European Journal of Medicinal Chemistry 45 (2010) 439–446 Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech Original article Antibacterial and DNA interaction studies of zinc(II) complexes with quinolone family member, ciprofloxacin Mohan Patel*, Mehul Chhasatia, Pradhuman Parmar Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, 388 120 Gujarat, India a r t i c l e i n f o a b s t r a c t Article history: Received 13 March 2009 Received in revised form 23 July 2009 Accepted 15 October 2009 Available online 29 October 2009 DNA binding and cleavage characteristics of Zn(II) complexes have been investigated. The DNA interaction property of the complexes has been investigated using absorption spectra, viscosity measurements, as well as gel electrophoresis studies. Intrinsic binding constant (Kb) has been estimated under similar set of experimental conditions. Absorption spectral study indicate that the Zn(II) complexes intercalate between the base pairs of the DNA tightly with intrinsic binding constant in the range of 1.0  104–4.0  104 M1 in phosphate buffer. The proposed DNA binding mode supports the large enhancement in the relative viscosity of DNA on binding. The antimicrobial activity of all the ligands and metal complexes has been examined by minimum inhibitory concentration method (MIC). Ó 2009 Elsevier Masson SAS. All rights reserved. Keywords: Zn(II) complexes Ciprofloxacin DNA interaction Intrinsic binding constant (Kb) MIC 1. Introduction Fluoroquinolones represent an important group of chemotherapeutic compounds, which exhibit high antibacterial activities. An efficient representative of this group, ciprofloxacin (Cip ¼ C17H18FN3O3, 1-cyclopropyl-6-fluoro-4-oxo-7-(1-piperazinyl)-1,4dihydroquinoline-3-carboxylic acid) [1] is widely used in clinical practice as a broad spectrum antimicrobial agent [2]. Quinolones comprise a group of well-known antibacterial agents and the first members being in clinical practice over 40 years [3,4]. They can act as antibacterial drugs that effectively inhibit DNA replication and are commonly used in treatment of many infections [5,6]. Studies on the biological properties of quinolone-metal complexes have been focused on the interaction with DNA, antibacterial activity tests on diverse microorganisms, cytotoxicity and potential antitumor activity [7–15]. In this context, we have studied the interaction of Zn(II) with ciprofloxacin, in the presence of nitrogen-donor ligands such as bpdmed(A1)/mtma(A2)/apq(A3)/ bpeed(A4)/dcnd(A5)/dpeda(A6). The coordination behaviors of the ligands towards transition metal salts have been investigated and the data have been correlated with their elemental analysis, thermal properties, magnetic measurements, IR and their DNA binding and cleavage behavior were also being examined using * Corresponding author. Tel.: þ91 2692226858x220. E-mail address: jeenen@gmail.com (M. Patel). 0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2009.10.024 spectroscopic techniques (UV spectroscopy), viscosity measurements and gel electrophoresis technique. The antimicrobial efficiency (MIC) of the compounds has been screened against five different microorganisms. 2. Results and discussion 2.1. 1 H NMR and 13 C NMR spectra of Schiff bases The 1H and 13C NMR spectra of the ligands were carried out in CDCl3/MeOD and reported along with the possible assignment. The 1 H NMR spectra of ligands exhibiting peaks at about 6.5–8.19 ppm were assigned to the aromatic protons. The singlet peak which appeared at 7.5 ppm was assigned to the azomethine proton (–CH]N–). In the 13C NMR spectra, the peaks observed at about 105.9–135.4 and 128.5–143.0 ppm were assigned to aromatic and thiophene carbons respectively. Peaks observed at about 140.0– 150.9, 165.3–167.3 ppm were assigned to C–N and C]N carbons respectively. 