Cytotoxic ruthenium(II) polypyridyl complexes with naproxen as NSAID: Synthesis, biological interactions and antioxidant activity

Accepted Manuscript Cytotoxic ruthenium(II) polypyridyl complexes with naproxen as NSAID: Synthesis, biological interactions and antioxidant activity Payal Srivastava, Ramranjan Mishra, Madhu Verma, Sri Sivakumar, Ashis K. Patra PII: DOI: Reference:

S0277-5387(19)30256-6 https://doi.org/10.1016/j.poly.2019.04.009 POLY 13869

To appear in:

Polyhedron

Received Date: Revised Date: Accepted Date:

24 January 2019 4 April 2019 8 April 2019

Please cite this article as: P. Srivastava, R. Mishra, M. Verma, S. Sivakumar, A.K. Patra, Cytotoxic ruthenium(II) polypyridyl complexes with naproxen as NSAID: Synthesis, biological interactions and antioxidant activity, Polyhedron (2019), doi: https://doi.org/10.1016/j.poly.2019.04.009

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Cytotoxic ruthenium(II) polypyridyl complexes with naproxen as NSAID: Synthesis, biological interactions and antioxidant activity Payal Srivastava a§, Ramranjan Mishra a§, Madhu Verma b, Sri Sivakumar b and Ashis K. Patra a*

Author address:

a

Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur

208016, Uttar Pradesh, India. E-mail: [email protected] b

Department of Chemical Engineering and Centre for Environmental Science and Engineering,

Indian Institute of Technology Kanpur, Kanpur 208016, Uttar Pradesh, India. §

P.S. and R.M. contributed equally to this work.

Dedicated to Prof. Akhil R. Chakravarty on his 65th Birthday

1

Abstract The non-steroidal anti-inflammatory drug (NSAID) naproxen (nap) bound to ruthenium(II) in the presence of a bidentate nitrogen donor heterocyclic ligands (bpy= 2,2’-bipyridine and phen= 1,10-phenanthroline), namely, [Ru(bpy)2(nap)][PF6] (1) and [Ru(phen)2(nap)][PF6] (2) have been synthesized and characterized using various physicochemical methods. Naproxen was coordinated to the Ru(II) centre through carboxylato oxygen atoms (-COO-) in a bidentate fashion. The compounds were evaluated for their photophysical properties, stability in solution, reactivity with 5’-guanosine monophosphate (5’-GMP) and GSH, interactions with CT-DNA and BSA. The complexes showed high binding affinity or reactivity towards these biological targets and bioanalytes. Both the compounds 1 and 2 showed moderate antioxidant activity by scavenging

DPPH

(1,1-diphenyl-picrylhydrazyl)

and

ABTS

(2,2′-azinobis(3-

ethylbenzothiazoline-6-sulfonic acid) radicals. The complexes 1 and 2 were highly cytotoxic against PC3 and MCF-7 cancer cells giving IC50 values ranging from 17µM- 27µM.

Keywords Medicinal Inorganic Chemistry, Non-steroidal anti-inflammatory drugs (NSAIDs), Ruthenium, Naproxen, Antioxidant activity, Cytotoxicity.

2

1. Introduction Despite the primary success of platinum(II) based anticancer compounds as clinically successful drugs in cancer chemotherapy, prolonged treatment with platins are associated with serious challenges. This include lower uptake at nucleus, high drug efflux, high rate of DNA repair, lack of tumor selectivity that ultimately leads to drug resistance and undesired off-target side effects such as nephrotoxicity, myelosuppression, peripheral neuropathy, ototoxicity and nausea [1-4]. Tackling all these major impediments for the betterment of cancer chemotherapeutics became the incentive for finding some alternative drugs, which can overcome aforementioned shortcomings. Over the years, ruthenium(II/III) based anticancer agents have emerged as exciting alternatives to their Pt(II) counterparts [5]. The most intensively studied Ru(III) anticancer drug candidates NAMI-A ([ImH][trans-RuCl4(DMSO-S)(Im)], where Im = imidazole and DMSO-S =sulfur bonded-dimethylsulfoxide) and KP1019 ([IndH][trans-RuCl4(Ind)2], where Ind = indazole) have reached phase-II clinical trials [6,7]. Ruthenium have many similarities to iron in terms of their redox potential and transferrin binding ability, that can be exploited for better drug internalization by piggyback mechanism or delivery, advantageous low drug efflux, longitivity of the drug action, more efficient approach to reach its biological targets [8-11]. Although as recently highlighted critically by Enzo Alessio, pioneer discoverer of NAMI-A, the area of ruthenium anticancer compounds is associated with many undemonstrated myths and undue over-simplification or generalization propagated in previous literature over the decades [12]. Therefore, contribution towards establishing various concepts or hypothesis, especially speciation, reactivity with the potential bioanalytes, and in situ fate of the Ru-drugs in tumor cells, in this challenging multi-faceted area is very important. Ruthenium(II) polypyridyl based complexes are chemically stable, exhibit effective binding and fluorescence imaging ability, redox chemistry, slow ligand exchange kinetics etc [13-18]. Ruthenium(II) polypyridyls are designed mainly to be used as DNA intercalators and photo-activated chemotherapeutic (PACT) agents involving reactive photoproducts or via ROS to trigger apoptosis [19-23]. Moreover, several Ru(II) polypyridyl complexes reported to be efficiently accumulated in potential pharmacological targets like nuclei or mitochondria and equipotent or more effective than cisplatin [24].

