International Journal of Medicine and Pharmaceutical Sciences (IJPMPS) ISSN 2250-0049 Vol.2, Issue 2, Sep 2012 10-18 Š TJPRC Pvt. Ltd.,
SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL ACTIVITIES OF COPPER(II) COMPLEX OF 2,9-DIMETHYL-1,10-PHENANTHROLINE VISHNU P. SONDHIYA, R.N. PATEL, DINESH K. PATEL, K.K. SHUKLA & Y. SINGH Department of Chemistry, A.P.S. University, Rewa , M.P. , India.
ABSTRACT One new copper(II) complex of 2,9-dimethyl-1,10-phenanthroline viz., [Cu(dmp)Cl2(H2O)] has been synthesized and structurally characterized by using single crystal x-ray diffraction studies. The complex crystallizes in monoclinic crystal system having space group P21. The copper(II) environment is distorted square pyramidal. Two molecules are present in the unit cell. Complex has been also characterized by elemental analysis, infrared, UV–visible, electron paramagnetic resonance and electrochemical studies. DNA binding activity of the complex has been measured by absorption spectroscopy. DNA cleavage activity of complex was measure using pBR322 DNA in the presence and absence of H2O2. The antibacterial activity of complex has been measured using agar disc diffusion method against E. coli, Citrobacter and Vibrio respectively.
KEYWORDS: Crystal Structure, Epr, Antibacterial Activity, DNA Cleavage, DNA Binding Activity INTRODUCTION 1,10-phenanthroline and its derivatives such as 2,9-dimethy-1,10-phenanthroline is one of the most studied ligand in coordination chemistry. Coordination compounds of transition metals with an N2Cl2 coordination sphere are in the focus of investigations not only of their interest in medicine but also in photochemistry1-5 and catalysis6. The easy replacement of the two chloro ligand makes these complexes to well studied intermediates on the synthetic pathway to a great variety of mixed ligand complexes with diimine ligands. Large numbers of copper(II) complexes have been synthesized and explored for their biological activities because of their biological relevance7,8. Survey of literature demonstrates that interest on the design of novel transition metal complexes capable of DNA cleaving duplex DNA with high sequence and structure selectivity9-11. Sigman and coworker12 have shown that copper complexes of 1,10-phenanthroline (phen) act as effective chemical nuclease with a high preference for double-stranded DNA in the presence of molecular oxygen and a reducing agent.
Synthesis, Characterization and Biological Activities of Copper(Ii) Complex of 2,9-Dimethyl-1,10-Phenanthroline
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METHODOLOGY All the solvents and chemicals were purchased from commercial sources. The elemental analysis of the complex was performed on an Elementar Vario ELIII Carlo Erba 1108 Elemental analyzer. Fast Atom Bombardment mass spectrum of the complex was recorded on a JEOL SX 102/DA 6000 mass spectrometer using xenon (6 kV, 10 mA) as the FAB gas. UV-vis spectrum was recorded at room temperature on Shimadzu 1601 spectrophotometer. Cyclic voltammetry was carried out on a BAS-100 Epsilon electrochemical analyzer having an electrochemical cell with a three-electrode system. Ag/AgCl was used as the reference electrode, glossy carbon as the working electrode and platinum wire as the auxiliary electrode. NaClO4 (0.1M) was used as supporting electrolyte and DMSO as solvent. All measurements were carried out at 298 K under a nitrogen atmosphere. Electron paramagnetic resonance (epr) spectra were recorded with a Varian E-line Century Series epr spectrometer equipped with a dual cavity and operating at X-band of 100 kHz modulation frequency. Tetracyanoethylene was used as field marker (g = 2.00277). Synthesis of [Cu(dmp)Cl2(H2O)] The compound was obtained by the interaction of hot dilute aqueous solution of CuCl2.2H2O and 2,9-dimethyl-1,10-phenanthroline in a 1:1 molar ratio. The solution was stirred for 3 hours at 50°C, after cooling at room temperature color changed from brown to parrot green in