The pharmacology of heterocyclic ligands and their metal chelates is focus of research for bioinorganic chemists [1]. The nitrogen containing organic compounds and their metal complexes display a wide range of biological activities [2-4] e.g. antitumor, antibacterial, antifungal and antiviral properties. Metal complexes that can bind to DNA are gaining considerable attention due to their diverse applications as new generation metallo-pharmaceuticals [5].

The production of new therapeutic modulaties for cancer chemotherapy is the subject of immense interest owing to the fact that many present treatment regimes (platinum-based drugs) in chemotherapy have failed or fall short either in terms of efficiency or toxicity problems associated with them [6,7]. Among the non-platinum complexes for metal based chemotherapy, copper complexes have been much explored due to the fact that copper is bio-essential element responsible for numerous bioactivities in living organism [8]. Copper being redox active exists in biologically accessible +2/+1 oxidation states and is an important co-factor of several enzymes involved in oxidative metabolism, ceruloplasmine, superoxide dismutase, ascorbic acid oxidase and tyrosinase [9].

In addition to this, in recent years there has been a rapid expansion in the development of metal complexes as extensive diagnostic agents [6]. In this regard therapeutic agents that pertain to less toxic and more effective metallic component like Cu(II) are of particular interest and include a plethora of compounds: antitumor, antioxidant, antimicrobial and antiflammatory agents [10]. The appropriate redox property of Cu(II) is essential for various metabolic pathways like mitochondrial respiration, free radical scavenging, and iron absorption where it acts as a catalytic cofactor [11]. The Cu(II) complexes follows different mode of action towards DNA (non-covalent) as compared to cisplatin (covalent). Therefore, Cu(II)-based generic complexes exhibit higher antineoplastic potency towards human ovarian carcinoma, leukaemia, and various cervicouterine carcinomas as compared to cisplatin [12]. A large number of chemotherapeutic agents imply potentially useful therapeutic strategies and elicit antitumor effect by inducing cancer cell apoptosis by generating large amount of noxious radicals into the cancer cells [13]. Keeping in view the applications of coordination complexes, we here in report the synthesis, DNA binding studies, cleavage activity, molecular docking studies and in vitro cytotoxicity of Bis [2-(benzothiazol-2-yl iminomethyl)-phenol] copper 1, Bis [2-(benzothiazol-2-yliminomethyl)-valine] copper 2, Bis [cyanoaceto (2-mercaptobenzylidene)-hydrazide] copper 3, Bis [2-(phenacyl bromide)-aminothiophenol] copper 4, Bis [(2-mercaptobenzaldehyde) thiosemicarbazone] copper 5, Bis [N-(phenacyl bromide)-2-yliminobenzothiazole] copper 6, Bis (2-aminobenzothiazole) copper 7, Bis [2-(benzothiazol-2-yliminomethyl)-phenol] copper 8 and Bis 2-[(2‘-aminobenzylidene)-amino]-benzenethiol copper 9 complexes.

Materials and methods

All chemicals were purchased from Sigma-Aldrich (India) and Merck (India). Melting points were determined on a Kofler apparatus and are uncorrected. The IR spectra were recorded on KBr pellets with Perkin Elmer RXI Spectrometer and values are given in cm^-1. 1 H and 13 C NMR spectra were run in CDCl₃ on a JEOL Eclipse (400 MHz) instrument with TMS as internal standard and values are given in ppm (δ). Mass spectra were recorded on a JEOL SX102/DA- 6000 Mass Spectrometer. Carbon, hydrogen and nitrogen contents were determined on Carlo Erba Analyzer Model 1106. Calcium chloride (anhydrous) was used as a drying agent. Electronic spectra were recorded on UV-1700 PharmaSpec UV-Vis spectrophotometer (Shimadzu). Fluorescence measurements were made on SCHIMADZU RF-5301 fluorescence spectrophotometer. Super coiled pBR322 DNA was purchased from GeNei (India) and was used for the agarose gel experiment without further purification. Double-stranded calf thymus DNA, purchased from Sigma, was dissolved in a 0.1 M Tris-buffer. The purity of DNA was verified by monitoring the ratio of absorbance at 260 nm to that of 280 nm, which was in the range 1.8-1.9. The concentration of the DNA was determined spectrophotometrically using ε260 = 6600 M^-1 cm-1. The human cancer cell lines used for the cytotoxicity experiment were SW480, HepG2, HT-29 and HL-60 which were obtained from National Cancer Institute (NCI), biological testing branch, Federick Research and Development Centre, USA. The treated and control cancer cells were viewed with a FluoView FV1000 (Olympus, Tokyo, Japan) confocal laser scanning microscope (CLSM) equipped with argon and HeNe lasers.

Procedure for the synthesis of new copper complexes (1-9)

Bis [2-(benzothiazol-2-yliminomethyl)-phenol] copper (1): It was prepared by a general synthetic method in which methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) was added to the methanolic solution of 2-aminobenzothiazole (0.150 g, 1 mmol) and 2-hydroxybenzaldehyde (0.122 g, 1 mmol) in a 1:1:1 molar ratio which was refluxed for 1.5 h to obtain a greenish colour solution. The coloured product was isolated, washed with water and dried in vacuo to get pure powder.

Yield 82%. M.p. 241°C. Anal. Calc (%) for C₂₈H₂₀CuN₄O₂S₂: C, 58.78; H, 3.52; N, 9.79. Found: C, 58.62; H, 3.35; N, 9.49; IR (KBr, ν cm^-1) 3335 (OH), 1649 (HC=N), 1630-1619 (aromatic), 1440 (C-N), 430 (Cu-N), 422 (Cu-S); ¹H NMR (CDCl₃, 400 MHz): δ 8.31, 7.92 (s, 1H, 2 × OH exchangeable with D₂O), 6.35-7.41 (m, 8H, 2 × 8 aromatic ring protons), 7.16, (s, 1H, 2 × HC=N); ¹³C NMR (CDCl₃, 100 MHz): δ 112-133 (2 × 12 aromatic carbon signals), 151, 149 (2 × HC=N); ESIMS (m/z): 572 [C₂₈H₂₀CuN₄O₂S₂]⁺.