3

Non-steroidal anti-inflammatory drugs (NSAIDs) which are used as analgesic, antiinflammatory and antipyretic agents, functions mainly through inhibiting cyclooxygenase (COX) isoforms, COX-1 and COX-2 which catalyze the production of prostaglandins from arachidonic acid [25,26]. Cancer related inflammation is an enhancing factor in proliferation of malignancies, metastasis and tissue invasion [27-30]. In recent years, it has been established that cyclooxygenases (COX) play an important role in tumor growth, progression and metastasis. COX-2 is involved in tumor invasion, tumorigenesis, cancer immune invasion which are challenging stumbling block towards designing effective anticancer drugs [31]. The biological role of NSAIDs mainly lies in inhibiting cyclooxygenases, thus attaching naproxen as NSAID to the Ru(II)-polypyridyl core in a concerted manner could be an interesting strategy to circumvent tumor metastasis and immune invasion. Moreover, conjugation of aromatic NSAIDs to Ru(II)polypyridyl cage can also enhance the lipophilicity of the resulting ruthenium complexes for better cellular uptake in tumors. The biological activity of different NSAID with other 3dtransition metals, e.g., Mn, Fe, Co, Ni, Cu and Zn in presence or absence of nitrogen-donor heterocyclic ligands, have been reported in literature [32-35]. Recently, various cytotoxic metal complexes of Pt(II), Pt(IV), Ru(II), Os(II) conjugated to NSAIDs are reported as an effective antitumour agents [36-41]. Herein, we synthesized and characterized two Ru(II) polypyridyl complexes conjugated to naproxen as NSAID, viz: [Ru(bpy)2(nap)][PF6] (1) and [Ru(phen)2(nap)][PF6] (2) and studied their photophysical properties, DFT-optimized structures, reactivity with 5’-GMP and GSH and binding ability with DNA and BSA. The antioxidant capability of the complexes was evaluated with respect to free naproxen. The complexes exhibited potential cytotoxicity towards MCF-7 and PC3 cancer cell lines. 2.1. Materials and methods Ruthenium chloride (RuCl3. xH2O), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2’-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) (ABTS), Butylated hydroxytoluene (BHT) and 6hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased from Sigma (U.S.A). Sodium (2S)-2-(6-methoxynaphthalen-2-yl)propanoate (Naproxen sodium), 2,2’Bipyridine

(bpy),

1,10-phenanthroline

(phen),

lithium

chloride

(LiCl),

ammonium

hexafluorophosphate (NH4PF6), was purchased from Alfa Aesar, India and were used as 4

received. Solvents used were either HPLC grade or were purified using standard procedures [42]. [Ru(bpy)2Cl2] and [Ru(phen)2Cl2] were prepared according to reported literature [43]. Calfthymus (CT) DNA, ethidium bromide (EB), bovine serum albumin (BSA), were obtained from Sigma-Aldrich, U.S.A. Tris(hydroxymethyl)aminomethane−HCl (Tris-HCl) buffer solution (pH 7.2) was prepared using Milli-Q water of resistivity of 18.2 MΩ.cm-1. Elemental analysis of the complexes was performed using a Perkin-Elmer 2400 Series-II elemental analyzer. FTIR was recorded from 4000-400 cm-1 on a Perkin-Elmer Model 1320 spectrometer. ESI-mass spectral measurements carried out on WATERS Q-TOF Premier mass spectrometer. The UV-Vis absorption spectra were measured using Perkin Elmer Lambda 25 UV–vis spectrophotometer at 298 K. The fluorescence measurements were obtained using Agilent Cary Eclipse fluorescence spectrofluorometer at 298 K. Electrochemical measurements was performed using CH Instrument Model CHI 610E potentiostat in DMF equipped with a glassy carbon working electrode, Ag/AgCl reference electrode and a platinum wire auxiliary electrode and TBAPF6 (0.1 M) was used as a supporting electrolyte. 2.2. Synthesis and characterization 2.2.1. Synthesis of [Ru(bpy)2(nap)][PF6] (1) To an ethanolic solution (20 mL) of [Ru(bpy)2Cl2] (0.250 g, 0.516 mmol), naproxen (0.119 g, 0.516 mmol) pretreated with triethylamine (0.052 g; d=0.725 g mL-1 at 298 K, 0.516 mmol) was added to it. The reaction mixture was refluxed overnight and the corresponding solution was concentrated