2.2. Spectroscopic studies of the mixed-ligand complexes 2.2.1. Infrared spectroscopy The prominent IR spectral data of the complexes are shown in Table 1. The n(C]O) stretching vibration band appears at 1708 cm1 for ciprofloxacin, where as for complexes it appear at 1615–1629 cm1; this shift towards lower energy suggests that 440 M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446 Table 1 Infrared spectral data of the Zn(II)complexes. Compounds I II III IV V VI n(C]O) cm1 pyridone n(COO)asy n(COO)sym Dn n(C–Cl) n(C]N) n(C]N) n(M–N) n(M–O) cm1 cm1 cm1 cm1 cm1 azomethine cm1 ring cm1 cm1 1629 1628 1620 1626 1615 1627 1590 1585 1601 1596 1605 1588 1382 1376 1380 1385 1384 1383 208 209 221 211 221 205 1117 1131 1142 1145 1136 1150 1579 1576 – 1564 1570 1576 – – 1602 – – – 545 546 540 535 540 542 505 509 510 512 508 510 coordination occurs through the carbonyl oxygen of pyridine ring [16]. The sharp band in ciprofloxacin at 3520 cm1 [17] is due to hydrogen bonding; which is attributed to ionic resonance structure and peak observed because of free hydroxyl stretching vibration. This band completely vanished in the spectra of the metal complexes indicating deprotonation of the carboxylic proton. The data were further supported by n(M–O) [18] band which appeared at 505–512 cm1. The strong absorption band obtained at 1624 cm1 and 1340 cm1 in ciprofloxacin were assigned to n(COO)asy and n(COO)sym respectively, while in the metal complexes these bands were observed at 1600 and 1380 cm1, respectively. The frequency separation (Dn) in the investigated complexes is greater than 200 cm1, suggesting an unidentate bonding nature for the carboxyl group [19–23]. The n(C]N) peak for the Schiff bases was observed at 1590–1620 cm1 which on complexation were shifted to 1564–1579 cm1, which indicates N– N coordination of the ligands [24–27]. This data was further supported by n(M–N) [28] which appeared at 535–546 cm1.In the case of the n(C–S) band of mtma observed at 700 cm1 is shifted lower to 670 cm1 in the spectra of the complex indicating the participation of the sulfur atom of the thiophene ring. This data was further supported by a new band observed at 425 cm1 which can be assigned to n(M–S) [29–31] mode. 2.2.2. Electronic spectra and magnetic measurements The UV–Vis. spectra of the complexes have been recorded using UV-160A UV–Vis. spectrophotometer, Shimadzu (Japan). The electronic spectra of the Zn(II) complexes exhibited only a high-intense band at w31,000 cm1 and were assigned [32] to a ligand-metal charge transfer transition. Zn(II) is belongs to d10 system so the complexes which synthesized are diamagnetic in nature because of unavailability of unpaired electrons in this system [33]. 2.2.3. TG analyses The water content in the complexes was determined by thermal analysis, using a 5000/2960 SDTA, TA instrument (USA) differential thermal analysis apparatus operating at a heating rate of 10  C per minute in the range of 20–800  C in N2 in order to establish their compositional differences as well as to ascertain the nature of associated water molecules. It was observed that all the complexes showed a loss in weight corresponding to three water molecules in the range 50–130  C, indicating the water of crystallization. In the second step weight loss during 130–180  C corresponding to two coordinated water molecules. For Zn(II) complexes a loss in weight was seen corresponding to a piperazine (pip) molecule in the temperature range 180–250  C, followed by liberation of Cip.(L) in the temperature range 250–500  C. Finally, decomposition of An occurred in the temperature range 520–800  C, and the remaining weight was consistent with metal oxide [34]. Probable structure of the complexes from the above analytical facts is given in Fig. 1. 