Antimicrobial Activities, Characterization and Synthesis of Organotin(IV) Complexes with Benzohydrazide Derivative

1Department of Chemistry, The Islamia University of Bahawalpur, 63100, Bahawalpur Pakistan. 2Department of Chemistry, Government Sadiq College Women University Bahawalpur, 63100, Bahawalpur Pakistan. 3Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, 63100, Bahawalpur, Pakistan. 4Institute of Chemical Sciences, Bahauddin Zakariya University, Multan, 60800, Multan Pakistan. 5Department of Environmental Sciences, Bahauddin Zakariya University, Multan, 60800, Multan Pakistan. 6Department of Biosciences, COMSATS Institute of Information Technology, Islamabad. 45550, Islamabad Pakistan.

The ligand design, its synthesis and characterization plays a significant role in addressing and transporting the molecule to the target, interactions with biomolecules and pathogenic resistance 1 .This growing field has always been at the esteem of synthetic, biological and medicinal perspectives.Among the organic compounds/ligands, the Schiff bases particularly derived from aroylhydrazones are regarded as the most privileged class of ligands owing to their versatile synthesis, novel structural features & flexibility and superior metal chelating ability.Aroylhydrazones, mostly can exhibit keto-enol tautomerism (Figure -1), that is primarily due to the solvent/reagent effect, temperature change, UV irradiations or pH change.These isomers can impart diverse chelating properties 2 and are proved to be the best antimicrobial, antioxidant, antiinflammatory, antitumour, antitubercular, anticoagulants, anticonvulsant agents [3][4][5] .Also the metal complexes of hydrazones are proved to be the best luminescent probes 6 , nonlinear optics, catalysts 7 and molecular sensors 8 etc.
The biocidal activities of the ligands increases upon chelation with suitable metal ions as it promotes their target oriented nature 2 .In this regard, organometallic derivatives/complexes of pblock elements, preferentially belonging to group 14, (e.g.tin(IV) and silicon(IV)), have been of significant interests.Organotin(IV) complexes with Schiff base ligands have fascinating chemical behavior, kinetically stable, relatively lipophilic in nature and less toxic than that of platinum drugs.Also the phenyl-and n-butyltin(IV) complexes display a larger array of biological activities as compared to their methyl-, hexyl-or octyltin(IV) analogs 9,10 .Although there are several limitations of tin metal complexes used as pharmacological agents, like high cost, toxicity, immunogenicity, low lipid solubility, little penetration into cells etc.But these therapeutic agents still include a plethora of compounds that are mostly antitumor, antimicrobial, antioxidant, antiflammatory and radioprotectants 11 .
On chelation the polarity of the tin metal declines to a much greater extent, this is due to the overlap of the ligand orbital/orbitals, partial sharing of the positive charges of the metal ions and delocalization This delocalization over the whole chelate ring enhances the lipophilic characters of the complexes.That actually promotes the penetration of the complexes into lipid layer of cell membrane and blocks the active sites on enzymes of microorganisms 12,13 .As a result of which the macromolecular or DNA synthesis inhibition, reduction of mitochondrial energy metabolism or elevation in oxidative and DNA damage occurs.Therefore, the organotin(IV) compounds appear to be very promising as potential drugs particularly active against cancers 14 .

EXPERIMENTAL
Chemicals and reagents used in this study were research grade products and purchased from the commercial sources like Merck and Aldrich.These were used either without any further purification or purified where necessary.Analytical grade solvents were dried via methods cited in the literature before use.To determine melting points of the synthesized moieties, GallenKemp melting point apparatus was used and are uncorrected.Bruker Tensor 27 Fourier Transform Infra-Red Spectrophotometer was used in the range of 4000-400 cm -1 to collect IR spectral data.The elemental analyses (CHNS) were carried out on Carlo Erba 2400 automatic analyzer.Multinuclear NMR ( 1 H and 13 C) spectra were recorded on Varian MR Instrument at 300 MHz and/or 400 MHz in DMSO, CDCl 3 or deutrated acetone using tetramethylsilane (TMS) as internal standard.The mass spectra were recorded on JEOL JMS 600-H series (EBE) MS spectrometer.

Biological Activities
Pertinent literature confirmed that, the organotin(IV) complexes depict remarkable in vivo and in vitro biocidal and antimicrobial activities, also these are frequently be used in cancer chemotherapy because of their best apoptotic properties.Usually, in vitro antibacterial or fungicidal activities of the organotin(IV) follow the general order of activity as: RSnX 3 < R 2 SnX 2 < R 4 Sn -R 3 SnX, where the anionic X exhibit little influence on activity 18,19 .Hubert et al. reported that the geometry and stereochemistry around tin atom while Hadjikakou et al., confirmed that it is the only ligand type not the geometry that is responsible for the antitumor activity of the organtin(IV) compounds [20][21][22] .Hence, to explore the therapeutic effects of the synthesized compounds various biological activities were performed.

Procedure for biological activities Antifungal assay
Antifungal assay of the ligands and their organotin(IV) complexes was carried out against various fungal strains i.e. C. albicans, A. flavus, M. canis using Miconazole as a standard drug.

