α-N-Heterocyclic Thiosemicarbazone Derivatives as

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Development of New Potential Anticancer Metal Complexes Derived from 2-Hydrazinobenzothiazole Shadia A. Elsayeda, Entsar A. Saad*a and Sahar I. Mostafa1

a

Chemistry Department, Faculty of Science, Damietta University, Damietta, Egypt; 1Chemistry Department, Faculty of Science, Mansoura University, Mansoura, Egypt

Abstract:

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Background: Due to the bad side effects of clinically approved anticancer drugs there is a great need to explore and develop new metal-based anticancer drug molecules of high efficiency with less or no side effects. Objective: To synthesize new metal complexes of 2-hydrazinobenzothiazole (hbt) and to investigate their potential anticancer characteristics. Method: New five complexes; [VO(hbt)2SO4].4H2O (1), [Ru(hbt)2Cl3(H2O)] (2), [M(hbt)2Cl2] [M(II) = Pd (3), Pt (4)] and [Ag(hbt)2].NO3 (5) were prepared and their structure was investigated by means of FT-IR, 1H NMR, ESI-MS and UV-Vis spectra, elemental and thermal analysis, magnetic and molar conductance measurements. The ligand and its complexes were examined as anticancer agents against Ehrlich ascites carcinoma (EAC) and human cancer cells (hepatocellular carcinoma Hep-G2, mammary gland breast cancer MCF-7 and colorectal carcinoma HCT-116). This feature is further supported by the DNA-metal complexes binding ability. In addition, anti-oxidation activity of the complexes was investigated. Conclusion: Complex (5) showed the highest antioxidation and anticancer activities. Keywords: Anticancer, Antioxidant, Cytotoxic, DNA, Palladium, Silver, Spectra.

1. INTRODUCTION Cancer is one of the most common causes of death worldwide [1,2]. Consequently, there is an extensive research to explore and develop new drug molecules of high efficiency with less or no side effects. Metal complexes have attracted increasing interest in medicinal chemistry field since cisplatin, cis-[PtCl2(NH3)2] was approved for clinical use [3,4]. However, its clinical application was limited due to its side effects and that initiated the research for other alternatives of platinumbased anticancer drugs [2]. Heterocyclic compounds comprising of nitrogen and sulfur atoms are of a remarkable interest from the researchers in chemistry and medicinal chemistry fields [5]. Among those, benzothiazole was found to be the most useful moiety, which used as a precursor for the synthesis of numerous compounds with a wide range of biological activities [6,7] as well as in radioactive amyloid imaging agents [8]. In addition, the change in their structure at 2-substituent position at C2 is of great interest to study the structure-activity relationship (SAR) which may result in a commonly change of its bioactivity and afford a new approach for the new therapeutic agents [9]. Benzothiazole based compounds have been used clinically to treat several types of diseases with therapeutic potency [10]. 2-Aminothiazole, 2aminobenzothiazole and their Schiff-bases (nitrogen-sulfur donors), have ability to be involved in coordination with metal ions with wide range of biological applications (antimicrobial, anti-inflammatory, anti-degenerative and anti-HIV agents) [11]. Recently, series of transition metal XXX-XXX/14 $58.00+.00

complexes with 2-mercaptobenzothiazole and 2aminobenzothiazole have been reported. The in vitro anticancer activities of these complexes have been investigated versus human breast cancer (MDA-MB231) and human ovarian cancer (OVCAR-8) cell lines. Moreover, CT-DNA binding mechanisms of these complexes have been discussed [12,13]. In continuation of our interest to develop new anticancer agents, this study is dealing with the preparation of V(IV), Ru(III), Ag(I), Pd(II) and Pt(II) complexes with 2hydrazinbezothiazole (hbt). Their structure was discussed by using physico-chemical techniques (IR, 1H NMR, ESI-MS and UV-visible spectroscopy), elemental analysis, thermal stability, magnetic and molar conductance measurements. The complexes were tested for their cytotoxicity against Ehrlich ascites carcinoma (EAC) and human tumor cells (hepatocellular carcinoma Hep-G2, mammary gland breast cancer MCF-7 and colorectal carcinoma HCT-116). Their DNA interaction and anti-oxidation activity were also examined. 2. MATERIALS AND METHOD 2.1 Materials All chemicals and solvents were purchased from Alfa-Aesar and used without further purifications. EAC cell line was obtained from the Nile Center for Experimental Research, Mansoura, Egypt. Human tumor cell lines (HepG2, MCF-7 and HCT-116) were obtained from American Type Culture Collection (ATCC) via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. © 2014 Bentham Science Publishers

