{Computational, antimicrobial, DNA binding and anticancer activities of pyrimidine incorporated ligand and its copper(II) and zinc(II) complexes} J. Serb. Chem. Soc. 84 (3) 277–291 (2019) UDC 546.562’472+519.677:547.963.32+ JSCS–5183 547.853:615.277 Original scientific paper 277 Computational, antimicrobial, DNA binding and anticancer activities of pyrimidine incorporated ligand and its copper(II) and zinc(II) complexes MURUGESAN SANKARGANESH1, NAGARAJ REVATHI2,3, JEYARAJ DHAVEETHU RAJA1*, KARUNGANATHAN SAKTHIKUMAR1, GUJULUVA GANGATHARAN VINOTH KUMAR1, JEGATHALAPRATHABAN RAJESH1, MANIKKAM RAJALAKSHMI4 and LIVIU MITU5** 1Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai, Ramanatha- puram, Tamil Nadu 623 806 India, 2Department of Chemistry, Ramco Institute of Technology, Rajapalayam, Virudhunagar, Tamil Nadu 626 117, India, 3Department of Chemistry, Manon- manium Sundaranar University, Tirunelvi, Tamil Nadu 627 012, India, 4Bioinformatics Centre, PG & Research Centre Department of Biotechnology and Bioinformatics, Holy Cross College (Autonomous), Tiruchirapalli, Tamil Nadu 620 002, India and 5Department of Nature Sciences, University of Pitesti, Pitesti 110 040, Romania (Received 6 June, revised 7 August, accepted 5 September 2018) Abstract: In this research article, the synthesis, structural characterization, and biological, DNA binding and anticancer properties of pyrimidine incorporated Schiff base ligand L and its [CuL2](ClO4)2 (1) and [ZnL2](ClO4)2 (2) complexes are reported. The isolated complexes 1 and 2 have significant antibacterial and antifungal properties, greater than those of ligand L. The interaction between protein and L were analyzed by an in silico method. The intercalative binding of the prepared compounds was proved from electronic absorption, fluorometric, cyclic voltammetric and viscometric methods. The calculated binding parameters such as, Kb (2.65×103, L; 7.74×103, 1 and 2.99×103, 2); Ksv (3.30×103, L; 4.31×103, 1 and 3.89×103, 2), and Kapp (2.15×105, L; 3.30×105, 1 and 2.82×105, 2) indicted that complex 1 has better interaction ability than L and complex 2. The in vitro anticancer properties of L, and complexes 1 and 2 against human cancer (MCF-7, HeLa and HEp-2) and normal (NHDF) cell lines were determined by the MTT assay method. The obtained results designated that complexes 1 and 2 exhibited substantial anticancer activity against the cancer cell lines, better than that of L. Keywords: metal complexes; DFT; antimicrobial; DNA interaction; anticancer studies. *,** Corresponding authors. E-mail: (*)jdrajapriya@gmail.com; (**)ktm7ro@yahoo.com https://doi.org/10.2298/JSC180609080S 278 SANKARGANESH et al. INTRODUCTION Pyrimidine is a heterocyclic compound and it forms the backbone of deoxy- ribonucleic acid (DNA) and ribonucleic acid (RNA). Pyrimidine derivative drugs reveal various pharmacological and biological activities, such as, antitumor,1,2 antiamoebic,3 antimalarial,4,5 antipneumocystis carinii pneumonia6 and antimic- robial.2,7,8 Moreover, the attachment of CF3 groups results in compounds that are lipophilically and pharmacologically more active than the corresponding non- -fluorinated compounds. These –CF3-substituted organic compounds have vari- ous biological competences, such as, herbicidal, fungicidal, analgesic, antihyper- glycemic and antipyretic, etc.9–13 Pyrimidine-containing drugs, such as gefitinib, erlotinib and afatinib were used in cancer-related treatments.14 Therefore, Schiff base ligands were prepared from biologically and pharmacologically active pyri- midine derivative compounds. These Schiff base ligands possess analytical, bio- logical and pharmacological applications.15 In earlier days, Schiff bases were employed to develop metal-based complexes16,17 because metal complexes acquire diverse biological abilities, such as antidiabetic, antimicrobial, antioxi- dant, DNA cleavage or binding and anticancer activities.18–20 Especially, cop- per(II) and zinc(II) complexes were synthesized with much