Bis [2-(benzothiazol-2-yliminomethyl)-valine] copper (2): It was synthesized by a general synthetic method in which methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) was added to the methanolic solution of 2-aminobenzothiazole (0.150 g, 1 mmol) and L-valine (0.177 g, 1 mmol) in a 1:1:1 molar ratio which was refluxed for 2 h to obtain a bluish green solution. The coloured product was isolated, washed with water and dried in vacuo to get pure powder.

Yield 75%. M.p. 232°C. Anal. Calc (%) for C₂₄H₃₀CuN₆O₂S₂: C, 51.27; H, 5.38; N, 14.95; S, 11.41. Found: C, 51.13; H, 5.19; N, 14.81; S,11.29. IR (KBr, ν cm-1) 3342 (OH), 3256 (NH₂) 1636-1616 (aromatic), 1657 (C=N), 1440 (C-N), 427 (Cu-N), 410 (Cu-S); 1H NMR (CDCl₃, 400 MHz): δ 8.11, 8.24 (s, 1H, 2 × OH exchangeable with D₂O), 6.63- 6.93 (m, 8H, 2 × 4 aromatic ring protons), 4.34 (br s, 2H, 2 × NH₂ exchangeable with D₂O), 2.44, 2.39 (d, 1H, 2 × CH-(CH₃)₂), 2.15, 2.11 (m, 1H, 2 × (CH₃)₂-CH, 1.3 (d, 6H, (CH₃)₂-CH); ¹³C NMR (CDCl₃, 100 MHz): δ 122-153 (2 × 6 aromatic carbon signals), 162.4-165 (4 × C=N), 62, 60.6 (C-N); ESI-MS (m/z): 561 [C₂₄H₃₀CuN₆O₂S₂]⁺.

Bis [2-(phenacyl bromide)-aminothiophenol] copper (4): It was also synthesized by a general synthetic method in which methanolic solution of 2-aminothiophenol (0.125 g, 1 mmol), phenacyl bromide (0.199 g, 1 mmol) and (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) were added to the in a 1:1:1 molar ratio and was refluxed for 1.5 h to obtain a yellow solution. The coloured product was isolated, washed with water and dried in vacuo to get pure powder.

Yield 75%. M.p. 224°C. Anal. Calc (%) for C₂₈H₂₄CuN₂O₂S₂: C, 61.35; H, 4.41; N, 5.11; S, 11.70. Found: C, 61.19; H, 4.28; N, 5.04; S, 11.52. IR (KBr, ν cm-1) 3213 (NH), 1675 (CO), 1625-1638 (aromatic), 1436 (C-N), 435 (Cu-N), 412 (Cu-S); ¹H NMR (CDCl₃, 400 MHz): δ 6.27-6.61 (m, 8H, 2 × 4 protons of aminothiophenol moiety), 6.83-7.12 (m, 12H, 2 × 5 protons of phenacyl moiety), 4.3, 4.2 (s, 1H, 2 × NH exchangeable with D₂O), 4.72 (s, 1H, 2 × H₂C-CO), 3.75 (s, 2H, 2 × CH₂); ¹³C NMR (CDCl₃, 100 MHz): δ 168, 170 (2 × C=O), 129-145 (2 × 6 aromatic carbon signals of phenacyl moiety), 116-132 (2 × 6 aromatic carbon signals of aminothiophenol moiety); 62, 65 (2 × CH₂); ESI-MS (m/z): 548 [C₂₈H₂₄CuN₂O₂S₂]⁺.

Bis [(2-mercaptobenzaldehyde) thiosemicarbazone] copper (5): It was prepared by a general synthetic method in which methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) was added to the methanolic solution of 2-merceptobenzaldehyde (0.138 g, 1 mmol) and thiosemicarbazide (0.091 g, 1 mmol) in a 1:1:1 molar ratio which was refluxed for 1 h to obtain an off white solution. The coloured product was isolated, washed with water and dried in vacuum to get pure powder.

Yield 76%. M.p. 247°C. Anal. Calc (%) for C₁₆H₁₆CuN₆S₄: C, 39.69; H, 3.33; N, 17.36; S, 26.49. Found: C, 39.56; H, 3.21; N, 17.14; S, 26.35. IR (KBr, ν cm-1) 3230, 3177 (NH, NH₂), 1654 (C=N), 1619-1631 (aromatic), 1436 (C-N), 435 (Cu-N), 414 (Cu-S), 537 (C=S); ¹H NMR (CDCl₃, 400 MHz): δ 7.7, 7.4 (s, 1H, 2 × HC=N), 7.1-7.51 (m, 8H, 2 × 4 aromatic protons), 3.6 (s, 1H, 2 × NH exchangeable with D₂O), 3.7 (s, 1H, 2 × NH₂ exchangeable with D₂O); ¹³C NMR (CDCl₃, 100 MHz): δ 181, 178 (2 × C=S), 154, 151 (2 × C=N), 125-134 (2 × 6 aromatic carbon signals); ESI-MS (m/z): 484 [C₁₆H₁₆CuN₆S₄]⁺.

Bis [N-(phenacyl bromide)-2-yliminobenzothiazole] copper (6): It was prepared by a general synthetic method in which methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) was added to the methanolic solution of 2-aminobenzothiazole (0.150 g, 1 mmol) and phenacyl bromide (0.199 g, 1 mmol) in a 1:1:1 molar ratio which was refluxed for 2 h to obtain a dark green solution. The coloured product was isolated, washed with water and dried in vacuo to get pure powder.

