Design, Synthesis, Spectral Characterization and Antimicrobial Studies of Metals Based Nitrogen Donor Schiff Bases

A new series of bidentate nitrogen donor type Schiff bases (L)-(L) were prepared by the condensation reaction of ethylene-1, 2-diamine with 3, 4, 6-trimethylacetophenone, 2-aminoacetophenone and 2, 4-dimethylacetophenone in 1:2 molar ratio. The ligands were further coordinated with Co(II), Cu(II), Ni(II) and Zn(II) metals to produce their new metal complexes having an octahedral geometry. These compounds were characterized on the basis of their physical, spectral and analytical data. Elemental analysis and spectral data of the uncomplexed ligands and their metal(II) complexes were found to be in good agreement with their structures, indicating high purity of all the newly synthesized compounds. All ligands and their metal complexes were screened for antimicrobial activity. The results of antimicrobial activity indicated that metal complexes have significantly higher activity compared to their corresponding ligands. This higher activity might be due to chelation process which reduces the polarity of metal ion by coordinating with ligands.


Introduction
Metals based nitrogen and oxygen containing Schiff bases have been of great interest due to their extensive pharmacological activities [1]. It is well known that these nitrogen and oxygen donor ligands are involved in the chelation of the metals with their active sites [2]. Recently, coordination compounds of biologically active nature [3] have received much attention. Chelation introduces drastic changes in the biological properties of the ligands and their metal fraction [4]. It has been previously reported that many diseases have been cured by different chelating agents. A number of Schiff base metal complexes have been studied for biological activities like antibacterial [5], antifungal [6], antitumor [7], anticancer [8], anti-inflammatory [9], anticonvulsant [10], antiviral [11], ant iproliferative [12] and analgesic [13]. Similarly metal complexes of ethylene-1,2diamine are known to possess following biological activities such as antibacterial [14], antifungal [15], anticancer [16] and ant idiabetic [17]. In view of the significant structural, biological behavior and applications of ethylenediamine compounds, we wish to report the synthesis of a new class of Schiff bases (L 1 )-(L 3 ), derived from the reaction of ethylene-1,2-diamine with 3,4,6-trimethylacetophenone, 2aminoacetophenone and 2,4-dimethylacetophenone respectively, and their Co(II), Cu(II), Ni(II) and Zn(II) metal Based Nitrogen Donor Schiff Bases complexes (1)-(12) (Scheme). The compounds were characterized on the basis of physical properties, elemental analysis, infrared and uv-visible spectra, and antimicrobial activities. The Schiff bases and their metal chelates were screened for in-vitro antibacterial activity against six bacterial strains; Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis and in-vitro antifungal activity against six fungal strains; Trichophyton mentogrophytes, Epidermophyton floccosum, Aspergillus niger, Microscopum canis, Fusarium culmorum and Trichophyton schoenleinii. The antimicrobial results indicated that increased antimicrobial activity of Schiff bases against certain bacterial and fungal strains was due to chelation.

Materials and Methods
Chemicals used were of analytical grade and purchased from commercial sources Sigma Aldrich and were used without further purification. All ligand synthesis reactions were carried out in solvents that were purified and dried before use, using standard literature methods. The redistilled and de-ionized water was used in all experiments. Gallenkamp apparatus was used to determine melting points of synthesized ligands and decomposition temperature of the metal complexes and were uncorrected. Infrared spectra of solids (in a KBr matrix) were recorded in the 3700-370 cm -1 region on a Nicolet FT-IR Impact 400D infrared spectrometer. 1 H and 13 CNMR spectra were run on a Bruker Advance 300 MHz instrument. Mass spectrometry work was carried out by Ms. B. Woods N.U.I. Maynooth using an Agilent Technologies 6210 Time-of-Flight LC/MS. UV spectra were obtained on a Hitachi UV-3200 spectrophotometer. Microanalysis (C, H and N %) of the synthesized compounds was carried out using a CHN Analyzer on Perkin Elmer 2400 series II. Molar conductances of the transition metal complexes were measured using an Inolab Cond 720 Conductivity Bridge at room temperature using 0.001 molar solutions in DMF. A Stanton SM12/S Gouy balance was used to measure the magnetic susceptibility of the metal complexes at room temperature using mercury acetate ligand as a standard.

Chemistry of Synthesis of Ligands
Different ketones such as 3,4,6-trimethylacetophenone, 2aminoacetophenone and 2,4-dimethylacetophenone in methanol (20 mL) were added to a refluxed solution of ethylene-1,2-diamine in same solvent in 1:2 molar ratio for 20 minutes followed by the few drops of H 3 PO 4 . The reaction mixture was refluxed for 6h by monitoring through TLC. When the reaction was completed, it was cooled to room temperature, filtered and volume reduced to about one-third using rotary evaporator. The solid product thus obtained was filtered, washed with methanol and dried. It was recrystallized in hot methanol/ether (2:1). The same method was used for the preparation of ligands (L 1 )-(L 3 ).