to

half

of

its

volume.

To

this

concentrated

solution,

ammonium

hexafluorophosphate (NH4PF6) (0.336 g, 2.06 mmol) was added and a black precipitate was obtained immediately. The precipitate was filtered, washed with diethylether (3 x 5 mL) and dried under vacuum over anhydrous CaCl2 overnight. Yield: 0.268 g (66%). Anal. Calcd for C34H29F6N4O3P1Ru1: C, 51.85; H, 3.71; N, 7.11; Found: C, 52.02; H, 3.85; N, 7.08. ESI-MS (m/z) in EtOH: [M-PF6]+ calcd: 643.13 (100.0%), 645.13 (59.2%), 642.13 (53.8%), 641.13 (39.9%), 640.13 (40.2%), 644.13 (36.8%). Found: 643.12 (100.0%), 642.12 (53.2%), 645.12 (46.5%), 641.12 (39.9%), 640.12 (30.2%), 644.12 (27.5%) (See Fig. S1, SI). FT-IR (KBr, νmax, cm-1): 1604 (s) (νasym(CO2-)), 1462(s) (νsym(CO2-)), Δν(CO2-) =142 cm-1, 3436 (w, br), 2927 (w), 1923 (w), 1710 (m), 1630 (w), 1604 (s), 1551 (m), 1504 (w), 1482 (w), 1462 (s), 1445 (m), 1420 (w), 1390 (m), 1311 (w), 1265 (m), 1229 (w), 1213 (w), 1159 (w), 1121 (w), 1062 (w), 1021 (w), 5

926 (w), 841 (vs) (νP-F, PF6), 762 (s), 729 (m), 658 (w), 557 (m), 476 (w), 422 (w) (vs, very strong; s, strong; m, medium; w,weak; br, broad) (Fig. S3, SI). UV-vis in ethanol [λmax, nm, 298 K), (ε, M-1cm-1)]: 293 (32380), 333 (5640), 360 (4710), 508 (4740). 2.2.2. Synthesis of [Ru(phen)2(nap)][PF6] (2) Complex 2 was prepared by a similar procedure using [Ru(phen)2Cl2] (0.250 g, 0.469 mmol), naproxen (0.108 g, 0.469 mmol), triethylamine (0.047 g, 0.469 mmol) and ammonium hexafluorophosphate (0.306 g, 1.88 mmol). Yield: 0.239 g (61%). Anal. Calcd for C38H29F6N4O3P1Ru1: C, 54.61; H, 3.50; N, 6.70; Found: C, 54.35; H, 3.68; N, 6.61. ESI-MS (m/z) in EtOH: [M-PF6]+ calcd: 691.13 (100.0%), 693.13 (59.2%), 690.13 (53.8%), 689.13 (39.9%), 692.13 (41.1%), 688.13 (40.2%). Found: 691.12 (100.0%), 690.12 (52.5%), 693.12 (46.9%), 689.12 (38.9%), 692.12 (31.4%), 688.12 (28.9%) (Fig. S2, SI). FT-IR (KBr, cm-1): 1604(s) (νasym(CO2-)), 1426 (s) (νsym(CO2-)), Δν(CO2-) =178 cm-1, 3432 (m, br), 2971 (w), 2930 (w), 1971 (w), 1727 (w), 1629 (m), 1604 (s), 1570 (m), 1504 (m), 1484 (m), 1447 (m), 1426 (s), 1408 (m), 1390 (m), 1265 (m), 1228 (m), 1201 (w), 1176 (w), 1121 (w), 1096 (w), 1052 (w), 1029 (m), 926 (w), 840 (vs, νP-F, PF6), 772 (w), 732 (w), 719 (s), 557 (s), 476 (m) (Fig. S3, SI). UV-vis in ethanol [λmax, nm, 298 K), (ε, M-1cm-1)]: 270 (13500), 318 (5390), 333 (5670), 452 (3420), 499 (3250). 2.3. Stability The stability of the ruthenium complexes 1 and 2 were studied in Tris-HCl buffer (5 mM, pH=7.2) using UV-vis absorption spectral changes with time for 8 h at 298 K. 2.4. Interaction with 5’-GMP and GSH The reactivity of the ruthenium(II) complexes with 5’-guanosine monophosphate (5’-GMP) and glutathione (GSH) was studied by incubating in (1:1) aqueous methanolic mixture for 72 h at 37 C. The Ru(II)-5’GMP/GSH adducts were further characterized by ESI-MS to establish their speciation in solution. 2.5. Theoretical Methods

