- Figure 3. Crystal structure of [Ru(bpy)(dppp)(dicl)]+ (3), showing the [RuCl 2 (dppb)(bpy)] with complex (1) against MCF-7 cells, the IC 50 n value increases 38-fold when coordinated with diclofenac. This result selected atom-labelling scheme and the 50% probability thermal ellipsoids. PF - and water molecule were omitted. shows the increase in the cytotoxicity after the complexation of o 6 diclofenac to the precursor complex. The complexes (1 – 4) show t greater cytotoxic activity against MCF-7 tumor cells than ruthenium l The heteroleptic Ru(II) complex with diclofenac exhibits a six- a complexes containing 1,1-bis(diphenylphosphine)methane (dppm) coordinated metal center that is bonded to one diphosphine (dppp), and diclofenac, reported in the literature5. Besides, complex (1) D the bipyridine (bpy) and to the monoanionic diclofenac ligand in O,O- presents a SI two times higher than that of cisplatin. bidentate mode, leading to a distorted octahedral geometry around The morphological changes of the MCF-7 cells upon incubation the metal. The oxygen atom O1 is trans coordinated to the with the complex (1) are investigated using an inverted microscope phosphorous atom P1, and the O2 oxygen atom of diclofenac ligand fitted with a camera. The incubation with concentrations of 5, 10 and coordinates trans to N2 nitrogen of the bpy (Fig. 3). The monocationic charge of the ruthenium(II)/diclofenac-based complex 15 M for 24 and 48 h resulted in the loss of adhesion and is neutralized by one PF  counterion, which presents an octahedral appearance of round cells, an indicative of cell death. In addition, a 6 decrease in cell density when the cells were treated with 1 and 2.5 geometry and fluorine disordered over two positions with 0.5 occupation site. The negative charge of the monoanionic diclofenac M, after 48 h of incubation was observed, which is consistent with This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 5 Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 6 of 11 ARTICLE Journal Name the cytotoxicity values obtained. In contrast, the control cells treated DNA and suggest the main type of interaction, we used both circular with 0.5% DMSO presented a normal density and spread morphology dichroism (CD) and competitive displacement assays. (Figure 4). The CD spectrum of CT-DNA exhibits two conventional bands, a positive at 275 nm, due to base stacking and a negative at 245 nm, due to the right-handed helicity. These bands are too sensitive towards any interaction of complex molecules with DNA. Thus, CD is the most conventional technique to monitor conformational changes of DNA in solution44. The analysis was performed employing a CT- DNA solution with fixed concentration (50 M) and the amount of ruthenium complexes (0 – 25 M) was increased continuously. The CD spectra of CT-DNA in the presence of complex (1) are show in t Figure 6(A). No significant changes in the ellipticity of the bands p (positive and negative) were observed in the presence of the i Figure 4. Effects of different concentrations of complex (1) on the complex (1), suggesting that this interaction did not induce any r c morphology of MCF-7 cells, after 24 and 48 h of incubation. conformational changes in CT-DNA structure. s Evaluation of cell apoptosis by flow cytometry u n Apoptosis, also known as programmed cell death, is an essential a physiological process for tissue homeostasis. Some special characteristics is observed during the apoptotic process, such as cell M shrinkage, chromatin condensation, protein cleavage, DNA breakdown and phagocytosis, as results of morphological and d biochemical changes42. e To investigate the capability of the complex (1) to induce cell death by apoptosis, the MCF-7 cells were treated with 