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Cisplatin (Myllal, France) was purchased from local pharmacy. RPMI-1640 medium, MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), dimethyl sulfoxide (DMSO), DNA/methyl green, L-ascorbic acid and 5-fluorouracil were obtained from Sigma Company, St. Louis, USA. Fetal bovine serum was purchased from GIBCO, UK. 2,2′-Azo-bis-(2-amidinopropane) dihydrochloride (AAPH) and 2,2′-azino-bis-3ethylbenzthiazoline-6-sulphonic acid (ABTS) were purchased from Wak. 2.2 Measurements IR and 1H NMR spectra were performed in Chemistry Department, McGill University, Montreal, Canada spectra on a Nicolet 6700 Diamond spectrometer (powder phase in 400-4000 cm-1 range) and VNMRS 400 or 500 MHz spectrometer in DMSO-d6 using TMS as an internal reference, respectively, also the mass spectra (ESIMS) were recorded using LCQ Duo and double focusing MS25RFA instruments, respectively. Elemental analyses were obtained from microanalysis unit in Cairo University, Egypt. Molar conductivity measurements were carried out at room temperature on YSI Model 32 Conductivity Bridge. UV-Vis spectroscopic data (max, nm) were recorded in DMF using JASCO V- 630 spectrometer. Thermal Gravimetric Analyses (TGA) data were collected in the temperature range 20 – 800 oC under nitrogen atmosphere 15.00 mL/min using Shimadzu TGA-50 instrument at heating rate of 20 o C/min. 2.3 Preparation of Complexes 2.3.1[VO(hbt)2SO4]. 4H2O (1) An aqueous solution of VOSO4.H2O (0.045 g, 0.25 mmol; 2 mL) was added to 5 mL ethanolic solution of hbt (0.082 g, 0.5 mmol). The dark green solution was stirred at 60 - 70 oC for 4 h. Upon evaporation at room temperature, a dark green precipitate was filtered off, washed with EtOH and Et2O, and then dried in vacuo. Yield: 65%; Elemental analysis for C14H22N6O9S3V: Calcd: C, 29.7; H, 3.9; N, 14.8 %, Found: C, 29.2; H, 3.5; N, 14.4 %. Conductivity data (10-3 M, DMF): M = 121 cm2 mol-1. IR (powder film,, cm-1): (NH2), 3300, 3180 (br); (NH), 3100; (NH2), 1631; (C=N)thiazole, 1540; (SO4), 1259, 1108, 955; (N-N), 1031; (V=O), 967; ν(MN), 480. ESI-MS (m/z): 492.05 (Calc. 492.9) and 327.33(Calc. 326). UV-Vis (DMSO, λmax, nm): 272, 329, 586. eff = 1.78 B.M. 2.3.2 [Ru(hbt)2Cl3(H2O)] (2) An ethanolic solution of hydrated (RuCl3.3H2O, 0.065 g, 0.25 mmol; 5 mL) was added to hbt (0.082 g, 0.5 mmol) in 5 mL ethanol. The reaction mixture was refluxed for 6 h, upon which a purple precipitate was formed. The isolated product was filtered off, washed with EtOH, Et2O then dried in vacuo. Yield: 63 %; Elemental analysis for C14H16Cl3N6ORuS2: Calcd: C, 30.3; H, 2.9; Cl, 19.1; N, 15.1%. Found: C, 30.0; H, 2.8; N, 14.7 %. Conductivity data (10-3 M, DMF): M = 22 -1 cm2 mol-1; IR (powder film, , cm-1): (NH2), 3330 (br); (NH), 3055; (NH2), 1616; (C=N)thiazole, 1558; (N-N), 1065; ν(M-N), 489. ESI-MS (m/z): 467.3 (Calc. 466.5) and 430.2 (Calc. 431.0). UV-Vis (DMSO, λmax, nm): 291, 411, 713. eff = 1.70 B.M.