Yield 80%. M.p. 253°C. Anal. Calc (%) for C₃₀H₂₆CuN₄O₂S₂: C, 59.83; H, 4.35; N, 9.30; S, 10.65. Found: C, 59.69; H, 4.20; N, 9.16; S, 10.43. IR (KBr, ν cm-1) 3224 (NH), 1693 (C=O), 1651 (C=N), 1621-1634 (aromatic), 1436 (C-N), 435 (Cu-N), 417 (Cu-S); ¹H NMR (CDCl₃, 400 MHz): δ 6.27-6.61 (m, 8H, 2 × 4 protons of 2-aminothiazole moiety), 6.83-7.12 (m, 10H, 2 × 5 protons of phenacyl moiety), 4.3 (s, 1H, 2 × NH exchangeable with D₂O), 4.72 (s, 1H, 2 × H₂C-CO); ¹³C NMR (CDCl₃, 100 MHz): δ 164, 167 (2 × C=O), 147, 144 (2 × C=N), 129-145 (2 × 6 aromatic carbon signals of phenacyl moiety), 121-128 (2 × 6 aromatic carbon signals of 2-aminothiazole moiety); 61, 63 (2 × CH₂); ESI-MS (m/z): 602 [C₃₀H₂₆CuN₄O₂S₂]⁺.

Bis (2-aminobenzothiazole) copper (7): It was also synthesized by a general synthetic method in which methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) was added to the methanolic solution of 2-aminobenzothiazole (0.150 g, 1 mmol) in a 1:1 molar ratio which was refluxed for 55 min. to obtain a leaflet green solution. The coloured product was isolated, washed with water and dried in vacuum to get pure powder.

Yield 81%. M.p. 264°C. Anal. Calc (%) for C₁₄H₁₂N₄O₁₀S₂Cu: C, 46.20; H, 3.32; N, 15.39; S, 17.62. Found: C, 46.12; H, 3.23; N, 15.33; S, 17.59. IR (KBr, ν cm-1) 3221 (NH₂), 1624-1617 (aromatic), 1641 (C=N), 1429 (C-N), 428 (Cu-N), 645 (C-S). ¹H NMR (CDCl₃, 400 MHz): δ 8.2-7.1 (m, 8 H, 2 × aromatic ring 4 protons), 3.72 (s, 4 H, 2 × NH₂ exchangeable with D₂O). ¹³C NMR (CDCl₃, 100 MHz): δ 122-156 (2 × 6 aromatic carbon signals), 145 (C=N), 134 (C-N), 120 (C-S). ESI-MS (m/z): 362 [C₁₄H₁₂N₄O₁₀S₂Cu]⁺.

Bis [2-(Benzothiazol-2-yliminomethyl)-phenol] copper (8): It was prepared by adding salicyaldehyde (0.122 g, 1 mmol), 2-aminobenzothiazole (0.150 g, 1mmol) to a methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) in a 1:1:1 molar ratio. The reaction mixture was refluxed for 50 min. at room temperature. The greenishyellow precipitate which formed was filtered off under vacuum, washed thoroughly with water and dried in vacuo over anhydrous CaCl₂.

Yield: 76%. M.p. 279°C. Anal. Calc (%) for C₂₈H₁₈CuN₄O₂S₂: C, 58.99; H, 3.18; N, 9.83; S, 11.25. Found: C, 58.92; H, 3.13; N, 9.78; S, 11.21. IR (KBr, ν cm-1) 1621-1615 (aromatic), 1647 (C=N), 1633 (HC=N), 1440 (C-N), 1082 (C-O), 448 (Cu-O), 647 (C-S). ¹H NMR (CDCl₃, 400 MHz): δ 8.2-7.1 (m, 16H, 2× aromatic ring 8 protons), δ 8.14 (s, 1H, HC=N), δ 8.11 (s, 1H, HC=N). ¹³C NMR (CDCl₃, 100 MHz): δ 121-158 (2×12 aromatic carbon signals), 158, 149 (C=N). ESIMS (m/z): 570 [C₂₈H₁₈N₄O₂S₂Cu]⁺.

Bis 2-[(2‘-Amino-benzylidene)-amino]-benzenethiol copper (9): It was prepared by adding 2-aminobenzaldehyde (0.121 g, 1 mmol), 2-aminothiophenol (0.125 g, 1mmol) to a methanolic solution (20 ml) of CuSO₄.5H₂O (0.233 g, 1 mmol) in a 1:1:1 molar ratio. The reaction mixture was refluxed for 50 min. at room temperature. The reddish precipitate which formed was filtered off under vacuum, washed thoroughly with water and dried in vacuo over anhydrous CaCl₂.

Yield: 79%. M.p. 284°C. Anal. Calc (%) for C₂₆H₂₂N₄S₂Cu: C, 60.27; H, 4.28; N, 10.81; S, 12.38. Found: C, 60.23; H, 4.19; N, 10.77; S, 12.35.IR (KBr, ν cm-1) 1628-1619 (aromatic), 1643 (HC=N), 1434 (C-N), 424 (Cu-N), 651 (C-S). ¹H NMR (CDCl₃, 400 MHz): δ 8.1-7.04 (m, 16H, 2 × aromatic ring 8 protons), δ 8.39 (s, 1 H, HC=N), δ 8.27 (s, 1 H, HC=N), δ 4.14 (s, 1 H, NH₂ exchangeable with D₂O), δ 3.92 (s, 1 H, NH₂ exchangeable with D₂O). 13C NMR (CDCl₃, 100 MHz): δ 117-157 (2 × 12 aromatic carbon signals), 163, 161 (C=N). ESI-MS (m/z): 518 [C₂₆H₂₂N₄S₂Cu]⁺.

