Synthesis, characterization, and<i>in vitro</i>antioxidant and anticancer studies of ruthenium(III) complexes of symmetric and asymmetric tetradentate Schiff bases

This article was downloaded by: [New York University] On: 15 June 2015, At: 05:38 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Journal of Coordination Chemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gcoo20 Synthesis, characterization, and in vitro antioxidant and anticancer studies of ruthenium(III) complexes of symmetric and asymmetric tetradentate Schiff bases a Ikechukwu P. Ejidike & Peter A. Ajibade a a Faculty of Science and Agriculture, Department of Chemistry, University of Fort Hare, Alice, South Africa Accepted author version posted online: 06 May 2015.Published online: 14 Jun 2015. Click for updates To cite this article: Ikechukwu P. Ejidike & Peter A. Ajibade (2015): Synthesis, characterization, and in vitro antioxidant and anticancer studies of ruthenium(III) complexes of symmetric and asymmetric tetradentate Schiff bases, Journal of Coordination Chemistry, DOI: 10.1080/00958972.2015.1043127 To link to this article: http://dx.doi.org/10.1080/00958972.2015.1043127 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. 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Terms & Downloaded by [New York University] at 05:38 15 June 2015 Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions Journal of Coordination Chemistry, 2015 http://dx.doi.org/10.1080/00958972.2015.1043127 Synthesis, characterization, and in vitro antioxidant and anticancer studies of ruthenium(III) complexes of symmetric and asymmetric tetradentate Schiff bases Downloaded by [New York University] at 05:38 15 June 2015 IKECHUKWU P. EJIDIKE and PETER A. AJIBADE* Faculty of Science and Agriculture, Department of Chemistry, University of Fort Hare, Alice, South Africa (Received 12 January 2015; accepted 26 March 2015) Ruthenium(III) complexes of three tetradentate Schiff bases with N2O2 donors formulated as [RuCl (LL1)(H2O)], [RuCl(LL2)(H2O)] and [RuCl(LL3)(H2O)] were synthesized and characterized by elemental analyses, molar conductance, FTIR, and electronic spectral measurements. The FTIR data showed that the tetradentate Schiff base ligands coordinate to Ru ions through the azomethine nitrogen and enolic oxygen. The antioxidant activities of the complexes were investigated through scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals. The DPPH activity for [RuCl(LL2)(H2O)] with IC50 = 0.031 mg mL−1 was higher than the values obtained for the other Ru(III) compounds. The study revealed that the synthesized Ru(III) complexes of the tetradentate Schiff base exhibited strong scavenging activities against DPPH and moderate against ABTS radicals. In addition, the antiproliferative studies of the complexes were also tested against human renal cancer cells (TK10), human melanoma cancer cells (UACC62), and human breast cancer cells (MCF7) using the SRB assay. The results indicated that the Ru(III) complexes showed low anticancer activities against the tested human cancer cell lines. *Corresponding author. Email: pajibade@ufh.ac.za © 2015 Taylor & Francis 2 I.P. Ejidike and P.A. Ajibade Keywords: Ru(III) complexes; Tetradentate Schiff base; Spectral studies; Antioxidant; Anticancer Downloaded by [New York University] at 05:38 15 June 2015 1. Introduction Synthesis of biologically active molecules is a vigorous task and the variables affecting biological activity are diverse [1–4]. Many studies on the molecular structure of metal complexes and their bioactivity have created much awareness in the ﬁeld of bio-inorganic chemistry [5, 6]. The interaction of DNA and transition metal complexes containing multidentate