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Journal of Coordination Chemistry

ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20

Heteroleptic ruthenium bioflavonoid complexes:from synthesis to in vitro biological activity

Adnan Zahirović, Emira Kahrović, Marina Cindrić, Sandra Kraljević Pavelić,Mirsada Hukić, Anja Harej & Emir Turkušić

To cite this article: Adnan Zahirović, Emira Kahrović, Marina Cindrić, Sandra KraljevićPavelić, Mirsada Hukić, Anja Harej & Emir Turkušić (2017): Heteroleptic ruthenium bioflavonoidcomplexes: from synthesis to in vitro biological activity, Journal of Coordination Chemistry, DOI:10.1080/00958972.2017.1409893

To link to this article: https://doi.org/10.1080/00958972.2017.1409893

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Accepted author version posted online: 26Nov 2017.Published online: 06 Dec 2017.

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Journal of Coordination Chemistry, 2017https://doi.org/10.1080/00958972.2017.1409893

Heteroleptic ruthenium bioflavonoid complexes: from synthesis to in vitro biological activity

Adnan Zahirovića, Emira Kahrovića, Marina Cindrićb, Sandra Kraljević Pavelićc, Mirsada Hukićd, Anja Harejc and Emir Turkušića

afaculty of science, department of Chemistry, university of sarajevo, sarajevo, Bosnia and herzegovina; bfaculty of science, department of Chemistry, university of Zagreb, Zagreb, Croatia; cdepartment of Biotechnology, Centre for high-throughput technologies, university of rijeka, rijeka, Croatia; dinstitute for Biomedical research and diagnostics nalaZ, sarajevo, Bosnia and herzegovina

ABSTRACTHeteroleptic ruthenium(II) bioflavonoid complexes of quercetin, morin, chrysin, and 3-hydroxyflavone were prepared and their interaction with CT DNA and BSA along with antioxidant and in vitro anticancer and antimicrobial activities was investigated. The formulation and characterization of complexes were achieved through elemental and thermal analysis, mass spectrometry, 1H NMR spectroscopy along with infrared, electronic absorption, and emission spectroscopy as well as square-wave voltammetry, and magnetic and conductivity measurements. Ruthenium(II) is octahedrally coordinated in cationic complex species to two bidentate diimine ligands (2,2′-bipyridine or 1,10-phenanthroline) and one bidentate monobasic flavonoid ligand through 3,4-site of quercetin, morin, and 3-hydroxyflavone or 4,5-site of chrysin. Complexes bind CT DNA by intercalation and binding constants comparable to ethidium bromide or 10 times higher. Binding constants of complexes to BSA were several times higher compared to ibuprofen and diazepam, and suggest that the complexes have a strong affinity to BSA. Antioxidant activity tests showed that the complexes are more potent in terms of radical inhibition compared to the parent flavonoids. Cytotoxic testing revealed that the Ru(II) complex of quercetin with 2,2′-bipyridine co-ligand has good selectivity to breast adenocarcinoma, while the complex of 3-hydroxyflavone with 2,2′-bipyridine co-ligand showed strong cytotoxicity toward all tested cell lines with IC50 ∼ 1 μM. All complexes showed moderate activity toward Acinetobacter baumannii, while the Ru(II) complex of 3-hydroxyflavone with 2,2′-bipyridine showed excellent activity toward MRSA and Candida albicans.

© 2017 informa uK limited, trading as taylor & francis Group

KEYWORDSruthenium; flavonoid; biomolecules; anticancer; antimicrobial

ARTICLE HISTORYreceived 18 september 2017 accepted 18 november 2017

CONTACT emira Kahrović emira@pmf.unsa.ba, emira_kahrovic@yahoo.com supplemental data for this article can be accessed at https://doi.org/10.1080/00958972.2017.1409893.

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1. Introduction

Over the last few decades, growing interest in the synthesis of novel biologically active compounds has not waned. The severe side-effects attached to conventional platinum chem-otherapeutics and emerging bacterial resistance to antibiotics demand new solutions to the treatment of disease. Some ruthenium compounds that have substantially lower cyto- and genotoxicity, different ligand exchange kinetics and transport, activation mechanisms, and stronger activity are prominent candidates [1–3]. The focus of research on metal-based drugs is oriented mostly to synthetic organic ligands, which are empirically confirmed as effective in the treatment of diseases. Nowadays, because molecular targets are known, well-estab-lished synthesis–activity relationship governs synthetic strategy design. Yet despite the fact that flavonoids and some metal–flavonoid complexes showed prominent properties, the synthesis and activity of ruthenium–flavonoid complexes have not been investigated [4–6].

Flavonoids are a diverse class of polyphenolic phytochemicals that belong to phenylpro-panoids; they formally are derived from 2-phenyl-4H-chromen-4-on. Their primary role lies in the acclimatization of plants and floral pigmentation; however, they may have significance as part of the photosystem I [7, 8]. The positive effect of flavonoids on health such as ROS (reactive oxygen species) and RNS (reactive nitrogen species) inhibition, antimicrobial, and antineoplastic activity along with their role in disease prevention are well documented [8–11].

From a chemical point of view, the synthesis and structure of ruthenium–bioflavonoid complexes are intriguing because of the strong antagonism between ruthenium and bio-flavonoids. There are at least five reasons for this antagonism: (i) the pronounced catalytic ability of ruthenium to induce decomposition in organic molecules under relatively mild reaction conditions; (ii) the stability of ruthenium in two neighboring oxidation states + 2 and + 3 makes it suitable for redox reactions that generate radicals (Fenton and Haber-Weis reaction) and can, thus, deactivate flavonoids and even trigger their oxidative decomposition; (iii) the soft character of Ru(II), which does not accommodate binding hard O-donor flavo-noids; (iv) flavonoids, as strong antioxidants, easily undergo oxidation or oxidative decom-position in contact with metal ions that do not have stabile electron configurations; (v) phenolic groups of flavonoids are weakly acidic (pKa1 ∼ 6–8) and become highly unstable upon deprotonation in an alkaline solution. Strong delocalization of cinnamoyl system causes the keto-group to become highly deactivated. A combination of these effects makes it hard to bond flavonoids to metal ions.

From a biological standpoint, the coordination of biologically active ligands with metal ions regularly results in a several-fold increase in activity. Some of the flavonoids, especially catechins, showed promising antimicrobial activity toward Vibrio cholera, Shigella, and MRSA [12, 13], while flavan-3-ol demonstrated an inhibiting effect on the human immunodeficiency viruses HIV-1 and HIV-2 [14, 15]. The antitumor activity of flavonoids could be associated with the inhibition of mutagenic p53, tyrosine kinase, and the expression of Ras protein [16–19]. Moreover, flavonoids are recognizable biologically as part of the daily human diet and have positive health effects.

Previous studies of metal–flavonoid complexes were mostly focused on heteroleptic complexes of 3-hydroxyflavone or 3-aminoflavone and (poly)amine/diimine ligands with divalent Cu [20–26], Zn [25, 27–30], Ni [25, 26, 29, 30], Co [25, 26, 29–33], Mn [25, 26, 29, 30],

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Fe [25, 26, 29, 34] and trivalent rare earth metal ions [35, 36]. Complexes of some polyphenolic flavonoids are also reported [37–46]. Most of the complexes were used as models of quercetin 2,3-dioxygenase or superoxide dismutase. For further reading on metal–flavonoid complexes we recommend the article by Kasprzak et al. [47].

It is evident that interest in ruthenium complexes with flavonoid-based ligands has grown over the last 5 years, yet to date there have been no extensive reports on the synthesis of ruthenium coordination compounds with polyhydroxyl bioflavonoids [48–51]. Only a limited number of papers on the biological activity of heteroleptic complexes of non-polyphenolic derivatives of flavone have been published [52–55]. Because the biological activity of flavo-noids arises from its hydroxylation, we aimed to prepare ruthenium complexes for some polyhydroxyl flavonoids. Recently, one organoruthenium(II) quercetin complex and one Ru(II)-kaempferol complex were reported [50, 51].

Here, we present a report on the synthesis and characterization of eight new heteroleptic Ru(II) complexes of bioflavonoids quercetin, morin, chrysin, and 3-hydroxyflavone with 2,2′-bipyridine and 1,10-phenanthroline as co-ligands. The interaction of complexes with CT DNA (calf-thymus DNA) and BSA (bovine serum albumin), along with their antioxidant and in vitro anticancer and antimicrobial activity, were also investigated.