2.2.4. Mass spectroscopy The FAB mass spectrum of complex-II shows the molecular ion peak at m/z 1248 due to [M þ 2Hþ] in absence of three lattice water molecules. The peak at m/z 1148 is due to removal of two coordinate water and two methoxy group [M-C2H10O4]. The peak Fig. 1. Probable structure of complex [Zn2(Cip)2(mtma)2(pip)(H2O)2]. 441 M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446 Fig. 2. Mass spectrum of complex [Zn2(Cip)2(mtma)2(pip)(H2O)2]. occurring at m/z 615 correspond to fragment produced by cleavage of Zn–N bond with piperazine [M-C26H26N2O7ClFSZn þ Hþ]. The appearance of peak at m/z 1077 is due to removal of two chlorine atom [M-C2H10O4Cl2 þ Hþ]. The peak at m/z 961 is due to removal of one ligand [M-C13H19NO4S  Hþ]. The peak arising at m/z 218 corresponds to the ligand [C12H11NOS]. The M þ 2 peak appears due to the isotopes of Cl and Zn. The mass spectrum is shown in Fig. 2. MTCC 121. In case of Serratia marcescens MTCC 86 complex III is more potent than all standard drugs; while for Pseudomonas aeruginosa MTCC 1688 complex VI is more potent than all standard drugs except gatifloxacin. In case of Escherichia coli MTCC 443 complexes II, III, V and VI are more potent than gatifloxacin, norfloxacin and pefloxacin. Enhancement in the activity can be explained on the basis of chelation theory and/or may be due to Overtone’s concept [37–39]. 2.3. Antibacterial screening of Zn(II) complexes The susceptibility of certain strains of bacterium towards mixed ligand–metal complexes was judged by measuring the minimum inhibition concentration (MIC). A stock solution of 2000 mM was prepared by dissolving each complex in DMSO solution. As assessed by colour, the complexes remain intact during biological testing. The results are shown in Table 2. MIC was determined with the help of progressive double dilution method [35,36] in liquid media containing compound with a varying range of 0.3–3200 mM, in all cases, from the experimental examination we have found that all the metal complexes were more active relative to metal salts and Schiff bases. All the complexes are more potent than all standard drugs against Staphylococcus aureus MTCC 1430 and Bacillus subtilis 2.4. DNA binding and cleavage study 2.4.1. Absorption spectroscopic studies In order to quantitatively compare the binding strength of the complexes, the intrinsic binding constants Kb of the complexes with sperm herring DNA were calculated using the following function equation [40]: ½DNA=ð3a L 3f Þ [ ½DNA=ð3b L 3f Þ D 1=K b ð3b L 3f Þ (1) where [DNA] is the concentration of DNA in base pairs, the apparent absorption coefficient 3a, 3f and 3b correspond to Aobsd/ [Zn], the extinction coefficient for the free zinc complex, for each Table 2 Minimum inhibitory concentration data (MIC) (mM). Compounds Zn(OAc)2 H2O Ciprofloxacin Gatifloxacin Norfloxacin Enrofloxacin Pefloxacin Levofloxacin Sparfloxacin Ofloxacin L1 L2 L3 L4 L5 L6 I II III IV V VI Gram positive Gram negative Staphylococcus aureus Bacillus subtilis Serratia marcescens Pseudomonas aeruginosa Escherichia coli 1240 1.63 5.06 2.51 1.95 2.10 1.66 1.27 1.94 2115 2531 2528 2080 1884 1248 0.75 0.46 0.75 0.72 0.67 0.50 992 1.09 4.00 2.51 3.90 2.40 2.21 2.04 1.38 1904 2301 2318 1891 1570 1387 0.37 0.31 0.60 0.36 0.33 0.50 1984 1.63 2.93 4.07 1.67 5.10 1.66 1.53 1.66 2539 2761 2739 2080 1884 1525 1.87 2.69 1.49 2.15 2.33 1.89 1736 1.36 1.01 3.76 1.39 5.70 1.66 1.53 2.21 2750 2991 2950 2458 2041 1664 1.49 1.92 1.86 1.79 1.66 1.26 1736 1.36 2.93 2.82 1.39 2.70 0.97 1.27 1.38 2750 2761 3161 2647 2041 1803 2.24 1.92 1.86 2.15 1.66 1.89 442 