SYNTHESIS , CHARACTERIZATION , THERMAL AND ANTIMICROBIAL STUDIES OF SCHIFF BASE M ( II ) COMPLEXES

Asha M S 1 , Othbert Pinto 1 , Joseph N Sebastian 1 , Sumana Naik 1 and Shaukath Ara Khanum 2 .  1. Department of Chemistry, St. Philomena’s College Bannimantap, Mysuru, Karnataka, India. 2. Department of Chemistry, Yuvaraja’s college, Mysuru, Karnataka, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The N, O type Schiff base ligand and its five new metal complexes of Co(II), Ni(II), Zn(II), Cd(II) and Cu(II) derivatives of 6-acetyl-7 hydroxy 4,8-dimethyl-2H-chromen-2-one have been synthesized from o-phenylenediamine in alcoholic medium. A series of metal complexes have been characterized quantitatively and qualitatively by using micro elemental analysis, FT-IR, 1 H NMR, Mass, UV-Vis, TGA/DTG, ESR, magnetic susceptibility data and molar conductance studies. From the spectral study, all the complexes obtained were octahedral in nature. Complexes exhibited enhanced anti-microbial activity in comparison to their ligand.

Introduction:-
Schiff bases are capable of forming coordinate bonds with many metal ions via azomethine group, and so they have been used for the synthesis of metal complexes due to their easy formation and strong metal binding ability. The Schiff base ligands and their corresponding metal complexes have expanded enormously and include a vast area of organometallic compounds and various aspects of bioinorganic chemistry [1-3] and have continued to play the role of one of the most important stereo chemical models in main group and transition-metal coordination chemistry due to their preparative accessibility, diversity and structural variability [4,5].
Schiff base and its metal complexes attract considerable interest and occupy an important role in the development of chemistry of chelate systems [6,7] due to the fact that especially these with N 2 O 2 tetradentate ligands, such systems closely resemble metallo-proteins. Some Schiff base complexes are also used as model molecules for biological oxygen carrier systems [8] as well as having applications in analytical fields [9] and have been reported to show a variety of biological actions by virtue of the azomethine linkage, which is responsible for various antibacterial, antifungal, antiviral, anti-inflammatory, antioxidant, anticancer, herbicidal, clinical and analytical activities [10][11][12].
On the other hand, azo compounds are very important molecules and have attracted much attention in both academic and applied research [13]. Azo compounds and their metal complexes are known to be involved in a number of biological reactions, such as inhibition of DNA, RNA, and protein synthesis, nitrogen fixation, and carcinogenesis [14,15]. Also, the azo compounds and their metal-azo complexes are extremely used in dyes and data storage [16][17][18].
As the continuation interest of our study of transition metal complexes, here we present the synthesis and characterization of new complex derivatives of 6-acetyl-7 hydroxy 4,8-dimethyl-2H-chromen-2-one. The antibacterial screening activities of the complexes obtained are carried out and the results are reported herein.  These cell suspensions were diluted with sterile MHA to provide initial cell counts of about 104 CFU/ml. The filamentous fungi were grown on sabouraud dextrose agar (SDA) slants at 28 o C for 10 days and the spores were collected using sterile doubled distilled water and homogenized.

Disc diffusion assay:-
Antibacterial activity was carried out using a disc diffusion method [21]. Petri plates were prepared with 20 ml of sterile Mueller Hinton Agar (MHA) (Himedia, Mumbai). The test cultures were swabbed on the top of the solidified media and allowed to dry for 10 mins. The tests were conducted at 1000 µg/disc. The loaded discs were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. Negative control was prepared using respective solvent. Streptomycin (10 µg/disc) was used as positive control. The plates were incubated for 24 h at 37 o C for bacteria and 48 h at 27 o C for fungi. A zone of inhibition was recorded in millimeters and the experiment was repeated twice.