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2.3.3 [M(hbt)2Cl2] (M(II) Pd (3), Pt (4)) An aqueous solution of K2PdCl4 or K2PtCl4 (0.25 mmol; 2 mL) was added to hbt (0.082 g, 0.5 mmol) in 5 mL EtOH. Upon stirring the reaction mixture for 3 h at room temperature, an orange (Pd) or green (Pt) precipitate was obtained. It was filtered off, washed with EtOH, Et2O and dried in vacuo. For [Pd(hbt)2Cl2]: Yield: 60%; Elemental analysis for C14H14Cl2N6PdS2: Calcd.: C, 33.1; H, 2.8; N, 16.5 %, Found C, 32.7; H, 2.6; N, 16.1; Conductivity data (10-3 M, DMF): M = 17 -1 cm2 mol-); IR (powder film, , cm-1): (NH2), 3206; (NH), 3041; (NH2); (NH2), 1619, (C=N)thiazole, 1587; (N-N), 1022, (M-N), 462; 1HNMR (, ppm): ((NH2), 9.15, 9.27 (s, 2H); (NH), 11.26 (s, 1H); (H6), 8.27, 8.72 (d, 1H); (H5), 7.50, 7.70 (t, 1H); (H4), 7.23, 7.26 (t,1H); (H7), 7.28 (d,1H). ESI-MS (m/z): 508.76 (Calc. 507.75), 471.91 (Calc. 472.25) and 435.9 (Calc. 436.75). UV-Vis (DMSO, λmax, nm): 270, 319, 469. For trans-[Pt(hbt)2Cl2]: Yield: 66 %; Elemental analysis for C14H14Cl2N6PtS2: Calcd.: C, 28.2; H, 2.4; N, 14.1 %, Found: C, 28.0; H, 2.2; N, 14.0 %; Conductivity data (10-3 M, DMF): M = 18 -1 cm2 mol-): IR (powder film, , cm-1): (NH2), 3217; (NH), 3040; (NH2), 1608, (C=N), 1584; (N-N), 1024, (M-N), 470 : 1HNMR (, ppm): ((NH2), 8.73 (s, 2H); (NH), 9.20 (s, 1H); (H6), 8.74 (d, 1H); (H5), 7.72 (t, 1H); (H4), 7.24 (t,1H); (H7), 7.10 (d,1H). ESI-MS (m/z): 596.1(Calc. 596.1) and 560.48 (Calc.560.0). UV-Vis (DMSO, λmax, nm): 292, 334, 442. 2.3.4 [Ag(hbt)2]NO3 (5) Silver nitrate (0.043 g, 0.25 mmol) was dissolved in 2 mL water then added to 5 mL ethanolic solution of hbt (0.082 g, 0.5 mmol). The reaction mixture was stirred at room temperature for 4 h in the dark. The white precipitate obtained was filtered off, washed with water, EtOH and airdried in dark. Yield: 65%; Elemental analysis for C14H14AgN7O3S2: Calcd. C, 33.6; H, 2.8; N, 19.6. Found: C, 33.4; H, 2.5; N, 19.2. Conductivity data (10-3 M, DMF): M =167 -1 cm2 mol-1; IR (, cm-1): (NH2), 3329, 3245; (NH), 3154; (NH2), 1637, (C=N), 1562; (N-N), 1022, (NO3), 1370, (M-N), 465; 1HNMR (, ppm): ((NH2), 5.75 (s, 2H); (NH), 9.71 (s, 1H); (H6), 7.84, (d, 1H); (H5), 7.53 (t, 1H); (H4), 7.33 (t,1H); (H7), 7.15 (d,1H). ESI-MS (m/z): 438.1 (Calc. 438.3). UV-Vis (DMSO, λmax, nm): 273, 304. 2.4 Biological Studies 2.4.1 Trypan Blue Assay The anticancer activity of the complexes against EAC was evaluated using trypan blue exclusion [14]. Compounds concentrations that cause 50% cell growth inhibition (IC50) were calculated. 2.4.2 MTT Assay MTT assay was applied to determine the inhibitory effects of the complexes against different human cell lines (Hep-G2, MCF-7 and HCT-116). MTT assay is based on the conversion of the yellow tetrazolium bromide to a purple formazan derivative by mitochondrial succinate dehydrogenase in viable cells. The cells were cultured in RPMI-1640 medium with 10% fetal bovine serum in presence of 100 units/mL penicillin and 100 µg/mL

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streptomycin antibiotics at 37 oC in a 5% CO2 incubator for 48 h in 96-well plate at a density of 1.0 X 104 cells/well [15]. After incubation, the cells were treated with different concentrations of the inhibitors (the complexes) and incubated again for 24 h. MTT solution (20 µL, 5 mg/mL) were added and incubated for 4 h, DMSO (100 µL) was added to each well to dissolve the purple formazan formed. The absorbance was measured at 570 nm using a plate reader (EXL 800, USA) and the percent relative cell viability was calculated using eq. 1. % Relative cell viability = (Atreated sample /Auntreated sample) X 100 (1) 2.4.3 DNA/Methyl Green Colorimetric Assay Methyl green reversibly binds polymerized DNA, and the complex is stable at neutral pH, whereas free methyl green fades. Incubation in the buffer used for displacement reactions for 24 h results in virtually a complete loss of methyl green absorbance. A colorimetric assay was used to measure the displacement of methyl green from DNA by compounds with ability to bind to DNA. The displacement was spectrophotometrically determined by decreasing in absorbance at 630 nm [16]. 20 mg of DNA/methyl green were suspended in 100 mL of 0.05 M Tris-HCl buffer, pH 7.5, containing 7.5 mM MgSO4 and stirred at 37 °C for 24 h. Compounds under test were dissolved in Eppendorf tubes. The solvent was removed under vacuum, and 200 μL of the DNA/methyl green solution were added to each tube. Reaction mixtures were incubated in the dark at ambient temperature. The final absorbance of the samples was determined at 630 nm after 24 h. IC50's were calculated. 2.4.4 Erythrocyte Hemolysis Assay Blood was obtained from adult male albino rats (160-180 g), housed under controlled standard conditions according to National Institute of Health (1996), by cardiac puncture and collected in heparinized tubes. The RBCs were separated from plasma and the buffy coat was washed with (3 x 10 mL) of 0.15 M NaCl. The RBCs were centrifuged at 2500 rpm for 10 min to obtain a constantly packed cell preparation. In this assay, RBCs hemolysis was mediated by peroxyl radicals. Equal volumes of RBCs (10% suspension) at pH 7.4 [phosphate-buffered saline (PBS)], and AAPH (200 mM in PBS) containing the tested compound at different concentrations were mixed. The obtained mixture was shaken gently while being incubated at 37 °C for 1 h, followed by dilution with eight volumes of PBS and centrifuged at 2500 rpm for 10 min. The absorbance (A) of the supernatant was measured at 540 nm. Similarly, the reaction mixture was treated with eight volumes of distilled water to achieve complete hemolysis, and the absorbance (B) of the supernatant was measured. % Hemolysis was calculated by eq. (2), and L-ascorbic acid was used as a positive control [17]. % Hemolysis = (1 -A/B) X 100 (2) 2.4.5 ABTS Assay ABTS (60 mM, 2 mL) was mixed with 3M MnO2 (25 mg/mL) in phosphate buffer (pH 7, 0.1 M). The resulting mixture was centrifuged and filtered to obtain green-blue solution (ABTS radical). The absorbance (Ac) of the resulting solution was adjusted at ca. 0.5 at 734 nm. To which the tested complex (50 µL, 2 mM) in methanolphosphate buffer (1:1) was added, and the absorbance (At)