6

The energy-minimized geometries of the ruthenium complexes (1, 2) were optimized by density functional theory (DFT) using B3LYP level of theory using LanL2DZ (for Ru atom) and 631G** (for other atoms) basis set by Gaussian 09 program [44-46]. The geometry optimizations of the complexes were performed without any constraints and frequency calculations were also done on both the optimized geometries. The detailed optimized coordinates are shown in Tables S1 and S2 in supplementary information for both the complexes. 2.6. DNA binding experiments DNA binding experiments were performed using UV-vis absorption titration and ethidium bromide (EB) competitive displacement assay in Tris-HCl buffer (5 mM, pH 7.2). The interaction of the complexes with the calf thymus DNA using UV-vis absorption titration was performed keeping fixed complex concentration while varying the DNA concentration. The ratio of UV absorbance (A260/ A280) is 1.8-1.9, suggesting that DNA is apparently free from protein. Each spectra was recorded after giving equilibration time of 5 min. The binding affinity of the ruthenium complexes to CT DNA was determined by the following equation [47]: [DNA]/(εa - εf) = [DNA]/(εb - εf) + 1/Kb(εb - εf) where [DNA] is the CT-DNA concentration in the base pairs, εa is the apparent extinction coefficient, εf and εb are the extinction coefficients of the respective complex in its free and fully bound form. The Kb refers to the intrinsic equilibrium binding constant, obtained from the linear plot of [DNA]/(εa - εf) vs. [DNA]. The competitive binding of the complexes with the CT-DNA is studied through ethidium bromide displacement assay. In the presence of CT-DNA, EB gives increased emission intensity showing strong intercalation which decreases gradually on titration with the complexes due to partial displacement in buffer media. The fluorescence spectra of EB were recorded at λex = 546 nm (λem = 605 nm) in the presence of complexes. The apparent binding constant (Kapp) was determined by the following equation [48]: Kapp x C50= KEB x [EB] Where Kapp refers to the apparent binding constant of the complexes, C50 is the concentration of the complex at 50% reduction of fluorescence emission intensity of the initial EB-bound DNA. 7

KEB is the binding constant of the EB (KEB = 1.0 x 107 M-1), and [EB] is the final concentration of ethidium bromide. 2.7. Protein binding The protein interaction study of the complexes by tryptophan fluorescence quenching titrations were performed using bovine serum albumin (BSA) in Tris-HCl buffer medium at pH=7.2. The emission spectra of the protein (7 μM) was recorded at em= 354 nm with an excitation wavelength of 295 nm and gradually titrating against the complexes as quenchers with increasing concentration. Due to the overlap in the emission spectra of the complexes with the protein, the titration measurements are reported after performing due correction with the compounds spectra. The quenching constant (KSV) was determined by the following Stern-Volmer equation [49]: I0 / I =1 + kqτ0[Q]= 1 + K[Q] where I0 and I refers to the steady-state emission intensities of BSA in the absence and presence of quencher [Q], KSV is the Stern-Volmer quenching constant for BSA, kq is the quenching rate constant, τ0 is the average lifetime of the biomolecule without quencher (~10-8 s). Using the Scatchard equation, log(I0 – I) / I = log K + nlog[Q], the binding constant (K) and the number of binding sites (n) for such static quenching interaction can be calculated [50].

