Scientific paper Synthesis, Characterization and Biological Activities of Ciprofloxacin Drug Based Metal Complexes Mohan N. Patel,* Promise A. Dosi and Bhupesh S. Bhatt Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388 120 Gujarat (INDIA) * Corresponding author: E-mail: jeenen@gmail.com; Phone number: (+912692) 226856*218 Received: 20-12-2011 Abstract The interaction of small molecules with DNA has attracted a great deal of attention. Mixed ligand copper(II) complexes of type [Cu(cpf)(Ln)Cl] [cpf = ciprofloxacin, Ln = phenanthroline derivatives] were synthesized and characterized by elemental analysis, reflectance, IR and mass spectra. Viscosity measurements, absorption titration and DNA melting temperature studies were employed to determine the mode of binding of complexes with DNA. DNA cleavage study showed better cleaving ability of the complexes compare to metal salts and standard drug. The SOD mimic study showed IC50 value of complexes in the range of 0.95 to 1.75 |M. Antibacterial activity was assayed against selective Gram(-ve) and Gram(+ve) microorganisms. Keywords: Neucleolytic activity, Calf thymus DNA, Phenanthroline, Absorption titration, DNA Melting temperature 1. Introduction Copper is an essential micronutrient and found in several proteins and enzymes. Numerous copper compounds are able to act as antioxidant, antimicrobial, antiparasitic, anti-inflammatory, anticonvulsant and antitumoral agents. Copper complexes with ligands containing nitrogenated aromatic rings have deserved a great interest since the complex of 1,10-phenanthroline proved its ability to break DNA chains.1,2 Cells and tissues injury (oxidative stress) results from the imbalance between pro and antioxidant species.3 Superoxide dismutase activity (SOD) in conjunction with catalase appears to be the most effective enzymatic defence system against the toxicity of oxygen metabolism.4,5 Among the known SOD enzymes, CuZn(SOD) is the most efficient catalytic species. Recently, there has been considerable interest in the DNA binding properties of transition metal complexes with small molecules on a molecules level.6 The design of complexes that bind and react with DNA becomes important as we begin to delineate the expression of genetic information on a molecular level. Recently, the complexes of vanadium(IV), copper(II), magnesium(II), ura-nium(VI), manganese(II), iron(III), cobalt(II), nickel(II), molybdenum(II) and europium(III) with fluoroquinolones have been synthesized and explored for their biological activities, because of its biological relevance.713 Targeting above facts and in continuation to our earlier work,14 herein, the interaction of Cu(II) with the ciprofloxacin in the presence of a nitrogen donor heterocyclic ligand (phe-nanthrolines) has been studied in an attempt to examine their biological behavior. 