more interest due to their enhanced biological applications.21–23 The interest in the biological properties of copper(II) and zinc(II) complexes has been continued in the present study through incorporation of a bio-active pyri- midine ligand. The formations of L, and complexes 1 and 2 were confirmed from analytical, spectroscopic and theoretical techniques. Moreover, their biological pro- perties, such as antimicrobial, DNA binding and in vitro anticancer activities were studied. EXPERIMENTAL Materials and methods 4-(4-Morpholinyl)benzaldehyde, 2-hydrazino-4-(trifluoromethyl)pyrimidine, Tris-HCl, sodium chloride and ethidium bromide were procured from Sigma Aldrich. Cu(ClO4)2·6H2O and Zn(ClO4)2·6H2O were received from Alfa Aesar company. Calf thymus (CT) DNA was purchased from GeNei Bangalore, India. Elemental analyses were realized on an Elementar Vario EL III analyzer. FTIR and NMR spectra were recorded using an FTIR Affinity-1 Shi- madzu instrument and Bruker (400 and 125 MHz) spectrometers. The mass spectra were obtained on an ESI-MS spectrometer. The ESR spectra were recorded at 300 and 77 K in an IIT, Mumbai. Cyclic voltammetric studies were recorded on a CHI650C instrument. The absorption and fluorescence spectra were recorded on a UV-1800 Shimadzu spectro- photometer and FluoroMax-4 spectrometer, HORIBA, respectively. Analytical and spectral data are given in Supplementary material to this paper. Synthesis of ligand L Ligand L was synthesized by refluxing 4-(4-morpholinyl)benzaldehyde (0.1912 g, 1 mmol) with 2-hydrazino-4-(trifluoromethyl)pyrimidine (0.1781 g, 1 mmol) in 25 mL of etha- nol for 6 h. The resulting solution was slowly evaporated on a water bath. The yellow col- PYRIMIDINE INCORPORATING METAL(II) COMPLEXES 279 oured product was collected by filtration and washed with 10 mL of cold methanol and recrys- tallized from hot chloroform. Synthesis of complexes 1 and 2 An ethanolic solution of ligand L (2 mmol) was mixed with metal perchlorates (1 mmol) in 10 mL of ethanol maintaining a metal : ligand ratio of 1 : 2. The resulting solution was ref- luxed under stirring for 5 h. Then the solution was evaporated on a water bath, the precipitated solid filtered and washed thoroughly with 10 mL of petroleum ether and dried in vacuo over CaCl2. Computational studies Density functional theory (DFT) calculations were performed with the hybrid exchange- -correlation function using the 6-311G(d,p) and LANL2DZ basis set by the Gaussian 09 pro- gram to understand the mode of complexation between the ligand and its complexes.24 Initially, the optimized geometries of both the ligand and its complexes were determined by the B3LYP functional but using 6-311G(d,p) and LANL2DZ basis set, respectively. Antimicrobial studies The antimicrobial activity of the prepared compounds were screened against the bacterial species, Escherichia coli, Klebsiella pneumoniae, Pseudomonas fluorescens, Shigella sonnei and Staphylococcus aureus, and the fungal species, Aspergillus niger, Candida albicans, C. tropicalis, Mucor indicus and Rhizopus by the well diffusion method.21 The reference drugs streptomycin and amphotericin were used for the antibacterial and antifungal studies, respectively. Anticancer studies The anticancer capabilities of ligand L, complexes 1 and 2 against various cancer cell lines (MCF-7, breast adenocarcinoma; HeLa, cervical, HEp-2, laryngeal) and a single normal cell line (NHDF, normal human dermal fibroblasts) were performed by the MTT assay25: Absorbance of sample Inhibition, % 100 100 Absorbamce of control = − (1) Protein–ligand docking The docking of ligand L with protein was studied by a previously reported method.26 The prepared proteins and ligand L were docked using the LibDock module of Accelrys Discovery Studio software, version 2.1, to acquire the drug with protein interaction. DNA interaction The CT-DNA interaction of ligand L and complexes 1 and 2 in 5 mM and 50 mM Tris- HCl buffer solution, respectively, under physiological conditions