M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446 Fig. 3. Absorption titration spectra of complex [Zn2(Cip)2(bpdmed)2(pip)(H2O)2] (4 mM) with sperm herring DNA; [DNA] ¼ 2–20 mM. Incubation time 10 min at 37  C. addition of DNA to the zinc complex and zinc complex in the fully bound form, respectively. In plots [DNA]/(3a  3f) vs [DNA], Kb is given by the ratio of slope to the y intercept. Fixed amounts (4 mM) of complexes were titrated with increasing amounts of DNA. The electronic spectral traces are given in Fig. 3. For complexes, the absorption spectra show clearly that the addition of DNA to the complexes yields hyperchromism and a blue shift to the ratio of [DNA]/[M]. Obviously, these spectral characteristics suggest that all the complexes interact with DNA most likely through a mode that involves a stacking interaction between the aromatic chromophore and the base pairs of DNA. Addition of increasing amounts of DNA resulted in hypsochromism of the peak maxima in the UV–visible spectra of the complexes. In the plot of [DNA]/(3a  3f) vs. [DNA], the binding constant Kb is given by the ratio of the slope to the y intercept. The binding constant for Zn(II) complexes varies in the range of 1.0–4.0  104 M1, all data are given in Table 3. The DNA-binding constant of the title complexes are comparable to those of some DNA intercalative polypyridyl Ru(II) complexes 1.0–4.8  104 M1 [41,42]. 2.4.2. Viscosity measurements The binding modes of the complexes were further investigated by viscosity measurements. Photophysical probes generally provide necessary, but not sufficient, clues to support a binding model. Hydrodynamic measurements that sensitive to length change (i.e. viscosity and sedimentation) are regarded as the least ambiguous and the most critical tests of binding mode in solution in the absence of crystallographic structural data [43,44]. A classical intercalation model results in lengthening the DNA helix, as base pairs are separated to accommodate the binding ligand, leading to the increase of DNA viscosity. However, a partial and/or non-classical intercalation of ligand may bend (or kink) DNA helix, resulting in the decrease of its effective length and, concomitantly, its viscosity. The Table 3 The binding constants (Kb) of Zn(II) complexes with DNA in Phosphate buffer pH 7.2. Complexes I II III IV V VI Kb (M1) 4 2.5  10 3.0  104 3.0  104 1.0  104 1.0  104 4.0  104 effects of the complexes, on the viscosity of rod-like DNA are shown in Fig. 4. It is observed that as the concentration of complexes increase, the viscosity of DNA increases steadily, which indicates that the complexes bind to DNA through a classical intercalation mode. Compare to all complexes, the binding ability of classical intercalator ethidium bromide is more. Among all complexes, complexes III and VI bind more strongly than other. This difference of DNA-binding mode between complexes should be caused by their different ancillary ligands. 2.4.3. Cleavage of plasmid pUC19 DNA There has been considerable interest in DNA cleavage reactions that are activated by transition metal complex [45,46]. The delivery of metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient DNA cleavage reaction. Fig. 5 illustrates the gel electrophoretic separations showing the cleavage of plasmid pUC19 DNA induced by the complexes under aerobic conditions and in presence of H2O2, respectively [47]. When circular plasmid DNA is conducted by electrophoresis, the fastest migration will be observed for the supercoiled form-I (SC). If one strand is cleaved, the supercoiled will relax to produce a slower-moving open circular form-II (OC). If both strands are cleaved, a nicked form-III (NC) will be generated that migrates in between. This clearly