2. Experiments 2.1. Materials-Instrumentation All the chemicals and solvents used were of analytical grade. Ciprofloxacin hydrochloride was purchased from Bayer AG (Wuppertal, Germany). Cu-pric chloride dihydrate, 3-chlorobenzaldehyde, 3-bro-mobenzaldehyde, 3-benzyloxybenzaldehyde, 4-benzy-loxybenzaldehyde, 9-anthraldehyde, pyridine-2-carbal-dehyde, pyridine-3-carbaldehyde, thiophene-2-carbal-dehyde, benzaldehyde, acetic acid and EDTA were purchased from Sd fine chemicals (India). Ethidium bromide and Luria Broth were purchased from Himedia (India). CT DNA was purchased from Sigma Chemicals Co. (India). Metal contents of the complexes were analyzed gravimetrically and volumetrically,15 after decompo- sing the organic matter with acid mixture (HClO4, H2SO4 and HNO3). C, H and N elemental analyses were performed on a Perkin-Elmer 240 elemental analyzer. Magnetic moments were measured by Gouy's method using mercury tetrathiocyanatocobaltate(II) as the calibrant (xg = 16.44 x 10-6 cgs units at 20 °C), on Citizen Balance. The diamagnetic correction was made using Pascal's constant. IR spectra were recorded on FT-IR Shimadzu spectrophotometer with sample prepared as KBr pellets in the range 4000-400 cm1. The reflectance spectra were recorded on a LAMBDA 19 UV/Vis/NIR spectrophotometer, Perkin Elmer (USA). The FAB mass spectra were recorded on a Jeol SX 120/Da-600 mass spectrometer/ Data system using Argon/Xenon (6 kV, 10 mA) as the FAB gas. The accelerating voltage was 10 kV and spectra were recorded at room temperature. 2. 2. Synthesis of Ligands 2-(3-Bromophenyl)-1H-imidazo[4,5-f][1,10]phenant-hroline (L1) A mixture of 3-bromobenzaldehyde (3.5 mmol), 1,10-phenanthroline-5,6-dione (3.5 mmol, 0.530 g), ammonium acetate (50 mmol), and glacial acetic acid (10 mL) were refluxed for about 2 h, then cooled to room temperature, and diluted with water (25 mL). Dropwise addition of concentrated aqueous ammonia (5 mL) till it gives yellow precipitates, followed by washing with water gave crude product. The crude products were purified by silica gel filtration (60-100 mesh, ethanol). The principal yellow band was collected. Yield: 36%; m.p.: 181-183 °C; Anal. Calc. for C19H11BrN4 (375.22 g/mol): Calc. (%):C, 60.82; H, 2.95; N, 1^1.93; Found (%): C, 60.72; H, 2.78; N, 14.83. 1H NMR (CDCl3, 400 MHz): 8/ppm: 13.773 (s, 1H, NH), 9.055 (d, 2H, H2>2„), 9.006 (d, 2H, H4>4„), 8.314 (s, 1H, HPh2), 8.274 (d, 1H, HPh6), 7.841 (t, 211, H3,3„), 7.681 (t, 1H, HPh5), 7.592 (d, 1H, HPh4), 13C NMR (CDCl3, 100 MHz): 156.15 (C6>6„), 155.27 (C2), 148.53 (C2>2„), 132.89 (Cph3), 132.66 ^4), 131.48 (CpU), 131.34 (C^, 130.16 (Cph2), 129.19 (Cph5), 126.54 (CpM), 125.41 (C5,5„), 124.91 (C4',4„), 122.15 (C3',3„). 2-(3-Chlorophenyl)-1H-imidazo[4,5-f][1,10]phenant-hroline (L2) Similar procedure was followed by taking 3-chloro-bezaldehyde.Yield: 32%; m.p.: 192-194 °C; Anal. Calc. for C19H11ClN4 (330.77 g/mol): Calc. (%):C, 68.99; H, 3.35; N, 16.94; Found (%): C, 68.78; H, 3.28; N, 16.81. 1H NMR (CDCl3, 400 MHz): 8/ppm: 13.770 (s, 1H, NH), 9.015 (d, 2H, H2>2„), 8.862 (d, 2H, H4>4„), 8.289 (s, 1H, Hph2), 8.226 (d, 1H, Hph6), 7.796 (t, 2H H3,3„), 7.616 (t, 1H, Hph5), 7.552 (d, 1H, Hph4), 13C NMR (CDCl3, 100 MHz): 154.16 (C66,), 152.89 (C2), 149.69 (C2>2„), 139.61 (Cph3), 134.83 ^4), 132.30 (Cph1), 130.2227' (Cp,^), 129.56 (CPh2), 128.89 (CPh5), 126.41 (CpM), 125.68 (CPh55„), 124.13 (C4'4„), 121.55 (C^,,). 2-(4-(Benzyloxy)phenyl)-1H-imidazo[4,5-f][1,10]phe-nanthroline (L3) Similar procedure was followed by taking 4-benzy-loxybezaldehyde. Yield: 38%; m.p.: 185-187 °C; Anal. Calc. for C26H18N4O (402.42 g/mol): Calc. (%):C, 77.59; H, 4.51; N, 13.92; Found (%): C, 77.42; H, 4.33; N, 13.81. 