were appraised using absorption spectroscopy and fluorescence, cyclic voltammetric and viscometric measure- ments.27-29 RESULTS AND DISCUSSION The synthetic route to ligand L, and complexes 1 and 2 are depicted in Schemes 1 and 2, respectively. The ligand L (4-(4-morpholinyl)benzaldehyde, 2- -[4-(trifluoromethyl)-2-pyrimidinyl]hydrazone) is lemon yellow in colour and well soluble in acetone, acetonitrile, DMSO and DMF solvents. The complexes 1 and 2 are brown and dark brown colour and soluble in DMSO and DMF solvents. The molar conductance at infinite dilution (Λm) of complexes 1 and 2 in DMSO 280 SANKARGANESH et al. was found at 104 Ω–1 cm2 mol–1 for complex 1 and 109 Ω–1 cm2 mol–1 for com- plex 2, which signified them to be electrolytes. Moreover, the obtained conduct- ance values confirmed that percholarate ions are present in the outside coordin- ation sphere of complexes 1 and 2. Scheme 1. Synthesis of ligand L. Scheme 2. Synthesis of complexes 1 and 2. NMR spectra The 1H-NMR spectra of ligand L and complex 2 were recorded in DMSO-d6 solution (Supplementary material). In the 1H-NMR spectrum of ligand L, the azomethine (–CH=N–) proton signal appeared at 8.10 (1H, s) and the NH proton signal at 9.90 (1H, s) ppm. The pyrimidine (–CH=C–CF3 and =CH–N=) protons signals were observed at 7.19 (1H, d) and 8.77 (1H, d) ppm, respectively. The signals of the aromatic protons were found at 6.99 (2H, d) and 7.55 (2H, d) ppm. The morpholine moiety protons (mor-CH2–N–CH2– and mor-CH2–O–CH2–) signals appeared at 3.20 (4H, t) and 3.75 (4H, t) ppm, respectively. In the 1H-NMR spectra of complex 2, the signals of the azomethine (–CH=N–) and pyrimidine moiety protons (=CH–N=) appeared at 8.64 (1H, s) and 8.83 (1H, d), respectively. These spectral results reveal that the azomethine and pyrimidine nitrogen atoms participate in the complexation. As per com- parison with the free ligand L, the signals of the pyrimidine (–CH=C–CF3 and –NH), aromatic and morpholine (mor-CH2–N–CH2– and mor-CH2–O–CH2–) protons remained more or less at the same positions in complex 2. The 13C-NMR spectra of ligand L and complex 2 were taken in DMSO-d6 solution and the obtained data are given in the Supplementary material. In the PYRIMIDINE INCORPORATING METAL(II) COMPLEXES 281 spectra of ligand L, the azomethine (–CH=N), pyrimidine C2 and C6 appeared at 138.60, 160.61 and 153.60 ppm, respectively. In complex 2, these peaks appeared at 139.45, 161.10 and 154.16 ppm, respectively. These results indicate that ligand L formed coordinate bonds with the central metal atom via azomethine and pyrimidine ring (C2) nitrogen atoms. FT-IR spectra The FT-IR spectrum of ligand L showed a characteristic band at 1544 cm–1 due to ν(–CH=N) stretching vibrations.30 The bands at 1591, 1465 and 1402 cm–1 are associated with ν(–NH) and ν(=CH–N=) aromatic ring nitrogen stretching vibrations, respectively. In the spectra of complexes 1 and 2, characteristic bands appeared at 1522 (1) and 1535 cm–1 (2), and 1388 (1) and 1384 cm–1 (2), which are due to ν(CH=N) and ν(aromatic, =CH–N=) group vibrations. This result indicates that nitrogen atoms of azomethine ν(CH=N) and the pyrimidine ring ν(aromatic, =CH–N=) participated in the complexation. Bands are observed at 1087 and 1088 cm–1 in complexes 1 and 2, respectively, due to the presence of perchlorates in the outer coordination spheres. Mass spectra In the ESI-MS spectrum of ligand L, the molecular ion peak was observed at m/z 350.9 and daughter peaks were observed for C12H7N4F3+, C6H5N4F3+ and C5H4N3F3+. In complexes 1 and 2, molecular ion peaks were obtained at m/z, 765 (1) and 767 (2). These values evidence that one mole of complex 1 or 2 con- tained with two moles of ligand L. Electronic absorption spectra In the UV–Vis spectra of ligand L, and complexes 1 and 2, bands were observed at 266 and 336 nm owing to π→π* and n→n* transitions of ligand L.31 The ligand to metal charge transfer (LMCT) spectra of complexes 1 and 2 showed bands at 272 and 329 nm for complex 1 