2. Experimental

2.1. Materials

Most of the chemicals were commercially available and used as received, if not otherwise indicated. Calf-thymus DNA (Type I, highly fibrous) was supplied by Merck and bovine serum albumin was purchased from Sigma. Ruthenium(III) chloride hydrate (β-RuCl3∙2H2O) was recrystallized several times from conc. hydrochloric acid under dry hydrogen chloride and then dried under vacuum over conc. sulfuric acid and sodium hydroxide (iodometrically 41.7% Ru, argentometrically 43.1% Cl). The 2,2′-bipyridine (bpy) was recrystallized from hot hexane and 1,10-phenanthroline (phen) from ethanol–water 1 : 1 mixture and precipitated as monohydrate. Tetraethylammonium perchlorate precipitated from an aqueous solution of its bromide salt with sodium perchlorate and was vacuum dried. Cis-[Ru(bpy)2Cl2]∙2H2O and cis-[Ru(phen)2Cl2]∙2H2O were prepared by refluxing a dimethylformamide mixture of RuCl3 and corresponding diimine in the presence of LiCl in excess, precipitated with acetone and recrystallized from an aqueous LiCl solution under nitrogen [56]. [Ru(phen)2(CO3)]∙2H2O was prepared by refluxing an aqueous solution of cis-[Ru(phen)2Cl2]∙2H2O in the presence of sodium carbonate in excess [57]. Reagent-grade quercetin dihydrate, morin dihydrate, and chrysin were purchased from Merck. An alkaline condensation of 2′-hydroxyacetophe-none and salicylaldehyde followed by Algar-Flynn-Oyamada oxidation and recrystallization from hot hexane afforded the 3-hydroxyflavone [58].

2.2. Physical measurements

Elemental analyses of C, H, and N were performed using a Perkin Elmer 2400 Series CHNS/O analyzer. Graphite furnace atomic absorption spectroscopy was used for ruthenium deter-mination from DMSO (dimethylsulfoxide) solution on a Varian 240ZAA. High-resolution matrix-assisted laser desorption ionization – time-of-flight/time-of-flight mass spectra were

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recorded on a 4800 Plus MALDI TOF/TOF analyzer in the positive ion reflector mode in 10 – 2000 Da region using α-cyano-4-hydroxycinnamic acid matrix. 1H and 13C NMR spectra were acquired using a Bruker BioSpin GmbH instrument from DMSO-d6 solution at 400 and 100 MHz, respectively. All shifts are given in ppm relative to tetramethylsilane. NMR spectra were processed by SpinWorks 3. The largest peak of the solvolysis effect was set as the baseline. Infrared spectra were collected as KBr pellets in the 4000–400 cm−1 region with a Perkin Elmer BX FTIR. Absorption spectra were acquired in the 200–1100 nm range in meth-anol solution using a Perkin Elmer Lambda 35. Emission spectra were measured in dry ace-tonitrile in the emission region 600–900 nm with excitation at π→π* and MLCT (metal-to-ligand charge transfer) bands using a Perkin Elmer LS 55 Luminescence. Thermogravimetric analysis was carried out in Al crucibles to 600 °C with a heating rate of 10 °C min−1 in dioxygen using a Mettler-Toledo TGA/SDTA851e. A SQUID magnetometer MPMS-XL5 (Quantum Design) was employed from magnetic measurements at 300 K. Electrochemical measurements were done using a three-electrode system with a Pt as working, a Pt wire as counter and Ag/AgCl as a reference electrode via a salt bridge in 10 mM tetraethylammonium perchlorate acetone solution using an Autolab potentiostat/galvanostat (PGSTAT 12) electrochemical worksta-tion. Conductivity of 1 mM solutions was determined in acetonitrile using a Phywe conductometer.

2.3. Interaction of complexes with CT DNA and BSA

Calf-thymus DNA (Type I, highly fibrous, A260/A280 = 1.3) was purified using phenol–chloro-form–isoamyl alcohol extraction and precipitated as sodium salt (A260/A280 = 1.84). Stock solution of CT DNA was prepared prior to measurements by resuspending the solid nucleic acid in 10 mM Tris–HCl (tris(hydroxymethyl)aminomethane) buffer pH 7.42 and then was left for good hydration overnight. The concentration was determined based on the extinction coefficient 6600 M−1 cm−1 at 260 nm.

The interaction of complexes with DNA was investigated at physiological pH 7.42 in 10 mM Tris–HCl buffer. The complexes were dissolved initially in methanol and then diluted to the required concentrations. The methanol content never exceeded 1%. Spectrophotometric titration of complexes (∼5 × 10−5 M) with CT DNA (∼7 × 10−3 M) was carried out by adding μL amounts of CT DNA (0–60 μL) to the solution complex (2 mL) and acquiring spectra in the 200–700 nm region with a 5 min equilibrium time. Titration of CT DNA (∼7 × 10−5 M) with complexes (∼2 × 10−3 M) was carried out in a similar manner by monitoring absorbance within the region of maximum DNA absorption. Viscosity measurements were done using an Ubbelohde ASTM viscometer at 25 ± 0.1 °C measuring time with a digital stopwatch (± 0.01 s). The viscosity of CT DNA (∼1 × 10−4 M) was measured according to the increased concentration of the complexes (0–4.20 × 10−5 M) with an equilibrium time of 5 min; a cor-rection was made to the viscosity of buffer in the presence of complex. A fluorescence binding assay was performed by measuring the fluorescence quenching of intercalated ethidium bromide (2.40 × 10−5 M CT DNA and 6 × 10-6 M EB) in the presence of increasing concentrations of the complexes (0–7.40 × 10−6 M) recording an emission spectra in the 520–700 nm region with excitation at 497 nm.

Stock solution of BSA was prepared prior to the measurements and the concentration was determined based on the extinction coefficient 43,824 M−1 cm−1 at 280 nm. Interaction of complexes with BSA (3.40 × 10−6 M) was carried out by monitoring the fluorescence

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quenching of BSA in the presence of increasing concentrations of the complexes (0–7.40 × 10−6 M). Emission spectra were acquired in the 300–400 nm range with excitation at 280 nm.

The behavior of complexes in aqueous solution at physiological pH 7.42 in 10 mM Tris–HCl buffer was investigated spectrophotometrically. Electronic spectra of complexes (5 × 10−5 M) were collected in the 200–700 nm region every 10 min during 2 h and after 24 h.

2.4. Antioxidant activity testing

Antioxidant activity was calculated by measuring the change in DPPH (2,2-diphenyl-1-pic-rylhydrazyl) absorbance at 517 nm in the presence of increasing concentrations of complexes and ligands (0–1.50 × 10−5 M) after 30 min incubation. The IC50 values are given as concen-trations of substances that cause a 50% reduction in DPPH absorbance after 30 min. They were calculated through the linear regression of the percentage of antioxidant activity versus concentration.

2.5. In vitro anticancer and antimicrobial activity testing

The cell lines HeLa (cervical carcinoma), SW620 (colorectal adenocarcinoma, metastatic), HepG2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) were cultured as monolayers and maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin in a humidified atmosphere with 5% CO2 at 37 °C.

The panel cell lines were inoculated onto a series of standard 96-well microliter plates on day 0 at 5000 cells per well according to the doubling times of a specific cell line. Test agents were then added in five 10-fold dilutions (0.01 to 100 μM) and incubated for a further 72 h. After 72 h of incubation, the cell growth rate was evaluated by performing the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [59]. The solvent (DMSO) was also tested for eventual inhibitory activity.

The IC50 values for each compound were calculated from dose–response curves using linear regression analysis. Each test point was performed in quadruplicate in two individual experiments.

Ruthenium(II) bioflavonoid complexes were tested for antimicrobial activity toward sev-eral Gram-positive bacteria: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 19433, Streptococcus beta-hemolytic group A, Methicillin-resistant Staphylococcus aureus, Gram-negative bacteria: Klebsiella pneumoniae ATCC 1705, Acinetobacter baumannii ATCC-BAA 747, Pseudomonas aeruginosa, Escherichia coli, and fungi Candida albicans.

Antimicrobial activity was tested by measuring the inhibition zone diameter using a disk diffusion method. Pathogens were inoculated on Mueller-Hinton agar and the holes were made by sterile Durham’s tubes. An aliquot (50 μL) of DMSO solution of the tested agent (2 mg mL−1) was inserted into the drilled wells. The diameter of the inhibition zone of bac-terial growth was measured after 24 h incubation at 37 °C. Vancomycin, gentamicin, and nystatin were used as positive controls and DMSO as negative control. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined through a serial two-fold dilution technique for those compounds that showed noteworthy activity.

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2.6. Synthesis of complexes

Complexes of general formula [Ru(bpy)2La–d](OTf )∙nH2O (1a–1d) and [Ru(phen)2La–d](OTf )∙nH2O (2a–2d), where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and La–d = monobasic anion of quercetin (HLa), morin (HLb), chrysin (HLc), and 3hydroxyflavone (HLd), were prepared as described below.

