Ruthenium complexes with mono- or bis-heterocyclic chelates: DNA/BSA binding, antioxidant and anticancer studies.

Journal of Biomolecular Structure and Dynamics ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20 Ruthenium complexes with mono- or bisheterocyclic chelates: DNA/BSA binding, Antioxidant and Anticancer studies Sanam Maikoo, Abir Chakraborty, Nyeleti Vukea, Laura Margaret Kirkpatrick Dingle, William John Samson, Jo-Anne de la Mare, Adrienne Lesley Edkins & Irvin Noel Booysen To cite this article: Sanam Maikoo, Abir Chakraborty, Nyeleti Vukea, Laura Margaret Kirkpatrick Dingle, William John Samson, Jo-Anne de la Mare, Adrienne Lesley Edkins & Irvin Noel Booysen (2020): Ruthenium complexes with mono- or bis-heterocyclic chelates: DNA/BSA binding, Antioxidant and Anticancer studies, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2020.1775126 To link to this article: https://doi.org/10.1080/07391102.2020.1775126 View supplementary material Accepted author version posted online: 28 May 2020. Submit your article to this journal View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tbsd20 Ruthenium complexes with mono- or bis-heterocyclic chelates: DNA/BSA binding, Antioxidant and Anticancer studies Sanam Maikooa, Abir Chakrabortyb, Nyeleti Vukeab, Laura Margaret Kirkpatrick Dingleb, William John Samsonb, Jo-Anne de la Mareb, Adrienne Lesley Edkinsb, Irvin Noel Booysen*a School of Chemistry and Physics, University of KwaZulu-Natal, Pietermaritzburg, South Africa Biomedical Biotechnology Research Unit, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, South Africa cr ip b t a an us *Corresponding author: E-mail: Booyseni@ukzn.ac.za M Abstract Deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) binding interactions for a series of ruthenium heterocyclic complexes were monitored using ultraviolet-visible (UV-Vis) d spectrophotometry, fluorescence emission spectroscopy and agarose gel electrophoresis. pt e Investigations of the DNA interactions for the metal complexes revealed that they are groovebinders with intrinsic binding constants in the order of 104 – 107 M-1. Electronic ce spectrophotometric DNA titrations of the bis-heterocyclic metal complexes illustrated hypochromism of their intraligand electronic transitions and the presence of diffuse isosbestic Ac points which are synonymous with homogeneous binding modes. Metal complexes with the mono-heterocyclic chelates also showed alterations in their intraligand transitions and changes in their metal-based electronic transitions which are suggestive of metal coordination to the CTDNA structure. Using agarose gel electrophoresis assessments, Hoechst DNA binding competition studies corroborate that the metal complexes are DNA groove-binders. Optimal uptake of these metal complexes by BSA was observed based on their optimal apparent association and Stern-Volmer constants (Kapp and KSV > 104 M-1). Radical scavenging studies revealed that the metal complexes have high activities towards the neutralization of NO and DPPH radicals. Data attained from the BSA electronic spectrophotometric titrations for the 1 majority of the metal complexes illustrated distinct hyperchromism accompanied with blue shifts which indicates unwinding of the protein strands. Predominately, the metal complexes showed moderate cytotoxicity against both triple-negative breast cancer and cervical cancer cell lines that was greater than that of 5-fluorouracil. Keywords: Ruthenium heterocyclic complexes, DNA/BSA binding, antioxidant activities, 1. ip t cytotoxicity. Introduction cr Ruthenium-based anticancer drugs have demonstrated cytotoxicity against a wide variety of cancer cells accompanied with minimal side effects to healthy cells (Lazarević, Rilak, & us Bugarčić Ž, 2017; Thota, Rodrigues, Crans, & Barreiro, 2018; Zhang & Sadler, 2017). It is an hypothesized that the biocompatibility of these potential metallopharmaceuticals culminates from the similar chemistry of ruthenium and the essential metal, iron, as these elements are group congeners (Merlino, 2016). In addition, ruthenium can induce cancer cell apoptosis M through utilization of its high coordination affinities to nucleotides (Pages, Ang, Wright, & Aldrich-Wright, 2015). Alteration of the co-ligands within the coordination sphere of ruthenium d have been shown to lead to intriguing structure-activity relationships and diverse mechanisms of pt e action (Zeng et al., 2017). In fact, the leading candidates of ruthenium chemotherapeutic agents, e.g. trans-[RuCl4(DMSO)(Im)](ImH) (ImH = protonated imidazole) (NAMI-A), are pro-drugs which are activated upon hydrolysis (Dwyer, Johnson, Cazares, McFarlane Holman, & Kirk, ce 2018). Furthermore, conjugated aromatic chelating ligands of metal complexes are able to promote DNA interaction through intercalation or groove-binding as the possible mechanism of Ac anticancer activity (Levina, Mitra, & Lay, 2009). Current research focuses on designing target-specific ruthenium anticancer drugs and involves encompassing biologically relevant moieties (BAMs) in ligand scaffolds where the meticulously selected BAMs may facilitate defined biodistribution patterns (Caruso et al., 2016). This design approach is exemplified by arene metal complexes with flavone or chromone analogs, where a correlation between the lipophilicity and the in vitro screening of melanoma cell lines was found (Pastuszko, Majchrzak, Czyz, Kupcewicz, & Budzisz, 2016). In addition, a fascinating bifunctional metal complex, (ethacrynic acid-g6-benzylamide)(1,3,5-triaza-7- 2 phosphaadamantane)dichloride (ethaRAPTA) induced death of MCF-7 breast cancer cells, which is regarded as a significant advancement considering that these cells are resistant to cisplatin (Chatterjee, Biondi, Dyson, & Bhattacharyya, 2011). The dual functionality of this metal complex stems from the inherent cytotoxicity of the RAPTA constituent and ethacrynic acid-g6benzylamide moiety’s glutathione S-transferase inhibiting capability which combats drug resistance. Heterocyclics such as benz(imidazole/othiazole) moieties are common to various ip t organopharmaceuticals and their derivatives have been shown to exhibit in vivo therapeutic activities to common human cancers (Taha et al., 2015; Yadav & Ganguly, 2015; Yamin & metal complexes have shown higher cr Teplow, 2017). Moreover, metal-ligand synergistic correlations have been observed where the activities than their corresponding antimicrobial bioassays conducted on the us benz(imidazole/othiazole)-derived free-ligands. This phenomena is illustrated by the metal complex, [Ni3(abb)3(H2O)3(μ- an ttc)](ClO4)3·3H2O·EtOH and its free-ligand, 1-(1H-benzimidazol-2-yl)-N-(1H-benzimidazol-2ylmethyl)methanamine (abb) which showed that the metal complex displayed higher M antimicrobial activity than the free-ligand (Kopel et al., 2015). In fact, complementary biological and anticancer activities of metal-based chemotherapeutic drugs can improve their efficacy by d negating the common side-effects associated with secondary infections (Ng, Wu, & Aldrich- pt e Wright, 2018). In this research study, we report the synthesis and characterization of new metal ce complexes bearing bis-heterocyclic ligands as well as exploring the structure-activity correlations of the aforementioned metal complexes and those with mono-heterocyclic chelates, Ac [RuCl(Hobz)2(PPh3)]Cl (3) (Hobz = 2-hydroxyphenylbenzimidazole) and [RuIIICl(obs)2(PPh3)] (4) (Hobs = 2-hydroxyphenylbenzothiazole). Two novel diamagnetic metal complexes, cis[RuIICl(PPh3)2(ombb)](PF6) (1) and trans-[RuIICl2(PPh3)2 (bbb)] (2) were synthesized from the reactions of trans-[RuCl2(PPh3)3] with bis-benzimidazole ligands, 2,2’-[oxybis(methylene)]-bis(1H-benzimidazole) (ombb) and 4,4’-bis(1H-benzimidazol-2-yl)-2,2’-bipyridine (bbb), respectively. The DNA and BSA interaction, antioxidant capabilities and in vitro anticancer activities of the metal complexes 1 – 4 were evaluated. 2. Experimental 3 2.1 Materials and methods The metal precursor, trans-[RuCl2(PPh3)3] and ammonium hexafluorophosphate as well as the organic precursors, including diglycolic acid, 2,2’-bipyridine-4,4’-dicarboxylic acid, ophenylenediamine, were all procured from Sigma-Aldrich. High purity ascorbic acid, 2,2diphenyl-1-picrylhydrazyl (DPPH), Griess reagent, sodium nitroprusside, phosphate buffered saline tablets (PBS), calf thymus (CT)-DNA, Bovine Serum Albumin (BSA) and electrochemical analysis grade tetrabutylammonium hexafluorophosphate were also obtained from Sigma ip t Aldrich. Organic solvents were purchased from Merck SA and used without additional purification. The bis-benzimidazoles, ombb and bbb, as well as the mono-heterocyclic metal cr complexes 3 and 4, were prepared according to literature trends (Adebisi, Booysen, Akerman, & us Xulu, 2016; Swarnalatha, Rathnamala, Babu, & Bhuvanesh, 2016; Tavman & Çinarli, 2014). The synthetic procedures of 1 and 2 are described in the accompanying supporting information an document. 31 P, 1H and 13C NMR spectra were obtained in DMSO-d6 using a Bruker Advance 400 MHz M spectrometer equipped with an autosampler. Solid-state infrared spectra were recorded using a Perkin-Elmer Spectrum 100 while electronic spectra were collected using a Perkin-Elmer d Lambda 25. Melting point ranges were determined with the aid of a Stuart SMP3 melting point pt e apparatus. Redox properties of the novel metal complexes 1 and 2 were probed using a Metrohm Autolab potentiostat in conjunction with a three electrode system: a glassy carbon working electrode (GCWE), a pseudo Ag|AgCl reference electrode and an auxiliary Pt counter electrode. ce Electrochemical grade tetrabutylammonium hexafluorophosphate (0.1 M) was added to the 2 mM dichloromethane solutions of the metal complexes as a supporting electrolyte. Fluorescence Ac measurements were carried out using a 1 cm quartz emission cell and a Perkin Elmer LS-45 fluorescence spectrometer equipped with a xenon lamp source. Elemental analysis data were obtained using a CHNS-O Flash 2000 Organic Elemental Analyzer. 2.2 UV-Vis spectrophotometric DNA titrations and agarose gel electrophoresis assessment of DNA binding 4 The CT-DNA interaction studies of the metal complexes were performed at a pH of 7.2 in phosphate-buffered saline (PBS). The CT-DNA solution in PBS gave rise to a ratio of 1.9:1 at 260 nm and 280 nm, which implies that the CT-DNA was sufficiently free of protein. Using the molar absorption coefficient (ε260 = 6600 M-1cm-1), the CT-DNA concentration per nucleotide was calculated (Reichmann, Rice, Thomas, & Doty, 1954). The final CT-DNA stock solution was kept at 4 ºC and used within 2 days. Solutions of the metal complexes and CT-DNA were incubated at 25 ºC for 24 hours preceding any UV-Vis measurements [21]. Subsequently, UV- ip t Vis spectra were collected of standard solutions for the respective metal complexes in DMSO and after the addition of varying concentrations of CT-DNA in PBS buffer. The intrinsic binding cr constant (Kb) was obtained by fitting the data obtained from the titration into the following – – (A) an – us equation: In the above equation, [DNA] is the concentration of DNA in base pairs, εa is the M extinction coefficient of the detected absorption band at the given DNA concentration [corresponding to Aobs/(complex)], εf is the extinction coefficient of the free metal complex in solution, and εb is the extinction coefficient of the fully bound metal complex to DNA. A slope pt e d of 1/(εa - εf) and