0.67 – 5.7 M t Figure 6. (A) CD spectra of CT-DNA (50 M) incubated with different p of the complex (1) for 24 h and then stained with PE Annexin V-7AAD concentrations of complex (1) for 18 h at 37 C and (B) Emission and analyzed using flow cytometry. e spectra of the DNAHoechst (2.5 M  125 M, λ = 343 nm) system The percentage of apoptotic cells (Q2 and Q3 quadrants) ex c in the absence and presence of increasing concentrations of complex increase in a concentration-dependent manner after the incubation (1) (0 – 125 M). c with the complex (1) (Figure 5). At 5.7 M the percentage of A apoptotic cells significantly increase to 44.8%, as compared to 17.9 % in the control. Thus, these results suggest that complex (1) is able Additionally, competitive binding studies between CT-DNA and s to induce cell death by apoptosis, according to the observations in Ru(II)-diclofenac complexes were investigated by monitoring the morphological assay. n emission intensity of DNA-Hoechst 33258, in different concentration o of complexes. It is well known the Hoechst is able to interact with DNA through the minor groove and then to exhibit intense i t fluorescence45. After the addition of Ru(II)-diclofenac complexes a c significant decrease in fluorescence was observed with increasing a concentrations of the complexes (Figure 6B). These results indicate that the complexes may displace the Hoechst from the DNA grooves, s causing a decrease in fluorescence, indicating that the Ru(II)- n diclofenac complexes are able to interact with DNA by minor groove. a r T Figure 5. (A) Apoptotic effects of complex (1) on MCF-7 cells analyzed by flow cytometry using PE Annexin V-7AAD. Data are representative n of two individual experiments. (B) Representations of apoptotic cells (early + late stages) for different concentrations of complex (1), after o 24 h of incubation. t l a DNA interaction studies D DNA is an important carrier of genetic information that can control the cell growth and proliferation and an important target for antitumor drugs. DNA damage can prevent the process of cell replication leading to cell death. Due to its structure, DNA macromolecule presents a number of possibilities for drugs to perform intermolecular interactions, such as covalent binding, reversible groove association or intercalation43,44. In order to investigate the ability of Ru(II)-diclofenac complexes to interact with 6 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A Page 7 of 11 PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Journal Name ARTICLE Table 2. In vitro cytotoxicity results (IC values) toward cell lines studied 50 IC (M) Compounds 50 A549 MRC-5 SI1 MDA-MB-231 MCF-10A SI2 MCF-7 SI3 [Ru(dicl)(dppb)(bpy)]PF 4.35 ± 0.45 0.56 ± 0.03 0.13 1.56 ± 0.55 4.42 ± 0.27 2.83 0.87 ± 0.08 5.08 6 [Ru(dicl)(dppe)(bpy)]PF 3.59 ± 0.10 1.80 ± 0.16 0.50 3.56 ± 0.09 5.47 ± 0.36 1.54 3.09 ± 0.09 1.77 6 [Ru(dicl)(dppp)(bpy)]PF 12.49 ± 0.12 1.00 ± 0.20 0.08 3.62 ± 0.41 6.51 ± 0.24 1.80 1.53 ± 0.36 4.25 6 [Ru(dicl)(dppf)(bpy)]PF 15.28 ± 2.17 2.81 ± 0.24 0.18 12.43 ± 0.15 8.93 ± 0.45 0.72 3.21 ± 0.07 2.78 6 cis-[RuCl (dppb)(bpy)]19 ND ND -- 27.55 ± 1.84 11.67 ± 1.44 0.42 33.84 ± 0.44 0.34 t 2 p cis-[RuCl (dppe)(bpy)]19 21.26 ± 0.43 ND -- 26.06 ± 4.84 25.51 ± 0.18 0.98 8.99 ± 3.14 2.83 2 i Diclofenac > 100 > 100 -- > 100 > 100 -- > 100 -- r Cisplatin 11.54 ± 1.19 29.09 ± 0.78 2.52 2.43 ± 0.20 29.45 ± 0.85 12.12 13.98 ± 2.02 2.10 c ND = Not determined. SI1 = IC MRC-5/IC A549, SI2 = MCF-10A/IC MDA-MB-231 e SI3 = IC MCF-10A/IC MCF-7. s 50 50 50 50 50 u BSA interaction study n Albumin is an important protein for the maintenance of many The fluorescence quenching indicates that complexes (1 - 4) a physiological processes. This protein is the most abundant in the