and 270 and 338 nm for complex 2. In the UV–Vis spectrum of complex 1, a broad band was displayed at 777 nm and the band assignments are 2B1g→2A1g, 2B1g→2B2g and 2B1g→2Eg transit- ions in a square planar arrangement.32 Complex 2 possess d10 electronic config- uration, which authenticates the absence of a d–d transition, and therefore com- plex 2 showed INCT bands at 270 and 338 nm, which confirms the complexation. ESR spectra ESR spectral data give knowledge of the environment of a metal ion. The ESR spectrum data of complex 1 recorded in DMSO at liquid nitrogen tempera- ture gave gII and gI values of 2.35 and 2.07, respectively. The spectrum of complex 1 showed that g-tensor value are gII > gI > 2.0027, indicating the unpaired electrons lie predominantly in the dx2–dy2 orbital, which suggests square 282 SANKARGANESH et al. planar geometry around the central metal ion.33–36 The gII/AII value is useful for predicting the structure of the complex 1 and the value of 154 suggests square planar arrangement of complex 1 around the central metal ion.37 According to Hathaway and Billing,38–40 the G value greater than 4 suggested that the local tetragonal axes are aligned parallel or only slightly misaligned. Computational studies The 3D structure of ligand L, and complexes 1 and 2 were optimized using Density functional theory (DFT) calculations accomplished through the (B3LYP/ /6-311G(d,p)/LANL2DZ) basis set using the Gaussian 09 program.24 The basis set 6-311G(d,p) was used for the N, O, C and H atoms and LANL2DZ was exploited for complexes 1 and 2. In ligand L, the HOMO is spread over the whole π-moiety, while the LUMO is spread over the pyrimidine and imine moi- eties. After the introduction of Cu(II), the HOMO is spread over the surrounding of the metal ion and its LUMO is spread on the pyrimidine moiety with little contribution of the metal ion. Similarly, in 2, the HOMO and LUMO are more spread over the pyrimidine moiety and slightly in the morpholine moiety with a metal ion contribution (Fig. 1). In addition, the calculated HOMO–LUMO energy gap for complexes 1 and 2 was lower than that for the free ligand. The order of the energy band gap is L > 2 > 1. The obtained results obviously indicate that the complexation of Cu(II) and Zn(II) to the ligand L result in a disruption of internal charge transfer, which may be responsible for the appearance of new absorption band in the visible region. Fig. 1. Frontier molecular orbitals of Ligand L, complexes 1 and 2 (from left to right). PYRIMIDINE INCORPORATING METAL(II) COMPLEXES 283 Biological studies The pharmacological, DNA binding and in vitro anticancer activities of lig- and L, complexes 1 and 2 were studied as follows. Antimicrobial studies. The antimicrobial activity of ligand L, and complexes 1 and 2 were screened against the bacterial species E. coli, K. pneumoniae, P. fluorescens, S. sonnei and S. aureus, and the fungal species A. niger, C. albicans, C. tropicalis, M. indicus and Rhizopus. The reference drugs streptomycin and amphotericin were used for the antibacterial and antifungal studies, respectively. The zone of inhibition values of isolated compounds are presented in Table I. The obtained results established that the synthesized complexes 1 and 2 acted as better antibacterial and antifungal agents than ligand L due to their chelation ability.41 Additionally, ligand L, and complexes 1 and 2 show superior activity against E. coli bacteria and C. albicans fungi than against the other pathogens. TABLE I. Antimicrobial activities (zone of inhibition, mm) of ligand L, and complexes 1 and 2 Compound Bacterial strains E. coli K. pneumoniae P. fluorescens S. sonnei S. aureus 1 9 8 8 8 8 2 8 6 6 7 6 L 6 5 4 5 4 Streptomycin 18 12 14 16 16 Fungal strains A. niger C. albicans C. tropicalis M. indicus Rhizopus 1 9 15 12 7 9 2 8 13 9 6 8 L 5 10 6 5 6 Amphotericin 18 17 31 9 24 Anticancer studies. The positive results received from the biological studies encouraged the investigation of the anticancer ability