aromatic ligands with a prescribed N4 or N2O2 coordination has been studied [7, 8]. Development of new therapeutic agents and DNA probes [9–11] stems from DNA binding studies, as it has inspired considerable interest in the study of the biochemical behavior of these metal compounds: interactions with DNA and serum proteins [12–15]. In the quest for small molecules that can efﬁciently bind to DNA and cleave it, Schiff bases and their metal complexes have gained recognition by various researchers and groups [16]. Schiff bases are important class of compounds widely studied for various applications [17–32]. Ruthenium Schiff base complexes have been widely studied, imperative as biochemical, analytical, and antimicrobial reagents [27, 28]. Metal complexes of Schiff bases have attracted considerable attention due to their antifungal, antibacterial, and antitumor activities [33, 34]. Physiological and biochemical processes are a pathway for generation of reactive oxygen species (ROS) through the living cells in the body [35–37]. Hence, antioxidants become important as they play vital roles toward protecting the human body against damage by ROS [38–40]. In view of growing interest in oxygenation and azomethination of Ru(III) complexes for development of new therapeutic agents and DNA probes for disease defense, we present the synthesis and characterization of some stable Ru(III) Schiff bases complexes of the type [RuCl(LL′)(H2O)]·2H2O (LL′ = H2LL1, H2LL2, H2LL3) and their antioxidant and anticancer studies. 2. Experimental 2.1. Materials and methods All reagents used were analytical grade and used as received, 2,4-pentanedione from Fluka, ethylenediamine and ascorbic acid from Merck, RuCl2·3H2O, 2′,4′-dihydroxyacetophenone and 1-phenylbutane-1,3-dione from Aldrich. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), rutin hydrate, and butylated hydroxytoluene (BHT) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Elemental analysis was obtained using a Perkin-Elmer elemental analyzer. Molar conductance of the Schiff base ligands and their Ru complexes were determined on freshly prepared 10−3 M solutions in CH2Cl2 at room temperature using a Crison EC-Meter Basic 30+ conductivity cell. IR spectra were recorded on an FT-IR spectrometer, Perkin-Elmer System (Spectrum 2000) from 4000 to 400 cm−1 using the KBr disk method. Electronic spectra were recorded on a Perkin-Elmer Lambda-25 spectrophotometer from 200 to 900 nm. Melting points were recorded with a Stuart melting point (SMP 11). Ruthenium(III) and tetradentate Schiff bases 3 2.2. General procedure for the synthesis of Schiff base ligands (H2LL1–H2LL3) Downloaded by [New York University] at 05:38 15 June 2015 2.2.1. [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)HOC6H3OH)], H2LL1. The ligand was prepared by modiﬁcation of a literature method [41]. The tetradentate Schiff base was synthesized via the method: an ethanolic solution (20 mL) containing ethane-1,2-diamine (0.01 mol, 0.601 g) was added slowly to a stirring ethanolic solution (50 mL) containing 2′,4′-dihydroxyacetophenone (0.02 mol, 3.043 g). The resulting light brown mixture was stirred and reﬂuxed for 3 h. The obtained precipitate was ﬁltered and washed with ethanol, followed by recrystallization in ethanol and air-drying to give a brownish yellow solid (Yield = 2.51 g, 76.52%). 2.2.2. [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)CH:C(CH3)OH)], H2LL2. The ligand was prepared by modiﬁcation of a literature method [42]. A typical procedure for the synthesis was as follows: ethylenediamine (0.015 mol, 0.902 g) in 30 mL ethanol was slowly added to an ethanolic solution (40 mL) containing 2′,4′-dihydroxyacetophenone (0.015 mol, 2.282 g), followed by slow addition of acetylacetone (0.015 mol, 1.502 g) dissolved in 30 mL ethanol. The resulting colored mixture was reﬂuxed with stirring for 4 h, cooled and the resultant precipitate was ﬁltered, washed several times with ethanol, followed by recrystallization in ethanol (Yield = 2.53 g, 61.23%). 