interact with BSA, through static or dynamic quenching, M circulatory system and is responsible for the transport and simultaneously or not. In order to distinguish these mechanisms of distribution of several compounds, including metal ions, steroid interaction the experiments were carried out at two temperatures. d hormones, vitamins, among others. Therefore, albumin can act as an From the fluorescence quenching results it is possible to obtain important carrier for metallodrug delivery. Thus, investigation of the parameters such as Stern-Volmer constant (K ), binding constant e sv interaction of metallodrugs with this protein has been widely (K ) and the number of binding sites (n), referent to the interaction t b explored46,47. of the complexes with BSA48. According to Stern-Volmer equation: p Bovine Serum Albumin (BSA) macromolecule exhibits high F /F = 1 + K [Q] = 1 + k τ [Q], where F is the fluorescence intensity e 0 sv q 0 0 structural homology with Human Serum Albumin (HSA) and, because of BSA free of complexes, F is fluorescence intensity of BSA in c of that, is widely used as the model protein to study the interaction presence of complexes (quencher), K is the Stern-Volmer constant, sv c between serum albumin and complexes. The BSA-compound [Q] is concentration of complexes, k is BSA-quenching constant and q A interaction study is based on the intrinsic fluorescence of tryptophan τ is the average lifetime of BSA without quencher (~ 10 s)48, the 0 and tyrosine residues47. The fluorescence spectra were recorded in parameters were obtained and are listed in Table 3. the absence and presence of increasing concentrations of Ru(II)- The K values increase with increasing temperature indicating s sv diclofenac complexes, at 298 and 310 K. A strong emission band at that the dynamic quenching may be the associated mechanism. n 350 nm (λ = 270 nm) in absence of complexes was observed. The Dynamic mechanism involves the interaction between the ex o fluorescence intensities decrease gradually when concentrations of fluorophore and the quencher (complexes) in the transient excited complexes increase (Figure 7A). state and the increase of the temperature favors this process. i t However, the k values are greater than the maximum value for c q dynamic quenching (2 × 1010 L M s, indicating the occurrence of a static mechanism in this process. Thus, both dynamic and static s mechanism may be occurring in the interaction between the n complexes and BSA. a r T n o t l a D Figure 7. (A) Fluorescence spectra of BSA (2.5 M, λ = 270 nm) in ex absence and presence of increasing concentrations of complex (1) in Tris-HCl buffer (pH 7.4) and 10% of DMSO. (B) Stern-Volmer plot and (C) Plot of log[(F -F)/F] vs. log [Q], for complex (1), at 310 K. 0 PPlleeaassee ddoo nnoott aaddjjuusstt mmaarrggiinnss .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A PleasDe adltoo nno Ttr aandsjaucstti omnasrgins Page 8 of 11 ARTICLE Journal Name Table 3. The values of K , k , K , n and thermodynamic parameters for Ru(II)-diclofenac complexes sv q b Complexes T (K) K sv ×103 (M-1) k q × 1011 (M-1 s-1) K b × 103 (M-1) n ∆H (kJ mol) ∆S (J mol K ∆G (kJ mol) 298 (6.03 ± 0.22) 6.03 (2.46 ± 0.59) 0.89 [Ru(dicl)(dppb)(bpy)]PF 6 310 (6.71 ± 0.28) 6.71 (3.04 ± 0.26) 0.93  98.94 267.11  19.34 289 (13.2 ± 0.4) 13.2 (55.0 ± 9.4) 1.15 [Ru(dicl)(dppe)(bpy)]PF 6 310 (14.4 ± 0.1) 14.4 (55.7 ± 3.5) 1.15  80.57 179.63  27.04 298 (17.8 ± 1.1) 17.8 (49.4 ± 1.5) 1.11 [Ru(dicl)(dppp)(bpy)]PF 6 310 (18.0 ± 0.9) 18.0 (54.4 ± 6.6) 1.12 10.34 55.17  26.78 298 (5.23 ± 0.60) 5.23 (4.36 ± 0.29) 0.95 [Ru(dicl)(dppf)(bpy)]PF 6 310 (5.42 ± 0.39) 5.42 (3.24 ± 0.41) 0.91  33.04  41.21  20.76 t p i r c The binding constant (K ) was obtained using the equation: promising in