SYNTHESIS OF SCHIFF BASES DERIVED FROM 2-HYDROXY-1-NAPHTH- ALDEHYDE AND THEIR TIN(II) COMPLEXES FOR ANTIMICRIBIAL AND ANTIOXIDANT ACTIVITIES Neelofar,

The current studies were designed to prepare tin(II) complexes of various Schiff base derivatives of 2-hydroxy-1-naphthaldehyde (HN) with L-histidine and sulfamethazine have been prepared and characterized by different physiochemical studies such as elemental analysis, atomic absorption, UV-Vis spectra, FTIR spectra, H–NMR, C-NMR and conductance studies. Antimicrobial and antioxidant activities were also calculated. Antibacterial activity was evaluated by the agar-well diffusion method. Two Gram-negative (Klebsiella pneumoniae and Escherichia coli) and three Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) bacterial strains were used. Antifungal activity was resolute against three fungal strains (Aspergillus niger, Aspergillus flavus and Alternaria solani) by using the agar tube dilution method. The antioxidant activity of ligands and their complexes was measured on the basis of the radical scavenging effect of 1,1-diphenyl-2-picryl-hydrazyl (DPPH)-free radical activity. Ligand HNSM exhibited excellent activities as antibacterial activity (22 mm), antifungal activity (55%) and antioxidant activity (119 ppm).


INTRODUCTION
Schiff bases are formed by reacting aldehydes or ketones with amines to form imine or azomethine group. These Schiff bases are extensively utilized as drugs which are biological active compounds due this reason it is widely used for industries [1,2]. Schiff bases are important due to carbon nitrogen double bond (C=N) which can coordinate with metal [3]. These important compounds have been reported to possess diverse biological activities such as antifungal, analgesic, anti-inflammatory, antibacterial, antioxidant, antitumor, cardiovascular, antitubercular [4][5][6][7] and as local anesthetic [8]. Apart from these synthetic imine groups are synthesized in labs various natural products compounds. The imine group present in such compounds has been shown to be critical in their biological activities. Schiff base ligands are able to coordinate in various oxidation states with many different metals [9]. Amino acids have importance in Schiff base complexes formation in which these can act as ligands due to their physiological and pharmacological activities. Moreover, these metal chelates appear to be involved in many of biological processes, such as transamination, racemization and carboxylation [10]. In particular histidine is a very important bioactive amino acid. The histidine binding to transition metal ions in biological systems has a major physicochemical role in several proteins as well as involved in the X-ray structural determination studies of metalloproteins like carbonic anhydrase, carboxypeptidase and plastocyanin. Additionally, histidine acting as the major zinc binding moiety in serum thus plays a major role in the zinc metabolism [11]. Hence, this histidine moiety is probably the most important metal-binding site in a large number of enzyme active centres in biological system. Furthermore, histidine seems to be involved in copper transport in blood [12]. Sulfonamides are an important class of antimicrobial agents owing to their low toxicity, low cost and excellent activity against bacterial diseases. They have the ability to prevent or slow down bacterial multiplication in wounds or infected systems without appreciable toxicity to the body tissues, thus sulfonamides are bacteriostatic rather than bactericidal [13]. Several sulfonamides Schiff bases were found to exhibit a wide spectrum of biological activities [14]. The chemistry of sulfonamides has been recently known as synthons in the preparation of various valuable biologically active compounds used as antibacterial, antitumor [15] and diuretic [16]. N-Substituted sulfonamides are recognized as anti-thyroid, anti-carbonic anhydrase, hypoglycaemic, and protease inhibitors [17].
We have reported herein the synthesis of Schiff bases derived from 2-hydroxy-1naphthaldehyde (HN) with L-histidine and sulfamethazine and their complexes with inorganic tin(II) chloride. The study of these synthesized Schiff base ligands and their metal complexes have been correlated to their biological and antioxidant activities with their structural aspects.

Chemicals and reagents
Solvents used in this study were of analytical grade. Tin(II) chloride, 2-hydroxy-1naphthaldehyde, L-histidine and sulfamethazine were of analytical grade and purchased from Sigma-Aldrich (Steinheim, Germany).

Instruments/equipments
1 H-NMR and 13 C-NMR were recorded at 400 MHz for 1 H and at 100 MHz for 13 C using TMS as internal standard with Bruker DPX-400 instrument in deuterated solutions. IR spectra were determined using a Jasco A-302 spectrophotometer. UV-Visible spectra were recorded using SP-3000 PLUS (Optima, Japan) spectrophotometers.