Procedure at 96 -Well Plates
Antifungal bioassay was performed by pre-described method 15 .Briefly, the test compounds were dissolved in 2% DMSO and 20µg/ well of this were loaded into wells of 96 well plates.In each well, 180µl overnight preserved fungal culture was poured.A volume in each well was retained up to 200 µl.The whole mixture was then incubated at 25 o C for 24 hours by covering microplate with a lid to ensure the aseptic environment.After incubation, the absorbance of each well was measured at 405 nm using ELISA micro-plate reader.The percentage inhibition was calculated by employing formula, Percentage inhibition = 100(X-Y)/X X = Absorbance for control with fungal culture without any antifungal agent Y= Absorbance for test sample with fungus

Cytotoxicity Assay
Cytotoxicity assay of the ligand (2) and its corresponding organotin(IV) complexes (3-11)  was performed by evaluating inhibitory effects on the growth of Nauplii (brine shrimps), by using brine shrimp lethality method 23 .The method includes following steps.The hatching tray was filled with artificial sea water (i.e.salt solution 38g/ l) and a porous membrane used to divide the tray into two halves.Brine shrimp eggs were sprinkled in one half of the tray and covered with a lid.The second half of the hatching tray was left

Antiurease Activity
The antiurease activity test was modified from Berthelot assay 24,25 .Briefly explained here as, in each well of 96-well plate, 200µl assay volume containing 55µl of phosphate buffer (0.2 M) with pH 7 followed by the addition of 10µl test solution and 10µl enzyme mixture (0.015 units) was used.All the contents were incubated at 37ºC for 10 minutes.Then 10µl of urea stock solution (80mM) was added in each well and incubated at 37ºC for further 10 minutes.A pre-reading was taken at 630 nm.After that 115µl of phenol hypochlorite reagent was added in each well.The Phenol hypochlorite reagent was made freshly by combining 45µl phenol solution with 70µl alkali solution.After incubation at 37ºC for 10 minutes, the color appeared.And the absorbance was taken at 630 nm using 96-well plate reader.The solution having no sample served as a negative control.Wells containing thiourea served as positive control.

Antibacterial Assay
Antibiotic sensitivity test was performed using a standard disc diffusion assay method by using E.coli, B. subtilis and S. aureus as indicator target strains [26] Briefly described, 50µl compound were loaded onto a sterile filter-paper disc (6 mm in diameter).A 50µl simple broth was used as a negative control, and imipenem (20µg/ml) was used as positive control.After application onto paper discs, the discs were air dried and placed onto the nutrient agar plate, which had been inoculated with a lawn of bacterial strain.After incubation for 24 h at 37 o C, the antibacterial activity was evaluated by measuring the diameter of the growth-inhibition zones from the edge of each filter paper.The inhibitive radii mean (the clear zone radii in which the tested strains did not grow) was noted.
The percentage inhibition was calculated by the following formula
The carbonyl (-C=O) group showed its presence at 1279 cm -1 in 2, No significant change is detected in all the complexes (1270-1286 cm -1 ), it also confirms the non-participation of carbonyl oxygen in complexation.
Sn-C absorption bands appeared in the range of 487-499 cm -1 and an absorption band in the range of 442-458 cm -1 indicates the presence of a Sn-O bond in all the coordination compounds.
Antifungal activity profile of compound 2 and its corresponding organotin(IV) complexes is graphed in figure-2.Ligand (2) reveals percent inhibition against all fungal strains in the range of 40.8-49.2%.Among its chelates, compound 3 displayed highest value of 48.3% against C. albicans and the compound 9 (55.3%)exhibits highest inhibition against A. flavus.While the compound 7 (59.3%)has shown highest value against M. canis.The result shows that the activity of the ligand enhanced upon chelation that is in accordance to the previous reports 28 .From the results, it is also clear that ll the compounds are moderately antifungal in nature.
Antibacterial assay profile of ligand (2) and its corresponding organotin(IV) complexes is represented in figure-3.The ligand (2) has shown percent inhibition of 12.2-14.4against E. coli, B. subtilis and S. aureus.Among tin complexes, compound 8 depicted maximum inhibitions of 19.1% against E. coli and the compound 10 exhibited 21.6% against B. subtilis.While the compound 7 remained best inhibitor against S. aureus (17.8%).Compared with the inhibitory zones of the standard, the synthesized compounds are good to best antibacterial in nature.Also the results confirm the previous reports that the diorganotin(IV) complexes (7,8,10) are good antibacterial as compared to triorganotin(IV) complexes [29].Figure-5 confirms that, the compound 9 (triphenyltin(IV)) has shown maximum cytotoxicity of 39.6%.The literature also confirms its worth i.e. to be the best inhibitor 29 .

Fig. 1 .
Fig. 1.Possible tautomeric forms of aroylhydrazones and E/Z configuration with respect to the C=N double bond

Fig. 2 .Fig. 3 .
Fig. 2. Antifungal activity profile of ligand (2) and its corresponding organotin(IV) complexes by using 96 -well plates method.Each bar represents the percentage inhibition (mm) for compounds and reference drug (10mM test concentration)