2.6.1. General procedure for the preparation of complexes 1a–1dStarting complex cis-[Ru(bpy)2Cl2]∙2H2O (100 mg, 0.19 mmol) was suspended in methanol (25 mL) and silver triflate (98 mg, 0.38 mmol) was added portion-wise. The reaction mixture was stirred magnetically for 1 h protected from light at room temperature before the AgCl was filtered off. The methanol solution (1 mL) of sodium methoxide (11 mg, 0.19 mmol) was added to the reaction mixture and the resulting purple solution was stirred for an additional hour at room temperature before the flavonoid ligand was added (0.19 mmol; HLa 64 mg, HLb 64 mg, HLc 48 mg, HLd 45 mg). The reaction mixture was refluxed for 3 h and then con-centrated to small volume (5 mL) after which diethylether (85 mL) was added dropwise. Solid red-to-purple complexes were filtered off, washed with ether (3 × 5 mL), water (3 × 15 mL) and then vacuum dried over silica. Recrystallization was carried out from the methanol/ether mixture.

Compound 1a. Yield: 98 mg (54%). Anal. Calcd (%) for C36H35F3N4O15SRu: C, 45.33; H, 3.70; N, 5.87; Ru, 10.60; Found (%): C, 44.92; H, 3.20; N, 6.48; Ru, 10.38. IR (KBr) ν(cm−1), (intensity): 3399, 3200(vb,s) ν(O–H), 1644(s) ν(C=O), 1599(m) ν(C=C), 1360, 1280(m) ν(C–O), 1260(s) ν(S=O), 882, 842, 818, 798(w) δ(C–H), 763(s) δ(bpy), 638(m) δ(bpy), 517(w) ν(C–S). HR MALDI-TOF/TOF MS for C35H25N4O7Ru Found (Calcd): 715.0728 (715.0799) [M]+, 559.0157 (559.0112) [M-bpy]+, 414.0542 (414.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 204 (4.73), 245 (4.48), 293 (4.76), 362 (4.08), 500 (4.03), 877 (3.09). ΛM (S cm2 mol−1) (1 mM, MeCN): 170. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 10.80 (s, 1H, Hc4′), 10.43 (s, 1H, Hc7), 10.04 (d, 1H, J = 5.44 Hz, Ha6′), 9.55 (d, 1H, J = 5.04 Hz, Ha′6′), 8.90–8.63 (m, 5H, Ha3′, Ha′3′, Ha3, Ha′3, Ha4′), 8.40 (t, 1H, J = 8.04 Hz, Ha′4′), 8.27–8.16 (m, 2H, Ha5′, Ha′5′), 8.10–7.96 (m, 3H, Ha6, Ha4, Ha′4), 7.89–7.54 (m, 5H, Hc6′, Hc2′, Hc8, Hc6, Ha′6), 7.45–7.22 (m, 2H, Ha5, Ha′5), 7.18 (d, 1H, J = 5.32 Hz, Hc5′), 6.17 (s, 1H, Hc3′), 5.99 (s, 1H, Hc5). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 119.6, 122.8, 123.9, 124.2, 124.8, 124.9, 125.1, 125.2, 126.4, 127.3, 127.5, 127.8, 127.9, 128.4, 128.6, 137.9, 138.2, 139.0, 139.1, 149.5, 150.2, 150.5, 151.7, 153.2, 153.6, 155.7, 157.7, 157.9, 158.2.

Compound 1b. Yield: 116 mg (65%). Anal. Calcd (%) for C36H33F3N4O14SRu: C, 46.20; H, 3.55; N, 5.99; Ru, 10.80; Found (%): C, 45.70; H, 3.00; N, 6.18; Ru, 10.47. IR (KBr) ν(cm−1), (inten-sity): 3433, 3205(vb, s) ν(O–H), 1640(s) ν(C=O), 1606(m) ν(C=C), 1357, 1281(m) ν(C–O), 1259(s) ν(S=O), 841, 805(w) δ(C–H), 765(s) δ(bpy), 638(m) δ(bpy), 516(w) ν(C–S). HR MALDI-TOF/TOF MS for C35H25N4O7Ru Found (Calcd): 715.0665 (715.0799) [M]+, 559.0241 (559.0112) [M-bpy]+, 414.0153 (414.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 203 (4.66), 245 (4.48), 294 (4.82), 351 (4.02), 502 (4.00). ΛM (S cm2 mol−1) (1 mM, MeCN): 178. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 12.83 (s, 1H, Hc4′), 10.93 (s, 1H, Hc7), 10.16 (s, 1H, Hc2′), 10.04 (d, 1H, J = 4.80 Hz, Ha6′), 9.54 (d, 1H, J = 6.40 Hz, Ha′6′), 8.84 (d, 1H, J = 8.00 Hz, Ha3′), 8.77–8.65 (m, 3H, Ha′3′, Ha3, Ha′3), 8.40 (td, 1H, J = 8.08, 1.48 Hz, Ha4′, 8.25 (td, 1H, J = 7.64, 1.32 Hz, Ha′4′), 8.18 (t, 1H, 7.84 Hz, Ha5′), 8.11–7.96 (m, 3H, Ha′5′, Ha4, Ha′4), 7.89–7.68 (m, 3H, Hc8, Ha6, Ha′6), 7.60–7.38 (m, 2H, Ha5, Ha′5), 7.32–7.23 (m, 1H, Hc6′), 7.18 d (1H,

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J = 4.88 Hz, Hc5′), 6.91 (s, 1H, Hc6), 6.16 (s, 1H, Hc3′), 5.99 (s, 1H, Hc5). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 105.7, 123.9, 124.2, 124.8, 125.2, 126.1, 126.3, 126.4, 126.9, 127.3, 127.8, 127.9, 128.3, 129.5, 129.6, 132.0, 135.6, 136.6, 136.9, 137.9, 138.2, 138.4, 139.0, 139.1, 149.5, 153.2, 153.5, 154.1, 155.7, 156.4, 157.7, 157.9, 158.0.

Compound 1c. Yield: 106 mg (67%). Anal. Calcd (%) for C36H37F3N4O13SRu: C, 46.80; H, 4.03; N, 6.06; Ru, 10.94; Found (%): C, 46.51; H, 3.14; N 6.65; Ru, 10.63. IR (KBr) ν(cm−1), (intensity): 3430, 3226(vb, s) ν(O–H), 1647(s) ν(C=O), 1594(m) ν(C=C), 1363, 1284(m) ν(C–O), 1237(s) ν(S=O), 887, 846, 815(w) δ(C–H), 763(s) δ(bpy), 637(m) δ(bpy), 517(w) ν(C–S). HR MALDI-TOF/TOF MS for C35H25N4O4Ru Found (Calcd): 667.0898 (667.0952) [M]+, 511.0674 (511.0264) [M-bpy]+, 414.0120 (414.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 204 (4.80), 245 (4.53), 293 (4.82), 381 (4.24), 487 (4.16). ΛM (S cm2 mol−1) (1 mM, MeCN): 173. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 11.20 (s, 1H, Hc7), 10.94 (s, 1H, Hc3), 10.28 (s, 1H, Hc6), 10.04 (d, 1H, J = 5.72 Hz, Ha6′), 9.91 (s, 1H, Hc8), 9.54 (d, 1H, J = 6.52 Hz, Ha′6′), 9.33 (d, 1H, J = 5.00 Hz, Ha3′), 8.86–8.62 (m, 4H, Ha′3′, Ha3, Ha′3, Ha4′), 8.30–7.98 (m, 3H, Ha′4′, Hc4′, Ha5′), 7.93–7.74 (m, 5H, Ha′5′, Ha4, Ha′4, Hc3′, Hc5′), 7.64–7.52 (m, 2H, Ha6, Ha′6), 7.41–7.22 (m, 2H, Ha5, Ha′5), 6.44 (d, 1H, J = 8.24 Hz, Hc2′), 6.20 (d, 1H, J = 7.96 Hz, Hc6′). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 92.8, 94.5, 99.8, 105.4, 107.7, 108.9, 124.1, 124.2, 124.8, 124.9, 126.2, 126.6, 127.3, 127.5, 127.7, 127.8, 127.9, 128.3, 130.0, 135.6, 137.0, 137.2, 138.2, 139.0, 151.0, 153.2, 153.5, 153.7, 153.8, 156.5, 158.2, 158.4, 164.1.