Bacterial and fungal strains
Two Gram-negative (Klebsiella pneumoniae and Escherichia coli) and three Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) for antibacterial activity and three fungal strains (Aspergillus niger, Aspergillus flavus and Alternaria solani) for antifungal activity were used for evaluation of microbial activities.

Preparation of ligands
Ligands were prepared according to the reported method in literature [18]. Equimolar ethanolic solution of 2-hydroxy-1-naphthaldehyde (0.01 mol, 1.7218 g) and L-histidine (0.01 mol, 1.553 g) and sulfamethazine (0.01 mol, 2.901 g) separately were refluxed with constant stirring for 40 min (Scheme 1). The precipitates formed were separated by sintered glass crucible and were dried in vacuum oven. Ligands were re-precipitated with ethanol. Physical properties of the synthesized ligands are given in Table 1.

Preparation of complexes
The complexes were prepared by the method in literature [19]. Equimolar ethanolic solution of ligands (HNLH and HNSM) (0.0025 mol) and SnCl 2 .2H 2 O (0.0025 mol) were refluxed with constant stirring for 2 h (Scheme 2). The precipitate formed was separated by filtering through   [20][21]. In order to evaluate the interfering effect of DMSO on the biological screening, alternate studies on DMSO solution showed no activity against any bacterial strains.

Antifungal assay
Antifungal activity against three fungal strains (Aspergillus niger, Aspergillus flavus and Alternaria solani) was determined by using the agar tube dilution method. Screw caped test tubes containing sabouraud dextrose agar (SDA) medium (4 mL) were autoclaved at 121 o C for 15 min. The tubes were allowed to cool at 50 o C and non-solidified SDA was loaded with 66.6 µL of compound from the stock solution (12 mg/mL in DMSO) to make a 200 µg/mL final concentration. The tubes were then solidifying a slanting position at room temperature. Each tube was inoculated with a 4mm diameter piece of inoculum from seven days old fungal culture. The media was supplemented with DMSO and Turbinafine (200 µg/mL) were used as a negative and a positive control, respectively [22].

Antioxidant activity (DPPH free radical scavenging activity)
The antioxidant activity of ligands and their corresponding tin(II) complexes and the standard were assessed on the basis of the radical scavenging effect of 1-diphenyl-2-picryl-hydrazyl (DPPH)-free radical activity by modified method. A freshly prepared DPPH solution exhibited a deep purple colour with a maximum absorption at 517 nm. This purple colour disappears when an antioxidant is present in the medium. Therefore, antioxidant molecules can quench DPPH free radicals and convert them to a colourless product, resulting in a decrease in absorbance at 517 nm.
The diluted working solutions of the test samples were prepared in methanol. Ascorbic acid was used as standard in 1-100 µg/mL solution. 0.002% of DPPH was prepared in methanol and 2 mL of this solution was mixed with 1 mL of sample solution and standard solution separately. These solution mixtures were kept in dark for 30 min and optical density was measured at 517 nm using Cecil-Elect Spectrophotometer. Methanol (1 mL) with DPPH solution (0.002%, 1 mL) Powder Maroon was used as blank. The free radical scavenging activities are expressed as the ratio percentage of the sample absorbance decrease and the absorbance of tested compound at 517 nm. The optical density was recorded and % inhibition was calculated using the formula [23]: where A = optical density of the blank and B = optical density of the sample. DPPH free radical scavenging activity % was proportional to the concentration of the tested compounds. Concentration of the sample at which the inhibition percentage reaches 50% is its IC 50 value. IC 50 value is negatively related to the antioxidant activity, as it expresses the amount of antioxidant needed to decrease its radical concentration by 50%.