2.2.3. [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)CH:C(C6H5)OH)], H2LL3. The ligand was prepared by modiﬁcation of a literature method [42]. A typical procedure for the synthesis was as follows: ethylenediamine (0.015 mol, 0.902 g) in 30 mL ethanol was slowly added to an ethanolic solution (40 mL) containing 2′,4′-dihydroxyacetophenone (0.015 mol, 2.282 g), followed by slow addition of 1-phenylbutane-1,3-dione (0.015 mol, 2.4329 g), (H2LL3) dissolved in 40 mL ethanol. The resulting colored mixture was reﬂuxed with stirring for 4 h, cooled and the resultant precipitate was ﬁltered, washed several times with ethanol, followed by recrystallization in ethanol (Yield = 3.75 g, 74.17%). 2.3. General procedure for the preparation of the complexes All operations were carried out under strictly anhydrous conditions. The various complexes were prepared by addition of RuCl3·3H2O (0.5 mmol) dissolved in 15 mL of absolute ethanol, into a hot ethanolic solution (20 mL) of H2LL1–H2LL3 ligands (0.5 mmol) in molar ratio (1 : 1). The color changed immediately. The resulting mixture was then reﬂuxed for 6 h. The precipitated solids were allowed to cool and ﬁltered off from the reaction mixture, thoroughly washed with absolute ethanol and then with diethyl ether (3 × 5 mL), and were dried over anhydrous calcium chloride. 2.4. Antioxidant assay 2.4.1. Scavenging activity of DPPH radical. The antioxidant activities for N2O2 Schiff base ligands and their synthesized Ru(III) complexes were studied spectrophotometrically by DPPH method. DPPH is known as a stable commercially available free radical, soluble in methanol to give a violet solution, which, upon reduction by an antioxidant, changes to a 4 I.P. Ejidike and P.A. Ajibade Downloaded by [New York University] at 05:38 15 June 2015 corresponding light yellow to yellow. The free radical scavenging effects of the Ru(III) compounds and Schiff base ligands with the DPPH radical were evaluated as previously described with slight modiﬁcation [43, 44]. A solution of 0.4 mM DPPH in methanol was prepared and 1.0 mL of this solution was mixed with 1.0 mL DMF solutions of Schiff base ligands and Ru(III) complexes with various concentrations (8, 17, 25, 33, and 42 μg mL−1). The reaction mixture was stirred thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm. Rutin and ascorbic acid (vitamin C) are used as standard drugs. The actual decrease in absorption was measured against that of the control. All tests and analyses were run in triplicate and the results obtained were averaged. The ability to scavenge DPPH radical was calculated by the following equation: DPPH radical scavenging activity ð%Þ ¼ Abscontrol Abssample 100 Abscontrol where Abscontrol is the absorbance of DPPH radical + DMF, and Abssample is the absorbance of DPPH radical + sample [test samples/standard]. 2.4.2. ABTS radical scavenging assay. ABTS scavenging ability of the Ru(III) compounds and Schiff base ligands was evaluated by the previously described method of Adedapo and co-workers [45]. The working solution was prepared by mixing stock solutions of 7 mM ABTS solution and 2.4 mM potassium persulfate solution in equal amounts (1 : 1) and allowing the solution to react in the dark for 12 h at room temperature. The resulting solution was further diluted by mixing 1 mL ABTS+ solution to obtain an absorbance of 0.706 ± 0.001 units at 734 nm using the