Minimum inhibitory concentration (MIC):-
Minimum inhibitory concentration studies of synthesized compounds were performed according to the standard reference method for bacteria [22] and filamentous fungi [23] (CLSI 2008). Required concentrations (1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml, 31.25 µg/ml and 15.62 µg/ml) of the compounds were dissolved in DMSO (2%), and diluted to give serial two fold dilutions that were added to each medium in 96 well plates. An inoculum of 100 ml from each well was inoculated. The antifungal agent's ketoconazole, fluconazole for fungi and streptomycin, ciprofloxacin for bacteria were included in the assays as positive controls. For fungi, the plates were incubated for 48-72 h at 28 o C and for bacteria the plates were incubated for 24 h at 37 o C. The MIC for fungi was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. 5 ml of tested broth was placed on the sterile MHA plates for bacteria and incubated at respective temperatures. The MIC for bacteria was determined as the lowest concentration of the compound inhibiting the visual growth of the test cultures on the agar plate. The yield, elemental analysis and molar conductance data of metal complexes are presented in Table 1 and 2.

IR Spectra:-
The IR spectra of the complexes were compared with those of the free ligands in order to determine the coordination sites that may be involved in chelation. There were some guide peaks in the spectra of the ligands, which were helpful in achieving this goal. The IR spectral bands of metal(II) complexes are listed in Table 3. Table 3:-The important diagnostic IR absorption bands (in cm -1 ) of ligands and their complexes: In the IR spectrum of o-phenylenediamine, a pair of bands corresponding to ν (NH 2 ) was present at 3210 and 3270 cm -1 but was absent in the IR spectra of all the complexes [24]. A single broad medium band at 3157 and 3134 cm -1 was observed in the spectra of ligand iiib and iv respectively, which may be assigned to intramolecularhydrogen

Magnetic susceptibility, electronic and ESR spectral studies:-
The electronic absorption spectra of the Schiff base ligand and its Co(II), Ni(II), Zn(II), Cd(II) and Cu(II) complexes were recorded in DMF solution in the range of 200 to 800 nm regions and data are presented in Table 4. (v M 6 ). The spectra resemble that reported for octahedral complex [35]. Thus the various bands can be assigned to: 4 T 1 g → 4 T 2 g, 4 T 1 g → 4 A 2 g, 4 T 1 g→ 4 T 1 g.
The magnetic moment of the nickel (II) lay in the range 3.45 and 3.39 BM (iiic M 1 and v M 6 ) which corresponds to 2 unpaired electrons. The solution spectra of the nickel (II) complexes exhibited absorption in the region 370, 390, 470, 440 (iiic M 1 ) and 360, 380, 460, 480 nm (v M 6 ). The spectra resemble that reported for octahedral complex. Thus the various bands can be assigned to: 4 T 1 g → 4 T 2 g, 4 T 1 g → 4 A 2 g, 4 T 1 g→ 4 T 1 g and L→M charge transfer respectively.
The electronic spectrum of the copper(II) complex shows broad band absorption in the region 675 nm, 647 nm and 515 nm (iiic M 5 ), 678 nm, 642 nm and 519 nm (v M 10 ), which may be assign to 2 B 1g → 2 A 1g , (dx 2 -y 2 → dz 2 )(ν 1 ), 2 B 1g → 2 B 2g , (dx 2 -y 2 → dzy ) (ν 2 ), and 2 B 1g → 2 E g , (dx 2 -y 2 → dzy, dyz) (ν 3 ) transition and it is in conformity with octahedral geometry [36]. The broadness of the band which are similar in energy give rise to only one broad absorption band, and the broadness of the band is due to dynamic Jahn-Teller distortion. These data suggest that the Cu(II) complex have distorted octahedral geometry [37]. The ESR spectrum of Cu(II) complex has been recorded on X-band at frequency 9.1GHz at low temperature. The spectrum of Cu(II) (viiM 9 and viiM 10 ) complex is characteristic of axial symmetry with g ║ , g ┴ , and Δg values of 2.40, 2.07, (viiM 9 ) and 2.48, 2.07 (viiM 10 ) 0.37 (viiM 9 ) and 0.39 (viiM 10 ). Anisotropy of the g-tensor is due to the Jahn-Teller effect, which reduces the symmetry from octahedral to distorted octahedral. The fact that g ║ > g ┴ >2.0023 (g e ) and G = 5.06, > 4 indicates octahedral geometry of the Cu(II) ion of d x 2 -y 2 ground state with negligible exchange interaction in the complex [39]. Zn and Cd(II) ion with d 10 electronic configuration permits a wide range of symmetries and coordination numbers. Since d 10 configuration affords no crystal field stabilization, the stereochemistry of a particular compound depends on the size and polarizing power of the M(II) cation and the steric requirement of the ligands and have no d-d transition and are diamagnetic. The electronic spectra of complexes reveal a charge transfer bands π-π and n-π transitions in the vicinity of the Schiff base ligand at 288, 285 and 293, 278 nm for both complexes; and 387, 389 and 360, 374 cm -1 , respectively suggesting an octahedral structure which is common for d 10 systems.