Compound 1d. Yield: 107 mg (67%). Anal. Calcd (%) for C36H29F3N4O13SRu: C, 51.74; H, 3.50; N, 6.70; Ru, 12.09; Found (%): C, 51.79; H, 3.15; N, 6.95; Ru, 11.85. IR (KBr) ν(cm−1), (inten-sity): 3434(vb,s) ν(O–H), 1612(s) ν(C=O), 1601(m) ν(C=C), 1358, 1315(m) ν(C–O), 1273(s) ν(S=O), 881, 847(w) δ(C–H), 764(s) δ(bpy), 636(m) δ(bpy), 516(w) ν(C–S). HR MALDI-TOF/TOF MS for C35H25N4O3Ru Found (Calcd): 651.0980 (651.1002) [M]+, 495.1029 (495.0315) [M-bpy]+, 414.1667 (414.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 203 (4.70), 245 (4.62), 295 (4.72), 364 (4.28), 520 (4.12). ΛM (S cm2 mol−1) (1 mM, MeCN): 149. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 8.92 (d, 1H, J = 4.96 Hz, Ha6′), 8.81 (t, 2H, J = 8.60 Hz, Hc3′, Hc5′), 8.75–8.69 (m, 3H, Ha′6′, Ha3′, Ha′3′), 8.42 (d, 2H, J = 7.36 Hz, Ha3, Ha′3), 8.16 (td, 1H, J = 8.04, 1.00 Hz, Ha4′), 8.11 (td, 1H, J = 8.16, 1.28 Hz, Ha′4′), 8.00 (dd, 1H, J = 8.20, 1.20 Hz, Hc5), 7.91 (td, 2H, J = 6.48, 1.20 Hz, Ha5′, Ha′5′), 7.84 (d, 1H, Hc8), 7.80–7.75 (m, 3H, Ha4, Ha′4, Ha6), 7.71–7.68 (m, 2H, Ha′6, Hc7), 7.43–7.27 (m, 6H, Ha5, Ha′5, Hc2′, Hc6′, Hc6, Hc4′). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 118.5, 118.9, 120.7, 123.8, 123.9, 124.0, 124.2, 125.4, 126.4, 126.6, 126.7, 126.9, 127.0, 127.3, 127.6, 128.9, 129.8, 132.7, 133.5, 135.0, 135.3, 136.5, 136.9, 149.1, 150.2, 153.2, 153.4, 153.9, 155.4, 157.8, 158.1, 159.6, 160.1, 185.9.

2.6.2. General procedure for the preparation of complexes 2a–2dSolid [Ru(phen)2(CO3)]∙2H2O (100 mg, 0.18 mmol) was dissolved in methanol solution (5 mL) of triflate acid (54 mg, 32 μL, 0.36 mmol) yielding an orange-brown solution. Sodium meth-oxide (10 mg, 0.18 mmol) dissolved in methanol (2 mL) was added and the reaction mixture was stirred for 1 h at room temperature after which flavonoid ligand was added (0.18 mmol; HLa 61 mg, HLb 61 mg, HLc 46 mg, HLd 43 mg). The resulting wine-red solution was refluxed for 3 h. Upon cooling to room temperature, diethylether (85 mL) was added dropwise and the dark red-purple complexes were filtered off, washed with ether (3 × 5 mL), ice cold meth-anol (2 × 2 mL), water (3 × 15 mL), and then vacuum dried over silica. Recrystallization was carried out from the methanol/ether mixture.

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Compound 2a. Yield: 99 mg (59%). Anal. Calcd (%) for C40H27F3N4O11SRu: C, 51.67; H, 2.93; N, 6.03; Ru, 10.87; Found (%): C, 51.35; H, 2.97; N, 6.60; Ru, 10.70. IR (KBr) ν(cm−1), intensity: 3192(vb,s) ν(O–H), 1643(s) ν(C=O), 1595(m) ν(C=C), 1324, 1277(m) ν(C–O), 1259(s) ν(S=O), 880, 841(w) δ(C–H), 841(s) δ(phen), 719(m) δ(phen), 517(w) ν(C–S). HR MALDI-TOF/TOF MS for C35H25N4O3Ru Found (Calcd): 763.0232 (763.0799) [M]+, 583.0099 (583.0112) [M-phen]+, 462.0215 (462.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 205 (4.85), 223 (4.83), 266 (4.87), 401 (4.06), 483 (4.14), 877 (3.32). ΛM (S cm2 mol−1) (1 mM, MeCN): 162. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 10.78 (s, 1H, Hc4′), 10.40 (s, 1H, Hc7), 9.46 (s, 1H, Hc8), 9.34–9.32 (m, 1H, Hc6′), 8.96 (s, 1H, Hc6), 8.81 (d, 1H, J = 7.80 Hz, Hb4), 8.76 (d, 1H, J = 8.00 Hz, Hb′4), 8.46–8.15 (m, 9H, Hb7, Hb′7, Hb5, Hb′5, Hb6, Hb′6, Hb2, Hb′2, Hb9), 7.97–7.93 (m, 2H, Hb′9, Hb3), 7.77 (d, 1H, J = 1.60 Hz, Hc2′), 7.57–7.42 (m, 3H, Hb′3, Hb8, Hb′8), 6.67 (d, 1H, J = 8.40 Hz, Hc5′), 6.46 (s, 1H, Hc3′), 6.11 (s, 1H, Hc5).

Compound 2b. Yield: 140 mg (70%). Anal. Calcd (%) for C40H51F3N4O20SRu: C, 43.76; H, 4.68; N, 5.10; Ru, 9.21; Found (%): C, 43.55; H, 3.65; N, 5.57; Ru, 8.74. IR (KBr) ν(cm−1), (intensity): 3430, 3222(vb,s) ν(O–H), 1647(s) ν(C=O), 1593(m) ν(C=C), 1363, 1284(m) ν(C–O), 1235(s) ν(S=O), 886, 840(w) δ(C–H), 840(s) δ(phen), 719(m) δ(phen), 518(w) ν(C–S). HR MALDI-TOF/TOF MS for C39H25N4O7Ru Found (Calcd): 763.0797 (763.0799) [M]+, 583.0104 (583.0112) [M-phen]+, 462.1266 (462.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 207 (4.98), 221 (4.96), 266 (5.02), 391 (4.35), 470 (4.39). ΛM (S cm2 mol−1) (1 mM, MeCN): 125. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 12.96 (s, 1H, Hc4′), 11.92 (s, 1H, Hc7), 11.6 (s, 1H, Hc2′), 9.87 (s, 1H, Hc8), 9.36 (dd, 1H, J = 5.24, 1.12 Hz, Hb4), 9.32 (dd, 1H, J = 5.24, 1.12 Hz, Hb′4), 8.87–8.82 (m, 2H, Hb7, Hb7′), 8.48–8.38 (m, 4H, Hb5, Hb′5, Hb6, Hb′6), 8.30–8.16 (m, 5H, Hb2, Hb′2, Hb9, Hb′9, Hb3), 8.06–8.02 (m, 1H, Hb′3), 7.92 (dd, 1H, J = 5.40, 1.00 Hz, Hc5′), 7.56 (d, 1H, J = 8.84 Hz, Hc6′), 7.50 (td, 2H, J = 8.36, 5.40 Hz, Hb8, Hb′8), 6.49 (s, 1H, Hc6), 6.17 (s, 1H, Hc3′), 5.92 (s, 1H, Hc5). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 92.5, 94.4, 97.6, 103.6, 104.5, 105.4, 107.7, 108.9, 111.5, 125.1, 125.5, 126.1, 126.6, 126.8, 127.1, 128.0, 128.1, 128.2, 128.3, 128.6, 129.6, 129.9, 136.7, 137.2, 137.8, 138.6, 142.0, 148.0, 148.1, 149.0, 151.0, 154.8, 155.0, 155.2, 157.5, 160.8, 161.3, 163.3, 180.6.

Compound 2c. Yield: 124 mg (63%). Anal. Calcd (%) for C40H53F3N4O20SRu: C, 43.68; H, 4.86; N, 5.09; Ru, 9.19; Found (%): C, 43.53; H, 3.68; N, 5.09; Ru, 8.81. IR (KBr) ν(cm−1), (intensity): 3432, 3262(vb, s) ν(O–H), 1638(s) ν(C=O), 1599(m) ν(C=C), 1366, 1279 m ν(C–O), 1262(s) ν(S=O), 878, 841(w) δ(C–H), 841(s) δ(phen), 720(m) δ(phen), 516(w) ν(C–S). HR MALDI-TOF/TOF MS for C39H25N4O4Ru Found (Calcd): 715.0894 (715.0952) [M]+, 535.0703 (535.0264) [M-phen]+, 462.1575 (462.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 205 (4.89), 222 (4.92), 266 (5.00), 400, 476 (4.28). ΛM (S cm2 mol−1) (1 mM, MeCN): 155. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 10.14 (s, 1H, Hc7), 9.32 (dd, 1H, J = 5.20, 1.04 Hz, Hb4), 9.25 (dd, 1H, J = 5.20, 0.88 Hz, Hb′4), 8.82 (d, 1H, J = 8.16 Hz, Hb7), 8.75 (d, 1H, J = 7.84 Hz, Hb′7), 8.45–8.34 (m, 4H, Hb5, Hb′5, Hb6, Hb′6), 8.28–8.19 (m, 3H, Hb2, Hb′2, Hb9), 8.12 (dd, 1H, J = 8.16, 5.24 Hz, Hb′9), 8.02 (d, 1H, J = 5.32, Hb3), 7.97–7.93 (m, 3H, Hb′3, Hc3′, Hc5′), 7.52–7.44 (m, 5H, Hb8, Hb8′, Hc2′. Hc6′, Hc4′), 6.90 (s, 1H, Hc3), 6.12 (s, 1H, Hc8), 5.86 (s, 1H, Hc6). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 90.8, 103.6, 105.7, 107.6, 125.2, 125.4, 126.1, 126.3, 126.4, 128.1, 129.5, 130.3, 130.5, 131.0, 134.0, 134.2, 135.5, 136.0, 148.8, 149.0, 150.2, 150.7, 151.7, 151.8, 154.4, 154.8, 158.5, 159.3, 163.7, 169.1, 178.1.