of the pyrimidine incor- porated compounds. The in vitro anticancer activities of the ligand L, and com- plexes 1 and 2 against human cancer cell lines (MCF-7, breast adenocarcinoma; HeLa, cervical; HEp-2, laryngeal) and NHDF (normal human dermal fibroblasts) cell line were examined by using the MTT assay (Fig. S-1). The 50 % inhibitory concentration (IC50, µg mL–1) values of the prepared compounds against human normal and cancer cell lines are given in Table II. From the obtained data, cis- platin can affect the cancer as well as normal cell lines at lower concentration (6.93±0.35 µg mL–1 MCF-7; 7.46±0.37 µg mL–1 HeLa; 10.28±0.51 µg mL–1 HEp-2). When compared to the above results, L had no significant anticancer activity against the cancer cell lines or the normal cell lines (75.82±3.79 µg mL–1 MCF-7; 76.26±3.81 µg mL–1 HeLa; 81.03±4.05 µg mL–1 HEp-2). Moreover, the IC50 values of the complexes 1 and 2 against cancer cell lines revealed that 284 SANKARGANESH et al. complex 1 has moderate anticancer ability on MCF-7 (54.51±2.73 µg mL–1) and on HeLa (55.40±2.77 µg mL–1) cell lines which was not so obvious on the HEp-2 cell line (77.57±3.88 µg mL–1). However, complex 2 expressed modest anticancer activities on all three selected cancer cell lines (58.89±2.94 µg mL–1, MCF-7; 59.98±2.99 µg mL–1, HeLa; 60.79±3.04 µg mL–1, HEp2). From these observations, the pyrimidine incorporated complexes 1 and 2 could control the growth of cancer cells. TABLE II. Anticancer activities of ligand L, and complexes 1 and 2 on cancer and normal cell lines Compound IC50 / µg mL-1 MCF-7 HeLa HEp-2 NHDF Cisplatin 6.93±0.35 7.26±0.36 7.46±0.373 10.28±0.51 L 75.82±3.79 76.26±3.81 81.03±4.05 100.48±5.02 1 54.51±2.73 55.40±2.77 77.57±3.88 105.67±5.28 2 58.89±2.94 59.98±2.99 60.79±3.04 107.04±5.35 Protein–ligand interaction The mode of interaction of ligand L with antioxidant enzymes and the estro- gen receptor alpha was analyzed using molecular docking studies. From the various obtained docked poses, the best pose was chosen based on the higher absolute energy and the LibDock score. The protein–ligand interaction was found to be stronger in the presence of hydrogen bond interactions, especially with bond length less than 0.3 nm (Table III).42,43 The present study also revealed the presence of H-bond interaction between ligand L and the proteins indicating a better protein–ligand interaction. Binding of ligand L with the antioxidant enzymes and the estrogen receptor alpha predicts the mode of action through which the compound acts as an anticancer agent by regulating their involvement in cancer progression. TABLE III. In silico results for protein–ligand interactions Interaction of ligand L with No. of poses Absolute energy, kcala mol-1 Libdock score, kcal mol-1 No. of H-bonds Bond length, nm Interacting residues 1SPD_A 12 74.225 60.174 1 0.24 VAL 81 1QQW_A 25 74.426 112.491 1 0.23 TYR 358 2HE3_A 19 74.426 101.122 1 0.25 SER 132 1A52_A 67 74.669 97.379 4 0.240 ARG 394 a1 kcal = 4184 J DNA binding Absorption titration. The interaction between the compounds and CT-DNA was ascertained by electronic absorption spectroscopy. The absorption spectra of complexes 1 and 2 (50 µM) in the presence and absence of CT-DNA (0–50 µM) PYRIMIDINE INCORPORATING METAL(II) COMPLEXES 285 were assessed by typical absorption spectral titrations (Fig. 2a and b). As shown in Fig. 2a and 2b, the incremental addition of CT-DNA to solutions of complex 1 and 2 led to hypochromism (6.27, L; 15.60, 1 and 13.57, 2) with bathochromic shifts of 3–6 nm. These observed spectral titration results clearly indicate that complexes 1 and 2 can bind with CT-DNA through intercalative interaction due to the presence of the pyrimidine substituent. In order to determine the intrinsic binding constants (Kb) of the prepared compounds, the following equation25 was used: ( ) 1DNA DNA b b f a f b f c c K ε ε ε ε ε ε − = +  −  − − (2) where cDNA is the concentration of CT–DNA. Fig. 2. Absorption spectra of complexes 1 (a) and 2 (b) in