HNMR spectra:-
The NMR spectra of Schiff bases were recorded in dimethyl sulfoxide (DMSO) solution, using tetramethylsilane (TMS) as an internal standard. Chemical shifts were reported as δ-values in parts per million (ppm) relative to Si (CH 3 ) 4 as relative reference (δ = 0 ppm) and to the solvent as internal reference.
The formation of the complex iiic M 3 from its ligand (iiib) is shown in Figure 1 and 2. The proton NMR spectrum shows two phenolic-OH groups at 10.40 ppm which are absent in the spectra of M(II) complexes. This indicates that the deprotonated phenolic-O atom is involved in chelation.

2198
The chemical shift data of the ligands and their metal complexes are presented in Table 6 and 7.

Mass spectral studies:-
The mass spectra of Schiff base ligands (iiib, iv) exhibited parent peaks due to molecular ions (M + ). The proposed molecular formula of these complexes was confirmed by comparing their molecular formula weights with m/z values. The mass spectrum for the Schiff base ligands iiib and iv ( Figure 3) showed a molecular ion peak at m/z = 440, 536 respectively. Thermal analysis:-In the present investigation, heating rates were suitably controlled at 10 • C min −1 under nitrogen atmosphere, and the weight loss was measured from the ambient temperature up to 800 • C. The data are provided in Table 8. The TGA and DTG curve of complex iiic M 1 is given in Figure 4.The weight loss for each chelate was calculated within the corresponding temperature ranges.
The stages of decomposition, temperature range, decomposition products as well as observed and calculated mass loss percentages of all the complexes are illustrated in table 8.  Racah parameters:-For the octahedral complexes the ligand field parameters Dq, B and C were found ( Table 9) by solving the secular equation and possible to simplify the problem to that of solving a quadratic equation in Dq, ν 1 , ν 2, ν 3 and ν 4 only.
These parameters indicate the significant covalent character of the metal ligand bonds.

Biology:-
The antimicrobial screenings of the synthesized compounds were undertaken using disc diffusion method. The screening results of the tested compounds against the gram positive bacteria, gram negative bacteria in addition to the pathogenic fungi microorganisms are summarized in Table 10-13.
The obtained data revealed that most of the compounds showed moderate to excellent activities against the tested microorganisms. Complexes ivM 1 , ivM 3 , ivM 9, ivM 2 , ivM 4 and ivM 10 were shown maximum zone of inhibition in both the bacterial and fungal strains compared to remaining compounds in the series with respect to standard Streptomycin and Ciprofloxacin in the case of bacterial, Fluconazole and Ketoconazole with respect to fungal strains. A comparative study of ligand and their metal complexes indicates that some of the metal chelates exhibits higher antimicrobial activity than the free ligands. The increase in antimicrobial activity of metal chelates is due to the presence of metal ion in the complexes.  Significant MIC values were observed against gram positive; gram negative bacteria and antifungal activity are summarized in Table 10-12. Compounds ivM 1 , ivM 2 , ivM 5, ivM 6 , ivM 7 and ivM 10 exhibited good MIC result among the series. In comparison, compound ivM 6 , ivM 7 and ivM 10 with halogen substituted is more potent and showed better activity for most of the tested bacteria and fungi. The antimicrobial activity of synthesized complex compounds was screened against eight bacteria and four fungi using in vitro disc diffusion method respectively. The results revealed that most of the synthesized complex compounds exhibited good biological activities due to the presence of metal ion in the complexes.