DNA binding studies

Cleavage experiments were performed with Axygen agarose electrophoresis [14] connected to a Genei 50-500 V power supply visualized and photographed by the Vilber-INFINITY gel documentation system. Cleavage experiments of supercoiled pBR322 DNA (300 ng) by complex 8 and 9 (1.0-7.0 μM) in a 5 mM Tris-HCl / 50 mM NaCl buffer at pH 7.2 were carried out and the reaction followed by Agarose gel electrophoresis. The samples were incubated for 1 h at 37°C. A loading buffer, containing 25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol, was added and electrophoresis was carried out at 60 V for 1 h in a Tris-HCl buffer using a 1% agarose gel containing 1.0 mg/mL of ethidium bromide.

Absorption and emission spectroscopy was used to investigate further the binding affinity of complexes. It involves the standard methods and practices reported in the literature [15-17]. While measuring the absorption spectra, an equal amount of DNA was added to both the complex solution and the reference solution to eliminate the absorbance of the DNA itself, Tris buffer was subtracted through base line correction.

Anticancer activity

Cell lines and culture conditions: Human cancer cell lines SW480 (colon adenocarcinoma), HepG2 (hepatic carcinoma), HT-29 (Colon carcinoma) and HL-60 (human leukaemia) were taken for the study. The cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 10U penicillin and 100 μg/mL streptomycin at 37°C with 5% CO2 in a humidified atmosphere. Fresh medium was given every second day and on the day before the experiments were done. Cells were passaged at pre-confluent densities, using a solution containing 0.05% trypsin and 0.5 mM EDTA.

Cell viability assay (MTT): The anticancer activity in vitro was measured using the MTT assay [18,19]. Exponentially growing cells were harvested and plated in 96-well plates at a concentration of 1 × 104 cells/well. After 24 h incubation at 37°C under a humidified 5% CO₂ to allow cell attachment, the cells in the wells were respectively treated with target complexes at various concentrations for 48 h. The concentration of DMSO was always kept below 1.25%, which was found to be non-toxic to the cells. A solution of 3-(4,5-dimethylthizao1-2-y1)- 2,5-diphenyltetrazolium bromide (MTT), was prepared at 5 mg/mL in phosphate buffered saline (PBS; 1.5 mM KH₂PO₄, 6.5 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl; pH 7.4). 20 μL of this solution were added to each well. After incubation for 4 h at 37°C in a humidified incubator with 5% CO₂, the medium/MTT mixtures were removed and the formazan crystals formed by the mitochondrial dehydrogenase activity of vital cells were dissolved in 100 μL of DMSO per well. The absorbance of the wells was read with a microplate reader (Bio-Rad Instruments) at 570 nm. Effects of the drug cell viability were calculated using cells treated with DMSO as control. Cancer cells were grown on glass cover slips in 12-well cell culture plates (CoStar). After incubation with the test complexes, the disks were flipped on glass plates and the treated and control cancer cells were observed with a FluoView FV1000 (Olympus, Tokyo, Japan) confocal laser scanning microscope (CLSM) equipped with argon and HeNe lasers.

Data analysis: Cell survival was calculated using the formula: Survival (%) = [(absorbance of treated cells-absorbance of culture medium) / (absorbance of untreated cells - absorbance of culture medium)] × 100 [20]. The experiment was done in triplicate and the inhibitory concentration (IC) values were calculated from a dose response curve. IC₅₀ is the concentration in ‘μM’ required for 50% inhibition of cell growth as compared to that of control. IC₅₀ values were determined from the linear portion of the curve by calculating the concentration of agent that reduced absorbance in treated cells, compared to control cells, by 50%. Evaluation is based on mean values from three independent experiments, each comprising at least six microcultures per concentration level.

Comet assay (Single cell gel electrophoresis)

The comet assay [21] was performed to evaluate the genotoxic effect of the copper complexes 8 and 9 in HT-29 cells. HT-29 (1 × 106) cells were treated with three different concentrations, 10, 25 and 50 mg/mL of copper complexes 8 and 9 in for 24 h. The cells were then washed and 200 mL of cell suspension in low melting agarose (LMA) were layered onto the labelled slides precoated with agarose (1.5%). The slides were placed on ice for 10 min and submerged in a lysis buffer (2.5% NaCl, 100 mM EDTA, 10 mM Tris, 10% DMSO and 1% Troton X-100) at 48°C at pH 10 for more than 1 h. The slides were then equilibrated in an alkaline buffer (30 mM NaOH, 1 mM EDTA) at pH 13 at 48°C, electrophoresed at 0.86 V/cm at 48°C, neutralized, washed, and dried. At the time of capturing the images, the slides were stained with ethidium bromide (EtBr, 150 mL 1X) and cover slips were placed over them. For visualization of DNA damage, EtBr-stained slides were observed under 209 objectives of a fluorescent microscope (Olympus BX-51, Japan). The images of 50-100 randomly selected cells were captured per slide using a CCD camera.

Molecular docking

The rigid molecular docking studies were performed using HEX 6.1 software [22]. The initial structure of the copper complexes 8 and 9 was generated by Discovery studio software. The molecules of complexes 8 and 9 were optimized for use in the following docking study. The crystal structure of the B-DNA dodecamer d(CGCAAATTTCGC)2 (PDB ID: 1BNA) were downloaded from the protein data bank. All calculations were carried out on an Intel CORE i5, 2.6 GHz based machine running MS Windows 7 as the operating system. Visualization of the docked pose have been done using PyMol molecular graphics program [23].

Determination of physicochemical properties: Bioactivity score and PASS (Prediction of biological activity spectra) analysis: The physicochemical parameters including octanol partition coefficients (miLogP), HBD, HBA, TPSA and Rotatable bonds were calculated using molinspiration server (http://www.molinspiration.com/cgi-bin/ properties) and ChemAxon (chemicalize.org). The PASS (Prediction of biological activity spectra) approach is based on the structure-activity correlation, Complex activity prediction is done by ‘‘comparing’’ the structure of query compound with the structure of well-known biological active substrate existing in database of the freely available PASS web service. Complex activity prediction is done by ‘‘comparing’’ the structure of query complex with the structure of well-known biological active substrate existing in database of PASS web service.