Chemistry of Synthesis of the Transition Metal(II) Complexes
A methanolic solution (20 mL) of respective metal(II) salt chloride.nH 2 O (5 mmol) was added (n = 0, 2 or 6) to hot magnetically refluxed methanolic solution (20 mL) of the respective Schiff base ligand (10 mmol). The mixture was refluxed for 3 h, during which a precipitated product was formed. It was then cooled to room temperature, filtered, washed with methanol and finally with diethyl ether. The precipitated product thus obtained was dried and recrystallized in a mixture of hot aqueous methanol (1:2) to obtain TLC pure product. All complexes were prepared according to the same above mentioned procedure.

In-Vitro Antibacterial Activity
All newly synthesized Schiff bases (L 1 )-(L 3 ) and their transition metal(II) complexes (1)- (12) were screened for their in-vitro antibacterial activity against (Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis) bacterial strains by the agar-well diffusion method [18] and recorded in Table 3. Small portion (10 mL) of nutrient broth was inoculated with the test organisms and incubated at 37°C for 24 h. Using a sterile pipette, 0.6 mL of the broth culture of the test organism was added to 60 mL of molten agar which had been cooled to 45°C, mixed well and poured into a sterile petri dish. Duplicate plates of each organism were prepared. The agar was allowed to set and harden and the required numbers of holes were cut using a sterile cork borer ensuring proper distribution of holes on the border and one in the center. Agar plugs were removed. Different cork borers were used for different test organisms. Using a 0.1 mL pipette, 100 µL of the test sample dissolved in an appropriate solvent was poured into appropriately labelled cups. The same concentrations of the standard antibacterial agent (streptomycin in 1 mg/mL) and the solvent (as control) were used. The plates were left at room temperature for 2 h to allow diffusion of the sample and incubated face upwards at 37°C for 24 h. The diameter of the zones of inhibition was measured to the nearest mm.

In-Vitro Antifungal Activity
Antifungal activities of all compounds were studied against six fungal strains Trichophyton mentogrophytes, Epidermophyton floccosum, Aspergillus niger, Microscopum canis, Fusarium culmorum and Trichophyton schoenleinii according to recommended procedure [19] and recorded in Table 4. Test sample was dissolved in sterile DMSO to serve as stock solution. Sabouraud dextrose agar was prepared by mixing Sabouraud 4% glucose agar and agar agar in distilled water. It was then stirred with a magnetic stirrer to dissolve it and a known amount was dispensed into screw capped test tubes. Test tubes containing media were autoclaved at 121°C for 15 min. Tubes were allowed to cool to 50°C and the test sample of desired concentrations pipetted from the stock solution into the non-solidified Sabouraud agar media. Tubes were then allowed to solidify in a slanting position at room temperature. Each tube was inoculated with a 4 mm diameter piece of inoculum removed from a seven day old culture of fungi.

Minimum Inhibitory Concentration (MIC)
Compounds containing promising antibacterial activity were selected for minimum inhibitory concentration (MIC) studies [20]. The minimum inhibitory concentration was determined using the disc diffusion technique by preparing discs containing 10, 25, 50 and 100 µg ml -1 concentrations of the compounds along with standards at the same concentrations.

Results and Discussion
The condensation reaction of ethylene-1, 2-diamine with 3,4,6-trimethylacetophenone, 2-aminoacetophenone and 2,4-dimethylacetophenone in 1:2 molar ratio afforded three Schiff base ligands (L 1 )-(L 3 ) (Scheme). These ligands were air and moisture stable compounds. All of them were colored compounds. These were microcrystalline solids which melted at 92-202 o C. All were soluble in DMSO and DMF at room temperature and soluble on heating in methanol and ethanol. These bidentate ligands reacted readily with Co(II), Cu(II), Ni(II) and Zn(II) metals as their chlorides [CoCl 2 .6H 2 O, NiCl 2 .6H 2 O, CuCl 2 .2H 2 O and ZnCl 2 ] in methanol to form their metal(II) complexes (Scheme). All the synthesized metal(II) complexes were intensely colored except Zn(II) complexes which were white and all complexes were microcrystalline in nature. The metal(II) complexes decomposed without melting. They were all insoluble in common organic solvents such as ethanol, methanol, dichloromethane and acetone but soluble in DMSO and DMF. The spectral data and elemental analysis of the prepared ligands and their metal(II) complexes were in good agreement with their structure, indicating the high purity of all the compounds. The analytical data of the complexes indicated a 1:2 metal: ligand stoichiometry.