Infrared studies
Relevant IR spectral bands of the ligands and their tin complexes along with their bands assignments are given in Table 3. In ligand HNLH and its complex [Sn(HNLH)Cl 2 (H 2 O)], the (C=N) bands of ligand was appeared at 1626 cm -1 and this important band was shifted (1620 cm -1 ) in the spectra of the complex as indicated by the spectra of the tin complex. This shift is inferenced that tin ion is coordinated to the nitrogen atom of azomethine in the ligand. The (COO -) bands at 1541 cm -1 and 1390 cm -1 for asymmetric and symmetric stretching in ligands are shifted to 1496 cm -1 and 1311 cm -1 in complexes. A new band at 3404 cm -1 is ascertained in the spectra of complex only and is assigned to υ(H 2 O) [8,10,12].
In ligand HNSM and its complex [Sn(HNSM)Cl 2 (H 2 O) 2 ], the azomethine bands of the free ligand appeared at 1589 cm -1 and was shifted (1564 cm -1 ) as indicated by the spectra of the tin complex. This shift suggests that tin ion is coordinated to the nitrogen atom of azomethine in the ligand. The bands for (SO 2 ) are observed at 1344 cm -1 and 1384 cm -1 in ligand and are assigned to (SO 2 ) asymmetric and symmetric stretching. These bands have been shifted to 1138 cm -1 and 1149 cm -1 respectively in the case of complex. The band for (S-N) is observed at 974 cm -1 in ligand and is shifted to 1018 cm -1 in the spectra of tin complex. A new band is observed at 3383 cm -1 appears only in complex and is assigned to (H 2 O) [15,17,24]. Table 3. FTIR data of ligands and their corresponding complexes.   13 C NMR Spectra of the compounds were recorded in DMSO-d 6 and these spectra also support the authenticity of the proposed structures ( Table 5). The signals observed in the range 156-171 ppm were due to azomethine carbons. These signals were shifted in the case of tin(II) complexes, indicating the coordination of azomethine nitrogen to the tin upon complexation [20].   Table 6. The molar conductance values have indicated that all the synthesized complexes were non-electrolytic in nature in DMF solvent. In non-electrolytic complexes the anion is bonded with metal and present within the coordination sphere while in electrolytic complexes the anion outside the coordination sphere. It has been supported by the low conductance data that the Schiff bases are coordinated to the tin atom in their deprotonated anionic forms and that the two chlorides ligands are also coordinated to the tin atom [20].

Electronic absorption spectral studies
The electronic absorption spectra of ligands and their Sn(II) complexes in DMF solution were carried out in the range of 200-1000 nm at room temperature as shown in Table 7. There is a shift of the bands to longer λ in spectra of all Sn(II) complexes which is a good evidence of complex formation. The bands observed at 270 nm and 280 nm in complexes and ligands both are attributed to intraligand π-π* transition of phenyl group. Band between 310 nm and at 400 nm in case of HNLH and HNSM ligands are due to n-π* transition. In case of complexes the band at 430 nm (in the case of HNLH) and at 390 nm (in the case of HNSM) is due to charge transfer transitions. It has been reported that the metal is capable of forming dп-pп* bonds with ligands containing nitrogen as the donor atom. The tin ion has vacant 5d orbital and consequently ligand-to-metal (L→M) binding can take place by the acceptance of pair of electrons from ligand to the tin metal. No d-d transition is expected for tin complexes [8,12,15,17,26,27]. antibacterial activity of the ligands and their corresponding tin complexes was tested positive ) bacteria. The antibacterial activity results clearly showed that the synthesized ligands and their tin complexes were biological active as shown in Table 8. The data obtained manifest that some of these compounds exhibited good activities against the tested organisms. Among the ligands and complexes, the highest activity were shown by HNLH against two bacterial species i.e. Klebsiella pneumonia and Staphylococcus aureus while its complex [Sn(HNLH)Cl 2 (H 2 O)] showed maximum activity against Escherichia coli among the tested bacteria. Ligand HNSM and its tin complex [Sn(HNSM)Cl 2 (H 2 O) 2 ] showed highest activity against Bacillus subtilis and Staphylococcus aureus, respectively.

Anti-fungal activity
The synthesized ligands and their tin(II) complexes were also subjected to anti-fungal activity against three fungal strains (Aspergillus niger, Aspergillus flavus and Alternaria solani) as shown in  Free radicals, which are involved in the process of lipid peroxidation, are considered to play a major role in numerous chronic pathologies, such as cancer and cardiovascular diseases. A compound with radical reducing power may serve as a potential antioxidant [28]. The IC 50 value, defined as the concentration of the sample leading to 50% reduction of the initial DPPH concentration, was calculated from the linear regression of plots of concentration of the tested compounds against the mean percentage of the antioxidant activity [29]. Antioxidants are free radicals scavengers that may prevent, protect, or reduce the extension of such damage. A number of chemical species including both synthetic and natural products may act as antioxidants [30]. The lower the IC 50 value, the higher is antioxidant activity of the tested samples. IC 50 values are given in