spectrophotometer. Test samples (1 mL) were allowed to react with 1 mL of the ABTS+ solution, followed by the absorbance reading at 734 nm after 7 min using the spectrophotometer. The ABTS scavenging capacities of the Ru(III) compounds and Schiff base ligands were compared with that of rutin and BHT (standard drugs). All tests were run in triplicate, and the results obtained were averaged. The percentage inhibition was calculated as ABTS radical scavenging activity using the following equation: ð%Þ Inhibition ¼ Abscontrol Abssample 100 Abscontrol where Abscontrol is the absorbance of ABTS radical + DMF, and Abssample is the absorbance of ABTS radical + sample [test samples/standard]. 2.5. Cell lines and culture conditions Human renal cancer cell line (TK10), human melanoma cancer cell line (UACC62), and human breast cancer cell line (MCF7) were obtained from NCI in the framework a collaborative research program between CSIR and NCI. Cell lines were routinely maintained as a monolayer cell culture at 37.0 °C with 5% CO2, 95% air and 100% relative humidity in RPMI medium which is supplemented with 5% fetal bovine serum, 2 mM L glutamine and 50 μg mL−1 gentamicin. Ruthenium(III) and tetradentate Schiff bases 5 Downloaded by [New York University] at 05:38 15 June 2015 2.6. Cell viability assay Cell viability was examined by Sulforhodamine B (SRB) assay as previously described [46, 47]. The cells (TK10, UACC62, and MCF7) (3–19 passages) were inoculated into 96-well microtiter plates at plating densities of 7–10,000 cells/well and were incubated for 24 h. After 24 h, the cells were treated with the experimental compounds which were previously dissolved in DMSO and diluted in medium to produce concentrations of 0.01, 0.1, 0, 10, and 100 μM. Cells without drug addition served as control. The blank contains complete medium without cells. Parthenolide was used as a standard. The plates were incubated for 48 h after addition of the compounds. Viable cells were ﬁxed to the bottom of each well with cold 50% trichloroacetic acid, washed, dried and dyed by SRB. Thereafter, the unbound dye was removed, and protein-bound dye was extracted with 10 mM Tris base for optical density determination at 540 nm using a multiwell spectrophotometer. Data analysis was performed using GraphPad Prism software; 50% of cell growth inhibition (IC50) was determined by non-linear regression. The Z′-factor coefﬁcient was adapted to monitor the quality of immunocytochemical assays such as the SRB. 3. Results and discussion 3.1. Synthesis Mononuclear ruthenium(III) complexes, [RuCl(LL′)(H2O)] (LL′ = H2LL1, H2LL2, H2LL3) (LL′ = dibasic tetradentate Schiff base ligand), were synthesized in good yields from reaction of RuCl3·3H2O with Schiff base ligands in 1 : 1 M ratio in absolute EtOH to give sixcoordinate ruthenium(III) Schiff base complexes according to the equation: RuCl3 3H2 O þ H2 LL0 ! ½RuClðLL0 ÞðH2 OÞ þ 2HCl þ 2H2 O where H2LL = H2LL1 = [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)HOC6H3OH)], H2LL2 = [OHC6H3OHC(CH3):N(C2H4)N:C(CH3)CH:C(CH3)OH)] and H2LL3 = [OHC6H3OHC (CH3):N(C2H4)N:C(CH3)CH:C(C6H5)OH)] (scheme 1). The synthesized mononuclear ruthenium(III) Schiff base complexes (ﬁgure 1) are stable in air at room temperature, nonhygroscopic and insoluble in water, partially soluble in common solvents such as dichloromethane, acetonitrile, chloroform, but easily soluble in polar solvents such as DMF and DMSO producing intense color in their solutions. The solubility of the complexes may be due to the presence of chlorides [48] and hydroxyl groups on the benzene ring [49]. The tetradentate N2O2 donor site of LL′ (H2LL1, H2LL2, H2LL3) is capable of forming complexes with ruthenium. The analytical data are listed in table 1 and are in agreement with the proposed formulations for the complexes. 