Gul S, Sultana N, Arayne MS, Shamim S, Akhtar M and Khan A (2013)

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Gul S, Sultana N, Arayne MS, Shamim S, Akhtar M and Khan A (2013)
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  1 Sparfloxacin-Metal Complexes as Urease Inhibitors: their Synthesis, Characterization, Antimicrobial and Anti-Enzymatic Evaluation Somia Gul 1 , Najma Sultana 2 , M. Saeed Arayne 2 , Sana Shamim 3 , Mahwish Akhtar 3  and Ajmal Khan 4   1. Faculty of Pharmacy, Jinnah University for Women. Karachi.74600 2. United Biotechnologies, Karachi-75270. Pakistan. 3. Dow college of Pharmacy, Dow University of Health Sciences. 4. H.E.J Institute of Chemistry, University of Karachi. Abstract Four new metal complexes (S12-S15) of SPFX (third-generation quinolones) via heavy metals have been synthesized in good yield and characterized by physicochemical and spectroscopic methods including TLC, IR, NMR and elemental analyses. Sparfloxacinato ligand binds with metals through pyridone and oxygen atom of carboxylic group. The biological actives of complexes have been tested against four Gram-positive and seven Gram-negative bacteria and six different fungi. Statistical analysis of antimicrobial data was done by one way ANOVA, Dunnett’s test ; it was observed that S13, S14 and S15 found to be most active complexes; Antifungal data confirm that all four synthesized complexes are most active and show significant activity against F.solani with respect to parent drug and none of complexes show activity against A. purasiticus, A. effuris and S. cervicis. To study inhibitory effects of newly formed complexes, enzyme inhibition studies have been conducted against urease, α - chymotrypsin and carbonic anhydrase. Enzymatic activity results of these complexes indicated them good inhibitors of urease enzyme while all complexes show mild activities against carbonic anhydrase enzyme. Further research may prove promising role of these synthesized complexes as Urease inhibitors. Keywords: Metal complexes, antimicrobial, anti-enzymatic, spectroscopic 1 Introduction For infectious diseases, multiple therapies are usually required and so the possibility of drug-drug interactions increased. Careful consideration of concomitant drug therapy is needed. Literature survey reveals that fluoroquinolones showed several important interactions with many  2 drugs [1]. Usually fluoroquinolones are prescribed for many diseases including respiratory and urinary tract infections. Sparfloxacin (SPFX) is an orally active synthetically broadspectrum third generation quinolones use for upper respiratory tract infection. Metals are considered essential to a human body in performing physiologically important and vital functions, in the  body [2]. The action of many drugs is dependent on coordination with metal ions or/and the inhibition on the formation of metalloenzymes [3]. The proposed mechanism of the interaction is chelation between the 4-oxo and adjacent carboxyl group of quinolone and metal cations [4-8]. Literature survey reveals that concurrent administration of magnesium and aluminium containing antacid with ciprofloxacin resulted in a nearly complete loss of activity of the drug [9] and  patients who orally administrated fluoroquinolones should avoid mixtures containing multivalent cations, because quinolones were chelate bonded to these metals, in consequence formed metal complex in the gastric system [10]. Alkaysi et al., [11] published norfloxacin interaction with aluminium, magnesium and calcium and Turel [12] compiled interaction of 16 metals with eight quinolones. Absorption of fluoroquinolones is manifestly reduced by antacids, calcium carbonate, ferrous sulphate and sucralfate. Despite the fact that quantitative differences between fluoroquinolones exist, these combinations should be avoided whenever possible [7]. A reasonable recommendation may be to avoid using sucralfate and norfloxacin concurrently, or avoid administration of norfloxacin and ciprofloxacin within two hours of sucralfate administration. Magnesium- and aluminum-containing antacids may also interfere with quinolones absorption. Survey assembled a number of different complexation of quinolones. Mononuclear dioxomolybdenum (VI) complexes with enrofloxacin and sparfloxacin were discovered by Efthimiadou and co-workers [13]. They also [14] discovered ciprofloxacin, cinoxacin, norfloxacin and nalidixic acid complexation with VO 2+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Zn 2+ , MoO 2  , Cd 2+  and UO 2+ , vanadyl complex with enrofloxacin [15] and copper complex with sparfloxacin [16] Kalliopi [17] reported nickel complex with sparfloxacin. Ciprofloxacin interaction with Mn 2+ , Fe 3+ , Co 2+ , Ni 2+  and MoO 2 was presented by Psomas et al.,[18] .  Alkaysi et al., [19] published norfloxacin interaction with aluminum, magnesium and calcium and Turel [20] compiled interaction of 16 metals with eight quinolones. He also published ciprofloxacin complex with Cu (II) [21] and Ionic complexes of protonated norfloxacin with Zn(II) and Cu(II) [22] .  Wallis and co-workers have reported complexes of ciprofloxacin with V (IV)O 2+ , Fe(III) [23] and copper (II) [24]. Complexes of norfloxacin with Zn(II) and Cu(II) were prepared by  3 Chen [25] and complexes of ofloxacin with Cu(II) was discovered by Macías et al.[26]. While Wang [27] reported norfloxacin complex with Mn in 2002. Interaction studies of SPFX metal complexes urged an idea of their synthesis [28]. Now, here we  present synthesis of these complexes to aid prove to interaction studies. My research group has worked on this clinically important field of metal interaction and complexation for last few years [29-30]. We have already published metal complexes of SPFX as antifungal agents [9]. In this section, spectroscopic characterization of these novel neutral mononuclear metal complexes has been conducted with spectroscopic techniques such as IR, 1 H-NMR and elemental analyses (CHN). Prior to synthesis, M: L ratios were determined by conductance. The antimicrobial activity of these complexes has been evaluated against four Gram-positive and seven Gram-negative bacteria while antifungal activity against six different fungi has been determined also. Statistical analysis of antimicrobial data was done by one way ANOVA, Dunnett’s test. Enzyme inhibition studies have been conducted against urease, carbonic anhydrase and α -chymotrypsin enzymes. As well as physiochemical parameters have also been recorded carefully. 