2.8. Antioxidant Studies Non-steroidal anti-inflammatory drugs are well known radical scavengers. The complexes known to posses antioxidant activity plays important role against inflammation. The presence of naproxen (NSAID) in the complexes 1 and 2, encourage to evaluate their antioxidant ability by scavenging free radicals. The ability of the Hnap and complexes to scavenge DPPH (1,1diphenyl-picrylhydrazyl) radicals and ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS+) were performed in ethanol. DPPH (0.1 mM) is a stable radical which absorbs at 517 nm, an equal volume of the compounds (0.1 mM) were added to the ethanolic solution of the DPPH. The change in the absorbance spectra at 517 nm was recorded after 20 min. Butylated hydroxytoluene (BHT) was used as a reference compound and ethanol as a control solution. 8

ABTS+ was produced in an aqueous medium by reacting 2 mM stock solution of ABTS with potassium persulfate (0.17 mM) and the reaction was allowed to stand for approx. 16 h in the dark at room temperature. The oxidation of ABTS was incomplete in the reaction as ABTS and potassium persulfate react at a stoichiometry of 1:0.5. ABTS+ was diluted to give an absorbance of 0.70 and the spectra was recorded at 734 nm. 10 µL each of the compounds to be tested were added in the reaction mixture and the absorbance were measured immediately. Trolox was used as a reference compound and ethanol as a control solution. The results were expressed as a percentage inhibition of the absorbance of the initial ABTS solution (ABTS%). 2.9. Cell cytotoxicity assay The MCF-7 and PC3 cells were cultured using DMEM medium supplemented with FBS (10%, v/v) and antibiotic (penicillin/streptomycin 1%, v/v) at 37 °C in an incubator containing 5% CO 2 atmosphere. The viability of MCF7 and PC3 cells, treated with 1 and 2 was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. For this, 5 x 103 cells were seeded in 96-well plate and incubated overnight at 37 °C with 5% CO2. Once the cells obtained their morphology, they were treated with different concentrations (5, 15, 25, 35, 45, 55 μM) of 1 and 2 for 24 h. Further cells were incubated with MTT (200 µl, 0.5 mg/ml) for 4 h, then media was removed and 200 µl of DMSO was added to each well. The data was represented as formazan absorbance at 570 nm, considering the control (untreated) cells as 100% viable. The absorbance of the formazan (purple solution) was measured after 20 min using a MultiSkan UVvis spectrometer at a wavelength of 570 nm. All readings were taken in triplicates. The IC50 values were calculated by nonlinear regression plot using GraphPad Prism software. 3. Results and discussion 3.1. Synthesis and characterization Two ruthenium(II) complexes namely [Ru(bpy)2(nap)][PF6] (1) and [Ru(phen)2(nap)][PF6] (2) were synthesized following a general synthetic procedure by reacting ethanolic solution of deprotonated naproxen with corresponding N,N’ donor-based precursors, viz. [Ru(NN)2Cl2] (NN= bpy (1) and phen (2)) and isolated as PF6 salts in high yields (Scheme 1, S1, SI). They were characterized spectroscopically using ESI-MS, UV-vis, FT-IR and fluorescence spectroscopy. The selected physicochemical data are shown in Table 1. The complexes are air 9

stable in the solid state and soluble in alcohols, acetonitrile, dimethyl formamide and dimethyl sulfoxide while sparingly soluble in chloroform and dichloromethane. The ESI-MS of the compounds in ethanol shows respective complex cation peaks, [M-PF6]+, which revealed m/z 643.12 for [C34H29N4O3Ru1]+ (1) and m/z 691.12 for [C38H29N4O3Ru1]+ (2) with matching theoretical isotopic distributions (Figs. S1, S2, SI). The FT-IR data shows strong characteristic bands at 1604 cm-1 and 1426-1462 cm-1 corresponding to asymmetric and symmetric stretching vibrations of the carboxylato group. The difference (Δ) = [νasym(C=O) - νsym(C=O)] = 142-178 cm-1 indicates a bidentate mode of binding of the carboxylate group of naproxen to the ruthenium(II) centre [51,52]. Additionally, strong absorption frequencies for both the complexes at approx. 840 cm-1 (νP-F, PF6-) verifies the existence of hexafluorophosphate counter-ion (PF6-) in the complexes (Fig. S3, SI). The electronic absorption spectra of the complexes shows distinct ligand-centered broad bands at 274 nm and a band ranging 318-333 nm due to n-π* and π-π* electronic transitions of the nitrogen donor heterocyclic bases and naphthyl moiety of naproxen. The complex 1 showed a broad peak at 360 nm attributed to π-π* transition and a peak at 508 nm due to the metal [Ru(II), d6]-to-ligand charge transfer transition (MLCT). While the complex 2 showed MLCT transition bands at 452 and 499 nm. Upon excitation at 318 nm, a strong emission band is observed at 354 nm characteristic to naphthyl moiety of naproxen (Fig. 1). The ruthenium(II) complexes are redox-active, complex 1 exhibited metal-centered quasi-reversible peak potential at 0.62 V corresponding to Ru (II/III) and ligand centered peaks at -1.64 V and 1.40 V vs. Ag/AgCl. Complex 2 exhibited metal centered quasi-reversible peak potential at 0.4 V and two peak potentials at -1.71 V and -1.47 V vs. Ag/AgCl due to ligand based reductions (Fig. S4, SI).