IR Spectra
Some of the characteristic IR peaks are reported in experimental part. The Schiff bases (L 1 )-(L 3 ) showed the characteristic azomethine (C=N) stretching [21] at 1633-1635 cm -1 , hence giving clue of condensation product. The ligand (L 2 ) displayed band at 3250 cm -1 resulting from NH 2 vibrations [22]. The comparison of the IR spectra of the Schiff bases (L 1 )-(L 3 ) with their metal(II) complexes (1)- (12) indicated that the Schiff bases were principally coordinated to the metal(II) ions bidentately. The vibrations of azomethine group appearing in the spectra of metal(II) complexes shifted to lower frequency (13-20 cm -1 ) at 1615-1622 cm -1 indicating the coordination of the azomethine nitrogen [23] with the metal(II) atoms. IR bands at 3250 cm -1 resulting from NH 2 vibrations of ligand (L 2 ) remained unchanged in the complexes showing their non involvement in the coordination. The following evidences further support the mode of chelation: Appearance of the new bands at 533-549 cm -1 assigned to v(M-N) vibrations [24] in their metal complexes and were absent in their ligands. All the metal(II) complexes displayed [25] N new broad peaks at 3468-3478 cm -1 which were assigned to water molecules. These new bands were only observed in the spectra of the complexes but absent in the spectra of the Schiff bases. Therefore, these clues supported the evidence of the participation of azomethine-N in the coordination. All these evidences compromise with the complexation of the metal(II) ions to the prepared Schiff bases.  .82 ppm as a singlet, respectively. The R 1 and R 4 protons of aromatic ring were observed at 6.75 and 7.14 ppm as a doublet, respectively. Ligand (L 2 ) displayed (CH 3 ) protons on azomethine linkage and (CH 2 ) protons of ethylene group at 2.16 and 3.87 ppm as a singlet, respectively. The (NH 2 ) protons present in the ligand (L 2 ) were observed at 5.33 ppm as a singlet. The R 2 and R 5 protons of aromatic ring were observed at 7.05 and 7.67 ppm as a doublet, and R 3 and R 4 protons appeared at 6.97 and 7.39 ppm as a triplet, respectively. The (CH 3 ) protons on azomethine linkage and aromatic ring, and (CH 2 ) protons of ethylene group present in the ligand (L 3 ) were observed at 2.24, 2.32 and 3.74 ppm as a singlet, respectively. The R 2 protons of aromatic ring were found at 6.75 ppm as double doublet, and R 4 & R 5 protons appeared at 7.14-7.37 ppm as a doublet, respectively. The coordination of the azomethine (C=N) nitrogen was confirmed by the downfield shifting of all the protons signal in their Zn(II) complexes. This downfield shifting of proton in Zn(II) complexes was attributed to the discharging of electronic cloud towards the Zn(II) ion. A singlet peak was observed in all the Zn(II) complexes at 10.5 ppm due to the presence of water molecules confirming the coordination of the ligands with the Zn metal atom. All other protons underwent downfield shift by 0.06-0.14 ppm owing to the increased conjugation on complexation with the zinc metal atom. Thus, the number of proton calculated from the integration curves [26] and obtained values of the expected CHN analysis agreed well with each others. 13 C NMR Spectra 13 C NMR spectra of the Schiff bases and their diamagnetic Zn(II) complexes were recorded in DMSO-d 6 . The 13 C NMR spectral data are reported along with their possible assignments in the experimental section and all the carbons were found in the expected regions. The 13 [27]. Furthermore, the conclusions drawn from these studies present further support to the modes of bonding discussed in their IR and 1 H NMR spectra.

Mass Spectra
The mass fragmentation pattern of the ligands (L 1 )-(L 3 ) followed the cleavage of C=N (exocyclic) and C=C bonds. The mass spectral data and the most stable fragmentation values of the ligands were depicted in experimental section. All the ligands showed pronounced molecular ion peaks. The data of the Schiff bases shown by mass spectra strongly confirmed the formation of the ligands possessing proposed structures and also, their bonding pattern.

Molar Conductances and Magnetic Measurements
Molar conductance studies of the complexes were carried out in DMF. The data of molar conductances (13.1-15.3 ohm -1 cm 2 mol -1 ) of metal(II) complexes (1)- (12) showed that these complexes were non electrolytic [28] in nature. The magnetic moment (B.M) values of all the metal(II) complexes, (1)- (12) were measured at room temperature. The observed magnetic moment values of Co(II) complexes were found in the range of 4.36-4.53 B.M indicating the Co(II) complexes as high-spin suggesting three unpaired electrons in an octahedral environment [29]. The Ni(II) complexes displayed magnetic moment values in the range of 3.33-3.57 B.M indicative of two unpaired electrons per Ni(II) ion suggesting these complexes to have an octahedral [30] geometry. The measured magnetic moment values 1.89-1.95 B.M for Cu(II) complexes are indicative of one unpaired electron per Cu(II) ion for d 9 -system suggesting octahedral [31] geometry. All the Zn(II) complexes were found to be diamagnetic [32] as expected.