1H NMR (CDCl3, 400 MHz): 8/ppm: 13.589 (s, 1H, NH), 9.030-9.019 (complex, 2H, H2>2„), 8.904 (d, 2H, H4>4„), 8.229 (d, 2H, Hph26), 7.854-72789 (complex, 2H, H3,,3„), 7.515 (d, 2H, HBz26), 7.430 (t, 2H, HBz53), 7.362 (t, 1H, Bz4), 7.266 (d, 2H, , Ph35), 5.221 (s, 2H, OCH2), 13C NMR (CDCl3, 100 MHz): 159.03 (CPh4) 154.09 (C6>6„), 153.14 (C2), 149.16 (C2,2„), 136.26 (CBz1), 130.01 (C45), 131.23 (CPh2,6), 128.9(5 (cbZ3,S), 127.6(5 (cBj, 127.53 (cBZ2,6), 126.54 (C5>5„), 124.42 (C4>4„), 121.20 (C3>3„), 115.26 (CPh3,5), 113:44 (cP,1), 69.96 (CH2). , 2-(3-(Benzyloxy)phenyl)-1H-imidazo[4,5-f][1,10]phe-nanthroline (L4) Similar procedure was followed by taking 3-benzy-loxybezaldehyde. Yield: 35%; m.p.: 188-190 °C; Anal. Calc. for C26H18N4O (402.42 g/mol): Calc. (%): C, 77.59; H, 4.51; N, 13.92; Found (%): C, 77.47; H, 4.39; N, 13.77. 1H NMR (CDCl3, 400 MHz): 8/ppm: 13.563 (s, 1H, NH), 9.053-9.039 (complex, 2H, H2>2„), 8.914 (d, 2H, H4>4„), 8.248 (d, 2H, Bz26), 7.895-7.801 (complex, 2H, Hy4"), 7.516 (d, 1H, HPh6), 7.497-7.469 (complex, 5h, HPh2 5Bz3 4 5), 7.261 (d, 1H, HPh4), 5.216 (2H, OCH2), 13C NMR (CDCl3, 100 MHz): 157.51 (CPh3), 154.13 (C6>6„), 152.95 (C2), 149.76 (C2,2„), 136.88 (C45), 132.51 (CBz1), 130.32 (CPh5), 128.71 (C^), 127.69 (C^, 127^.18 (cBZ2 6), 126.47 (C5'5„), 125.25 (cPU), 124.41^4,4,,), 121.55 (C3,3„), 119.81 (CPh6), 114.20 (CPh4), 111.31 (CPh2), 70.81 (CH2). 2-(Anthracen-9-yl)-1H-imidazo[4,5-f][1,10]phenant-hroline (L5) Similar procedure was followed by taking 9-anthral-dehyde.Yield: 35%; m.p.:179-181 °C; Anal. Calc. for C27H16N4 (396.44 g/mol): Calc. (%):C, 81.80; H, 4.07; N, 14.13; Found (%): C, 81.67; H, 3.92; N, 14.01. 1H NMR (CDCl3, 400 MHz): 8/ppm: 13.587 (s, 1H, NH), 8.974-8.991 (complex, 2H, H2,2„), 8.885 (d, 2H, H4,4„), 8.521 (s, 1H, HA9), 8.454 (d, 4H, HA1A4A5A8), 7.833 (t, 2H, H3>3„), 7.440 (t, 4H, HA2A3A6A7), 13C NMR (CDCl3, 100 M3^z): 154.15 (C6,6„), 152.91 (C2), 149.71 (C2,2„), 134.33 (CA9), 132.32 (C45), 132.20 (CA11A12), 130241 (CA13A14), 129.80 (cA10), 1:28.17 (CA4A5), 126.45 (C^,,), 125.21 (C 52,53,56,57), 124.10 (cA1,A8), 124.00 (C4.4,,), 121.41 (C33„) 2-(Pyridin-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroli-ne [L6] Similar procedure was followed by taking pyridine-2-carbaldehyde. Yield: 23% m.p.:145-147 °C, anal. calc. for: C18H11N5 (297.31 g/mol): calc. (%): C, 72.72; H, 3.73; N, 23.5(5, found (%): C, 72.64; H, 3.79; N, 23.49. 1H NMR (CDCl3, 400 MHz) 5„); 123T2, (C4,4,); 121.65, (C4,,,); 120.12(C3,,3„); 120.12, (CpM). 2-(Pyridin-3-yl)-1H-imidazo[4,5-f][1,10]phenanthroli-ne [L7] Similar procedure was followed by taking pyridine- 3-carbaldehyde. Yield: 29% m.p.:141-143 °C, anal. calc. for: C18H11N5 (297.31 g/mol): calc. (%): C, 72.72; H, 3.73; IN, 23.56, found (%): C, 72.79; H, 3.67; N, 23.48. . 1H NMR (CDCl3, 400 MHz) 6„); 149.653 (C2); 148.94, (C6",); 135.44, (C2,2,,); 132.92, (C4,,,); 132.41, (C3,,,); 125.85, (C45); md, (Cyy.H24.00, (C5,,,); 123.24, (C4. 4..); 12L21, (C3. 3..). , 2-(Thiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenant-hroline [L8] Similar procedure was followed by taking thiophene-2-carbaldehyde. Yield: 23% m.p.:148-150 °C, anal. calc. for: C17H10N4S (302.35 g/mol): calc. (%): C, 67.53; H, 3.33; N, 18.53, found (%): C, 67.58; H, 3.29; N, 18.48. 