few days. Parrot green block shaped crystals were collected by filtration. These were dried in air at room temperature and stored in a CaCl2 desiccator. Anal. Calc. for C14H12Cl2CuN2O: C,46.88; H,3.37; N,7.81%. found:C,46.75; H,3.74; N,8.88; FAB Mass (m/z) Calc.: 358.71. Found: 359. Single crystal X-ray data were collected on CCD detector based diffractometer, SMART APEX from Bruker-Nonius Axs and CrysAlisPro, Oxford Diffractometer using graphite monochromatized MoKα (λ=0.71073Å). The diffraction data was solved using SIR-9213 with GUI control and structure was solved by SHELXL-9714 Refinement of F2 against all reflections. Non-hydrogen atoms were refined anisotropicaly. All hydrogen atoms were geometrically fixed and allowed to refine using a riding model. Molecular graphics was generated using different software such as ORTEP-3for WINDOWS15, PLATON and Mercury16. The superoxide dismutase activity (SOD) of the present copper(II) complex was evaluated using alkaline DMSO as source of superoxide radicals (O2-) generating system in association with nitro blue tetrazolium chloride (NBT) as a scavenger of superoxide17. In certain concentration of the complex solution 2.1 ml of 0.2 M potassium phosphate buffer (8.6 pH) and 1 ml of 56 µM NBT solutions was added. The mixtures were kept in ice for 15 minutes and then 1.5 ml of alkaline DMSO solution was added while stirring. The absorbance was monitored at 540 nm against a sample prepared under similar condition except NaOH was absent in DMSO. The in vitro antibacterial activities were tested against E.coli. Citrobacter and Vibrio. The agar disk diffusion was adopted for determination of antibacterial activity18 of the complex and comparison was made
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Vishnu P. Sondhiya, R.N. Patel, Dinesh K. Patel, K.K. Shukla & Y. Singh
with standard antibiotc. Autoclaved Nutrient agar medium was poured in to sterile petri-dish and allowed to solidify. Petri-dishes were seeded with bacterial species. Paper disc was placed at the dish after dipping test compound (DMSO solution). The width of the growth inhibition zone around the disc was measured after 24 h incubation at 37°C. Electronic absorption experiments were performed with a fixed concentration of metal complexes (25 µM) but variable nucleotide concentration ranging from 0 to 25 µM and after each addition of DNA to the metal complex, reading were noted. The data were then fit to the following equation to obtain intrinsic binding constant Kb19:
where [DNA] is the concentration of DNA base pairs, εa is the extinction coefficient of the complex at a given DNA concentration, εf is the extinction coefficient of the complex in free solution and εb is the extinction coefficient of the complex when fully bound to DNA. A plot of [DNA]/[εa-εf] versus [DNA] gave a slope and the intercept equal to 1/[εa-εf] and (1/Kb)[εb-εf], respectively. The intrinsic binding constant Kb is calculated from the ratio of the slope to the intercept. DNA cleavage experiment was performed by agarose gel electrophoresis method20. The efficiency of DNA cleavage was measured by determining the ability of the complexes to form open circular (OC) and nicked circular (NC) DNA from its super coiled (SC) form. The DMSO solution (1 × 10-3 M) containing metal complexes (5 µL, 250 µM) were taken in a clean Eppendroff tube and 1 µg of pBR 322 DNA was added. The content were incubated for 30 min at 37 °C and loded on 0.8 % agarose gel after mixing 3 µl of loading buffer (0.25 % bromophenol blue + 0.25 % xylene cynaol + 30 % glycerol sterilized distilled water). The electrophoresis was performed at constant voltage (75 V) until the bromophenol blue reached upto ¾ length of the gel. Further, the gel was stained for 10 min by immersing it in ethidium bromide solution (5 µg/ml of water) and then de-stained for 10 min by keeping it in sterile distilled water. The plasmid bands were visualized by photographing the gel under a UV transilluminator.