Scheme 1: Structural representation of the complexes [Ru(NN)2(nap)][PF6] (NN= bpy, 1; phen, 2) containing N,N-donor heterocyclic bases 10

Fig. 1 (a) UV-visible spectra of 1, 2 and (b) emission spectra of complexes 1, 2 and naproxen (Hnap) in ethanol (125 M), λex = 318 nm with a slit width of 5 nm at 298 K. Table 1. The physicochemical data and DNA and protein binding parameters for the complexes 1 and 2. Complex

[Ru(bpy)2(nap)][PF6] (1)

IRa (cm-1)/ ν(CO2) λmaxb/nm, (ε/M−1 cm−1)

1604, 1462

[Ru(phen)2(nap)][PF6] (2) 1604, 1426

293 (32380), 333 (5640), 360 (4710), 508 (4740)

270 (13500), 318 (5390), 333 (5670), 452 (3420), 499 (3250) 4 c -1 (2.36 ± 0.02) x 10 (2.90 ± 0.02) x 104 Kb /M 7.90 x 106 9.55 × 106 Kappd/M-1 5 e -1 (1.06 ± 0.04)x 10 (0.91 ± 0.04) x 105 KBSA /M 13 f -1 -1 1.06 x 10 0.91 x 1013 kq / M s 6 g -1 2.45 x 10 2.11 x 106 K/M h 1.79 1.45 n a b IR stretching frequency in KBr phase. UV-visible spectral bands in ethanol. cKb intrinsic DNA binding constant with CT DNA. dKapp, apparent DNA binding constant determined from ethidium bromide displacement assay. eKBSA, Stern-Volmer quenching constant. fkq, quenching rate constant. gK, binding constant. hn, number of binding sites.

3.2. Interaction with 5’-GMP and GSH The reactivity and speciation studies of the ruthenium(II) compounds with the 5’-guanosine monophosphate (5’-GMP) was performed in 1:1 MeOH/H2O mixture after incubation at 72 h at 37 C and subsequently analyzed by ESI-MS. The ESI-MS data shows the formation of Ru(II)GMP adduct, [Ru(bpy)2(5’-GMP)]+ (m/z = 776.09) and [Ru(phen)2(5’-GMP)]+ (m/z= 824.09), probably via binding with the N7 of the guanine bases (Fig. 2(a) and (b)). The formation of such 11

[Ru(NN)2(GMP)]+ under ESI-MS is indicative of labile carboxylate bound Ru-nap linkage which may allows the of Ru(II)-DNA crosslinks thereby possibly triggering apoptosis via DNAdamage activity. The formation of pentacoordinated Ru(II) species as evidenced through ESIMS, though unusual but considering mass spectral conditions and GMP being a bulky ligand could be the possible reason for such an observation. Glutathione, one of the most abundant molecule present in mM level in the cancer cells with several important roles in maintaining cellular homeostasis. GSH are responsible for both protective and pathogenic roles in the cancer cells [53, 54]. Glutathione are often overexpressed in cancer cells and binds with several anticancer agents. The ruthenium(II) complexes were incubated with glutathione (GSH) in 1:1 MeOH/H2O mixture for 72 h at 37 C and analyzed using ESI-MS which possibly shows the formation of Ru(II)-sulfinato adducts, i.e., [Ru(bpy)2(GSO2)]+ (m/z= 752.10) and [Ru(phen)2(GSO2)]+ (m/z= 800.11) (Fig. 2(c) and (d)), that can result from an aerial oxidation of the ruthenium bound GS- to GSO2-, resulting in the formation of pentacoordinated Ru(II) species in the mass spectral condition, and proceeds via release of a naproxen moiety suggesting potential affinity of the Ru(II) complexes towards the thiols. S. Bonnet et al. reported analogous reacivity of Ru(II) complex with N-acetyl cysteine, which is structurally similar to glutathione and considering the various possible oxidation states of sulfur, such kind of Ru(II) sulfinato species can possibly form here [55].