Y intercept of 1/Kb(εb - εf) was found from a plot of [DNA]/(εa – εf) versus [DNA]. The ratio of the slope to the intercept is estimated to be the intrinsic binding constant (Kb) (Kaplanis et al., 2014). ce In addition, the ability of metal complexes to interact with human genomic DNA (gDNA) isolated from cancer cell lines was assessed by agarose gel electrophoresis. Metal complexes at Ac two different concentrations (50 µM and 200 µM, selected in relation to the IC50 values) or the vehicle control (DMSO) were incubated with 100 ng of gDNA in a total reaction volume of 20 µL. Reaction mixtures were incubated at 37°Ϲ for 2-4 hours, followed by electrophoresis for 1 hour at 90V in 1x Tris-acetic acid EDTA (TAE) buffer using 0.8% (w/v) agarose containing 0.5 µg/mL EtBr. The DNA was visualized under UV light. The assay was conducted as independent triplicates and the average fluorescence intensity of DNA treated with metal complexes was determined by ImageJ relative to the DMSO control. 5 The abilities of the metal complexes to compete with Hoechst for binding to DNA were assessed. Three concentrations of the metal complexes (5 µM, 50 µM and 100 µM) were incubated with a final concentration of 1 µg/mL of Hoechst-33342 in the presence or absence of DNA in a 100 µL reaction volume in a black-walled clear bottom 96 well plate at 25 °C. The fluorescence emission over the range of 400-600 nm was collected after excitation at 350 nm. Antioxidant studies ip t 2.3 Experimental methodologies for the radical scavenging measurements were adapted from cr those in the literature (Krishnamoorthy et al., 2011; Ramachandran & Viswanathamurthi, 2013). us Data reproducibility was confirmed by conducting each experiment in triplicate and the standard equation shown below was used to determine the experimental percentage radical scavenging ) (B) M ( an activities: where the absorbance of the control is Ac (NO or DPPH radicals) and Af is the absorbance upon d addition of the individual metal complexes to the control. The metal complex concentrations that pt e induce 50% radical scavenging activity (IC50 values) could be readily calculated from their distinctive experimental percentage radical scavenging activities. An experiment was initiated by collecting the UV-Vis spectrum of the control [DPPH (0.2 mM in MeOH)] followed by the ce addition of 0.1 mL of a metal complex (30 μM in DMSO) and then the sample solution was mixed to ensure homogeneity. Thereafter, the sample solution was incubated in the dark for 20 Ac minutes and its UV-Vis spectrum was run. (Atha et al., 2010)The following experimental technique was implemented for the NO radical assay: firstly, a 10 mM solution of sodium nitroprusside was prepared in PBS buffer and incubated for a 3-hour period at room temperature. Afterwards, Griess reagent (1 mL) was added to a 0.5 mL of the nitroprusside solution and the resultant solution constituted the control. Then the UV-Vis spectrum of the control was collected. A sample solution was prepared by the addition of a metal complex (30 μM in DMSO) to a 0.5 mL aliquot of sodium nitroprusside. 6 After a 3-hour incubation period, 1 mL of the Griess reagent was added to a sample solution and its UV-Vis spectrum was run. 2.4 BSA binding interaction studies The BSA stock solution was prepared in PBS buffer at a pH of 7.2 and the concentration of this stock solution was determined spectrophotometrically using the extinction coefficient of ip t 43824 M-1 cm-1 at 280 nm (Atha et al., 2010). Due to the considerable insolubility of 1 - 4 in the buffer, stock solutions (1.0 mM) of the metal complexes were prepared in acetonitrile. Data cr correction was implemented to the quenching titration studies to compensate for the inner filter us effect using the following equation (Nehru et al., 2020): Fcor = Fobs x 10(A1+A2)/2 (C) an where, Fcor and Fobs are designated as the corrected and observed fluorescence intensities, respectively, whereas A1 and A2 are defined as the absorbance values of each ratio of BSA and a M metal complex at peak maxima of the excitation and emission wavelengths, respectively. 2.5.1 Electronic spectrophotometric titrations d The BSA interaction experiments were conducted by maintaining the BSA concentration pt e (~16 µM) whilst varying the concentrations of the respective metal complexes (0 – ~40 µM). An incubation time of 2 minutes was used for each sample mixture. Equal volumes of a metal ce complex were added to both the reference and sample cells. The data from the absorbance titrations were fitted to the following equation: ] ( Ac [ )+( ) ( ) (D) where A and A0 are the absorbance values of BSA at 280 nm in the presence and absence of a metal complex, εBSA and εB are the extinction coefficients of BSA and the bound complex (viz. adduct of a metal complex and BSA), Cmetal complex is the concentration of a metal complex and Kapp is the apparent association constant. From the equation (D), the following double reciprocal plot can be generated and the apparent association constant (Kapp) is determined from the ratio of the intercept to the slope (Zhong et al., 2004). 