Compound 2d. Yield: 121 mg (73%). Anal. Calcd (%) for C40H33F3N4O10SRu: C, 52.23; H, 3.62; N, 6.09; Ru, 10.99; Found (%): C, 52.55; H, 2.80; N, 6.22; Ru, 10.77. IR (KBr) ν(cm−1), (inten-sity): 3467(vb, s) ν(O–H), 1611(s) ν(C=O), 1584(m) ν(C=C), 1355, 1316(m) ν(C–O), 1261(s)

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ν(S=O), 875, 841(w) δ(C–H), 841(s) δ(phen), 719(m) δ(phen), 516(w) ν(C–S). HR MALDI-TOF/TOF MS for C39H25N4O3Ru Found (Calcd): 699.0989 (699.1002) [M]+, 519.0844 (519.0315) [M-phen]+, 462.1379 (462.0451) [(M-L)•]+. UV–vis (MeOH) λ(nm) (log[ε/M−1 cm−1]): 204 (4.82), 224 (4.79), 266 (4.87), 361 (4.11), 510 (4.22). ΛM (S cm2 mol−1) (1 mM, MeCN): 135. 1H NMR (400 MHz; DMSO-d6; Me4Si, 298 K), δ(ppm): 9.96 (dd, 1H, J = 5.20, 1.22 Hz, Hb4), 9.23 (dd, 1H, J = 5.24, 1.04 Hz, Hb′4), 8.80 (dd, 1H, J = 8.24, 1.04 Hz, Hb7), 8.75 (dd, 1H, J = 8.20, 0.96 Hz, Hb′7), 8.47–8.33 (m, 6H, Hc3′, Hc5′, Hb5, Hb′5, Hb6, Hb′6), 8.29–8.24 (m, 2H, Hb2, Hb′2), 8.18 (dd, 1H, J = 8.20, 5.20 Hz, Hb9), 8.09 (dd, 1H, J = 8.20, 5.28 Hz, Hc5), 8.03–7.97 (m, 3H, Hb′9, Hb8, Hb′8), 7.83 (d, 1H, J = 8.48 Hz, Hc8), 7.75 (td, 1H, J = 8.60, 1.52 Hz, Hc4′), 7.54–7.49 (m, 2H, Hc7, Hb3), 7.37–7.30 (m, 4H, Hc6, Hb′3, Hc2′, Hc6′). 13C NMR (100 MHz; DMSO-d6), δ(ppm): 120.8, 123.9, 125.3, 125.4, 125.6, 126.3, 126.6, 126.7, 126.9, 127.4, 127.8, 128.1, 128.2, 128.4, 128.9, 129.7, 130.3, 130.4, 130.5, 132.7, 133.4, 133.9, 134.2, 135.4, 135.9, 148.7, 148.9, 149.1, 150.3, 150.8, 151.6, 151.7, 153.2, 154.7, 155.1, 155.8, 186.3.

3. Results and discussion

3.1. Synthesis

Ruthenium(II) bioflavonoid complexes (1a–1d and 2a–2d) were prepared by Ag+ or triflic acid assisted substitution of chloro or carbonato ligand from cis-[Ru(bpy)2Cl2] or [Ru(phen)2(CO3)] with deprotonated flavonoid in 1 : 1 M ratio in dry methanol at reflux tem-perature. All complexes were isolated as triflate salts upon addition of ether (Scheme 1).

The formulation and characterization of the complexes were made by elemental and thermal analysis, mass spectrometry, 1H NMR spectroscopy along with infrared, electronic absorption, and emission spectroscopy as well as square-wave voltammetry, magnetic and conductivity measurements. Ruthenium(II) is octahedrally coordinated in cationic complex species by two bidentate diimine ligands and one bidentate monobasic flavonoid ligand through 3,4-site of quercetin, morin, 3-hydroxyflavone and 4,5-site of chrysin.

Flavonoids mostly bind metal ions through carbonyl oxygen and neighboring deproto-nated phenolic oxygen O3 or O5 [60–62]. Quercetin and morin, having both O3 and O5, coordinate Ru(II) through deprotonated O3 because the corresponding proton is more acidic than the one at O5 [63].

Scheme 1. reaction scheme: (i) 2 eq agotf, meoh, rt, 1 h, stirring; (ii) 1 eq naome, meoh, rt, 1 h, stirring; (iii) 1 eq hla–d, meoh, reflux, 3 h; (iv) 2 eq hotf, meoh, rt, 1 h, stirring.

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The isolated complexes were dark red-purple substances that remain stable for at least six months in solid state, soluble in dimethylformamide, acetonitrile, dimethylsulfoxide, methanol, and ethanol, moderately soluble in dichloromethane, chloroform, tetrahydrofuran and water, and insoluble in apolar organic solvents such as ether, hexane, or toluene.

3.2. Elemental, thermal and mass analysis

Chemical analysis of C, H, N, and Ru confirmed the composition and purity of the complexes. Thermogravimetric analysis was used to study the thermal stability of complexes in dioxygen upon heating. The typical thermogravimetric curve did not distinguish any decomposition steps (Figure 1). Small mass increment corresponding to oxidation of Ru(II) by dioxygen was observed below 250 °C. Above 250 °C, the organic ligands decomposed in a one-step process. Ru(II)-bioflavonoid complexes with 2,2′-bipyridine (1a–1d) showed somewhat higher ther-mal stability with decomposition temperatures in the 450–495 °C range, compared to 350–410 °C for 1,10-phenanthroline analogs (2a–2d). Simultaneous differential thermogram showed one exothermic peak above 280 °C, which suggests that the complexes decomposed before melting. Mass spectra showed typical ruthenium isotopic distribution for molecular ion [M]+ of 1a–1d and 2a–2d (Figures S3–S10). The same fragmentation pattern of [M]+ ion corresponding to a loss of flavonolato ligand [(M-L)•]+ and the loss of one diimine ligand [M-diimine]+ was found in all spectra.

3.3. Spectroscopic characterization1H NMR spectra of the complexes confirmed coordination of Ru(II) with two diimine ligands and one monobasic flavonoid ligand. A low-field aromatic region (7–10 ppm) was crowded with signals of diimine protons; however, due to overlap with flavonoid proton signals, in all cases the signal was not well-resolved. The position of the diimine proton signals is

Figure 1. tG (full line) and sdt (dotted line) curves for 1a and 2a. Inset: infrared spectra of residues.

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affected by the proximity of metal center, the van der Waals interactions and diamagnetic anisotropic effect discussed widely elsewhere [64, 65]. The most obvious features of the 1H NMR spectra of complexes were the flavonoid phenolic hydrogen singlets. Binding of querce-tin, morin, and 3-hydroxyflavone to Ru(II) through O3-phenolic oxygen was confirmed by the absence of the Hc3 singlet in the spectra of complexes, a consequence of deprotonation upon coordination [66, 67]. The coordination of chrysin to Ru(II) resulted in the disappearance of the Hc5 singlet for the same reason and, thus, confirmed its coordination through depro-tonated O5-phenolic oxygen. A representative 1H NMR spectrum with annotation of different H atoms is shown in Figure 2.

The most downfield-shifted and well-resolved signal of diimine ligand, assigned to Ha6′ for 1a–1d and Hb4 for 2a–2d corresponding to the 1H atom, was taken as the internal standard for the integration. In all cases, integration indicates the presence of 25 H atoms per molecular unit of the complexes.

In the 13C NMR spectra of complexes, most of the signals were found in the 120–150 ppm region. The signals of interest were those of flavonoid carbon atoms neighboring the oxygen atoms included in the coordination to Ru(II). In the spectra of complexes, the signal of car-bonyl C4 was found in the 158–186 ppm region. The signal of C3 bearing deprotonated phenolic oxygen in complexes with quercetin, morin, and 3-hydroxyflavone was found in the 157–163 ppm region, while the signal of non-substituted C3 in complexes with chrysin was positioned near 105 ppm. The signal of C5 bearing oxygen in complexes with quercetin, morin, and chrysin was positioned in the 153–169 ppm region, while the signal of non-sub-stituted C5 in complexes with 3-hydroxyflavone was positioned at ∼125 ppm. In all spectra, the signal of triflate carbon was found near 136 ppm.

Figure 2. 1h nmr spectrum of 2b. Inset: atom numbering scheme.