1H NMR (CDCl3, 400 MHz) 2 > 1 > 3 > 4 > 5 > ciprofloxacin. The binding ability of complexes is more compare to ciprofloxacin and less compare to classical intercalator ethidium bromide.38 Figure 1: Effect on relative viscosity of DNA under the influence of increasing amount of complexes at 27 ± 0.1 °C in in 5 mM Tris-HCl buffer (pH 7.2) as a medium. 3. 7. Absorption Titration Complex binding with CT-DNA through intercalation usually result in hypochromism and bathochromism, due to intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA.39,40 The binding of Cu(II) complexes to duplex DNA led to decrease in the absorption intensities with a small amount of red shifts in the UV-Vis absorption spectra. After intercalation, the n* orbital of the intercalated ligand can couple with the n orbital of nucleoba-ses of DNA base stacks, thus decreasing the n- n* transition energy and resulting in the bathochromism.41,42 On the other hand, the coupling n orbital is partially filled by electrons, thus decreasing the transition probabilities resulting in the hypochromism. The binding constant (^b) of the complexes to DNA were determined by monitoring the changes of absorbance at 281-284 nm with increasing concentration of DNA (Figure 2). The appreciable decrease in absorption intensity and significant red shift of the n- n * band of complexes is similar to that observed for its interaction with DNA in DMSO solution. The binding constant (Kfc) of complexes (Table 4) are in the range of 1.12 x 104 -3.05 x 104. Comparing the intrinsic binding constant of complexes with those of DNA-intercalative [Ru(dmb)2(ipbp)]2+ (1.18 x 104 M-1) complex[43] [where, dmb = 4,4'-dimethyl-2,2'-bipyridine and ipbp = 3-(1H-imidazo[4,5-f][1,10]phenanthroline-2-yl)-4H-1-ben-zopyran-2-one]. The Kb value of complexes, is greater than Co(II) complexes of terpyridines reported by Indu-mathy et al.44 and Cu(II) complexes of type [Cu(phen)2Cl2] 45 while comparable to ruthenium complexes reported by Tan et al.46. So we can deduce that all the complex bind moderately to DNA by intercalation. These spectral characteristics obviously suggest that complexes interact with chromophore and the nuc-leobases of DNA base stacks. 240.0 Wavelength (nm) 400. Figure 2: Electronic absorption titration curve of [Cu(cpf)(L1)Cl] in absence and in presence of increasing amount of DNA; 50-150 |M in in 5 mM Tris-HCl buffer (pH 7.2), [Complex] = 15 ||M, [DNA]= 50-150 |M with incubation period of 30 min. at 37 °C, Inset: Plot of [DNA]/(ea-ef) versus [DNA]. Table 4: Binding constant and IC50 values of copper(II) complexes Compounds Kb (M-1) IC5o (^M) T m Complex 1 2.08 x 104 0.70 79.4 Complex 2 3.05 x 104 0.60 80.1 Complex 3 2.05 x 104 0.90 78.9 Complex 4 1.59x 104 1.20 78.5 Complex 5 1.12 x 104 1.45 78.3 Complex 6 1.65 x 104 0.95 78.9 Complex 7 1.57 x 104 1.20 78.6 Complex 8 1.31 x 104 1.50 78.2 Complex 9 1.12 x 104 1.75 78.5 3. 8. Thermal Denaturation Experiment The melting of DNA is an important parameter to study the interaction of transition metal complexes with nucleic acids. Thermal behaviours of DNA in the presence of complexes can give an insight into their conformation changes when temperature is raised, and offer information about the interaction strength of complexes with DNA. The melting temperature Tm, at which 50% of the DNA has become single strand, can be determined from the thermal denaturation curves of DNA by monitoring the absorption changes at 260 nm. According to the literature,47-49 the intercalation of metallointercalators generally results in a considerable increase in melting temperature (Tm). In the absence of the complex, a DNA-melting experiment showed that Tm of CT DNA (100 pM) is 74.2 ±0.1 °C under experimental conditions (Supply. 