RESULTS AND DISCUSSIONS Conventional solution method was adopted for the synthesis of present complex. Interaction of cupric chloride with 2,9-dimethyl-1,10-phenanthroline in aqueous medium lead the formation of [Cu(dmp)Cl2(H2O)]. X-ray diffraction measurements of the [Cu(dmp)Cl2(H2O)] were made at 293K using suitable crystal size for data collection. Crystallographic data and structure refinement parameters are summarized in Table 1. The selected bond lengths and angles are listed in Table 2.
Synthesis, Characterization and Biological Activities of Copper(Ii) Complex of 2,9-Dimethyl-1,10-Phenanthroline
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Complex crystallizes as green crystals that belong to the monoclinic crystal system having space group P21with two molecules in the unit cell. The ORTEP view of the complex is given in Fig. 1. X-ray analysis shows that the copper(II) center in complex is pentacoordinated with two nitrogen from neocuprine and one oxygen atom from water molecule and two chlorine atoms. The related bond length and angles are presented in Table 2. The compound exhibits a distorted square pyramidal geometry. Most of the angles involving the center copper atom are widely different from 90° and 180°, indicating significant distortion from square pyramidal geometry. The three dimentional packing diagram is presented in Fig. 2. It can be observed that the molecules are packed in two dimensional manners with the parallel arrangement of the ring. Two molecules are present in the unit cell. The trigonally index τ is calculated using the equation τ = (β-α)/6021 (for perfect square pyramidal and trigonal bipyramidal geometries the value of τ are zero and unity, respectively). The value of τ is 0.262, which is indicating distorted square pyramidal geometry.
ELECTRON PARAMAGNETIC RESONANCE SPECTROSCOPY The epr spectrum of complex is recorded in DMSO at 77 K showed four well resolved hyperfine lines corresponding to coupling of the electron spin with the nuclear spin (63,65Cu, I = 3/2) are obtained in the parallel region (Fig. 3). The epr parameters of the complex is presented in Table 3. The solution epr spectrum of [Cu(dmp)Cl2(H2O)] at 77 K is axially symmetric (g║ = 2.256 and g┴ = 2.078) having a quartet hyperfine structure (Fig. 3) on the parallel component arising from the interaction of an unpaired electron with a single copper nucleus. This signal is typical of copper mononuclear complex. Apart from this, a weak signal observed at lower field suggests a dimeric structure for the complex in solution. Epr parameters and d-d transition energies were used to evaluate α2, β2, and γ2 which may be regarded as measures of the covalency of the in-plane σ-bonding, in-plane π and out-of-plane π-bonding respectively. The in plane σ-bonding parameter α2 was calculated22 from the expression.
The orbital reduction factor K2║ and K2┴ were calculated from the expression23. Significant information about the nature of bonding in the copper(II) complexes can be derived from the magnitude of the K║ and K┴.
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Vishnu P. Sondhiya, R.N. Patel, Dinesh K. Patel, K.K. Shukla & Y. Singh
where K║ = α2β2, K┴ = α2γ2 and λ0 represents the one electron spin-orbit coupling constant for the free ion, equal to -828 cm-1. Hathaway24 has pointed out that for pure σ-bonding K║ ≈ K┴ ≈ 0.77 and for in plane πbonding K║ < K┴ while for out-of-plane π-bonding K║ > K┴. It is observed that the relationship g║ > g┴ is typical of axially symmetric d9 copper (SCu = ½) having one unpaired electron in dx2-y2 orbital25. The g║ value of the complex is found to be g║ < 2.256, which indicate considerable covalent character of the M-L bond26. It is observed K║ < K┴ for present complex indicates in-plane bonding. The evaluated values of α2, β2 and γ2 of the complexes are consistent with strong in-plane π-bonding.