3.2. Infrared spectra Important IR absorptions for the complexes are shown in table 2. The observed bands have been classiﬁed into those originating from the ligands and those arising from the bands formed between ruthenium(III) and the coordinating sites. IR spectra of the free ligands showed bands at 3475–3479, 2873–3076, 1605–1616, 1470–1588, and 1171–1288 cm−1 6 I.P. Ejidike and P.A. Ajibade H3C H2N NH2 O H3C C OH + N NH2 Ethanol OH Stirred, Reflux OH HO H3C CH3 C N N OH HO H 8O 3 lux Ref ol, n a Eth Downloaded by [New York University] at 05:38 15 June 2015 C8 N NH2 OH HO 1 OH H3C H3C C H2 LL HO C CH3 C N N C OH HO C C5 H8 O2 C Ethanol, Reflux C H2 LL HO 10 H Et 10 O ha no 2 l, Re fl u x CH3 2 H3C CH3 C N N C OH HO C C HO H2 LL 3 Scheme 1. Synthesis of H2LL1–H2LL3. assignable to ν(OH), ν(CH3/CH2), ν(C=N), ν(C=C), and ν(C–O), respectively [20, 49, 50]. H2LL1, H2LL2 and H2LL3 show a very strong absorption at 1605–1616 cm−1 in their IR spectra, characteristic of the azomethine ν(C=N) (table 2). In the Schiff base complexes, this absorption shifted to 1621–1623 cm−1 indicating coordination of the Schiff bases through nitrogen in accord with coordination of the azomethine function to the metal ion for all the complexes [28, 50, 51]; this shift of wavenumber is expected due to coordination of nitrogen of the azomethine group to ruthenium, thereby reducing electron density in the azomethine [50, 52]. A medium band corresponding to phenolic oxygen ν(C–O) is observed at 1171– 1288 cm−1 for the free ligands. Upon chelation, this band shifted to lower frequency (1470–1543 cm−1) for all the ruthenium(III) Schiff base complexes [28, 53]. This indicates enolization of >C=O followed by deprotonation and complexation with metal and the destruction of keto group presumably viz., enolization and ketolization bonding of the ligand through the resulting enolate and ketolate oxygen. This is further supported by the disappearance of ν(OH) at 3475–3479 cm−1 in the complexes. The presence of coordinated water at 3419–3435 and 846–861 cm−1, due to ν(O–H) stretching and ν(O–H) rocking vibrations, respectively, further conﬁrmed the presence of water [49, 54, 55]. In the low frequency Ruthenium(III) and tetradentate Schiff bases H3C C Cl N CH3 N C Ru O 7 . 2H 2O O H2O HO OH [a] H3C Downloaded by [New York University] at 05:38 15 June 2015 C Cl N CH3 N C O C Ru O C H2O . 2H 2O CH3 HO [b] H3C C N Cl CH3 N C O C Ru O C . 2H 2O H2O HO [c] Figure 1. Proposed structure for the Ru(III) complexes (a) [RuCl(LL1)(H2O)]·2H2O, (b) [RuCl(LL2) (H2O)]·2H2O, and (c) [RuCl(LL3)(H2O)]·2H2O. region, the observed bands at 519–535 and 415–437 cm−1 are probably due to the formation of ν(M–N) and ν(M–O) vibrations, respectively [41, 56, 57]. 3.3. Molar conductivity measurements The molar conductivity (Λμ) values of the Ru(III) complexes in 10−3 M DMF solution (table 1) at room temperature are 23.8–47.4 μS cm−1, indicating the essential non-electrolytic character of the compounds [1, 58]. 3.4. The antioxidant assay Oxidative reactions of biological molecules induce a variety of pathological events such as cellular injury and aging process, and these damaging events are caused by free radicals [59, 60]. Therefore, to prevent free radical damage in the body, it is important to administer drugs that may be rich in antioxidants. The antioxidant activity of ligands and their metal complexes have been investigated using the in vitro method [43, 61]. However, the antioxi- 516.92 275.32 463.88 337.40 525.95 C18H24N2O7RuCl C15H19N2O3 C15H23N2O6RuCl C20H21N2O3 C20H25N2O6RuCl [RuCl(LL1) (H2O)]·2H2O H2LL2 [RuCl(LL2) (H2O)]·2H2O H2LL3 [RuCl(LL3) (H2O)]·2H2O 328.36 C18H20N2O4 H2LL1 F.wt Compound Orange-brown Darkish-green Golden-yellow Darkish-green Brownishyellow Dark-green Color 74.17 53.67 61.23 76.20 60.28 76.52 Yield (%) 70.96 (71.20) 45.88 (45.67) 65.26 (65.44) 38.75 (38.84) 42.08 (41.82) 5.13 (5.27) 4.56 (4.79) 7.13 (6.96) 4.81 (5.00) 4.43 (4.68) 6.28 (6.14) H % Found (Calcd) 65.73 (65.84) C Analytical data and physical properties of the N2O2 Schiff bases and their Ru complexes. Empirical formula Table 1. 