3). The observed melting temperatures of DNA in presence of complexes (Tm) are shown in Table 4. The increase amount of Tm is comparable to that observed for classical intercalators.47-49 3. 9. Gel Electrophoresis Isolation of plasmid DNA from pure culture of E. coli was carried out by conventional method.50 The basic principle employed is "alkali-lysis", in which at the alkaline pH, both the genomic and plasmid DNA are denatured. On reduction of the pH the plasmid DNA molecule being small in size, quickly reanneals itself while the large genomic DNA is not. The denatured genomic DNA is then sedimented while the plasmid DNA remains in solution. This is then precipitated. Cleavage of plasmid pUC19 DNA by synthesized complexes was monitored by agarose gel electrophoresis technique. When plasmid DNA was subjected to electrophoresis after interaction, upon illumination of gel (Figure 3) the fastest migration was observed for super coiled (SC) Form I, where as the slowest moving was open circular (OC) Form II and the intermediate moving is the linear (L) Form III generated on cleavage of open circular. The data of plasmid cleavage are presented in Table 5. All the complexes show higher DNA cleavage ability compare to the drug and metal salt. Figure 3: Photogenic view of interaction of pUC19 DNA (450 |g/mL) with series of copper(II) complexes (200 ||M) using 1% agarose gel containing 0.5 |g/mL ethidium bromide. All reactions were incubated in TE buffer (pH 8) in a final volume of 15 |L, for 3 h. at 37 °C. : Lane 1, DNA control; Lane 2, CuCl2 • 2H2O; Lane 3, ciprofloxacin; Lane 4, [Cu(cpf)(L1)Cl]; Lane 5, [Cu(cpf)(L2)Cl] ; Lane 6, [Cu(cpf)(L3)Cl]; Lane 7, [Cu(cpf)(L4)Cl]; Lane 8[Cu (cpf)(L5)Cl], [Cu(cpf)(L6)Cl], [Cu(cpf)(L7)Cl], [Cu(cpf)(L8)Cl], [Cu(cpf)(L9)Cl]. Table 5: Gel electrophoresis data. Compounds % SC % OC % L % Cleavage DNA Control 75 25 - DNA + Metal salt 72 28 - 4.00 DNA + Ciprofloxacin 67 33 - 10.66 DNA + 1 18 52 30 76.00 DNA + 2 16 52 32 78.66 DNA + 3 20 51 29 73.33 DNA + 4 26 54 20 65.33 DNA + 5 30 55 15 60.00 DNA + 6 22 57 20 74.41 DNA + 7 26 55 17 69.76 DNA + 8 28 53 19 67.44 DNA + 9 30 42 28 65.11 3. 10. SOD Mimic Activity Superoxide is one of the main reactive oxygen species (ROS) in the cell and as such, SOD serves a key antioxidant role. SOD outcompete damaging reactions of superoxide, thus protecting the cell from superoxide toxi-city. The reaction of superoxide with non-radicals is spin forbidden in biological systems; this means, its main reactions are with itself (dismutation) or with another biologi- 2 3 t (min) Figure 4: plot of absorbance values(Aèi560) against time (t). cal radical such as nitric oxide (NO). The superoxide anion radical (O2^) spontaneously dismutes to O2 and hydrogen peroxide (H2O2) quite rapidly (~105 M-1s-1 at pH 7). SOD is biologically necessary because superoxide reacts even faster with certain