shows that the relative binding efficacy of the complexes to DNA is much higher than the binding efficacy of metal salt itself (Table 4). The different DNA-cleavage efficiency of the complexes was due to the different binding affinity of the complexes to DNA, which has been observed in other cases. One of the most interesting electrophoretic results of the complexes takes place when experiment is done in presence of H2O2 in TAE buffer. The DNA þ complex þ H2O2 systems (Fig. 6) cleave the supercoiled DNA form (I) and convert into nicked form (III) and open circular form (II) more than complex alone. Therefore, we concluded that the mixture of complex with H2O2 have been found to be efficient oxidant. 3. Conclusion Six binuclear Zn(II) complexes using ciprofloxacin and neutral bidentate ligands, were synthesized and characterized. Antibacterial study indicates that all the metal complexes were more active compared to metal salts and Schiff bases. All the complexes were found to be more potent than all standard drugs against S. aureus and B. subtilis. For S. marcescens, complex III and for P. aeruginosa, complex VI is more potent than all standard drugs. The strong antimicrobial M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446 443 were purchased from E. Merck (India) Ltd. Mumbai. Xylene cyanol FF, ethidium bromide and Luria Broth were from Himedia, India. Agarose was purchased from Sisco research lab., India. Bromophenol blue, acetic acid and EDTA were purchased from Sd fine chemicals, India. Sperm herring DNA was purchased from Sigma Chemical Co., India. Organic solvents were purified by standard methods [48]. 4.2. Instrumentation Fig. 4. The effect of the increasing amount of complexes on the relative viscosity of DNA at 27  0.1  C. activities of these complexes against tested organisms suggest further investigation on these complexes. DNA-binding study indicate that intrinsic binding constant of all complexes varies in the range of 1.0  104–4.0  104 M1, which is comparable to those of some DNA intercalative polypyridyl Ru(II) complexes1.0  104–4.8  104 M1. The complexes bind to DNA by classical intercalative mode. Gel electrophoresis data suggests that presence of H2O2 enhances the DNA cleavage to a significant extent. Hence, the proposed work seems to be worth for generating database to develop new effective useful DNA probes. 4. Experimental 4.1. Materials and methods All the chemicals used were of analytical grade. Thiophene2-carboxaldehyde, 1,8-diaminonaphthalene were purchased from Lancaster, England. Ciprofloxacin hydrochloride was purchased from Bayer AG (Wyppertal, Germany). Anthranilic acid, benzoyl chloride, ethylenediamine, 2,3-butanedione, aniline, hydrazine hydrate, pyridine, cyclohexanone, benzil, p-anisidine, glycerol, and zinc acetate Infrared spectra were recorded on a FT-IR Shimadzu spectrophotometer as KBr pellets in the range 4000–400 cm1. C, H and N elemental analyses were performed with a model 240 Perkin Elmer elemental analyzer. The reflectance spectra of the complexes were recorded in the range of 1700–350 nm (as MgO discs) on a Beckman DK-2A spectrophotometer. The metal contents of the complexes were analyzed by EDTA titration [49] after decomposing the organic matter with a mixture of HClO4, H2SO4, and HNO3 (1:1.5:2.5). MIC study was carried out by means of laminar air flow cabinet, Toshiba, Delhi, India, Thermogravimetric analyses was obtained with a model 5000/2960 SDTA, TA instrument (USA). The 1H NMR and 13C NMR was recorded on a Bruker Avance (400 MHz). The electronic spectra were recorded on a UV-160A UV–Vis. spectrophotometer, Shimadzu (Japan). The magnetic moments were measured by Gouy’s method using mercury tetrathiocyanatocobaltate(II) as the calibrant (cg ¼ 16.44  106 cgs units at 20  C), Citizen Balance. The diamagnetic correction was made using Pascal’s constant [50]. The FAB mass spectra were recorded on a Jeol SX 102/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. M-Nitro benzyl alcohol (NBA) was used as the matrix. The matrix peaks appear at m/z 136, 137, 154, 289, 307. 