7 ( ) 2.5.2 Fluorescence emission spectroscopic titrations The effects of increasing the metal complex concentrations on the emission spectrum of BSA were assessed. Fluorescence emission spectra were recorded at 293 K with the width of emission ip t and excitation slits adjusted to 5 nm. The spectra were recorded in the wavelength range of 300 – 500 nm at an excitation wavelength of 280 nm. The resulting data was used to calculate the cr Stern-Volmer constant (KSV) using the Stern-Volmer relationship (Paul et al., 2013): us (E) an where Fo and F are the emission intensities in the absence and presence of the metal complexes, respectively. The concentration of the respective metal complex is designated by [Q]. The KSV d M values were obtained from the slope of the plot: pt e The quenching rate constant (kq) can then be determined from equation (F): (F) ce where τ0 is the lifetime of the protein (10-8 s) without a quencher. The binding constants (Kb) and the binding number (n) can be quantified using equation (G) Ac (Nehru et al., 2020): (G) where F0, F and [Q] are the same as in equation (E), Kb is the binding constant of each metal complex with BSA and n refers to the number of binding sites per BSA molecule, which can be determined from the following plot based on equation (G): 8 2.6 In vitro anticancer cell line studies The cytotoxicity of the metal complexes against the HeLa and HCC70 triple negative breast carcinoma cell lines were determined using the MTT assay as previously described (de la Mare et al., 2012). In this assay, metabolically active cells convert the MTT dye to a blue formazan product that can be detected spectrophotometrically. Briefly, cells were seeded in a 96 well plate in complete medium, allowed to adhere overnight and incubated with a selective concentration range for the metal complexes (including a vehicle control and positive controls: ip t 5-Fluorouracil and Paclitaxel in triplicate for 96-hours). Thereafter, the cells were incubated with MTT reagent for 4 hours, the resulting formazan crystals solubilized in SDS solution overnight cr and the absorbance at 595 nm measured. The IC50 values were calculated using non-linear an us regression in GraphPad Prism 4.0. Results and discussion 3.1 Synthesis, spectral characterization and redox properties of 1 and 2 M 3. The diamagnetic metal complexes 1 and 2 were isolated in moderate yields by separate d equimolar reactions of ombb and bbb with trans-[RuCl2(PPh3)3] in methanol. The source of the pt e PF6- counter-ion of 1 emanates from the addition of ammonium hexafluorophosphate in an equivalent molar amount with respect to the metal precursor. For 1, the bis-heterocyclic ligand (ombb) functions as a neutral tridentate chelator whereby coordination occurs through the ce benzimidazole nitrogen and bridging ether oxygen donor atoms. In the case of 2, the ligand bbb coordinates via its neutral NpyNpy donor set affording a constrained five-membered chelate ring Ac trans-orientated to the cis-chloride co-ligands. These diamagnetic metal complexes were found to be stable in air as well as soluble in high boiling point aprotic solvents (viz. DMSO and DMF), while they exhibit moderate solubility in chlorinated solvents. Generally, the signals of 1 and 2 in their 1H NMR spectra display shifts with respect to analogous signals found in the proton spectra of their free ligands. These spectral differences resemble coordination of organic chelators to transition metal centers, see Figs. S6 and S7 (Haddad, Yousif, & Ahmed, 2013). In particular, the aromatic protons associated with the benzimidazole phenyl rings are shielded upon coordination of the ombb (in 1) and bbb (in 2) 9 organic chelators. Common to both proton spectra of the metal complexes is the presence of the intense multiplets within the region of 7.65 – 7.04 ppm, which are characteristic of the triphenylphosphine co-ligands (Satyanarayana & Reddy, 1987). Furthermore, the singlet associated with the methylene protons adjacent to the ethereal oxygen (viz. H5, H5’, H6 and H6’) in 1 displays a noticeable shift from 5.39 to 5.31 ppm, which is attributed to ring current effects of the chelating aromatic benzimidazole, and these proton NMR spectral changes provide tangible evidence of O-coordination (Mikata, Fujimoto, Fujiwara, & Kondo, 2011; Stojcevic & ip t Baird, 2009). A single peak was observed in each decoupled 31P NMR spectra, which confirmed the presence of magnetically equivalent phosphorous atoms within the coordination spheres of cr the respective metal complexes, see Figs. S8 and S9. us The solid-state infrared spectra of 1 and 2 illustrate their intense intracyclic ʋ(C=N) [1431 cm-1 for 1 and 2] which are found at lower wavenumber in comparison to those found in an the IR spectra of their free-ligands, ombb (at 1459 cm-1) and bbb (at 1441 cm-1), see Figs. S10 and S11. The same trend is observed when comparing the coordinated (at 1088 cm-1) and M uncoordinated (at 1142 cm-1 for ombb) ether stretches of 1 and its corresponding free-ligand, which is also another influential feature of coordinative bonding. The dominating infrared d experimental stretches (at 690 cm-1 for 1 and 692 cm-1 for 2) in the respective IR spectra of the pt e metal complexes are typical of ʋ(Ru-P) and are similar to those found in other metal complexes (Irvin N. Booysen, Adebisi, Akerman, Munro, & Xulu, 2016; Irvin Noel Booysen, Maikoo, Piers Akerman, Xulu, & Munro, 2013). ce As expected, the electronic spectra of the metal complexes show several intense intraligand π-π* electronic transitions below 300 (for 1) and 400 nm (for 2), which mostly Ac originate from the pi-conjugated moieties of their respective organic chelators, see Figs. S12 and S13. At more red-shifted regions between 400 and 600 nm, p(Cl) → d(Ru) Ligand-to-Metal Charge Transfer (LMCT) bands are found. A distinctive metal-based electronic transition is found in the UV-Vis spectrum of 2 (at 712 nm) converse to the absence of a d-d electronic transition in the UV-Vis spectrum of 1, which is largely ascribed to the low spin d6 electron configuration of its central metal ion (Adebisi et al., 2016). The redox properties of the metal complexes were investigated by means of voltammetric experiments. The cyclic voltammogram (CV) of 1 displays a one irreversible redox process (Epa 10 = 1.36 V vs Ag | AgCl), which is attributed