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was measured. and % inhibition was calculated by applying eq. (3) [18]. % Inhibition = (Ac – At)/Ac X 100 (3) Vitamin C was used as the standard antioxidant (positive control), while blank sample was carried out using methanol/phosphate buffer (1:1) without ABTS. Negative control sample was carried out with methanol/phosphate buffer (1:1) instead of the tested compound. Our study was approved by the Animal House of Biochemistry, Chemistry Department, Faculty of Science, Damietta University, Egypt. 2.5 Statistical Analysis Data are expressed as mean ± SD, from 3 – 5 separate determinations. Student’s-test was used to evaluate the statistical significance and p < 0.05 was considered significant.

4. RESULTS AND DISCUSSIONS The complexes were prepared by following a general procedure by dissolving hbt in EtOH and the corresponding metal salt in water in molar ratio (1:2) at room temperature. In case of ruthenium complex (2), the reaction mixture was taken place in EtOH under reflux. All preparations were performed under aerobic conditions. All isolated complexes are colored however complex (5) is white, stable at normal conditions, insoluble in common organic solvent such as methanol, EtOH, acetonitrile, and dichloromethane, but readily soluble in DMF and DMSO. Attempts performed to grow a single crystal were unsuccessful. The molar conductivity measurements (ΛM) in DMF, suggest the non-electrolytic nature of the complexes (12 -22 -1 cm2 mol-1) except for (5) (167 -1 cm2 mol-1) which shows 1:1 behavior. 4.1 Vibrational Spectra IR spectral data of hbt ligand (Fig. 1) and its complexes were presented in the experimental section. The assignments of hbt have been reported for benzothiazole derivatives [19], which shows strong bands near 3317 and 3210 cm-1 assigned to as(NH2), s(NH2), respectively, while the band at 3127 cm-1 is attributed to (NH). Bands observed at 1647 and 1596 cm-1 are assigned to  (NH2) and (C=N), respectively. In addition, an intense band appeared near 1060 cm-1 is assigned to the hydrazinic (N-N) stretch [20].

Fig. 1 Structure of 2-hydrazinobenzothiazole with atom numbering scheme

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In the complexes, hbt has incorporated to the metal ion as a neutral monodentate N-donor and two coordination modes were observed; Complexes (1), (2) and (5) displays the other coordination mode, while they exhibit a lower frequency shift in (C=N) and (N-N) stretches (by ~ 34 cm1 ) due to the participation of N-cyclic azomethine nitrogen in complexation [12], additionally, there is no considerable change in (NH) bands. This lower frequency shift of azomethine group is due to decreasing the electron density around coordinated nitrogen atom through complexation [21]. In compounds (3) and (4), the bands identified for NH stretching vibration were shifted to lower frequency by 87 cm-1, indicating the coordination through NH2 nitrogen atom [22]. This feature was also supported by no significant shifts on (C=N) and (C-S) stretches upon complexation [23]; In complex (1), the bands observed at (1259, 1108, 955) and 967cm-1 are assigned to coordinated SO4 and (V=O) strecthes, respectively [24]. For complex (5), the new strong band at appeared at 1380 cm-1 is characteristic of ionic nitrate [25]. All complexes showed new band appeared in 489 - 462 cm-1 region belonging to (M-N) stretching vibration [26]. 4.2 1H NMR Spectra 1 H NMR spectral data of hbt and its diamagnetic complexes; (3), (4) and (5) were recorded in DMSO-d6 (reported in the experimental section; Fig.1 above shows the atom labelling Scheme). The 1H NMR spectrum of hbt exhibits two singlets (4.88 ppm for NH2 and 8.83 ppm for NH protons), two doublets (7.55 ppm for H(4) and 7.15 ppm for H(7) protons) [19]. The triplets as 7.07 and 6.85 are attributed to H(5) and H(6), respectively. In the complexes, (3) (Fig. 2a, b) and (4), there is a significant downfield shift of NH2 (by 4.27 – 3.94 ppm). Moreover, the signals of adjacent NH proton were also affected by coordination, this confirm that the ligand is coordinated to the metal center through NH2 group [22]. On the other hand, in the spectrum of complex (5) (Fig. 2c) there is an insignificant shift observed for NH 2, while that of NH proton shifted downfield demonstrating the coordination of hbt through cyclic nitrogen of benzothiazole moiety [27]. The 1HNMR spectrum for complex (3), suggest the presence of a mixture of cis- and trans-configuration in 3:17, respectively (Fig. 3) as two signals are observed for each proton [28].