8.09 (8.30) 5.18 (5.33) 9.98 (10.17) 5.83 (6.04) 5.31 (5.42) 8.71 (8.53) N Downloaded by [New York University] at 05:38 15 June 2015 211 231 235 228 239 244 Decomp. temp (°C) – 23.8 – 30.6 47.4 – Conductance (μS cm−1) 8 I.P. Ejidike and P.A. Ajibade Ruthenium(III) and tetradentate Schiff bases Table 2. FTIR spectral data of the N2O2 Schiff bases and their Ru complexes (cm−1). Compound ν(OH) ν(CH3/CH2) ν(C=N) ν(C=C) ν(C–O) ν(Ru–N) ν(Ru–O) 1 3475 3435 3475 3419 3479 3430 2929, 2873 2977, 2845 2928, 2911 2959, 2844 3076, 2981 2997, 2901 1616 1622 1612 1621 1605 1623 1588, 1532 1530, 1479 1534, 1480 1543, 1525 1543, 1470 1543, 1508 1267, 1173 1244, 1170 1243, 1171 1243, 1168 1288, 1241 1258, 1138 – 535 – 522 – 519 – 437 – 428 – 415 H2LL [RuCl(LL1)(H2O)]·2H2O H2LL2 [RuCl(LL2)(H2O)]·2H2O H2LL3 [RuCl(LL3)(H2O)]·2H2O Downloaded by [New York University] at 05:38 15 June 2015 9 dant mechanism of the complexes has not been explained so far [62]. The antioxidant assay study was carried out using different concentrations of the test samples (Schiff base ligands and the Ru(III) complexes) with DPPH and ABTS radicals, while ascorbic acid, rutin, and BHT were used as standards in order to establish some structure antioxidant activity relationship. 3.4.1. DPPH radical scavenging assay. DPPH is a compound widely used to examine the ability of a given sample to act as free radical scavengers or hydrogen donors and to evaluate antioxidant activity of foods [63]. The results of the DPPH radical scavenging abilities of H2LL1, H2LL2 and H2LL3 and the Ru complexes were studied and compared with the standard (ascorbic acid and rutin). The Ru(III) complexes exhibited signiﬁcant DPPH radical scavenging ability in all the concentrations used, i.e. chelated Ru(III)-Schiff base complexes were more effective free radical scavengers than the corresponding free H2LL1, H2LL2, and H2LL3 Schiff bases; this could be attributed to the acquisition of additional superoxide dismutating centers [64]. However, the Ru(III) complexes showed comparable or higher scavenging activity compared to the standards (ascorbic acid and rutin) with [RuCl(LL2)(H2O)] showing signiﬁcantly higher scavenging ability. The DPPH radical scavenging ability of the Ru(III) complexes can be ranked, [RuCl(LL2)(H2O)] > [RuCl(LL3)(H2O)] > [RuCl(LL1)(H2O)]. IC50 values of Schiff bases H2LL1, H2LL2, and H2LL3 on DPPH radical are 0.067 ± 0.006, 0.065 ± 0.001, and 0.055 ± 0.002 mg mL−1, respectively, whereas [RuCl(LL1)(H2O)], [RuCl(LL2)(H2O)], and [RuCl(LL3)(H2O)] showed IC50 values at 0.041 ± 0.003, 0.031 ± 0.006, and 0.036 ± 0.002 mg mL−1, respectively (table 3). Therefore, the scavenging effect of the free ligand is lower compared to that of their corresponding Ru(III) complexes, Table 3. DPPH and ABTS radical scavenging capacities (IC50 ± SD, mg mL−1) of standard drugs, N2O2 Schiff bases, and their Ru complexes. Compound DPPH ABTS 1 0.067 ± 0.006 0.041 ± 0.003 0.065 ± 0.001 0.031 ± 0.006 0.055 ± 0.002 0.036 ± 0.002 0.045 ± 0.005 0.037 ± 0.009 0.008 ± 0.0003 0.011 ± 0.0005 0.009 ± 0.0004 0.036 ± 0.0027 0.009 ± 0.0006 0.025 ± 0.0047 0.023 ± 0.0035 0.004 ± 0.0003 H2LL [RuCl(LL1)(H2O)] H2LL2 [RuCl(LL2)(H2O)] H2LL3 [RuCl(LL3)(H2O)] Vitamin C Rutin Note: (n = 3, X ± SEM), IC50 – inhibitory concentration. 