to the single electron oxidation of the metal center, while a quasi-reversible redox process [ΔEp(2) = 80 mV and ΔEp(ferrocene) = 90 mV] is found in the CV of 2 (Epa = 0.42 V and Epc = 0.34 V vs Ag| AgCl) attributed to the d5/d6 system interconversion, see Figs. S14 and S15. Literature trends corroborate the assignment of the redox processes, e.g. the CVs of ruthenium(II) complex salts, [Ru(η5-C5H5)(PPh3)2(L)](PF6) (HL = 5phenyl-1H-tetrazole and imidazole) display one irreversible oxidation process each at Epa = 1.22 Antioxidant studies cr 3.2 ip t V and 1.25 V, respectively (Moreno et al., 2010). us Oxidative stress has been implicated as one of the major causes of DNA mutation and excessive free radical concentrations in the blood stream can escalate cancer progression an (Pizzino et al., 2017). Natural antioxidants such as Vitamin C are often not potent enough to inhibit the exponential growth of cancerous tumors induced by free radicals. Metal complexes have been shown to be highly effective scavengers of various free radicals, which largely stems M from the redox active metal center and its organic ligand’s capabilities to donate an electron and proton, respectively (Bal-Demirci et al., 2015; Sathiya Kamatchi, Chitrapriya, Kim, Fronczek, & d Natarajan, 2013). The radical scavenging activities of the metal complexes 1 – 4 were pt e investigated towards the NO and DPPH radicals, respectively, refer to Table 1. The low IC50 values (Table 1) of the metal complexes strongly supports their exceptional antioxidant activities, which are shown to be more potent than the standard antioxidant vitamin C ce [IC50 (DPPH) = 141 µM; IC50 (NO) = 210 µM]. These favorable IC50 values could be ascribed to the attached ligands acting synergistically to enhance the radical scavenging activity of 1 – 4, as Ac benzimdazole and benzothiazole derivatives are known to possess good antioxidant properties (Ayhan-Kilcigil et al., 2005; Prouillac, Vicendo, Garrigues, Poteau, & Rima, 2009). The higher antioxidant activities for the paramagnetic metal complex 3 can be explained by the unpaired electron in the low spin d5 orbital, which increases the propensity of the metal complex cation of 3 to neutralize the DPPH and NO free-radicals more effectively. The acidity of any available protons on the ligands may also be increased due to the electron deficient Ru(III) metal center, which may also positively impact the radical scavenging capability of the metal complex (Prakash, Manikandan, Viswanathamurthi, Velmurugan, & Nandhakumar, 2014). The obtained 11 IC50 values are within the range of those obtained for other documented metal complexes, for example, the IC50 values for some metal complexes were reported to be within the ranges of 24.0 – 59.7 µM and 6.7 – 11 µM for the DPPH and NO radicals respectively (Ramachandran & Viswanathamurthi, 2013). 3.3 DNA interaction studies t The potential of transition metal complexes as chemotherapeutic drugs can be readily ip gauged from their affinities to DNA (Galindo-Murillo, García-Ramos, Ruiz-Azuara, Cheatham, cr & Cortés-Guzmán, 2015). These metal complexes have illustrated diverse DNA interaction modes; for instance, their metal centers can directly coordinate to donor atoms within the DNA us double helix or these metal complexes can facilely form DNA adducts via DNA intercalation between the DNA base pairs, resulting in major- or minor DNA groove binding (Pages et al., an 2015). Numerous methods have been utilized to monitor the progressive formation of DNAmetal complex adducts, however one of the most versatile and simplest techniques is UV-Vis M spectrophotometry (Hajian & Guan Huat, 2013). Electronic spectral changes associated with DNA intercalation usually entails the d decrementing absorbance of an aromatic chromophore’s intra-ligand transition (viz. pt e hypochromism) accompanied with a progressive red shift of the corresponding wavelength maximum (viz. bathochromism) (Bhattacharya & Mandal, 1997). These UV-Vis spectral ce alterations are accounted to pi-stacking interactions between a pi-conjugated chelator and the DNA base pairs (Shahabadi, Mohammadi, & Alizadeh, 2011). The contradictory UV-Vis spectral observations, the hyperchromic effect can arise from groove-binding as well as Ac electrostatic attractions between the DNA and metal complex. Hypochromism is known to compromise the structural integrity of DNA by inducing denaturation or cleavage (Sirajuddin, Ali, & Badshah, 2013). The UV-Vis spectral profiles depicting the DNA binding titrations for 1 and 2 reveal hypochromism (2.15 % at λmax = 347 nm and 21.14 % at λmax = 379 nm) with red shifts of 3 nm and 4 nm, respectively, see Fig. 2. In addition, diffuse isosbestic points are observed at 295, 361 and 511 nm in the UV-Vis spectral profile of 2, which indicates that the metal complex exhibits a homogenous binding mode towards DNA. Synonymously, the arene metal complex, [(ɳ612 C12H18)RuCl(PFPdpm)] [PFPdpm = 5-(penta-fluoro)phenyldipyrromethene] showed red shifts (of approximately 2 nm) for its intra-ligand and MLCT bands accompanied with gradual decreases in the absorbance values (under 10% hypochromism) during its CT-DNA binding electronic spectrophotometric experiment (Paitandi et al., 2017). The steric demands of the cis[Ru(PPh3)2]2+ core affords a significantly lower intrinsic binding constant for 1 (Kb =1.5 x 105 M1 ) (Motswainyana & Ajibade, 2015). Similarly, a carbonyl metal complex, [RuCl(CO)(dppb)(bipy)]PF6 illustrated hypochromic behavior, however its sterically crowded t bis(diphenylphosphine)butane (dppb) co-ligand impeded DNA intercalation and favored groove- ip binding based on its lower Kb (3.78 x 104 M-1) value in comparison to ethidium bromide with a cr Kb value of 106 M−1 