Fig. 2 Structure of: (a) trans-[Pd(hbt)2Cl2] (3), (b) cis-[Pd(hbt)2Cl2] (3) and (c) [Ag(hbt)2].NO3 (5)

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Fig. 3 1H NMR spectrum of [Pd(hbt)2Cl2] (3) complex

4.3 Electronic Spectra and Magnetic Properties The UV-Vis spectra of hbt and some of its complexes were recorded in DMSO in the range of 200 – 900 nm. The absorption bands of the ligand at 270 – 302 nm region are assigned to n-* and -* transitions [29]. In the spectra of all complexes, these bands were shifted to higher wavelength (275 - 305 nm). In case of the octahedral oxovanadium complex (1) (3d1 configuration), the spectrum shows low intensity band at 600 nm and two more bands at 330 and 404 nm. This complex was found to be paramagnetic with eff = 1.78 B.M. which is close to spinonly value (1.73 B.M) that is expected for S = 1/2 for dxy based ground state for oxovanadium(IV) complexes [30]. The complex (2) of Ru(III) is paramagnetic (d5, t2g) with eff 1.7 B.M., exhibited two absorption bands at 418 and 733 nm due to LMCT and 2T2g- 2A2g transitions, respectively, indicating low spin octahedral geometry [31]. The spectra of Pd(II) (3) and Pt(II) (4) complexes displayed low intensity bands around (320, 469) and (334, 442), which are assigned to d-d transition; 1A1g  1B1g and 1A1g  1E1g, respectively, confirming square planar environment around the metal centers.12 4.4 Mass Spectra In the experimental section, the mass spectral data of hbt complexes are reported. It is clear that the molecular ion peaks of the complexes (M+) coincide with their proposed formula. The mass spectrum of (1), [VO(hbt)2SO4].4H2O, exhibits its molecular ion peak at m/z 492.05 (Calc. 492.9) with relative abundance 100%, corresponds to [VO(hbt)2SO4]+. The second peak observed at m/z 327.33(Calc. 326) with relative intensity 18% due to [VO(hbt)SO4]+ fragment. The spectrum of (2), [Ru(hbt)2Cl3(H2O)], showed its molecular ion peak at m/z 467.3 (Calc. 466.5) (relative abundance = 21%), attributed to [Ru(hbt)2Cl]+. One more peak was observed at m/z 430.2 (20%), (Calc. 431.0), assigned to [Ru(hbt) 2]+. Complex (3), [Pd(hbt)2Cl2], showed peaks at m/z 508.76 (48%) (Calc. 507.75), 471.91 (50%) (Calc. 472.25) and 435.9 (Calc. 436.75) correspond to [Pd(hbt)2Cl2]+, [Pd(hbt)2Cl]+ and [Pd(hbt)2]+, respectively. The molecular ion peaks of (4), [Pt(hbt)2Cl2], observed at m/z 596.1(Calc. 596.1) and 560.48 (Calc.560.0) attributed to [Pt(hbt)2Cl2]+ and [Pd(hbt)2Cl]+, respectively. Finally, complex (5), [Ag(hbt)2]NO3 shows molecular ion peak at 438.1 (Calc. 438.3), assigned to [Ag(hbt)2] + [8-11]. 4.5 Thermal Analysis

