Sparfloxacin-Metal Complexes as Urease Inhibitors : Their Synthesis , Characterization , Antimicrobial , and Antienzymatic Evaluation

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 sevenGram-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 were 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. parasiticus, 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 to be good inhibitors of urease enzyme while all complexes show mild activities against carbonic anhydrase enzyme. Further research may prove the promising role of these synthesized complexes as urease inhibitors.


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 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][5][6][7][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 binds with these metals through chelation, in consequence formed metal complex in the gastric system [10].Ma et al. [11] published norfloxacin interaction with aluminium, magnesium and calcium and Alkaysi et al. [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].
Interaction studies of SPFX metal complexes urged an idea of their synthesis [28].Now, here we present synthesis of these complexes to aid in proving interaction studies.My research group has worked on this clinically important field of metal interaction and complexation for the 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   metal ions.For this purpose, 1 mM alcoholic solutions 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).Figure 1 represents conductometric ratio.

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 were carried out in different solvents as water, methanol, chloroform, and dimethyl sulfoxide.

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].

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 100 gmL −1 and diluted in four concentrations of 40, 20, 10, and 5 gmL −1 .Three mm filter paper discs were impregnated with 20 mL 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 ∘ C for 4-6 hrs until the turbidity exceeded that of 0.5 MacFarland standards.Nutrient agar was prepared, autoclaved at 121 ∘ C for 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 36 ∘ C ± 1 ∘ C, for 24 h, 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 gmL −1 ) were placed on SDS medium plates previously seeded with fungal culture and incubated for seven days at 36 ∘ C ± 1 ∘ C, for 48 hours.Zones of inhibition were carefully measured using Vernier caliper.

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+ was soluble 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 the abovementioned enzymes.

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][36][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 a bidentate ligand and its coordination occurs through the metal via the pyridone and one carboxylato oxygen atom and forms slightly distorted octahedral geometry.

Infrared Analysis.
IR spectra of SPFX M.complex (S12-S15) revealed that the absorption at 1720 cm −1 observed in the spectrum of sparfloxacin, attributed to the (C = O) carb , has been replaced with two very strong characteristic bands in the range of 1636-1641 cm −1 and 1368-1387 cm −1 assigned as (O-C-O) asymmetric and symmetric stretching vibrations, respectively (Table 2) [31].The Δ[ asym (CO 2 ) −  sym (CO 2 )] values fall in the range of 249-271 cm −1 indicating a monodentate coordination mode of the carboxylato group via the pyridone and one carboxylato oxygen atom [7].The vibration (C = O) p , pyridone stretch is slightly shifted from 1641 to 1636-1641 cm −1 upon bonding [35].Broad split band at 3094-3347 cm −1 can be assigned to the O-H stretching vibrations of water molecules and also includes the N-H stretching vibration of the piperazinyl moiety [38,39].New bands around 470-490 cm −1 seemed in the spectra of complexes can be assigned to (M-O) [40].

1 H NMR Analysis.
The proton NMR spectrum of complexes showed a set of signals which were almost identical to those of SPFX, while changes occurred particularly at carboxylic protons as well as protons of aromatic C-NH.Singlet at  3.90-3.98ppm was assigned to C-NH 2 group.The spectra showed multiplet at  1.03-1.53ppm for cyclopropyl protons, multiplet at  3.04-3.31ppm for iperazinyl protons, and singlet at  8.46-8.62ppm for -CH 2 protons (Table 3).No broad weak band for acidic proton at : 11 ppm seen in spectra of complexes indicating that this targeted moiety took part in complexation of metals with SPFX and SPFX acts as bidentate deprotonated ligands bound to the metal through the pyridone oxygen and one carboxylate oxygen [9].

Elemental Analysis.
The results obtained from elemental analysis CHN point toward that all of the complexes are formed from the reaction of the metal salt with drug in 1 : 2 molar ratio (Table 4).

Antimicrobial Studies
3.4.1.Antibacterial Activity.Comparison of antibacterial activity data of novel SPFX metal complexes suggests that almost all complexes are active antimicrobial agents (Tables 5(a)-5(k), Figure 2) and most of them exhibit better activity than parent drug.SPFX complexes including S13, S14, and S15 were found to be the most active complexes possessing higher antimicrobial activity against B. subtilis and M. luteus in all four tested concentrations.All synthesized complexes show moderate activity against S. aureus and S. features as compared to parent drug as well as other advance fluoroquinolones.Spectrum of Gram-negative activity indicated that all complexes show remarkable (excellent) activity against P. aeruginosa, E.coli, and S. typhe while S13, S14, and S15 exhibit good activity against P. mirabilis and citrobacter in comparison to SPFX and other standards, M. luteus and S. typhe.All complexes show almost same or less activity against K. pneumoniae and S. flexneri.3.4.2.Antifungal Activity.These synthesized complexes were also evaluated for antifungal activity.The average results are shown in Tables 6(a)-6(c) (Figure 3).All of the complexes show excellent activity against F. solani as compared to the parent drug and other standards, while all synthesized complexes are less active against T. rubrum and more or less equally potent against C. albican in comparison to parentdrug.None of complexes show activity against A. parasiticus, A. effuris, and S. cervicis.In general, antifungal data and its statistical analysis confirm that all four synthesized complexes are most active and show significant activity against F. solani with respect to parent drug as well as other advance fluoroquinolones.S14, and S15 were found to be most active complexes and possess higher antimicrobial activity against B. subtilis and M. luteus in all four tested concentrations but less active than the parent drug, while all complexes show almost same or less activity against K. pneumoniae and S. flexneri.Antifungal data confirm that all four synthesized complexes are most active and show significant activity against F.solani with respect to parent drug as well as other advance fluoroquinolones and none of complexes show activity against A. parasiticus, A. effuris, and S. cervicis.Enzymatic activity results of these complexes indicated them to be 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.

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).

Figure 3 :
Figure 3: Graphical representation of inhibition zone against fungi.

Table 4 :
Elemental analyses of the complexes.