SYNTHESIS, CHARACTERISATION, THERMAL ANALYSIS AND DNA CLEAVAGE ACTIVITY OF A NOVELZINC (II) COMPLEX OF PYRAZOLE SCHIFF BASES

A hydrazone Schiff base Zn(II) metal complex is synthesised from the Shiff base ligand Thiophene-2-carboxylic acid hydrazide and 1, 3-diphenyl-1H-pyrazole-4-carboxaldehyde reacted together in 1:1 mole ratio to obtain Schiff base ligand (HL) which was subsequently, allowed to react with Zn(CH3COO)2.2H2O. The Schiff base ligand and its Zn (II) complex prepared were characterized on the basis of elemental nalysis,thermogravimetry, UV-Visible spectroscopy, FT-IR spectroscopy and NMR spectroscopy. IR spectrum of the zinc complex shows that the ligand (HL) is coordinated to the metal ion in monoanionicbidentate fashion with the 1:2 metal to ligand stoichiometry. The thermal behaviour of thecomplex shows a single step decomposition pattern leaving the respective ZnO residue. The DNA cleavage activity of the complex ismonitored using agarose gel lectrophoresis method which indicates the potential of the complex to cleave supercoiled DNA.


INTRODUCTION
Schiff base Hydrazone are of significant interest and attention because of their biological activity including anti-tumour, anti-bacterial, antifungal and anti-carcinogenic properties and catalytic activity (1). Schiff bases readily coordinate with metal ions with various modes of coordination under different reaction conditions and their metal complexes have potent chemical, physical, biological and catalytic properties (2,3,4).Schiff bases of pyrazoleheterocyles found their place in different fields of chemistry because of their wide biological activity like antimicrobial (5), antiinflammatory (6), anti tubercular (7), anti tumor (8), anti angiogensis (9), anti parasitic (10), anti viral (11) and also possesses analgesic and anxiolytic activity (12). Many transition metal pyrazole Schiff base complexes are reported to have biological importance (13). Copper pyrazole complexes were found to be effective apoptosis inducers and inhibited angiogenesis on Matrigel and HUVEC migration in vitro (14). Some palladium pyrazole Schiff base complexes were synthesized and characterised for their cytotoxic effect against the fast growing head and neck squamous carcinoma cells SQ20B and SCC-25 and were found to have higher clonogenic cytotoxic effect than cisplatin when tested on SQ20B cell line (15). Among the transition metal complexes, zinc complex have a significant biological importance due to their presence in various enzymes and proteins (16). In this work, we synthesised zinc complex of pyrazole heterocyclic and characterised by elemental analysis, FT-IR, UV visible and NMR techniques. From the data obtained, a 1:2 metal to ligand tetrahedral coordination geometry was proposed for Zn complex.

MATERIALS AND METHODS
Reagent grade chemicals were procured commercially and used without subsequent purification.
[Zn(Ac)2.2H2O] were purchased from Lobachem and Rankem. The commercial solvents were used without further purification.

Physical measurements
Melting Points of the samples were determined using Raaga apparatus. FT-IR spectra of solid sample of ligands and complex were recorded using KBr pellets on a Nicolet Avatar instrument in the frequency range 400-4000 cm -1 . Microanalyses (C, H & N) were performed on a Vario EL III CHNS analyser. Electronic absorption spectra of the samples were recorded using a Jasco V-630 spectrophotometer. 1 H NMR spectrum of the ligand was recorded on a Bruker Avance-3 spectrometer at 400 MHz.

Synthesis of schiff base
The Schiff base was prepared by reacting a mixture of thiophene carboxylic acid hydrazide (0.273 g, 1 mM) and 1,3-diphenyl-1H-pyrazole-4carboxaldehyde (0.173g , 1mM) in 50 mL of aqueous methanol. A few drops of glacial acetic acid were added to the reaction mixture. The resulting *Correspondence: Jayanthi Eswaran, Department of Chemistry, Kongunadu Arts and Science College, Coimbatore, Tamil Nadu, solution was refluxed for 6 h, cooled and the precipitate obtained was checked for purity. The analysis of the product by TLC revealed the formation of the ligand. Synthetic scheme for the preparation of the Schiff base is given in below.

RESULTS AND DISCUSSION
The Schiff base ligand is synthesised by reacting equimolar quantities of thiophene carboxylic acid hydrazide and 1,3-diphenyl-1Hpyrazole-4-carboxaldehyde in methanol medium to yield pale yellow colour ligand thiophene 2carboxylic acid ( solvents and completely soluble in DMF and DMSO.The ligand and the complex are characterized using IR, UV-visible and NMR spectroscopic techniques and elemental analysis method. Thermal analysis of the Zinccomplex was done to discover its formation as proposed.