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Thermal degradation behaviour of the reported complexes, (1) – (5) was recorded using thermo-gravimetric technique (TGA) at the temperature range of 25 – 800 oC under nitrogen atmosphere. The TG diagram of (1), [VO(hbt)2SO4].4H2O, exhibits four endothermic steps; the first step at 50 – 100 oC corresponds to the weight loss of four molecules of crystal lattice water (Calcd. 12.8, Found 13.1%) [32,33]. The second weight loss at 100 – 300 oC is attributed to the release of SO2, N2 and two H2 fragments (Calcd. 17.0, Found 17.0%). The third step observed at 300 – 425 oC is due to the loss of two CHNS and O2 species (Calcd. 26.5, Found 26.4%), while the fourth step (426 - 480 o C) is due to the loss of C6H4 fragment (Calcd. 13.5, Found 13.5%), leaving a residual weight percent of 30.3% of vanadium oxides and carbides. The TG thermogram of (2), [Ru(hbt)2Cl3(H2O)], shows four endothermic mass loss steps at 50 - 300, 310 500 and 500 – 700 oC due to the release of H2O, 3/2 Cl2 and 2N2 (Calcd. 33.5, Found 32.2%), 2C6H4 (Calcd. 27.3, Found 26.3%) and CS2 (Calcd. 13.6, Found 13.05%) fragments, followed by the loss of 3H2 (Calcd. 1.5, Found 1.07), respectively, leaving a residue of 25.3% of ruthenium(III) nitride [34]. The TG diagram of (3), [Pd(hbt)2Cl2], displays three decomposition steps at the temperature ranges of 150 250 oC, 251 - 350 oC, 351 - 450 oC, corresponding to the mass loss of Cl2 and two C6H4 (Calcd. 43.9, F. 42.9%), SCN, -NH and 2H2 (Calcd. 15.1, Found 15.4%) and CN, -NH (Calcd. 8.1, found 8.6), respectively, leaving a residue of palladium(II) with 27.1%.30 The TG diagram of (4), [Pt(hbt)2Cl2] displays three decomposition steps at the temperature ranges of 150 - 260 oC, 261 - 370 oC and 371 – 475 oC, which corresponds to the loss of Cl2 and two C6H4 fragments (Calcd. 37.4, Found 37.0%), two CS units (Calcd.

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14.8, Found 14.4%), 2N2 and 3H2 (Calcd. 10.4, Found 10.7), respectively, leaving platinum metal (32.7%). The TG diagram of (5), [Ag(hbt)2NO3] shows three decomposition steps at 50 - 180 oC, 181 - 270 oC and 271 - 450 oC, corresponding to the release of NO2, ½O2 and 2H2 (Calcd. 13.4, 13.7%), 2C6H4, 2NH and N2 (Calcd. 41.9, 41.3%), and CS (Calcd 8.8, found 9.0%), respectively, leaving a residue silver nitride and carbide residues (Calcd. 35.9%) [12]. 4.6 Biological Applications 4.6.1 Cytotoxicity Activities Medicinal inorganic chemistry is a field of increasing prominence as metal-based compounds offer possibilities for the design of therapeutic agents, which is not available for organic compounds. The geometries, metal ion redox states, thermodynamic and kinetic characteristics and the intrinsic properties of the cationic metal ion as well as the ligand offer a wide spectrum of reactivity. Recently, we have reported that benzothiazole derivatives display extensive diversity of biological activities; antimicrobial, anti-inflammation and anti-cancer [12,13]. In comparison, complex (5) is the most powerful cytotoxic agent with respect to the other reported complexes. It exhibits high cytotoxic activity against EAC with IC50 of 5.15 (Fig. 4) and human cell lines: Hep-G2, MCF-7 and HCT-116 cells with 9.9, 13.1 and 17.7 µg/mL, respectively (Fig. 5 and Table 1). On the other hand, complexes (2) and (3) show promising cytotoxicity against EAC and Hep-G2 cells with IC50 5.49 and 16.2 µg/mL, respectively. The high cytotoxic activity of Ag(I) complex, (5), is expected, since we have been early reported that Ag(I) complexes are high active anti-cancer agents as well as wound healing stimulators [25,36,37].

Fig. 4 Cytotoxic activity of the compounds {[VO(hbt)2SO4] (1), [Ru(hbt)2Cl3(H2O)] (2), [M(hbt)2Cl2] [M(II) = Pd (3), Pt (4)] and [Ag(hbt)2]NO3 (5)} against Ehrlich ascites carcinoma (EAC) cells. Data are expressed as mean ± SD, n = 3 - 5 separate determinations. IC50 (µg/mL): 1 – 10 (very strong), 11 – 20 (strong), 21 – 50 (moderate), 51 – 100 (weak) and above 100 (non-cytotoxic). p < 0.001for all versus cisplatin. IC50 for the ligand (hbt) was 22.4±1.93.

In fact, the metal-ligand (M–N) coordinate bond, in Pd(II) (3) and Pt(II) (4) complexes, are greatly weaker than those C–C and C–N covalent bonds. Moreover, the relatively fast ligand exchange behaviour in complex (3) with cisconfiguration [38], may be responsible for the high activity with respect to complex (4). In addition, Ru(II) complexes

are known as strong DNA binding agents with remarkable antitumor activity [39]. They are suitable as alternatives to Pt(II) anticancer agents as they are able to mimic the binding of iron to biologically significant molecules [40]. Moreover, complexes (1) and (4) show optimistic cytotoxicity against EAC with IC50 of 9.63 and 11.25 µg/mL,

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respectively (Fig. 4). In all doses and different oxidation states, vanadium compounds are considered toxic. Vanadium(IV) in the form of VOSO4 is about 5 times more toxic compared to vanadium(III) in the form of V 2O3. In spite of this, no vanadium compound has been categorized as carcinogenic which is necessary for its potential use in cancer therapy. Vanadium in various forms damages DNA; one of the proposed mechanisms of DNA damage involves

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H e P G -2

M C F -7 80

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R e la tiv e V ia b ility o f C e lls (% )