depth investigation using additional in vitro and in b log[(F -F)/F] = logK + nlog[Q], by plotting log[(F -F)/F] versus log[Q]. vivo assays. s 0 b 0 The K values (Table 3) suggest an efficient interaction between u b complexes and BSA and the n values implies in an interaction by only Conflicts of interest n one binding site. These values are similar to those found for other There are no conflicts to declare. ruthenium complexes in the literature18. Nevertheless, these values a are lower than that obtained for cobalt-diclofenac complexes (105- M Acknowledgements 106 M)14 and Ru(II)/arene/diclofenac complexes (104-107 M)6. However, this interaction may be enough for the BSA to be able to d carry the Ru(II)-diclofenac complexes. The authors would like to thank the financial support of e The interaction between complexes and BSA can occur through Brazilian Research Agencies: FAPEMIG, FAPESP, CNPq and different types of molecular interactions, such as hydrogen bonds, CAPES. This study was financed in part by the Coordenação de t p electrostatic, van der Waals or hydrophobic interactions. To Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - distinguish which of these types of interactions are occurring, Finance Code 001. K.M.O. is supported by a postdoctoral e thermodynamic parameters such as enthalpy (∆H), entropy (∆S) and fellowship grand from CAPES (PNPD program). G.R.G. thanks c Gibbs energy (∆G) were determined, using equations (1) and (2): CAPES for a Master fellowship. R.S.C. would like to thank the c financial support provided by PROPP/UFOP, FAPEMIG (APQ- A ln(K b1 /K b2 ) = (1/T 1 – 1/T 2 ) × ΔH°/R (1) 01674-18) and CNPq (grants 403588/2016-2 and 308370/2017- 1). We thank Dr. Juan C. Tenorio, Dr. Christian Lehmann and all ΔG° =  RTlnK = ΔH°  TΔS° (2) the staff of the Chemical Crystallographic Department from s b Max Planck Institute for Research Coal (Mülheim a.d.Ruhr- n Where K b1 and K b2 are the binding constants at T 1 (298 K) and T 2 (310 Germany) for the use of the single-crystal X-ray diffraction o K), respectively. R is the gas constant50. The thermodynamic equipment. Authors gratefully acknowledge the generous i parameters are listed in Table 3. financial support from the Universidade Federal de Ouro Preto t As a result, the negative ∆H and ∆S values for complexes (1), (2) (UFOP) and the “Laboratório Multiusuário de Caracterização de c and (4) reveal that the main source of ΔG values are derived from a Moléculas”/UFOP for the NMR facilities. We would like to thank a large contribution of ΔH term with a little contribution from factor Professor Dr. Jason Guy Taylor from Federal University of Ouro s ΔS, suggesting the occurrence of van der Waals and hydrogen bond Preto (UFOP) for help in improving the English of the n interactions. On the other hand, the negative ∆H and positive ∆S manuscript. values for complex (3) suggest an electrostatic interaction between a complex (3)/BSA. All compounds exhibit negative values of ∆G, References r indicating that the interaction of Ru(II)-diclofenac complexes with T BSA occurs by spontaneous process. 1 C. C. 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Loganathan, E. Suresh, A. Riyasdeen, M. A. Akbarsha and M. Palaniandavar, Dalton Trans., 2014, 43, 1203–19. t p i r c s u n a M d e t p e c c A s n o i t c a s n a r T n o t l a D 10 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A Page 11 of 11 Dalton Transactions Ru t p i r c s u n a M d The well-known anti-inflammatory diclofenac drug can form new ruthenium e t p compounds with promising anti-tumor properties. e c c A s n o i t c a s n a r T n o t l a D .MP 35:21:21 0202/51/8 no ytisrevinU yendyS nretseW yb dedaolnwoD .0202 tsuguA 51 no dehsilbuP View Article Online DOI: 10.1039/D0DT01591A