4.3. Synthesis and physical properties of Schiff bases 4.3.1. N,N0 -bis-(phenyl)-1,2-dimethyl-ethane-1,2-diimine (A1wbpdmed) An ethanolic solution (100 mL) of aniline (1.86 g, 20 mmol) was added drop wise to ethanolic solution (100 mL) of 2,3-butanedione (0.86 g, 10 mmol) and refluxed on water bath for 8 h. The resulting mixture was filtered. The obtained yellow crystalline product was washed with n-hexane, recrystallized in ethanol and dried in air. Yield: 58%, m.p.: 114  C, Found %: C, 81.49, H, 6.71, N, 11.69. C16H16N2 (236.31) requires %: C, 81.32, H, 6.82, N, 11.85. Ir (cm1): 1613 (C]N), 1572 (C]C); 1H NMR (ppm): 6.81–7.42 (10H, m, Ar–H), 2.19 (6H, s, Al–H); 13C NMR (ppm): 118.8–129.0 (Ar–C), 168.3 (C]N), 150.9 (C–N), 15.4 (Al–C). 4.3.2. 4-Methoxy-N-(thiophene-2-ylmethylene)aniline (A2wmtma) An ethanolic solution (100 mL) of thiophene-2-carbaldehyde (1.12 g, 10 mmol) was added drop wise to an ethanolic solution Fig. 5. Agarose gel electrophoresis of pUC19 plasmid DNA (0.12 mg) with different complexes in TE buffer using 1% agarose gel containing 1 mg/mL ethidium bromide: Lane 1: DNA (control); Lane 2: DNA þ Metal salt; Lane 3: DNA þ cip; Lane 4: DNA þ I; Lane 5: DNA þ II; Lane 6: DNA þ III; Lane 7: DNA þ IV; Lane 8: DNA þ V; Lane 9: DNA þ VI. 444 M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446 Table 4 Gel electrophoresis data. Compounds % SC(I) % NC(III) % OC(II) DNA Control DNA þ Metal salt DNA þ cip DNA þ I DNA þ II DNA þ III DNA þ IV DNA þ V DNA þ VI DNA þ H2O2 DNA þ H2O2 þ Metal salt DNA þ H2O2 þ cip DNA þ H2O2 þ I DNA þ H2O2 þ II DNA þ H2O2 þ III DNA þ H2O2 þ IV DNA þ H2O2 þ V DNA þ H2O2 þ VI 58 49 44 45 44 43 45 42 48 35 33 34 42 41 42 41 42 41 31 39 32 40 41 44 42 43 39 22 22 25 42 40 39 39 40 38 11 12 24 15 15 13 13 15 13 43 45 41 16 19 19 20 18 21 (100 mL) of 4-methoxyaniline (1.43 g, 10 mmol) and refluxed on water bath for 8 h. The resulting mixture was filtered. The obtained crystalline product was washed with n-hexane, recrystallized in ethanol and dried in air. Yield: 68%, m.p.: 275  C, Found %: C, 66.38, H, 5.18, N, 6.47. C12H11NOS (217.29) requires %: C, 66.33, H, 5.10, N, 6.45. Ir (cm1): 1620 (C]N), 700 (C–S), 1580 (C]C); 1H NMR (ppm): 6.95–7.31 (4H, m, Ar–H), 7.1–7.2 (3H, m, Thio-H), 3.72 (3H, s, OCH3), 7.5 (1H, s, CH]N); 13C NMR (ppm): 113.9–124.4 (Ar–C), 157.3 (C–O), 128.5–130.0 (Thio-C), 165.4 (C]N), 142.4 (C–N), 55.3 (OCH3). 4.3.3. 3-Amino-2-phenyl-3H-quinazolin-4-one (A3wapq) The solution of anthranilic acid (1.37 g, 0.1 mol) was prepared in pyridine (100 mL) followed by addition of benzoyl chloride (2.814 g, 0.2 mol). The resulting mixture was stirred for 0.5 h., and finally treated with 5% NaHCO3 (15 mL). The separated solid was crystallized in ethanol. Yield: 80%, m.p.: 120  C, The obtained 2-phenyl-3,1-benzoxazin-4-one (0.557 g, 0.05 mol) in ethanol (50 mL) was mixed with hydrazine hydrate (0.125 g, 0.05 mol) in ethanol (50 mL) and refluxed for 3 h. The obtained product was crystallized in ethanol. Yield: 85%, m.p.: 196  C, Found %: C, 70.67, H, 4.62, N, 17.59. C14H11N3O (237.26) requires %: C, 70.87, H, 4.67, N, 17.71. Ir (cm1): 1680 (C]O), 1590 (C]N), 1545 (C]C); 1 H NMR (ppm): 7.28–8.19 (9H, m, Ar–H); 13C NMR (ppm): 127.02–133.02(Ar–C), 164.7.6(C]O), 165.3 (C]N, ring), 145.5 (C–N). 