Tris-HCl/NaCl buffer at room temperature in the presence of CT-DNA solutions. Dotted lines refer to the free compound; the solid lines to absorption spectra of the compound in the presence of different concentrations of DNA. The Kb values were obtained from the ratio of slope to the intercept from plots of cDNA/(εa–εf) vs. cDNA. The obtained Kb values of the synthesized com- pounds (2.65×103, L; 7.74×103, 1 and 2.99×103, 2) are given in Table IV. The obtained Kb values are low as compared to that for ethidium bromide (EB) (1.4×106). The lower Kb values for synthesized compounds are due to the flexible, versatile morpholine moiety in the complexes that greatly facilitate TABLE IV. Absorption spectral properties of the synthesized compounds on interaction with CT-DNA Compound λmax / nm Δλ / nm Hypochromism, % Kb / 103 Free Bound 1 329.0 335.0 6.0 15.60 7.74 2 338.0 343.0 5.0 13.57 2.99 L 336.0 339.0 3.0 6.27 2.65 286 SANKARGANESH et al. intercalation with the base pairs. The binding strengths of the studied compounds with DNA show that complex 1 has a higher binding affinity as compared to ligand L and complex 2, in the following order: 1 > 2 > L. Competitive binding. The fluorescence spectra of EB–DNA in the presence of the prepared compounds are presented in Fig. 3. As shown in Fig. 3a and b, the concentration of complexes 1 and 2 enlarges to the EB–DNA, the fluores- cence intensity of the complexes 1 and 2 steadily decreases. These results con- firm that competitive interactions occurred. The intercalative binding of ligand L, complexes 1 and 2 with EB–DNA could be summarized by the Stern–Volmer equation:44 I0/I = 1 + KsvcQ (3) where I and I0 are the emission intensities in the presence and absence of the quenchers (ligand L, complexes 1 and 2), respectively, Ksv is the linear Stern– –Volmer quenching constant and cQ is the concentration of the quencher. The Ksv values were obtained from the slope of plots of I0 / I vs. cQ (Fig. 3). Addit- ionally, the binding affinity (Kapp) of ligand L complexes 1 and 2 in contrast to that of EB was evaluated using the following equation:45 Fig. 3. Emission spectra of EB–CT-DNA in the presence of increasing amounts of com- plex 1 (a) and 2 (b). PYRIMIDINE INCORPORATING METAL(II) COMPLEXES 287 KEBcEB = Kappccomplex (4) where, ccomplex is the concentration of the newly prepared compounds necessary for a 50 % reduction in the fluorescence intensity of EB and KEB = 1.0×107. The binding constant (Ksv) and binding affinity (Kapp) values of the present com- pounds are given in Table V. The Ksv and Kapp values of the prepared com- pounds are in the following order: 1 (Ksv 4.31×103 and Kapp 3.30×105) > 2 (Ksv 3.89×103 and Kapp 2.82×105) > L (Ksv 3.30×103 and Kapp 2.15×105). TABLE V. The quenching constants and binding affinity of ligand L, and complexes 1 and 2 with EB–DNA Compound Ksv / 103 Kapp / 105 1 4.31 3.30 2 3.89 2.82 L 3.30 2.15 Electrochemical studies The results of cyclic voltammetric measurements of complexes 1 and 2 in the presence and absence of CT-DNA (0–50 µM) are presented in Fig. 4a and b. Here, as the concentration of CT-DNA rises, the cathodic and anodic peak cur- rents of the prepared complexes 1 and 2 increase, and their peak potentials are shifted to negative values. These findings confirm that the newly prepared com- pounds can interact with CT-DNA. Viscosity measurements The viscometric studies were very useful in the clarification of the nature of the binding of the synthesized compounds to CT-DNA. The graphs of the relative viscosity vs. ccomplex/cDNA are presented in Fig. 5. As the concentration of com- pounds increases, the viscous flow of CT-DNA increases. These results correlate Fig. 4. Cyclic voltammograms of complexes 1 (a) and 2 (b) in Tris-HCl buffer at 25 °C in the presence of increasing amounts of CT-DNA. 288 SANKARGANESH et al. with the spectroscopic and cyclic voltammetric results that suggested the prepared compounds should interact with CT-DNA through an intercalation mode. Fig. 