Preparation, Biological Activity and Characterisation of Novel Macroacyclic (N2O4 and N2O2) Schiff Base Ligands and Their Zn(II), Cd(II) and Hg(II) Complexes

New macroacyclic N2O4(L1) and N2O2(L2) Schiff base ligands have been synthesized from 1,4-di-(4-fluoro-2-aminophenoxy)butane(DFAB)withsalicylaldehyde and 1,4-di-(4fluoro-2-aminophenoxy)butane(DFAB)with anthracene-9-carbaldehyde, respectively. Zn(II), Cd(II) and Hg(II) complexes of the Schiff base ligands have also been prepared and all compounds have been characterised by IR, 1HNMR , 13CNMR spectroscopy and mass spectrometry. We are also especially interested in the antibacterial activity of these new complexes. The in vitro antibacterial activity of the metal ions, free ligands and their complexes were tested against the gram-positive bacteriaand gram-negative bacteriaby paper disc diffusion and minimum inhibitory concentration (MIC)methods. It is apparent the metal complexeshave good antibacterial activity but related free ligands and metal ions have not antibacterial activity.

Schiff bases and their complexes, a typically of chelators are capable of forming coordinate bonds with many metal ions through azomethine group and phenolic group or via its azomethine or phenolic groups [1][2] . The chemistry of Schiff base ligands and their metal complexes have attracted increasing interest owing to their role in the understanding of molecular processes occurring in biochemistry, antifungal, antibacterial, anticancer, catalytic fields and as encapsulating ligands for radiopharmaceuticals [2][3][4][5][6][7][8][9][10][11] . Therefore, The chemistry of Schiff base ligands and their metal complexes have attracted a lot of interest due to their facile synthesis and wide range of applications including pigments, intermediates in organic synthesis and as polymer stabilizers [12][13][14][15] .

4-fluorophenolis commercially available
from Merck and is used without any changes.All other solvents and materials were of reagent grade and used without further purification. IR spectra were recorded (KBr) on a BrukerVERTEX 70 spectrometer. 1 H NMR and 13 C NMR spectra were recorded on a Bruker-AV400MHz. Mass spectra were recorded on a 5973 Tecnology Agilent (HP) spectrometer (EI = 70 eV).

Synthesis
In this work, a new diamine 1,4-di-(4fluoro-2aminophenoxy)butane has been synthesized by modified previous procedure 16 . Then, macroacyclic (N 2 O 4 and N 2 O 2 ) Schiff base ligands and their complexes have been synthesized and characterized by IR, 1 H-NMR and 13 C-NMR spectroscopy and mass spectrometry.

Synthesis of macroacyclic Schiff base ligand (L 1 )
To a solution of 0.12 g salicylaldehyde (1 mmol) in 20 cm 3 ethanol was added dropwise a solution of 0.2 g diamine salt (0.5 mmol) in 10 cm 3 water and 30 cm 3 ethanol over 30 minutes. Then the solution of NaOH (5M) was added drop by drop until pH of the mixture of reaction reached 7. The reaction was stirred at room temperature for 24 h to be completed according to scheme 3. After completion of the reaction, the whity precipitate was filtered and washed with acetonitrile and cold methanol. To a solution of 0.12 g salicylaldehyde (1mmol) in 20 cm 3 ethanol was added appropriate amount of M 2+ (1 mmol) and it was stirred at room temperature for 2 h. After that dropwise a solution of 0.2 g diamine salt (0.5mmol) in 10 cm 3 water and 30 cm 3 ethanol over 30 minutes. Then the solution of NaOH (5M) was added drop by drop until pH of the mixture of reaction reached 7. The mixture of reaction was refluxed for 36 h. After completion of the reaction, the whity precipitate was filtered and washed with acetonitrile and cold methanol.   13 C NMR (ä, DMSO-d 6 , MHz): 25.88 (2C; C a ), 68.86 (2C; C b ), 106.33-156.36 (24C; C c , C d , C e , C f , C g , C h , C j , C k , C l , C m , C n, C O ), 163.86 (2C; C i ).