12

Fig. 2 ESI-MS of Ru-GMP/GSH adduct formed from the complex 1 (a) and 2 (b) with 5’-guanosine monophosphate (5’-GMP) and 1 (c) and 2 (d) with GSH after 72 h incubation at 37 C.

3.3. Theoretical Study A computational study based on density functional theory (DFT) was carried out to release the optimized structural features of the complexes. The structures were optimized using B3LYP level of theory [LanL2DZ (for Ru-atom) and 6-31G** (for other atoms) basis set] performed by Gaussian 09 program [56]. The energy-minimized structures of the complexes are shown in Fig. 3, and computational data were given in Table S1 and S2. The obtained frontier molecular orbital diagrams shows that the HOMO for complex 1 and 2 is located over the naproxen moiety whereas LUMO is occupied by the metal and N,N donor ligands. The energy gaps between HOMO and LUMO of complexes 1 and 2 were 2.37 and 2.41 eV respectively.

13

Fig. 3 Energy optimized structures of [Ru(bpy)2(nap)]+ (1) (a) and [Ru(bpy)2(nap)]+ (2) (b). Frontier molecular orbitals (HOMO and LUMO) of complexes 1 (c) and 2 (d).

3.4. DNA binding studies Ruthenium polypyridyl complexes are well known DNA intercalators with high binding affinity and used as structural probes, cellular bioimaging agents and PACT agents for cancer chemotherapy [57-60]. The DNA binding interactions with metallodrugs is therefore is an important aspect to evaluate their action mechanism. The absorption spectral titration were carried out to determine the binding interaction of the ruthenium(II) complexes with CT-DNA by monitoring the corresponding spectral changes during the course of titration. It shows that both the complexes leads to significant hypochromism of the ligand-centered absorption bands. The hypochromicity of the electronic band indicates intercalative mode of binding with stacking interactions between planar aromatic moiety of the ligands and aromatic base pairs of DNA. From the linear plot, intrinsic binding constant (Kb) values were calculated to be (2.36 ± 0.02) × 104 and (2.90 ± 0.02) × 104 M−1 respectively (Fig. 4)

14

Additionally, fluorescence titrations measurements were also carried out to obtain the extent of interaction between the complexes and CT-DNA using competitive displacement of ethidium bromide (EB) by determination of apparent DNA-binding affinity (Kapp). EB is nonemissive in buffer medium, however shows enhanced emission intensity when bound to CTDNA on intercalating with ds-DNA base pairs. Upon titration with the complex solution into the EB-DNA complex, decrease in the emission intensity of EB at 605 nm (λex=546 nm) is observed indicating competitive displacement of EB and subsequent binding of the metal complexes to the CT-DNA. The calculated apparent binding constant Kapp for complexes 1 and 2 are 7.90 x 106 and 9.55 x 106 M-1 respectively suggesting partial intercalative binding of the complexes towards DNA.

Fig. 4 (a) Absorption spectral traces of complex 1 in Tris-HCl buffer (5 mM, pH 7.2) on titration with CT-DNA. Inset: [DNA] versus {[DNA]/(Δεaf)}. (b) Emission spectra of EB-bound DNA in presence of complex 2 in Tris-HCl buffer (5 mM, pH 7.2), λex= 546 nm. Inset: Plot of I/I0 vs. [Complex] (μM).

3.5. Protein binding studies To determine molecular interactions between the ruthenium(II) complexes and proteins, intrinsic tryptophan florescence quenching of BSA was performed in the presence of the complexes. Fluorescence of BSA originates due to the presence of tryptophan residues, Trp-134 and Trp-212 located at two subdomains and quenching arises due to variety of molecular interactions like 15

energy transfer, ground state complex formation, collision quenching, structural deformation etc. [61]. Therefore emission quenching of tryptophan in BSA is an important tool to determine binding affinity of low molecular weight pharmacologically active compounds. Serum albumins are major transporter for exogenous drugs and play important roles in their metabolism [62]. On increasing the concentration of the complexes, emission intensity of BSA gets quenched at 354 nm upon excitation at ex = 295 nm. Such quenching is probably due to the changes in the secondary structure of protein associated with diverse molecular interaction, showing effective binding with the complexes. The Stern-Volmer quenching constants (KSV) for the complexes are (1.06± 0.04) x 105 M-1 (1) and (0.91± 0.04) x 105 M-1 (2) respectively. The binding constants (K) were found to be in the range of 2.11-2.45 x 106 M-1 and number of binding sites (n) = 1.79 (1) and 1.45 (2) obtained from the Scatchard equation indicating existence of predominantly static quenching mechanism (Fig. 5).