5. Effect of increasing amount of complexes 1 and 2 on the relative viscosity of DNA. 1/R = ccompound/cDNA. CONCLUSION The Schiff base ligand L, and complexes 1 and 2 were isolated from pyri- midine backbones. The analytical data confirmed that the prepared complexes 1 and 2 possess 1:2 stoichiometry ratios. Square planar geometry of complexes 1 and 2 were finalized from the results of spectroscopic techniques. The prepared compounds possess better antibacterial and antifungal activity against E. coli (bacteria) and C. albicans (fungi) than against the other tested pathogenic species. The molecular docking results revealed that ligand L can interact with proteins. The interacting ability of ligand L, and complexes 1 and 2 with CT-DNA were explored by spectroscopic, cyclic voltammetric and viscometric techniques and the results suggested that the prepared complexes can bind with CT-DNA by intercalatation. The in vitro antitumor results suggested that the pyrimidine incor- porating complexes 1 and 2 have moderate anticancer activities. SUPPLEMENTARY MATERIAL Spectral and analytical data of the synthesized compounds, as well as graphical representation of anticancer activities, are available electronically at the pages of journal website: http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgements. The authors acknowledge the Department of Science and Techno- logy (DST)-Science and Engineering Research Board (SERB-Ref. No.: SR/FT/CS-117/2011 dated 29.06.2012), Government of India, New Delhi for the financial support. The authors express their sincere and heartfelt thanks to Managing Board, Dean, Principal, Head and staff members, Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai for their constant encouragement and providing the research facilities. PYRIMIDINE INCORPORATING METAL(II) COMPLEXES 289 И З В О Д КОМПЈУТЕРСКА АНАЛИЗА, ДНК ИНТЕРАКЦИЈЕ, АНТИМИКРОБНА И АНТИКАНЦЕРСКА АКТИВНОСТ БАКАР(II) И ЦИНК(II) КОМПЛЕКСА СА ЛИГАНДОМ КОЈИ САДРЖИ ПИРИМИДИНСКИ ПРСТЕН MURUGESAN SANKARGANESH1, NAGARAJ REVATHI2,3, JEYARAJ DHAVEETHU RAJA1, KARUNGANATHAN SAKTHIKUMAR1, GUJULUVA GANGATHARAN VINOTH KUMAR1, JEGATHALAPRATHABAN RAJESH1, MANIKKAM RAJALAKSHMI4 и LIVIU MITU5 1Chemistry Research Centre, Mohamed Sathak Engineering College, Kilakarai, Ramanathapuram, Tamil Nadu 623 806 India, 2Department of Chemistry, Ramco Institute of Technology, Rajapalayam, Virudhun- agar, Tamil Nadu 626 117, India, 3Department of Chemistry, Manonmanium Sundaranar University, Tirunelvi, Tamil Nadu 627 012, India, 4Bioinformatics Centre, PG & Research Centre Department of Bio- technology and Bioinformatics, Holy Cross College (Autonomous), Tiruchirapalli, Tamil Nadu 620 002, India и 5Department of Nature Sciences, University of Pitesti, Pitesti 110 040, Romania У овом раду описана је синтеза, структурна карактеризација, антимикробна и анти- канцерска својства, као и испитивање интеракција са молекулом ДНК, комплекса [CuL2] .(ClO4)2 (1) и [ZnL2] .(ClO4)2 (2) који као лиганд (L) садрже Шифову базу. У односу на лиганд, одговарајући комплекси (1 и 2) су показали значајну антибактеријску и анти- фунгалну активност. На основу електронских апсорпционих, флуорометријских и цик- лично-волтаметријских мерења, као и на основу мерења вискозитета, потврђено је интеркалативно везивање комплекса за хеликс ДНК. Израчунати су следећи параметри везивања испитиваних комплекса за ДНК: Kb (2.65×103 (L); 7.74×103 (1) и 2.99×10 3 (2)), Ksv (3.30×103 (L); 4.31×103 (1) и 3.89×103 (2)) и Kapp (2.15×105 (L); 3.30×105 (1) и 2.82×105 (2)). Ове вредности показују да комплекс 1 има бољу интеркалациону способ- ност везивања за ДНК у односу на лиганд L и комплекс 2. In vitro антиканцерска својства лиганда L и комплекса 1 и 2 према хуманим ћелијама тумора (MCF-7, HeLa и HEp-2) и нормалним (NHDF) ћелијским линијама одређена су помоћу MTT методе. Добијени резултати су показали да комплекси 1 и 2 показују значајно већу активност према ћелијама канцера у односу на лиганд L. 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