H e P G -2 H C T -1 1 6 M C F -7 80

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C o n c e n tr a tio n o f 2 h b t-R u (µ g /m L )

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H e P G -2 H C T -1 1 6

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H e P G -2 H C T -1 1 6

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M C F -7 80

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C o n c e n tr a tio n o f P d -h b t (µ g /m L )

120

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C o n c e n tr a tio n o f A g -h b t (µ g /m L )

C o n c e n tr a tio n o f 5 -F U (µ g /m L )

100

H C T -1 1 6

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R e la tiv e V ia b ility o f C e lls (% )

R e la tiv e V ia b ility o f C e lls (% )

120

R e la tiv e V ia b ility o f C e lls (% )

vanadium and vanadium-containing compounds generation of reactive oxygen species (ROS) [41]. Among human cell lines, it is worthy to mention that all complexes exhibit higher activities against Hep-G2 suggesting that it is the most susceptible human cell line (Table 1).

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Fig. 5 Dose-dependent effect of the five newly synthesized complexes {[VO(hbt)2SO4] (1), [Ru(hbt)2Cl3(H2O)] (2), [M(hbt)2Cl2] [M(II) = Pd (3), Pt (4)] and [Ag(hbt)2]NO3 (5)} versus 5-fluorouracil (5-FU, the reference drug) on human hepatocellular carcinoma (Hep G2), colorectal carcinoma (HCT-116), and breast cancer (MCF-7) cells viability

Table 1 Cytotoxic activity of the compounds against human tumor cells Compounds 5-FU Ligand

Compound symbol -

In vitro cytotoxicity IC50 (µg/mL)@ Hep G2 MCF-7 7.9±0.28 5.4±0.20

HCT-116 5.3±0.17

hbt

53.3±3.45***

46.06±3.75***

63.6±6.30***

[Ag(hbt)2]NO3

(5)

9.9±0.89**

13.1±1.12***

17.7±1.67***

[Pd(hbt)2Cl2]

(3)

16.2±1.35***

25.8±1.98***

29.2±2.56***

[Ru(hbt)2Cl3(H2O)]

(2)

49.5±3.14***

37.0±2.37***

41.4±3.25***

[VO(hbt)2SO4]

(1)

31.6±2.60***

58.8±3.35***

67.0±2.81***

[Pt(hbt)2Cl2]

(4)

71.5±4.23***

81.5±3.93***

85.7±4.52***

Hep G2 is a human hepatocellular carcinoma cell line; HCT-116 is a human colorectal carcinoma cell line; MCF-7 is a human breast cancer cell line; 5-FU is 5-fluorouracil (the reference drug). Data are expressed as mean ± SD, n = 3 - 5 separate determinations. @IC50 (µg/mL): 1 – 10 (very strong), 11 – 20 (strong), 21 – 50 (moderate), 51 – 100 (weak) and above 100 (non-cytotoxic). * p < 0.05, ** p < 0.01 and ***p < 0.001, respectively versus 5-FU.

4.6.2 DNA Binding Abilities Since DNA replication is a key event for cell division, it is a critically important target in cancer chemotherapy. Cisplatin forms crosslink interactions with chromosomal DNA that interfere with cell division by

mitosis and leads to programmed cell death. This interference activity contributes to the antitumor activity of cisplatin. Damage to DNA elicits DNA repair mechanisms and activates a programmed cell death process when the damage is too severe to be repaired. According to X-ray crystallographic studies, cisplatin induces the duplex to bend

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toward the major groove, resulting in significant widening of the minor groove. Most cytotoxic platinum drugs form strong covalent bonds with the DNA bases [42]. However, a variety of platinum complexes act as DNA intercalators upon coordinating the appropriate ancillary ligands [43]. There are also reports on palladium derivatives interacting with DNA in covalent [44] and noncovalent ways [45]. As DNA is the primary pharmacological target of many antitumor compounds [4], DNA metal complex interactions have paramount importance in understanding the mechanism of tumor inhibition in the treatment of cancer. The interaction of the complexes (Table 2) suggested their ability to arrest DNA synthesis in cell lines

7

under investigation in the order (5) > (3) > (2) > (1) > (4). These results are in agreements with those obtained from our cytotoxicity study and support that the complexes bind DNA not only through coordination but also via hydrogen bond and hydrophobic interactions; i.e., intercalation [3,4]. It has been reported that the intercalation into DNA mainly involves the aromatic moiety of ligand. In addition, complexes containing aromatic ligands can intercalate into the DNA duplex with/without direct central metal ion–DNA bases coordination. This feature suggests that hbt ligand intercalates between DNA bases and the metal ions may coordinate directly to a DNA base.