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Infrared spectra of complexes showed symmetrical stretching of C=O in the 1647–1611 cm−1 region. Compared to the spectra of parent flavonoids this vibration shifted to lower wavenumbers by 5–21 cm−1, confirming, thus, the coordination of flavonoid through carbonyl oxygen. Bands appearing in the spectra of flavonoids in the 1260–1350 cm−1 region arise through a combination of C–O stretching (mainly), in-plane C–H and in-plane C–C vibrations of the flavonoid ring. Due to flavonoid coordination through deprotonated phe-nolic oxygen, these bands shifted to higher wavenumbers in the spectra of complexes. Three medium-intensity bands in the 1460–1410 cm−1 region in the spectra of complexes arise from in-plane deformations of diimine rings and were insensitive to changes in the flavonoid ligand. Bands around 1260 and 520 cm−1 in the spectra of complexes were assigned to symmetrical stretching of S=O and C-S bonds of the free triflate anion, respectively.

The electronic absorption spectra of complexes 1a–1d and 2a–2d showed five major absorption bands in the 200–700 nm region (Figure 3). Most of the bands were Gaussian in shape and so well-resolved that it was possible to calculate the oscillator strength. Bands I-III, positioned in the ultraviolet region, arise predominantly through intraligand transitions. Band I (∼205 nm) was assigned to mixed π→π* transitions of diimine and flavonoid, while band II (220–245 nm) was attributed to dπ(Ru)→π*(diimine) transition. Purely Gaussian in shape with a large extinction coefficient (log ε > 4.70) a constant position and almost con-stant width at half height (19 ± 1 nm) band III was assigned to n→π* transition of diimine ligand. Bands IV and V proved to be solvatochromic, pH-dependent, broad, and slightly skewed to lower energies. Their shape and position was affected by both ligands, thus, indicating their metal-to-ligand charge-transfer (MLCT) character.

Emission spectra of complexes 1a–1d and 2a–2d showed red-shifted emission bands in the red and near-infrared region upon excitation at MLCT bands (Figure 3). Large values of Stokes shift (> 12 000 cm−1) indicated significant electronic change between ground and the excited state caused by (de)protonation of phenolic groups of flavonoid.

Figure 3. electronic absorption (full line) and emission spectra (dotted line) of 1a and 2a (2.50 × 10−5 m) and hla (5.00 × 10−5 m).

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The behavior of complexes 1a–1d and 2a–2d in aqueous solution in physiological con-ditions was investigated as a perquisite for proper attribution of biological activity to the originally formulated complexes or their hydrolytic products. Ruthenium(II)-bioflavonoid complexes proved inert in substitution of bidentate flavonoid ligand and consequently any type of biological activity was ascribed to the authentically formulated complex species.

3.4. Magnetic, conductivity, and electrochemical measurements

Negative values of magnetic susceptibility for complexes 1a–1d and 2a–2d confirmed the presence of low-spin diamagnetic t2g

6 Ru(II) center (Figure S26 and Table 1). Molar conduc-tivities of 1 mM acetonitrile solutions of bioflavonoid Ru(II) complexes supported their 1 : 1 electrolyte nature (Table 1).

Electrochemical characterization of novel compounds with potential biological activity is of huge importance, since redox properties are one of the determining factors for their possible application. Two main features of square-wave voltammograms of complexes are: (i) irreversible flavonoid-centered oxidation (0.2–0.6 V) corresponding to the formation of flavonoxyl radical and (ii) the quasi-reversible one-electron redox process involving the Ru(II)/Ru(III) couple in the 0.6–0.8 V region (Figure 4). The values of E1/2 for the Ru(III)/Ru(II) couple in complexes 1a–1d and 2a–2d, having RuN4O2 core, shifted toward more positive potentials compared to corresponding dichloro-species with RuN4Cl2 core, in accord with the better

Table 1. electrochemical, conductivity, and magnetic measurements data for 1a–1d and 2a–2d.

E′p/2/V E″p/2/V E1/2/V ΔEp/2/V Λm/S cm2 mol−1 χm/10−9 m3 mol−1

1a 0.685 0.882 0.804 0.197 170 −9.371b 0.755 0.838 0.795 0.083 178 −10.751c 0.609 0.773 0.685 0.164 173 −9.741d 0.528 0.646 0.592 0.118 149 −12.652a 0.689 0.887 0.807 0.198 162 −11.542b 0.622 0.771 0.700 0.149 125 −14.072c 0.526 0.652 0.591 0.129 155 −15.202d 0.526 0.648 0.592 0.122 135 −10.33

Figure 4. sW voltammograms of 1a, 2a and hla (∼10−5 m) in 0.1 m et4nClo4/me2Co at Pt electrode.

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stabilization of Ru(II) with π-accepting flavonoid compared to pπ-donor chloride. Values of E1/2 for the Ru(III)/Ru(II) couple in complexes became more positive in the order 3-hydroxy-flavone ∼ chrysin < morin < quercetin. This is because Ru(II) is stabilized by stronger reso-nance in flavonoid core. Reversibility of electron transfer for the Ru(III)/Ru(II) couple was lower in complexes with polyhydroxyl flavonoids compared to chrysin and 3-hydroxyflavone analogs.

3.5. Interaction with CT DNA

Nucleic acids along with proteins are still considered the main targets of potentially biolog-ically active substances. Therefore, investigation of the interaction of complexes with DNA is usually the first step in biological evaluation.

The health benefits of the interaction of flavonoids with DNA are considered to originate from protecting DNA from oxidative stress and, therefore, this area has been studied exten-sively. Different authors proposed different binding modes to DNA ranging from intercalative and groove-binding to long-range interactions over hydrogen bonding [68, 69]. The diversity of results stems from the strong dependence on binding on pH and the concentration of flavonoid. However, generally, flavonoids are considered intercalators.

Bipyridine and phenanthroline complexes of Ru(II) showed a strong affinity to binding DNA. Groove-binding is possible (e.g. [Ru(phen)3]2+) [70–72], but in the absence of readily leaving groups intercalation is most likely.

Complexes 1a–1d and 2a–2d meet some of the requirements of intercalators such as the presence of planar π-extended aromatic rings and cationic nature, but due to the hydroxyl groups their hydrophobicity is lowered to ensure good stacking between nucleobases.

3.5.1. Electronic spectroscopy studiesSpectrophotometric titration of complexes 1a–1d and 2a–2d with increasing concentrations of CT DNA revealed moderate hypochromism at MLCT bands with 1–18 nm of bathochromic shift; the exception was 1a and 1b where hypsochromic shift was observed (Figure 5).

Binding constants (Kb) were calculated to be of the 104 M−1 order, with the exception of 2d with 105 M−1 constant (Table 2). The ability of the bis(diimine)ruthenium(II) complexes to bind DNA depends on the ligands occupying fifth and sixth coordination position on Ru(II) [73–75]. Ruthenium(II)-polypyridyl complexes having sterically demanding or bulky ligands tend to bind DNA in groove or by partial intercalation, having binding constants of the 103–104 M−1 order [76]. On the other hand, complexes with a heterocyclic fused π-con-jugated ligand system are typically strong DNA-intercalators with 105–106 M−1 binding constants [77, 78]. Ruthenium(II)-bioflavonoid complexes 1a–1d and 2a–2d bind to DNA with constants Kb > 104 M−1 comparable to partial or moderately strong intercalators or groove-binders. However, hypochromism at MLCT bands along with bathochromic shift are typical for the intercalative binding mode.

Correlation between binding constants of Ru(II)-bioflavonoid complexes with those of flavonoids was not observed (Kb for morin, quercetin and chrysin were 1.10 × 103, 7 × 104, and 5.44 × 105 M−1, respectively) [79–81].

In order to gain a better insight into the interaction of complexes with DNA, a second approach considering changes to the DNA absorption maximum (∼260 nm) with increasing concentrations of the complex was used. Since the extinction coefficient of double-stranded

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DNA (6,600 M−1 cm−1) is lower than that of single-stranded (7,100 M−1 cm−1), changes in the intensity of the absorption maximum can give information about the impact of complexes on hydrogen-bonding between complementary nucleobases. Titrations of CT DNA with 1a–1d and 2a–2d showed hyperchromism, which suggests an unwinding of the DNA helix; this is typical for intercalation or covalent binding (Figure 6). Measurements showed that band shifting was minor ( ± 1.5 nm) and rather random and therefore a clear conclusion could not be derived. The binding constant was found to be 104 M−1 order, again with the exception of 2d (Table 2).

3.5.2. Fluorometric studiesIn order to confirm the intercalative binding mode, the competence of complexes 1a–1d and 2a–2d to displace ethidium bromide (EB), as strong intercalator, from its binding sites

Figure 5. spectroscopic titration of 2a (5.00 × 10−5 m) with Ct dna (0–2.00 × 10−4 m, A260/A280 = 1.84) in 10 mm tris–hCl buffer ph 7.42, t = 295 K, t = 5 min. Inset: Graphical determination of binding constant.