10 I.P. Ejidike and P.A. Ajibade Downloaded by [New York University] at 05:38 15 June 2015 related to chelation of the organic molecules with the metal ions. Also, the violet color of DPPH radical changed to yellow upon addition of Ru(III) compounds because proton from the test samples were transferred to DPPH, converting it into the corresponding hydrazine form [40]. Thus, these compounds could be a promising therapeutic agents to treat stress-induced pathological conditions such aging, cancer, and cardiovascular and neurodegenerative diseases. 3.4.2. ABTS radical scavenging activity. ABTS cation radicals (ABTS+) are produced by oxidation of ABTS with potassium persulfate and, thus, are reduced in the presence of hydrogen-donating antioxidants [65]. This has been the basis of one of the spectrophotometric methods applied to measurement of the total antioxidant activity of solutions of pure substances and aqueous extracts [66, 67]. The method described gives a measure of the antioxidant activity of test samples determined by the decolorization of ABTS+ through measuring the reduction of the radical cation as the percentage inhibition of absorbance at 734 nm [68]. H2LL1, H2LL2 and H2LL3 and their Ru complexes exhibited low-to-moderate scavenging ability of the ABTS radical (ﬁgure S2) and showed comparable or slightly lower activity to that of rutin and BHT (standard drugs). At a concentration of 4.35 μg mL−1, the percentage inhibition was 60.2, 66.4, 59.8, 54.8, and 50.6% for [RuCl(LL1)(H2O)], [RuCl(LL2)(H2O)], [RuCl(LL3)(H2O)], rutin, and BHT, respectively. Nevertheless, the ABTS activities of the Ru(III) complexes were signiﬁcantly enhanced compared to their corresponding free Schiff base ligands. Lowest concentration of the H2LL1, H2LL2 and H2LL3 and its Ru complexes were more effective in quenching ATBS radicals in the system. IC50 value of H2LL1, H2LL2, and H2LL3 on ABTS radical is 0.067 ± 0.006, 0.065 ± 0.001, and 0.055 ± 0.002 mg mL−1, respectively, while the IC50 values at 0.011 ± 0.0005, 0.036 ± 0.0027, and 0.025 ± 0.0047 mg mL−1 are for [RuCl(LL1) (H2O)], [RuCl(LL2)(H2O)], and [RuCl(LL3)(H2O)], respectively (table 3). Furthermore, the synthesized compounds scavenged the ABTS radical in a concentration-dependent pattern. Generally, the scavenging activities of the DPPH radical by H2LL1, H2LL2, and H2LL3 and the Ru(III) complexes are higher than that of ABTS radical. Wang and co-workers [69] reported that some compounds which exhibited ABTS scavenging activity did not possess DPPH scavenging activity. Hence, this study revealed the synthesized tetradentate Schiff base Ru(III) complexes exhibited strong scavenging activities against DPPH and moderate activity against ABTS radicals. This result shows that the compounds in this study can scavenge different free radicals in different systems, indicating that they may be useful as therapeutic agents for treating pathological damage associated with radical generation. 3.5. Antiproliferative activity evaluation The in vitro anticancer activities of Ru(III) complexes and parthenolide (at various concentrations ranging from 0.01 to 100 μM) were evaluated using three cancer cell lines: human renal cancer cell (TK10), human melanoma cancer cell (UACC62), and human breast cancer cell (MCF7) using the SRB assay and parthenolide was used as standard. Parthenolide demonstrated high levels of antiproliferative effect against all cell lines, in accord with previous reports [70]. The values of the concentration of the compounds for 50% inhibition (IC50) were obtained from non-linear regression analysis of dose response data for the compounds tested and are presented in table 4. The Ru(III) complexes demonstrate lowto-moderate in vitro antiproliferative effect compared to parthenolide (standard agent) Ruthenium(III) and tetradentate Schiff bases Table 4. lines*. 