Chemistry

Development of highly functional coordination complexes from simple building blocks has always been the curiosity of synthetic inorganic chemists. So we here in report the convenient synthesis of Bis [2-(benzothiazol-2-yl iminomethyl)-phenol] copper 1, Bis [2-(benzothiazol-2-yliminomethyl)-valine] copper 2, Bis [cyanoaceto (2-mercaptobenzylidene)-hydrazide] copper 3, Bis [2-(phenacyl bromide)-aminothiophenol] copper 4, Bis [(2-mercaptobenzaldehyde) thiosemicarbazone] copper 5, Bis [N-(phenacyl bromide)-2- yliminobenzothiazole] copper 6, Bis (2-aminobenzothiazole) copper 7, Bis [2-(benzothiazol-2-yliminomethyl)-phenol] copper 8 and Bis 2-[(2‘-aminobenzylidene)-amino]-benzenethiol copper 9 by reacting 2-(benzothiazol-2-yl iminomethyl)-phenol, 2-(benzothiazol-2-ylimino methyl)-valine, cyanoaceto (2-mercaptobenzylidene)-hydrazide, 2-(phenacyl bromide)-aminothio- phenol, (2-mercaptobenzaldehyde) thiosemicarbazone, N-(phenacyl bromide)-2-yliminobenzo- thiazole, 2-aminobenzothiazole, benzothiazol-2-yliminomethyl)-phenol and 2-[(2‘-amino benzylidene)-amino]-benzenethiol with copper sulphate, respectively in 1:1 ratio in methanol for the period of about 1-2.5 h under reflux conditions (Scheme 1) and on the completion of reaction, new complexes 1-9 were obtained in potential yield of 75-82%.

Scheme 1: Formation of Bis [2-(benzothiazol-2-yliminomethyl)-phenol] copper 1 and Bis [2-(benzothiazol-2-yliminomethyl)-valine] copper 2, Bis [cyanoaceto (2-mercaptobenzylidene)-hydrazide] copper 3, Bis [2-(phenacyl bromide)-aminothiophenol] copper 4, Bis [(2-mercaptobenzaldehyde) thiosemicarbazone] copper 5, Bis [N-(phenacyl bromide)-2-yliminobenzothiazole] copper 6, Bis (2-aminobenzothiazole) copper 7, Bis [2-(benzothiazol-2-yliminomethyl)-phenol] copper 8 and Bis 2-[(2ʹ-aminobenzylidene)-amino]-benzenethiol copper 9.

All the new complexes were stable towards air and moisture and soluble in DMSO. The complexes were thoroughly characterized from spectroscopic (IR, ¹H NMR, ¹³C NMR, TGA and MS) and analytical data (Experimental section). The spectral data is in good agreement with the proposed structures of copper heterocyclic complexes. In the IR spectra, the absorption bands in the range 3177-3256 cm-1 show the presence of NH₂ in the compounds while as the absorption bands at 3335-3342, 1633-1657, 3213-3230, 1675-1693, 424-435 and 410-422 cm-1 confirm the presence of OH, C=N, NH, C=O, Cu-N and Cu-S, respectively in complexes (1-9). In ¹H NMR study, the absorption at δ 8.31-7.92 confirmed the presence of OH while as the absorption peaks at δ 4.34-3.6 revealed the presence of NH₂ in complexes (1-9). The presence of signals (multiplet) at δ 6.35-7.41 depicted the aromatic moiety in complexes (1-9) while as absorption at δ 4.3-4.2 and δ 8.3-8.1 showed the presence of NH attached to groups primary and secondary carbon atoms in complexes (1-9). In ¹³C NMR study, the signals at δ 144-165, δ 1164-173, δ 181-178 revealed the presence of C=N, C=O and C=S while as signals in the range of δ 112-145 confirm the presence of aromatic ring carbons in complexes (1-9). Finally the presence of distinct molecular ion peak [M⁺.] at m/z: 572, 561, 499, 548, 484, 602, 362, 570 and 518 in the MS also proved the formation of complexes 1-9, respectively.

Thermo gravimetric analysis (TGA)

To examine the thermal stability of complexes 1 and 2, the thermogravimetric analysis were carried out under nitrogen atmosphere in the temperature range 700°C at a heating rate of 20°C min-1 (Figure 1). The TGA analysis of complexes 1-9 shows weight loss starting at 250, 280, 238, 115, 338, 198, 252, 378 and 294oC, respectively, depicting the stability of complexes.

Figure 1: Thermo gravimetric analysis showing the thermal stability of complexes 1-9.

DNA Binding studies

The DNA cleavage was controlled by the relaxation of supercoiled circular form of pBR322 DNA into the nicked and linear form. When a circular plasmid DNA is subjected to agarose gel electrophoresis, the fastest migration will be observed for supercoiled form (Form I). If one strand is cleaved, the supercoils will relax to produce a slower moving open circular form (Form II). If both strands are cleaved, a linear form (Form III) will be generated that migrates in between Form I and Form II. The DNA cleaving ability of complex 8 and 9 was investigated using pBR322 DNA. In the absence of any external additives the complex 8 and 9 cleaved double stranded supercoiled plasmid DNA (SC form: Form I) (300 ng) in 5 mM Tris–HCl / 50 mM NaCl buffer into nicked circular form (NC form: Form II) after 1 h of incubation at physiological pH 7.2 and temperature 25°C [24]. Keeping the DNA concentration constant (300 ng) the concentration of complex 8 and 9 was varied (1.0-7.0 μM) and the cleavage reaction was further monitored by gel electrophoresis. The results revealed concentration-dependent electrophoretic cleavage clearly showing the conversion of SC form (Form I) to NC form (Form II) with increase in concentration of complex 8 and 9.

At 3 μM concentration, complex 8 and 9 exhibited efficient nuclease activity. At still higher concentrations there was complete conversion of SC form into NC form with the concurrent formation of LC form. Presence of Form I, II, and III of pBR322 DNA indicated that complex 8 and 9 are involved in double strand DNA cleavage (Figures 2 and 3).