targets such as the NO radical, which makes peroxynitrite. The system used as a basis of superoxide radical generator in order to check SOD like activity of the synthesized complexes was NBT/NADH/PMS system. Absor-bance at a function of time was plotted to have a straight line obeying equation Y = mX + C (Figure 4). Figure 5 shows percentage inhibition of reduction of nitro blue te-trazolium (NBT) plotted against concentration of the complex 1. Compounds exhibit SOD-like activity at biological pH with their IC50 values ranging from 0.60 to 1.75 |M. The superoxide scavenging data are presented in Table 4. The higher IC50 can only be accredited to the vacant coordination site facilitating the binding of superoxide anion, electrons of aromatic ligands that stabilize Cu-O2 - interaction and not only to the partial dissociation of complex in solution. Figure 5: Plot of percentage of inhibiting NBT reduction with an increase in the concentration of complex 1. 4. Conclusion The second generation fluoroquinolone drug, ciprofloxacin, based neutral mixed ligand complexes of copper(II) with various phenanthroline derivatives have been synthesized. The spectroscopic evidences suggest square pyramidal geometry around Cu(II) ions. All the complexes were screened for diverse biological activities to check their possible applications as antibacterial or SOD mimic agents. Antibacterial activity shows better MIC value of all complexes than ciprofloxacin against different microorganisms. Complexes show moderate activity for DNA interaction assay. Also complexes show good SOD mimic activity for nonenzymatic NADH/ PMS/NBT assay. Although complexes does not have very much structural differences, but based on the trends observed in the biological activities, we can say that complexes with electron withdrawing groups and with larger ring system show good biological activities by stabilizing the charge of central metal ion over the extended ring systems. 6. References 1. D. S. Sigman, M. D. Kuwabara, C. B. Chen, T. W. Bruice, Methods Enzymol. 1991, 208, 414. 2. S. N. Morehouse, H. Suliman, J. Haff, D. Nguyen, Inorg. Chim. 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Naravo vezanja bakrovih (II) kompleksov na DNK smo študirali z merjenjem viskoznosti ter merjenjem titracijskih in talilnih krivulj nastalih kompleksov s pomočjo UV-Vis absorpcije. Raziskave cepitve DNK so pokazale, da imajo sinte-tizirani bakrovi (II) kompleksi večjo sposobnost cepitve DNK kot soli kovin in običajna zdravila. Za proučevane komplekse smo izvedli tudi raziskave mimike superoksid dismutaze (SOD) ter določili vrednosti IC50 v območju med 0,95 in 1,75 |M. Antibakterijska aktivnost smo testirali na izbranih grampozitivnih in gramnegativnih mikroorganizmih. Supplementary material Supplementary 1. Structure and name of ligands 2-(3-bromophenyl)-1 //-imidazole-/' ][ 1,10]phetianthroline Ligand 1 (L1) -N N- 2-(3-chloropheny I)-IH-imidazo{4(5-/ ] [ 1,1 OJphenanthroline Ligand 2 (L2) PhHoCO 2-(4-{benzy loxy)pheny 1>- lif-imidazo[4,5/] [ 1,1 OJphenanthroline 2-{ 3-( ben zy 1 oxy)phcny I) -1W - i m ida zo [4,5 ] f 1,10] phonan th ro I i n c Ligand 3 (L3) Ligand 4 (L4) ~N N- 2-(anthracen-9-yl)-1 ¿/-imidazu [4,5 ] [ 1,1O]phenanthrolioe Ligand 5 (L5) Ligand 6 (L6) 2-(pyridin-3-yl)- 1//-imid