4.3.4. N, N0 -bis-(1-phenylethylidene)-ethane-1,2-diamine (A4wbpeed) An ethanolic solution of ethylenediamine (0.60 g, 10 mmol) and acetophenone (2.40 g, 20 mmol) were refluxed on water bath for 6 h., and concentrated up to one-third volume after that kept it overnight over desiccator. Fine crystalline particles obtained on filtration, washed with 1:1 absolute ether:hexane and dried in air. Yield: 66%, m.p.: 135  C, Found %: C, 81.70, H, 7.68, N, 10.67. C18H20N2 (264.36) requires %: C, 81.78, H, 7.63, N, 10.60. Ir (cm1): 1630 (C]N), 1565 (C]C); 1H NMR (ppm): 7.40–7.70 (10H, m, Ar– H), 2.07 (6H, s, Al–H), 3.9 (4H, t, Al–H); 13C NMR (ppm): 125.9–131.4 (Ar–C), 18.2 (Al–C), 169.0 (C]N), 52.5 (C–N). 4.3.5. N, N0 -dicyclohexylidene-naphthalene-1,8-diamine (A5wdcnd) An ethanolic solution (100 mL) of cyclohexanone (1.96 g, 20 mmol) was added to ethanolic solution (100 mL) of 1,8-diaminonaphthalene (1.58 g, 10 mmol). The mixture was stirred continuously for 4 h to get fine yellow crystalline product. Obtained crystalline product was washed with n-hexane. The product was recrystallized in ethanol and dried in air. Yield: 68%, m.p.: 135  C, Found %: C, 83.00, H, 8.09, N, 8.74. C22H26N2 (318.45) requires %: C, 82.97, H, 8.23, N, 8.80. Ir (cm1): 1600 (C]N), 1570 (C]C); 1H NMR (ppm): 6.50–7.28 (6H, m, Ar-H), 1.31–2.37 (20H, m, Al–H); 13C NMR (ppm): 105.9–134.7 (Ar–C), 169.0 (C]N), 140.0 (C–N), 22.3 (Al–C), 25.3 (Al–C), 36.9 (Al–C). 4.3.6. N,N0 -(1,2-diphenyl-ethane-1,2-diylidene)dianiline (A6wdpeda) An ethanolic solution of benzil (2.10 g, 10 mmol) and aniline (1.86 g, 20 mmol) were refluxed on water bath for 12 h., and concentrated up to one-third volume after that kept it overnight over sulfuric acid in desiccator. Fine crystalline particles obtained on filtration, washed with 1:1 absolute ether:hexane and dried in air. Yield: 64%, m.p.: 165  C, Found %: C, 86.60, H, 5.57, N, 7.70. C26H20N2 (360.45) requires %: C, 86.64, H, 5.59, N, 7.77. Ir (cm1): 1610 (C]N), 1530 (C]C); 1H NMR (ppm): 6.80–7.60 (20H, m, Ar–H); 13C NMR (ppm): 124.2–135.4 (Ar–C), 167.3 (C]N), 141.5 (C–N). 4.4. Synthesis and physical properties of the mixed-ligand complexes 4.4.1. [Zn2(Cip)2(mtma)2(pip)(H2O)2]$3H2O (II) A methanolic solution of Zn(OAc)2$H2O (2.01 g, 10 mmol) was added to methanolic solution of mtma (1.12 g, 10 mmol), followed by addition of a previously prepared solution of Cip$HCl (3.67 g, 10 mmol) in water; pH was adjusted to 6–7.5 pH using dilute NaOH solution. During reaction the piperazine ring of ciprofloxacin was substituted by chloride ion in the presence of NaOH [51]. The resulting red solution was refluxed for 8–10 h on a steam bath, and then was kept overnight at room temperature. Fig. 6. Agarose gel electrophoresis of pUC19 plasmid DNA (0.12 mg) with different complexes in TE buffer using 1% agarose gel containing 1 mg/mL ethidium bromide: Lane 1: DNA (control); Lane 2: DNA þ H2O2; Lane 3: DNA þ H2O2 þ Metal salt; Lane 4: DNA þ H2O2þcip; Lane 5: DNA þ H2O2 þ I; Lane 6: DNA þ H2O2 þ II; Lane 7: DNA þ H2O2 þ III; Lane 8: DNA þ H2O2 þ IV; Lane 9: DNA þ H2O2 þ V; Lane 10: DNA þ H2O2 þ VI. 445 M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446 Table 5 Experimental and physical parameters of the complexes. Complexes empirical formula Elemental analysis % found (required) C C62H66Cl2F2N8O11Zn2 (I) C54H56Cl2F2N6O13S2Zn2 (II) C58H56Cl2F2N10O13Zn2 (III) C66H74Cl2F2N8O11Zn2 (IV) C74H86Cl2F2N8O11Zn2 (V) C82H74Cl2F2N8O11Zn2 (VI) 55.61 49.87 51.94 56.86 59.10 62.04 H (55.62) (49.86) (51.96) (56.82) (59.12) (62.05) 4.98 4.37 4.25 5.32 5.76 4.73 (4.97) (4.34) (4.21) (5.35) (5.77) (4.74) A fine amorphous product was obtained which was washed with ether and dried in a vacuum desiccator. The complexes (I and III– VI) were prepared by same method. The physical parameters of the complexes are shown in Table 5 and probable reaction scheme is shown in Scheme 1. 