11 IC50 values (μM) of Ru(III) complexes and parthenolide against human cell Antiproliferative activity IC50 (μM) 48 h Compound 1 [RuCl(LL )(H2O)]·2H2O [RuCl(LL2)(H2O)]·2H2O [RuCl(LL3)(H2O)]·2H2O Parthenolide TK-10 UACC-62 MCF-7 >100 >100 >100 4.64 ± 1.43 >100 >100 >100 11.37 ± 2.18 >100 90 ± 8 90 ± 5 3.52 ± 2.02 Downloaded by [New York University] at 05:38 15 June 2015 *Cells were treated with various concentrations of tested compounds for 48 h. IC50 values were calculated as described in Section 2. Each value represents the mean ± SD of three independent experiments (Z′ factor > 0.5). against selected tumor cell lines. [RuCl(LL1)(H2O)]·2H2O displayed non-selective antiproliferative activity against all tumor cells tested, while [RuCl(LL2)(H2O)]·2H2O and [RuCl(LL3)(H2O)]·2H2O had a low antiproliferative effect with IC50 at 90 μM against MCF-7 (Z′ factor > 0.5). The inhibition effects were enhanced by increasing the concentration of the Ru(III) complexes. The results showed that [RuCl(LL2)(H2O)]·2H2O was more active against all the selected tumor cells than [RuCl(LL1)(H2O)]·2H2O and [RuCl(LL3) (H2O)]·2H2O (Z′ factor > 0.5), which is in agreement to their order of in vitro DPPH scavenging ability of the Ru(III) complexes. Binding of the three N2O2 Schiff base Ru(III) complexes to biological targets other than DNA could be responsible for the observed antiproliferative activity of the complexes. 4. Conclusion Ru(III) complexes of symmetric and asymmetric Schiff base ligands derived from ethane1,2-diamine, 4-acetylresorcinol, acetylacetone, and 1-phenylbutane-1,3-dione were synthesized and characterized. Conductance measurements revealed non-electrolytes. The ligands are dibasic, ONNO tetradentate, coordinated to the Ru(III) through the phenolic oxygen and imino nitrogen; octahedral geometry around the Ru(III) ions is completed by H2O and a Cl−. In vitro anticancer studies of the Ru complexes show that they are inactive against human cancer cells (TK10) and human melanoma cancer cell (UACC62) but show mild activity against human breast cancer cell line (MCF7). The synthesized complexes exhibited low biological activities as potential anticancer agents. Derivatization of the Schiff base by substituting CH3 on the C2 and C4 position of acetylacetone could increase the activity of the complexes. Nevertheless, antioxidant activities of the complexes exhibited moderate to strong free radical inhibitors or scavenger for treating pathological damage associated with radical generation. Disclosure statement No potential conﬂict of interest was reported by the authors. Funding This work was ﬁnancially supported by the Govan Mbeki Research and Development (GMRD), University of Fort Hare. 12 I.P. Ejidike and P.A. Ajibade Supplemental data Supplemental data for this article can be accessed here [http://dx.doi.10.1080/00958972.2015.1043127]. Downloaded by [New York University] at 05:38 15 June 2015 References [1] C.A. Bolos, A.T. Chaviara, D. Mourelatos, Z. Iakovidou, E. Mioglou, E. Chrysogelou, A. Papageorgiou. Bioorg. Med. Chem., 17, 3142 (2009). [2] X. Zou, L. Zou, Y. He, C. Bünger. Cancer Treat. Rev., 34, 527 (2008). [3] J. Matysiak. Eur. J. Med. Chem., 42, 940 (2007). [4] M. Šoškić, V. Magnus. Bioorg. Med. Chem., 15, 4595 (2007). [5] B. Gillon, C. Mathonière, E. Ruiz, S. 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Synthesis, characterization, andin vitroantioxidant and anticancer studies of ruthenium(III) complexes of symmetric and asymmetric tetradentate Schiff bases