as well as other metal-based DNA intercalators (Carnizello et al., 2016) (Cory, McKee, Kagan, Henry, & Miller, 1985; Gao et al., 2008). Comparatively, the larger us intrinsic binding constant (Kb) of 2 (1.2 x 107 M-1) is higher than that obtained for [(ɳ6C12H18)RuCl(PFPdpm)] (6.5 x 104 M-1), which hints at multimodal interactions of 2 with CT- an DNA. Thus, the metal complexes 1 and 2 can be regarded as preferential groove-binders. Diverse spectral alterations were observed in the overlay electronic spectra of 4 upon its M titration with CT-DNA, attesting to this metal complex’s variable DNA interactive modes. Individual decreasing (λmax = 294 nm) and increasing (λmax = 329 nm) intraligand transitions are d bridged by an isosbestic point appearing at 308 nm, while similarly, a broad shoulder centralized pt e at 390 nm disappears with the appearance of a low intensity MLCT band at 517 nm. These UVVis spectral changes are symbolic of stereoelectronic redistributions with Hobz organic chelators ce of 3 upon interaction with CT-DNA. Of particular interest is the appearance of the metal-based electronic transition, which is suggestive of the metal center reducing and, hypothetically, this Ac arises from coordinative bonding of the ruthenium to selected DNA base pairs. Analogous to the bis-heterocyclics (1 and 2), hypochromism (18 %) of 3’s intra-ligand transition (monitored at 331 nm) is observed, which is accompanied by the disappearance of a very low-intensity d-d transition band at 639 nm. In addition, a clear appearance of a distinct MLCT band is detected at 416 nm, which again indicates reduction of the metal center upon coordination with the CTDNA structure (as in the case of 3). The calculated intrinsic binding constants (9 x 104 M-1 for 3 and 2 x 105 M-1 for 4) of the mono-heterocyclic metal complexes are both smaller in magnitude than classical intercalators, which suggest that metal complexes 3 and 4 are predominately DNA groove-binders (Gao et al., 2008). Furthermore, the metal complexes 1 – 4 have similar intrinsic 13 binding constants (Kb) to the classical groove-binder, Hoechst-33342, and its analogues with Kb 3 x 105 M-1 to 7 x 106 M-1 (Bazhulina et al., 2009; Loontiens, values ranging from Regenfuss, Zechel, Dumortier, & Clegg, 1990; McGowan et al., 1988; Stokke & Steen, 1985). The UV Agarose gel electrophoresis of human genomic DNA isolated from cancer cell lines demonstrated that each of the metal complexes could interact with DNA and resulted in a reduction in the fluorescence intensity of ethidium bromide (EtBr) staining. In particular, metal complexes 1 and 2 demonstrated a dose-dependent and significant reduction in fluorescence ip t intensity of EtBr staining (Fig. 3). The extent of the metal complexes’ DNA interactive capabilities can be directly correlated to the corresponding apparent DNA binding constants cr where 1 and 2 have higher intrinsic DNA binding constants and afford substantial reduction in us the fluorescence intensity of EtBr. Furthermore, the Hoechst DNA binding competition studies corroborate that the metal complexes 1 – 4 are DNA groove-binders, see Fig. S16. In particular, an the concentration-dependent studies affirm that the respective metal complexes exhibit gradually BSA interaction studies d 3.4 M higher DNA binding affinities than the classical DNA groove-binder, Hoechst-33342. pt e Serum albumins are major dissoluble proteins in the circulatory system and serve as primary transport media for numerous pharmaceuticals and physiologically relevant metal complexes (Milutinović et al., 2017). In particular, the affinity of a pharmaceutical to albumin ce has a significant influence on its pharmacokinetics. Mechanistically, the drug carrier protein can induce the careful reversible uptake and release of pharmaceuticals through altering its flexible Ac structure while retaining its structural integrity. Primarily, medicinal inorganic complexes and organic compounds have been shown to bind to BSA domains IIA and IIIA subdomains (Karami, Alinaghi, Amirghofran, & Lipkowski, 2018). Biomolecular titrations between the in vivo drug carrier protein, BSA and the respective metal complexes 1 - 4 were monitored using UV-Vis spectrophotometry and fluorescence emission spectroscopy. Gradual variations in intensities are indications of the conformational changes in the BSA structure induced by the additions of the individual metal complexes whilst any shifts in the electronic spectral bands reveals whether a metal complex is bound to the BSA 14 chromophore in its hydrophobic or hydrophilic environments (Paul et al., 2013; Zhong et al., 2004). Data attained from the electronic spectrophotometric titrations illustrated distinct hyperchromism accompanied by slight blue-shifts which are observed in the separate UV-Vis spectra of 1, 2 and 3, see Fig. 4 and S17. These spectral observations concur with literature trends which suggests that minor unwinding of the protein strands occur leading to non-exposure of the polar tryptophan residues’ microenvironment of BSA, for hydrogen-bonding interactions with water molecules (Cheng, Liu, & jiang, 2013). ip t Similarly, the distinguishing absorption band of BSA gradually increased upon interaction with 4, however a minor red-shift of the peak maxima was noted. The aforementioned cr electronic spectral changes are attributed to the structural BSA integrity being compromised by us its interaction with the metal complex. Consequently, the unfolding protein backbone exposes the tryptophan residues to aqueous media rendering increasing polarity and hydrophilicity for the an BSA chromophore (Suryawanshi, Walekar, Gore, Anbhule, & Kolekar, 2016; Wu, Lin, Zhai, Zhuo, & Zhu, 2013). An explanation for the difference