Figure 2: Agarose gel electrophoresis patterns of pBR322 plasmid DNA (300 ng) cleaved by complex 8 (1.0-7.0 μM), after 1 h incubation time (concentration dependent) Lane 1: control; Lane 2: 1.0 μM 1 + DNA; Lane 3: 2.0 μM 8 + DNA; Lane 4: 3.0 μM 8 + DNA. Lane 5: 4.0 μM 8 + DNA; Lane 6: 5.0 μM 8 + DNA, Lane 7: 6.0 μM 8 + DNA, Lane 8: 7.0 μM 8 + DNA in buffer (5 mM Tris-HCl/50 mM NaCl, pH 7.2 at 25°C).

Figure 3: Agarose gel electrophoresis patterns of pBR322 plasmid DNA (300 ng) cleaved by complex 9 (1.0-6.0 μM), after 1 h incubation time (concentration dependent) Lane 1: control; Lane 2: 1.0 μM 9 + DNA; Lane 3: 2.0 μM 9 + DNA; Lane 4: 3.0 μM 9 + DNA. Lane 5: 4.0 μM 9 + DNA; Lane 6: 5.0 μM 9 + DNA, Lane 7: 6.0 μM 9 + DNA in buffer (5 mM Tris-HCl/50 mM NaCl, pH 7.2 at 25°C).

It is a well-known fact that DNA is the primary pharmacological target of number of anticancer drugs, and hence, the interaction between DNA and metal complexes are of paramount importance in understanding the mechanism. The interaction of transition metal complexes with DNA takes place via both covalent and / or noncovalent interactions. In the case of covalent binding, the labile ligand of the complexes is replaced by a nitrogen base of DNA such as guanine N7 while the non-covalent DNA interactions include intercalative, electrostatic and groove binding of metal complexes outside of a DNA helix [25]. The interaction of complexes 8 and 9 with CT DNA was examined by titrating the fixed amount of complexes with increasing concentration of CT DNA (5.3-2.6 × 10-5 M) as shown in Figure 4. The absorption spectra of complexes 8 and 9 indicated a blue shift in the wavelength on interaction with CT DNA as shown in Figure 4. This shift may be attributed to the change in environment around the metal centre [Cu(II)] as it may exhibit a preferential selectivity towards phosphate backbone. The spectral changes are typical of modes of interactions of DNA to substrate(s) that include hyperchromicity (blue shift) and hypochromicity (red shift). Hypochromic effect is attributed to the intercalative binding mode whereas hyperchromic effect might be ascribed to the electrostatic binding mode. The present data suggest a hyperchromism, which usually involve electrostatic interactions. It is also found that the binding of these complexes occur via the classical intercalation between the planar aromatic chromophores and DNA base pairs nevertheless the hyperchromic effect observed arises mainly due to the presence of Cu(II) ion which bind to DNA via electrostatic attraction to the phosphate group of DNA backbone and thereby causing a contraction and overall damage to the secondary structure of DNA.

Figure 4: Absorption spectra of complexes 8 and 9 in Tris-HCl buffer upon the addition of calf thymus DNA [complex] = 6.67 × 10^-6 M, [DNA] = 0.70 - 4.24 × 10^-5 M. Arrow shows change in intensity with increasing concentration of DNA.

To compare quantatively the effect of binding strength, the intrinsic binding constants (Kb) of the complexes 1 and 2 were determined by using Wolfe-Shimer equation [26] (Equation 1)

[DNA] / ε_a–ε_f = [DNA] / ε_b–ε_f + 1 / K_b ¦ε_b–ε_f¦ (1)

where [DNA] represents the concentration of DNA, and ε_a, ε_f and ε_b the apparent extinction coefficient (A_obs / [M]), the extinction coefficient for free metal complex (M), and the extinction coefficient for the free metal complex (M) in the fully bound form, respectively. The K_b values for complexes 8 and 9 are 2.35 × 10³ M^-1 and 2.12 × 10³ M^-1, respectively, which suggest that complex 8 has strong binding affinity for CT DNA as compared to complex 9.

In fluorescence spectral studies at room temperature, the complexes 8 and 9 emit luminescence at 374 nm respectively in 0.01 tris-HCl / 50 mM NaCl buffer when excited at 260 nm. Fixed amount of complexes 8 and 9 were titrated with increasing amount of CT DNA, over a range of 5.3-2.6 × 10^-5 M DNA concentration. The addition of DNA caused a gradual increase in fluorescence emission (Figure 5) intensity being consistent with the non-intercalative binding mode such as electrostatic binding mode (surface binding) and be protected by DNA efficiently, since the hydrophobic environment inside the DNA helix reduces the accessibility of solvent molecule to the complex, which as a consequence restrict the complex mobility at binding site, leading to a decrease of the vibrational modes of relaxation and thus higher emission intensity [27]. Also, the number of bound copper complex per DNA (n) calculated [28] for complexes 8 and 9 were found to be 1.19 and 1.04, respectively from fluorescence spectral data.

Figure 5: Emission spectra of compounds 8 and 9 in the presence of DNA in 5 mM Tris-HCl / 50 mM NaCl buffer. Arrows show the intensity changes upon increasing concentration of the DNA. 1

The binding constant K, determined from the Scatchard equation for complexes 8 and 9 was calculated to be 1.83 × 10³ and 1.45 × 10³ M^-1, respectively which is in agreement with the similar trend of DNA binding ability (8 > 9) as observed in case of absorption spectral studies.