ELECTRONIC SPECTRAL STUDY Electronic spectra of complex were recorded in DMSO at room temperature. The d-d band observed at 788 nm which is assigned to π→π * transition of phenyl ring27. The charge transfer band was observed at 322 nm due to N→Cu LMCT transition28. The complex has a new band at 451 nm in solution. The polarized absorption spectra of this complex has shown that three band at 322, 451 and 788 nm are present a direct copper-to-copper interaction exists in the complex29. The 788 nm (dxy→dx2-y2) was assigned to the normal metal-ligand interaction29 so called copper band30. The band at 322 nm is assigned as a charge transfer band. The band due to the copper-to-copper interaction at 451 nm is based on the fact that it is a new band which is not shown in most of the copper(II) complexes29 Furthermore, polarized absorption spectrum shows that the absorption at 451 nm is much stronger along the copper-to-copper band than in the plane of the complex. Such type of copper-to-copper interaction in solution is due to the solvent effect. Due to this effect the molecule dimerizes (formation of dimeric molecule).
CYCLIC VOLTAMMETRY The electrochemical behavior has been studied by cyclic voltammetry under nitrogen atmosphere in DMSO solution containing NaClO4 as supporting electrolyte, using a glassy carbon working electrode and Ag/AgCl reference electrode. The cyclic voltammetric behavior of complex was determined between -0.96 to + 1.2 V (Fig. 4) and cathodic response (Peak A) in the range 270 to 295 mV with its counterpart (Peak D) in the range -549 to -593 mV. In this complex two electron redox process were observed (CuII/CuI and CuI/Cu0)31,32. Such type of electrochemical behavior was observed in present complex due to copper-tocopper interaction in solution (solvent effect). Due to solvent effect molecule dimerizes.
SUPEROXIDE DISMUTASE ACTIVITY IC50 value for SOD activity has been defined as the chromophore concentration required to yield 50% inhibition of the reduction of NBT. The IC50 value of the complex is 39 µM (Fig. 5). The complex showed appreciable superoxide dismutase due to its distorted square pyramidal geometry as established by single crystal X-ray diffraction studies. The distorted geometry of the complex may favor the geometrical
Synthesis, Characterization and Biological Activities of Copper(Ii) Complex of 2,9-Dimethyl-1,10-Phenanthroline
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change, which is essential for the catalysis. The SOD activity of the complex may be attributed to the flexible nature of ligand, which is able to accommodate the geometrical change from Cu(II) to Cu(I)33. The higher IC50 can only be accredited to the vacant coordination site, which facilitate the binding of superoxide anion, electron of aromatic ligands that stabilize Cu-O2•- interaction and not only to the partial dissociation of complex in solution.
ANTIBACTERIAL ACTIVITY The in-vitro antibacterial activity was measured by agar disc diffusion method against E. coli, Citrobacter and Vibrio. The antibacterial activity data are shown in Table 4. The antibacterial activity of complex is shown in Fig. 6 and the inhibition zone is given in Table 5. The activity was measured in graded concentration (750-60 µg/ml). The comples was most active against Viberieo (MIC = 100 µg/ml) and less active against E. coli. and Citrobactor (MIC = 250 µg/ml). Antibacterial activity of the complex depends on the ligand and helation facilitates the activity of a complex to cross a cell membrane and can be explained by Tweedy’s chelation theory34. Similar antibacterial activity was measured by our school previously35.
DNA BINDING STUDIES The binding strength of the complex with CT-DNA could be expressed in terms of intrinsic constant Kb, which represents the binding constant per DNA base pair. The experimental results are shown in Fig. 7. The intensity of the peak at ~350 nm got decreased due to the addition of CT-DNA. The binding constant Kb was found to be 7.5 × 104 M-1. The observed value of Kb is closer to other copper(II) complexes containing chloro ligand36 and the higher value of Kb was observed due to electron withdrawing nature of chloro ligand37.
