SYNTHESIS, CHARACTERIZATION, ANTI-MICROBIAL AND CYTOTOXIC APPLICATIONS OF ZINC(II) COMPLEXES

In the present research, three zinc(II) complexes were synthesized using 3-(3-fluorophenyl)-2methylacrylic acid (HL), 1,10-phenanthroline and 2,2-pyridine as ligands. The composition, structural confirmation, coordination way of ligand and assignment of geometry to the complexes were made by different analytical tools like elemental analysis, FTIR and H-NMR. The ligand 3-(3-fluorophenyl)-2-methylacrylate appeared to coordinate the metal (Zn) atom via COO moiety in all of the complexes (1-3) while 1,10phenanthroline and 2,2-pyridine co-ordinated to Zn(II) atom through N-donor sites in complexes 2 and 3, respectively. The complex 1 showed four while 2 and 3 showed six co-ordinated geometry. The synthesized complexes were evaluated for antimicrobial and cytotoxic activities. The obtained results showed that complexes are active against microbial agents and exhibited significant cytotoxicity.


INTRODUCTION
Zn(II) as cation has a very promising ability to catalyse many biochemical reactions [1]. Zn plays an inhibitory role in the growth of bacterial strains like E. coli, S. faecalis, etc [2]. This may be attributed to Zn(II) cation interaction with cell membrane protein resulting in its deactivation and disturbance in transport of nutrients [3]. Interaction of Zn(II) with O-donor ligand and N-donor ligand bring a valuable change in the biological activities [4]. N-donor ligands having heterocyclic rings like 1,10-phenanthroline, 2,2 ' -pyridine enhance anti-microbial activities [5][6][7][8].
Zinc(II) carboxylates have been studied extensively for the last three decades due to their bioactive role in anti-microbial activities [9][10][11]. Carboxylic acids have very important role to enhance lipophilic character and biochemical processes in metabolic pathways [12]. Substituted carboxylic acids are more promising in enhancing bioactive role of metal and have rich mode of co-ordination with metal centre [13][14][15][16]. Various modes of co-ordination like monodentate, bidentate leading to tetrahedral and octahedral geometry have been reported [17,18].
The research work on metal carboxylate complexes is a growing area to explore some novel anti-bacterial, anti-fungal and cytotoxic compounds in order to cope resistance problems. With this continuation and growing research field, we have synthesized three complexes of Zn(II) with both carboxylic acid and N-donor ligands and found out their anti-microbial and cytotoxic activities.
international chemical supplier company Sigma-Aldrich and utilized as received without further purification. The organic solvents were bought from Merck (Germany). The solvents were dried by applying typical procedures [19]. Gallenkamp electrothermal melting point apparatus made in UK was used to find out melting point of ligand and synthesized compounds. Thermo Nicolet-6700 FT-IR instrument was utilized to have FT-IR spectra in the range of 4000-400 cm -1 . 1 H NMR spectra were recorded by 300 MHz NMR spectrometer made up of Bruker (Switzerland).

Syntheses
The 3-(3-fluorophenyl)-2-methylacrylic acid (HL) was prepared using 3-fluorobenzaldehyde (14 mmol, 1.736 g), methylmalonic acid (28 mmol, 3.305 g) and piperidine (28 mmol, 2.38 g) taken in 1:2:2 molar ratio (Scheme 1). The reactant mixture was refluxed in double neck flask using pyridine as solvent for 24 h at 100 o C. After refluxing, the reactant mixture was cooled at room temperature and then put into ice water with addition of concentrated HCl. The solution pH was maintained at 3. The solid product was obtained by filtration which was then washed with water and dried in air.  Step 1.
The complex 3 was synthesized by the reacting the mixture of zinc(II) chloride (0.136 g, 1.0 mmol), sodium salt of ligand (0.404 g, 2.0 mmol) and 2,2 ' -pyridine (0.156 g, 1.0 mmol) (Scheme 5) in dry methanol at 25 o C for 5 h. The insoluble NaCl was detached via filtration and product was acquired by evaporating the solvent through rota-vapour. + 2LNa Step 1.

RESULTS AND DISCUSSION
The ligand 3-(3-fluorophenyl)-2-methylacrylic acid (HL) was synthesized successfully using procedure given in experimental section. The results along with characterization data are represented as

Fourier transform-infrared spectroscopy
The newly prepared ligand and complexes were characterized firstly by FT-IR Spectrophotometer (Nicolet-6700 FT-IR) in the array of 4000-400 cm 1 . The FT-IR spectra of ligand and complexes (1-3) are represented in Figures 1-4. The characteristic values of vibrational frequencies of complexes were correlated with their precursors to confirm syntheses of complexes [20]. The decrease in  value in complexes in comparison to higher  values in ligand (HL) support the formation of complexes, the  asym (COO -) frequencies move to lesser while sym (COO -) to higher frequency [21,22]. The divergence of asymmetric and symmetric frequencies of COOmoiety (Δν) indicates the co-ordinating way of COOmoiety with Zn atom. If the value of Δν difference is larger than 200 cm -1 , it represents a monodentate coordination while Δν value smaller than 200 cm -1 indicates bidentate co-ordinating way of COOmoiety with metal atom [23,24]. In present study, the Δν in zinc(II) complexes have been observed as 158 (1)