4.5. Dilution method Before inoculation all the bacteria were incubated and activated at 30  C for 24 h into Luria Broth. The compounds were dissolved in DMSO and then diluted using cautiously adjusted Luria Broth. Two-fold serial concentrations of the compounds were employed to determine the (MIC) ranging from 0.3 to 3200 mM. Test cultures were incubated at 37  C (24 h). The lowest concentrations of antimicrobial agents that result in complete inhibition of microorganisms were represented as (MIC) mM. The control experiments were performed with only equivalent volume of solvents without added test compounds. All experiments were performed in triplicate and the average was taken as the final reading [52]. The ciprofloxacin, gatifloxacin, norfloxacin, enrofloxacin, pefloxacin, levofloxacin, sparfloxacin and ofloxacin were used as a standard drug. 4.5.1. Inoculation procedure The target micro-organism cultures were prepared separately in 15 mL of 2% Luria Broth medium. First the Luria Broth was N M 8.34 (8.37) 6.44 (6.46) 10.43(10.45) 8.01 (8.03) 7.43 (7.45) 7.15(7.12) 9.75 (9.77) 10.08 (10.05) 9.73(9.75) 9.33 (9.38) 8.74 (8.70) 7.44(7.49) m.p.  C % Yield Formula weight (gm/mol) 315 340 220 270 >360 280 69.5 67.2 69.5 67.8 66.4 68.0 1338.96 1300.86 1340.81 1395.06 1503.24 1587.23 autoclaved for 20 min at 120  C and at 15 lb/in2 pressure before inoculation. Inoculation of 10 mL of glycerol stock was done to the previously prepared corning tubes with the help of a micropipette using sterilized tip under sterile condition. The bacteria were then cultured for 24 h at 37  C in an incubator. 4.6. DNA-binding and cleavage assay 4.6.1. UV–Vis. spectroscopy All experiments involving the interaction of the complexes with DNA were carried out in buffer (0.2 M Na2HPO4, 0.2 M NaH2PO4, pH 7.2). A solution of DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of about 1.68, indicating that the DNA was sufficiently free of protein [53]. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6600 M1 cm1) at 260 nm [54]. 4.6.2. Viscosity study Relative viscosity measurements were carried out using an Ubbelohde viscometer maintained at a constant temperature of 27.0 (0.1)  C in a thermostatic bath [55]. Flow time was measured with a digital stopwatch. Each sample was measured three times and an average flow time was calculated. Data were presented as (h/h0)1/3 versus binding ratio [M]/[DNA] [56], where h is the relative viscosity of DNA in the presence of complex and h0 is the viscosity of DNA alone. Viscosity values were calculated from the observed flow time of DNA-containing solutions (t > 100 s) corrected for the flow time of buffer alone (t0), h ¼ t  t0. 4.6.3. Gel electrophoresis Isolation of plasmid DNA from pure culture of E. coli was carried out by conventional method [57]. For the gel electrophoresis experiments, supercoiled pUC19 DNA (0.12 mg) in TE buffer (10 mM Tris–HCl, pH 8.0 and 1 mM EDTA) was treated with different Zn(II) complexes; and in the presence of hydrogen peroxide in the reaction solution. The samples were incubated for 30 min at 37  C, then a loading buffer containing 10 mM TE (pH 7.5), 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol and 60 mM EDTA was added and electrophoresis was performed at 100 V for 2 h in 1X TAE buffer (0.04 M Tris–Acetate, pH 8, 0.001 M EDTA) using 1.0% agarose gel containing 1.0 mg/mL ethidium bromide. Bands were visualized by UV light and photographed on a capturing system (AlphaDigiDocÔ RT. Version V.4.1.0 PC-Image software). Acknowledgement Scheme 1. Reaction scheme of complex [Zn2(Cip)2(mtma)2(pip)(H2O)2]$3H2O. We wish to express our gratitude to Prof. (Miss) R. G. 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