Cytotoxicity mechanism was confirmed by studying the treatment of supercoiled plasmid pBR322 DNA with different concentrations of complex 8 and 100 μM copper. Our nucleolytic experiments suggest that cell death may be due to cleavage or fragmentation of DNA of these cancer cells and that the active species responsible for this are ROS (?OH) which resulted from the in vitro reaction of different concentrations of complex 8 with copper in presence of thiobarbituric acid. We observe from gel electrophoresis that after adding copper (100 μM) the concentration of radicals increase which in presence of different concentrations of complex 8 show the nicking of plasmid pBR322 DNA from its supercoiled form (Form I) to open circular form (Form II). As shown in lane 6, 7 and 8 (Figure 6), the nicking is quite obvious by the disappearance of form I and appearance of form II and with the increase in concentration of complex 8 (lane 8), the band intensity (form II) became maximum, depicting the more pronounced cleavage at high concentration.

Figure 6: Fragmentation pattern of supercoiled plasmid pBR322, Lane 1 contains DNA only, lane 2 contains DNA and copper, lane 3, 4 and 5 contain DNA and complex 8 (100, 200 and 300 μM respectively), lane 6, 7 and 8 contain DNA and complex 8 (100, 200, 300 μM respectively) plus 100 μM copper and lane 9 contains DNA, Cisplatin (20 μM) plus 100 μM copper added to it.

In the DNA cleavage reactions mediated by various antioxidants in the presence of Cu (II), it has been established that Cu (II) is reduced to Cu (I) by the antioxidants and that Cu (I) is an essential intermediate in the DNA cleavage reactions. It is also generally understood that DNA cleavage by various antioxidants and Cu (II) is the result of the generation of hydroxyl radicals. Since Cu (II) is reduced to Cu (I) and the re-oxidation of Cu (I) to Cu (II) by molecular oxygen gives rise to superoxide anion which in turn leads to the formation of H₂O₂. Presumably Cu (I) is oxidized to Cu (II) by H₂O₂ in a Fenton type reaction giving rise to hydroxyl radicals. To determine the hydroxyl radical production and the role of copper ions in DNA cleavage, an experiment was performed where progressively increasing concentrations of complex 8 and Cisplatin (12.5-600 μM) were tested on thiobarbituric acid induced DNA breakage (Figure 7) and from these results we may conclude that the DNA cleavage by thiobarbituric acid involves endogenous copper ions (Cu (I) acts an intermediate) that leads to DNA cleavage.

Figure 7: Comperative determination of hydroxyl radical production by complex 8 (A) and Cisplatin (B)by the assay of Thiobarbituric acid.

The complex 8-Cu (II) (Figure 7A) and Cisplatin-Cu (II) (Figure 7B) are shown to generate the hydroxyl radicals (?OH) which react with Calf thymus DNA, resulting in strand breaks. The assay is based on the fact that degredation of DNA by hydroxyl radical results in the release of TBA reactive material, which forms a coloured adduct readable at 532 nm. Increasing concentrations of complex 8 or Cisplatin in presence of Cu (II) showed a corresponding increase in the generation of hydroxyl radicals. The results in Figure 7 confirmed the relatively higher rate of formation of hydroxyl radicals and correlated with the rate of DNA degredation by the complex 8 as well as Cisplatin.

In vitro cytotoxicity

The anticancer activity in vitro was measured using the MTT assay during which the conversion of the soluble yellowish MTT to the insoluble purple formazan by active mitochondrial lactate dehydrogenase of living cells has been used to develop an assay system for measurement of cell proliferation [18,19]. The data reported in Table 1 suggests the During the cytotoxic screening of complexes 1-9, the potential behaviour was depicted against given cancer cells, during which the complex 1 showed IC₅₀ = 4.29 μmol L⁻¹ (SW–480), 4.91 μmol L⁻¹ (HepG2), 5.64 μmol L⁻¹ (HT-29) and 7.16 μmol L⁻¹ (HL-60). Complex 2 also showed minimum IC50 value in the range of IC₅₀ = 5.76 μmol L⁻¹ (SW–480), 3.51 μmol L⁻¹ (HepG2), 3.26 μmol L⁻¹ (HT-29) and 6.61 μmol L⁻¹ (HL-60) cell line. Complex 3 showed minimum IC₅₀ value in the range of IC₅₀ = 3.83 μmol L⁻¹ (SW–480), 5.71 μmol L⁻¹ (HepG2), 3.12 μmol L⁻¹ (HT-29) and 4.18 μmol L⁻¹ (HL-60) cell line. Complex 4 depicted minimum IC₅₀ value in the range of IC₅₀ = 4.82 μmol L⁻¹ (SW– 480), 5.28 μmol L⁻¹ (HepG2), 3.31 μmol L⁻¹ (HT-29) and 5.19 μmol L⁻¹ (HL-60) cell line. Complex 5 depicted IC₅₀ value in the range of IC₅₀ = 4.88 μmol L⁻¹ (SW–480), 4.64 μmol L⁻¹ (HepG2), 4.87 μmol L⁻¹ (HT-29) and 4.29 μmol L⁻¹ (HL-60) cell line. Complex 6 depicted IC₅₀ value in the range of IC50 = 6.47 μmol L⁻¹ (SW–480), 7.34 μmol L⁻¹ (HepG2), 5.92 μmol L⁻¹ (HT-29) and 7.43 μmol L⁻¹ (HL-60) cell line. Complex 7 depicted IC₅₀ value in the range of IC₅₀ = 3.77 μmol L⁻¹ (SW–480), 4.37 μmol L⁻¹ (HepG2), 5.23 μmol L⁻¹ (HT-29) and 5.11 μmol L⁻¹ (HL-60) cell line. While as Complex 8 and 9 depicted IC₅₀ value in the range of IC₅₀ = 5.31, 4.63 μmol L⁻¹ (SW–480), 6.51, 5.33 μmol L⁻¹ (HepG2),5.91, 4.29 μmol L⁻¹ (HT-29) and 6.32, 5.11 μmol L⁻¹ (HL-60) cell line. The screening data reveals that all the complexes showed promising anticancer activity by depicting inhibition count values close to the standard drug, Cisplatin. But complexes 2-5, 9 suppressed the cancer cell growth of HT-29 cells effectively than Cisplatin.