Fig. 5 Emission spectral traces of bovine serum albumin (BSA) protein (7 μM) in the presence of complex 1 in Tris buffer (5 mM, pH 7.2) at 298 K. Inset: (a) (I0/I) vs. [complex] (μM) and (b) Scatchard plot: log ([I0- I]/I) vs. log[complex] for complexes 1 and 2 with least-square linear curve fitting.

3.6. Antioxidant Studies The oxidative stress in cell is induced due to excessive production of reactive oxygen species (ROS) that disrupts the intracellular redox balance. It is associated with the damage of nucleic acids, proteins and membrane lipids, over–expression of oncogenes, results in inflammation that leads to various types of cancer, neurodegenerative or cardiovascular diseases [63]. In response 16

to inflammatory signals, leukocytes and other phagocytic cells which are part of human immune system congregate near the site of inflammation and generate ROS to mitigate the infection but ultimately lead to DNA damage of normal cells in the vicinity of cancer cells. Therefore, studying the radical scavenging activity of the present complexes to prevent carcinogenesis of the normal cells will play important role in evaluating their anti-inflammatory and anticancer activity [29, 64, 65]. The ability of the NSAID containing ruthenium(II) complexes 1 and 2 to scavenge in vitro DPPH and ABTS radicals was studied in ethanol. The complexes are more active radical scavengers than the free naproxen, suggesting enhancement in antioxidant activity with coordination to Ru(II) centre (Fig. 6). Complex 2 shows marginally higher DPPH scavenging activity (41%) than complex 1 (35%) and the complexes were found to be moderately effective with respect to the reference compounds, viz. butylated hydroxyl toluene (BHT) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)

Fig. 6 Interaction of Hnap, complexes 1 and 2 and reference compounds (BHT and Trolox) (0.1 mM) with (a) % DPPH radical scavenging ability (b) % superoxide radical scavenging activity measured at λ= 517 and 734 nm respectively at 298 K with 5% error limit.

3.7. Cytotoxicity assay The cytotoxicity of the ruthenium(II) NSAID complexes were performed in vitro to determine the IC50 value against breast cancer MCF7 cells and human prostate cancer PC3 cell lines. The complexes were incubated for 24 h and the cell viability was determined by MTT assay. The complexes showed significant toxicity at micromolar concentrations with IC50 values for the 17

complexes 1 and 2 to be 26.80 ± 6.4 and 23.01 ± 3.3µM against MCF-7 cells and 20.42 ± 1.7 and 17.38 ± 2.1 µM against PC3 cell lines respectively (Fig. 7). The complex 2 with phenanthroline moiety showed higher cell death activity than the complex 1.

Fig.7 Cell viability plots showing cytotoxicity of the complexes 1 and 2 in (a) MCF7 and (b) PC3 cancer cells by MTT assay after 24 h of incubation.

4. Conclusion We have synthesized and characterized two Ru(II) polypyridyl-NSAID complexes, viz. [Ru(bpy)2(nap)][PF6] (1) and [Ru(phen)2(nap)][PF6] (2), where naproxen is coordinated to the ruthenium(II) centre in bidentate fashion through carboxylate oxygens. The photophysical properties, interaction with DNA, GMP, GSH and serum protein, antioxidant activity were studied. The complexes showed efficient binding towards DNA via partial intercalative mode. Both the complexes were able to form covalent adduct with 5’-GMP, a truncated version of DNA, thereby suggesting different modes to trigger apoptosis. Ruthenium(II) complexes also reacts with GSH with concomitant release of naproxen which possibly can scavenge ROS, thereby possibly preventing carcinogenesis of normal cells. The antioxidant activity was evaluated through DPPH and ABTS radical scavenging assay and were found to be moderate free radical scavengers. The complexes showed in vitro cytotoxicity against the MCF-7 and PC3 cancer cell lines.

Acknowledgements 18

We thank Science and Engineering Research Board (SERB), Government of India (Project no. SB/FT/CS-062/2012), for the financial assistance. P.S. and R.M. acknowledges UGC and CSIR for research fellowships respectively.

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Graphical Abstract: Synopsis Text: Two cytotoxic ruthenium(II) polypyridyl complexes containing naproxen (nap) as NSAID, [Ru(NN)2(nap)][PF6] (NN= bpy (1), phen (2)) were synthesized and characterized. The physicochemical properties, theoretical aspects, binding interactions with DNA and BSA and antioxidant activities were investigated.

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