in trends could be explained by the larger M number of hydrogen-bonding sites on metal complexes 1 – 3, which results in more stable adduct formations with BSA. As a result, less unfolding of the protein backbone occurs which keeps the tryptophan residues unexposed and well-hidden in the hydrophobic protein cavity (Herskovits, pt e d Gadegbeku, & Jaillet, 1970; Mishra, Malakar, Biswal, Barman, & Krishnamoorthy, 2015). From the double reciprocal plots of 1 / (A0 – A) versus 1 / Cmetal complex, the apparent association constants (Kapp) for 1, 2, 3 and 4 were calculated to be 1.80 x 105 M-1, 7.47 x 106 M-1, ce 4.66 x 104 M-1 and 2.00 x 104 M-1, respectively. These apparent association constants (Kapp) are of similar magnitude to those obtained for other metal complexes and moreover, the Kapp values can Ac also denote ideal binding affinities towards BSA, which are considered to be between 104 – 106 M-1 (Ravi Kumar et al., 2017; Topală, Bodoki, Oprean, & Oprean, 2014; Wong et al., 2014). The larger association constants linked with the bis-heterocyclic metal complexes 1 and 2 can be attributed to their larger sizes which allows more favourable hydrophobic interaction with the BSA interfacial surface. The presence of the chloride co-ligands also promotes higher reactivities as the halides are known to undergo in vivo ligand substitution to form aqua adducts that generate active coordination centres (Messori, Orioli, Vullo, Alessio, & Iengo, 2000). 15 BSA readily undergoes fluorescence quenching if one or more of its accessible domains are occupied by a quencher, which in the process leads to increasing concentrations of the nonfluorescent active BSA-quencher. Similarly to the UV-Vis spectrophotometry titration analysis, any changes in the conformation or environment of BSA due to interactions with a metal complex can result in quenching, with shifts in the characteristic BSA emission peak (Varlan & Hillebrand, 2010). Steady-state fluorescence quenching of BSA peak maximum occurs in the presence of all metal complexes, which affirms the formation of the BSA-metal complex ip t aggregates, see Figs. 5 and S18. Blue-shifts in the BSA peak maxima occurs upon interaction with 1, 2 and 3, whereas a cr red-shift is observed when the protein is titrated against 4. These findings are consistent with us those found in the molecular absorption spectrophotometric titrations, whereby metal complexes 1 – 3 induces less unfolding of the protein thus keeping the BSA chromophore in a more an hydrophobic environment, while metal complex 4 bares the BSA chromophore to an enhanced hydrophilic environment by increasing protein unfolding (Ojha & Das, 2011). The Stern-Volmer M (KSV) and quenching rate (kq) constants were derived from the Stern-Volmer relationship and are shown in Table 2, see Fig. 6. d The obtained kq values indicated that the fluorescence decay is a process of static pt e quenching since these values are of a larger magnitude than that of quenchers involved in a dynamic equilibrium process (2 x 1010 M-1 s-1) (Ramachandran & Viswanathamurthi, 2013; Varlan & Hillebrand, 2010). Since KSV is the Stern-Volmer constant for static quenching, it can ce be used to examine the binding behaviour of the respective metal complexes towards BSA. The results conclude that the bis-heterocyclic metal complexes (1 and 2) exhibit higher binding Ac affinities for BSA than the mono-heterocyclic metal complexes (3 and 4), which is consistent with the findings presented in the UV-Vis spectrophotometric analysis. Stern-Volmer (KSV) and quenching rate (kq) constants of similar magnitude were documented for other metal complexes, for example, cis-[Ru(quin)(dppm)2]PF6 [quin = quinaldate; 5 -1 dppm = 13 bis(diphenylphosphino)methane] display KSV and kq values of 0.77 x 10 M and 1.10 x 10 M-1 s-1, respectively (da Silva et al., 2017). In the case of static quenching mechanisms, it can be presumed that there are comparable and independent binding sites within the BSA biomolecule. Therefore, the binding constant (Kb) 16 and the number of binding sites (n) could be determined using a modified version of the SternVolmer equation (Equation G). The calculated Kb suggests robust binding interactions for 1 (8.34 x 104 M-1) and 4 (1.27 x 103 M-1) with BSA, as metal complexes having binding constants ranging from 103 - 105 M-1 are considered strong binders (Kamtekar et al., 2013). Consequently, these results emphasize stable adduct formations for 1 and 4 when binding to BSA, respectively. Similar Kb values have been reported for the interaction of metal complexes with albumins in literature, for example, [(η6-p-cymene)Ru(ATSC)Cl]PF6 (ATSC = 9-anthraldehyde t thiosemicarbazone) exhibited a Kb value of 12.1 x 104 (Beckford, 2010). In contrast, the lower ip Kb values for 2 (6.65 x 102 M-1) and 3 (5.79 x 101 M-1) were not expected as they were seen to be cr effective quenchers of BSA in the previous study, which implies weaker binding with BSA due to possible low plasma distribution (Nišavić et al., 2018). The n values obtained for all metal us complexes are approaching unity, which indicate only one binding site for each metal complex In vitro anti-cancer activity M 3.5 an occurred when binding to the BSA structure (Cheng et al., 2013). The cytotoxicity of the metal complexes 1 – 4 against the two cancer cell lines were assessed d using an MTT assay and the results are shown in Table 3. The commonly used pt e chemotherapeutics 5-FU and paclitaxel were included as positive controls and displayed cytotoxicity against HCC70 cells, with IC50 values of 127.07 (± 8.61) µM and 3.06 ± 1.11 nM, respectively. The metal complexes showed greater cytotoxicity than 5-FU but lower cytotoxicity ce than paclitaxel in HCC70 cells with IC50 values of 88.20 (± 1.10), 44.07 (± 1.12) and 39.37 (± 1.15) µM, for metal complexes 1, 3 and 4, respectively. The metal complexes were, however, Ac more toxic than paclitaxel in HeLa cervical cancer cells (IC50 values of 17.42 ± 1.14, 16.59 ± 1.16 and 26.13 ± 1.16 µM, respectively). On the other hand, metal complex 2 was non-toxic to HCC70 and HeLa cells. The non-toxicity of 2 could be due to its lower cell membrane permeability despite it having comparable DNA interactive capabilities to the other metal complexes (Refat, Sharshar, Elsabawy, El-Sayed, & Adam, 2016). In addition, the metal complexes 1 - 4 exhibited significantly lower anti-cancer cellular activities than that of the antineoplastic drug, paclitaxel. 