Synthesis of macroacyclic Schiff base ligand (L 2 )
To a solution of 0.2 g anthracene-9carbaldehyde (1 mmol) in 20 cm 3 ethanol was added dropwise a solution of 0.2 g diamine salt (0.5 mmol) in 10 cm 3 water and 30 cm 3

Template synthesis of metal complexes of L 2 with M 2+ (M = Zn, Cd and Hg)
To a solution of 0.2 g anthracene-9carbaldehyde (1 mmol) in 20 cm 3   C e , C f , C g , C h , C j , C k , C l , C m , C n, C o ,C p , C q , C r , C s, C t , C u , C v , C w ), 178.13 (2C; C i ).

Complex (5)
[   2C; C b ), 103.18-171.66 (40C; C c , C d , C e , C f , C g , C h , C j , C k , C l , C m , C n, C o ,C p , C q , C r , C s, C t , C u , C v , C w ), 180.14 (2C; C i ).

Complex (6)
[ .54-171.27 (40C; C c , C d , C e , C f , C g , C h , C j , C k , C l , C m , C n, C o ,C p , C q , C r , C s, C t , C u , C v , C w ), 178.17 (2C; C i ).

Antibacterial activity
The in vitro antibacterial activity of the metal ions, free ligands and their complexes were tested against the gram-positive bacteria; Bacillus anthracis (RTCC 1036), Staphylococcus epidermidis (PTCC 1114), Staphylococcus aureus (RTCC 1885), Enterococcus faecalis (RTCC 2121), Tetracycline and Gentamycine were used as standard compounds to determine the sensitivityof one strain/isolate in each microbial species tested. Antibacterial activity in the disc diffusion assay was evaluated by measuring the zone of inhibition against the test organisms. Each assay in this experiment was repeated twice. For investigation of antimicrobial activity of as prepared metal ions, free ligands and their complexes. The inhibitory effect of complexes on the growth of microbes were studied. The results can be seen in Table 1 and Table 2.
The results showed that antibacterial activity of the complexes exceeded the Tetracycline and Gentamycine used as standard compounds.
Because single crystals of these complexes could not be isolated from any solvents, no definitive crystal structures could be assigned. However, on the basis of characterization results, the molecular ratio of the L 1 and L 2 to metal ions could be confirmed as 1:1.

Antibacterial activity test
The inhibition effect on bacteria growth was determined by disc diffusion method [17][18][19][20]. Each compound was dissolved in methanol as a solvent (1g/10 ml) and 50 ìl of each solution applied on the paper disc (the disc diameter was 6 mm). The impregnated discs with different solutions were left for complete evaporation of the solvent. Then disc papers were placed on the inoculated plates with the bacteria of interest. After incubation in the standard upside down position in 40 °C for 24 h, zones of growth inhibition around each of the discs were measured to the nearest millimetre. A blank, containing only methanol, showed no inhibition in a preliminary test. The macrodilutionbrot susceptibility assay was used for the evaluation of minimal inhibitory concentration (MIC). It is apparent the metal complexes have greater antibacterial activity but related free ligands and metal ions have not antibacterial activity. The results showed that in some cases the antibacterial activity of this complexes exceeded of other Schiff base complexes [21][22][23][24]. Tweedy's chelation theory is a good clarification for this phenomenon.

CONCLUSION
A new macroacyclic Schiff base ligands derived from condensation of 1,4-di-(4-fluoro-2-aminophenoxy)butane withsalicylaldehyde and 1,4-di-(4-fluoro-2-aminophenoxy)butane withanthracene-9-carbaldehyde have been synthesized and it's complexation capacity towards Zn 2+ , Cd 2+ and Hg 2+ has been studied by adopting one-pot template method. The structures of the complexes were confirmed by 1 HNMR, 13 CNMR, IR spectroscopy and mass spectrometry. We are also especially interested in the antibacterial activity of these new complexes, free ligands ( L 1 and L 2 ) and metal ions. The results showed that in the metal complexes have greater antibacterial activity but related free ligands and metal ions have not antibacterial activity.