Table 1: The IC₅₀ values of complexes 1-6 against given cancer cell lines.

Microscopic examination of gross morphology of cancer cells and comparison with copper complex 8 treated normal and cancer cells is shown in Figure 8. The treatment of HT29 cells treated with the complex 8 10 μM (8A-8C) and complex 9 8 μM (8D-8F) is shown during which the decrease in the cell growth occur after 24 h but after 36 h treatment the cancer cells were almost dead.

Figure 8: Confocal microscopy of HT29 cells treated with the complex 8 (8A- 8C) and 9 (8D-8F) for 24 and 36 h.

Comet assay

In the comet assay, the images of HT-29 cells treated with complexes 8 and 9 showed the formation of comets. There was dosedependent increase in tail length when treated with complexes 8 and 9 (Figure 9). Complexes 8 and 9 showed genotoxic nature (formation of comet) which is in accordance with its maximum cytotoxicity as seen in MTT assay. The quantified increase in DNA damage suggested that the complexes 8 and 9 induced dose-dependent fragmentation of chromosomal DNA leading to apoptosis. The images of comet assay for positive control (cisplatin), cells treated with complexes 8 and 9 (10 μg/ mL) are depicted in Figure 9. Slides were analysed for parameter like tail length (TL), using image analyzer CASP software version 1.2.2. The results of the tail length are shown in Figure 10.

Figure 9: Detection of DNA damage in HT-29 cells. Treated cells (24 h) were layered over agarose gel, lysed, electrophoresed in alkaline buffer and stained with propidium iodide. The DNA fragmentation resulting in a comet-like appearance in cells treated with Cisplatin and complex 8 and 9.

Figure 10: Diagram comparing the effect of copper complex 8 and 9 on the tail length in comet assay. Complex 8 and 9 caused maximum DNA damage among the two complexes in the comet assay. The extent of damage caused by copper complex 9 was more than the Cisplatin.

Molecular docking studies with DNA

In our experiment, molecular docking studies of complex 8 and 9 with DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) were performed in order to predict the chosen binding site along with preferred orientation of the molecules inside the DNA groove. It is evident from the Figure 11 that the complex 8 recognizes the narrow minor groove region of DNA groove mainly through oxine ring and situated within slim A-T regions due to the planarity of the molecule because of two aromatic rings and preferential binding of oxine moiety to A-T regions leads to van der Waals and hydrophobic interaction with DNA functional groups which stabilizes the groove as well as the complex. The complex 9 showed the groove fit behaviour and arranged in a perpendicular manner with respect to the minor groove walls of the helix and stabilized by hydrogen bonding between the carbonyl group of oxine and NH of 6^th Thiamine at a distance of 2.72 Å. The resulting binding energies of docked complexes 8 and 9 were found to be –278.1 KJ mol^-1 and –284.94 KJ mol^-1, respectively which determine the stable binding as between DNA receptor and target ligand (Figure 11).

Figure 11: Docking models of DNA with complex 8 and 9 respective ly, The N- and O termini of the [2-(Benzothiazol-2-yliminomethyl)-phenol and 2-[(2ʹ-Aminobenzylidene)-amino]-benzenethiol of the complexes are shown as blue and red sticks, respectively.

Physicochemical properties

Bioactivity score: The bioactivity scores of the complex 8 and 9 were also calculated for different parameters, GPCR (G proteincoupled receptor) ligand activity, ion channel modulation, kinase inhibition activity, protease inhibitor, enzyme inhibitor and nuclear receptor ligand activity. As we know for metal heterocyclic complexes, if the bioactivity score is more than 0.00 then the complex is active, but if it is between -0.50 and 0.00 then the complex is moderately active and if the complex has -0.50 then it is inactive. The potential bioactivity score of the complexes 8 and 9 is given in Table 2 which clearly shows that complexes show those properties which are required for the characteristics of complex for acting as a drug.

Table 2: Bioactivity score of the synthesized metal complex 8 and 9.

PASS analysis: Molecular properties such as membrane permeability and bioavailability are always associated with some basic molecular descriptors such as log P (partition coefficient), molecular weight (MW), hydrogen bond acceptors and donors count in a complex. Lipinski used these molecular properties in formulating his ‘‘Rule of Five’’. This rule states that most metal complexes with good membrane permeability have log P ≤ 5, number of hydrogen bond acceptors ≤ 10 and number of hydrogen bond donors ≤ 5. This rule is widely used as a filter for drug-like properties. The required complexes 8 and 9 showed good permeability by showing log P < 5 also the other parameters like topological surface area, molecular weight, hydrogen bond acceptor and hydrogen bond donor depicted that both complexes has potential to act as drug (Table 3). Also by considering the bioactivity score, the overall potential of the complexes can be said to be physiologically active.

Table 3: Physicochemical properties of the synthesized complex 8 and 9.

In summary the development and operationally simple strategy for the better synthesis of copper complexes was successful. The reactions completed in almost 1-2 h and on completion, promising yields (75-82%) were obtained. From in vitro cytotoxicity screening, it is clear that copper complexes were found to be potential cytotoxic agents in comparison with standard drugs Cisplatin. Absorption and fluorescence studies reveal the stabilization of the energy levels of the complexes in presence of DNA. The cleavage and molecular docking studies undertaken in the present work are in total agreement with the primary intercalative mode of binding, although the van der Waals and other types of interactions can also be argued. Bioactivity score and PASS analysis also depicted the drug nature of these complexes. Hence, the present study has shown that these synthesized complexes can be used as template for future development through modification and derivatization to design more potent and selective cytotoxic agents.

Authors thank the Chairman, Department of Chemistry, A.M.U., Aligarh, for providing necessary research facilities, the UGC for a research fellowship and SAP (DRS-I) for research support are also gratefully acknowledged. Authors thank Department of Biochemistry JNMC Aligarh for biological studies.