17 4. Conclusion This research study demonstrated the DNA/ BSA interaction and antioxidant studies of diamagnetic and paramagnetic metal complexes bearing mono- or bis-heterocyclic chelates. The paramagnetic metal complex 3 showed the highest radical scavenging activities, which was ascribed to its d5 metal center enhancing its propensity to neutralize NO and DPPH radicals. t Steric factors of the metal complexes, such as their triphenylphosphine co-ligands, impeded ip DNA intercalation and led to the preferential DNA groove binding mode for these metal cr complexes. Groove binding interactions of the bis-heterocyclic metal complexes are dictated by electrostatic attractions whereas the DNA adducts of the metal complexes with the mono- us heterocyclic chelates could also be stabilized by direct coordination to the DNA base pairs. Electronic and fluorescence BSA titration spectral data of 1 – 3 portrayed unwinding of the an protein strands while that of 4 is characteristic of exposure of the tryptophan residues to aqueous media due the unfolding protein backbone. Steady-state quenching experiments show that the M interaction of each metal complex with BSA adhere to a static mechanism with single binding sites. In addition, the mono-heterocyclic metal complexes showed weaker binding affinities than d the bis-heterocyclic metal complexes based on the trends in the calculated KSV and Kapp values. pt e Low cytotoxicity of the metal complex 2 which was accounted to poor cell permeability, while ce the other metal complexes showed superior in vitro anticancer activities than 5-FU. Supporting Information Document Ac CCDC 1866502 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Supplementary figures S1-S26 associated with this article can be found in the online version. Acknowledgements: 18 This research was supported by funding from the South African Research Chairs Initiative of the Department of Science and Technology (DST) and National Research Foundation of South Africa (NRF) (Grant No. 98566), NRF CPRR (Grant No. 105829), Thuthuka NRF (Research Grant No. 94020), Incentive Funding for Rated Researchers NRF (Research Grant No. 114737), Rhodes University and University of KwaZulu-Natal. The views expressed are those of the authors and should not be attributed to the DST, NRF, Rhodes University or University of KwaZulu-Natal. 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Journal of Pharmaceutical Ac ce Sciences, 93(4), 1039-1046. doi:10.1002/jps.20005 28 Ac ce pt e d M an us cr ip t Figure 1: Structures of the respective metal complexes. 29 Figure 2: Overlay UV-Vis spectra of metal complexes 1 (a), 2 (b), 3 (c) and 4 (d) in the absence and presence of increasing amounts of CT-DNA. A dashed line indicates the initial spectrum. Ac ce pt e d M an us cr ip t Volumes of DNA solutions ranges from 0 to 36 µL. 30 Fig. 3: Analysis of interaction of the metal complexes with human genomic DNA by agarose gel electrophoresis. The average fluorescence intensity of the DNA bands was quantified by ImageJ and is shown relative to the intensity of the DMSO control (± SEM, n=3). Statistical significance Ac ce pt e d M an us cr ip t was assessed by two-way ANOVA with Bonferroni post-tests, where * indicates p < 0.05 31 Fig. 4: UV-Vis spectral profile depicting the titration between metal complex 1 and BSA. The inset is the corresponding double reciprocal plot of 1 / (A0 – A) versus 1 / Cmetal complex. Volumes Ac ce pt e d M an us cr ip t of the standardized metal complex solutions range from 0 to 59 µL. 32 Figure 5: Fluorescence emission spectral profile depicting the titration between the metal Ac ce pt e d M an us cr ip t complex 2 and BSA. Volumes of DNA solutions range from 0 to 34 µL. 33 for the metal complexes. Ac ce pt e d M an us cr ip t Figure 6: The plots of 34 Table 1: Antioxidant activities of 1 – 4 and Vitamin C against the DPPH and NO radicals as well as their corresponding intrinsic binding constants (Kb). Kb (M-1) NO Radical IC50 (µM)* IC50 (µM)* 1 45 10 1.5 x 105 2 47 12 1.2 x 107 3 29 8 9 x 104 4 40 10 2 x 105 Vitamin C 141 210 t DPPH Radical ip Metal complex - us cr *standard deviation is less than 8 % of mean values Table 2: Stern-Volmer, quenching rate and intrinsic binding constants obtained of the kq (M-1 s-1) Kb (M-1) 1 1.48 x 105 1.48 x 1013 8.34 x 104 2 1.98 x 105 1.98 x 1013 6.65 x 102 3 1.85 x 104 1.85 x 1012 5.79 x 101 4 1.11 x 104 1.11 x 1012 1.27 x 103 M KSV (M-1) ce pt e d Metal complex an composites comprised of BSA and the corresponding metal complexes. Table 3: Cytotoxicity analysis of the metal complexes against the different cancer cells. Ac Metal complexes IC50 values against HCC70 cells 2 IC50 values against HeLa cells 1 88.20 ± 1.10 µM (R = 0.9656) 17.42 ± 1.14 µM (R2 = 0.9491) 2 Non-toxic Non-toxic 3 44.07 ± 1.12 µM (R2 = 0.9702) 16.59 ± 1.16 µM (R2 = 0.9328) 4 39.37 ± 1.15 µM (R2 = 0.9593) 26.13 ± 1.16 µM (R2 = 0.9215) 5-FU 127.07 ± 8.61 µM (R2 = 0.8667) - Paclitaxel 3.06 ± 1.11 nM (R2 = 0.9595) 31.03 ± 1.15 nM (R2 = 0.9760) 35