Table 2. data on interaction of 1a–1d and 2a–2d with Ct dna.

b = bathochromich = hypsochromic*r = [complex]/[dna] = 0.40.

Spectroscopic titration of complex

Spectroscopic titration of DNA Fluorescence titration of CT DNA-EB Viscometry

λ/nm Δλ/nm Kb/104 M−1 λ/nm Δλ/nm K/104 M−1 λ/nm Δλ/nm Ksv/104 M−1 Kapp/106 M−1 (η/η0)1/3*1a 490 2h 1.87 258 0.5h 1.20 591 1h 9.33 5.50 1.311b 488 5h 7.06 258 1.0h 3.14 591 3b 6.97 4.14 1.351c 476 1b 5.59 258 0.3h 1.72 591 2b 5.13 3.00 1.331d 508 3b 7.25 258 0.4b 6.99 591 1b 8.52 4.96 1.372a 478 5b 8.56 258 1.0b 7.91 591 2b 14.4 8.28 1.402b 462 3b 2.31 258 1.5b 1.51 591 2b 14.2 8.08 1.332c 479 9b 9.87 258 1.0h 9.83 591 1b 16.0 8.89 1.402d 501 18b 53.8 258 1.0h 14.9 591 1h 164 93.3 1.43

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on DNA was investigated fluorometrically. EB showed strong fluorescence in aqueous solu-tion when intercalated into DNA [82]. The intensity of fluorescence of EB bound to DNA significantly decreased, suggesting its displacement in the presence of increasing concen-trations of Ru(II)-bioflavonoid complexes (Figure 7). This linear dependence of changes in fluorescence according to the concentration of complexes is in agreement with the Stern–Volmer relation (Figure S36). Stern–Volmer quenching constant (KSV) values were higher than 104 M−1, which suggests a strong affinity of complexes to DNA (Table 2). Apparent binding constants showed that the complexes (Kapp > 106 M−1) are intercalators comparable to EB, with the exception of the Ru(II) complex of 3-hydroxyflavone with phenanthroline co-ligand which intercalates DNA 10 times stronger than EB (Kapp = 9.33 × 107 M−1).

3.5.3. Viscometric measurementsHydrodynamic properties of DNA like sedimentation and viscosity are highly sensitive to change in the length of DNA. In the absence of structural data, they are considered the most ambitious test for determining the binding mode of small molecules to DNA. The significant increase in DNA viscosity in the presence of Ru(II)-bioflavonoid complexes confirms the intercalative binding mode (Figure S37). In the case of 1a–1c, the decrease in viscosity at the lowest concentrations suggests electrostatic binding, which is a facilitating step for intercalation in higher concentrations.

Electronic spectroscopy and fluorometric studies along with viscometric measurements reached certain findings. Firstly, when compared to quercetin, higher hydrophobicity of chrysin and 3-hydroxyflavone increases the binding constant of a corresponding complex. Secondly, compared to bipyridine, a more extended π-system in phenanthroline increases

Figure 6. spectroscopic titration of Ct dna (∼7 × 10−5 m) with 2a (0–3.00 × 10−5 m) in 10 mm tris-hCl buffer ph 7.42, t = 295 K, t = 5 min. Inset: Graphical determination of binding constant.

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the binding constant of a corresponding complex. Thirdly, compared to the parent flavonoid ligands, the corresponding complexes bind DNA more strongly. Fourthly, both ligands (diimine and flavonoid) affect the binding constant of a complex to DNA.

3.6. Interaction with BSA

The binding of biologically active molecules to proteins has great significance for the trans-port, absorption, bioavailability and activity of a compound. Albumins are the most abundant proteins in plasma and are most commonly used in the study of the interaction of drugs with proteins. Bovine serum albumin is structurally similar to human serum albumin and therefore its interaction with complexes was studied through fluorescence spectroscopy. Upon excitation at 280 nm, two tryptophan (Trp) residues, Trp-212, located within a hydro-phobic binding pocket in the subdomain IIA, and Trp-134, located on the surface of the albumin molecule in the first subdomain IB, showed intrinsic fluorescence [83]. In the pres-ence of increasing concentration of Ru(II)-bioflavonoid complexes emission band of BSA at 340 nm showed a significant decrease (Figure 8).

The linear change of fluorescence intensity with increasing concentrations of complexes is in agreement with the classical Stern–Volmer relation (Figure S40). KSV values of the 105 M−1 order suggested that strong interaction of complexes with BSA occurs (Table 3). All KSV values were higher than the parent flavonoids, KSV (105 M−1) = 1.34, 0.65, 1.29 and 1.10 for quercetin, morin, chrysin and 3-hydroxyflavone, respectively [84–87]. Plotting log[(I0−I)/I] versus log[complex] gave values of binding constant (Kb) and the number of binding sites (n) (Figure S41). The Kb values were higher than 106 M−1 in all cases and comparable to those of ibuprofen (3.6 × 106 M−1) and diazepam (1.6 × 106 M−1), which are known to bind albumin very tightly,

Figure 7. fluorescence decrease of Ct dna-eB ([dna] = 2.40 × 10−5 m, [eB] = 6.00 × 10−6 m) in presence of increasing concentrations of 2a (0–7.40 × 10−6 m). Inset: Graphical determination of stern–Volmer constant.

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and thus suggesting high affinity of complexes toward BSA [83]. Complexes with morin and quercetin bind to BSA stronger than chrysin and 3-hydroxyflavone analogs, indicating the significance of hydroxyl groups for interaction, possibly due to the additional formation of hydrogen bonds between complexes and BSA. In addition, the binding of morin complexes (1b and 2b) to BSA was stronger when compared to quercetin analogs (1a and 2a), suggest-ing the importance of the flavonoid B ring hydroxylation pattern for interaction. The number of binding sites was 1–1.6. The rate of quenching (kq) was higher than 1013 M−1s−1, suggesting that static quenching occurs. Flavonoids quench tryptophan fluorescence mainly through static quenching. Plotting I0/I versus [complex] revealed that at higher concentrations of Ru(II)-bioflavonoid complexes, the curve slightly deviates toward the y-axis, which is char-acteristic of static quenching (Figure S41).

Minor blue-shift (1–4 nm) was observed in all cases, indicating that polarity around tryp-tophan decreased and hydrophobicity increased. Since the intrinsic fluorescence of BSA comes mainly from Trp-212, significant fluorescence quenching indicates that binding occurs near the subdomain IIA. It was also observed that there is a negative correlation between the binding constants of complexes to BSA and DNA. This clearly indicates that opposite

Table 3. data on interaction of 1a–1d and 2a–2d with Bsa.

Δλ/nm Ksv/105 M−1 Kb/107 M−1 n

1a 3.0 5.11 5.15 1.411b 2.0 4.41 7.71 1.471c 1.0 4.37 6.01 1.451d 4.0 3.43 6.44 1.482a 2.5 3.29 0.24 1.182b 4.0 4.59 29.8 1.562c 2.0 5.25 4.55 1.392d 1.0 2.69 6.84 1.46

Figure 8. fluorescence quenching of Bsa (3.40 × 10−6 m) in the presence of increasing concentrations of 2b (0–7.40 × 10−6 m).

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effects, hydrophilicity for BSA versus hydrophobicity for DNA, are responsible for binding Ru(II)-bioflavonoid complexes to these biomolecules.

3.7. Antioxidant activity

The positive effects on health of flavonoids can be attributed at least in part to their antiox-idant activity. Structure-activity studies showed that antioxidant activity depends on the presence of double bond in the C ring, 4-oxo group and hydroxylation pattern, increasing in the presence of 3-, 3′- and 4′-hydroxyl groups [88].

The antioxidant activity of complexes was determined spectrophotometrically using the DPPH method (Figures S42 and S43). Ruthenium-bioflavonoid complexes 1a–1d and 2a–2d showed stronger antioxidant activity than the parent flavonoids, which could be a conse-quence of easier H atom transfer upon complexation [89]. Quercetin demonstrated stronger antioxidant activity compared to morin and the same order of activity was maintained upon binding to Ru(II). On the other hand, complexes with chrysin showed better activity com-pared to 3-hydroxyflavone analogs owing to the presence of one OH group and the fact that potent 3-hydroxyl group of 3-hydroxyflavone is deprotonated upon coordination. Complexes with chrysin and 3-hydroxyflavone showed significantly higher activity than free flavonoids, suggesting that complexes do not exert their antioxidant activity solely through hydrogen atom transfer mechanism. When compared to bipyridine analogs, the lower IC50 values of Ru(II)-bioflavonoid complexes with phenanthroline co-ligand suggested that the π-extended system of diimine has significance for the stabilization of flavonoxyl radical.

Furthermore, a correlation between E1/2 values and IC50 values was found (Figure 9). As the potential of the Ru(III)/Ru(II) couple increases, IC50 values decrease, suggesting that anti-oxidant activity of complexes originates from flavonoid ligand, but also that the whole mol-ecule has an impact on the stabilization of generated flavonoxyl radical.