Analytical data
Analytical data of the ligand (HL) and complexaregiven in table.1 and they are in good agreement with the expected values. Generally DNA damage is indicated by the conversion of supercoiled form of plasmid DNA to circular form. The potential of newly synthesized complex to cause oxidative plasmid DNA breakage was assessed by the plasmid DNA breakage assay (17).The 20, 30 and 40 µMconcentration of the test compounds were added to 500 ng of pBR322 supercoiled plasmid DNA along with the blank and incubated for 6h at ambient temperature under dark. Then, the samples were mixed with 6X orange loading dye (Fermentas, Mumbai) and loaded into 1% agarose gel containing Ethidium bromide. After 30 min of gel run, the extent of damage caused by the test compounds were visualized under UV light

FT-IR spectral data of the ligand and complex
IR spectrum of the ligand showed a sharp band in the region 3232 cm -1 due to the presence of ν(N-H) stretching vibrations. A very strong band found around 1647 and 1598 cm -1 was assigned as due to amide carbonyl symmetric and asymmetric stretching vibration. The other bands at 1547 and 1073 cm -1 were assigned to the ν(C=N) and ν(N-N) stretching frequencies of the ligand.
The bands due to ν(C=O) and ν(N-H) stretching vibrations of the hydrazones were absent in the IR spectra of these complex with the appearance of two new bands between 1547-1493 cm - ν(C=N-N=C) function generated with the enolisation followed by deprotonation and attested the coordination of hydrazone ligand in the enol form. Furthermore, a decrease in the ν(C=N) stretching frequency involving the azomethine nitrogen indicated its involvement in the coordination to the metal ion (18).
The analytical data and IR characteristics are in good agreement with the proposed structure of Zinccomplex. The important IR stretching frequencies of the ligand and complexare given in the Table 2.The IR spectrum of ligand and complex is shown in Figure 1 and 2.

ELECTRONIC SPECTRUM
The electronic spectra of the ligand and complex were recorded in DMSO solution. The ligand spectrum exhibited one broad band in the range 240-360 nm were assigned to the n→π* and π→π* intra ligand transitions which were of higher energy transitions. The spectrum of complex exhibited two bands in the range 240-380 nm region. The higher energy bands below 300 nm are attributable to n→π* and π→π* intra ligand transitions [16]. Other broad band that was observed in the 300-370 nm regions can be assigned to a ligand to metal charge transfer (LMCT) transitions of the imine group.

Proton NMR spectrum of the ligand
1 H-NMR spectrum of the free hydrazone ligand recorded using CDCl3 as solvent was assigned on the basis of observed chemical shift. The spectrum displayed a singlet due to an NH proton in 9.0 ppm. The ligand showed a sharp singlet for azomethine (HC=N) at 8.87 ppm. Signals corresponding to the protons of benzene proton and thiophene proton of the ligand were observed as multiplets in the range of 7.22-8.58 ppm. NMR spectrum of the ligand ascertained its formation as expected.

Thermal analysis of the complex
Thermo-gravimetric analysis of the zinc complex showed a single step decomposition pattern in the temperature range 180-356°C.The coordinated ligand present in the complex decomposed exothermically to yield ZnO

DNA cleavage study
To check the role of synthesized complex on DNA breakage, plasmid DNA damage assay was performed using the pBR322 plasmid DNA and the efficiency of the cleavage was monitored by agarose gel electrophoresis.The DNA cleavage efficiency of the complex was due to the difference in the binding affinity of the complex to DNA. Results of the experiment revealed that complex significantly damaged the plasmid DNA upon treatment for 30 min (Fig.5). The efficiency of studied complex to cleave super-coiled DNA to linear form is the characteristic of anticancer drugs those could effectively bind to the nuclear DNA and impart damage to it and thus arrest the proliferation of cancerous cells.
residue.The percentage weight loss for the decomposition was found to be 89.97 % for the decomposition of the two ligands.The remaining 10.03% matches with the residue ZnO.Thus it confirmed the formation of the complex as proposed in the Scheme 2.

Fig. 8. TG-DTA curve of Zinc complex
Based on the above facts, a four coordinate tetrahedral geometry is proposed for the Zinc complex with 1:2 metal to ligand stoichiometry and the structure is given below.

CONCLUSION
Interesting coordination modes of hydrazone and their biological perspective provoked us to synthesize new zinc hydrazone complex by using the ligand prepared from 1,3 diphenyl pyrazole-1H 4carbaldehyde and thiophene carboxylic hydrazide (HL).The ligand was characterised by FT-IR,UV-visible and NMR spectral method showed its formation as expected. The elemental data of the ligand and the complex are in good agreement with the proposed molecular formulae of them. The IR spectral data of the zinc complex indicated the absence of N-H and carbonyl stretching vibrations and formation of new C꞊N vibrations. This shows the coordination of the ligand in monoanionicbidentate fashion in complex. UV spectral data of the complex showed four bands, two due to ligand centred transitions at 240-310 nm and another two metal centred transition in the range 320-450 nm. Based on the above spectral data tetrahedral geometry was proposed with 1:2 co-ordination of metal to ligand in which ligand formed NO chelate with the metal centre.The DNA cleavage studies showed that the complex have the potential to cleave DNA.