Proton-nuclear magnetic resonance studies
Proton-NMR of ligand and its zinc(II) complexes (1-3) were recorded using Bruker 300 MHz NMR and the representative spectra are represented in Figures 5-6. DMSO was used as a solvent. The 1 H-NMR interpretation has been done by using the names and numbering in precursors as given in Scheme 6. The pattern of intensity and peak multiplicity are used to allot

Synthesis, characterization, anti
the values to proton resonance signals [25,26]. The formation of zinc complexes was confirmed by the disappearance of acidic proton peak at 12.36 ppm. The olefinic proton (R 1 CH=C(R 2 )COOH)] appeared at 6.85, 6.44, 6. and 3, respectively. While aromatic protons and their expected values. the values to proton resonance signals [25,26]. The formation of zinc complexes was confirmed by the disappearance of acidic proton peak at 12.36 ppm. The olefinic proton )COOH)] appeared at 6.85, 6.44, 6.46 and 6.60 ppm in ligand HL, complexes While aromatic protons and heterocyclic organic rings protons appeared at NMR spectrum of 3-(3-flourophenyl)-2-methylacrylic acid (HL).
complexes 293 the values to proton resonance signals [25,26]. The formation of zinc complexes was confirmed by the disappearance of acidic proton peak at 12.36 ppm. The olefinic proton , complexes 1, 2 rings protons appeared at

Anti-microbial and cytotoxic investigations
Ligand and the Zn(II) complexes (1-3) were subjected for In vitro anti-bacterial potential evaluation. Four bacterial strains named as Bordetella bronchi septica, Escherichia coli, Micrococcus luteus and Staphylococcus aureus were used in the experiment using Agar welldiffusion process [27]. Two anti-biotic drugs Cefixime and Roxithromycin were taken as +ve control while DMSO as a solvent and negative control. The outcomes are represented in Table  1. Anti-bacterial activity was evaluated by measuring zone of inhibition in millimetre (mm). There are three main parameters, significant ( > 20 mm), better (< 20 mm) and insignificant (< 10 mm) to explain the results [28]. The results showed that zinc(II) complexes (1-3) are comparatively more active than ligand HL but comparable or less than reference drugs used. The microbe's killing was may be due to enzyme retardation, impairment in cell membrane or Tweedy ' s Chelation Theory [29]. The chelates (metal complexes) decrease the polarity and increase lipophilicity of metal complexes which help in crossing the cell membrane made up of lipid layer [30]. The newly prepared ligand and i t s z inc(II) complexes (1-3) were tested against four fungal strains named as Aspergillus flavus, Fusarium solani, Aspergillus fumigates and Aspergillus niger. This activity was determined using Agar Tube Dilution method [31] and the results in the form of % inhibition are represented in Table 2. Terbinafine was taken as +ve control. The criteria to explain results is % growth inhibition in four slabs, > 70, 70-60, 60-50 and < 50% growth inhibition measured as significant, good, moderate and non-significant, respectively [28]. The results revealed that zinc(II) complexes (1-3) are more active than ligand acid (HL). The complex 3 showed highest antifungal activity among the complexes. The nitrogen containing ligand enhance antifungal activity that may be due to higher permeation ability into cell. This is attributed to proper overlapping of metal ion and nitrogen donor ligand orbital which results partial sharing of positive charge on metal ion with ligand orbital. Thus reduction of polarity on metal ion lead to more lipophilic character of metal complex, allowing it to cross lipid membrane of fungus and show more potent activity [32][33][34]. Table 2. Antifungal activity data of ligand and its Zn(II) complexes (1-3).

In vitro cytotoxic assessment
The cytotoxic studies of ligand and Zn(II) complexes (1-3) were evaluated via brine-shrimp lethality procedure [31] and standard drug was MS-222 (Tricaine Methane sulfonate). The results are shown in Table 3.The results exhibited that generally, the tested Zn(II) complexes have shown more cytotoxicity than ligand but less than their reference drug used.

CONCLUSION
Three new Zn(II) complexes using O and N-donor mixed ligands were prepared and characterized by elemental analysis, FT-IR, 1 H and 13 C NMR spectroscopic methods. The FT-IR studies showed that carboxylate ligand co-ordinate with Zn(II) ion in bidentate fashion. The complex 1 exhibited four co-ordinated while complexes 2 and 3 six co-ordination behaviour around metal atom. The antimicrobial results showed that Zn(II) complexes 1-3 exhibit good antimocrobial activity than the ligand. The cytotoxic studies revealed that complexes 2 and 3 were more toxic than complex 1 and ligand (HL).