Figure 9. Correlation of E1/2 for ru(iii)/ru(ii) couple with iC50 values of antioxidant activity of 1a–1d and 2a–2d.

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3.8. In vitro anticancer activity

Ruthenium(II)-bioflavonoid complexes were tested for cytotoxic activity toward HeLa (cer-vical carcinoma), SW620 (colorectal adenocarcinoma, metastatic), HepG2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) cell lines (Table 4). Taking into account that the starting ruthenium complexes, cis-[Ru(bpy)2Cl2] and [Ru(phen)2(CO3)], did not show sig-nificant antiproliferative activity, it is reasonable to ascribe the activity of Ru(II)-bioflavonoid complexes primarily to flavonoid ligand. Yet, free flavonoids showed lower activity than the corresponding Ru(II) complexes. Free quercetin showed only weak activity toward hepato-cellular carcinoma (IC50 = 60 μM), while chrysin and 3-hydroxyflavone were moderately active toward all tested cancer cell lines. Upon coordination of chrysin to Ru(II), activity of the corresponding complex 2c toward cervical carcinoma improved five-fold compared to free chrysin. The antiproliferative activity of Ru(II) complex of 3-hydroxyflavone with 2,2′-bipyri-dine co-ligand (1d) toward breast adenocarcinoma was found to have an eighty-fold increase compared to free 3-hydroxyflavone. Considering the fact that the number of hydroxyl groups on flavonoid ligand could influence the antiproliferative activity of corresponding complexes, it was found that complexes with polyphenolic flavones (quercetin and morin) had lower antiproliferative activity compared to complexes of non-polyphenolic analogs (chrysin and 3-hydroxyflavone). On the other hand, complexes of polyhydroxyl flavones showed greater selectivity. It is noteworthy that the ruthenium(II)-quercetin complex with 2,2′-bipyridine co-ligand (1a) showed strong activity with good selectivity toward breast adenocarcinoma (IC50 = 0.39 μM). Of all the tested compounds, the Ru(II) complex of 3-hydroxyflavone with 2,2′-bipyridine co-ligand (1d) showed very strong antiproliferative properties, several times stronger than most of the bioactive Ru(II) complexes [90–95], with IC50 values in the low micromolar range (IC50 < 1 μM). It was observed that, in most cases, ruthenium(II)-bioflavo-noid complexes with 2,2′-bipyridine had stronger activity than their 1,10-phenanthroline analogs.

3.9. Antimicrobial activity

Ruthenium(II)-bioflavonoid complexes were tested for antimicrobial activity toward several Gram-positive bacteria strains: S. aureus ATCC 25923, E. faecalis ATCC 19433, S. beta-hemolytic

Table 4. the iC50 values (μm) of in vitro anticancer activity of 1a–1d and 2a–2d.

Cell lines

SW620 HepG2 MCF-7 HeLa1a > 100 > 100 0.39 ± 70.64 > 1001b > 100 > 100 85.87 ± 70.64 > 1001c 62.78 ± 19.36 45.02 ± 6.67 12.55 ± 18.18 26.80 ± 1.211d 0.75 ± 0.15 2.51 ± 0.67 0.52 ± 0.38 0.78 ± 0.202a > 100 > 100 > 100 > 1002b > 100 > 100 7.64 ± 70.43 > 1002c 53.39 ± 36.52 43.26 ± 21.31 43.67 ± 37.06 8.42 ± 20.232d 8.23 ± 46.41 11.42 ± 66.02 8.32 ± 0.86 19.32 ± 65.891 4.53 ± 60.11 > 100 2.10 ± 70.64 > 1002 > 100 > 100 92.38 ± 44.00 > 100hla > 100 59.54 ± 20.48 > 100 93.22 ± 22.97hlb > 100 > 100 > 100 > 100hlc 85.81 ± 51.93 33.82 ± 9.95 63.81 ± 25.45 43.01 ± 16.34hld 50.73 ± 22.29 8.88 ± 17.68 42.06 ± 21.08 5.44 ± 31.22

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group A, Methicillin-resistant S. aureus, Gram-negative bacteria strains: K. pneumoniae ATCC 1705, A. baumannii ATCC - BAA 747, P. aeruginosa, E. coli, and fungi C. albicans.

In vitro antimicrobial testing showed that the Ru(II)-bioflavonoid complexes possess only modest antimicrobial activity (Table S1). The complexes were mostly inactive toward Gram-negative pathogens; the exception was A. baumannii where moderate activity was recorded (inhibition zone 13–17 mm) compared to gentamicin (35 mm). This lack of activity in Ru(II) complexes toward Gram-negative bacteria is well known [95, 96]. It can be attributed to the fact that Gram-negative bacteria possess a thicker cell wall than Gram-positive bacteria and, have an additional lipopolysaccharide layer along the outer membrane, which is an aggra-vating factor in the diffusion controlled transport of the antimicrobial agent.

Ruthenium(II) complexes of non-polyphenolic flavones (chrysin and 3-hydroxyflavone) with 2,2′-bipyridine co-ligand showed activity toward Gram-positive bacteria. Yet no signif-icant activity was found in the parent flavonoid ligands; only quercetin was moderately active. Upon binding of quercetin to Ru(II), activity toward S. aureus, E. faecalis and MRSA was lost, which indicates the significance of carbonyl and 3-hydroxyl group for antimicrobial activity. On the other hand, activity of Ru(II) complex of 3-hydroxyflavone (1d) toward MRSA and C. albicans is improved compared to parent ligand, while activity toward A. baumannii remained almost the same (Table S1). Moreover, 1d showed a larger inhibition zone (26 mm) of MRSA-growth than the reference antibiotic gentamicin (20 mm). The inhibition zone of C. albicans growth for 1d was equal to nystatin as reference drug (28 mm). The results show that the replacement of 2,2′-bipyridine with 1,10-phenanthroline in Ru(II)-bioflavonoid com-plexes leads to a loss of antimicrobial activity in the corresponding complex.

Taking into account the affinity of complexes 1a–1d and 2a–2d to bind DNA and BSA and their antimicrobial and antiproliferative activity, it seems that strong biological activity correlates to the possibility to attack and bind DNA. However, the importance of binding to proteins or other bimolecular targets for antiproliferative or antimicrobial activity cannot be excluded based on our results.

4. Conclusion

Eight new heteroleptic ruthenium(II) complexes of the bioflavonoids quercetin, morin, chry-sin, and 3-hydroxyflavone with 2,2′-bipyridine and 1,10-phenanthroline as co-ligands were prepared and characterized. Considering the antagonism that exists between ruthenium and bioflavonoids, the synthetic strategy consisted of ruthenium(II) stabilization by two bidentate diimine ligands followed by assisted substitution of chloro or carbonato ligands from Ru(II) complex species by deprotonated flavonoid. In cationic complex species, Ru(II) is octahedrally coordinated to two diimine ligands and one monobasic bidentate flavonoid ligand, through 3,4-site in the case of quercetin, morin, 3-hydroxyflavone, and 4,5-site in the case of chrysin. The complexes showed high binding affinity to biomolecules such as DNA and BSA. Binding to DNA occurred via intercalation. Higher hydrophobicity of chrysin and 3-hydroxyflavone ligand compared to polyhydroxyl flavonoids and the more extended π-sys-tem in phenanthroline compared to bipyridine contribute to an increase in the binding constants of the corresponding complexes to DNA. The ruthenium(II) complexes of morin and quercetin bind to BSA more strongly than the corresponding complexes of non-polyphe-nolic flavonoids, which indicates importance of the hydroxyl groups in binding to BSA. Stronger antioxidant activity of the complexes compared to the parent flavonoids can be

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considered a consequence of easier H-atom transfer and better stabilization of the flavonoxyl radical. Antiproliferative properties of the complexes ranged from moderate to very strong. Complexes with polyphenolic flavones had lower activity but higher selectivity compared to their non-polyphenolic analogs. Good selectivity accompanied by strong activity (IC50 = 0.38 μM) toward breast adenocarcinoma was found in the Ru(II)-quercetin complex with 2,2′-bipyridine co-ligand. The ruthenium(II) complex of 3-hydroxyflavone with 2,2′-bipyridine co-ligand demonstrated strong cytotoxicity toward all tested cell lines with IC50 values lower than 1 μM. The same complex showed promising antimicrobial activity toward the opportunistic fungal pathogen C. albicans. Complexes of non-polyphenolic fla-vones were effective against Gram-positive bacteria, but showed only moderate activity toward Gram-negative A. baumannii. These prominent results encourage further design and development of ruthenium–flavonoid complexes as biologically active compounds.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Federal Ministry of Education and Science of Bosnia and Herzegovina [grant number 05-39-3936-1/15] designated for authors from Bosnia and Herzegovina.

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