2. Experimental: 2.1 Materials and reagents Sparfloxacin was a kind gift by Abbott Pharmaceuticals (Karachi) while solvents and chemicals of analytical grade were purchased from the market. Metal salts (Al (OH) 3 , As 2 O 3 , AgCl and PbCO 3 ) were of pious grade from E. Merck. All solutions were prepared fresh before work. 2.2 Instruments The melting points were taken on an electro thermal melting point apparatus (Gallenkamp) in open capillary tubes and are uncorrected. TLC spots were detected by UV lamp. Infrared spectra were recorded as KBr pellets on Shimadzu 470 instrument. 1 H NMR spectra were obtained by using Bruker /XWIN NMR spectrometer with TMS as internal standard. Complexes were dissolved in CDCl 3 , D 2 O or MeOD for NMR. An elemental analysis is done by Carlo Erba Strumentazione Elemental analyzer-MOD 1106 instrument.  4 2.3 Stoichiometric study Conductometric titration was performed to inspect the stoichiometric ratio of the ligand and metal ions. For this purpose, 1mM alcoholic solution of drug (SPFX) and metal salts were  prepared individually. In 20 mL of drug (SPFX) solution, 2 mL of metal solution was added each time, after every 2 min the conductance value was carefully noted. All the values of conductance were noted until state of chemical equilibrium is achieved. Graph was plotted between corrected conductivity and the volume of titrant added and the end point was determined. Results show that all complexes have stoichiometries of 2:1 (drug: metal). Fig: 1 represent conductometric ratio. 2.4 Synthesis of complexes  A warm methanolic, unimolar solution of metal salts was mixed with a bimolar solution of SPFX in methanol (1:2) in round bottomed flask and was refluxed for 4 h, above 80°C on a water bath with constant stirring. The solution was filtered and the product left for slow evaporation and then crystallized at room temperature. After a few days, crystals deposited were collected, washed with methanol and dried. % yield, color, melting points and solubility of all the complexes was carried out in different solvents as water, methanol, chloroform, and dimethyl sulfoxide. 2.5 Antimicrobial activity For antibacterial and antifungal studies, disk susceptibility technique was used. The diffusion technique developed by Bauer et al., [31] recommended by the FDA [32] has been adopted which has most extensively been used in the clinical laboratories [33]. 2.6 Preparation of dried paper disk The stock solutions of standard drug (SPFX) and SPFX-metal complexes were prepared in water to get the concentration of 100µgmL -1  and diluted in four concentrations of 40, 20, 10 and 5µgmL -1 . 3mm filter paper discs were impregnated with 20mL of each of the different dilutions. Discs were allowed to remain at room temperature till complete diluents evaporation and kept under refrigeration (ready to be used). 2.7 Procedure for antimicrobial activity Organisms studied were taken from the slant with the help of wire loop and were immersed in the tube containing nutrient broth which was incubated at 37 o C for 4-6 hrs until the turbidity exceeded that of 0.5 MacFarland standards. Nutrient agar was prepared; autoclaved at 121ºC for  5 15 minutes then poured in dry, sterile Petri dishes, cooled and set. The bacterial inoculum was uniformly spread using sterile cotton swab on a sterile Petri dish with agar. Discs soaked with metal complexes and derivatives were placed onto the surface of the agar with bacterial inoculum and sparfloxacin disk was used for control. These were then incubated at 36ºC ± 1ºC, for 24h while the water paper discs were used as a positive control. Three replicate trials were conducted against each organism for each concentration. Statistical analysis was used for data interpretation included calculation of the mean values, standard deviation and investigation of significant differences in results. Similar procedures were adopted for antifungal activities. Derivative discs (5, 10, 20 and 40 μgmL -1 ) were placed on SDS medium plates previously seeded with fungal culture and incubated for seven days at 36ºC ± 1ºC, for 48 hours. Zones of inhibition were carefully measured using Vernier caliper. 2.8 Statistical study Statistical analysis of antimicrobial data was done by one way ANOVA, Dunnett’s test through SPSS software version 10.0 (Carry, NC, USA). 3. Results and discussion: 3.1 Synthesis of SPFX- metal complexes with heavy metals Four metal complexes were synthesized by refluxing metal salt solutions of Al(OH) 3 , As 2 O 3 , AgCl and Pb 2 CO 3 in methanol with SPFX in the ratio of 1: 2 [M: L] (determined by conductance), for 4 hours and the volume was reduced by evaporation. Moreover, their melting  points and solubility were noted. Solubility facts of these complexes show that Al 3+  and As 3+ were soluble in CdCl3, Ag1+ was soluble in MeOH, and Pb 3+  in both MeOH and CdCl3. Physicochemical parameters of SPFX and SPFX-metal complexes are given in table 1. The antimicrobial activity of these complexes has been evaluated against mentioned bacteria and fungi and analysis of data was done by one way ANOVA. Enzyme inhibition studies have been conducted against above mentioned enzymes. 3.2 Proposed structure of SPFX metal complexes The coordination chemistry of some quinolones (including sparfloxacin) antibiotics with transition and d10 metal ions has been reported [34, 35-37]. In this case, the SPFX has several  potential donor sites but, due to steric hindrances, the ligand can provide a maximum of two donor atoms to any one metal centre. The spectroscopic changes suggested that the SPFX acts as
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