DNA CLEAVAGE ACTIVITY Gel electrophoresis experiments using pBR322 plamid DNA was performed with two different concentrations of complex in the presence and absence of H2O2 as an oxidant (Fig. 8). It was observed that complex cleave DNA more effectively (lane 5 and 6) at higher concentration (500 µM) while at lower concentration (250 µM) it was less active (lane 3 and 4). The superior cleavage potential exhibited by complex at higher concentration in the presence of H2O2 (lane 6) thereby producing hydroxyl radicals or molecular oxygen, both of which are capable of damaging DNA by Fenton type chemistry38. The objective was achieved by monitoring the transition from the naturally occurring, covalently closed circular form (Form I) to the nicked circular relaxed form (Form II) by means of gel electrophoresis of plasmid DNA. The cleavage is inhibited by free radical scavengers implying that hydroxyl radical or peroxy derivatives mediate the cleavage reaction. The reaction is modulated by a metallo-complex bound hydroxyl radical or a peroxo species generated from the co-reactant H2O2. As can be seen from the results (Fig. 9), complex exhibits nuclease activity in the presence and absence of H2O239. The requirement for a reluctant for the cleavage of
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Vishnu P. Sondhiya, R.N. Patel, Dinesh K. Patel, K.K. Shukla & Y. Singh
DNA by copper complexes suggests that Cu(II) ions are being reduced to Cu(I) ions, which are susceptible to subsequent oxidation by bioxygen in air40. The resulting reactive oxygen species diffuses into the double standard DNA and cleaves it. The mechanistic pathway for the above reaction as follows: Cu2+ + reductant → Cu+ Cu+ + O2→ Cu2+ + O2•– O2•– + DNA → Non-specific cleavage in DNA
SUPPLEMENTARY DATA CCDC 870915 contain the supplementary crystallographic data for These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif or from the Director, CCDC, 12 Union Road, Cambridge, CB2, IEZ, UK (Fax: +44 1223 336 033; e-mail: deposit@ccdc.cam.ac.uk).
CONCLUSIONS We have synthesized a new copper(II) complexes derived from 2,9-dimethyl-1,10-phenanthroline and characterized by various physicochemical techniques. In present copper(II) complex g║ > g┴ > 2.0023 which is consistent with a dx2-y2 ground state in a distorted square pyramidal geometry. These complexes showed superoxide dismutase, antibacterial, CT-DNA binding and pBR 322 DNA cleavage activity.
ACKNOWLEDGEMENTS Our grateful thanks to School of Chemistry, University of Hyderabad, Hyderabad for Single Crystal X-ray data collection, RSIC (SAIF) IIT, Bombay for epr measurements and SAIF, Central Drug Research Institute, Lucknow is also thankfully acknowledged for providing elemental and mass spectral analysis. Financial assistance from CSIR [Scheme no. 01(2451)/11/EMR-II] and UGC [Scheme no.36-28/2008 (SR)] New Delhi are also thankfully acknowledged.
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APPENDICES Fig. 1 ORTEP view of [Cu(dmp)Cl2(H2O)] along with numbering scheme. Fig. 2 3D packing diagram of [Cu(dmp)Cl2(H2O)]. Fig. 3 EPR spectra of [Cu(dmp)Cl2(H2O)] at 77 K in DMSO. Fig. 4 Cyclic voltammogram of complex [Cu(dmp)Cl2(H2O)] scan rate 200 mV/s, 0.1 M NaClO4 was used as supporting electrolyte. Fig. 5 SOD activity of [Cu(dmp)Cl2(H2O]. Fig. 6 Antibacterial activity chart of [Cu(dmp)Cl2(H2O)]. Fig. 7 Electronic absorption spectra of [Cu(dmp)Cl2(H2O)] in the absence and presence of increasing amounts of DNA (0-25 µM) and plot of [DNA]/εa-εf) vs. [DNA]. Fig. 8 Gel electrophoresis showing the chemical nuclease activity of the complex 1 on pBR 322 plasmid DNA, incubation at 37 °C for 1 hr in the presence and absence of H2O2; Lane 1- Control DNA, Lane 2- DNA + H2O2, Lane 3- DNA + complex 1 (250 µM), Lane 4- DNA + complex 1 (250 µM) + H2O2, Lane 5- DNA + complex 1 (500 µM), Lane 6- DNA + complex 1 (500 µM) + H2O2.