Electronic Spectra
The electronic spectra of Co(II) complexes generally showed [33] three absorption bands in the region 8627-8699, 17736-17859 and 29674-29744 cm -1 which may be assigned to 4T 1 g→4T 2 g(F), 4T 1 g→4A 2 g(F) and 4T 1 g→4Tg(P) transitions respectively, and are suggestive of octahedral geometry around the Co(II) ion. The electronic spectral data of Ni(II) complexes showed [25] the bands in the region 8639-8659, 17679-17694 and 25716-257710 cm -1 assigned respectively, to the d-d transitions of 3A 2 g(F)→3T 2 g(F) and 3A 2 g(F)→3T 1 g(F). Also a strong band due to metal to ligand charge transfer was appeared at 29767-29816 cm -1 . The electronic spectra of all the Cu(II) complexes exhibited [34] absorption bands in the region at 8670-8735 and 17437-17543 cm -1 which may be assigned to the transitions 2Eg→2T 2 g. The high energy band at 29673-29731 cm -1 was due to forbidden ligand to metal charge transfer. On the basis of electronic spectra, octahedral geometry around the Cu(II) ion was suggested. The Zn(II) complexes did not show any d-d transition thus showing diamagnetic nature and their spectra were dominated only by a charge transfer band [35] at 28715-28785 cm -1 .

Biological Screening Antibacterial Bioassay (in-vitro)
The newly synthesized Schiff bases (L 1 )-(L 3 ) and their metal(II) complexes (1)- (12) have been subjected for the screening of their in-vitro antibacterial activity against Escherichia coli, Streptococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis bacterial strains according to standard procedure [18] and results were reported in Table 3. The obtained results were compared with those of the standard drug streptomycin. The synthesized ligand (L 1 ) showed a significant (16-20 mm) activity against all bacterial strains except Bacillus subtilis strain which possessed moderate (15 mm) activity. Ligand (L 2 ) displayed overall significant (17-20 mm) activity against all strains. Ligand (L 3 ) demonstrated significant (16-18 mm) activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis, and moderate (11-14 mm) activity was observed by Streptococcus faecalis, Pseudomonas aeruginosa. The metal complexes (1)-(4), (6)-(8) and (10)-(12) displayed overall significant (16-25 mm) activity against all the bacterial strains. Compound (5) exhibited a significant (17-21 mm) activity against Streptococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus subtilis bacterial strains except Escherichia coli and Staphylococcus aureus which possessed moderate (12-14 mm) activity. Beside this, the compound (9) exhibited a significant (16-20 mm) activity against all bacterial strains except Streptococcus faecalis which possessed moderate (14 mm) activity. The data reported in Table 3 clearly indicated that (L 2 ) showed overall good bacterial activity as compared to other two ligands. The Zn(II) complexes (4) and (8) of (L 2 ) were found to be the most active complexes. The metal(II) complexes showed [36] higher activity results upon complexation rather than their uncomplexed Schiff bases.

Minimum Inhibitory Concentration (MIC)
The synthesized ligands and their transition metal(II) complexes showing promising antibacterial activity (above 80%) were selected for MIC studies and obtained results are reported in Table 5. The antibacterial results indicated that the metal(II) complexes (1)-(4), (6)-(8) and (10)-(12) were found to display activity more than 80%, therefore, these complexes were selected for their MIC screening. The MIC values of these compounds fall in the range 23.11 to 48.43 µg/mL. Amongst these, the compound (4) was found to be the most active possessing maximum inhibition 23.11 µg/mL against bacterial strain P. Aeruginosa. Table 5. Minimum Inhibitory Concentration (µg/mL) of the Selected Compounds (1)-(4), (6)- (8) and (10)

Conclusions
Three bidentate N, O donor type Schiff base ligands were prepared by the condensation reaction of ethylene-1,2diamine with 3,4,6-trimethylacetophenone, 2aminoacetophenone and 2,4-dimethylacetophenone in 1:2 molar ratio These ligands were further complexed with transition metals to produce their new metal complexes. Elemental analysis and spectral data of the uncomplexed ligands and their metal(II) complexes were found to be in good agreement with their structures, indicating high purity of all the compounds with octahedral geometry. All ligands and their metal complexes were screened for antimicrobial activity. The results of antimicrobial activity indicated that metal complexes have significantly higher activity than their uncomplexed ligands. This higher activity might be due to chelation process which reduces the polarity of metal ion by coordinating with ligands.