Table 2 DNA/methyl green colorimetric assay of the compounds

Compound symbol

DNA/methyl green (IC50, µg/mL)

[Ag(hbt)2]NO3

(5)

39.5±2.6*

[Pd(hbt)2Cl2]

(3)

48.7±3.0**

[Ru(hbt)2Cl3(H2O)]

(2)

54.3±3.2**

[VO(hbt)2SO4]

(1)

67.6±3.7**

[Pt(hbt)2Cl2]

(4)

75.0±4.1**

DNA-active compound

Data are expressed as mean ± SD, n = 3 - 5 separate determinations. IC50 values represent the concentration required for a 50 % decrease in the initial absorbance of the DNA/methyl green solution. * p < 0.01 and ** p < 0.001, respectively versus hbt.

4.6.3 Antioxidant Activities Under normal conditions, ROS are usually scavenged by enzymatic antioxidants as SOD and catalase, and non-enzymatic antioxidants as well such as GSH, thus participating in oxidative stress prevention [46-49]. If not properly scavenged; ROS can cause cell death as they react with proteins, lipids, DNA and other biological molecules. ROS can trigger lipid peroxidation, inhibit antioxidant enzymes, speed up DNA damage, motivate procarcinogenesis, and alter the expression of tumor promotion related genes [50-53]. Therefore, developing new dual acting antitumor/free radicals scavenging complexes appeared of interest.

Accordingly, one of our objectives is testing the reported complexes for their antioxidation activity through their ability to inhibit lipid peroxidation and rat erythrocyte haemolysis (Table 3). Among the complexes, complex (5) shows the highest and the best anti-oxidation activity either using ABTS or erythrocyte haemolysis methods. In addition, the activity of the other complexes decreases in the order (3)  (2)  (1)  (4), using ABTS method, while in case of erythrocyte haemolysis method, the order is (3)  (1)  (2)  (4). These results are in a great agreement with the obtained cytotoxic activity and add explanation, that free radical scavenging could contribute in the complexes activity against different tumor cells.

Table 3 Antioxidant assays of the compounds by ABTS (2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid) method and by erythrocyte hemolysis method ABTS method

Erythrocyte hemolysis method

% Inhibition

% Erythrocyte hemolysis

-

89.2±2.5

4.3±0.32

[Ag(hbt)2]NO3

(5)

65.7±3.7*

9.0±0.44*

[Pd(hbt)2Cl2]

(3)

58.3±3.2*

11.1±0.53*

[Ru(hbt)2Cl3(H2O)]

(2)

48.8±3.0*

29.4±1.5*

[VO(hbt)2SO4]

(1)

31.7±2.1*

15.0±0.9*

[Pt(hbt)2Cl2]

(4)

22.4±1.7*

50.5±3.3*

Compounds Vitamin C

Compound symbol

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Data are expressed as mean ± SD, n = 3 – 5 separate determinations. * p < 0.001 versus vitamin C. benzothiazoles: a review. RSC Advances, 2014, 4, 60176CONCLUSION 60208. [10] Keri, R.S.; Patil, M.R.; Patil, S.A.; Budagumpi, S. A A series of new complexes of V(IV), Ru(III), comprehensive review in current developments of Pd(II), Pt(II) and Ag(I) derived from 2benzothiazole-based molecules in medicinal chemistry. Eur. J. hydrazinobenzothiazole have been synthesized and Med. Chem., 2015, 89, 207-251. 1 characterized based on spectral IR, H NMR, mass and UV[11] Nalawade, A.; Nalawade, M.R.; Patange, M.S.; Tase, M.D.; Vis, elemental analysis, magnetic, thermal and conductivity Badawy, A.; Omer, A.; Saksena, D.P.; Demaki, G.; measurements. hbt shows neutral monodentate behaviour Anyaegbunam, F.; Allahdadi, H. Thiazole Containing Schiffs through cyclic azomethine-N or amino-N atom. The Bases and Their Transition Metal Complexes. IJESI, 2013, 1, 1-4. anticancer activities of the complexes have been investigated [12] Shabana, A.A.; Butler, I.S.; Gilson, D.F.; Jean-Claude, B.J.; against EAC and human cancer cells (Hep-G2, MCF-7 and Mouhri, Z.S.; Mostafa, M.M.; Mostafa, S.I. Synthesis, HCT-116) and complex (5; Ag(I)) shows the highest activity characterization, anticancer activity and DNA interaction with IC50 of 5.15, 9.9, 13.1 and 17.7 µg/mL for EAC, Hepstudies of new 2-aminobenzothiazole complexes; crystal G2, MCF-7 and HCT-116, respectively. The DNA binding structure and DFT calculations of [Ag(Habt)2]ClO4. ability and antioxidant activities have been also studied and Inorganica Chimica Acta, 2014, 423, 242-255. the order of activity is (5) > (3) > (2) > (1) > (4). [13] El-Asmy, H.A.; Butler, I.S.; Mouhri, Z.S.; Jean-Claude, B.J.; Emmam, M.; Mostafa, S.I. 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*Address correspondence to Entsar A. Saad at the Department of Chemistry, Faculty of Science, Damietta University, P.O. Box: 34517, Damietta, Egypt; Tel/Fax: +2-057-240-3866, +2-057-240-3868; E-mails: [email protected]

Revised: April 16, 2014

Accepted: April 20, 2014

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Principal Author Last Name et al.